Indian Pharmacopoeia 2010 Volume 3

I
OPOEI
010
Volume III
lliQfI'lVlllit
Government of India
Ministry of Health & Family Welfare
Published by
THE INDIAN PHARMACOPOEIA COMMISSION
GHAZIABAD
2010, Indian Pharmacopoeia Commission

Application for reproduction should be made to
The Secretary-cum-Scientific Director
INDIAN PHAR1vlACOPOEIA COMMISSION
Sector-23, Raj Nagar,
Ghaziabad-201 002, India
Tel: (91-120)-2783401
Fax: (91-120)-2783311
Website: www.ipc.gov.in
E.mail: ipclab@vsnl.net
ISBN 81-903436-8-8 (Vol. III)
ISBN 81-903436-9-6 (Set)
Sixth Edition (6.0)

Effective from 1st September, 2010
Government of India
Ministry ofHealth & Family Welfare
On behalfof
Designed, produced & published by
The Indian Pharmacopoeia Commission

Indian Pharmacopoeia Laboratory
Govt. ofIndia, Ministry of Health & Family Welfare
. Sector 23, Raj Nagar, Ghaziabad-201 002
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Information Resources (NISCAIR)
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INDIAN
PHARMACOPOEIA
2010
Volume III
INDIAN PHARMACOPOEIA 2010

CONTENTS
VOLUME I
Notices
Preface
Vl1
Indian Pharmacopoeia Commission
IX
Acknowledgements
xv
Introduction
XVl1
General Chapters
VOLUME II
General Notices
711
General Monographs on Dosage Forms
719
Monographs on Drug substances, Dosage forms and

Pharmaceutical aids (A to M)
755
VOLUME III
1729
General Notices
Monographs on Drug substances, Dosage forms and
Pharmaceutical aids (N to Z)
Monographs on Vaccines and Immunosera for Human Use
Monographs on Herbs and Herbal Products
Monographs on Blood and Blood-related Products
Monographs on Biotechnology Products
Monographs on Veterinary Products
Index
1725
1737
2345
2463
2555
2591
2619
2755
VolumeIIl
CONTENTS
VOLUME 3
1729
General Notices
Monographs on Drug substances, Dosage fOTITIS and
Phannaceutical aids (N to Z)
Monographs on Vaccines and Immunosera for Human Use
Monographs on Herbs andHerbal Products
Monographs on Blood and Blood-related Products
Monographs on Biotechnology Products
Monographs on Veterinary Products
Index
1727
1737
2345
2463
2555
2591
2619
2755
GENERAL NOTICES
GENERAL NOTICES
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General Statements
Name
Official and OfficialArticles
Official Standards
Added Substances
Alternative Methods
Meanings ofTerms
1732
Provisions Applicable to Monographs and Test Methods
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Expression ofContents
Expression ofConcentrations
Abbreviated Statements
...
Weights and Measures
1732
1733
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Monographs
General Monographs
Production
Manufacture ofDrug Products
Excipients
1"
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Individual Monographs
Titles
Chemical Formulae
1733
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Atomic and Molecular Weights

Definitions
1734
1734
Statement ofContent
Category
1734
1734
Dose
Usual Strength
1734
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Description
Solubility
1734
1734
Test Methods
Identification
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GENERAL NOTICES
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Tests and Assays

Tests
Other Tests
Limits
Quantities
Apparatus
Reagents and Solutions
fudicators
Reference Substances
TestsAnimals
Calculation ofResults
Storage
Storage Containers
Labelling
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GENERAL NOTICES
IP 2010
General Notices
use but not necessarily to articles that may be sold under the
same name for other purposes.
General Statements
The General Notices provide the basic guidelines for the
interpretation and application of the standards, tests, assays,
and other specifications of the Indian Phannacopoeia (IP), as
well as to the statements made in the monographs and other
texts of the Phannacopoeia.
A monograph is to be constructed in accordance with any
general monograph or notice or any appendix, note or other
explanatory material that is contained in this Phannacopoeia
and that is applicable to that monograph. All statements
contained in the monograph, except where a specific general
notice indicates otherwise and with the exceptions given
hereafter, constitute standards for the official articles. An article
is not ofpharmacopoeial quality unless it complies with all of
the requirements stated.
Exceptions to the General Notices do exist, and where they
do, the wording in the individual monograph or an appendix
takes precedence and specifically indicates directions or the
intent. Thus, the specific wording of standards, tests, assays
and other specifications is binding wherever deviations from
the General Notices exist. Likewise, where there is no specific
mention to the contrary, the General Notices apply.
Name. The full name or title ofthis book, including addenda

thereto, is Indian Phannacopoeia 2010, abbreviated to IP 2010.
In the texts, the term "Pharmacopoeia" or "IP" without
qualification means the Indian Phannacopoeia 2010 and any
addenda thereto.
Official and Official Articles. The word 'official' wherever
used in this Pharmacopoeia or with reference thereto, is
synonymous with 'pharmacopoeial', with 'IP' and with
'compendial'. The designation IP in conjunction with the
official title on the label of an article is an indication that the
article purports to comply with IP standards.
The following tenns are used where the articles for which
monographs are provided are to be distinguished.
An official substance is a single drug or a drug entity or a

pharmaceutical aid for which the monograph title includes no
indication of the nature of a dosage fonn.
An official preparation is a drug product (dosage form) and is
the finished or partially finished preparation or product ofone
or more official substances fonnulated for use on the patient.
An article is an item for which a monograph is provided,
whether an official substance or an official preparation.
Official Standards. The requirements stated in the

monographs apply to articles that are intended for medicinal
The active pharmaceutical ingredients (drug substances),

excipients (pharmaceutical aids), phannaceutical preparations
(dosage forms) and other articles described in the monographs
are intended for human and veterinary use (unless explicitly
restricted to one of these uses).
The requirements given in the monographs are not framed to
provide against all possible impurities, contaminants or
adulterants; they provide appropriate limitation of potential
impurities only.
A preparation must comply throughout the shelf-life assigned
to it by the manufacturer; for opened or broached containers
the maximum period of validity for use may sometimes be
stated in the individual monograph. Nevertheless, the
responsibility for assigning the period of validity shall be
with the manufacturer.
Added Substances. An official substance, as distinguished

from an official preparation, contains no added substances
except when specifically pennitted in the individual monograph.
Unless otherwise specified in the individual monograph, or
elsewhere in the General Notices, suitable substances may be
added to an official preparation to enhance its stability,
usefulness or elegance, or to facilitate its preparation. Such
auxiliary substances shall be hannless in the amounts used,
shall not exceed the minimum quantity required to provide
their intended effect, shall not impair the therapeutic'efficacy
or the bioavailability or safety ofthe preparation and shall not
interfere with the tests and assays prescribed for detennining
compliance with the official standards. Particular care should
be taken to ensure that such substances are free from hannful
organisms. The freedom to the manufacturers to add auxiliary
substances imposes on them the responsibility of satisfying
the licensing authorities on the purpose of the addition and
the innocuity of such substances.
Alternative Methods. The tests and assays described are the
official methods upon which the standards of the
Pharmacopoeia are based. Alternative methods of analysis
may be used for control purposes, provided that the methods
used are shown to give results of equivalent accuracy and
enable an unequivocal decision to be made as to whether
compliance with the standards of the monographs would be
achieved if the official methods were used. Automated
procedures utilising the same basic chemistry as the test
procedures given in the monograph may also be used to
determine compliance. Such alternative or automated
procedures must be validated.
In the event of doubt or dispute, the methods of analysis of
the Phannacopoeia are alone authoritative and only the result
obtained by the procedure given in this Phannacopoeia is
conclusive.
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IP 2010
GENERAL NOTICES
Meanings ofTerms
Alcohol. The term "alcohol" without qualification means
ethanol (95 per cent). Other dilutions of ethanol are indicated
by the term "ethanol" or "alcohol" followed by a statement of
the percentage by volume of ethanol (C 2H60) required.
Desiccator. A tightly-closed container of suitable size and
design that maintains an atmosphere oflow moisture content
by means of silica gel or phosphorus pentoxide or other
suitable desiccant.
Drying and ignition to constant weight. Two consecutive
weighings after the drying or igniting operations do not differ
by more than 0.5 mg, the second weighing following an
additional period of drying or of ignition respectively
appropriate to the nature and quantity of the residue.
Ethanol. The term "ethanol" without qualification means
anhydrous ethanol or absolute alcohol.
Filtration. Unless otherwise stated, filtration is the passing of
a liquid through a suitable filter paper or equivalent device
until the filtrate is clear.
Freshly prepared. Made not more than 24 hours before it is
issued for use..
per cent v/v (percentage, volume in volume) expressing

the number ofmillilitres of substance in 100 millilitres of
final product.
The expression "parts per million" refers to the weight in
weight, unless otherwise stated.
Where the content of a substance is expressed in terms of the
chemical formula for that substance an upper limit exceeding
100 per cent may be stated. Such an upper limit applies to the
result ofthe assay calculated in terms ofthe equivalent content
ofthe specified chemical formula. For example, the statement
'contains not less than 99.0 per cent and not more than 101.0
per cent of C 7H 60 2 implies that the result of-the assay is not
less than 99.0 per cent and not more than 101.0 per cent,
calculated in terms ofthe equivalent content of C 7H 60 2 .
Where the result ofan assay or test is required to be calculated
with reference to the dried, anhydrous, ignited substance, or
the substance free from solvent, the determination of loss on
drying, water content, loss on ignition, content ofthe specified
solvent, respectively is carried out by the method prescribed
in the relevant test in the m o n o g r a p h .
Expression of Concentrations. The following expressions in
addition to the ones given under Expression of Content are
also used:
Label. Any printed packing material, including package inserts

that provide information on the article.
per cent w/v (percentage, weight in volume) expressing

the number of grams of substance in 100 millilitres of
product
Negligible. A quantity not exceeding 0.50 mg.

Solution. Where the name of the solvent is not stated,
"solution" implies a solution in water. The water used complies
with the requirements of the monograph on Purified Water.
The term 'distilled water' indicates Purified Water prepared by
distillation.
Temperature. The symbol 0 used without qualification
indicates the use of the Celsius thermometric scale.
Water. Ifthe term is used without qualification it means Purified
Water of the Pharmacopoeia. The term 'distilled water'
indicates Purified Water prepared by distillation.
Water-bath. A bath of boiling water unless water at another
temperature is indicated. Other methods of heating may be
used provided the required temperature is approximately
maintained but not exceeded.
Provisions Applicable to Monographs and Test Methods
Expression of Contents. Where the content of a substance is
defined, the expression "per cent" is used according to
circumstances with one of two meanings:
per cent w/w (percentage, weight in weight) expressing
the number of grams of substance in 100 grams of fmal
product,
per cent v/w (percentage, volume in weight) expressing

the number of millilitres of substance in 100 grams of
product.
Usually, the strength of solutions of solids in liquids is
expressed as percentage weight in volume, ofliquids in liquids
as percentage volume in volume, of solids in semi-solid bases
(e.g. creams) and of gases in liquids as percentage weight in
weight.
When the concentration of a solution is expressed as parts of
dissolved substance in parts of solution, it means parts by
weight (g) ofa solid in parts by volume (m!) ofthe final solution;
as parts by weight (g) of a gas in parts by weight (g) of the
final solution.
When the concentration of a solution is expressed in molarity
designated by the symbol M preceded by a number, it denotes
the number ofmoles ofthe stated solute contained in sufficient
Purified Water (unless otherwise stated) to produce 1 litre of
solution.
Abbreviated Statements. Incomplete sentences are employed
in parts of the monographs for directness and brevity (for
; Relative Density.
example, Iodine Value. Not more than
.......to ........) Where the tests are abbreviated, it is to be
understood that the test method referred to in brackets
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GENERAL NOTICES
IP 2010
provides the method to be followed and that the values

specified are the applicable limits.
Weights and Measures. The metric system of weights and
measures is employed in the Pharmacopoeia. All measures are
required to be graduated at 25 and all measurements in tests
and assays, unless otherwise stated, are to be made at that
temperature. Graduated glass apparatus used in analytical
operations shall comply with the requirements stated in
Chapter 2.1.6
Monographs
(;eneralMonographs
General monographs on dosage fonus include requirements
ofgeneral application and apply to all preparations within the
scope of the Introduction section of the general monograph,
except where a preamble limits the application. The
requirements are not necessarily comprehensive for a given
specific preparation; additional requirements may sometimes
be given in the individual monograph for it.
Production. Statements given under the heading Production
relate to particular aspects of the mimufacturing process and
are not necessarily comprehensive. However, they are
mandatory instructions to manufacturers. They may relate,
for example, to source materials, to the manufacturing process
and its validation and control, to any in-process testing that
is to be carried out by the manufacturer on the final product
either on selected batches or on each batch prior to release.
All this cannot be verified on a sample ofthe final product by
an independent analyst. It is for the licensing authority to
verify that the instructions have been followed.
The absence of a section on Production does not imply that
attention to features such as those given above is not required.
An article described in a monograph of the Pharmacopoeia is
to be manufactured in accordance with the principles of good
manufacturing practice and in accordance with the
requirements of the Drugs and Cosmetics Rules, 1945. The
general principles applicable to the manufacture and quality
assurance of drugs and preparations meant for human use
apply equally to veterinary products as well.
Manufacture of Drug Products. The opening definitive
statement in certain monographs for drug products is given in
terms ofthe active ingredient(s) only. Any ingredient(s) other
than those included in the statement, must comply with the
general notice on Excipients and the product must conform to
the Pharmacopoeial requirements.
Official preparations are prepared only from ingredients that
comply with the requirements of the pharmacopoeial
monographs for those individual ingredients for which
monographs are provided.
Excipients. Any substance added in preparing an official

preparation shall be innocuous, shall have no adverse influence
in the therapeutic efficacy of the active ingredients and shall
not interfere with the tests and assays of the Pharmacopoeia.
Care should be taken to ensure that such substances are free
from halwful organisms.
Individual Monographs
Drug products that are the subject ofan individual monograph
are also required to comply with the tests given in the general
monographs.
Titles. The main title for a drug substance is the International
Non-proprietary Name (INN) approved by the World Health
Organization. Subsidiary names and synonyms have also been
given in some cases; where included, they have the same
significance as the main title.
The main titles of drug products are the ones commonly
recognised in practice. Synonyms drawn from the full nonproprietary name of the active ingredient or ingredients have
also been given. Where, however, a product contains one or
the other ofdifferent salts ofan active molecule, the main title
is based on the full name ofthe active ingredient. For example,
Chloroquine Phosphate Tablets and Chloroquine
SulphateTablets.
Chemical Formulae. When the chemical structure ofan official
substance is known or generally accepted, the graphic and
molecular formulae are normally given at the beginning ofthe
monograph for. information. This information refers to the
chemically pure substance and is not to be regarded as an
indication of the purity of the official material. Elsewhere, in
statement of purity and strength and in descriptions of
processes of assay, it will be evident from the context that the
formulae denote the chemically pure substances.
Where the absolute stereochemical configuration is specified,
the International Union of Pure and Applied Chemistry
(IUPAC) RlS and E/Z systems of designation have been used.
If the substance is an enantiomer of unlmown absolute
stereochemistry, the sign ofthe optical rotation, as determined
in the solvent and under the conditions specified in the
monograph, has been attached to the systematic name. An
indication ofsign ofrotation has also been given where this is
incorporated in a trivial name that appears on an IUPAC
preferred list.
Atomic and Molecular Weights. The atomic weight or
molecular weight is shown, as and when appropriate at the
top right hand corner of the monograph. The atomic and
molecular weights and graphic formulae do not constitute .
analytical standards for the substances described.
Definition. The opening statement of a monograph is one
that constitutes an official definition of the substance,
preparation or other article that is the subject of the
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IP 2010
GENERAL NOTICES
monograph. In certain monographs for pharmaceutical

preparations the statement is given in terms of the principal
ingredient(s).
In monographs on vegetable drugs, the definition indicates
whether the subject of the monograph is, for example, the
whole drug or the drug in powdered fonn.
Certain pharmaceutical substances and other articles are
defined by reference to a particular method ofmanufacture. A
statement that a substance or article is prepared or obtained
by a certain method constitutes part of the official definition
and implies that other methods are not permitted. A statement
that a substance may be prepared or obtained by a celiain
method, however, indicates that this is one possible method
and does not imply that other methods are not permissible.
Statement of content. The limits of content stated are those
detennined by the method described under Assay.
Category. The statement of category is provided for
information and is indicative ofthe medical or pharmaceutical
basis for recognition in the Pharmacopoeia. It generally
represents an application of the best known pharmacological
action of the article or of its active ingredient. In the case of
pharmaceutical aids it may indicate the more common usage
ofthe article. The statement is not intended to limit iIi any way
the choice or use of the article nor to indicate that it has no
other activity or use.
Dose. Doses mentioned in the Pharmacopoeia are intended
merely for general guidance and represent, unless otherwise
stated, the average range of quantities which are generally
regarded as suitable for adults when administered by mouth.
They are not to be regarded as binding upon the prescribers.
The medical practitioner will exercise his own judgment and
act on his own responsibility in respect of the amount of any
therapeutic agent he may prescribe or administer or the
frequency of its administration. If it is usual to administer a
drug by a method other than by mouth, the single dose suitable
for that method of administration is mentioned. In the case of
some preparations notes have been given below the statement
of doses to show the approximate quantities of active
ingredients contained in the maximal doses as information for
the prescriber.
Usual Strength. The statement on the usual strength(s) of a
preparation given in the individual monograph indicates the
strength(s) usually marketed for information ofthe pharmacist
and the medical practitioner. It does not imply that a strength
other than the one(s) mentioned in the individual monograph
meeting all the prescribed requirements cannot be
manufactured and marketed with the approval of the
appropriate authority.
Description. The statements under the heading Description
are not to be interpreted in a strict sense and are not to be
regarded as official requirements.
Solubility. Statements on solubility are given in Chapter 2.4.26

and are intended as infonnation on the approximate solubility
at a temperature between 15 and 30, unless otherwise stated,
and are not to be considered as official requirements. However,
a test for solubility stated in a monograph constitutes part of
the standards for the substance that is the subject of that
monograph.
Test Methods
References to general methods of testing are indicated by test
method numbers in brackets immediately after the heading of
the test or at the end of the text.
Identification. The tests given under the heading Identification
are not necessarily sufficient to establish absolute proof of
identity. They provide a means of verifying that the identity
of the material under examination is in accordance with the
label on the container.
In celtain monographs alternative series ofidentification tests

are given; compliance with either one or the other set of tests
is adequate to verify the identity of the aIticle.
When tests for infrared absorption are applied to material
extracted fi'om fonnulated preparations, strict concordance
with the specified reference spectrum may not always be
possible, but nevertheless a close resemblance between the
spectrum ofthe extracted material and the specified reference
spectrum should be achieved.
Tests andAssays
The tests and assays are the official methods upon which the
standards of the Pharmacopoeia depend. The requirements
are not framed to take into account all possible impurities. It is
not to be presumed, for example, that an impurity that is not
detectable by means of the prescribed tests is tolerated.
Material found to contain such an impurity is not of
pharmacopoeial quality ifthe nature or amount ofthe impurity
found is incompatible with good pharmaceutical practice.
Pharmacopoeial methods and limits should be used merely as
compliance requirements and not as requirements to guarantee
total quality assurance. Tests and assays are prescribed for
the minimum sample available on which the attributes ofthe
alticle should be measured. Assurance of quality must be
ensured by the manufacturer by the use of statistically valid
sampling and testing programmes.
Tests. Unless otherwise stated, the assays and tests are carried
out at a temperature between 20 and 30.
Where it is directed that an analytical operation is to be carried
out 'in subdued light', precautions should be taken to avoid
exposure to direct sunlight or other strong light. Where a
procedure is directed to be performed 'protected from light'
precautions should be taken to exclude actinic light by the
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IP 2010
GENERAL NOTICES
use oflow-actinic glassware, working in a dark room or similar

procedures.
For preparations other than those of fixed strength, the
quantity to be taken for a test or an assay is usually expressed
in terms of the active ingredient. This means that the quantity
ofthe active ingredient expected to be present and the quantity
of the preparation to be taken are calculated from the strength
stated on the label.
Other Tests. In the monographs on dosage forms and certain
preparations, under the sub-heading 'Other tests' it is stated
that the article complies with the tests stated under the general
monograph ofthe relevant dosage form or preparation. Details
of such tests are provided in the general monographs.
Limits. The limits given are based on data obtained in normal
analytical practice. They take into account nonnal analytical
errors, of acceptable variations in manufacture and of
deterioration to an extent that is acceptable. No further
tolerances are to be applied to the limits for determining whether
or not the article under examination complies with the
requirements of the monograph.
Quantities. Unless otherwise stated, the quantities to be taken
for assays, limit tests and other tests are of the substance
under examination.
In tests with numerical limits and assays, the quantity stated
to be taken for testing is approximate. The amount actually
used, which may deviate by not more than 10 per cent from
that stated, is accurately weighed or measured and the result
ofanalysis is calculated from this exact quantity. In tests where
the limit is not numerical but usually depends upon
comparison with the behaviour of a reference in the same
conditions,. the stated quantity is taken for testing. Reagents
are used in the prescribed amounts.
Quantities are weighed or measured with an accuracy
commensurate with the indicated degree of precision. For
weighings, the precision is plus or minus 5 units after the last
figure stated. For example, 0.25 g is to be interpreted as 0.245
g to 0.255 g. For the measurement of volumes, if the figure
after the decimal point is a zero or ends in a zero, e.g. 10.0 ml or
0.50 ml, the volume is measured using a pipette, a volumetric
flask or a burette, as appropriate; in other cases, a graduated
measuring cylinder or a graduated pipette may be used.
Volumes stated in microlitres are measured using a micropipette
or microsyringe.
The term 'transfer' is used generally to indicate a quantitative
operation.
Apparatus. Measuring and weighing devices and other
apparatus are described in the chapter entitled'Apparatus for
Tests and Assays'. A specification for a definite size or type
of container or apparatus in a test or assay is given merely as
a recommendation.
Unless otherwise stated, comparative tests are carried out

using identical tubes of colourless, transparent, neutral glass
with a flat base, commonly known as Nessler cylinders.
Reagents and Solutions. The reagents required for the tests
and assays of the Pharmacopoeia are defined in the various
chapters showing their nature, degree of purity and the
strengths of the solutions to be made from them. The
requirements set out are not intended to imply that the materials
are suitable for use in medicine; reagents not covered by
monographs in the phannacopoeia shall not be claimed to be
ofIP quality.
The term' analytical reagent grade of commerce' implies that
the chemical is ofa high degree ofpurity wherein the limits of
various impurities are known. Where it is directed to use a
'general laboratory reagent grade ofcommerce' it is intended
that a chemically pure grade material, not necessarily required
to be tested for limiting or absence of certain impurities, is to
be used.
Indicators. Where the use ofan indicator solution is mentioned
in an assay or test, approximately 0.1 ml of the solution shall
be added, unless otherwise directed.
Reference Substances. Certain monographs require the use
of a chemical reference substance or a biological reference
preparation or a reference spectrum These are authentic
specimens chosen and verified on the basis oftheir suitability
for intended use as prescribed in the Pharmacopoeia and are
not necessarily suitable in other circumstances.
IP Reference Substances, abbreviated to IPRS (and referred
to as RS in the individual monographs) are issued by the
Indian Pharmacopoeia Commission (IPC). They are the official
standards to be used in cases of arbitration. Secondary
Standards (Working Standards) may be used for routine
analysis, provided they are standardized at regular intervals
against the Reference Substances
Biological Reference Substances, .also abbreviated to IPRS
and Standard Preparations of antibiotics are issued by
agencies authorised by the IPC. They are standardized against
the International Standards and Reference Preparations
established by the World Health Organization (WHO). The
potency of these preparations is expressed in International
Units.
.
Reference spectra are published by the IPC and they are
accompanied by information concerning the conditions used
for sample preparation and recording of the spectra.
Test Animals. Unless otherwise directed, animals used in a
test or an assay shall be healthy and are drawn from a unifOlID
stock, and have not previously been treated with any material
that will interfere with the test or the assay.
Calculation of Results. In determining compliance with a
numerical limit in assay or test, the result should be calculated
1735
IP 2010
GENERAL NOTICES
to one decimal place more than the significant figures stated

and then rounded up or down as follows: if the last figure
calculated is 5 to 9, the preceding figure is increased by 1; if it
is 4 or less, the preceding figure is left unchanged.
Where no specific storage directions or limitations are given

in the monograph or by the manufacturer, it is to be understood
that the storage conditions include protection from moisture,
freezing and excessive heat (any temperature above 40).
Storage. Statements under the side-heading Storage constitute

non-mandatory advice. The articles ofthe Pharmacopoeia are
to be stored under conditions that prevent contamination and,
as far as possible, deterioration. Precautions that should be
taken in relation to the effects of the atmosphere, moisture,
heat and light are indicated, where appropriate, in the individual
monograph.
Storage Containers. The requirements, guidance and

information on containers for pharmaceutical use are given in
the chapter entitled Containers (6.1)
Specific directions are given in some monographs with respect

to the temperatures at which Pharmacopoeial articies should
be stored, where it is considered that usage at a lower or
higher temperature may produce undesirable results. The
storage conditions are defined by the following terms:
Store in a dry, well ventilated place at a temperature not
exceeding 30
Store in a refrigerator (20 to 8). Do not freeze
Store in a freezer (-2 to -18)
Store in a deep freezer (Below _18)
Storage conditions not related to temperature are indicated in
the following terms:
Store protected from light
Store protected from light and moisture
In general, an article should be packed in a well-closed

container i.e. one that protects the contents from contamination
by extraneous solids, liquids or vapours and from loss of the
article under normal conditions of handling and storage.
Where, additionally, loss or deterioration of the article from
effervescence, deliquescence or evaporation under normal
conditions ofstorage is likely, the container must be capable
of being tightly closed, and re-closed after use.
In certain cases, special requirements of pack have been
indicated in some monographs under Storage, using
expressions that have been defined in chapter 6.1.
Labelling. The labelling of drugs and pharmaceuticals is

governed by the Drugs and Cosmetics Rules, 1945. The
statements that are given in the monographs under the sideheading 'Labelling' are not comprehensive. Only those that
are necessary to demonstrate compliance or otherwise with
the monograph have been given and they are mandatory. For
example, in the monograph on Betamethasone Sodium Tablets
the labelling statement is "The label states the strength in
terms ofthe equivalent amount ofbetamethasone". Any other
statements are included as recommendations.
1736
MONOGRAPHS
DRUG SUBSTANCES, DOSAGE FORMS

AND
PHARMACEUTICAL AIDS
NtoZ
....
1737
1739
MONOGRAPHS
N
1743
1743
1744
1745
1745
1747
1748
1749
1750
1751
1751
1752
1753
1754
1755
1756
1757
1758
1758
1759
1760
1761
1762
1763
1764
NalidixicAcid
Nalidixic Acid Tablets
Nalorphine Hydrochloride
Nalorphine Injection
Naloxone Hydrochloride
Naloxone Injection
Naltrexone Hydrochloride
Naltrexone Tablets
Nandrolone Decanoate
Nandrolone Decanoate Injection
Nandrolone Phenylpropionate
Nandrolone Phenylpropionate Injection
Naphazoline Nitrate
Naproxen
Naproxen Oral Suspension
Naproxen Suppositories
Naproxen Sustained-release Tablets
Naproxen Tablets
Nebivolol Hydrochloride
Nebivolol Tablets
NelfinavirMesylate
Nelfinavir Mesylate Oral Powder
Nelfinavir Tablets
Neomycin Sulphate
Neomycin Eye Drops
1765
1766
1767
1767
1768
Neomycin Eye Ointment

Neostigmine Bromide
Neostigmine Tablets
Neostigmine Methylsulphate
Neostigmine Injection
1739
MONOGRAPHS
1769
1770
1771
1772
1773
1774
1775
1776
Neotame
Nevirapine
Nevirapine Oral Suspension
Nevirapine Tablets
Niclosamide
Niclosamide Tablets
Nicotinamide
Nicotinamide Tablets
1776
1777
1777
1778
1779
1780
1781
1782
NicotinicAcid
Nicotinic Acid Tablets
Nicoumalone
Nicoumalone Tablets
Nifedipine
Nifedipine Capsules
Nifedipine Sustained-release Tablets
Nifedipine Tablets
1783
1784
1784
1785
Nikethamide
Nikethamide Injection
Nitrazepam
Nitrazepam Tablets
1786
1787
1787
1788
1789
1790
1791
1792
Nitrofurantoin
Nitrofurantoin Tablets
Nitrofurazone
Nitrous Oxide
Noradrenaline Bitartrate
Noradrenaline Bitartrate Injection
Norethisterone
Norethisterone Tablets
1793
1794
1794
Norfloxacin
Norfloxacin Eye Drops
Norfloxacin Tablets
1795
1796
Norgestrel
Norgestrel and Ethinyloestradiol Tablets
1740
MONOGRAPHS
1797
1797
1798
1800
1800
1801
1802
Nortriptyline Hydrochloride
Nortriptyline Tablets
Noscapine
Noscapine Linctus
Novobiocin Sodium
Nystatin
Nystatin Ointment
Nystatin Pessaries
1803
1803
Nystatin Tablets
1741
IP 2010
NALIDIXIC ACID TABLETS

Reference solution (a). A 0.002 per cent w/v solution of the
substance under examination in dichloromethane.
Nalidixic Acid
Reference solution (b). A 0.0008 per cent w/v solution of the

Reference solution (c). A 0.1 per cent w/v solution of nalidixic
acid RS in dichloromethane.
Mol. Wt. 232.2

Nalidixic Acid is l-ethyl-l ,4-dihydro-7-methyl-4-oxo-l ,8naphthyridine-3-carboxylic acid.
Nalidixic Acid contains not less than 99.0 per cent and not
more than 101.0 per cent of ClzHI2Nz03, calculated on the
dried basis.
Apply to the plate 10 III ofeach solution. After development,

dry the plate in air and examine in ultraviolet light at 254 nm.
Any secondary spot in the chromatogram obtained with test
solution (a) is not more intense than the spot in the
chromatogram obtained with reference solution (a) and not
more than one such spot is more intense than the spot in the
chromatogram obtained with reference solution (b).
Heavy metals (2.3.13).1.0 g complies with the limit test for
heavy metals, Method B (20 ppm).
Sulphated ash (2.3.18) Not more than 0.1 per cent.
Category. Antibacterial.
Loss on drying (2.4.19). Not more than 0.5 per cent, determined
on 1.0 g by drying in an oven at 105 0
Dose. 2 to 4 g daily, in divided doses.

Description. A white to slightly yellow, crystalline powder.
Assay. Weigh accurately about 0.15 g, dissolve in 10 ml of
Identification
Test A may be omitted iftests B, C andD are carried out. Tests
B, C and D may be omitted if test A is carried out.
A. Determine by infrared absorption spectrophotometry (2.4.6).

Compare the spectrum with that obtained with nalidixic acid
RS or with the reference spectrum ofnalidixic acid.
B. When examined in the range 230 nm to 360 nm (2.4.7), a
0.0005 per cent w/v solution in 0.1 M sodium hydroxide shows
absorption maxima at about 258 nm and 334 nm; ratio of the
absorbance at about 258 nm to that at about 334 nm, 2.2 to 2.4.
dichloromethane, add 30 ml of 2-propanol and 10 mlof carbon

dioxide-free water and titrate with 0.1 M ethanolic sodium
hydroxide, determining the end-point potentiometrically
(2.4.25) and using a glass electrode as the indicator electrode

and a silver-silver chloride reference electrode with a sleeve
diaphragm or a capillary tip filled with a saturated solution of
lithium chloride in ethanol. Throughout the titration keep
the temperature ofthe solution at 15 0 to 20 0 and pass a current
of nitrogen through the solution.
1 ml of 0.1 M ethanolic sodium hydroxide is equivalent to
0.02322 g ofClzHlzNz03.
C. In the test for Related substances, the principal spot in the

chromatogram obtained with test solution (b) corresponds to
that in the chromatogram obtained with reference solution (c).
Storage. Store protected from light and moisture.
D. Dissolve 0.1 gin 2 ml of hydrochloric acid and add 0.5 ml

of a 10 per cent w/v solution of 2-naphthol in ethanol (95 per
cent); an orange-red colour develops.
Nalidixic Acid Tablets
Tests
Related substances. Determine by thin-layer chromatography
(2.4.17), coating the plate with silica gel HF254. '
Mobile phase. A mixture of 70 volumes of ethanol (95 per
cent), 20 volumes of dichloromethane and 10 volumes of5 M
ammonia.
Test solution (a). Dissolve 0.2 g of the substance under
examination in 10 ml of dichloromethane.
Test solution (b). A 0.1 per cent w/v solution of the substance
under examination in dichloromethane.
Nalidixic Acid Tablets contain not less than 95.0 per cent and
not more than 105.0 per cent ofthe stated amount ofnalidixic
acid, ClzHlzNz03.
Usual strengths. 250 mg; 500 mg.
Identification
To a quantity ofthe powdered tablets containing 1g ofNalidixic
Acid add 50 ml of chloroform, shake for 15 minutes, filter and
evaporate the filtrate to dryness. The residue, after drying at
105 0 , complies with the following tests.
Compare the spectrum with that obtained with nalidixic acid
RS or with the reference spectrum ofnalidixic acid.
1743
NALIDIXIC ACID TABLETS
IP 2010

absorbance at about 258 nm to that at about 334 nm, 2.2 to 2.4.
Nalorphine Hydrochloride contains not less than 97.0 per cent

and not more than 103.0 per cent ofCI9HzIN03,HCl, calculated
on the dried basis.
Tests
Dose. By intravenous injection, 5 mg, repeated twice at three

minute intervals, if necessary.

(2.4.17), coating the plate with silica gel HF254.

cent), 20 volumes of dichloromethane and 10 volumes of
5 Mammonia.
Test solution. Shake a quantity of the powdered tablets
containing 0.1 g ofNalidixic Acid with 50 ml of chloroform for
15 minutes, filter, evaporate the filtrate to dryness and dissolve
the residue in 5 ml of chloroform.
Reference solution. Dilute 1 volume of the test solution to
200 volumes with chloroform.
Apply to the plate 10 III of each solution. After development,
Any secondary spot in the chromatogram obtained with the
test solution is not more intense than the spot in the
chromatogram obtained with the reference solution.
Other tests. Complies with the tests stated under Tablets.
Assay. Weigh and powder 20 tablets. Weigh accurately a
quantity of the powder containing about 0.1 g of Nalidixic
Acid, add 150 ml of 0.1 M sodium hydroxide, shake for 3
minutes, dilute to 200.0 ml with 0.1 M sodium hydroxide, mix
and allow to stand for 15 minutes. Dilute 2.0 ml ofthe solution
to 100.0 ml with water and measure the absorbance of the
resulting solution at the maximum at about 334 nm (2.4.7),
using 0.1 M sodium hydroxide as the blank Calculate the
content of C 12H 12N z0 3 taking 494 as the specific absorbance
at 334 nm.
Category. Narcotic antagonist.
Description. A white or almost white, crystalline powder;

odourless. It slowly darkens on exposure to air and light.
Identification
Test A may be omitted if tests B, C, D and E are carried out.
Tests C and D may be omitted if tests A, Band E are carried
out.
Compare the spectrum with that obtained with nalorphine
hydrochloride RS.

an absorption maximum only at about 298 nm; absorbance at
about 298 nm, about 0.6.
C. To 10 ml of a 2 per cent w/v solution add 0.05 ml of dilute
ammonia solution; a white precipitate soluble in sodium
hydroxide solution is produced.
D. Dissolve 2 mg in 2 ml of water, add 0.15 ml ofpotassium
fen-icyanide solution containing, in each ml, 0.05 mlofferric
chloride solution; a deep bluish green colour is produced
immediately.
E. Gives reaction A ofchlorides (2.3.1).
Tests
Melting range (2.4.21). 260 0 to 263 0
Acidity. Dissolve 0.2 g in 10 ml offreshly boiled and cooled
water and titrate with O. 02 M sodium hydroxide using methyl
red solution as indicator; not more than 0.2 ml of 0.02 M
sodium hydroxide is required to change the colour of the
solution.
Specific optical rotation (2.4.22). -122 0 to -125 0 , determined

in a 2.0 per cent w/v solution.
Sulphated ash (2.3.18). Not more than 0.1 percent.
on 1.0 g by drying in an oven at 1000 at a pressure not exceeding
0.7 kPa for 2 hours.
CI9HzIN03,HCl
Mol. Wt. 347.8
Nalorphine Hydrochloride is 17-allyl-7,8-didehydro-4,5aepoxymorphinan-3,6a-diol hydrochloride.
Assay. Weigh accurately about 25 mg and dissolve in sufficient

water to produce 250 ml. Measure the absorbance of the
resulting solution at the maximum at about 285 nm (2.4.7).
Calculate the content ofCI9HzIN03,HCl from the absorbance
1744
NALOXONE HYDROCHLORIDE
IP 2010
obtained by repeating the operation with nalorphine

hydrochloride RS in place of the substance under
examination.
by repeating the operation with nalorphine hydrochloride

RS.
Storage. Store protected from light.
Naloxone Hydrochloride
Naloxone Hydrochloride Dihydrate
Nalorphine Hydrochloride Injection

Nalorphine Injection is a sterile solution of Nalorphine
Hydrochloride in Water for Injections containing suitable
buffering agents.
HO
Nalorphine Injection contains not less than 90.0 per cent and
not more than 110.0 per cent ofthe stated amount ofnalorphine
hydrochloride, CI9H21N03,HCl.
,-'
N~CH2
1Ilo...00..-'"
Usual strength. 10 mg per ml.

CI9H2IN04,HCI,2H20
Identification
A. To a volume containing 50 mg ofNalorphine Hydrochloride
add dilute ammonia solution until the solution is alkaline and
extract with 25 ml ofa mixture of 1 volume of ethanol (95 per
cent) and 3 volumes of chloroform and evaporate the extract
to dryness. Dry the residue at a pressure not exceeding 2 kPa.
The residue complies with the following test.
Determine by infrared absorption spectrophotometry (2.4.6).
Compare the spectrum with that obtained with nalorphine
hydrochloride RS.
B. To a volume containing 0.1 g ofNalorphine Hydrochloride

add 0.05 ml of dilute ammonia solution; a white precipitate
soluble in sodium hydroxide solution is produced.
Mol. Wt. 399.9
Naloxone Hydrochloride is 17-allyl-4,5-a-epoxy-3,14dihydroxymorphinan-3-one hydrochloride dihydrate.

Naloxone Hydrochloride contains not less than 98.0 per cent
and not more than 102.0 per cent of C19H22ClN04, calculated
on the anhydrous basis.
Category. Antidote for opioids poisoning.
Description. A white to almost white, hygro$copic, crystalline
powder.
Identification
Test A may be omitted if tests Band C are carried out. Test B
may be omitted if tests A and C are carried out.
A. Determine by infrared absorption spectrophotometry
(2.4.6).Compare the spectruin with that obtained with naloxone
hydrochloride dihydrate RS or with the reference spectrum
of naloxone hydrochloride.
C. Gives reaction A ofchlorides (2.3.1).

Tests
pH (2.4.24).6.0 to 7.5.
Other tests. Complies with the tests stated under Parenteral
Preparations (Injections).
Assay. Transfer an accurately measured volume containing
about 10 mg of Nalorphine Hydrochloride to a separating
funnel, add 1 ml of dilute hydrochloric acid and dilute to
10 ml with water. Extract with five successive quantities, each
of 5 ml, of chloroform, allowing the layers to separate before
drawing offeach cWoroform extract and discard the chloroform
extracts. Transfer the aqueous layer to a 100-ml volumetric
flask with the aid of small quantities of water and dilute to
volume with water. Measure'the absorbance of the resulting
solution at the maximum at about 285 nm (2.4.7). Calculate
the content of C19H21N03,HCI from the absorbance obtained
B. Detennine by thin-layer chromatography (2.4.17), coating

the plate with silica gel G
Mobile phase. Mix 5 volumes of methanol and 95 volumes of
the upper layer from a mixture of60 ml of ammonia and 100 m1
of butan-I-ol.
Test solution. Dissolve 8 mg of the substance under
examination in 0.5 ml of water and dilute to 1 m1 with methanol.
Reference solution. Dissolve 8 mg of naloxone hydrochloride
dihydrate RS in 0.5 ml of water and dilute to 1 ml with methanol.
Apply to the plate 5 I.d of each solution, Allow the mobile
phase to rise 8 cm. Dry the plate in air and spray with a freshly
prepared 0.5 per cent w/v solution ofpotassium ferricyanide
in ferric chloride solution and examine in daylight. The
1745
NALOXONE HYDROCHLORIDE
IP 2010
principal spot in the chromatogram obtained with the test

solution corresponds to that in the chromatogram obtained
with the reference solution.
to pH 2.0 with
-
flow rate. 1.5 ml per minute,

a linear gradient programme using the conditions given
_below,
spectrophotometer set at 230 urn,
- injection volume. 20 /-ll.
Tests
Appearance ofsolution. A2.0 per cent w/v solution in carbon
dioxide-free water (Solution A) is clear (2.4.1) and colourless
(2.4.1).
Acidity or alkalinity. To 10 ml of solution A add 0.05 ml of

methyl red solution. Not more than 0.2 ml of 0.02 M sodium
hydroxide or 0.02 M hydrochloric acid is required to change
the colour of the indicator.
Specific optical rotation (2.4.22). -170 to -181, detennined

in solution A.
Related substances. Determine by liquid chromatography

(2.4.14).
Test solution. Dissolve 0.125 g of the substance under

examination in 25 m1 of 0.1 M hydrochloric acid.
Reference solution (a). Dissolve 5 mg of naloxone for peak
identification RS (containing naloxone impurity A (4,5cx -epoxy3, 14-dihydroxymorphinan-6-one) (noroxymorphone),
naloxone impurity B (4,5cx-epoxy-14-hydroxy-17-(prop-2-enyl)3-(prop-2-enyloxy)morphinan-6-one) (3-0-allylnaloxone),
naloxone impurity C (4,5cx -epoxy-3,1Oli,14-trihydroxy-17-(prop2-enyl)morphinan-6-one) (1 Ocx -hydroxynaloxone), naloxone
impurity D (7,8-didehydro-4,5cx -epoxy-3,14-dihydroxy-17(prop-2-enyl)morphinan-6-one) (7 ,8-didehydronaloxone),
naloxone impurity E (4,5 cx:4',5' cx-diepoxy-3,3',14,14'tetrahydroxy-17,IT-bis(prop-2-enyl)-2,2'-bimorphinanyl-6,6'dione) (2,2'-bisnaloxone) and naloxone impurity F (4,5cxepoxy-3,1013,14-trihydroxy-17- (prop-2-enyl)morphinan-6-one)
(lOcx-hydroxynaloxone) in 1 m1 of 0.1 M hydrochloric acid.
Reference solution (b). Dilute 1.0 m1 ofthe test solution to 20

m1 with 0.1 Mhydrochloric acid. Dilute 1.0 ml ofthis solution
to 25.0 ml with 0.1 M hydrochloric acid.
Chromatographic system
- a stainless steel column 12.5 cm x 4 mm, packed with
octylsilane bonded to porous silica (5 /-lm),
- column temperature. 40,
- mobile phase: A. a mixture of20 volumes of acetonitrile,
40 volumes of tetrahydrofuran and 940 volumes of the
solution prepared by dissolving 1.1 g of sodium
octanesulphonate in 1000 ml of water. Adjust to pH 2.0
with a 50 per cent v/v solution of orthophosphoric acid,
B. a mixture of 40 volumes of
tetrahydrofuran , 170 volumes of acetonitrile and 790
volumes of the solution prepared by dissolving 1.1 g of
sodium octanesulphonate in 1000 ml of wate/: Adjust
a 50 per cent v/v solution of
orthophosphoric acid,
Time
(in min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
0~40
100~0
0~100
40~50
100
Inject reference solution (a). Adjust the sensitivity of the

system so that the peak-to-valley ratio is minimum 2.0, where
Hp is height above the baseline of the peak due to impurity D
and H v is the height above the baseline of the lowest point of
the curve separating this peak from the peak due to naloxone.
The relative retention time with reference to naloxone, for
impurity C, impurity A, impurity F, impurity D, impurity E and
impurity B is about 0.6 minute, 0.8 minute, 0.9 minute, 1.1
minutes, 3.0 minutes and 3.2 minutes respectively.
Inject reference solution (b) and the test solution. In the
chromatogram obtained with the test solution, the area of
each secondary peak corresponding to naloxone impurities
A, B, C, E, F is not more than the area ofthe principal peak in
the chromatogram obtained with reference solution (b) (0.2
per cent). The area of secondary peak corresponding to
naloxone impurity D is not more than 1.5 times the area ofthe
principal peak in the chromatogram obtained with reference
solution (b) (0.3 per cent).The area' of any other impurities is
not more than 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (b) (0.1 per
cent). The sum ofthe areas of all other secondary peaks is not
more than 4 times the area of the principal peak in the
cent). Ignore any peak with an area less than 0.25 times the
area of the principal peak in the chromatogram obtained with
the reference solution (b) (0.05 per cent).
Water (2.3.43). 7.5 per cent to 11.0 per cent, determined on

O.2g.
Sulphated ash (2.3.18). Not more than 0.2 per cent, determined
on 0.5 g.
ethanol (95 per cent) and add 5.0 ml of O. 01 M hydrochloric
acid. Titrate with 0.1 M ethanolic sodium hydroxide,
determining the end-point potentiometrically (2.4.25). Carry
out a blank titration.
0.03638 g ofC19H22ClN04.
1746
IP 2010
NALOXONE INJECTION
Naloxone Injection
Naloxone Injection is a sterile solution of Naloxone
Hydrochloride in Water for Injections.
Naloxone Injection contains not less than 95.0 per cent and
not more than 105.0 per cent ofthe stated amount ofnaloxone
hydrochloride, CI9H2IN04,HCI.
Usual strength. 400 Jlg per ml.

Identification
A. In the Assay, the principal peak in the chromatogram
obtained with the test solution conesponds to the peak in the
B. Detennine by thin-layer chromatography (2.4.17), coating
Mobile phase. A mixture of 5 volumes of methanol and 95

volumes ofthe upper layer from a mixture of60 ml ofMammonia
and 100 ml of butan-I-ol.
the combined extracts over anhydrous sodium sulphate, filter,

evaporate the filtrate to dryness and dissolve the residue in 1
ml of methanol.
Reference solution. Dilute 1 ml of the test solution to 200 ml

with methanol.
Apply to the plate 20 Jll of each solution. Allow the mobile
phase to rise 10 cm, protecting the plate from light. After
development, dry the plate in a cunent of air, spray with a
freshly prepared 0.5 per cent w/v solution of potassium
hexacyanoferrate(III) in iron(IIl) chloride solution and
examine in daylight. Any secondary spot in the chromatogram
obtained with the test solution is not more intense than the
spot in the chromatogram obtained with the reference solution
(0.5 per cent). Ignore any spot remaining on the line of
application.
Bacterial endotoxins (2.2.3). Not more than 70 ill perml ofthe

injection, diluted ifnecessary, with water BETto give a solution
containing 0.04 per cent w/v of anhydrous Naloxone
Hydrochloride.
Test solution. Add 1 mlofammoniabufferpH 1O.0toavolume

ofthe injection containing the equivalent of2 mg ofanhydrous
naloxone hydrochloride, extract with three 20 ml quantities of
a mixture of 1 volume of propan-2-o1 and 3 volumes of Assay. Detennine by liquid chromatography (2.4.14).
chloroform, illy the combined extracts over anhydrous sodium
Solvent mixture. 0.1 volume of orthophosphoric acid, 45
sulphate, filter, evaporate the filtrate to illyness and dissolve
volumes of methanol and 55 volumes of water.
the residue in 1 ml of methanol. Dilute 1 ml ofthis solution to
Test solution. Dilute the injection' equivalent to 0.001 per cent
20 ml with methanol.
w/v ofNaloxone Hydrochloride with the solvent mixture.
Reference solution. A 0.01 per cent w/v solution of naloxone
hydrochloride RS in methanol.
Reference solution (a). A 0.001 per cent w/v solution of
naloxone hydrochloride RS in the solvent mixture.
phase to rise 10 cm, protecting the plate from light. After Reference solution (b). A 0.001 per cent w/v solution of
development, illy the plate in a cunent of air, spray with a naloxone hydrochloride RS and 0.0005 per cent w/v solution
freshly prepared 0.5 per cent w/v solution of potassium of noroxymorphone in the solvent mixture.
hexacyanoferrate (Ill) in iron(III) chloride solution and
examine in daylight. The principal spot in the chromatogram
- a stainless steel column 25 cm x 4.6 mm, packed with
obtained with the test solution corresponds to that in the
end-capped octadecylsilane bonded to porous silica (5
to 10 Jlm)(such as Zorbax C18, 7 to 8 Jlm),
mobile phase: a solution containing 0.068 per cent w/v
Tests
of sodium octanesulphonate and 0.1 per cent w/v of
pH (2.4.24).3.0 to 4.5.
sodium chloride in the solvent mixture,
- flow rate. 1 ml per minute,
Related substances. Detennine by thin-layer chromatography
spectrophotometer set at 229 nm,
(2.4.17), coating the plate with silica gel G
- injection volume. 20 Jll.
Mobile phase. A mixture of 5 volumes of methanol and 95
volumes of the upper layer from a mixture of 60 ml of 2 M Inject reference solution (b). The test is not valid unless the
resolution between the peaks due to naloxone and
ammonia and 100 ml of butan-I-of.
noroxymorphone in the chromatogram obtained with reference
Test solution. Transfer a volume of the injection containing solution (b) is not less than 1.3.
about 2 mg of Naloxone Hydrochloride in 1 ml of ammonia
bufferpH I 0.0, extract with three 20 ml quantities ofa mixture Injection reference solution (a) and the test solution.
of 1 volume ofpropan-2-o1 and 3 volumes of chloroform, dry Calculate the content ofCI9H2IN04,HCl in the injection.
1747
NALTREXONE HYDROCHLORIDE
IP 2010
Specific optical rotation (2.4.22). - 187 to - 195, determined

in a 2.0 per cent w/v solution in water.
Labelling. The label states the quantity ofactive ingredient in

terms of the equivalent amount of anhydrous naloxone
hydrochloride.When naloxone is prescribed for neonatal use,
Neonatal Naloxone Injection (containing the equivalent of20
micrograms per ml ofanhydrous naloxone hydrochloride) shall
be dispensed.

(2.4.14).

examination in 10 ml of 0.1 M hydrochloric acid.
Reference solution (a). Dissolve 5 mg of 17-but-3-enyl-4,5 aepoxy-3,14-dihydroxymorphinan-6-one RS (naltrexone
impurity C RS) in 2.5 ml of 0.1 M hydrochloric acid.
Reference solution (b). Dilute 1.0 ml of the test solution and
1.0 ml of reference solution (a) to 100 ml with 0.1 M
hydrochloric acid. Dilute 1.0 ml ofthis solution to 10 ml with
0.1 M hydrochloric acid.
, Hel
.........._N~
Mol. Wt. 377.9

Naltrexone Hydrochloride is 17-(cyclopropylmethyl)-4,5aepoxy-3, 14-dihydroxymorphinan-6-one hydrochloride.
Naltrexone Hydrochloride contains not less than 98.0 per cent
and not more than 102.0 per cent of CzoHz4ClN04, calculated
on the anhydrous and ethanol free basis.
Category. Antidote for opioids poisoning.
octadecylsilane bonded to porous silica (5 Ilm),
- mobile phase: A. a 0.11 per cent w/v solution ofsodium
octanesulphonate, adjust the pH to 2.3 with
B. acetonitrile,
- a linear gradient programme using the conditions given
below,
- flow rate. 1.2 ml per minute,
- spectrophotometer set at 230 nm,
injection volume. 10 Ill.
Time
Mobile phase A
Mobile phase B
Description. A white or almost white powder, very

hygroscopic.
Identification
A. Dissolve 20 mg ofthe substance under examination in 5 ml
of water and make allmline with dilute ammonia. Shake with
10 ml of dichloromethane, separate the organic layer and
evaporate the solvent. On the residue, determine by infrared
absorption spectrophotometry (2.4.6). Compare the spectrum
with that obtained with naltrexone hydrochloride RS or with
the reference spectrum of naltrexone hydrochloride.
B. Gives reaction A ofchlorides (2.3.1).
Tests
Appearance of solution. A2.0 per cent w/v solution in carbondioxide free water is clear (2.4.1) and not more intensely
coloured than reference solution YS5 or BS5 (2.4.1).
Acidity or alkalinity. To 10 ml of2.0 per cent w/v solution in
carbon-dioxidefree water, add 0.05 ml of methyl redsolution.
Not more than 0.2 ml of 0.02 M sodium hydroxide or 0.02. M
hydrochloric acid is required to change the colour of the
indicator.
(in min.)
(per cent v/v)
(per cent v/v)
0-45
9H55
1~5
45-47
55~90
45~10
47-55
10
Inject reference solution (b). The test is not valid unless the
resolution between the peaks due to naltrexone and naltrexone
impurity C is not less than 2.0. The relative retention time with
reference to naltrexone for 17-formyl-4,5a-epoxy-3,14dihydroxymorphinan-6-one (naltrexone impurity A) is about
0.4, for 4,5a-epoxy-3,14-dihydroxymorphinan-6-one
(noroxymorphone) (naltrexone impurity B) is about 0.7, For
17-(cyclopropylmethyl)-4,5a-epoxy-3, lOa, 14trihydroxymorphinan-6-one (naltrexone impurity F) is about
0.8, for 17-(cyclopropylmethyl)-4,5a-epoxy-3,10~,14trihydroxymorphinan-6-one (naltrexone impurity G) is about
0.9, for 17-but-3-enyl-4,5a-epoxy-3,14-dihydroxymorphinan6-one (naltrexone impurity C) is about 1.05,for l7-butyl-4,5aepoxy-3,14-dihydroxymorphinan-6-one (naltrexone impurity H)
is about 1.1, for 17-(cyclopropylmethyl)-4,5a-:epoxy-3,14dihydroxymorphinan-6,1O-dione (naltrexone impurity I) is
about 1.2, for 7-(cyclopropylmethyl)-4,5a-epoxy-14-hydroxy3-methoxymorphinan-6-one (naltrexone impurity 1) is about
1.3, for 17,17'-bis(cyclopropylmethyl)-4,5a:4',5'-a-diepoxy-
1748
IP 2010
NALTREXONE TABLETS
3,3' ,14,14' -tetrahydroxy-2,2' -bimorphinanyl-6,6' -dione

(pseudonaltrexone) (naltrexone impurity D) is about 1.4, for3(cyclopropylmethoxy)-17-(cyclopropylmethyl)-4,5(X-epoxy~ 14hydroxymorphinan-6-one (naltrexone impurity E) is about 1.7.
chromatogram obtained with the test solution the area any
peak corresponding to naltrexone impurity C, D, E, F and G is
not more than twice the area of the principal peak in the
cent); the area ofany peak corresponding to naltrexone impurity
A, B, H, I and J is not more than the area ofthe principal peak
in the chromatogram obtainedwith reference solution (b) (0.1
per cent); the area of any other secondary peak is not more
than the area of the principal peak in the chromatogram
obtained with reference solution (b)(0.1 per cent). The sum of
the areas of all secondary peaks is not more than 10 times the
area ofthe principal peak in the chromatogram obtained with
reference solution (b) (l.0 per cent). Ignore any peak with an
area less than 0.5 times the area of the principal peak in the
cent).
Ethanol (2.3.45). Not more than 3.0 per cent v/v, determined by
Method I using the following solutions.

examination in 10 ml of wate/:
Reference solution. Dilute 0.75 g of anhydrous ethanol to
1000 ml with water.
Sulphated ash (2.3.18). Not more than 0.1 per cent. .
Water (2.3.43). Not more than 10.0 per cent, determined on
O.2g.
Usual strength. 50 mg.
Identification
In the Assay, the principal peak in the chromatogram obtained
with the test solution corresponds to the peak in the
Tests
Dissolution (2.5.2).
Apparatus No.1,
Medium. 900 ml of water,
Speed and time. 50 rpm and 60 minutes.
Withdraw a suitable volume of the medium and fllter.
Detennine by liquid chromatography (2.4.14).
Test solution. Use the flltrate.

Reference solution. A solution of naltrexone hydrochloride
RS in water to obtain the same concentration as given in the
test solution.
- mobile phase: a mixture of600 volumes of0.05 M buffer
solution prepared by dissolving 7 g of monobasic
sodium phosphate in 1000 ml of water, add 1.1 g of
sodium 1-octane sulphonate monohydrate and 400 ml
of methanol, adjust the pH to 6.7 with dilute sodium
hydroxide,
~ injection volume. 100 Ill.
Assay. Weigh accurately about 0.2 g of the substance under

examination, dissolve in 60 ml of ethanol (95 per cent) and
add 1.0 ml of 0.1 M hydrochloric acid. Titrate with 0.1 M
sodium hydroxide, determining the end-point
potentiometrically (2.4.25). The curve shows 3 points of
inflexion. Read the volume added between the flrst 2 points of
inflexion.
Inject the reference solution and the test solution.
1 ml of 0.1 M sodium hydroxide is equivalent to 0.03779 g of
Calculate the content of CzoHz3N04.HCl in the tablets.
CzOHZ4ClN04.
Inject the reference solution. The test is not valid unless the
relative standard deviation for replicate injections is not more
than 2.0.
D. Not less than 80 per cent of the stated amount of

CzOHZ3N04.HCl.
Uniformity of content.-Comply with the test stated under

Tablets.
Naltrexone Tablets
Determine by liquid chromatography (2.4.14), as described

under Assay, using the following solution as the test solution.
Naltrexone Hydrochloride Tablets

Naltrexone Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount ofnaltrexone
hydrochloride, C ZO H23N04.HCl.
Test solution. Disperse 1 tablet in 100 ml of 0.1 M

orthophosphoric acid.
Calculate the content ofCzoHz3N04.HCl in the tablets.
1749
NALTREXONE TABLETS
IP 2010
Other tests. Comply with the tests stated under Tablets.

Assay. Determine by liquid chromatography (2.4.14).
Test solution. Weigh and powder 20 tablets. Shake a quantity

of powdered tablets containing about 250 mg ofNaltrexone
with 80 ml of 0.1 M orthophosphoric acid, sonicate for 30
minutes and dilute to 100 ml with the same solvent, filter.
H3 C O)lCH 2 (CH 2 hCH 3
Reference solution (a). Dissolve 22.5 mg of naltrexone RS in

1.5 ml of methanol and 0.6 ml of 0.1 M hydrochloric acid.
Dilute to 10 ml with 0.1 M orthophosphoric acid.
Reference solution (b). Dissolve about 3 mg of N-(3-butenyl)noroxymorphone hydrochloride RS (naltrexone impurity A
RS) in 3.0 ml of methanol and dilute to 10 ml with 0.1 M
orthophosphoric acid. To 0.5 ml ofthis solution, add 5.0 ml of
reference solution (a) and dilute to 10 ml with 0.1 M
orthophosphoric acid.
- a stainless steel column 15 cm x 3.9 mm packed with
octadecylsilane bonded to porous silica (5 ).1m),
column temperature. 45,
mobile phase: A. dissolve about 1.08 g of sodium 1octanesulphonate and 23.8 g of sodium acetate in 800
ml of water. Add 1.0 ml of triethylamine and 200 mlof
methanol, adjust the pH to 6.5 with glacial acetic acid,
B. dissolve about 1.08 g sodium 1octanesulphonate and 23.8 g sodium acetate in 400 ml
of water. Add 1.0 ml of triethylamine and 600 ml of
methanol, adjust the pH to 6.5 with glacial acetic acid,
below,
flow rate. 1 ml per minute,
injection volume. 20 ).11.
Time
(min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
100
1-35
100-70
0-7100
35-36
0-7100
100-70
Calculate the content ofCzoHz3N04.HCl in the Tablets.
CZ8~03
Mol. Wt. 428.7
Nandrolone Decanoate is 3-oxo-4-estren-1713-yl decanoate.

Nandrolone Decanoate contains not less than 97.0 per cent
and not more than 103.0 per cent of CZ8H4403, calculated on
the dried basis.
Category. Anabolic steroid.
Dose. By intramuscular injection, 25 to 50 mg, every 3 weeks.
Description. A white to creamy-offwhite, crystalline powder;
odour, faint and characteristic.
Identification

Compare the spectrum with that obtained with nandrolone
decanoate RS or with the reference spectrum of nandrolone
decanoate.
0.001 per cent w/v solution in ethanol (95 per cent) shows an
absorption maximum only at about 239 nm; absorbance at
C. Dissolve 25 mg in 1 ml of methanol, add 2 ml of
semicarbazide acetate solution, heat under a reflux condenser
for 30 minutes and cool; the precipitate, after recrystallisation
from ethanol (95 per cent), melts at about 175 (2.4.21).
Tests
resolution between the peaks due to naltrexone and naltrexone
impurity A is not less than 2.0, the tailing factor is not more
than 1.4, and the relative standard deviation for replicate
injections is not more than 2.0 per cent. The relative retention
time with reference to naltrexone for noroxymorphone is about
0.55, for 10-hydroxynaltrexone is about 0.7, for naltrexone
impurity A is about 1.26, for 2,2a-bisnaltrexone is about 1.80
and for 10-ketonaltrexoneis about 1.99.
Inject reference solution (a) and the test solution.
Specific optical rotation (2.4.22). +32.0 to +36.0, determined

in a 2.0 per cent w/v solution in dioxan.
(2.4.17), coating the plate with silica gel GF254.
Mobile phase. A mixture of 70 volumes of heptane and

30 volumes of acetone.
examination in 10 ml of chloroform.
substance under examination in chloroform.
Reference solution (b). A 0.01 per cent w/v solution of
nandrolone RS in chloroform.
1750
IP 2010
NANDROLONE PHENYLPROPIONATE
Apply to the plate 5 f.Ll of each solution. After development,

dry the plate in air and examine in ultraviolet light at 254 run. In
the chromatogram obtained with the test solution any spot
corresponding to nandrolone is not more intense than the
spot in the chromatogram obtained with reference solution
(b) and any other secondary spot is not more intense than the
spot in the chromatogram obtained with reference solution (a).
on 1.0 g by drying over phosphorus pentoxide at a pressure
not exceeding 0.7 kPa for 4 hours.
ethanol (95 per cent) to produce 100.0 mL Dilute 5.0 ml to
50.0 ml with ethanol (95 per cent) and measure the absorbance
ofthe resulting solution at the maximwn at about 239 run (2.4.7).
Calculate the content of C 2s H44 0 3 taking 407 as the specific
absorbance at 239 nm.
Tests
Assay. To an accurately measured volume containing about
0.1 g ofNandrolone Decanoate add sufficient chloroform to
produce 100.0 mL Dilute 3.0 ml ofthe solution to 50.0 ml with
chloroform. To 5.0 ml of this solution add 10 ml of isoniazid
solution and sufficient methanol to produce 20.0 ml. Allow to
stand for 45 minutes and measure the absorbance of the
using as the blank 5 ml of chloroform treated in the same
manner. Calculate the content ofC2sH4403 from the absorbance
obtained by repeating the operation using a suitable quantity
of nandrolone R8.
1 mg ofCIsH2602 is equivalent to 1.562 mg ofC2sH4403.
Nandrolone Phenpropionate

Nandrolone Decanoate Injection is a sterile solution of
Nandrolone Decanoate in Ethyl Oleate or other suitable ester,
in a suitable fixed oil or in any mixture ofthese.
Nandrolone Decanoate Injection contains not less than 90.0
per cent and not more than 110.0 per cent ofthe stated amount
ofnandrolone decanoate, C2sH4403.
Usual strength. 25 mg per mL
Mol.Wt. 406.6
Identification
Detennine by thin-layer chromatography (2.4.17), coating the
plate with silica gel GF254.
Nandrolone Phenylpropionate is 3-oxo-4-estren-17~-yI3

phenylpropionate.

Nandrolone Phenylpropionate contains not less than 97.0 per

cent and not more than 103.0 per cent ofC27H3403, calculated
on the dried basis.
Test solution. Dilute a suitable volume of the injection with

carbon tetrachloride to give a solution containing 0.5 percent w/v solution of Nandrolone Decanoate.
Category. Anabolic steroid.
Reference solution. A 0.5 per cent w/v solution ofnandrolone

decanoate RS in carbon tetrachloride.
Description. A white to creamy-white, crystalline powder;

odour, characteristic.
Apply to the plate 5 f.LI of each solution. After development,

dry the plate in air until the odour of solvent is no longer
detectable, spray with a 10 per cent v/v solution of sulphuric
acid in ethanol (95 per cent), heat at 105 for 30 minutes and
examine in ultraviolet light at 365 run. The principal spot in the
chromatogram obtained with the test solution corresponds to
that in the chromatogram obtained with the reference solution.
Ignore any subsidiary spots due to the vehicle.
Identification
Dose. By deep intramuscular injection, 25 to 50 mg weeldy.
A. Detennine by infrared absorption spectrophotometry (2.4.6).

phenylpropionate RS or with the reference spectrum of
nandrolone phenylpropionate.
B. When examined in the range 230 run to 360 nm (2.4.7), a
1751
IP 2010
NANDROLONE PHENYLPROPIONATE

C. Dissolve 25 mg in I ml of methanol, add 2 ml of
semicarbazide acetate solution, heat under a reflux condenser
for 30 minutes and cool; the precipitate, after recrystallisation
from ethanol (95 per cent) melts at about 182 (2.4.21).
Tests
examination in 10 ml ofchloroform.
Nandrolone Phenylpropionate Injection contains not less than

92.5 per cent and not more than 107.5 per cent of the stated
amount of nandrolone phenylpropionate, C27H3403'
Usual strengths. 25 mg per ml; 50 m per ml.
Identification
Dissolve a volume of the injection containing 50 mg of
Nandrolone Phenylpropionate in 8 ml of light petroleum
(40 to 60) and extract with three 8-ml quantities ofa mixture
of 7 volumes of glacial acetic acid and 3 volumes of water.
Wash the combined extracts with 10 ml of light petroleum
(40 to 60), dilute with water until the solution becomes
turbid, allow to stand for 2 hours in ice and filter. The precipitate,
after washing with water and drying over phosphorus
pentoxide at a pressure not exceeding 0.7 kPa, complies with
the following test.

Determine by thin-layer chromatography (2.4.17), using a

silica gel GF254 precoated plate the surface of which has
been modified by chemically-bonded octadecylsilyl groups.

nandrolone RS in chloroform.
Mobile phase. A mixture of20 volumes ofwater, 40 volumes

of acetonitrile and 60 volumes ofpropan-2-01.
Apply to the plate 5 Jll of each solution. After development,

dry the plate in air and examine in ultraviolet light at 254 nm. In
corresponding to nandrolone is not more intense than the
(b) and any other secondary spot is not more intense than
the spot in the chromatogram obtained with reference
solution (a).
Test solution. A 0.5 per cent w/v solution of the dried

precipitate in chloroform.
Sulphated ash (2.3.18). Not more than 0.1 per cent.

Assay. Weigh accurately about 10 mg, dissolve in sufficient
ethanol to produce 100.0 ml, dilute 5.0 ml to 50.0 ml with ethanol
and measure the absorbance of the resulting solution at the
maximum at about 240 nm (2.4.7). Calculate the content of
C27H3403 taking 430 as the specific absorbance at 240 nm.

nandrolone phenylpropionate RS in chloroform.
Reference solution (b). A mixture of equal volumes ofthe test
solution and the reference solution.
dry the plate in air until the solvent has evaporated and heat it
at 100 for 10 minutes. Allow to cool and examine in ultraviolet
light at 254 nm. The principal spot in the chromatogram
chromatogram obtained with reference solution (a). The
principal spot in the chromatogram obtained with reference
solution (b) appears as a single spot.
Tests
Nandrolone Pbenylpropionate
Injection
Nandrolone Phenylpropionate Injection is a sterile solution
of Nandrolone Phenylpropionate in Ethyl Oleate or other
suitable ester, in a suitable fixed oil or in a mixture ofthese.

0.1 g of Nandrolone Phenylpropionate add sufficient
chloroform to produce 100.0 ml. Dilute 3.0 ml ofthis solution
to 50.0 ml with chloroform. To 5.0 ml ofthe resulting solution
add 10 ml of isoniazid solution and sufficient methanol to
produce 20.0 ml. Allow to stand for 45 minutes and measure
the absorbance of the solution at the maximum at about
380 nm (2.4.7), using as blank 5 ml ofchloroform treated in the
same manner. Calculate the content of C27H3403 from the
1752
IP 2010
NAPHAZOLINE NITRATE
absorbance obtained from a 0.006 per cent w/v solution of

nandrolone phenylpropionate RS treated in the same manner.
Tests
Appearance of solution. A 1.0 per cent w/v solution in carbon

dioxide-free water is clear (2.4.1) and colourless (2.4.1).
Labelling. The label states that the preparation is for

intramuscular injection only.
pH (2.4.24).5.0 to 6.5, determined in a 1.0 per cent w/v solution.

(2.4.14).

examination in 100 ml ofthe mobile phase.
N aphazoline Nitrate
Reference solution (a). Dissolve 5 mg of 1-naphthylacetic

acid in the mobile phase, add 5 ml of the test solution and
dilute to 100 ml with the mobile phase.
Reference solution (b). Dissolve 5 mg ofnaphazoline impurity
A RS in 100 ml ofthe mobile phase. Dilute 5.0 ml ofthis solution
to 100.0 ml with the mobile phase.
Mol. Wt. 273.3
Naphazoline Nitrate is 2-(l-napthylmethyl)-2-imidazoline
nitrate.
Naphazoline Nitrate contains not less than 99.0 per cent and
not more than 101.0 per cent of C4HI4N2,HN03 calculated on
the dried basis.
Category. Sympathomimetic.
Description. A white or almost white crystalline powder.
Identification
Band C may be omitted if tests A and D are carried out.

Compare the spectrum with that obtained with naphazoline
nitrate RS.
0.002 per cent w/v solution in 0.01 Mhydrochloricacidshows
absorption maxima at about 270 nm, 280 nm, 287 nm and
291 nm; absorbances at these maxima are about 0.43,0.50,0.35
and 0.34 respectively.
C. Dissolve about 0.5 mg in 1 ml ofmethanol, add 0.5 ml ofa

freshly prepared 5 per cent w/v solution of sodium
nitroprusside and 0.5 ml ofa 2 per cent w/v solution ofsodium
.hydroxide, allow to stand for 10 minutes and add 1 ml of a
8 per cent w/v solution ofsodium bicarbonate; a violet colour
is produced.
D. Dissolve about 10 mg in 5 ml ofwater, add 0.2 g ofmagnesium
oxide, shake mechanically for 30 minutes, add 10 ml of
chloroform and shake vigorously. Allow to stand, separate
the chloroform layer, filter and evaporate the aqueous layer to
dryness. The residue gives reaction A for nitrates (2.3.1).
Reference solution (c). Dilute 2.0 ml of the test solution to

10.0 ml with the mobile phase. Dilute 1.0 ml ofthis solution to
100.0 ml with the mobile phase.
octadecylsilane bonded to porous silica (4 Jlm),
mobile phase: dissolve 1.1 g of sodium
octanesulphonate in a mixture of 5 volumes ofglacial
acetic acid, 300 volumes ofacetonitrile and 700 volumes
ofwater,
injection volume. 20 Jll.
Inject reference solution (a). The test is not valid unless the
resolution between the peaks due to naphazoline and
naphazoline impurity B is not less than 5.0. The relative
retention time with reference to naphazoline for
naphthylacetylethylenediamine (naphazoline impurity A) is
about 0.76, for I-naphthylacetic acid (naphazoline impurity B)
is about 1.27, for 1-naphthylacetonitrile (naphazoline impurity
C) is about 2.8, for B-naphazoline (naphazoline impurity D) is
about 1.28.
Inject the test solution, reference solution (b) and (c). Run the
chromatogram 3 times the retention time ofthe principal peak.
In the chromatogram obtained with the test solution the area
ofthe peak due to naphazoline impurity A is not more than the
reference solution (b) (0.5 per cent). The area of any other
secondary peak is not more than 0.5 times the area of the
solution (c) (0.1 per cent). The sum ofall the secondary peaks
is not more than 5 times the area of the principle peak in the
chromatogram obtained with reference solution (c) (1.0 per
cent), Ignore any peak with an area less than 0.25 times the
1753
NAPHAZOLINE NITRATE
IP 2010

reference solution (c) (0.05 per cent).
Naproxen contains not less than 99.0 per cent and not more
than 101.0 per cent OfC14H1403, calculatedon the dried basis.
Naphthylacetylethylenediamine. D.etermine by thin-layer

chromatography (2.4.17), coating the plate with silica gel G.
Category. Nonsteroidal antiinflammatory.

Description. A white oralmost white crystalline powder.
Mobile phase. A mixture of 100 volumes of methanol and

1.5 volumes of strong ammonia solution.
Identification

examination in 10 ml of methanOL
Test A may be omitted iftests Band C are carried out. Tests B

and C may be omitted if test A is carried out.
Reference solution. A solution containing 2 per cent w/vof

naphazoline nitrate RS and 0.01 per cent w/v of
naphthylacetylethylenediamine hydrochloride RS.

Compare the spectrum with that obtained with naproxen RS

dry the plate at 105 for 5 minutes, spray with a 0.5 percent
w/v solution of ninhydrin in methanol and heat at 105 for
10 minutes. Any spot corresponding to naphthylacetylethylenediamine hydrochloride in the chromatogram obtained
with the test solution is not more intense than the
corresponding spot in the chromatogram obtained with the
reference solution. The test is not valid unless the
chromatogram obtained with the reference solution shows
two clearly separated spots.
B. When examined in the range 230 urn to 350 nm (2.4.7), a 0.04

per cent w/v solution in methanol shows absorption maxima
at about 262 nm, 271 run, 316 urn and 331 nm. The absorbance
at the maxima are216 to 238, 219 to 241, 61 to 69 and 79 to 87
respectively.
Chlorides (2.3.12). 15.0 ml ofl.O per cent w/v solution in carbon

dioxide-free water complies with the limit test for chlorides
(375 ppm).
or with the reference spectrum of naproxen.
C. Melting range (2.4.21). 154 to 158.
Tests
Appearance of solution. A 5.0 per cent w/v solution in
methanol is clear (2.4.1) and not more intensely coloured than
reference solution BYS6 (2.4.1).
Specific optical rotation (2.4.22). + 59 to + 62, determined in

a 2.0 per cent w/v solution in ethanol (95 per cent).
Loss on drying (2.4.19). Not more than 0.5 per cent, detennined
on 1.0 g by drying in an oven at 105 for 3 hours.
Enantiomeric purity. Determine by liquid chromatography

(2.4.14).

anhydrous glacial acetic acid. Titrate with 0.1 M perchloric
acid, detenriining the end-point potentiometrically (2.4.25).
Carry out a blank titration.
NOTE-Protect the solutions from light.
I ml of 0.1 M perchloric acid is equivalent to 0.02733 g of

C4HI4N2,HN03.

examination in 50 ml of tetrahydrojitran.Dilute 2,0 ml ofthis
.solution to 20 ml with the mobile phase.
Reference solution (a). Dilute 2.5 ml ofthe test solution to 100
ml with the mobile phase.
Reference solution (b). Dissolve 5 mg of racemic naproxen
RS in 10 ml of tetrahydrofitran, dilute to 100 ml with the mobile
phase.
Naproxen
CI~1403
Mol. Wt. 230.3
Naproxen is (2S)-2-(6-methoxynaphthalen-2-yl)propionic
acid.
silica gel1t-acceptorht-donor for chiral separations (5
JllTI),
- mobile phase: a mixture of 5 volumes of glacial acetic
acid, 50 volumes of acetonitrile, 100 volumes of 2propanol and 845 volumes of hexane,
- injection volume. 20 Ill.
1754
IP 2010
NAPROXEN ORAL SUSPENSION
resolution between the peaks due to (2R)-2-(6methoxynaphthalen-2-yl) propanoic acid (naproxen impurity
G) ((R)-enantiomer) and naproxen is not less than 3.0.
solution (a) (0.1 per cent). The sum ofthe areas ofall impurities
is not more than 3 times the area of the principal peak in the
chromatogram obtained with reference solution (a) (0.3 per
cent). Ignore any peak with an area less than 0.5 times the area
of the principal peak in the chromatogram obtained with
reference solution (a) (0.05 per cent).
Inject reference solution (a) and the test solution. Run the
chromatogram 1.5 times the retention time of the principal
peak. The area of the peak due to naproxen impurity G is not
more than the area ofthe principal peak in the chromatogram
obtained with reference solution (a) (2.5 per cent).

(2.4.14).
Assay. Weigh accurately about 0.2 g and dissolve in a mixture

of25 ml of water and 75 ml of methanol. Titrate with 0.1 M
sodium hydroxide using 1 ml of phenolphthalein solution as
indicator.

Reference solution (a). Dilute 1.0 ml of the test solution to 50
ml with the mobile phase. Dilute 1.0 ml ofthis solution to 20.0
Reference solution (b). Dissolve 6 mg of 2-bromo-6methoxynaphthalene RS (Naproxen impurity N RS), 6 mg of
1-(6-methoxynaphthalen-2-yl)ethanone RS( Naproxen
impurity L RS) and 6 mg of (1RS)-1-(6- methoxynaphthalen2-ylJethanol RS (Naproxen impurity K RS) in 10 ml of
acetonitrile. To 1.0 ml of this solution, add 1.0 ml ofthe test
solution and dilute to 50 ml with the mobile phase. Dilute 1.0
ml ofthis solution to 20 ml with the mobile phase.
- mobile phase: a mixture of 42 volumes of acetonitrile
and 58 volumes of a 0.136 per cent w/v solution of
potassium dihydrogen phosphate adjust the pH to 2.0
with orthophosphoric acid,
resolution between the peaks due to naproxen impurity K and
naproxen is not less than 2.2. The relative retention time with
reference to naproxen for naproxen impurity K is about 0.9, for
naproxen impurity L is about 1.4 and naproxen impurity N is
about 5.3.
Inject reference solution (a), (b) and the test solution. Run the
chromatogram 1.5 times the retention time ofnaproxen impurity
N. The area ofthe peak due to naproxen impurity L is not more
than the area ofthe corresponding peak in the chromatogram
obtained with reference solution (b) (0.1 per cent); the area of
any other secondary peak is not more than the area of the
Heavy metals (2.3.13). 1.0 g complies with the limit test for

C,JII403'

Naproxen Oral Suspension is an aqueous suspension of
Naproxen in a suitable flavoured vehicle.
Naproxen Oral Suspension contains not less than 90.0 per
cent and not more than 110.0 per cent ofthe stated amountof
naproxen, C14H1403.
Identification
Evaporate 50 ml ofsolution A obtained in the Assay, to dryness
using a rotary evaporator. The residue complies with the
following tests.
(2.4.6).Compare the spectrum with that obtained with naproxen
RS or with the reference spectrum ofnaproxen.

0.004 per cent w/v solution in methanol exhibits maxima at 262
nm,27l nm,316nmand331 nm.
Tests
pH (2.4.24).2.1 to 4.0.
Mobile phase. A mixture of3 volumes of glacial acetic acid,

9 volumes of tetrahydrojU,:an and 90 volumes of toluene.
1755
NAPROXEN ORAL SUSPENSION
IP 2010
Test solution. Evaporate solution A obtained in the Assay to

dryness on a rotary evaporator and dissolve the residue in
sufficient methanol to produce a solution containing 5.0 per
cent w/v ofnaproxen.

0.004 per cent w/v solution in methanol exhibits maxima at 262
nm,271 nm,316nmand331 nm.

with methanol.
Tests

dry the plate in air and examine under ultraviolet light at 254
nm. Any secondary spot in the chromatogram obtained with
the test solution is not more intense than the spot in the
chromatogram obtained with the reference solution (0.5 per
cent).

(2.4.17), coating the plate \yith silica gel GF254.
Mobile phase. A mixture of 3 volumes of glacial acetic acid,
9 volumes of tetrahydrofuran and 90 volumes of toluene.
Assay. To a quantity of the oral suspension containing 0.5 g

ofNaproxen add 20 ml of3.5 M hydrochloric acid; mix, extract
with three 50 ml quantities of chloroform, filter each extract
through anhydrous sodium sulphate, combine the filtrates
and add sufficient chloroform to produce 200 ml (solution A).
To 5 ml of solution A add sufficient methanol to produce 250
ml and measure the absorbance of the resulting solution at
the maximum at about 331 nm (2.4.7).
Test solution. Weigh 20 suppositories and cut into small

pieces. Dissolve a quantity of the suppositories containing
0.5 g ofNaproxen in 50 ml of 2,2,4-trimethylpentane and extract
with four 25-ml quantities of methanol (80 per cent). Combine
the extracts, add 100 ml ofa2 per cent w/v solution of sodium
chloride, extract with four 25-ml quantities of chloroform
filtering each extract through a layer of anhydrous sodium
sulphate on an absorbent cotton plug moistened with
chloroform. Evaporate the combined filtrates to dryness using
a rotary evaporator with the aid of gentle heat. Shake the
residue with 10 ml of methanol, centrifuge and use the
supernatant liquid.
Calculate the content of Cl4Hl403 taking 81 as the specific

Reference solution. Dilute1 ml of the test solution to 200 ml

with methanol.
Other tests. Complies with the tests stated under Oral Liquids.

N aproxen Suppositories
Naproxen Suppositories contain Naproxen in a suitable
suppository base.
Naproxen Suppositories contain not less than 95.0 per cent
and not more than 105.0 per cent of the stated amount of
naproxen, C14H1403.
Identification
Dissolve a quantity of the suppositories containing 0.5 g of
Naproxen in 50 ml of 2,2,4-trimethylpentane and extract with
four 25 ml quantities of methanol (80 per cent). To the
combined extracts add 100 rnl of a 2 per cent w/v solution of
sodium chloride, extract with four 25 ml quantities of
chloroform, dry the combined extracts over anhydrous sodium
sulphate, filter and add sufficient chloroform to produce 200
ml (solution A). Evaporate 100 ml of solution A to dryness
using a rotary evaporator. The residue complies with the
following tests.
Compare the spectrum with that obtained with naproxen RS
9r with the reference spectrum of naproxen.
Other tests. Comply with the tests stated under Suppositories.

Test solution. Disperse ten suppositories in 500 ml of methanol

on a water-bath for 40 minutes with the aid of ultrasound and
by swirling the flask. Cool at 50 for 1 hour, centrifuge and use
the clear, supernatant liquid; if the solution is still cloudy
filter through glass-fibre paper (such as Whatrnan OFIe). To 5
ml ofthe filtrate add sufficient ofthe mobile phase to produce
a solution containing 0.01 per cent w/v ofnaproxen.
naproxen RS in the mobile phase.
Reference solution (b). A 0.01 per cent w/v each ofnaproxen
RS and 2-naphthylacetic acid in the mobile phase.
a stainless steel column 20 cm x 4 mm, packed with endcapped octadecylsilane bonded to porous silica (5 Ilm)
(such as Nucleosil C18),
mobile phase: a mixture of 1 volume of a 0.52 per cent
w/v solution of sodium acetate, adjusted to pH 5.8
using glacial acetic acid and 1 volume of methanol,
1756
IP 2010
NAPROXEN SUSTAINED-RELEASE TABLETS
the absorbance obtained from a solution of known

concentration of naproxen RS in the same medium.

D. Not less than 80 per cent ofthe stated amount ofC14H1403.
resolution between the peaks due to naproxen and 2naphthylacetic acid in the chromatogram obtained with the
reference solution (b) is more than 3.0.
Uniformity of content. Comply with the test stated under

Tablets.
under Assay using the following solutions.
Test solution. Disperse 1 tablet in about 140 ml of solvent

mixture B. Shake for 15 minutes, sonicate for 15 minutes, dilute
to 200 ml with solvent mixture B, filter. Dilute 2.0 ml of the
filtrate to 50 ml with the mobile phase.
Calculate the content of CI4HI403 in the suppositories.

Reference solution. A 0.025 per cent wIv solution of naproxen

RS in solvent mixture A. Dilute 10 ml ofthis solution to 25 ml
with solvent mixture B.
Naproxen Sustained-release Tablets

Naproxen Sustained-release Tablets contain not less than 90.0
ofnaproxen, C14H1403.
Calculate the content ofCI4HI403 in the tablet.

Solvent mixture A. 90 volumes of acetonitrile and 10 volumes

of water.
Identification
A. When examined in the range 200 TIm to 400 TIm (2.4.7), the
solution used in test B of dissolution, exhibits an absorption
maximum at about 332 TIm.
B. In the Assay, the principal peak in the chromatogram
chromatogram obtained with the reference solution (b).
Solvent mixture B. 50 volumes of acetonitrile and 50 volumes

of water.
Test solution. Weigh and powder 20 tablets. Disperse a quantity
of powder containing about 250 mg ofNaproxen in 70 ml of
solvent mixture B, sonicate for 15 minutes and dilute to 100.0
ml with solvent mixture B and filter. Dilute 2.0 ml ofthe filtrate
to 50 ml with the mobile phase.
Reference solution (a). A 0.05 per cent wlv solution of
naproxen RS in solvent mixture A.
Tests
Reference solution (b). Dilute 10 ml of reference solution (a)

A. Apparatus No.1,
Medium. 1000 ml of0.1 M hydrochloric acid,
Speed and time. 50 rpm and 2 hours.
Withdraw a suitable volume ofthe medium and filter. Measure
the absorbance of the filtered solution, suitably diluted with
the medium if necessary, at the maximum at about 332 TIm
(2.4.7). Calculate the content ofC,4H1403in the medium from
D. Not more than 10 per cent ofthe stated amount ofC,4H,403.
B. Apparatus No.1,
Medium. 1000 ml of 0.2 M phosphate bujferpH 6.8,
the absorbance of the filtered' solution, suitably diluted with
the medium if necessary, at the maximum at about 332 nm
(2.4.7). Calculate the content ofC,4H1403 in the medium from
a stainless steel column 25 cm x 4.6 rom, packed with
octadecylsilane bonded to porous silica (5 11m),
mobile phase: a mixture of 55 volumes of 1.0 per cent
vlv solution of acetic acid and 45 volumes of
acetonitrile,
- flow rate. 1ml per minute,
tailing factor ofthe principal peak is not more than 1.5 per cent
and the relative standard deviation for replicate injections is
not more than 2.0 per cent.
Inject the test solution and reference solution (b).
Calculate the content of CI4HI403 in the tablet.
Storage. Store protected from moisture, at a temperature not
exceeding 30.
1757
NAPROXEN TABLETS
IP 2010
Naproxen Tablets
Naproxen Tablets contain not less than 95.0per cent and not
more than 105.0 per cent of the stated amount ofnaproxen,
C14H1403;
Identification
A. Extract a quantity of the powdered tablets containing 20
mg ofNaproxen with 100 ml of methanol and filter. Evaporate
and dry the residue at 105. On the residue, determine by
infrared absorption spectrophotometry (2.4.6). Compare the
spectrum with that obtained with naproxen RS or with the
reference spectmm of naproxen.
Assay. Weigh and powder 20 tablets. Shake a quantity of the

powder containing about 50 mg ofNaproxen with 70 ml of
methanol for 30 minutes, add sufficient methanol to produce
100 ml and filter. Dilute 10 ml of the filtrate to 50 ml with
methanol and measure the absorbance at the maximum at about
331nm (2.4.7) using a 0.01 per cent wlv solution of naproxen
RS in methanol.
Calculate the content of CI4HI403 in the tablets.
.

0.002 per cent w Iv solution in methanol exhibits maxima at 262
nm,271 nm,316nmand331 nm.
OH.
OH
~O~~~O~ ,Hel
~F
F~
Tests
Mol. Wt. 441.9
Apparatus No.1;
Medium. 900 ml ofa phosphate buffer prepared by dissolving
2.62 g of sodium dihydrogen orthophospate monohydrate
and 11.5 g of anhydrous disodium hydrogen orthophosphate
in sufficient water to produce 1000 ml, adjusted to pH 7.4 with
0.1 M sodium hydroxide or 0.1 M hydrochloric acid,
Nebivolol Hydrochloride is (IRS, 1'RS)-I, 1'-[(2RS,2'SR)-bis (6flurochroman-2-yl)]-2,2'-iminodiethanol hydrochloride.

the absorbance of the filtrate, suitably diluted with the
dissolution medium ifnecessary, at the maximum at about 332
mn (2.4.7). Calculate the content ofC,4H,403, in the mediUlp
from the absorbance obtained from a solution of known
Description. A white to off-white powder.
D. Not less than 70 percent ofthe stated amount ofC14H1403.

Mobile phase. A mixture on volumes of glacial acetic acid,

9 volumes of tetrahydrofitran and 90 volumes of toluene.
containing about 0.5 g ofNaproxen with 10 ml of methanol for
15 minutes, centrifuge and use the supernatant liquid:
with methanol.
Apply to the plate 10 /ll of each solution. After development,
cent).
Nebivolol Hydrochloride contains not less than 98.0 per cent

and not more than 102.0 per cent of C22H2sF2N04,HCl,
calculated on the anhydrous basis.
Category. Antihypertensive.
Identification
Detennine by infrared absorption spectrophotometry (2.4.6).
Compare the spectrum with that obtained with nebivolol
hydrochloride RS or with the reference spectrum ofnebivolol
hydrochloride.
Tests
Related substances. Determine by .liquid chromatography
(2.4.14).

examination in 5 ml of acetonitrile and dilute to 100.0 ml with
the mobile phase.
Reference solution (a). Dissolve 30 mg of nebivolol
hydrochloride RS in 5 ml of acetonitrile and dilute to 100.0 ml
with the mobile phase.
Reference solution (b). Dilute 1 ml ofreference solution (a) to
porous silica (5 /lm) with chemically bonded phenyl
groups,
1758
NEBIVOLOL TABLETS
IP 2010
- mobile phase: a mixture of28 volumes of acetonitrile,

72 volumes of a buffer solution prepared by dissolving
3.4 g of tetrabutyl ammonium hydrogen sulphate in
1000 ml wate]; and 0.3 volume of diethylamine,
- injection volume. 20 ,..t.l.
column efficiency in not less than 2000 theoretical plates and
the tailing factor is not more than 2.0.
Inject the test solution and reference solution (b). In the
chromatogram obtained with the test solution, the area of any
secondary peak is not more than 0.5 times the area ofthe peak
in the chromatogram obtained with reference solution (b) (0.5
per cent) and the sum of areas of all the secondary peaks is
not more than the area of the peak in the chromatogram
obtained with reference solution (b) (1.0 per cent).
Sulphated ash (2.3 .18). Not more than 0.2 per cent.
Water (2.3.43). Not more than 1.0 per cent, determined on 0.3 g.

examination in 5 ml of acetonitrile and dilute to 50.0 ml with
the mobile phase. Dilute 10.0 ml of this solution to 100.0 ml
Reference solution. Dissolve 35 mg of nebivolol
hydrochloride RS in 5 ml of acetonitrile and dilute to 50.0 ml
with the mobile phase. Dilute 10.0 ml ofthis solution to 100.0
Use the chromatographic system described under Related
substances.
than 2.0 per cent.
Inject the test solution and the reference solution.
Calculate the content ofC22H25F2N04,HCl.
exceeding 30.
Nebivolol Tablets
Nebivolol Hydrochloride Tablets
Nebivolol Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount ofnebivolol,
C22H25F2N04.
Usual strength. 5 mg
Identification
Tests
Apparatus No.1,
Medium. 500 ml 0,1 M hydrochloric acid,
Withdraw a suitable volume ofthe medium and filter.
Detennine by liquid chromatography (2.4.14) as described
under Assay using the following solutions and a flow rate of
1.5 ml per minute.
Test solution. The filtrate obtained as given above.

Reference solution. A solution containing nebivolol
hydrochloride RS equivalent to 0.001 per cent w/v of
nebivolol, in the dissolution medium.
C22H2sF2N04.
Tablets.
Determine by liquid chromatography (2.4.14), using the
chromatographic conditions and the reference solution
described in the Assay.
Test solution. Disperse one tablet in the mobile phase, dilute

to obtain a solution containing 0.005 per cent w/v ofnebivolol
in the mobile phase and filter.
Calculate the content of C22H25F2N04 in the tablets.
Test solution. Weigh and powder 20 tablets. Weigh accurately

a quantity of the powder containing about 5 mg ofnebivolol,
disperse in 100.0 ml ofthe mobile phase and filter.
Reference solution. A solution containing nebivolol
hydrochloride RS equivalent to 0.005 per cent w/v of
nebivolol, in the mobile phase.
a stainless steel column 25 cm x 4.6 mm, packed with
octadecylsilane bonded to porous silica (5 /lm),
mobile phase: a mixture of45 volumes ofa buffer solution
prepared by dissolving 6.8 g of potassium dihydrogen
orthophosphate in 1000 ml of wate]; adding 2 ml of
triethylamine and adjusting the pH to 3.0 with 10 per
cent v/v orthophosphoric acid, 25 volumes of
acetonitrile and 30 volumes of methanol,
1759
NELFINAVIR MESYLATE
IP 2010

- injection volume. 20 111.
Tests
Specific optical rotation (2.4.22). -105 0 to -120 0 , determined
in a 1.0 per cent w/v solution in methanol.
than 2.0 per cent.
Test solution. A 0.1 per cent w/v solution of the substance

under examination in the mobile phase.
Calculate the content of C22H2SF2N04 in the tablets,

Labelling. The label states the strength in terms of the
equivalent amount of nebivolol.

substance under examination in the mobile phase.
methanesulphonic acid in the mobile phase.
column efficiency determined from the nelfinavir peak is not
less than 4000 theoretical plates and the tailing factor is not
more than 2.0.
Nelfinavir Mesylate ,
C32~sN304S,C~03S

(2.4.14), using the chromatographic system described in the
Assay.
Mol. Wt. 663.9
Nelfinavir Mesylate is (3S,4aS,8aS)-N-(tert-butyldecahydro2-[(2R,3R)-3-(3-hydroxy-o-toluamido)-hydroxy-4(phenylthio)butyl]isoquinoline-3-carboxamide methyl

sulphonate.
Nelfinavir Mesylate contains not less than 98.0 per cent and
not more than 101.0 per cent of C32H4SN304S,CH403S,
Category. Antiretroviral.
Separately inject reference solution (b) and record the

chromatograms. Separately inject the test solution and
continue the chromatography for at least three times the
retention time of the principal peak. In the chromatogram
obtained with the test solution, the area of any peak other
than the principal peak is not greater than half of the area of
the principal peak in the chromatogram obtained with reference
solution (a) (0.5 per cent) and the sum of the areas of all such
peaks is not greater than the area of the principal peak in the
cent). Ignore any peak due to methanesulphonic acid
corresponding to the retention time of the principal peak in
the chromatogram obtained with reference solution (b).
Methanesulphonic acid. 13.5 per cent to 15.5 per cent w/w,
calculated on the anhydrous basis, determined by the following
method. Weigh accurately about 0.6 g, dissolve in 50 ml of
dimethylformamide and titrate with 0.1 M sodium hydroxide,
1 ml of 0.1 Msodium hydroxide is equivalentto 0.00961 g of
CH3S03H.
Dose. 1.25 g twice daily.

Description. A white or almost white powder.
Identification

Compare the spectrum with that obtained with neljinavir
mesylate RS or with the reference spectrum of nelfinavir
mesylate.
obtained with the test solution corresponds to the peak in the
Water (2.3.43). Not more than 3.0 percent, determined on 0.5 g.

Test solution. A 0.01 per cent w/v solution of the substance,

Reference solution. A 0.01 per cent w/v solution ofneljinavir
mesylate RS in the mobile phase.
1760
IP 2010
NELFINAVIR MESYLATE ORAL POWDER
Use the chromatographic system described under Assay.
a stainless steel column 25 em x 4.6 rom, packed with
mobile phase: a filtered and degassed mixture of D. Not less than 75 per cent of the stated amount of
45 volumes of acetonitrile, 20 volumes of methanol C32~5N304S,
and 35 volumes ofa buffer prepared by dissolving 4.0 g
of sodium dihydrogen phosphate in 1000 ml of water,
(2.4.14).
to which 1 ml of dimethylamine solution and 1 g of
sodium octanesulphonate are added and mixed to "Test solution. Weigh accurately a quantity of the oral powder
containing 50 mg ofNelfinavir Mesylate, disperse in 10 ml of
dissolve,
methanol,
dilute to 50 ml with the mobile phase and filter.
Reference solution (a). Dissolve 10 mg of nelfinavir mesylate
RS in 2 ml of methanol and dilute to 10 ml with the mobile
Inject the reference solution. The test is not valid unless the phase.
column efficiency determined from the nelfinavir peak is not
less than 5000 theoretical plates, the tailing factor is not more
than 2.0 and the relative standard deviation for replicate
injections is not more than 2.0 per cent.
Separately inject the test solution and the reference solution
and measure the responses for the principal peak. Calculate
the content ofC32H45N304S,CH403S.
Storage. Storeprotected from light.
Nelfinavir Mesylate Oral Powder

Nelfinavir Mesylate Oral Powder contains not less than
amount ofnelfinavir, C32~5N304S,
Usual strength. 50 mg per g.

100 ml with the mobile phase.
a stainless steel column 15 em x 4.6 mm, packed with
mobile phase: a mixture of 28 volumes ofa buffer solution
prepared by dissolving 4.88 g of anhydrous sodium
dihydrogen phosphate in 1000 ml of water, adjusting
the pH to 3.4 with phosphoric acid and filtering,
27 volumes of acetonitrile, 20 volumes of methanol
and 25 volumes of water. Adjust the pH to 4.8 with
0.1 M sodium hydroxide or orthophosphoric acid.
Inject the reference solution (a). The test is not valid unless
the tailing factor is not more than 2.0 and the column efficiency
in not less than 4000 theoretical plates.
Identification
Tests
Apparatus. No 1
Medium. 900 ml of 0.1 M hydrochloric acid.

secondary peak is not more than the area of the peak in the
chromatogram obtained with the reference solution (b)
(1.0 per cent) and the sum of areas of all the secondary peaks
is not more than twice the area ofthe peak in the chromatogram
obtained with the reference solution (b) (2.0 per cent).

0.5g.
Assay. De~'rmine by liquid chromatography (2.4.14).
Determine by liquid chromatography (2.4.14).
Solvent mixture. 30 volumes of water and 70 volumes of

methanol.
Test solution. Use the filtrate and, ifnecessary, dilute with the
dissolution medium.
Reference solution. A 0.065 per cent w Iv solution of nelfinavir
mesylate RS in methanol. Dilute 10 ml ofthe solution to 100 ml
with the dissolution medium.
Test solution. Weigh accurately a quantity of the powder

containing 50 mg ofNelfinavir Mesylate, disperse in 50 ml of
0.1 M hydrochloric acid, dilute to 250.0 ml with the solvent
mixture and filter.
1761
NELFINAVIR MESYLATE ORAL POWDER
IP 2010
Reference solution. Dissolve 10 mg ofneljinavir mesylate RS

in 10 ml ofO.1Mhydrochloric acid and dilute to 50.0 ml with
the solvent mixture.
a stainless'steel column 15 cm x 4.6 mm,packed with
- column temperature 40,
prepared by dissolving 4 g of sodium dihydrogen
phosphate dihydrate and 1g of I-octane sulphonic
acid sodium salt into 1000 ml of water, adding 1ml of
dimethylamine and filtering, 45 volumes acetonitrile
and 20 volumes of methanol,
iIljection volume. 10 Ill.
tailing factor is not more than 2.0, the column efficiency in not
less than 2000 theoretical plates and the relative standard
deviation for replicate injections is not more than 2.0 per cent.
Calculate the content of C32H4SN304S in the oral powder.
exceeding 30.
equivalent amount ofnelfinavir.
Nelfinavir Tablets
Nelfinavir Mesylate Tablets
Nelfinavir Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of nelfinavir
mesylate, C32H4SN304S,CH403S.
Identification
A. Shake a quantity ofthe powdered tablets containing about
0.1 g of Nelfinavir Mesylate with 80 ml of methanol for
10 minutes, add sufficient methanol to produce 100 ml, mix
and filter. Dilute 5 ml ofthe filtrate to 100 ml with methanol.
When examined in the range 200 nm to 300 urn the resulting
solution shows an absorption maximum only at about 254 nm
(2.4.7).
Tests
Apparatus No.1,
Medium. 900 mlofO.OI Mhydrochloric acid,
Withdraw a suitable volume ofthe medium and filter promptly
through a membrane filter disc with an average pore diameter
not greater than 1.0 /lm. Reject the first few ml ofthefiltrate
and dilute a suitable volume of the filtrate with the same
solvent. Measure the absorbance of the resulting solution at
the maximum at about 250 nm (2.4.7). Calculate the content of
C32H4SN304S,CH403S from the absorbance of a solution of
known concentration of neljinavir mesylate RS.
D. Not less thau 75 per cent of the stated amount of
C32~sN304S,C~03S,

(2.4.14).
Test solution. Weigh accurately a quantity of the powdered

tablets containing about 100 mg ofNelfinavir Mesylate, add
about 20 ml of methanol, mix with the aid of ultrasound for
10 minutes and dilute to 100 ml with.the mobile phase.
Reference solution. Weigh accurately about 10 mg of
neljinavir mesylate RS, add about 10 ml of methanol, shake
for 10 minutes and dilute to 50 ml with the mobile phase.
octadecylsilane bonded to porous silica particles or
ceramic microparticles (5 /lm),
- mobile phase: a filtered and degassed mixture of
45 volumes of acetonitrile, 20 volumes of methanol
and 35 volumes ofa buffer prepared by dissolving 4.0 g
of sodium dihydrogen phosphate in 1000 ml of water,
to which are added 1 ml of dimethylamine solution and
1 g of sodium octanesulphonate and mixing to dissolve,
- spectrophotometer set at 215 urn,
column efficiency determined from the nelfinavir mesylate peak
is not less than 4000 theoretical plates and the tailing factor is
not more than 2.0.
Inject separately the diluent (10 ml of methanol diluted to
50 ml with the mobile phase) and the test solution and continue
the chromatography for 4 times the retention time of the
principal peak. Examine the diluent chromatogram Jor any
extraneous peaks and ignore the corresponding peaks
observed in the chromatogram obtained with the test solution.
Any secondary peak observed in the chromatogram obtained
with the test solution should not be more than 1.0 per cent
1762
IP 2010
NEOMYCIN SULPHATE
and the sum of the areas of all the secondary peaks should
not be more than 2.0 per cent when calculated by percentage
area normalisation. Inhibit integration of peak due to
methanesulphonic acid.
Mobile phase. A freshly prepared 3.85 per cent w/v solution

of ammonium acetate.
examination in 10 ml of water.
Reference solution. A 2.0 per cent w/v solution of neomycin
sulphate RS in water.

tablets containing about 200 mg ofNelfinavir Mesylate, add
10 minutes and dilute to 100.0 ml with the mobile phase. Filter
not greater than 1.0 11m, rejecting the first few ml ofthe filtrate.
Further dilute 5.0 ml ofthe filtrate to 100.0 ml with the mobile
phase.
neifinavir mesylate RS, add about 10 ml ofmethanol, mix with
the aid ofultrasound to dissolve and dilute to 50.0 ml with the
mobile phase. Dilute 5.0 ml ofthis solution to 50.0 ml with the
mobile phase.
Use the chromatographic system described in the test for
Related substances.
column efficiency determined from the nelfmavir mesylate peak
is not less than 5000 theoretical plates, the tailing factor is not
more than 2.0 and the relative standard deviation for replicate
Inject separately the test solution and the reference solution
and measure the responses for the major peale Calculate the
content ofC32H4sN304S,CH403S in the tablets.

dry the plate in air for 10 minutes, heat at 100 0 for 1 hour and
spray with a 0.1 per cent w/v solution of ninhydrin in
I-butanol saturated with water. Heat again at 100 0 for
5 minutes. The principal spot in the chromatogram obtained
with the test solution corresponds to that in the chromatogram
obtained with the reference solution.
B. Dissolve about 10 mg in 5 ml ofwater, add 0.1 ml ofpyridine
and 2 ml of a 0.1 per cent w/v solution of ninhydrin and heat
on a water-bath at a temperature of about 70 0 for 10 minutes;
a deep violet colour is produced.
C. A 5 per cent w/v solution gives the reactions of sulphates
(2.3.1).
Tests
pH (2.4.24). 5.0to 7.5, determined in a 1.0 percentw/v solution.
Specific optical rotation (2.4.22).+53.5 0 to +59.0 0 , determined
Neamine. Determine by thin-layer chromatography (2.4.17),
coating the plate with silica gel H
Mobile phase. A mixture of 30 volumes of methanol, 20

volumes of strong ammonia solution and 10 volumes of
dichloromethane.
Neomycin Sulphate
Neomycin Sulphate is a mixture ofthe sulphates ofsubstances
obtained by the growth of certain selected strains of
Streptomyces fradiae.
Neomycin Sulphate has a potency of not less than 600 Units
per mg, calculated on the dried basis.
Category. Antibacterial (topical and systemic).
Dose. 1 g every 4 hours.
Description. A white or yellowish-white powder; odourless
or almost odourless; hygroscopic.
Reference solution. A 0.05 per cent w/v solution of neamine

RSin water.
Apply to the plate as 5-mm bands 5 III of each solution. Dry
the bands; allow the mobile phase to rise at least 8 cm. Dry the
plate in a current ofwann air, heat at 1100 for 10 minutes, spray
the plate with ninhydrin and stannous chloride reagent and
heat at 1100 for 15 minutes. Spray the plate again with the
same reagent and heat at 110 0 for 15 minutes. Any band
corresponding to neamine in the chromatogram obtained with
Identification
Neomycin C. Determine by thin-layer chromatography (2.4.17),

coating the plate with silica gel of a suitable grade.
A. Determine by thin-layer chromatography (2.4.17), coating

the plate with silica gel H
Mobile phase. A mixture of 80 volumes of a 20 per cent w/v

solution of sodium chloride and 20 volumes of methanol.
1763
NEOMYCIN SULPHATE
IP 2010

examination in water and dilute to 5 ml with the same solvent.
Reference solution (a). Dissolve 30 mg ofjramycetin sulphate
RS in water and dilute to 25 ml with the same solvent.
25 ml with water.
Reference solution (c). Dissolve 40 mg of neomycin sulphate
RS in water and dilute to 5 ml with the same solvent.
Apply to the plate as 5 mm bands 5 J..Ll of each solution. Dry
the bands; allow the mobile phase to rise at least 12 cm. Dry
the plate at 100 to 105 for 10 minutes. Spray the plate with
ethanolic ninhydrin solution and heat at 100 to 105 for
10 minutes. In the chromatogram obtained with the test
solution the principal band corresponds to the principal band
in the chromatogram obtained with reference solution (c) and
the band due to neomycin C with Rf value slightly less than
that of the principal band is not more intense than the band
obtained with reference solution (a) (15 per cent) but is more
intense than the band in the chromatogram obtained with
reference solution (b) (3 per cent). The test is not valid unless
in the chromatogram obtained with reference solution (c) a
band appears with R f value slightly less than that of the
principal band.
Mobile phase. A mixture of 60 volumes of methanol,

40 volumes of strong ammonia solution and 20 volumes of
chloroform.
Test solution. Dilute ifnecessary a volume ofthe eye drops to
produce a solution containing 0.5 per cent w/v of Neomycin
Sulphate in water.
neomycin sulphate RS in water.
Reference solution (b). A mixture of equal volumes ofthe eye
drops and reference solution (a).
Apply to the plate 1 J..Ll of each solution. After development,
dry the plate in air, spray with a 1 per cent w/v solution of
ninhydrin in I-butanol and heat at 105 for 2 minutes. The
principal red spot in the chromatogram obtained with the test
with reference solution (a) and the principal red spot in the
chromatogram obtained with reference solution (b) appears
as a single spot.
Tests
coating the plate with silica gel H
.Mobile phase. A mixture of 30 volumes of methanol,

Loss on drying (2.4.19). Not more than 8.0 per cent, determined dichloromethane.
on 0.5 g by drying in an oven at 60 over phosphoruspentoxide Test solution. A volume of the eye drops containing 5 J..Lg
at a pressure not exceeding 0.7 kPa for 3 hours.
(3.5 Units).
Assay. Determine by the microbiological assay ofantibiotics,

Method A (2.2.1 0).
Reference solution. The same volume of water containing

0.1 J..Lg ofneamine RS.
Apply to the plate each solution. After development, dry the

plate in a current ofwarm air, heat at 110 for 10 minutes, spray
the plate with ninhydrin and stannous chloride reagent and
heat at 11 0 for 15 minutes. Spray the plate again with the
same reagent and heat at 110 for 15 minutes. Any spot
Labelling. The label states the strength in terms of Units of

neomycin per mg.
Neomycin Eye Drops

Neomycin Sulphate Eye Drops
Neomycin Sulphate Eye Drops are a sterile solution of
Neomycin Sulphate in Purified Water.
Neomycin Sulphate Eye Drops contain not less than 90.0 per
cent and not more than 115.0 per cent w/v ofthe stated amount
of neomycin sulphate.
Usual strength. 0.5 per cent w/v (3500 Units per ml).
Identification
Determine by thin-layer chromatography (2.4.17), coating the
plate with silica gel.
Neomycin C. Determine by liquid chromatography (2.4.14).
Test solution. Dilute the eye drops with 0.02 M borax to

contain 1 mg (700 Units) per mI. To 0.5 mI ofthe diluted solution
add 1.5 ml of a freshly prepared 2 per cent w/v solution of
I-jluoro-2,4-dinitrobenzene in methanol, dilute to 25 ml with
the mobile phase, allow to stand and use the clear lower layer.
Reference solution. Add 1.5 ml of the 1-fluoro-2,4dinitrobenzene solution to 0.5 ml ofa 0.1 per cent w/v solution
of neomycin sulphate RS in 0.02 M borax, heat in a waterbath at 60 for 1 hour and cool; dilute the solution to 25 ml with
the mobile phase, allow to stand and use the clear lower layer.
1764
IP 2010
NEOMYCIN EYE OINTMENT
porous silica particles (5 /-lm) (such as NucleosiI100-5),
- mobile phase: a mixture of 97 ml of tetrahydrofuran,
1.0 ml of water and 0.5 ml of glacial acetic acid diluted
with sufficient of a 2.0 per cent v/v solution of ethanol
in ethanol-free chloroform to produce 250 ml,
flow rate. 1.6 m1 per minute,
If necessary the tetrahydrofuran and water content of the
mobile phase may be adjusted so that the chromatogram
obtained with the reference solution shows resolution similar
to that in the specimen chromatogram supplied withframycetin
sulphate RS. The mobile phase should be passed through the
column for several hours before the solutions are injected.
Continue the chromatography for 1.4 times the retention time
ofthe peak due to neomycin B.
The column efficiency, determined using the peak due to
Neomycin B in the chromatogram obtained with the test
solution, should be not less than 13,000 theoretical plates.
of the peak corresponding to neomycin C is not less than
3.0 per cent and not more than 15.0 per cent ofsum ofthe areas
ofthe peaks corresponding to Neomycin B and Neomycin C.
Usual strength. 0.5 per cent w/w (3500 Units per g).
Identification
Mobilephase. A mixture of60 volumes of methanol, 40 volumes

of strong ammonia solution and 20 volumes of chloroform.
Test solution. Disperse a quantity of the eye ointment
containing 20 mg ofNeoinycin Sulphate in 20 ml of chloroform,
extract with 5 ml of water and use the aqueous extract.
neomycin sulphate RS in water.
Reference solution (b). A mixture of equal volumes of test
solution and reference solution (a).
Apply to the plate 1 /-ll of each solution. After development,
ninhydrin in i-butanol and heat at 105 for 2 minutes. The
principal red spot in the chromatogram obtained with the test
with reference solution (a) and the principal red spot in the
chromatogram obtained with reference solution (b) app~ars
as a single spot.
Tests
Other tests. Complies with the tests stated under Eye Drops.

coating the plate with silica gel H.
Assay. Measure accurately a quantity containing 5 mg of

Neomycin Sulphate and dilute to 50.0 ml with sterile phosphate
bufferpH 8.0 and mix. Dilute 10.0 ml ofthe resulting solution
to 100.0 ml with the same solvent.

dichloromethane.
Determine by the microbiological assay ofantibiotics, Method

A (2.2.10)
The upper fiducial limit of error is not less than 90.0 per cent
and the lower fiducial limit oferror is not more than 115.0 per
cent of the stated number of Units per ml.
Labelling. The strength is stated in terms of percentage w/v
as well as the number ofUnits per ml.

Neomycin Sulphate Eye Ointment

containing 20 mg ofNeomycin Sulphate in 20 m1 of chloroform,
shake gently with 8 ml of water, allow the layers to separate
and use the aqueous layer.
Reference solution. A 0.005 per cent w/v solution of neamine
RSin water.
Apply to the plate 2 /-l1 of each solution. After development,
dry the plate in a current ofwarm air, heat at 110 for 10 minutes,
spray with ninhydrin and stannous chloride reagent and
heat at 110 for 15 minutes. Spray the plate again with the
same reagent and heat at 1l0 for 15 minutes. Any spot
Neomycin C. Determine by liquid chromatography (2.4.17)
Neomycin Sulphate Eye Ointment is a sterile preparation

containing Neomycin Sulphate in a suitable basis.
Neomycin Sulphate Eye Ointment contains not less than
amount of neomycin sulphate.

containing 5 mg of Neomycin Sulphate in 20 ml of light
petroleum (120 to 160), add 5 m1 of 0.02 Mborax, shake,
separate the aqueous layer and centrifuge. To 0.5 m1 of the
separated aqueous layer add 1.5 ml ofa freshly prepared 2 per
1765
IP 2010
NEOMYCIN EYE OINTMENT
cent w/v solution of I-jluoro-2,4-dinitrobenzenein methanol,

heat on a water-bath at 60 for 1 hour and cool. Dilute the
resulting solution to 25 ml with the mobile phase, allow to
stand and use the clear lower layer.
Neostigmine Bromide
Refaence solution. Add 1.5 ml of the I-fluoro-2,4dinitr,obenzene solution to 0.5 ml ofa 0.1 per cent w/v solution
of neomycin sulphate RS in 0.02 M borax and proceed as for
the test solution.
porous silica particles (5 Ilm),
mobile phase: 97 ml of tetrahydrojitran, 1.0 ml of water
and 0.5 ml of glacial acetic acid with sufficient of a
2.0 per cent v/v solution of ethanol in ethanol-free
chloroform to produce 250 ml,
- spectrophotometer set at 350 run,
If necessary the tetrahydrofuran and water content of the
mobile phase may be adjusted so that the chromatogram
obtained with reference solution shows resolution similar to
that in the specimen chromatogram supplied withji-amycetin
sulphate RS. The mobile phase should be passed through the
column for several hours before the solutions are injected.
Continue the chromatography for 1.4 times the retention time
of the peakdue to neomycin B.
Neomycin B in the chromatogram obtained with the test
solution, should be not less than 13,000 theoretical plates.
Mol. Wt. 303.2

Neostigmine Bromide is 3-(dimethylcarbamoyloxy)
trimethylanilinium bromide.
Neostigmine Bromide contains not less than 98:0 per cent and
not more than 101.0 per cent ofClzH19BrNzOz, calculated on
the dried basis.
Category. Anticholinesterase.
Dose. 15 to 30 mg, repeated at suitable intervals; total daily
dose, 75 to 300 mg.
Description. Colourless crystals or a white, clystalline powder;
odourless; hygroscopic.
Identification
Tests B, C and D may be omitted if tests A and E are carried
out.
A. Detennine by infrared absorption spectrophotometly (2.4.6).
Compare the spectrum with that obtained with neostigmine
bromide RS.

of the peak corresponding to neomycin C is not less than
3.0 per cent and not more than 15.0 per cent ofthe sum ofthe
areas ofthe peaks corresponding to Neomycin B and Neomycin
C.

0.02 per cent w/v solution in 0.5 M sulphuric acid shows
absorption maxima at about 260 nm and 266 nm.
Other tests. Complies with the tests stated under Eye

Ointments.
C. Warm about 50 mg with 1 ml of dilute sodium hydroxide

solution; an odour of dimethylamine develops slowly.
Assay. Weigh accurately a quantity containing 5 mg of

Neomycin Sulphate, dissolve in 25 ml of chloroform, extract
with four quantities, each of20 ml, of sterile phosphate buffer
pH 8.0, combine the extracts and add sufficient of the buffer
solution to produce 100.0 ml.
D. Warm about 50 mg with 0.4 g ofpotassium hydroxide and

2 ml of ethanol (95 per cent) on a water-bath for 3 minutes,
replacing the evaporated ethanol. Cool, add 2 ml of dilute
diazobenzenesulphonic acid solution; an orange-red colour
is produced.
Cany out the microbiological assay of antibiotics, Method A

(2.2.10).
E. Gives the reactions ofbromides(2.3.1).
and the lower fiducial limit ofelTor is not more than 115.0 per
cent of the stated number of Units per g.
Tests
Acidity. Dissolve 0.2 g in20 ml of carbondioxide-free water

and titrate to pH 7.0 with 0.02 Msodium hydroxide (carbonatefree); not more than 0.1 ml is required.
Labelling. The strength is stated in terms of percentage w/v

as well as the number of Units per ml.
Appearance of solution. A 0.5 per cent w/v solution is clear

(2.4.1), and colourless (2.4.1).
1766
IP 2010
NEOSTIGMINE METHYLSULPHATE
3-Hydroxytrimethylanilinium bromide. Dissolve 50 mg in a

mixture of 1 ml of sodium carbonate solution and 9 ml of
water. Absorbance of the resulting solution at about 294 nm,
measured immediately after preparation, not more than
0.25 (2.4.7).
.
Sulphates (2.3.17). 0.75 g complies with the limit test for
sulphates (200 ppm).
on 1.0 g by drying in an oven at 105.
anhydrous glacial acetic acid, add 5 ml of acetic anhydride.
Titrate with 0.1 M perchloric acid, using crystal violet
solution as indicator. Carry out a blank titration.
1 ml of 0.1 M perchloric acid is equivalent to 0.03032 g of
C,2HI9BrN202.
Bromide, transfer to a semi-micro ammonia-distillation

apparatus, add 20 ml of a 50 per cent w/v solution of sodium
hydroxide and 0.5 ml ofa 2 per cent w/v solution of 2-octanol
in liqUidparaffin. Pass a current ofsteam through the mixture,
collect the distillate in 50 ml of 0.01 Msulphuric acid until the
volume is about 200 ml and titrate the excess of acid with
0.02 M sodium hydroxide using methyl red solution as
indicator. Repeat the operation without the substance unde~'
examination. The difference between the titrations represents
the amount of sulphuric acid required to neutralise the
dimethylamine produced.
1 ml of 0.01 M sulphuric acid is equivalent to 0.006064 g of
C12H19BrN202'
Neostigmine Methylsnlphate
Mol. Wt. 334.4

Neostigmine Methylsulphate is 3-(dimethylcarbamoyloxy)trimethylanilinium methyl sulphate.
Neostigmine Tablets
Neostigmine Bromide Tablets
Neostigmine Bromide Tablets contain not less than 92.5 per
cent and not more than 107.5 per cent of the stated amount of
neostigmine bromide, CI2H,9BrN202.
Usualstrength. 15 mg.
Neostigmine Methylsulphate contains not less than 98.5 per

cent and not more than 101.0 per cent of CI3H22N206S,
calculated on the dried basis.
Dose. By subcutaneous or intramuscular injection, 1 to 2.5
mg, repeated at suitable intervals; total daily dose,S to 20 mg.
Description. Colourless crystals or a white, crystalline powder;
hygroscopic.
Identification
Triturate a quantity of the powdered tablets containing about
0.3 g of Neostigmine Bromide with three quantities, each of
5 ml of ether and discard the ether. Macerate the residue with
several quantities, each of 10 ml of ethanol (95 per cent),
filtering after each maceration. Evaporate the combined filtrates
on a water-bath and dry the residue at 105 for 1 hour. The
residue melts at about 167, with decomposition. The residue
complies with the following tests.
A. Wann about 50 mg with 0.4 g ofpotassium hydroxide and
replacing the evaporated ethanol. Cool, add 2 ml of dilute
is produced.
B. Gives the reactions ofbromides (2.3.1).
Identification

Compare the spectrum with that obtained with neostigmine
methylsulphate RS or with the reference spectrum of
neostigmine methylsulphate.
absorption maxima, at about 261 nm and 267 nm. The ratio of
the absorbance at the maximum at about 267 run to that at the
maximum at261nm is 0.84 to 0.87.
C. Dissolve 0.1 g in 5 ml of distilled water and add 1 ml of a

6 per cent w/v solution of barium chloride; no precipitate is
produced. Add 2 ml of hydrochloric acid and heat in a waterbath for 10 minutes; a white precipitate is produced.

quantity ofthe powder containing about 0.15 g ofNeostigmine
D. Wann about 50 mg with 0.4 g ofpotassium hydrOXide and

Tests
1767
IP 2010
NEOSTIGMINE METHYLSULPHATE
replacing the evaporated ethai1Ol. Cool, add 2 ml of dilute

is produced.
Tests
Appearance ofsolution. A 5.0 per cent w/v solution in distilled
water is clear (2.4.1), and colourless (2.4.1).
Acidity or alkalinity. To 4.0ml ofa 5.0 per centw/v solution in
distilled water add 6.0 ml of water and 0.1 ml of phenolphthalein solution; the solution is colourless. Add
0.3 ml of O. 01 M sodium hydroxide; the solution becomes red.
Add 0.4 ml of 0.01 M hydrochloric acid; the solution becomes
colourless. Add 0.1 ml of methyl red solution; the solution
becomes red or yellowish-red.
3-Hydroxytrimethylanilinium methyl sulphate. Dissolve
50 mg in a mixture of 1 ml of sodium carbonate solution and
9 ml of water. Absorbance of the resulting solution at about
294 urn, measured immediately after preparation, not more than
0.20(2.4.7).
Usualstrengths. 0.5 mgperml; 2.5 mg per ml.
Identification
A. Dilute, if necessary, a volume of the injection containing
2.5 mg of Neostigmine Methylsulphate to 5 ml with water,
shake with three quantities, each of 10 ml, of ether and discard
the ether extracts.
When examined in the range 230 urn to 360 urn (2.4.7), a 2 cm
layer of the resulting solution shows absorption maxima at
about 260 urn and 267 urn.
B. Determine by thin-layer chromatography (2.4.17), coating
Mobile phase. A mixture of 50 volumes of chloroform,

35 volumes of methanol, 10 volumes of formic acid and
5 volumes of water.
Test solution. Dilute the injection under examination, if
necessary, with water to produce a solution containing
0.05 per cent w/v ofNeostigmine Methylsulphate.
Chlorides (2.3.12). 1.0 g complies with the limit test for

chlorides (250 ppm).

neostigmine methylsulphate RS in water.


Apply to the plate 10 /!l ofeach solution. After development,

dry the plate in air, spray with dilute potassium
iodobismuthate solution. The principal spot in the
that in the chromatogram obtained with reference solution (a).
The principal spot in the chromatogram obtained with
reference solution (b) appears as a single, compact spot.

Assay. Weigh accurately about 0.3 g and dissolve in 150 m1 of
water. Add 100 ml of2 M sodium hydroxide, distill and collect
the distillate in 50 ml ofa 4 per cent w/v solution of boric acid
until a total volume of250 ml is reached. Titrate the distillate
with 0.1 M hydrochloric acid using 0.25 ml of methyl redmethylene blue solution as indicator. Repeat the operation
without the substance under examination. The difference
between the titrations represents the amount of hydrochloric
acid required.
C. To 1 ml add 0.5 ml of sodium hydroxide solution and

evaporate to dryness on a water-bath. Heat quickly in an oilbath to about 250 and maintain at this temperature for about
30 seconds. Cool, dissolve the residue in 1 ml of water, cool in
ice water and add 1 ml of diazobenzenesulphonic acid
solution; an orange-red colour is produced.
1 ml of 0.1 M hydrochloric acid is equivalentto 0.03344 g of

C13H22N206S.
Tests
pH. (2.4.24) 4.5 to 6.5.

3-Hydroxy trimethylanilinium methyl sulphate. Determine
by liquid chromatography (2.4.14).
Neostigmine Methylsulphate Injection
Test solution. Dilute the injection if necessary, with water to

contain a 0.05 per cent w/v solution of Neostigmine
Methylsulphate.
Neostigmine Injection is a sterile solution of Neostigmine

Methylsulphate in Water for Injections.
Reference solution (a). Dilute 1 volume ofthe test solution to

100 volumes with water.
Neostigmine Injection contains not less than 90.0 per cent

neostigmine methylsulphate, C13H22N206S.
Reference solution (b). Add 0.05 ml of5 M sodium hydroxide

to 1 ml of the test solution and allow to stand for 5 minutes.
Add 0.1 ml of 5 M hydrochloric acid and use immediately.
1768
IP 2010
NEOTAME
a stainless steel column 25 em x 4.6 rom packed with
octadecylsilane chemically bonded to porous silica
particles (5 f.!m)(such as Lichrosphere 60 RP-selectB),
mobile phase: 0.0015 M solution of sodium
heptanesulphonate in a mixture of 15 volumes of
acetonitrile and 85 volumes of 0.05 M potassium
dihydrogen orthophosphate adjusted to pH 3.0 with
flow rate. of 1.1 m1 per minute,
injection volume. 10 f.!l.
In the chromatogram obtained with reference solution (b) the
principal peak has a retention time of about 6.8 minutes
(neostigmine methylsulphate) and there is a peak with a
relative retention time of about 0.5 (3-hydroxy)
trimethylani1inium methylsulphate. In the chromatogram
obtained with the test solution, the area of any secondary
peak with a retention time corresponding to that ofthe peak
due to (3-hydroxy)trimethylanilinium methylsulphate in the
chromatogram obtained with reference solution (b) is not
greater than the area ofthe principal peak in the chromatogram
obtained with reference solution (a) (1 per cent).
Assay. Dilute an accurately measured volume containing about
25 mg ofNeostigmine Methy1sulphate to 50.0 ml with water.
Measure the absorbance of the resulting solution at the
C 13 H zzNz0 6 S taking 14.35 as the specific absorbance at
260nm.
Neotame
Category. Non-nutritive sweetener.

Description. A white crystalline powder.
Identification
(2.4.6). Compare the spectrum with that obtained with neotame
RS or with the reference spectrum ofneotame.
Tests
Specific optical rotation (2.4.22). -40 to-43.4, determined in
a 0.5 per cent w/v solution.
(2.4.14).
Test solution. Dissolve 100 mg ofsubstance under examination
in 50.0 ml ofthe mobile phase.
neotame RS in the mobile phase.
neotame impurity A RS in the mobile phase.
Use the chromatographic system as described under Assay.
than 5.0 per cent.
Inject the test solution and reference solution (a). In the
chromatogram' obtained with the test solution, the area of
peak corresponding to neotame impurity A is not more than
1.5 times the area of the principal peak in the chromatogram
obtained with reference solution (a) (1.5 percent) and the sum
of the areas of all other secondary peaks is not more than
twice the area of the principal peak in the chromatogram
Loss on ignition (2.4.20). Not more than 0.2 per cent.
Water (2.3.43). NotmorethaiJ. 5.0 per cent, determined on 1.0 g.
in 25.0 ml ofmobile phase. Dilute 5.0 ml ofthis solution to 10.0
Mol.Wt. 378.4
Neotame isN-(3,3-dimethylbuty1)-L-a-aspartyl-Lphenylalanine 2-methy1 ester.
Neotame contains not less than 97.0 per cent and not more
than 102.0 per cent of CzoH30NzOs, calculated on the dried
basis.
Reference solution. A 0.1 per cent w/v solution ofneotame RS

in the mobile phase.
- a stainless steel column 10 em x 4.6 mm, packed with
endcapped octadecylsilane bonded to porous silica (5
f.!ID),
1769
NEVlRAPINE
IP2010

mobile phase: dissolve 3 gm of sodium 1heptanesulphonate and 3.8 ml of tri~thylamine in 750
ml of water, adjust the pH to 3.5 with orthophosphoric
acid. Add 250 ml of acetonitrile, adjust the pH to 3.7
Inject the reference solution; The test is not valid unless the
tailing factor is not more than 2.0 and the relative standard
Calculate the content of C2oH30N20s.
Storage. Store protected from moisture.

examination in 100 ml of methanol.
with methanol.
octadecylsilane bonded to porous silica or ceramic
microparticles (5 Ilm),
mobile phase: a filtered and degassed mixture of
20 volumes of methanol, 20 volumes of acetonitrile
and 60 volumes of a buffer prepared by dissolving
12.0 g of sodium dihydrogen phosphate in about 800 ml
of water, adjusting the pH to 3.0 with phosphoric acid
and diluting to 1000.0 ml with water;
column efficiency determined from the nevirapine peak is not
more than 2.0.
Nevirapine
Mol. Wt. 266.3
Separately inject the test solution and the reference solution.

In the chromatogram obtained with the test solution, the area
of any peak other than the principal peak is not greater than
half of the area of the principal peak in the chromatogram
obtained with the reference solution (0.5 per cent) and the
sum of the areas of all such peaks is not greater than the area
of the principal peak in the chromatogram obtained with the
reference solution (1.0 per cent).
Nevirapine is ll-cyclopropyl-5,11-dihydro-4-methy-6Hdipyrido[3,2-b:2' ,3' -e][l,4]diazepin-6-one.
Nevirapine contains not less than 98.0 per cent and not more
than 102.0 per cent ofClsHl4N40, calculated on the dried basis.
on 1.0 g by drying at 105 for 3 hours.
Dose. 200 mg once or twice daily.
Assay: Determine by liquid chromatography (2.4.14).

under examination in methanol.
Identification
Compare the spectrum with that obtained with nevirapine RS
or with the reference spectrum of nevirapine.
Tests
(2.4.14).
Reference solution. A 0.005 per cent w/v solution of

nevirapine RS in methanol.
- a stainless steel column 25- cm x 4.6 mm, packed with
microparticles (5Ilm),
12.0 g of sodium dihydrogen phosphate in about 800 ml
of water, adjusting the pH to 3.0 with orthophosphoric
acid and diluting to 1000.0 ml with wate/;
1770
IP 2010
NEVIRAPINE ORAL SUSPENSION

injection volume. 20 ,.ti.
than 2.0 and the relative standard deviation for the replicate
and measure the responses for the principal peak. Calculate
the content ofClsHl4N40.

Nevirapine Oral Suspension is a suspension ofNevirapine in
a suitable flavoured vehicle.
Test solution. To an accurately measured volume of the

preparation under examination containing about 25 mg of
Nevirapine add about 10 ml of methanol, mix with the aid of
ultrasound for 10 minutes, dilute to 50 ml with water, mix and
filter.
nevirapine RS, add about 10 ml of methanol, mix with the aid
of ultrasound to dissolve and dilute to 50 ml with water.
octylsilyl silica gel for chromatography (5Ilm) (Such as
Hypersil C8),
mobile phase: A. 0.1 M ammonium acetate,
B. methanol,
below,
injection volume. 20 ,.ti.
Nevirapine Oral Suspension contains not less than 90.0 per

cent and not more than 110.0 per cent ofthe stated amount of
nevirapine, ClsH14N40.
Time
(in min.)
Usual strength. 50 mg in 5 ml.
5
25
30
31
40
Identification
A. Detennine by thin-layer chromatography (2.4.17), coating
the plate with silica gel GF254.
Mobile phase. A mixture of 40 volumes of 1-butanol,

30 volumes of heptane, 30 volumes of acetone and 10 volumes
of strong ammonia solution.
Test solution. Dilute the preparation under examination with
methanol to obtain a solution containing 1 mg ofNevirapine
perml.
Reference solution. A 0.1 per cent w/v solution of nevirapine

RS in a mixture of75 volumes of methanol and 25 volumes of
water.
dry the plate in air and examine in ultraviolet light at 254 urn.
The principal spot in the chromatogram obtained with the test
solution corresponds to that in the -chromatogram obtained
Tests
pH (2.4.24). 5.0to 7.0.
(2.4.14).
Mobile phase A
(per cent v/v)
95
95
20
20
95
95
Mobile phase B
(per cent v/v)
5
5
80
80
5
5
more than 2.0.
Inject separately the diluent (dilute 10 ml of methanol to 50 ml
with water) and the test solution. Examine the diluent
chromatogram for any extraneous peaks and ignore the
corresponding peaks observed in the chromatogram obtained
with the test solution. Ignore any peaks due to preservatives
also.
with the test solution should not be more than 1.0 per cent
and the sum of the areas of all the secondary peaks should
not be more than 2.0 per cent when calculated by percentage
area normalisation.
Other tests. Comply with the tests stated under Oral Liquids.
Assay. Detennine by liquid chromatography (2.4.14).
Test solution. Weigh accurately a quantity of the preparation

under examination containing 25 mg ofNevirapine, add abol1t
10 ml ofmethanol, mix with the aid ofultrasound for 10 minutes,
dilute to 50.0 ml with water, mix and filter. Further dilute 10.0 ml
ofthe :filtrate to 25.0 ml with water.
1771
NEVIRAPINE TABLETS
IP 2010

nevirapine RS, add about 20 ml ofmethanol, mix with the aid
of ultrasound to dissolve and dilute to 100.0 ml with water.
Dilute 10.0 ml ofthis solution to 25.0 ml with water.
Use the chromatographic system described under the test for
Related substances.
than 2.0 per cent.
and measure the responses for the princip~l peak.
Determine the weight per ml of the oral suspension (2.4.29)
and calculate the content of CIsHI4N40 weight in volume.
Nevirapine Tablets
Nevirapine Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount ofnevirapine,
ClsH14N40.
Identification
A. When examined in the range 200 urn to 400 nm (2.4.7) a
0.001 per cent w/v solution in the mobile phase described
under Assay, shows an absorption maximum at about 230 urn.
containing 0.05 per cent w/v ofNevirapine and filter through

a membrane filter disc with an average diameter not exceeding
1.0 /lm, rejecting the first few ml ofthe filtrate.
Reference solution. A 0.05 per cent w/v solution ofnevirapine
RS in the mobile phase.
octadecylsi1ane bonded to porous silica or ceramic
microparticles (5/lm),
12.0 g ofsodium dihydrogen phosphate in about 800 ml
of water, adjusting the pH to 3.0 with orthophosphoric
acid and diluting to 1000.0 ml with water;
injections is not more than 2 per cent.
Inject the test solution and continue the chromatography for
at least five times the retention time of the principal peak.
Determine the amount of related substances by the area
normalisation method. Any individual impurity is not more
than 1.0 per cent and the sum ofall impurities is not more than
2.0 per cent.
Apparatus No.1,
Medium. 900 ml of 0.1 M hydrochloric acid,

of powder containing about 100 mg of Nevirapine with
. sufficient of the mobile phase to obtain a mixture containing
0.05 per cent w/v of Nevirapine. Mix and filter through a
membrane filter disc with an average pore diameter not greater
Withdraw a suitable volume of the medium and filter through
than 1.0 /lm, rejecting the first few ml ofthe filtrate.
a membrane filter disc with an average pore diameter not greater
than 1.0 /lm, rejecting the first few ml of the filtrate. Dilute Reference solution. A 0.05 per cent w/v solution ofnevirapine
suitably, ifnecessary. Measure the absorbance ofthe resulting RS in the mobile phase.
solution, at the maximum at about 313 urn (2.4.7).
Calculate the content of CIsHI4N40 from the absorbance

obtained from a solution ofknown concentration ofnevirapine
RS in 0.1 M hydrochloric acid.
D. Not less than 70 per cent ofthe stated amount ofC,sH'4N40.
(2.4.14).
Test solution. Shake a quantity ofthe powdered tablets with a
suitable quantity of the mobile phase to obtain a mixture
1772

microparticles (5 /lm),
12.0 g ofsodium dihydrogen phosphate in about 800 ml
of water, adjusting the pH to 3.0 with phosphoric acid
and diluting to 1000.0 ml with water;
IP 2010
NICLOSAMIDE

and measure the responses for the principal peale Calculate
.
the content ofCIsHI4N40 in the tablets.
Storage. Store protected from moisture at a temperature not
exceeding 30.
Niclosamide
cent w/v solution of ammonium sulphamate, shake, allow to

stand for 3 minutes and add 2 ml ofa 0.5 per cent w/v solution
ofN-(l-naphthyl) ethylenediamine dihydrochloride; a violet
colour is produced.
C. Heat the substance under examination on a copper wire in
a non-luminous flame; a green colour is imparted to the flame.
Tests
(2.4.14).

examination in 50 ml ofthe methanol.
Reference solution. Dilute 1.0 ml ofthe test solution to 100.0
ml with acetonitrile. Dilute 1.0 ml of this solution to 20.0 ml
with acetonitrile.
- a stainless steel column 12.5 cm x 4.0 mm, packed with
mobile phase: a mixture of 50 volumes of acetonitrile,
50 volumes ofa solution containing 0.2 per cent w/v of
potassium dihydrogen orthophosphate, 0.1 per cent
w/v of disodium hydrogen phosphate and 0.2 per cent
w/v of tetrabutylammonium hydrogen sulphate,
Anhydrous Niclosamide
~~xrN02
yH
CI
Mol. Wt. 327.1

Niclosamide is 2' ,5-dichloro-4' -nitrosalicylanilide.
Niclosamide contains not less than 98.0 per cent and not more
than 101.0 per cent ofC 13H gChNz0 4, calculated on the dried
basis.
Category. Anthelmintic.
Dose. 2 g as a single dose after a light breakfast followed by a
purgative 2 hours later.
Description. A yellowish white to yellowish, fine crystals or
powder; odourless.
Identification
Test A may be omitted if tests Band C are carried out. Tests B


Compare the spectrum with that obtained with niclosamide
RS or with the reference spectrum of nic1osamide.
B. Heat 50 mg with 5 ml of 1 Mhydrochloric acid and 0.1 g of
zinc powder in a water-bath for 10 minutes, cool and filter. To
the filtrate add 1 ml of a 0.5 per cent w/v solution of sodium
nitrite and allow to stand for 3 minutes. Add 2 ml of a 2 per
Inject the reference solution. Adjust the sensitivity so that

the height of the principal peak is not less than 20 per cent of
full scale ofthe recorder.
Inject the test solution and the reference solution. Run the
chromatogram twice the retention time of the principal peak.
In the chromatogram obtained with the test solution, the sum
ofthe areas ofall the secondary peaks is not more than 4 times
the area of the principle peak in the chromatogram obtained
with the reference solution. Ignore any peak with an area less
than 10 per cent of the area of the principal peak in the
Chlorides (2.3.12). T02.0 g add a mixture of40 ml of water and
1.2 ml of 5 M acetic acid, boil for 2 minutes, cool and filter;
10 ml ofthe filtrate diluted to 15 ml with water complies with
the limit test for chlorides (500 ppm).
2-Chloro-4-nitroaniline. Not more than 100 ppm, determined
by the following method. Boil 0.25 g with 5 ml of methanol,
cool, add 45 ml of 1 M hydrochloric acid, heat to boiling,
coot filter and dilute the filtrate to 50.0 ml with 1 M
hydrochloric acid. To 10.0 ml ofthis solution add 0.5 ml of a
0.5 per cent w/v solution of sodium nitrite and allow to stand
for 3 minutes. Add 1.0 ml of a 2 per cent w/v solution of
ammonium sulphamate, shake, allow to stand for 3 minutes
1773
NICLOSAMIDE
IP 2010
and add 1.0 ml ofa 0.5 per cent w/v solution ofN-(I-naphthyij
ethylenediamine dihydrochloride. Any.pinldsh violet colour
produced is not more intense than that obtained in a solution
prepared at the same time and in the same manner using
10.0 ml of a solution prepared by diluting 2.0 ml of a
0.00050 per cent w/v solution of 2-chloro-4-nitroaniline in
methanol to 20 ml with 1 M hydrochloric acid and beginning
at the words "add 0.5 ml of a 0.5 per cent w/v solution of
sodium nitrite.....".
5-Chlorosalicylic acid. Not more than 60 ppm, detenniiled by
the following method. Boil 1.0 g with 15 ml of water for
2 minutes, cool, filter through a membrane filter (pore size
0.45 !lm), wash the filter and dilute the combined filtrate and
washings to 20 ml with water: (solution A). Dissolve 30 mg of
5-chlorosalicylic acid in20 ml of methanol and add sufficient
water to produce 100.0 ml. Dilute 1.0 ml of this solution to
100.0 ml with water (solution B). To 10.0 ml ofeach ofsolutions
A and B add separately 0.1 ml offerrie chloride solution; any
violet colour produced in solution A is not more intense than
that obtained in solution B.
Assay. Weigh accurately about 0.3 g, dissolve in 80 ml of a
mixture of equal volumes of acetone and methanol. Titrate
with 0.1 M tetrabutylammonium hydroxide, determining the
end-point potentiometrically (2.4.25). Carry out a blank titration.
1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to
0.03271 gofCl3HsChN204'
B. Heat 50 mg with 5 ml of 1 Mhydrochlodc acid and 0.1 g of

the filtrate add 1 ml of a 0.5 per cent w/v solution of sodium
nitrite and. allow to stand for 3 minutes. Add 2 ml of a 2 per
stand for 3 minutes apd add 2 ml of a 0.5 per cent w/v solution
ofN- (I-naphthyl) ethylenediamine dihydrochloride; a violet
colour is produced.
Tests
Related substances. Detennine by liquid chromatography
(2.4.14).

containing about 100 mg ofanhydrous nicolsamide with 80 ml
of methanol for 15 minutes and dilute to 100.0 ml with the
same solvent.
ml with acetonitrile and further dilute 1.0 ml ofthis solution to
20.0 ml with acetonitrile.
octadecylsilane bonded to porous silica (5 !lm) ,
and 50 volumes ofa solution containing about 0.2 per
cent w/v ofpotassium dihydrogen orthophosphate, 0.2
per cent w/v of tetrabutylammonium hydrogen sulphate
and 0.1 per cent w/v of disodium hydrogen
orthophosphate,
flow rate. I ml per minute,
Inject the reference solution. Adjust the sensitivity so that
the height of the principal peak is not less than 20 per cent of
full scale of the recorder.
Niclosainide Tablets
Niclosamide Tablets contain not less than 95.0 per cent and
not more than 105.0 per cent of the stated amount of
niclosamide, C13HsC12N204' The tablets may contain
sweetening and flavouring agents.
Identification
Heat a quantity of the powdered tablets containing 0.5 g of
Niclosamide with 25 ml of hot ethanol (95 per cent), filter
while hot and evaporate the filtrate to dryness on a waterbath. The residue complies with the following tests.
A. Detennine by infrared absorption spectrophotomeuy (2.4.6).

Compare the spectrum with that obtained with niclosamide
RS or with the reference spectrum of niclosamide.
chromatogram twice the retention time of the principal peak.
In the chromatogram obtained with the test solution, the sum
ofthe areas of all the secondary peaks is not more than 4 times
the area of the principle peak in the chromatogram obtained
with the reference solution. Ignore any peak with an area less
than 10 per cent of the area of the principal peak in the
2-Chloro-4-nitroaniline. Not more than 100 ppm, Boil a
quantity of the powdered tablets containing 0.1 g of
Niclosamide with 20 ml of methanol and 20 ml ofa solution in
methanol containing 10 !lg of 2-chloro-4-nitroaniline, cool,
add 45 ml of 1 M hydrochloric acid, heat to boiling, cool, filter
and dilute the filtrate to 50.0 ml with 1 M hydrochloric acid.
To 10.0 ml of this solution add 0.5 ml of a 0.5 per cent w/v
solution of sodium nitrite and allow to stand for 3 minutes.
1774
IP 2010
NICOTINAMIDE
Add 1.0 ml of a 2 per cent w/v solution of ammonium

sulphamate, shake, allow to stand for 3 minutes and add
1.0 ml of a' 0.5 per cent w/v solution of N-(l-naphthyl)
ethylenediamine dihydrochloriqe. Any pinkish violet colour
produced is not more intense than that obtained in a solution
prepared at the same time and in the same manner using
10.0 ml ofa solution prepared by diluting 2.0 ml ofa 0.0005 per
cent w/v solution of 2-chloro-4-nitroaniline in methanol to
20.0 ml with 1 M hydrochloric acid and beginning at the
words "add 0.5 ml of a 0.5 per cent w/v solution of sodium
nitrite.....".
5-ChlorosaUcyUc acid. Boil a quantity ofthe powdered tablets
containing 0.5 g of Niclosamide with 10 ml of water for
2 minutes, cool, filter and to the filtrate add 0.2 ml offerric
chloride solution; no red or violet colour is produced.
quantity of the powdered tablets containing about 0.3 g of
Niclosamide dissolved in 60 ml of dimethylformamide. Titrate
with 0.1 M teirabutylammonium hydroxide, determining the
0.03271 gofC13HsC12N204'
Labelling. The label states that the tablets should be chewed
thoroughly before swallowing.
Identification

Compare the spectrum with that obtained with nicotinamide
RS or with the reference spectnllTI of nicotinamide.
B. Heat about 5 mg in a dry tube; pyridine is evolved.
C. Boil 0.1 g with 1 ml of dilute sodium hydroxide solution;

ammonia is evolved.
D. To 2 ml ofa 0.1 per cent w/v solution add 6 ml of cyanogen
bromide solution and I ml of a 2.5 per cent w/v solution of
aniline; a golden yellow colour develops.
Tests
dioxide-fi-ee water is clear (2.4.1), and not more intensely
coloured than reference solution BYS7 (2.4.1).
pH(2.4.24). 6.0 to 7.5, determined in a 5.0 per centw/v solution.

45 volumes of ethanol and 4 volumes of water.
examination in 10 ml of ethanol (50 per cent).
Reference solution. A 0.02 per cent w/v solution of the
substance under examination in ethanol (50 per cent).
Apply to the plate 5 f..ll of each solution. Allow the mobile
phase to rise 10 cm. Dry the plate in air and examine in ultraviolet
light at 254 mTI. Any secondary spot in the chromatogram
spot in the chromatogram obtained with the reference solution.
Nicotinamide
Niacinamide
OyNH
o
C6H6N 20
Mol. Wt. 122.1
Heavy metals (2.3.13). Dissolve 0.67 gin 10 mlofwater, 7.5 ml

of 1 M hydrochloric acid and sufficient water to produce
25 ml. The solution complies with the limit test for heavy metals,
Method A (30 ppm).
Nicotinamide ispyridine-3-carboxamide.
Nicotinamide contains not less than 99.0 per cent and not
more than 101.0 per cent ofC6H6N 20, calculated on the dried
basis.

Category. B-group vitamin.
of 1.5 to 2.7 kPa for 18 hours.
Dose. Orally, prophylactic, 15 to 30 mg daily; therapeutic, 50

to 250 mg daily. By intravenous injection, .50 to 250 mg daily.
DescriptioR Colourless crystals or a white, crystalline powder;

anhydrous glacial acetic acid, heating slightly if necessary.
Add 5 ml of acetic anhydride. Titrate with 0.1 M perchloric
1775
IP 2010
NICOTINAMIDE TABLETS
acid, using crystal violet solution as indicator. Carry out a

blank titration.

C6H6NzO.
Niacinamide Tablets
Nicotinamide Tablets contain not less than 92.5 per cent and
nicotinamide, C6H 6N zO.
light at 254 urn. Any secondary spot in the chromatogram

Assay. Weigh and powder 20 tablets. Weigh. accurately a
quantity of the powder containing about 50 mg of
Nicotinamide, shake with 50 ml of ethanol (95 per cent) for 15
minutes and dilute to 100.0 ml with ethanol (95 per cent). Mix,
filter, dilute 5.0 ml ofthe filtrate to 100.0 ml with ethanol (95
per cent) and measure the absorbance ofthe resulting solution
at the maximum at about 262 nm (2.4.7). Calculate the content
of C6H 6N zO taking 241 as the specific absorbance at 262 nm.
Identification
Nicotinic Acid
Shake a quantity of the powdered tablets containing 0.2 g of

Nicotinamide with 50 ml of ethanol for 15 minutes, filter and
evaporate the filtrate to dryness on a water-bath. The residue
Niacin

Compare the spectrum with that obtained with nicotinamide
RS or with the reference spectrum of nicotinamide.
B. Boil 0.1 g with I ml of dilute sodium hydroxide solution;

ammonia is evolved.
C. To 2 ml ofa 0.1 per cent w/v solution add 6 ml of cyanogen
bromide solution and 1 ml of a 2.5 per cent w/v solution of
aniline; a golden yellow colour develops.
D. When examined in the range 230 nm to 360 nm (2.4.7), the
solution obtained in the Assay shows an absorption maximum
only at about 262 nm and two shoulders at about 258 urn and
269nm.
~")
~COOH
C6HsNOz
Mol. Wt. 123.1
Nicotinic Acid is pyridine-3-carboxylic acid.

Nicotinic Acid contains not less than 99.5 per cent and not
more than 100.5 per cent ofC 6H sNOz, calculated on the dried
basis.
Dose. Prophylactic, 15 to 30 mg daily; therapeutic, 50 to 250
mgdaily.
Description. A white or creamy-white, crystalline powder.
Tests
Identification



45 volumes of ethanol and 4 volumes of water.

Compare the spectrum with that obtained with nicotinic acid
RS or with the reference spectrum of nicotinic acid.

containing 0.1 g of Nicotinamide with 15 ml of ethanol for
15 minutes, filter, evaporate to dryness on a water-bath and
dissolve the residue as completely as possible in 1 ml of
ethanol.
400 volumes with ethanol
Apply to the plate 5 III of each solution. Allow the mobile
B. Heat a small quantity with twice its weight of soda lime;

pyridine is evolved.
C. Dissolve about 50 mg in 20 ml of water, neutralise to litmus

paper with 0.1 M sodium hydroxide, add 3 ml of copper
sulphate solution; a blue precipitate is gradually produced.
D. To 2 ml ofa 0.1 per cent w/v solution add 6 ml of cyanogen
bromide solution and I ml of a 2.5 per cent w/v solution of
aniline; a golden yellow colour is produced.
1776
IP 2010
NICOUMALONE
Tests

containing 50 mg ofNicotinic Acid with 50 ml of hot ethanol
(95 per cent), filter and allow the filtrate to cool.

Mobile phase. A mixture of 85 volumes of I-propanol,

10 volumes of anhydrousformic acid and 5 volumes of water.
Reference solution. A 0.1 per cent w/v solution of nicotinic

acid RS in ethanol (95 per cent).

examination in 10 ml of water. Warm slightly, ifnecessary.
Apply to the plate 5 !!l of each solution. After development,

dry the plate in air and examine in ultraviolet light at 254 om.

substance under examination in water.
Apply to the plate 5 !!l of each solution. After development,
dry the plate at 105 for 10 minutes and examine in ultraviolet
light at 254 nm. Any secondary spot in the chromatogram
Heavy metals (2.3.13). Mix 1.0 g with 1.5 ml of dilute
hydrochloric acid and sufficient water to produce 25 ml, heat
gently and cool to room temperature. The resulting solution
complies with the limit test for heavy metals, Method B
(20 ppm).
B. Triturate a quantity of the powdered tablets containing

50 mg ofNicotinic Acid with 10 ml of water and filter. To 2 ml
ofthe filtrate add 6 ml of cyanogen bromide solution and 1 ml
of a 2.5 per cent w/v solution of aniline; a golden yellow
precipitate is produced.
C. Shake a quantity of the powdered tablets containing 0.1 g

of Nicotinic Acid with ethanol (95 per cent), filter and
evaporate the filtrate to dryness. Add to the residue 10 mg of
citric acid and 0.15 ml of acetic anhydride and heat on a
water-bath; a reddish-violet colour is produced.
Tests
on 1.0 g by drying in an oven at 105 for 1 hour.

quantity of the powder containing about 0.25 g of Nicotinic
Assay. Weigh accurately about 0.25 g, dissolve in 50 ml of Acid, add 40 ml of hot ethanol (95 per cent), previously
carbon dioxide-free water and titrate with 0.1 M sodium neutralised to phenolphthalein solution, and shake. Allow to
hydroxide using phenol red solution as indicator. Repeat the stand for 15 minutes, swirling occasionally, and then shake
operation without the substance under examination. The for 10 minutes. Filter through a plug of cotton and wash the
difference between the titrations represents the amount of filter with ethanol (95 per cent). Add 50 ml of carbon dioxidesodium hydroxide required.
free water and titrate with 0.1 M sodium hydroxide using
1 ml of 0.1 M sodium hydroxide is equivalent to 0.01231 g of phenol red solittion as indicator.
C6H sNOz.
1 ml of 0.1 M sodiilm hydroxide is equivalent to 0.01231 g of
C6H sNO z.
'

Niacin Tablets
Nicoumalone
Nicotinic Acid Tablets contain not less than 92.5 per cent and
not more than 107.5 per cent ofthe stated amount ofnicotinic
acid, C6H sNO z.
Acenocoumarol
NOz
Usual strengths. 25 mg; 50 mg; 100mg.
Identification
45 volumes of ethanol (95 per cent) and 8 volumes of water.
CI9HISN06
1777
Mol. Wt. 353.3
NICOUMALONE
IP 2010
Nicoumalone is (RS)-4-hydroxy-3-[ 1-(4-nitrophenyl)-3oxobutyl]coumarin.
Nicoumalone contains not less than 98.5 per cent and not
more than 100.5 percent ofC19HlsN06, calculated on the dried
basis.
Category. Anticoagulant.
Dose. Initial dose, first day, 8 to 12 mg; second day, 4 to 8 mg;
maintenance dose, 1 to 8 mg daily.
Description. A white to brownish-white powder; odourless or
almost odourless.
Loss on drying (2.4.19). Not more than 0.5 per cent; determined
on 1.0 g by dIying in an oven at 105.
acetone and titrate with 0.1 M sodium hydroxide using
bromothymol blue solution as indicator.. Repeat the operation
between the titrations represents the amount of sodium
hydroxide required.
C19HlsN06.
Identification
Compare the spectrum with that obtained with nicoumalone
RS or with the reference spectrum of nicoumalone.
0.001 per cent w/v solution in a mixture of 9 volumes of
methanol and 1 volume of 1 M hydrochloric acid shows
absorption maxima at about 283 mn and 306 nm; absorbances
at the maxima, about 0.65 and about 0.52, respectively.
C. Heat 50 mg with 2.5 ml of glacial acetic acid, 0.5 ml of

hydrochloric acid and 0.2 g of zinc powder on a water-bath
for 5 minutes, cool and filter. To the filtrate add 0.05 ml of
sodium nitrite solution and add the mixture to 10 ml ofal per
cent wlvsolution of 2-naphthol containing 3 ml of5 M sodium
hydroxide; a bright red precipitate is produced.
Tests
Appearance of solution. A 2.0 per centw/v solution in acetone
is clear (2.4.1).
B. A 2.0 per cent w/v solution in 0.1 M sodium hydroxide is

clear (2.4.1), and yellow.


50 volumes of cyclohexane and 20 volumes of glacial acetic
acid.
examination in 10 ml of acetone.
substance under examination in acetone.
Apply to the plate 20 J.ll of each solution. After development,
illy the plate in air and irmnediately examine in ultraviolet light
at 254 nm. Any secondary spot in the chromatogram obtained
with the test solution is not more intense than the spot in the
Nicoumalone Tablets
Acenocoumarol Tablets
Nicoumalone Tablets contain not less than 90.0 per cent and
nicoumalone, C19HlsN06.
Usual strengths. 1 mg; 2 mg; 3 mg; 4 mg.
Identification
A. Heat a quantity of the powdered tablets containing 50 mg
ofNicoumalone with 30 ml of acetone under a reflux condenser
for 5 minutes, filter and wash the residue with two quantities,
each of 10 ml, of acetone. Evaporate the combined filtrate and
washings to 5 ml, add water dropwise until the solution
becomes turbid, heat on a water-bath until the solution is
clear and allow to stand. Filter, wash the crystals with a mixture
of equal volumes of acetone and water and dry at 100 at a
pressure of2 kPa for 30 minutes.
On the residue determine by infrared absorption
spectrophotometry (2.4.6). Compare the spectrum with that
obtained with nicoumalone RS or with the reference spectrum
ofnicoumalone.
B. When examined in the range 230 nm to 360 nm (2.4.7), the
final solution obtained in the Assay shows absorption maxima
at about 283 nm and 306 nm.
C. Heat 50 mg ofthe residue obtained in testA, with 2.5 ml of

glacial acetic acid, 0.5 ml of hydrochloric acid and 0.2 g of
zinc powder on a water-bath for 5 minutes, cool and filter. To
the filtrate add 0.05 ml of sodium nitrite solution and add the
mixture to 10 ml of a 1 per cent w/v solution of 2-naphthol
containing 3 ml of 5 M sodium hydroxide; a bright red
1778
IP 2010
NIFEDIPINE
Nifedipine
Tests

acid.
OCH 3

containing 20 mg of Nicoumalone with 5 ml of acetone,
centrifuge and use the supernatant liquid.
200 volumes with acetone.
Apply to the plate 20 J1.1 of each solution. After development,
dry the plate in air and immediately examine in ultraviolet light
at 254 nm. Any secondary spot in the chromatogram obtained
C17HISN206
Mol. Wt. 346.3
Nifedipine is dimethyll,4-dihydro-2,6-dimethyl-4-(2nitrophenyl)pyridine-3,5-dicarboxylate.
.
Nifedipine contains not less than 98.0 per cent and not more
than 102.0 per cent of CI7HISN206, calculated on the dried
basis.
Category. Antianginal (calcium channel blocker).

Tablets.
Finely crush one tablet, add 30 ml of methanol, stir the mixture
for 30 minutes and filter through sintered glass, washing the
residue with three quantities, each of 15 ml, of methanol. To
the combined. filtrate and washings add 10 ml of 1 M
hydrochloric acid and sufficient methanol to produce
100.0 ml. Ifnecessary, dilute further with a solvent prepared
by diluting 1 volume of 1 M hydrochloric acid to 10 volumes
with methanol to produce a solution containing about
0.001 per cent w/v solution of Nicoumalone. Measure the
absorbance ofthe resulting solution at the maximum at about
306 nm (2.4.7). Calculate the content OfC19HlSN06 taking 521
as the specific absorbance at 306 run.
Dose. Initial dose, 5-20 mg daily, in divided doses; subsequent

doses, in accordance with the needs of the patient but total
daily dose not to exceed 100 mg.
Description. A yellow, crystalline powder; readily affected by
exposure to light.
NOTE- Nifedipine, when exposed to daylight and certain

wavelengths of artificial light, readily converts to a
nitrosophenyl derivative. Exposure to ultraviolet light leads
to the formation of a nitrophenyl derivative. Pelform the
tests and assay in the dark or under long-wavelength light
(greater than 420 nm). Use low-actinic glassware.
Identification
Test A may be omitted iftests B, C and D are carried out. Tests

B, C and D may be omitted iftest A is carried out.
Assay. Weigh and powder 20 tablets. Weigh accurately a A. Detennine by infrared absorption spectrophotometry (2.4.6).
quantity of the powder containing about 10 mg of Compare the spectrum with that obtained with nifedipine RS
Nicoumalone, add 30 ml of methanol, stir the mixture for or with the reference spectrum of nifedipine.
30 minutes and filter through sintered-glass, washIng the
..
.
'd
.h h
..
1 f 15 1 f
h I 'T'
B. In the test
for Related substances, the pnnclpal peak m the
resl ue WIt tree quantttles, eac 1 0 m , 0 met ana. 10
."
.
d f'l
d
l'
dd 10 1 f 1 M . chromatogmm obtamed WIth the test solutIon corresponds to
th e com b me I trate an was Hngs a
mo....
.
.
'
'd d ff"
f
I
d
the peak due to mfedlpme 111 the chromatogram obtamed WIth
hy droc hionc aCI an su IClent met tano to pro uce tl
fi
1 t'
100.0 ml. Dilute 5.0 ml ofthis solution to 50.0 ml with a solvent
le re erence so u Ion (a).
prepared by diluting 1 volume of 1 M hydrochloric acid to C. Detennineby thin-layer chromatography (2.4.17), coating
10 volumes with methanol and measure the absorbance ofthe the plate with silica gel GF254.
resulting solution at the maximum at about 306 nm (2.4.7). Mobile phase. A mixture of 60 volumes of cyclohexane and
Calculate the content ofC19HIsN06 taking 521 as the specific 40 volumes of ethyl acetate.
Storage. Store protected from light and moishlre.
1779
NIFEDIPINE
IP 2010
Reference solution. A 0.1 per cent w/v solution of nifedipine

RS in methanol.
Apply to the plate 5 !-t1 of each solution. After development,
D. To 25 mg add 10 ml of a mixture of 5 volumes of ethanol
(95 per cent), 3.5 volumes of water and 1.5 volumes of
hydrochloric acid and dissolve with gentle heating. Add
0.5 g of granulated zinc and allow to stand for 5 minutes,
swirling occasionally. Filter, add 5 ml ofa 1per cent w/v solution
ofsodium nitrite to the filtrate and allow to stand for 2 minutes.
Add 2 m1 ofa 5 per cent w/v solution of ammonium sulphamate,
shake vigorously with care and add 2 ml of a 0.5 per cent w/v
solution ofN-(I- naphthyl) ethylenediamine dihydrochloride;
an intense red colour develops which persists for more than
5 minutes.
Tests
(2.4.14).

examination in 20 ml of methanol and dilute to 50 ml with the
mobile phase.
Reference solution (a). Dissolve an accurately weighed
quantity of nifedipine RS in sufficient methanol to produce a
1.0 per cent w/v solution and dilute quantitatively with the
mobile phase to obtain a 0.4 per cent w/v solution.
dimethyl-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3, 5dicaboxylate RS (nitrophenylpyridine analogue) in methanol.
Reference solution (c). A 0.04 per cent w/v solution of
dimethyl- 2, 6-dimethyl-4-(2-nitrosophenyl)pyridine-3, 5dicarboxylate RS (nitrosophenylpyridine analogue) in
methanol.
Reference solution (d). Mix 1 volume of each of reference
solutions (b) and (c) and 0.1 volume ofthe test solution, dilute
to 10 volumes with the mobile phase and then dilute 2 volumes
ofthe resulting solution to 10 volumes with the mobile phase.
octadecylsilane bonded to porous silica (5 !-tm),
- mobile phase: 55 volumes of water, 36 volumes of
methanol and 9 volumes of acetonitrile,
injection volume. 20 !-tl.
Inject reference solution (d). The peaks appear in the order
nitrophenylpyridine analogue, nitrosophenylpyridine
analogue and nifedipine (retention time about 15.5 minutes).

The test is not valid unless, in the chromatogram obtained
with reference solution (d), (a) the resolution factor between
the peaks due to the nitrophenylpyridine analogue and the
nitrosophenylpyridine analogue is greater than 1.5, (b) the
resolution between the peaks due to the nitrosophenylpyridine
analogue and nifedipine is greater than 1.5, and (c) the height
of the peak due to the nitrophenyl-pyridine analogue is at
least 20 per cent ofthe full-scale deflection.
Inject the test solution and reference solutions (a) and (d) and
record the chromatograms for twice the retention time of
nifedipine. In the chromatogram obtained with the test solution
no secondary peak other than any peaks corresponding to
the nitrophenylpyridine analogue and the nitrosophenylpyridine
analogue has an area greater than that of the peak due to
nifedipine in the chromatogram obtained with reference solution
(d) and the areas of any peaks corresponding to the
nitrophenylpyridine analogue and the nitrosophenylpyridine
analogue are not greater than the areas of the corresponding
peaks in the chromatogram obtained with reference solution
(d). The total amount of related substances is not greater than
0.3 per cent. Ignore any peak with an area less than 10 per cent
ofthe area ofthe peak due to nifedipine in the chromatogram
obtained with reference solution (d).
Heavy metals (2.3.13). 2.0 g complies with limittest for heavy
metals, Method B (l0 ppm).
Assay. Weigh accurately about 0.13 g, dissolve in a mixture of
25 ml of 2-methyl-2-propanol and 25 ml of 1 M perchloric
acid and titrate with 0.1 M ceric ammonium sulphate, using
0.1 ml offerroin solution as indicator until the pink colour is
discharged, titrating slowly towards the end-point. Carry out
a blank titration.
1 ml of 0.1 M ceric ammonium sulphate is equivalent to
0.01732 g ofCI7HlsN206.
Nifedipine Capsules
Nifedipine Capsules contain not less than 90.0 per cent and
not more than 110.0 per cent ofthe stated amount ofnifedipine,
CI7HISN206.
Usualstrengths. 5 mg; 10 mg.
NOTE - Nifedipine, when exposed to daylight and certain

1780
IP 2010
NIFEDIPINE SUSTAINED-RELEASE TABLETS

to the formation of a nitrophenyl derivative. Perform the
Identification
with the aid ofsmall quantities of methanol. Dilute to volume

with methanol and mix. To 20.0 ml add sufficient methanol to
produce 100.0 ml and mix. Measure the absorbance of the
Calculate the content ofC,7H,sN206 in the capsules from the
absorbance obtained by repeating the operation with a
0.005 per cent w/v solution of nifedipine RS in methanol.
Mobile phase. A mixture of equal volumes of ethyl acetate

and cyclohexane.
Test solution. Transfer a quantity ofthe contents ofthe capsules
containing 30 mg of Nifedipine into a centrifuge tube containing
0.1 M sodium hydroxide, add 25 ml of dichloromethane,
stopper the tube and shake gently for 1 hour. Centrifuge for
10 minutes' at 2000 to 2500 rpm. Remove the supernatant
aqueous layer by aspiration with a syringe and transfer 5 mlof
the clarified lower layer to a suitable vial.
nifedipine RS in dichloromethane.
Reference solution (b). A mixture of equal volumes of test
Apply to the plate 500 III ofeach solution as bands 20 mm by
3 mm. After development, dry the plate in air until the solvent
is not detectable and immediately examine in ultraviolet light
at 254 Dm. The principal band, appearing as a dark blue band,
in the chromatogram obtained with the test solution
corresponds to that in the chromatogram obtained with the
reference solution (a). Spray with a solution prepared in the
following manner. Dissolve 3 g of bismuth subnitrate and 30 g
of potassium iodide in 10 ml of 3 M hydrochloric acid and
dilute with water to 100 m1; dilute 10 ml to 100 ml with 0.3 M
hydrochloric acid. In the chromatogram obtained with test
solution the principal band, appearing as a compact light
orange band against a yellow background, corresponds to
The band obtained with reference solution (b) appears as a
single band under both visualisation procedures.
Tests
Capsules.
Transfer the contents of a capsule quantitatively to a 200-ml
volumetric flask with the aid of methanol, dilute to volume
with methanol and mix. Complete the Assay beginning at the
words "Measure the absorbance...." and calculate the content
ofC17HlsN206 in the capsule.
Other tests. Comply with the tests stated under Capsules.
Assay. Transfer the contents of 5 capsules containing about
50 mg ofNifedipine quantitatively to a 200-ml volumetric flask

Nifedipine Sustained-release Tablets contain not less than
amountofnifedipine, CI7HISN206.

wavelengths of artifiCial light, readily converts to a
to the formation of a nitrophenyl derivative. Perform the
tests and the assay in the dark or under long-wavelength
light (greater than 420 nm). Use low-actinic glassware.
Identification

and cyclohexane.
Test solution. Transfer a quantity of the powdered tablets
containing 30 mg ofNifedipine into a centrifuge tube containing
0.1 M sodium hydroxide, add 25 ml of dichloromethane,
stopper the tube and shake gently for 1 hour. Centrifuge for 10
minutes at 2000 rpm to 2500 rpm. Remove the supernatant
aqueous layer by aspiration with a syringe and use 5 ml ofthe
clarified lower layer.
Reference solution. A 0.12 per cent w/v solution of nifedipine
RS in dichloromethane.
Apply to the plate 500 III of each solution as bands 20 mm by
3 mm. After development, dry the plate in air until the odour of
the solvent is not detectable and immediately examine in
ultraviolet light at 254 Dm. The principal band, appearing as a
dark blue band, in the chromatogram obtained with the test
with the reference solution. Spray with a solution prepared in
the following manner. Dissolve 3 g of bismuth subnitrate and
30 g of potassium iodide in 10 ml of 3 M hydrochloric acid
and dilute to 100 ml with water; dilute 10 ml ofthis solution to
100 ml with 0.3 M hydrochloric acid. In the chromatogram
obtained with the test solution the principal band, appearing
1781
NIFEDIPINE SUSTAINED-RELEASE TABLETS
IP 2010 .
as a compact light orange band againsta yellow background,

corresponds to that in the chromatogram obtained with
reference solution.
Tests
Nifedipine Tablets
Nifedipine Tablets contain not less than 90.0 per cent anduot
more than 110.0 per cent of the stated amount ofnifedipine,
C17HISNz06' The tablets may be coated.
A. Apparatus No.1,
dissolution medium, ifnecessary, at the maximum at about 340
run (2.4.7).

to the formation of a nitrophenyl derivative. Pelform the
Identification
Calculate the content ofC17HlsNz06 in the medium from the

absorbance obtained from a solution oflmown concentration
of nifedipine RS prepared by dissolving in minimum volume
of methanol and then diluting with the dissolution medium.

D. Not less than 25 per cent and not more than 45 per cent of
the stated amount of C17HISNz06.
Test solution. Transfer a quantity of the powdered tablets

containing 30 mg ofNifedipine to a centrifuge tube containing
20 ml of 0.1 M sodium hydroxide, add 25 mlof dichloromethane,
stopper the tube and shake gently for 1 hour. Centrifuge for 10
minutes at 2000 to 2500 rpm. Remove the supernatant aqueous
layer by aspiration with a syringe and transfer 5.0 ml of the
clarified lower layer to a suitable vial.
B. Apparatus No.1,
Medium. 900 ml of phosphate buffer pH 6.8 prepared by
dissolving 30 g of sodium lawyl sulphate, 24.8 g of disodium
hydrogen phosphate anhydrous, 2.85 g of citric acid
monohydrate and 0.75 ml of orthophosphoric acid in sufficient
water to produce 6000 ml. Adjust the pH to 6.8, if necessary,
Replace 0.1 M hydrochloric acid with phosphate buffer pH
6.8 and run the apparatus at 150 rpm for 6 hours. Withdraw a
suitable volume of the medium and filter. Measure the
absorbance ofthe filtrate, suitably diluted with the dissolution
medium, ifneceSSaIy, at the maximum at about 340 nm (2.4.7).
Calculate the content ofC17HlsNz06 in the medium from the
absorbance obtained from a solution oflmown concentration
of nifedipine RS prepared by dissolving in minimum volume
of methanol and then diluting with the dissolution medium.
C17HISNz06.
quantity ofthe powder containing about 25 mg ofNifedipine,
disperse in methanol, shake and dilute to 100.0 ml with
methanol, filter. Dilute 20.0 ml ofthe filtrate to 100.0 ml with
methanol. Measure the absorbance of the resulting solution
OfC17HISNz06 from the absorbance obtained with a 0.005 per
cent w/v solution of nifedipine RS in methanol.

and cyclohexane.

nifedipine RS in dichloromethane.
Apply to the plate 500 f..Ll of each solution as bands 20 mm by
3 mrn. After development, dry the plate in air until the solvent
is not detectable and immediately examine in ultraviolet light
at 254 run. The principal band, appearing as a dark blue band,
reference solution (a). Spray with a solution prepared in the
following manner. Dissolve 3 g of bismuth subnitrate and 30 g
of potassium iodide in 10 ml of 3 M hydrochloric acid and
dilute with water to 100 ml; dilute 10 ml to 100 ml with 0.3 M
hydrochloric acid. In the chromatogram obtained with the
test solution the principal band, appearing as a compact light
orange band against a yellow background, corresponds to
The band obtained with reference solution (b) appears as a
single band under both visualisation procedures.
Tests
Tablets
1782
IP 2010
NIKETHAMIDE
Shake one tablet with methanol in a 200-ml volumetric flask,

dilute to volume with methanol, mix and filter. Complete the
Assay beginning at the words "Measure the absorbance...."
and calculate the content ofC'7H,sNz06 in the tablet.
quantity of the powder containing 50 mg ofNifedipine into a
200 ml volumetric flask. Dissolve with the aid of 50 mt of
methanol. Dilute to volume with methanol, mix and filter. Dilute
20 ml ofthe filtrate to 100 ml with methanol and mix. Measure
the absorbance of the resulting solution at the maximum at
about 350 nm (2.4.7). Calculate the content ofC17HlsNz06 i'om
the absorbance obtained by repeating the operation with a
0.005 per cent w/v solution of nifedipine RS in methanol.
C. Heat 0.1 g with I ml of 2 M sodium hydroxide; diethylamine,

recognisable by its odour, is evolved progressively; the fumes
tum red litmus paper blue.
D. To 2 ml ofa 0.1 percent w/v solution add 2 ml of cyanogen
bromide solution and 3 ml of a 2.5 per cent w/v solution of
aniline and mix; a yellow colour is produced.
Tests
Appearance of solution. The ;ubstance, in liquid form or
liquefied by gentle heating, is clear (2.4.1), and not more
intensely coloured than reference solution YS5 (2.4.1).
pH (2.4.24). 6.0 to 7.8, detelmined in a 25.0 per cent w/v solution.
Congealing temperature (2.4.10).23 to 24.so.
Refractive index (2.4.27). 1.522 to 1.526.
Nikethamide
Mobile phase. A mixture of 75 volumes of chloroform and

25 volumes of I-propanol.
Test solution. Dissolve 0.4 g of tile su'bstance under
ethylnicotinamide RS in methanol.
.
Mol. Wt. 178.2

Nikethamide isN,N-diethylpyridine-3-carboxamide.
Nikethamide contains not less than 99.0 per cent and not more
than 101.0 per cent ofC IDH I4N zO, calculated on the anhydrous
basis.
Category. CNS and Respiratory stimulant.
Dose. By slow intravenous injection, 500 mg to 2 g repeated at
intervals of 15 to 30 minutes as necessary.
Description. A colourless or slightly yellowish, oily liquid or
crystalline mass; odour, slight and characteristic.
Identification
C and D may be omitted iftests A and B are carried out.

Compare the spectrum with that obtained with nikethamide
RS or with the reference spectrum ofnikethamide.
0.002 per cent w/v solution shows an absorption maximum
only at about 263 nm; absorbance at about 263 nm, about 0.57.
Apply to the plate 10 J.lI. of each solution. After development,

Any spot corresponding to ethylnicotinamide in the
chromatogram obtained with the test solution is not more
intense than the spot in the chromatogram obta~ned with
reference solution (a) and any other secondary spot is not
more intense than the spot in the chromatogram obtained
with reference solution (b).
Heavy metals (2.3.13).2.0 g complies with limit test for heavy
metals, Method B (10 ppm):
Water (2.3.43). Not more than 0.3 per cent, detennined on
2.0g.
anhydrous glacial acetic acid and 5 ml of acetic anhydride.
Titrate with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.4.25). Carry out a blank titration.
CIDHI~ZO.
Storge. Storeprotected from light.
1783
IP 2010
NIKETHAMIDE INJECTION
Nikethamide Injection is a sterile solution containing 25 per
cent w/v solution ofNikethamide in Water for Injections.
Nikethamide Injection contains not less than 24.0 per cent
and not more than 26.0 per cent w/v solution of nikethamide,

Calculate the content ofC IOH l4N zO taking 282 as the specific
Storage. Store protected from light, in single dose containers.
CIOHI~zO.
Nitrazepam
Identification
A. Make I ml alkaline with 5 M sodium hydroxide, extract with
5 ml of dichloromethane and evaporate the solvent.
obtained with nikethamide RS or with the reference spectrum
ofnikethamide.
B. Gives a voluminous precipitate with alkaline potassium

mercuri-iodide solution and a greyish-brown flocculent
precipitate with tannic acid solution. Gives no precipitate
with iodine solution or with potassium mercuri-iodide
solution.
C. Heat I ml with 0.2 g of sodium hydroxide; diethylamine,
recognisable by its odour, is evolved.
Tests
Mol. Wt. 281.3

Nitrazepam is 1,3-dibydro-7-nitro-5-phenyl-2H-1 ,4benzodiazepin-2-one.
Nitrazepam contains not less than 99.0 per cent and not more
than 101.0 per cent ClsHIIN303, calculated on the dried basis.
Category. Hypnotic; sedative.
pH (2.4.24).6.0 to 8.0.
Dose. 5 to 10 mg daily, at bed time.


25 volumes of I-propanol.
Test solution. Dilute 1 ml ofthe injection to 5 ml with methanol.
Any spot corresponding to ethylnicotinamide in the
intense than the spot in the chromatogram obtained with
reference solution (a) and any other secondary spot is not
Assay. Dilute 5.0 ml to 500.0 m1 with water. To 5.0 ml ofthe
solution add 5 ml of I M hydrochloric acid and sufficient
water to produce 500.0 ml. Measure the absorbance of the
Description. A yellow, crystalline powder; odourless or almost

odourless.
Identification
Compare the spectrum with that obtained with nitrazepam RS
or with the reference spectrum of nitrazepam.
B. Carry out the following procedure in subdued light.

When examined in the range 230 nm to 360 nm (2.4.7), a freshly
prepared 0.0005 per cent w/v solution in a 0.5 per cent w/v
solution of sulphuric acid in methanol shows an absorption
maximum only at about 280 nm; absorbance at about 280 nm,
about 0.45.
C. Dissolve 10 mg in 1 ml of methanol, warming ifnecessary,
and add 0.05 ml of 2 M sodium hydroxide; an intense yellow
colour is produced.
D. Dissolve 20 mg in a mixture of 5 ml of hydrochloric acid
and 10 ml of water, boil for 5 minutes, cool and add 2 ml of a
0.1 per cent w/v solution of sodium nitrite. Allow to stand for
1 minute, add 1 ml ofa 0.5 percent w/v solution of sulphamic
1784
IP 2010
NITRAZEPAM TABLETS
acid, mix, allow to stand for 1minute, add 1ml ofa 0.1 per cent
w/v solution of N-(l-naphthyl)ethylenediamine
dihydrochloride; a red colour is produced.
Tests
Mobile phase. A mixture of 85 volumes of nitromethane and
15 volumes of ethyl acetate.

acetic anhydride. Titrate with 0.1 M perchloric acid,
ClsHIlN303.
Storage. Store protectedfrom light and moisture.
Nitrazepam Tablets
Nitrazepam Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount ofnitrazepam,
ClsHIIN303.

10 volumes of methanol.
Test solution. Shake a quantity of the powdered tablets with
sufficient methanol to produce a solution containing 5 mg of
Nitrazepam per ml, allow to settle and decant the supernatant
liquid.
Reference solution. A 0.5 per cent w/v solution of nitrazepam
RS in methanol.

dry the plate in air, spray it with ethanolic sulphuric acid
(10 per cent v/v), heat at 105 for 10 minutes and examine in
ultraviolet light at 365 nm. The principal spot in the
C. To a quantity of the powdered tablets containing 5 mg of
Nitrazepam add 5 ml of hydrochloric acid and 10 ml of water,
heat on a water-bath for 15 minutes, filter and cool. To the
clear filtrate add 1 ml ofa 0.1 per cent w/v solution of sodium
nitrite, allow to stand for 3 minutes and add 1 ml of a 0.5 per
cent w/v solution of sulphamic acid. Allow to stand for
3 minutes and add 1 ml ofa 0.1 per cent w/v solution ofN-(1naphthyl) ethylenediamine dihydrochloride; a red colour is
produced.
Tests
Apparatus No.1,
the absorbance of the filtrate, suitably diluted ifnecessary, at
ClsHIIN303, in the medium from the absorbance obtained from
a solution of known concentration of nitrazapam RS in. the
same medium.
ClsH11N303.
Mobile phase. A mixture of 40 volumes of nitromethane,
40 volumes of toluene and 20 volumes of chloroform.
Identification
Carry out the following procedure in subdued light.
A. When examined in the range 230 nm to 360 nm (2.4.7), the

final solution obtained in the Assay shows an absorption
maximum only at about 280 nm.

containing 40 mg ofNitra'zepam with 25 ml of chloroform,
filter, carefully evaporate the filtrate to dryness and dissolve


1785
NITRAZEPAM TABLETS
IP 2010

Tablets.
NOTE - CarlY out thefollowing procedure in subdued light.
Nitrofurantoin contains not less than 98.0 per cent-and not

morethan 102.0 per cent ofCgH6N40 s, calculated on the dried
basis.
Dose. 50 to 100 mg four times daily.
Description. Lemon yellow crystals or a crystalline powder;
odourless or almost odourless.
Powder 1 tablet, add 5 ml of water, mix and allow to stand for Identification
15 minutes protected fi'om light. Add 70 ml of a 0.5 per cent Carry out the following test in subdued light.
v/v solution of hydrochloric acid in methanol, shake for
15 minutes protected from light, add sufficient of the A. When examined in the range 230 nm to 400 nm (2.4.7), the
hydrochloric acid solution to produce 100.0 ml and filter. final solution obtained in the Assay shows absorption maxima
Dilute 10.0 ml ofthe filtrate with sufficient ofthe hydrochloric at about 266 nm and 367 nm; the ratio ofthe absorbance at the
acid solution to produce a solution containing 0.0005 per maximum at about 367 nm to that at the maximum at about
cent w/v solution of Nitrazepam. Measure the absorbance of 266nmis 1.36 to 1.42.
the resulting solution immediately at the maximum at about B. To 1 ml ofa 0.1 per cent w/v solution in dimethylformamide
280 urn (2.4.7). Calculate the content ofC,sHIIN303 in the tablet add 0.1 ml of 0.5 M ethanolic potassium hydroxide; a brown
taking 910 as the specific absorbance at 280 nm.
colour develops.
Tests
Assay. CarlY out the following procedure in subdued light.

Weigh and powder 20 tablets. Weigh accurately a quantity of
the powder containing about 5 mg ofNitrazepam, add 5 ml of
water, mix and allow to stand for 15 minutes protected from
light. Add 70 ml of a 0.5 per cent v/v solution of hydrochloric
acid in methanol, shake for 15 minutes protected from light,
add sufficient of the hydrochloric acid solution to produce
100.0 ml and filter. Dilute 10.0 ml ofthe filtrate to 100.0 rnl with
the same solvent arid measure the absorbance of the resulting
solution immediately at the maximum at about 280 nm (2.4.7).
Calculate the content ofCIsHIIN303 taking 910 as the specific
Nitrofurantoin


examination in minimum volume of dimethylformamide and
dilute to 10 ml with acetone.
dry the plate in air, heat at 105 for 5 minutes and examine in
ultraviolet light at 254 nm. Spray with phenylhydrazine
hydrochloride solution and heat the plate at 105 for further
10 minutes. Any secondaty spot in the chromatogram obtained
chromatogram obtained with the reference solution by both
methods of visualisation.
Loss on drying (2.4.19). 5.0 to 7.1 per cent (hydrous fonn) and
not more than 1.0 per cent (anhydrous fonn), determined on
1.0 g by drying in an oven at 105.
CgH6N40 S
CgH6N40S,HzO
Mol. Wt. 238.2 (anhydrous)

Mol. Wt. 256.2 (hydrous)
Nitrofurantoin is 1-(5-nitrofurfurylideneamino)imidazolidine2,4-dione. It is anhydrous or contains one molecule of water

of hydration.
Assay. CarlY out the following procedure in subdued light.

Weigh accurately about 75 mg and dissolve in 25.0 ml of
dimethylformamide, add sufficient water to produce 500.0 ml
and mix. Dilute 5.0 ml to 100.0 ml with a solution containing
1.8 per cent w/v solution of sodium acetate and 0.14 per cent
v/v of glacial acetic acid. Measure the absorbance of the
1786
IP 2010
NITROFURAZONE

using as the blank the sodium acetate-acetic acid solution.
Calculate the content ofCsH 6N40 s taking 765 as the specific
acid. Measure the absorbance of the resulting solution at the

maximum at about 367 urn (2.4.7), using as the blank the sodium
acetate-acetic acid solution. Calculate the content ofCsH6N 40 s
taking 765 as the specific absorbance at 367 run.
Labelling. The label states whether the material is anhydrous

or hydrous.
.
Nitrofurazone
Nitofural
Nitrofurantoin Tablets contain not less than 90.0 per cent and
not more than I 10.0 per cent of the stated amount of
nitrofurantoin, CSH 6N4 0 S '
Identification
C6H6N40 4
CarlY out the following proceduJ'e in subdued light.
Mol. Wt. 198.1
Nitrofurazone is 5-nitro-2-furaldehyde semicarbazone.

final ~olution obtained in the Assay shows absorption maxima
Tests
Nitrofurazone contains not less than 97.0 per cent and not
more than 103.0 per c.ent ofC 6H 6N 40 4 , calculated on the dried
basis.
Related substances. Detelmine by thin-layer chromatography


Description. A yellow to brownish-yellow, crystalline powder;

Identification

containing 0.1 g ofNitrofurantoin with 10 ml of a mixture of
9 volumes of acetone and 1 volume of dimethylformamide
and filter.
A. Detennineby infrared absorption spectrophotometry (2.4.6).

Compare the spectrum with that obtained with nitrojitrazone
RS or with the reference spectrum ofnitrofurazone.

B. Dissolve 1 mg in 1 ml of dimetliylfohnamide and add 0.05 ml

'of 1 M ethanoUc potassium hydroxide; a ruby red colour is
produced.

dry the plate in air, heat at 105 for 5 minutes and examine in
ultraviolet light at 254 nm. Spray with phenylhydrazine
hydrochloride solution and heat the plate at 105 for further
10 minutes. Any secondary spot in the chromatogram obtained
with the test solution, is not more intense than the spot in the
chromatogram obtained with the reference solution by both
methods of visualisation.
Assay. Carry out the following procedure in sub.dued light.
the powder containing about 0.15 g of Nitrofurantoin, add
50.0 ml of dimethylformamide, shake for 5 minutes, add
sufficient water to produce 1000.0 ml and mix. Dilute 5.0 rnI to
100.0 ml with a solution containing 1.8 per cent w/v solution
of sodium acetate and 0.14 per cent v/v of glacial acetic
Tests
pH (2.4.24).5.0 to 7.0, detennined in the filtrate obtained by
shaking 1.0 g with 100 ml of carbon dioxide-free water and
filtering.
(2.4.14).

examination in 100.0 ml ofthe mobile phase.
Reference solution (a). A 0.0005 per cent w/v solution of(5nitro-2-fil1yl)methylene diacetate (Nitrofurazone impurity
B) in the mobile phase.
Reference solution (b). Dissolve 10 mg each ofnitrojitral RS
and nitrojitrantoin in 100 ml of the mobile phase. Dilute 5 ml
of this solution to 100 ml with the mobile phase.
1787
IP 2010
NITROFURAZONE
octadecylsilane bonded to porous silica (5 /!m),
and 60 volumes of water,
resolution between the peaks due to nitrofural and
nitrofurantoin is not less than 2.0.
Inject the test solution and reference solution (a). Run the
chromatogram 10 times the retention time ofnitrofural. In the
secondary peak is not more than the area ofthe principal peak
in the chromatogram obtained with reference solution (a) (0.5
per cent) and the sum of the areas of all the secondary peaks
is not more than twice the area of the principal peak in the
cent). Ignore any peaks with the area less than 0.05 times the
Assay. Carry out the following procedure in subdued light.
Weigh accurately about 60 mg, add 20.0 ml of
dimethylformamide, swirl to dissolve and add sufficient water
to produce 500.0 m!. Dilute 5,0 ml ofthe solution to 100.0 ml
with water and mix. Measure the absorbance of the resulting
solution at the maximum at about 375 nm (2.4.7). Calculate the
content ofC 6H 6N 40 4 taking 822 as the specific absorbance at
375nm.
Nitrous Oxide
Category. General anaesthetic.

Dose. By inhalation, 60 to 80 per cent, with oxygen 20 to 40 per
cent, as required.
Description. A colourless gas; odourless.
Identification
A. A glowing splinter of wood bursts into flame on contact
with the gas.
B. Shake with alkaline pyrogallol solution; the gas being
examined is not absorbed and the solution does not become
brown.
Tests
Acidity or alkalinity. Use hermetically-closed, flat-bottomed,
glass cylinders with dimensions such that 50 ml of liquid
reaches a height of 12 to 14 em, fitted with an outlet tube and
with an inlet tube with an orifice of 1 mm in internal diameter
reaching to within 2 mm of the bottom of the cylinder. For
solution (1) pass 2.0 litres ofthe gas under examination through
a mixture of 0.1 ml of O. 01 M hydrochloric acid and 50 ml of
carbon dioxide-free water. For solution (2) use 50 ml of carbon
dioxide-free water. For solution (3) add 0.2 ml of 0.01 M
hydrochloric acid to 50 ml of carbon dioxide-free water. To
each solution add 0.1 ml of a 0.02 per cent w/v solution of
methyl red in ethanol (70 per cent). The intensity ofthe colour
of solution (1) is between those of solutions (2) and (3).
Arsine and phosphine. Through a mercuric chloride paper
attached to a glass tube as in the limit test for arsenic (2.3.10),
pass 2.0 litres of the gas; no visible stain is produced.
Carbon dioxide. Not more than 300 ppm v/v determined by the
following method. Use the apparatus described in the test for
Acidity or alkalinity. Pass 1.0 litre through 50 ml ofclear barium
hydroxide solution. Any turbidity produced in the resulting
solution is not more. than that obtained in a reference solution
prepared at the same time by adding 1 ml of a 0.11 per cent
w/v solution of sodium bicarbonate in carbon dioxide-free
water to 50 ml of barium hydroxide solution.
Mol. Wt. 44.0

Nitrous Oxide contains not less than 98.0 per cent v/v ofNzO.
NOTE - Carry out thefollowing tests on afull cylinderfrom

which no gas has been withdrawn. The cylinder from which
the gas is taken should be kept at room temperature for not
less than 6 hours before carrying out the tests. Keep the
cylinder in the vertical position with the outlet valve
uppermost and deliver the gas at a rate of 4 litres per hour,
unless otherwise directed. The test for Carbon monoxide
should be carried out on the first portion ofgas drawn from
the cylinder and that for Nitric oxide and nitrogen dioxide
immediately thereafter.
Carbon monoxide. Notmore than 10 ppm v/v, determined by

the following method. Connect in series a U-tube containing
silica gel impregnated with chromium trioxide, a drechsel
bottle containing 100 ml of a 40 per cent w/v solution of
potassium hydroxide, a U-tube containing pellets of
potassium hydroxide, a U-tube containing phosphorus
pentoxide dispersed on previously granulated, fused pumice,
a tube containing iodine pentoxide in granules, previously
dried at 200 0 and kept at a temperature of 1200 , packed in I-em
columns separated by I-em columns of glass wool giving an
effective length of 5 em, and a flask containing 2.0 ml of 1 M
potassium iodide and 0.15 ml of starch solution.
1788
IP 2010
NORADRENALINE BITARTRATE
Flush the apparatus with 5.01itres of carbon dioxide-free air

and, if necessary, discharge the blue colour in the iodide
solution by adding a small quantity of freshly prepared
0.002 M sodium thiosulphate. Continue flushing until not
more than 0.045 ml of0.002 M sodium thiosulphate is required
after passing 5.0 litres ofcarbon dioxide-free air. Pass 5.0 litres
ofthe gas under examination through the apparatus and flush
the last traces of liberated iodine into the reaction flask by
passing through the apparatus 1.0 litre of carbon monoxidefree air. Titrate the liberated iodine with 0.002 M sodium
thiosulphate. Carry out a blank titration under the same
conditions, using 5.0 litres of carbon dioxide-free air. The
difference between the volumes of 0.002 M sodium
thiosulphate used in the two titrations is not greater than
1.0rnl.
Halogens and hydrogen sulphide. Pass a volume containing
1.0 litre measured at 25 and at 101.3 kPa through a mixture of
100 ml of water and 1 ml of silver nitrate solution; neither
opalescence nor darkening is produced.
Nitric oxide and nitrogen dioxide. Not more than 2 ppm v/v in
both the liquid and gaseous phases, determined by the
following method. Use two of the cylinders described in the
test for Acidity or alkalinity connected in series. Examine
separately both the liquid and gaseous phases of the gas
under examination. To obtain the liquid phase invert the
cylinder. The liquid vaporises on leaving the valve.
For solution A dissolve 1 g ofsulphanilic acid in a mixture of
10 ml ofglacial acetic acid and 180 ml ofwater. For solution
B dissolve 0.2 g of N-(l-naphthyl)ethylenediamine
dihydrochloride in 10 ml of a 50 per cent v/v solution of
glacial acetic acid, heating gently, and dilute to 200 ml with
water. Mix 9 volumes ofsolution A with 1 volume ofsolution
B (reagent A).
In the first cylinder place 15 ml ofa solution containing 2.5 per
cent w/v solution of potassium permanganate and 1.2 per
cent v/v ofsulphuric acid (96 per cent). Place 20 ml ofreagent
A in the second cylinder and connect the outlet tube of the
first cylinder to the inlet tube of the second cylinder. Pass
2.5 litres ofthe gas under examination through the reagents at
a rate of 15 litres per hour. Prepare a reference solution by
adding 0.25 ml of a 0.00616 per cent w/v solution ofsodium
nitrite to 20 ml of reagent A. Allow both the sample and
reference solutions to stand for 10 minutes. For both liquid
and gaseous phases, any red colour in the sample solution is
not more intense than that in the reference solution.
Oxidising substances. Pass a volume containing 2.0 litres
measured at 25 and at 101.3 kPa through a freshly prepared
solution of 0.5 g of soluble starch and 0.5 g of potassium
iodide in 100 ml ofwater containing 0.05 ml ofglacial acetic
acid; the colour of the liquid is not changed.
Water. Pass a measured quantity at a rate of6litres per hour

through an absorption tube containing magnesium
perchlorate; the increase in weight ofthe tube does not exceed
2 mg per litre ofgas, the initial and final weighings ofthe tube
being made when the air in it has been displaced by the nitrous
oxide.
Assay. Carry out the assay of nitrous oxide (2.3.32), using
100 ml ofthe gas under examination. Use a cylinder ofthe gas
under examination from which at least 1 per cent w/w of the
contents have been removed.
Storage. Store under pressure in metal cylinders of the type
conforming to the appropriate safety regulations and at a
temperature not exceeding 37.
Labelling. The cylinder is painted blue and carries a label
stating "Nitrous Oxide". In addition, ''Nitrous Oxide" or the
symbol ''N20'' should be stencilled in paint on the shoulder of
the cylinder.
.
Noradrenaline Acid Tartrate; Levarterenol Bitartrate;
Norepinephrine Bitartrate
OH
0
HO~
NH2
.
H OH
HOOe)( eOOH ,H 20
. HXOH
OH
CSHIIN03,C4Ht;06,H20
Mol. Wt. 337.3
Noradrenaline Bitartrate is (R)-2-amino-l-(3,4dihydroxyphenyl)ethanol tartrate monohydrate.

Noradrenaline Bitartrate contains not less than 98.5 per cent
and not more than 101.0 per cent of CSHllN03,C4H606,
Dose. By intravenous infusion, 2 to 20 /Lg per minute, according
to the blood pressure of the patient.
odourless. It gradually darkens on exposure to air and light.
Identification
Test A may be omitted iftests B, C, D, E andF are carried out.
Tests C, D, E may be omitted iftests A, BandF are carried out.
A. Dissolve 0.2 g in 2 ml ofwater containing about 10 mg of
sodium sulphite and add sufficient dilute ammonia solution
to give an alkaline reaction. Keep the mixture at about 4 for
1 hour and filter.
1789
NORADRENALINE BITARTRATE
IP 2010
On the residue (residue R) determine by infrared absorption

spectrophotometry (2,4,6). Compare the spectrum with that
obtained with noradrenaline acid tartrate RS treated in the
same manner.
0.005 per cent w/v solution in 0.01 M hydrochloric acid shows
an absorption maximum at about 279 nm; absorbance at about
279 nin, about 0.40.
C. Wash residue R obtained in test A with three quantities,
each of2 ml, of water, followed by 5 ml of ethanol (95 per
cent) and 5 ml of ether and dry the precipitate under preSSUTe
of 1.5 to 2.5 kPa for 3 hours. The specific optical rotation
(2.4.22), determined in a 2.0 per cent w/v solution,ofthe dried
precipitate in 0.5 M hydrochloric acid is -44 ,to -48.
D. To 1 ml of a 1 per cent w/v solution,add 0,05 ml offerric
chloride solution; an intense green colour is produced. Add,
drop by drop, sodiwn bicarbonate solution; ,the colour
changes to blue and then red.
E. To 1 ml ofa 0.1 per cent w/v solution add 10 ml ofphthalate
btifferpH 3.6, add 1 ml of O. 05 M iodine, setaside for 5 minutes
and add 2 ml of 0.1 M sodium thiosulphate; not more than a
faint red colour is produced; Repeat the test using buffei
solution pH 6.6 instead of phthalate buffer pH 3.6; a strong
reddish violet colour is produced (distinction from adrenaline
and isoprenaline).
F. The filtrate obtained in test A gives the reactions oftartrates

(2.3.1).
Tests
Appearance of solution. A freshly prepared 2.0 per cent w/v
solution is clear (2.4.1), and not more intensely coloured than
bands 20mm by 2 mm. Allowthe applied bands to dry in air,

spray them with a saturated solution of sodium bicarbonate,
allow to dry in air and spray the' bands twice with acetic
anhydride, drying between the two sprayings. Heat the plate
at. 50 for 90 minutes and develop the chromatograms. After
removal of the plate, allow it to dry in air and spray with a
freshly prepared mixture of8 volumes of methanol, 2 volumes
of ethylenediamine and 2 volumes of a 0.5 per cent w/v
solution of potassium ferricyanide. Dry the plate at 60 for
10 minutes and examine in ultraviolet light at 254 and 365 nm.
, In the chromatogram obtained with the test solution any band
with a slightly higher R r value than the principal band is not
more intense than the corresponding band in the chromatogram
obtained with reference solution (b). The chromatogram
obtained with reference solution (c) shows a clearly separated
band corresponding to the most intense band in the
chromatogram obtained with reference solution (a) at a higher
R r value than the most intense band.
Noradrenalone. Absorbance of a 0.2 per cent w/v solution in
0.01 M hydrochloric acid at 310 nm, not more than 0.40 (2.4.7).
, .
Sulph~ted
'
ash (2.3.18). Not more than 0.1 percent.
Water (2.3.43). 4.5 to 5.8 per cent, determined on 0.5 g.

anhydrous glacial acetic acid, warming if necessary.Titrate
with 0.1 M perchloric acid, using Clystal violet solutiOn as
indicator, until a bluish green colour is obtained. Carry out a
blank titration.
CgHIIN03,CJf606.
pH (2.4.24). 3.5 to 5.0, detennined in a 1.0 per centw/v solution.

Melting range (2.4.21). 100 to 106, with decomposition.
Adrenaline. Determine by thin-layer chromatography (2.4.17),

coating the plate with silica gel G
Noradrenaline Acid Tartrate Injection; Noradrenaline

Injection; Levarterenol Bitartrate Injection;
Norepinephrine Bitartrate Injection
Mobile phase. A mixture of50 volumes of acetone, 50 volumes

of dichloromethane and 0.5 volume ofanhydrousformic acid.
Prepare the following solutions immediately before use.
Test solution. Dissolve 0.25g of the substance under
adrenaline tartrate RS in water.
Noradrenaline Bitartrate Injection is a sterile solution of

Noradrenaline Bitartrate. It is prepared by diluting Sterile
Noradrenaline Concentrate to 250 times its volume with Sodium
Chloride and Dextrose Injection or with Dextr'ose Injection
(5 per cent w/v) immediately before use.

adrenaline tartrate RS in water.
Noradrenaline Bitmtrate Injection contains in 1 ml 8 Ilg of

Noradrenaline Bitmtrate equivalent to approximately 4 Ilg of
noradrenaline.
Reference solution (c). A mixture of equal volumes ofthe test

solution and reference solution (b).
Tests
Apply to the plate 6 III of each of test solution, reference

solutions (a) and (b) and 12 III of reference solution (c) as

1790
IP 2010
NORETHISTERONE
Sterile Noradrenaline Concentrate

Sterile Noradrenaline Concentrate is asterile, isotonic solution
containing 0.2 per cent w/v of Noradrenaline Bitartrate in
Water for Injections.
..
,.
Sterile Noradrenaline C,onceiltr.ate contains not less than
0.18 per cent and not more than 0.23 per cent w/v of
noradrenaline bitartrate, CgHIIN03,C4H606,HzO.
Identification
Norethisterone contains not less than 98.0 per cent and not
more than 102.0 per cent of CZOHZ60Z, calculated on the dried
basis.
Category. Progestogen.
Dose. 5 to 20 mg daily, in single or divided doses.
Description. A white or yellowish-white, crystalline powder;
odourless.
Identification
Mix 0.5 ml with 10 ml ofphthalate bufferpH 3.6, add l'ml of

0.05 M iodine, allow to stand for 5 minutes and add 2 ml of
0.1 M sodium thiosulphate; not more than a velY faint red
colour is produced. Repeat the test using phosphate buffer
pH 6.6 instead of phthalate buffer pH 3.6; a strong reddish
violet colour is produced;
A. Detennine by inli"ared absorption spectrophotometry (2.4.6).

Compare the spectrum with that obtained with norethisterone
RS or with the reference spectrum of norethisterone.
B. Determine by thin~layer chromatography (2.4.17), coating
the plate with silica gel G:
'
Solvent n1ixture. A mixture of 90 volumes of acetone and

10 volliines offormamide.
Tests
pH (2.4.24). 3.0 to 4.6.
Preparations (Concentrated Solutions for Injection).
Assay. Dilute 5.0 ml to 200.0 ml with water and measure the
279 nm (2.4.7). Calculate the content ofCgH"N03,C4H606,HzO
taking 80 as the specific absorbance at 279 nm..
Labelling. The label states (1) "Sterile Noradrenaline
Concentrate"; (2) that 1 voillme of the solution diluted to
250 volumes with Sodium Chloride and Dextrose Injectionor
with Dextrose Injection (5 per cent w/v) produces
Noradrenaline Bitartrate Injection, which must be used
immediately after preparation; (3) that ifthe solution isbrbwn
it should not be used.
Norethisterone
Norethindrone
Mobile phase. A mixture of 40 volumes of hexane and

10 volumes of dioxan.
Reference solution. Dissolve 10 mg of norethisterone RS in
10 nil of chloroform.
Place the dry plate in a tank containing a shallow layer of the
solvent mixture, allow the solvent mixture to ascend to the
top, remove the plate from the tank and allow the solvent to
evaporate. Use within 2 hours, with the flow of the mobile
phase in the direction in which the aforementioned treatment
was done.
Apply to the plate 2 J..lI of each solution. Allow the mobile
phase to l~ise 12 cm. DIy the plate in a current ofwann air, allow
the solvent to evaporate, heat at 1200 for 15 minutes and spray
the hot plate with ethanolic sulphuric acid (20 per cent v/v).
Heat at l20~ for a further 10 minutes, allow to cool and examine
in daylight and in ultraviolet light at 365 nm. The principal
spot in the chromatogram obtained with the test solution
corresponds to and exhibits fluorescence similar to that in tIie
C. Dissolve about 2 mg in 2 ml of ethanol (95 per cent) and
add 1 ml of a 1 per cent w/v solution of butylated
hydroxytoluene in ethanol (95 per cent) and 2 ml of I M
sodium hydroxir;fe. Heat in a water~bath for 30 minutes and
'
cool; a yellowi,sh pink colour is produced.
OH
-C=CH
Tests
Mol. Wt. 298.4
Norethisterone is 17~-hydroxy-19-nor-17 a-pregn-4-en-20-yn3-one.
Appearance of solution. Dissolve 0.2 g in sufficient dioxan to

produce 10 ml (solution A). The solution is clear (2.4.1), and
not more intensely coloured than reference solution YS6 (2.4.1).
1791
NORETHISTERONE
IP 2010
Specific optical rotation (2.4.22). -33.0 to -37.0, determined

in a solution prepared by diluting 5.0 ml of solution A to
10.0 ml with dioxan.
Light absorption. Dissolve 10 mg in sufficient ethanol
(95 per cent) to produce 100 ml, dilute 10 ml ofthe solution to
100 ml with methanol (98 per cent). Absorbance of the
resulting solution at the maximum at about 240 nm, 0.55 to 0.59
(2.4.7).
(2.4.14).
Assay. Weigh accurately about 0.4 g," dissolve in 40 ml of

tetrahydrofuran, add 10 ml of a 10 per cent w/v solution of
silver nitrate and titrate with 0.1 M sodium hydroxide using
2 ml of bromocresol green solution as indicator. Repeat the
operation without the substance under examination. The
difference between the titrations represents the amount of
sodium hydroxide required.
CZOHZ60Z'
Solvent mixture. A mixture of 40 volumes of water and 60

volumes of acetonitrile.
examination in 10 ml ofthe solvent mixture.
ml with the solvent mixture. Dilute 1.0 ml ofthis solution to
10.0 ml with the solvent mixture.
end-capped octylsilane bonded to porous silica (5 J!m),
mobile phase: A. water,
B. acetonitrile,
a linear gradient programme using the condition given
below,
spectrophotometer set at 254 nrn,
injection volume. 20 J!l.
Time
(in min.)
Mobile phase A
(per cent w/v)
Mobile phase B
(per cent w/v)
0-20
63
37
20-25
63-20
37-80
25-35
20
80
35-36
20-63
80-37
36-50
63
37
Norethindrone Tablets
Norethisterone Tablets contain not less than 90.0 per cent
norethisterone, CZOHZ60Z'
Identification
Place a quantity of the powdered tablets containing 25 mg of
Norethisterone on a small filter, wash with three quantities,
each of 5 ml, of light petroleum (60 to 80) and discard the
washings. Extract the residue with 15 ml of chloroform,
evaporate the extract to dryness and recrystallise from aqueous
methanol. The residue complies withthe following test.
plate with silica gel G.
Solvent mixture. A mixture of 90 volumes of acetone and

10 volumes of 1,2-propanediol.
Mobile phase. A mixture of40 volumes of cyclohexane and 10
volumes of toluene.
Inject the test solution and the reference solution. In the

secondary peak is not more than twice the area ofthe principal
peak in the chromatogram obtained with the reference solution
(0.2 per cent) and the sum of the areas of all the secondary
peaks is not more than 3 times the area ofthe principal peak in
the chromatogram obtained with reference solution (0.3 per
cent). Ignore any peaks with the area less than 0.5 times the
the reference solution (0.05 per cent).

Reference solution (a). Dissolve 25 mg of norethisterone RS
in 10 ml ofthe solvent mixture.
Reference solution (b). Mix equal volumes ofthe test solution
and reference solution (a).
was done.
1792
NORFLOXACIN
IP 2010
Apply to the plate 2 J.ll of each solution. Allow the mobile

phase to rise 12 cm. Dry the plate in a current ofwarm air, allow
the solvent to evaporate, heat at 120 0 for 15 minutes and spray
Heat at 1200 for a further 10 minutes, allow to cool and examine
reference solution (a). The principal spot in the chromatogram
obtained with reference solution (b) appears as a single,
compact spot.
N orfloxacin
Mol. WUI9.3
Tests
Uniformity ofcontent. (For tablets containing 10 mg or less)Comply with the test stated under Tablets.
Carry out the procedure described under Assay but using the
following test solution.
Test solution. Powder one tablet and dissolve as completely
as possible in 2 ml of water with the aid of ultrasound for 15
minutes and dilute to 5.0 ml with methanol. Centrifuge for 15
minutes and use the clear supernatant liquid.
Norfloxacin is l-ethyl-6-fluoro-4-oxo-7-(piperazin-l-yl)-l ,4dihydroquinoline-3-carboxylic acid.

Norfloxacin contains not less than 99.0 per cent and not more
than 101.0 per cent ofC16H18FN303, calculated on the dried
basis.
Dose. 400 mg to 800 g daily, in divided doses.
Description. A white to pale yellow, crystalline powder.
Calculate the content of C2oH2602 in the tablet.
Identification

Compare the spectrum with that obtained with norfloxacin
RS or with the reference spectrum ofnorfloxacin.

Test solution. Weigh and powder 20 tablets. Disperse a
quantity ofpowder containing about 5 mg of Norethisterone
in 10 ml of water, sonicate for 15 minutes and dilute to 25.0 ml
with methanol, filter.
norethisterone RS in methanol.
octadecylsilane bonded to porous silica (10 J.lm) (Such
as Spherisorb ODS 1),
mobile phase: a mixture of28 volumes of water and 72
. volumes of methanol,
- injection volume. 20 J.ll.
than 2.0 per cent.
Calculate the content of C2oH2602 in the tablets.
B. When examined in the range 230 nm to 360 urn (2.4.7), a

an absorption maximum at about 273 urn.
Tests
(2.4.17), coating the plate with silica gel G, previously washed
with methanol and dried.
Mobile phase. A mixture of 40 volumes of dichloromethane,
40 volumes of methanol, 20 volumes of toluene, 14 volumes
of diethylamine and 8 volumes of water.
examination in 100 rnl ofa mixture ofequal volumes ofmethanol
and dichloromethane.
Reference solution. Dissolve 4.0 mg ofnorfloxacin RS in 1 ml
of glacial acetic acid, add 4 ml of methanol and mix; dilute
1 mlofthe solution with 9 ml ofamixture ofequal volumes of

methanol and dichloromethane (reference solution A). Dilute
a portion ofreference solution A with an equal volume ofthe

methanol-dichloromethane mixture (reference solution B).
Apply separately to the plate spots of the three solutions in

quantities indicated below. For spot 1 use 5 J.lI of the test
1793
NORFLOXACIN
IPZ010
solution; for spots 2, 3 and 4 use 1 J.ll, 1.5 J.ll and2 J.ll respectively
ofreference solution A; for spot 5 use 5 J.ll ofreference solution
B. Place the plate in a paper-lined chamber previously
equilibrated with the mobile phase and allow the solvent front
to move about nine-tenths of the length of the plate. After
development, dry the plate in air and examine in ultraviolet
light at 254 nm and 365 run. Compare the intensities of any
secondary spots in the chromatogram obtained with the test
solution with those ofthe principal spots (2), (3), (4) and (5).
The sum of the intensities of secondary spots obtained with
the test solution is not more than 0.5 per cent of impurities.
(The spots (2) (3) (4) and (5) represent 0.2 per cent, 0.3 per
cent, 0.4 per cent and 0.5 per cent respectively of impurities).
Sulphated ash (2.3.18). Not more than 0.1 per cent, detennined
on 1.0 g in a platinum crucible.
0.7kPa.
Assay. Weigh accurately about 0.3 g and dissolve in 100 ml of
acid, detennining the end-point potentiometrically (2.4.25) and
using a suitable anhydrous electrode system. (The electrode
system may be rendered anhydrous by filling the electrode
with 0.1 M lithium perchlorate in acetic anhydride after
removing any aqueous solution contained in it). Carry out a
blank titration.
Other tests. Comply with the tests stated under Eye Drops.
Test solution. Dilute a suitable volume ofthe eye drops with a

0.1 per cent v/v solution of orthophosphoric acid to produce
a solution containing 0.Q05 per cent w/v ofNorfloxacin.
nOifloxacin RS inO.1per cent v/v solution of orthophosphoric
acid.
as Bondapack C 18),
column temerature. 50,
mobile phase: a mixture of 300 volumes of methanol
and 700 volumes of 0.1 per cent v/v orthophosphoric
acid, .
Precondition the column using 0.01 M anhydrous sodium
dihydrogen orthophosphate, adjusted to pH 4.0 with
orthophosphoric acid, at a flow rate of0.5 ml per minute for 8
hours. Equilibrate the column with the mobile phase for about
30 minutes before starting the chromatography.
tailing factor is not more than 2.0. The relative standard
Inject altemately the test solution and the reference solution.
1 ml of 0.1 Mperchloric acid is equivalent to 0.03193 g of

C16HISFN303'
Calculate the content of C16H.ISFN303 in the eye drops.
Norfloxacin Tablets
Norfloxacin Eye Drops are a sterile solution ofNorfloxacin in

PUlified water.
Norfloxacin Tablets contain not less than 90.0 per cent and
norfloxacin, C16HISFN303'
Norfloxacin Eye Drops contain not less than 90.0 percent and
not more than 110;0 per cent of the stated amount of
norfloxacin, C16HlsFN303'
Usual strengths. 200 mg; 400 mg; 800 mg.
Identification
Usual strength. 0.3 per cent w/v.
Identification



40 volumes of methanol, 20 volumes of toluene, 14 volumes
of diethylamine and 8 volumes of water.
Tests
pH (2.4.24).4.6 to 5.5.
Test solution. Shake a quantity ofthe fine~y powdered tablets

containing 75 mg ofNorfloxacin with 50 ml of a mixture of
equal volumes of acidified methanol (containing 0.9 per cent
1794
IP 2010
NORGESTREL
v/v of hydrochloric acid) and dichloromethane, centrifuge

and use the clear supernatant solution.
Reference solution. A 0.15 per cent w/v solution of nOlj!oxacin
RS in the same solvent mixture.
Apply to the plate 50 fll of each solution. After development,

Theprincipal spot in the chromatogram obtained with the test
obtained with the test solution cOlTesponds to the peak due
to norfloxacin in the chromatogram obtained with the reference
solution.

- injection volume. 20 fll.
lriject the test solution and the reference solution. The assay
is not valid unless the capacity factor is not less than 2, the
column efficiency is not less than 1500 theoretical plates, the
tailing factor for the norfloxacin peak is not more than 2.0 and
the relative standard deviation for replicate injections is not
more than 2.0 per cent.
Calculate the content ofCI6HIsFN303 in the tablets.
Norgestrel
Tests
Apparatus No.1,
Medium. 750 ml of acetate bufferpH4.0,
the absorbance of the filtrate, suitably diluted with acetate
buffer pH 4.0, if necessary, at the maximum at about 278 nm
(2.4.7). Concomitantly measure the abs.orbance of a solution
oflmown concentration of nOlj!oxacin RS in the same medium.
Calculate the total content ofCI6HIsFN303 in the medium.
C16HlsFN303.
Mol. Wt. 312.5

Norgestrel is rac-13-ethyl-17-hydroxy-18,19-dinor-17Cf.,pregn-4-en-20-yn-3-one.
Dose. As a contraceptive, 150 to 300 flg in combination with
20 to 50 flg ofethinyloestradiol daily.
Description. A white or almost white, clystalline powder;
practically odourless.
Test solution. Weigh and powder 20 tablets. Add 80 ml ofthe

mobile phase to an accurately weighed quantity of the
powdered tablets containing about 100 mg ofNorfloxacin, mix
with the aid ofultrasound for 10 minutes, dilute with a 0.1 per
cent v/v solution of phosphoric acid to 200.0 ml and mix.
Dilute 10.0 ml ofthis solution to 25.0 ml with the mobile phase,
mix and use the resulting solution after filtration through a
filter with porosity ofnot more than 0.1 fllU.
B. When examined in the range 220 nm to 360 111TI (2.4.7), a

0.001 per cent w/v solution in methanol shows an absorption
Reference solution. A 0.02 per cent w/vsolution ofnOlj!oxacin

C. Melting range (2.4.21). 205 to 212, but the range between

beginning and end of melting does not exceed 4.
- a stainless steel colulUn 30 cm x 3.9 mm, packed with
octadecylsilane bonded to porous silica (5 flm),
mobile phase: 85 volumes ofa 0.1 per cent v/v solution
of phosphoric acid and 15 volumes of acetonitrile,
- temperature. COlutm140 1, after preconditioning with
degassed 0.01 M sodium dihydrogen phosphate
adjusted to pH 4.0 with phosphoric acid flowing at a
rate of0.5 ml per minute for 8 hours,
Identification
A. Determine by infi'ared absorption spectrophotometry (2.4.6).
Compare the spectTUm with that obtained with norgestrel RS.
Tests
Specific optical rotation (2.4.22). -0.1 to +0.1 0, determined in
a 5.0 per centw/v solution in chloroform.
Mobile phase. A mixture of 80 volumes of dichloromethane
and 20 volumes of ethyl acetate.
1795
IP 2010
NORGESTREL

Apply to the plate 10 III of each.solution. After development,
dry the plate in air and spray with phosphomolybdic acid
solution. Any secondary spot in the chromatogram obtained
more than two such spots are more intense than the spot in
Reference solution. A solution containing 0.06 per cent w/v

solution of norgestrel RS and 0.006 per cent w/v solution of
ethinyloestradiol RS.
dry the plate in air, spray with ethanolic sulphuric acid
(80 per cent v/v) ,heat at 110 for 10 minutes and examine in
ultraviolet light at 365 nm. The principal spots in the
chromatogram obtained with the test solution correspond to
the spots for norgestrel (red fluorescence) and
ethinyloestradiol (orange-yellow fluorescence) in the
Tests
Sulphated ash. (2.3.18) Not more than 0.3 per cent.

Tablets.
Carry out the procedure described under Assay but using the
following test solution.
Assay. Weigh accurately about 0.1 g, dissolve in sufficient

ethanol (95 per cent) to produce 100.0 ml, dilute stepwise
with ethanol (95 per cent) to obtain a solution containing
0.001 per cent w/v of levonorgestre1 and measure the
absorbance of the resulting solution at the maximum at about
241 nm, (2.4.7). Calculate the content of CZIHzsOz from the
absorbance obtained with a 0.001 per cent w/v solution of
norgestrel RS in ethanol (95 per cent).
Test solution. Add 2.0 ml of methanol (70 per cent) and 2.0 ml
of a 0.00002 per cent w/v solution of diphenyl in methanol
(70 per cent) (internal standard solution) to one tablet, shake
for 20 minutes, centrifuge, filter the supernatant liquid through
a membrane filter with a pore size ofnot more than 0.2 mm and
use the filtrate.
Calculate the contents of norgestrel CZIHzsOz, and

ethinyloestradiol, CZOHZ40Z' in the tablet.
Assay. Detennine by liquid chromatography (2.4.14) using the
chromatographic system described under Uniformity of
content.
Norgestrel and Etbinyloestradiol

Tablets
Norgestrel and Ethinyloestradiol Tablets contain not less than
amounts of norgestrel, CZIHzsOz and ethinyloestradiol,
CzOHZ40 Z.
Category. Oral contraceptive.
Dose. One tablet daily for 21 days starting from the fifth day of
menstrual cycle.
Usual strength. Norgestrel, 300 Ilg and Ethinyloestradiol,
30 Ilg.
Identification
plate with silica gel G

and 4 volumes of ethanol (95 per cent).
Test solution. Powder 20 tablets finely, triturate with 20 ml of
dichloromethane, allow the solids to sediment and use the
clear supernatant liquid.
Test solution. Weigh and powder 20 tablets. To a quantity of

the powder equivalent to one tablet add 2.0 ml of methanol
(70 per cent) and 2.0 ml of a 0.00002 per cent w/v solution of
diphenyl in methanol (70 per cent) (internal standard
solution), shake for 20 minutes, centrifuge, filter the
supernatant liquid through a membrane filter with a pore size
afnot more than 0.2 mm and use the filtrate.
Reference solution. A solution in methanol (70 per cent)
containing 0.15 mg per ml of norgestrel RS and 0.015 mg per
ml of ethinyloestradiol RS. Take 2.0 ml of this solution and
add 2.0 ml of a 0.00002 per cent w/v solution of diphenyl in
methanol (70 per cent) and use the resulting solution.
- a stainless steel column 15 cm x 4.6 rntD, packed with
octadecylsilane bonded to porous silica (5 to 7 Ilm),
- mobile phase: a mixture of35 volumes of acetonitrile,
15 volumes of methanol and 45 volumes of water,
- flow rate. 1 to1.5 ml per minute,
1796
IP 2010
NORTRIPTYLINE TABLETS
Inject the reference solution. The resolution between the two

major peaks is not less 2.5 and the relative standard deviation
for replicate injections is not more than 2.0 per cent.
Inject the test solution and the reference solution. The relative
retention times are about 0.7 for ethinyloestradiol and about
1.0 for norgestrel.
Calculate the contents of norgestrel, C21H2S02, and
ethinyloestradiol, C2oH2402 in the tablets.
C. To about 50 mg dissolved in 3 ml ofwarm water, add 1drop

of a 2.5 per cent w/v solution of quinhydrone in methanol; a
red colour is produced after a few minutes (distinction from
amitriptyline).
D. Gives the reactions ofchlorides (2.3.1).
Tests
Mobile phase. A mixture of 85 volumes of cyclohexane,
15 volumes of ethyl acetate and 3 volumes of diethylamine.
examination in 10 ml of ethanol (95 per cent) prepared in
subdued light.

dibenzosuberone RS in ethanol (95 per cent) prepared in
subdued light.
, Hel
Mol. Wt. 299.8

Nortriptyline Hydrochloride is 3-(10, II-dihydro-5H-dibenzo
[a,dJcyclohept-5-ylidene)propyl(methyl)amine hydrochloride.
Nortriptyline Hydrochloride contains not less than 98.0 per
cent and not more than 101.5 percent ofC ,9H2I N,HC1, calculated
on the dried basis.
Category. Antidepressant.

phase to rise 14 cm in an unsaturated tank protected from
light. Dry the plate in air, spray with a freshly prepared solution
of sulphuric acid containing 4 per cent v/v offormaldehyde
solution and examine immediately in ultraviolet light at
365 nm. Any secondary spot in the chromatogram obtained
Dose. Initially, 50 to 150 mg daily; maintenance dose, 30 to 75

mgdaily.
Description. A white to off-white powder; odour slight and
characteristic.
Identification
Band C may be omitted if tests A and, D are carried out.
A. Dissolve 0.1 gin 10 ml of water, make allmline with 1 M

sodium hydroxide, extract with 5 ml of chloroform and
evaporate to dryness using a current of nitrogen.
Compare the spectrum with that obtained with nortriptyline
hyrdochloride RS treated in the same manner or with the
reference spectrum ofnortriptyline.
about 0.48.
anhydrous glacial acetic acid, warm slightly, if necessary, to
effect solution. Cool, add 5 ml of mercuric acetate solution.
CI9H2I N,HCl.
Nortriptyline Hydrochloride Tablets
Nortriptyline Tablets contain less than 90.0 per cent and not
more than 110.0 per cent ofthe stated amount ofnortriptyline,
C I9H2I N. The tablets are coated.
1797
IP 2010
NORTRIPTYLINE TABLETS
Usual strengths. The equivalent of 10 mgand 25mg of

nortriptyline.
methanol and shake for 30 minutes. Add sufficient water to

produce 10 ml, centrifuge and use the clear supernatant liquid.
Identification

nortriptyline hydrochloride RS in methanol (50 per cent).

5 mg ofnortriptyline with 20 ml of methanol and filter. To 1 ml
ofthe filtrate add 1 ml of a 2.5 per cent w/v solution of sodiurn
bicarbonate, 1 ml of a 2 per cent w/v solution of sodium
periodate and 1 ml ofa 0.3 per cent w/v solution ofpotassium
permanganate. Allow to stand for 15 minutes, acidifY with
1 M sulphuric acid and extract with 10 ml of 2i2,4~
trimethylpentane.
When examined in the range 230 nm to 360 nm (2.4.7), the
resulting trimethylpentane solution shows an absorption
0.1 g of nortriptyline with 10 ml of chloroform, filter and
evaporate the filtrate to a low volume. Add ether until a
turbidity is produced and allow to stand. Dissolve 50 mg of
the precipitate in 3 ml ofwann water, cool and add 1 drop ofa
2.5 per cent w/v solution of quinhydrone in methanol; a red
colour is produced after a few minutes (distinction from
amitriptyline):
Tests
Related substances. Detel1lline by thin71ayer chromatography

15 volumes of ethyl acetate and 3 volunies of diethylamine.
Test solution. Extract a quantity of the powdered tablets
containing 20 mg of nortriptyline with 5 ml of a mixture of
9 volumes of ethanol (95 per cent) and 1 volume of 2 M
hydrochloric acid, centrifuge and use the supernatant liquid.
dibenzosuberone RS in ethanol (95 per cent) prepared in
subdued light.
Follow the procedure given in the Assay. Calculatethe content

ofCzo Hz3 N in the tablet.
Other tests. Comply with the tests stated under Tablets,
Test solution. Shake vigorously 20 tablets with 50 ml of water

until the tablets disintegrate completely, add 100.0 ml of
methanol and shake for 30 minutes. Add sufficient water to
produce 200.0 ml, filter and dilute a volume of the filtrate
containing about 25 mg of nOltriptyline to 100.0 ml with
methanol (50 per cent).
nortriptyline hydrochloride RS in methanol (50 per cent).
octadecylsilane bonded to porous silica (l0 Ilm),
mobile phase: a 0.56 per cent w/v solution of sodium
hexanesulphonate in a mixture of equal volumes of
water and acetonitrile adjusted to pH 4.5 with glacial
acetic acid;
Calculate the content of C I9Hz1 N in the tablets.
Noscapine
Narcotine

phase to rise 14 cm in an unsaturated tank protected from
light. Dry the plate in air, spray with a freshly prepared solution
of sulphuric acid containing 4 per cent v/v offormaldehyde
solution and examine immediately in ultraviolet light at
365 nm. Any secondary spot in the chromatogram obtained
Uniformity of content (For tablets ~ontaining 10 mg or less).
Comply with the test stated under Tablets.
Detel1lline by liquid chromatography (2.4.14).
Test solution. Powder one tablet, add 2.5 ml of water, shake

vigorously to completely disperse the tablet, add 5 ml of
1798
Mol. Wt. 413.4
IP 2010
NOSCAPINE
Noscapine is (38)-6,7-dimethoxy-3-[(5R)-5,6, 7,8-tetrahydro4-methoxy-6-methyl-1 ,3-dioxolo[4,5-g]isoquinolin-5yl]phthalide, an alkaloid obtained from opium.
Reference solution (c). Dissolve 1.5 mg of papaverine

hydrochloride in 10 ml ofthe test solution and dilute to 50 ml
Noscapine contains not less than 98.5 per cent and not more
than 100.5 per cent ofC22H23N07, calculated on the dried basis.
nitrile groups chemically bonded to porous silica (5 11m),
- mobile phase: a mixture of35 volumes of methanol and
65 volumes of phosphate buffer pH 6.0,
Category. Cough suppressant.

Dose. 15 to 30 mg three to four times daily.
Description. Colourless crystals or a white crystalline powder.
Identification

Compare the spectrum with that obtained with noscapine RS
or with the reference spectrum of noscapine.
0.005 per cent w/v solution in methanol shows absorption
maxima at about 291 nm and 310 nm; ratio ofabsorbance at the
maximum at about 310 nm to that at the maximum at about
291 nm, 1.2 to 1.3.
C. To 0.1 g in a porcelain dish add a few drops of sulphuric
acid and stir; a greenish-yellow solution is formed which on
warming becomes red and finally violet.
D. Dissolve 50mg inS mlof5 Mhydrochloricacid, add 10ml
of a mixture of equal volumes of ethanol (95 per cent) and a
saturated solution of sodium acetate, mix and allow to stand
for about 3 minutes; shining crystals separate.
Tests
Appearance ofsolution. A 2.0 per cent w/v solution in acetone
examined immediately after preparation is clear (2.4.1), and not
more intensely coloured than reference solution YS6 (2.4.1).
at 20 in a solution prepared by dissolving 0.5 g in sufficient
0.1 M hydrochloric acid to produce 25.0 ml.
Related substances: Determine by liquid chromatography
(2.4.14).
examination with gentle heating in 14 ml of methanol, cool,
and dilute to 20 ml with phosphate buffer solution pH 6.,0.
Reference solution (a). Dilute 1.0 ml of the test solution to
20.0 ml with the mobile phase. Dilute 1.0 ml ofthe solution to
Reference solution (b). Dissolve 5 mg of papaverine
hydrochloride in 50 ml of the test solution. Dilute 1.0 ml of
this solution to 20 ml with the mobile phase.
Inject reference solution (c). The test is not valid unless the
resolution between the peaks due to noscapine and papaverine
(noscapine impurity A) is not less than 2.0. The relative
retention time with reference to noscapine for noscapine
Impurity Ais about 1.3.
Inject the test solution, reference solution (a) and (b). Run the
peak. In the chromatogram obtained with the test solution the
area ofthe peak due to noscapine impurity A is not more than
the area ofthe principal peak in the chromatogram obtained
with reference solution (b) (0.5 per cent), the area ofthe any
oth~r secondary peak is not more than 0.4 times the area of
solution (a) (0.2 per cent), and the sum of the areas of
secondary peaks other than noscapine impurity A is not more
obtained with reference solution (a) (0.5 per cent). Ignore any
peak with an area less than 0.1 times the area of the principal
peak in the chromatogram obtained with reference solution
(a) (0.05 per cent).
Morphine. Dissolve 0.1 gin 10 m1 of 0.1 M hydrochloric acid.
To 1 m1 of the resulting solution add a mixture of 1 ml of
potassium ferricyanide solution, 0.05 m1 of ferric chloride
test solution and 4 m1 of water; no blue or dark green colour
develops within 1 minute.
Sulphated ash (2.3.1 8). Not more than 0.1 per cent.
Assay. Weigh accurately about 0.35 g, dissolve in 40 m1 of
anhydrous glacial acetic acid, warming gently. Titrate with
0.1 M perchloric acid, determining the end-point
potentiOluetrically (2.4.25). Carry out a blank titration.
1 m1 of0.1 M perchloric acid is equivalentto 0.04134 g of
C22H23N07
1799
IF 2010
NOSCAPINE LINCTUS
Noscapine Linctus
Novobiocin Sodium
Narcotine Linctus
Noscapine Linctus is a solution of Noscapine in a suitable
flavoured vehicle. It may contain up to 1per cent w/v solution
ofCitric Acid.
Noscapine Linctus contains not less than 90.0 per cent and
noscapine, C22H23N07'
Usual strength. 7 mg per 5 ml.
Mol. Wt. 634.6
Identification
Novobiocin Sodium is the monosodium salt of novobiocin,
To a quantity containing 60 mg ofNoscapine add 20 ml of

water, 2 g of sodium chloride and 2 ml of 5 M sodium
hydroxide. Extract with successive quantities of50, 50, 25 and
25 ml ofether. Combine the extracts, wash with three quantities,
each of5 ml, of water and evaporate to dryness. Dissolve the
residue in 20 ml of chloroform. Wash with three quantities,
each of20 ml, of water, dry the chloroform layer with anhydrous
sodium sulphate, filter and evaporate the solvent. Ifnecessary,
induce crystallisation by scratching with a glass rod. The
crystals comply with the following tests.
Compare the spectrum with that obtained with noscapine RS
or with the reference spectrum of noscapine.
maxima at about 291 nmand 310 nm; ratio ofabsorbance at the
maximum at about 310 nm to that at the maximum at about
291 nm, 1.2 to 1.3.
Tests
Assay. Weigh accurately a quantity containing 60 mg of
Noscapine, add 20 ml of water, 2 g of sodium chloride and
2 ml of 5 M sodium hydroxide and extract with successive
quantities of50, 50,25 and 25 ml ofether. Combine the extracts,
wash with three quantities, each of5 ml, of water and evaporate
to dryness. To the residue add 50.0 ml of 0.1 M hydrochloric
acid, warm on a water-bath to dissolve and to remove any
traces ofether and dilute to 100.0 ml with water. Dilute 3.0 ml
to 50.0 ml with water and measure the absorbance of the
resulting solution at the maximum at about 310 nm (2..4.7).
Calculate the content ofC22H23N07 taking 90.7 as the specific
Determine the weight per ml ofthe linctus (2.4.29), and calculate
the content ofC22H23N07, weight in volume.
N-[7- {3-0-( aminocarbonyl)-6-deoxy-5-C-methyl-4-0-methyl~-L-Iy.xo-hexopyranosyl}-oxy-4-hydroxy-8-methyl-2-oxo-2H
I-benzopyran-3-yl]-4-hydroxy-3-(3-methyl-2butenyl)benzamide, an antimicrobial substance produced by

the growth of certain strains of Streptomyces niveus or
related organisms or by other means.
Novobiocin Sodium contains the equivalent of not less than
850 Ilg of novobiocin per mg, calculated on the dried basis.
Dose. The equivalent of 1 to 2 g ofnovobiocin daily, in divided
doses.
Description. A white or yellowish white, crystalline powder.
Identification
the plate with silica gel G. .
25 volumes of methanol and 1 volume of strong ammonia
solution.
Test solution. Dissolve a quantity of the substance under
examination in methanol so as to obtain a solution containing
0.1 per cent w/v solution of novobiocin.
Reference solution. A 0.1 per cent w/v solution of novobiocin
RS in methanol.


0.001 per cent w/v solution in a 0.4 per cent w/v solution of
potassium hydroxide shows an absorption maximum only at
about 307 nm.
C. The residue obtained by igniting it gives the tests for sodium
salts (2.3~ 1).
1800
IP 2010
NYSTATIN
Tests
Dose. In the treatment of alimentary candidiasis, 500,000 to

1,000,000 Units daily, in divided doses.
pH (2.4.24).6.6 to 8.5, determined in a 2.5 percentw/v solution.

Specific optical rotation (2.4.22). -50.0 to-58.0, determined
in a 5.0 per cent w/v solution in methanol containing I per
cent v/v of hydrochloric acid.
Loss on drying (2.4.19). Not more than 6 per cent, determined
on 0.2 g by drying in an oven at 60 over phosphoruspentoxide
Method A (2.2.1 0), and express the results in J.lg ofnovobiocin
per mg.
Description. A yellow to slightly brown powder; odour,

characteristic; hygroscopic.
Identification
final solution obtained in the test for Light absorption shows
absorption maxima at about 230 nm, 291 nm, 305 nm and
319 nm. The ratios of the absorbances at the maxima at about
291 nm and about 319 nm to the absorbance at the maximum at
about 305 nm are 0.61 to 0.73 and 0.83 to 0.96, respectively.
Use as the blank a solution prepared in the same manner
without the substance under examination.
Novobiocin Sodium intended for use in the manufacture of

Parenteral Preparations complies with the following
B. Shake 30 mg with 5 ml of water for 2 minutes, add 2 ml of
additional requirements.
sodium molybdotungstophosphate solution, and allow to
Bacterial endotoxins (2.2.3). Not more than 0.7 Endotoxin units
stand for I hour; the green colour produced is darker than
per mg.
that produced by repeating the test without the substance
Sterility. Complies with the test for sterility (2.2.11).
under examination.
Storage. Store protected from light and moisture at a
temperature not exceeding 30. If it is intended for use in the
manufacture of parenteral preparations, the container should
be sterile and sealed so as to exclude micro-organisms.
Labelling. The label states whether or not the contents are
intended for use in the manufacture ofparenteral preparations.
C. Shake 30 mg with 5 ml of water for 2 minutes, add 2 ml ofa

solution prepared by dissolving 0.1 g ofpyrogallol in 100 ml
of decolorised magenta solution, heat on a water-bath until a
dark pink colour is produced, cool and allow to stand for
1 hour; the pink colour is retained.
D. To 2 mg add 0.1 ml of hydrochloric acid; a brown colour is
produced.
E. To 2 mg add 0.1 ml of sulphuric acid; a brown colour is
produced which becomes violet on standing.
Nystatin
Tests
pH (2.4.24). 6.5 to 8.0, determined in a 3.0 per cent w/v
suspension in water.
OH
Light absorption (2.4.7). Dissolve 0.1 g in a mixture of5.0 ml of

glacial acetic acid and 50 ml of methanol, add sufficient
methanol to produce 1 00.0 ml and dilute 1.0 ml ofthe resulting
solution to 100.0 ml with methanol. Absorbance ofthe resulting
solution, measured within 30 minutes of preparation, at the
maximum at about 305 nm, not less than 0.60. Use as the blank
a solution prepared in the same manner without the substance
under examination.
COOH
o
\--o-\CH 3
~OH
OH
NH 2
Mol. Wt. 926.1

Nystatin is an antifungal substance produced by the growth
of certain strains of Streptomyces noursei or by any other
means. It consists mainly ofpolyenes, the principal component
being nystatin AI.
Nystatin has a potency of not less than 4400 Units per mg,
Category. Antifungal.
Composition. Determine by liquid chromatography (2.4.14).
Note-Carry out the test protectedfrom light.

examination in 50 ml of dimethyl sulphoxide.
Reference solution (a). A 0.04 per cent w Iv solution of nystatin
RS in dimethyl sulphoxide.
Reference solution (b). Dissolve 20 mg ofthe substance under
examination in 25 ml ofthe methanol and dilute to 50 ml with
1801
NYSTATIN
IP 2010
water. To 10.0 ml of this solution add 2.0 ml of dilute

hydrochloric acid. Allow to stand at room temperature for 1
hour.
Reference solution (c). Dilute 1.0 ml of reference solution (a)
in 100.0 rnl of dimethyl sulphoxide. Dilute 1.0 rnl ofthis solution
to 10.0 ml with dimethyl sulphoxide.
base-deactivated end-capped octadecylsilane bonded
to porous silica (5 /lm),
- mobile phase: A. a mixture of29 volumes ofacetonitrile
and 71 volumes of 0.385 per cent w/v solution of
ammonium acetate,
B. a mixture of 40 volumes of 0.385 per
cent w/v solution ofammonium acetate and 60 volumes
of acetonitrile.
below,
- injection volume. 20 /ll.
Time
(in min.)
0-25
25-35
35-45
45-50
50-55
Mobile phase A
(per cent w/v)
100
100-0
0
0-100
100
Determine by the microbiological assay ofantibiotics (2.2.10).

Nystatin intendedfor oral administration complies with the
following additional requirement.
Abnormal toxicity (2.2.1). Complies with the test for abnormal
toxicity, using a quantity containing not less than 600 Units
suspended in not more than 0.5 ml ofa 0.5 per centw/v solution
of acacia and injecting the suspension intraperitoneally.
Labelling. The label states the strength in terms ofthe number
of Units ofNystatin per mg.
Nystatin Ointment
Nystatin Ointment is a dispersion of Nystatin in microfine
powder in a suitable ointment basis.
Nystatin Ointment contains not less than 90.0 per cent and
not more than 130.0 per cent ofthe stated number ofUnits of
nystatin.
Usual strength. 100,000 Units per g.
Mobile phase B
(per cent w/v)
Identification
o
0-100
100
100-0
Disperse a quantity containing 25,000 Units in 10 ml of

chloroform, add 40 ml ofmethanol and shake. Filter and dilute
1 ml ofthe filtrate to 25 ml with methanol.

resulting solution shows absorption maxima at about 291 nm,
305 nm and 319 nm. The ratios ofthe absorbances at the maxima
at about 291 nm and about 319 nm to the absorbance at the
maximum at about 305 nm are 0.61 to 0.73 and 0.83 to 0.96,
respectively. Use as the blank a solution prepared exactly in
the same manner without the substance under examination.
resolution between the two principal peaks is not less than
3.5.
Inject the test solution, reference solution (a) and (c). In the
chromatogram obtained with the test solution the area of the
peak due to nystatin A 1 is not less than 85 per cent the area of
the plincipal peak in the chromatogram obtained with reference
solution (a), the area of the any other compound is not more
than 4.0 per cent the area of the principal peak in the
chromatogram obtained with reference solution (a). Ignore
any peak with a retention time of less than 2 minutes.
Assay. Protect the solution from light throughout the assay.
Weigh accurately about 75 mg and dissolve in sufficient
dimethylformamide to produce 50.0 ml; dilute 10.0 ml of the
resulting solution to 200.0 rnl with buffersolutionNo4 (2.2.10).
Tests
Other tests. Complies with the tests stated under Ointments.
Weigh accurately a quantity containing 400,000 Units, disperse
in 20 ml of ether in a stoppered flask, add 70 ml of
dimethylformamide, shake for a few minutes, add 10 ml of
water, shake vigorously for a few minutes and add sufficient
dimethylformamide to produce 100.0 ml. Mix well, filter and
dilute 10.0 ml of the filtrate to 100.0 ml with buffer solution
No 4 (2.2.10).
of Units of Nystatin per g.
1802
NYSTATIN TABLETS
IP 2010
Nystatin Pessaries
Nystatin Tablets
Nystatin Vaginal Tablets
Nystatin Tablets contain not less than 90.0 per cent and not
Nystatin Pessaries contain not less than 90.0 per cent and not
more than 130.0 per cent of the stated number of Units of
nystatin.
more than 130.0 per cent of the stated number of Units of

nystatin. The tablets are coated.
Usual strength. 500,000 Units.
Usual strength. 100,000 Units.
Identification
Identification
Extract a quantity of the powdered pessaries containing
300,000 Units with a mixture of50 ml of methanol and 5 ml of
glacial acetic acid, add sufficient methanol to produce
methanol. The resulting solution complies with the follwing
test.
Disperse a quantity containing 25,000 Units in 10 ml of
chlor%nn, add 40 ml of methanol and shake. Filter and dilute
1 ml ofthe filtrate to 25 ml with methanol.
When examined in the range 230 urn to 360 urn (2.4.7), the
resulting solution shows absorption maxima at about 291 urn,
305 urn and 319 urn. The ratios ofthe absorbances at the maxima
at about 291 urn and about 319 nm to the absorbance at the
maximum at about 305 urn are 0.61 to 0.73 and 0.83 to 0.96,
Tests
Other tests. Comply with the tests stated under Pessaries.
Extract a quantity of the powdered tablets containing

300,000 Units with a mixture of50 ml of methanol and 5 ml of
glacial acetic acid, add sufficient methanol to produce
methanol. The resulting solution complies with the follwing
test.
solution shows absorption maxima at about 291 nm, 305 nm
and 319 urn. The ratios of the absorbances at the maxima at
about 291 nm and about 319 nm to the absorbance at the
maximum at about 305 nm are 0.61 to 0.73 and 0.83 to 0.96,
Tests
Disintegration (2.5.1). 30 minutes, but using a 0.6 per cent
v/v solution of hydrochloric acid in place of water. If the
tablets fail to disintegrate, wash them rapidly by immersion in
water and continue the test using phosphate buffer pH 6.8;
the tablets then disintegrate within a further 30 minutes.

on 1.0 g ofthe powdered pessaries by drying in an oven at 60
on 1.0 g ofthe powdered tablets by drying in an oven at 60 at
a pressure not exceeding 0.7 kPa for 3 hours.
Weigh and powder 20 pessaries. Weigh accurately a quantity Assay. Weigh and powder 20 tablets. Weigh accurately a
ofthe powder containing 200,000 Units and shake with 50.0 ml quantity of the powder containing 200,000 Units and shake
of dimethylfonnamide for 1 hour. Centrifuge, dilute 10.0 ml of with 50.0 ml of dimethylfonnamide for 1 hour. Centrifuge, dilute
the clear, supernatant liquid to 200.0 ml with buffer solution 10.0 ml ofthe clear, supernatant liquid to 200.0 ml with buffer
solution No 4 (2.2.10).
No 4 (2.2.10).

of Units ofNystatin.
Labelling. The label states the strength in tenns ofthe number

of Units of Nystatin.
1803
MONOGRAPHS
0
1807
1807
1808
1809
1809
1810
1811
1812
1812
1813
1814
1815
1816
1816
1818
1819
1820
1821
1822
1823
1824
1825
1826
1827
1827
1828
1829
1830
1831
1832
Oestradiol Benzoate
Oestradiol Injection
Ofloxacin
Ofloxacin Infusion
Ofloxacin Opthalmic Solution
Ofloxacin Tablets
Olanzapine
Olanzapine Tablets
Oleic Acid
Omeprazole
Omeprazole Capsules
Ondansetron Hydrochloride
Ondansetron Injection
Ondansetron Orally Disintegrating Tablets
Ondansetron Oral Solution
Ondansetron Tablets
Oral Rehydration Salts
Ormeloxifene Hydrochloride
Ormeloxifene Hydrochloride Tablets
Ornidazole
Ornidazole Tablets
Orphenadrine Citrate
Orphenadrine Hydrochloride
Orphenadrine Tablets
OseltamivirPhosphate
Oseltamivir Capsules
Oseltarnivir Oral Suspension
Oxazepam
Oxazepam Tablets
Oxcarbazepine
1805
MONOGRAPHS
1833
1834
1834
1835
1835
1836
1838
1838
1840
1841
1842
1842
1845
1846
Oxcarbazepine Tablets
Oxprenolol Hydrochloride
Oxprenolol Tablets
Oxygen
Oxygen 93 Per cent
Oxytetracycline Dihydrate
Oxytetracycline Injection
Oxytetracycline Hydrochloride
Oxytetracycline Capsules
Oxytetracycline Eye Ointment
Oxytetracycline Hydrochloride Injection
Oxytocin
Oxytocin Injection
Oxytocin Nasal Solution
1806
IP 2010
OESTRADIOL INJECTION

examination in 10 ml ofa mixture of90 volumes of chloroform
and 10 volumes of methanol.
Oestradiol Benzoate
Test solution (b). Dissolve 0.1 g of the substance under

examination in 100 ml ofthe same solvent mixture.

substance under examination in the same solvent mixture.
~O

oestradiol benzoate RS in the same solvent mixture.
Mol. Wt. 376.5
Oestradiol Benzoate is
benzoate.
17~-hydroxyestra-1
,3,5(1 0)-trien-3-yl
Oestradiol Benzoate contains not less than 97.0 per cent and
not more than 103.0 per cent ofC2sH2s03, calculated on the
dried basis.
Category. Oestrogen.

dry the plate in air until odour of the solvent is no longer
detectable, heat at 1100 for 10 minutes, spray the plate while
hot with ethanolic sulphuric acid (20 per cent), heat again at
1100 for 10 minutes and examine in ultraviolet light at 365 nm.
chromatogram obtained with reference solution (a).
Dose. By intramuscular injection, I to 2 mg daily.
on 0.5 g.

odourless.
on 0.5 g by drying in an oven at 105 0 for 3 hours.
Identification
Test A may be omitted if tests Band C are carried out. Test C
may be omitted if tests A and B are carried out.
Compare the spectrum with that obtained with oestradiol
benzoate RS or with the reference spectrum of oestradiol
benzoate.
B. In the test for Related substances, the principal spot in the
that in the chromatogram.obtained with reference solution (b)
when examined in daylight and in ultraviolet light at 365 nm.
C. To about 1 mg add 0.5 ml of a 5 per cent w/v solution of
ammonium molybdate in sulphuric acid; a yellowish green
colour develops which exhibits an intense green fluorescence
when examined in ultraviolet light at 365 nm. Add 1 illl of
sulphuric acid and 9 ml of water; the solution becomes pink
with a yellowish fluorescence.
Tests

ethanol (95 per cent) to produce 250.0 ml. Dilute 10.0 ml to
100.0 ml with ethanol (95 per cent) and measure the
231 nm (2.4.7). Calculate the content ofC2sH2s03 taking 500 as
the specific absorbance at 231 nm.
Oestradiol Benzoate Injection
Oestradiol Injection is a sterile solution ofOestradiol Benzoate
in Ethyl Oleate or other suitable ester, in a suitable fixed oil or
in any mixture of these. It may contain suitable alcohols.
Oestradiol Benzoate Injection contains not less than 90.0 per
oestradiol benzoate, C2sH2S03'
Specific optical rotation (2.4.22). +57.0 to +63.0 , determined

in a 1.0 per cent wN solution in dioxan.
Identification


Mobile phase. A mixture of 90 volumes of toluene and

10 volumes of ethanol (95 per cent).

1807
IP 2010
OESTRADIOL INJECTION
Test solution. Add 10 ml of2, 2, 4-trimethylpentane to a volume
of the injection containing 2 mg of Oestradiol Benzoate and
extract with three quantities, each of 10 ml, of ethanol (70 per
cent). Wash the combined extracts with 15 ml of 2,2,4trimethylpentane, evaporate the ethanolic extract to dryness
using a rotary evaporator and dissolve the residue in 2 ml of
chloroform.
Labelling. The label states (1) the nature and composition of

the solvent; (2) that it is meant for intramuscular injection
only; (3) that any solid matter that may have separated on
standing should be redissolved by warming before use.
Ofloxacin
Reference solution. A 0.1 per cent w/v solution of oestradiol

benzoate RS in chloroform.
Apply to the plate 5 f.!l of each solution. After development,

(20 per cent), heat at 105 for 10 minutes and examine in
Tests
Test solution (a). Dilute an accurately measured quantity of
the injection containing about 1 mg of Oestradiol Benzoate to
10.0 ml with a mixture of 90 volumes of cyclohexane and
Test solution (b). Add 1 ml ofa solut~on prepared by dissolving
15 mg of 4-hydroxybenzaldehyde (internal standard) in
10.0 ml of dioxan, adding sufficient cyclohexane to produce
100.0 ml (solution A), to an accurately measured quantity of
the injection containing about 1 mg of Oestradiol Benzoate
and dilute to 10.0 ml with sufficient ofa mixture of90 volumes
of cyclohexane and 10 volumes of dioxan.
Reference solution. Add 10 ml of solution A to 10 mg of
oestradiol benzoate RS, accurately weighed, and dilute to
100.0 ml with the same solvent mixture.
- a stainless steel column 30 cm x 4 mm, packed with porous
silica particles{1O f.!m),
- mobile phase: 90 volumes of cyclohexane and
10 volumes of dioxan,
- injection volume. 20 f.!l.
Inject test solutions (a), (b) and the reference solution. The
assay is not valid unless the resolution between the peaks
due to benzyl alcohol (ifpresent) and oestradiol benzoate and
between the peaks due to oestradiol benzoate and the internal
standard is more than 1.5.
Calculate the content of C2sH2s03 in the injection.
Mol. Wt. 361.4

Ofloxacin is (RS)-9- fluoro-3-methyl-l0-(4-methylpiperazin-lyl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3,-de]-1 ,4-benzoxazine6-carboxylic acid
Ofloxacin contains not less than 98.5 per cent and not more
than 101.5 per cent of ClsH2oFN304. calculated on the dried
basis.
Dose. 200 to 400 mg daily.
Description. A pale yellow or bright yellow, cryst~lline powder.
Identification
Compare the spectrum with that obtained with ofloxacin RS
or the spectrum obtained with the reference solution.
Tests
Light absorption. Absorbance at 440 urn (2.4.7), of0.5 per cent
w/v solution in 0.1 M hydrochloric acid is not more than 0.25.
(2.4.14).
ofloxacin RS in methanol.
Reference solution (b). Dilute I ml ofreference solution (a) to
octadecylsilane bonded to porous silica (5 f.!m),
mobile phase: a mixture of 10 volumes of acetonitrile
and 90 volumes ofphosphate bufferpH 2.4 prepared by
dissolving 27.2 g of monobasic potassium phosphate
in 1000 ml of water, adjust the pH to 2.4 with
1808
IP 2010
OFLOXACIN OPHTHALMIC SOLUTION
NOTE -Protect the solutions from the light.
Test solution. Measure accurately a volume containing 50 mg

ofOfloxacin in 100.0 ml with mobilephase. Dilute 5.0 ml ofthe
solution to 50.0 ml with mobile phase.
column efficiency in not less than 1400 theoretical plates.
Inject the test solution and reference solution (b). Run the
chromatogram three times of the principal peak. In the
not more than the area of the peak in the chromatogram
Heavy Metals (2.3.13). 2.0 g complies with the limit test for
heavy metals, Method B (l0 ppm).
anhydrous glacial acetic acid. l)trate with 0.1 M perchloric
acid, determining the end-point potentiometrically (2.4.25).
Reference solution. A 0.005 per cent w/v solution of ojloxacin

RS in mobile phase.
- mobile phase: a mixture of80 volumes ofbuffer solution
orthophosphate and 0.47 g sodium 1-hexane
sulphonate in 1000 ml of water, add 1 ml of triethylamine
and adjust the pH to 3.0 with orthophosphoric acid
and 20 volumes of acetonitrile,
-
injection volume. 20 fll.
Calculate the content ofClsH2oFN304 in infusion.

ClsH20FN304.
Ofloxacin Ophthalmic Solution

Ofloxacin Ophthalmic Solution is a sterile aqueous solution of
Ofloxacin.
Ofloxacin Infusion
Ofloxacin Infusion is a sterile solution of Ofloxacin in 5 per
cent Dextrose Injection or in Sodium Chloride Injection.
Ofloxacin Infusion contain not less than 90.0 per cent and not
more than 120.0 per cent of the stated amount of ofloxacin,
ClsH20FN304'
Usual strength. 0.3 per cent w/w.
Identification
Usual strengths. 25 mg; 50 mg; 100 mg; 200 mg; 400mg.
Identification
Tests
pH (2.4.24).6.0 to 7.2.
Sterility (2.2.11). Comply with the test for sterility.
Tests
pH (2.4.24). 3.8to 7.5.

Preparation (Infusions).
Ofloxacin Ophthalmic Solution contains not less than 90.0 per

ofloxacin, ClsH20FN304.
Solvent mixture. Phosphate buffer pH 7.25 prepared by

dissolving 3.56 g of disodium hydrogen phosphate in 1000 ml
water, adjusted pH to 7.25 using orthophosphoric acid or
sodium hydroxide solution.
1809
OFLOXACIN TABLETS
IP 2010
Test solution. Measure accurately a volume of Opthalmic

Solution, containing 30 mg ofOfloxacin in 100.0 ml ofsolvent
mixture. Dilute 1.0 ml of the solution to 10.0 with solvent
mixture.
obtained from a solution of known concentration of

ojloxacin RS in the same medium.
ClsH20FN304.

RS in solvent mixture.

(2.4.14).
octadecylsilane bonded to porous silica (5 !Jm) (Such
as TSK GEL),
.
- mobile phase: a mixture of 20 volumes of acetonitrile,
80 volumes of phosphate buffer pH 7.25 prepared by
dissolving 2.54 g of tetrabutyl ammonium hydrogen
sulphate and 3.56 g of disodium hydrogen phosphate
in 1000 ml water,
- injection volume. 20 !Jl.

a quantity ofpowdered tablet containing 100 mg ofOfloxacin,
disperse in 60 ml of methanol and dilute to 100 ml with methanol
and filter.
than 2.0 per cent.
Calculate the content of CJsH2oFN304.

ojloxacin RS in methanol.
a stainless steel column 15 cm x 4.6 mmpacked with
octadecylsilane bonded to porous silica (5 !Jm),
and 92 volumes ofphosphate bufferpH 2. 4 prepared by
in 1000 ml of water, adjust the pH to 2.4 with
- injection volume. 10 !Jl.
column efficiency in not less than 1400 theoretical plates.
Ofloxacin Tablets
Ofloxacin Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount ofofloxacin,
CJSH20FN304'
Identification
Tests
Apparatus No.1,
Medium. 900 ml of 0.1 M hydrochloride acid,
cent) and the sum of areas of all the secondary peaks is not
more than twice the area of the peak in the chromatogram
Other tests. Comply with the tests stated under the Tablets.

a quantity ofpowdered tablet containing 25 mg ofOfloxacin,
disperse in 60 m1 of methanol and dilute to 100 ml with methanol
and filter.
RS in methanol.

the absorbance ofthe filtrate, suitably diluted with the medium
ifnecessary, at the maximum at about 294 nm (2.4.7). Calculate
the content ofCJsH2oFN304 in the medium from the absorbance
octadecylsilane bonded to porous silica (5 !Jm),
prepared by dissolving 27.2 g ofpotassium dihydrogen
1810
IP 2010
OLANZAPINE
phosphate in 1000 ml of water and adjust the pH to 2.4

with orthophosphoric acid and 8 volumes of
acetonitrile,
-

Reference solution (aj. A 0.2 per cent w/v solution of

olanzapine RS in solvent mixture.
Reference solution (bj. Dilute 1 ml ofreference solution (a) to
100 ml with solvent mixture.
-
than 2.0 per cent.
Calculate the content ofClsHzoFN304.
Olanzapine
Mol. Wt. 312.4

Olanzapine is 2-methyl-4-(4-methyl-l-piperazinyl)-10Hthieno[2,3-b][l ,5]benzodiazepine.
Olanzapine contains not less than 98.0 per cent and not more
than 102.0 per cent ofC 17H zoN 4S, calculated on the anhydrous
basis.
Category. Antipsychotic.
Dose. 10 mg daily.
a stainless steel column 25 cm x 4.6 mm packed with

mobile phase: A. a mixture of 80 volumes of buffer
solution pH 6.8 prepared by dissolving 4.825 g of
sodium dihydrogen orthophosphate monohydrate in
1000 ml of water, adjust pH to 6.8 with 10 per cent w/v of
sodium hydroxide and 20 volumes of acetonitrile,
B. acetonitrile,
Time
(in min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
100
20
63
37
30
45
55
32
100
38
100
o
o
tailing factor is not more than 2.0 and the column efficiency in
not less than 2000 theoretical plates.
chromatogram obtained with the test solution, the area ofany
not more than twice the area of the peak in the chromatogram
Description. A yellow crystalline powder.
Identification

Compare the spectrum with that obtained with olanzapine RS
or with the reference spectrum of olanzapine.
Tests
glacial acetic acid. Titrate with 0.1 M perchloric acid,

(2.4.14).


acetonitrile.

C 17HzoN 4S.

examination in 25 ml ofsolvent mixture.
Storage. Store protected from light and moisture, at a

LOg.
1811
OLANZAPINE TABLETS
IP 2010
orthophosphate in 900 ml water, add 2 ml of

triethylamine and dilute to 1000 ml with wate/: Adjust
the pH to 2.5 with orthophosphoric acid and 30 volumes
of methanol,
Olanzapine Tablets
Olanzapine Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent ofthe stated amount of olanzapine,
C 17HzoN 4S.
Usual strengths. 2.5 rng; 5 mg; 7.5 mg; 10 mg.
Identification
tailing factor is not more than 2.0, the column efficiency is not
less than 2500 theoretical plates. The relative standard
deviation for replicate injections is not more than 2 per cent.
Tests
Calculate the content ofC 17H zoN 4S.

Apparatus No.1,
Medium. 900 ml of O. 01 M hydrochloric acid,
Speed and time. 50 rpm for 45 minutes.
Withdraw a suitable volume ofthe medium and filter. Determine
Oleic Acid
NOTE - Protect all the solutions ji-om light.
H3C(H2C)6V=V(CH2)6COOH
Test solution. Use the filtrate, if necessary dilute with

dissolution medium.
Reference solution. Weigh 16 mg of olanzapine RS, dissolve
in about 2.5 ml of acetonitrile and dilute to 25 ml with 0.01 M
hydrochloroc acid.Dilute suitabely to get 0.00016 per cent
w/v in dissolution medium.
Chromatographic system as described under Assay, using
Injection volume 50 ).1.1.
tailing factor is not more than 2.0. The column efficiency is not
less than 2500 theoretical plates.
Calculate the content ofC 17H zoN 4S.
D. Not less than 70 per cent ofthe stated amount ofC 17H zoN 4S.
Test solution. Take 10 intact tablets to a suitable volumetric

flask, disperse in acetonitrile and dilute with 0.01 M
hydrochloric acid to get a final concentration of 0.01 per cent
w/v ofOlanzapine.
Reference solution. Weigh 10 mg of olanzapine RS, dissolve
in about 25 rnl of acetonitrile and dilute to 100 ml with 0.01 M
hydrochloric acid.
- a stainless steel column 25 em x 4.6 mm packed with
- mobile phase: a mixture of70 volumes of buffersolution
prepared by dissolving 3 g of ammonium dihydrogen
Mol. Wt. 282.5

Oleic Acid consists mainly of (Z)-octadec-9-enoic acid,
ClsH340Z, together with varying amounts of saturated and
other unsaturated fatty acids and is obtained by the hydrolysis
of fats or fixed oils and separation of the liquid acids by
expression or other suitable means. It may contain a suitable
antioxidant.
Category. Pharmaceutical aid (emulsion adjuvant).
Description. A clear, yellowish to pale brown, oily liquid; odour,
characteristic. On exposure to air it darkens in colour and the
odour becomes more pronounced.
Identification
A. To 1 ml add 1 ml of ethanol (95 per cent,),' the solution is
clear. It turns orange or red on addition of 0.1 ml of methyl
orange solution.
B.Take a mixture of 1 ml of nitric acid and 1 ml of water in a
test-tube with an internal diameter of about 12.5 mm and add
1 ml ofthe substance under examination on the surface of the
mixture. Introduce 0.5 g of copper turnings into the lower
layer and allow to stand under a hood for 4 hours; the upper
layer solidifies.
C.Complies with the test for Iodine value (2.3.28).
Tests
Weight per ml (2.4.29).0.889 g to 0.895 g.
Peroxide value (2.3.35). Not more than 10.0.
Acid value (2.3.23). 195 to 202, determined on 0.5 g..
1812
IP 2010
OMEPRAZOLE
Iodine value (2.3.28). 85 to 95, determined by MethodA..
Identification
Water-solubie acids. Shake 5 ml with 5 ml of water for

2 minutes, allow the liquids to separate and filter the aqueous
layer through paper moistened with water. To the filtrate add
0.05 ml ofmethyl orange solution; the liquid does not become
red.

Neutral fats and mineral oils. Boil I ml with 5 ml of 0.5 M

sodium carbonate and 25 ml of water in a large flask. The
solution, while still hot, is not more opalescent than
opalescence standard OS2 (204.1).
Congealing point. Dry about 10 ml by heating to 110 with
frequent stirring, transfer to a test-tube about 20 rom in diameter,
cool and when at 15 immerse the tube in a suitable water-bath
so that the cooling takes place at the rate of2 per minute. Stir
the sample with a thermometer; it does not become cloudy
until the temperature has fallen to 10. On further cooling it
congeals to a white solid or semi-solid mass at about 4.
Storage. Store protected from light and moisture in a refrigerator
esc to 15).
Labelling. The label states (1) where applicable, that it is used

for external use only; (2) the name and concentration of any
added antioxidant.
B. When examined in the range 230 nm to 360 nm (204.7), a

absorption maxima at about 276 nm and 305 nm; the ratio of
the absorbance at about 305 nm to that at about 276 nm, 1.6
to 1.8.
Tests
.
dichloromethane is clear (204.1).
Light absorption (204.7). Absorbances of a freshly prepared
2.0 per cent w/v solution in dichloromethane at 400 nm,
500 nm and 600 nm are not more than 0.25, 0.10 and 0:10
respectively.
Related substances. A. Detennine by thin-layer chromatography (204.17), coating the plate with silica gel OF254.
Mobile phase. Arnixture of50 volumes ofbenzene, 30 volumes
of ethyl acetate and 10 volumes of methanol.
Omeprazole

examination in 100 ml ofethanol.
O~N:/'
II
I CH3
rr,)-8
OCH
:;/

Compare the spectrum with that obtained with omeprazole
RS or with the reference spectrum of omeprazole.
H
N
HCO~N
3
CH 3
C17H19N303S
Reference solution (a). A 004 per cent w/v solution of

omeprazole RS in ethanol.
Mol. Wt. 34504
Omeprazole is 5-methoxy-2-[[(4-methoxy-3,5dimethylpyridin-2-yl)methyl]sulfinyl]-IH-benzirnidazole.
Omeprazole contains not less than 99.0 per cent and not mbre
than 101.0 per cent of C17HI9N303S, calculated on the dried
basis.
NOTE - Peiform the tests and assay in subdued light and
use low-actinic glassware.
Category. Antiulcerative (proton pump inhibitor).
Dose. For duodenal and gastric ulcers and for reflux
oesophagitis, 20 to 40 mg daily; for Zollinger-Ellison syndrome,
initially 60 mg once daily; usual range, 20 to 120 mg daily.

dry the plate in air and examine in ultraviolet light at 254 nm
immediately. Any secondary spot in the chromatogram
spot in the chromatogram obtained with reference solution (c)
and the total intensity of all such spots in the chromatogram
obtained with the test solution is not more than the intensity
of the spot obtained with reference solution (b).
B. In the Assay, the sum of the areas of all the secondary

peaks is not greater than 1.5 rer cent of the total area of all
peaks.
Heavy metals (2.3.13). The residue obtained in the test for
Sulphated ash, complies with the limit test fo~ heavy metals,
Method B (20 ppm).
1813
OMEPRAZOLE
IP 2010


omeprazole RS in the mobile phase.
octadecylsilane bonded to porous silica (5 ILm),
- mobile phase: a mixture of 65 volumes of phosphate
buffer pH 7.4 and 35 volumes of acetonitrile,
- injection volume. 20 ILL
Inject the reference solution. Repeat the procedure at least
five times and measure the peak responses ofthe peak due to
omeprazole. The relative standard deviation of the replicate
Calculate the content ofC17HJ9N303S,
Storage. Store protected from light and moisture in a refrigerator
(2 to 8).
NOTE - A combination of elevated temperatures (3JO-500)

and high humidity degrades Omeprdzole. It rapidly degrades
under acidic conditions.
Omeprazole Capsules
Omeprazole Capsules contain enteric-coated granules of
Omeprazole.
Omeprazole Capsules contain not less than 90.0 per cent and
omeprazole, C17HI9N303S,
NOTE - Perform the tests and assay in subdued light and

use low-actinic glassware.
filter. Dilute 2 ml of the filtrate to 1QO mI with O.lM sodium

hydroxide.
When exarninedin the.range 230 nm to 360 nm (2.4.7); the
resulting solution shows absorption maxima at about 276 nm
and305nm.
obtained with the test solution corresponds to the peak due
to omeprazole in the chromatogram obtained with the reference
solution.
Tests
A. Apparatus No.1,
Medium. 900 ml of 0.1 M hydrochloric acid, .
Speed and time. 100 rpm and 2 hours.
Tap the granules from a capsule slightly with a glass rod to
make them settle to the bottom. Rotate the paddle at 100 rpm
for 2 hours, drain the solution slowly without losing any
granules. Transfer them quantitatively to a 1OO-ml volumetric
flask, add 20 ml of 0.1 M sodium hydroxide and mix with the
aid of ultrasound. Dilute to volume with 0.1 M sodium
hydroxide, centrifuge about 15 ml for 5 minutes and dilute
5.0 mI ofthe clear supernatant liquid to 50.0 ml with the mobile
phase. Using the resulting solution as the test solution, carry
out the determination as described in the Assay. Calculate the
content of C17H19N303S in the supernatant liquid. Calculate
the percentage of omeprazole released in the acid medium by
subtracting the content of C17H19N303S in the test solution
from the total content ofomeprazole determined in the Assay.
Not more than 15 per cent ofthe stated amount ofC 17HJ9N303S
is dissolved in 2 hours.
B. Apparatus No.1,
Medium. 900 ml ofphosphate bufferpH 6.8,
Tap the granules from a capsule slightly with a glass rod to

make them settle to the bottom. Rotate the paddle at 100 rpm
for 45 minutes and filter the solution. Using the filtered medium
as the test solution, carry out the determination as described
in the Assay. Calculate the content of C17H19N303S in the
medium.
C17HI9N303S,
Identification
A.To a quantity of the contents of the capsules containing

50 mg of Omeprazole in a 1OO-ml volumetric flask add about
70 m1 of O. I M sodium hydrOXide. Mixin an ultrasonic bath for
about 5 minutes and heat on a water-bath for 10 minutes.
Cool, make up to volume with 0.1 M sodium hydroxide and
on 0.5 g of the contents of the capsules by drying in an oven
at 60 at a pressure not exceeding 0.7 kPa for 4 hours.
1814
IP 2010
ONDANSETRON HYDROCHLORIDE
Test solution. Mix the contents of 20 capsules. Weigh and

transfer the granules containingabout 20 mg of Omeprazole
to a 100-ml volumetric flask, add 20 ml of 0.1 M sodium
hydroxide, mix with the aid ofultrasound and dilute to volume
with 0.1 M sodium hydroxide. Centrifuge for 5 minutes and
dilute 5.0 ml ofthe clear supernatant liquid to 50.0 ml with the
mobile phase.
Reference solution. Take 20 mg of omeprazole RS in a dry,
stoppered test-tube, add 20.0 rnl of 0.1 M sodium hydroxide,
shake vigorously for 5 minutes and dilute 1.0 ml ofthe solution
with the mobile phase to produce 50.0 ml.
Inject the reference solution. Repeat the procedure at least
five times and measure the peak responses of the peak due to
omeprazole. The relative standard deviation of the replicate
Calculate the content ofCi7H 19N 30 3S in the capsules.
ondansetron hydrochloride RS or with the reference spectrum

of ondansetron hydrochloride.
B. It gives the reactions ofchlorides"(2.3.12).
Tests
(2.4.14).
Use the chromatographic system,the test solution and
reference solution (b) described under Assay.
in the chromatogram obtained with reference solution (b)
peaks is not more than 0.5 times the area of the peak in the
cent).
Water (2.3.43).9.0 to 10.5 per cent, determined on 0.2 g.
examination in 50 ml of the mobile phase and filter. Dilute
5.0 ml ofthe solution to 50.0 ml with the mobile phase.
ondansetron hydrochloride RS in the mobile phase.

Reference solution (c). A solution containing 0.01 per cent
w/v of ondansetron hydrochloride RS and 0.002 per cent w/v
of 3[Dimethylaminomethylj-1, 2, 3, 9-tetrahydro-9-methyl4H-carbozol-4-one RS (ondansetron impurity A RS) "in the
mobile phase.
ClsH19N30,HCl,2HzO
Mol. Wt. 365.9
Ondansetron HydrocWoride is (RS)-9-methyl-3-[(2-methyl-lHimidazol-l-yl)methyl]-1 ,2,3,9-tetrahydro-4H-carbazol-4-one

hydrochloride dihydrate.
Ondansetron Hydrochloride contains not less than 98.0 per
cent and not more than 102.0 per cent of ClsH19N30,HCl,
Category. Antiemetic. (5-HT3 receptor antagonist)
Identification
(2.4.6).Compare the spectrum with that obtained with
porous silica (5 11m) with chemically bonded nitrile
groups (Such as Supelco LC-CN),
- mobile phase: a mixture of 50 volumes of 0.02 M
monobasic potassium phosphate with the pH
previously adjusted to 5.4 with 1 M sodium hydroxide
relative retention time for ondansetron is about 1.0 and for
ondimsetron impurity A is about 1.1. The resolution between
ondansetron and ondansetron impurity A is not less than 1.5.
1815
ONDANSETRON INJECTION
IP 2010
Inject reference solution (a). The tailing factor is not more

Inject the test solution and reference solution (a).
Calculate the content ofClsHI9N30,HCI.
Ondansetron Hydrochloride Injection
Ondansetron Injection contains not less than 95.0 per cent
ondansetron, ClsH19N30.
Identification
chromatogram obtained with the reference solution (a).

of 3[Dimethylaminomethyl}-1, 2, 3, 9-tetrahydro-9-methyl4H-carbozol-4-one RS (ondansetron impurity A RS) in the
mobile phase.
- a stainless steel column 20 cm x 4.6 lTIlll, packed with
porous silica (5 11m) bonded to nitrile groups (such as
Supelco LC-CN),
- flow rate. 1.5 rn1 per minute,
ondansetron impurity A is about 1..1. The resolution between
Tests

pH (2.4.24).3.3 to 4.0.

(2.4.14).
Calculate the content ofCIsHI9N30 in the injection.
Use the chromatographic system, test solution and reference

solution (b) described under Assay.
secondary peale is not more than 0.2 times the area ofthe peak
per cent) and the sum ofthe areas of all the secondary peaks
is not more than 1.5 times the area of the peak in the
cent).
Bacterial endotoxins (2.2.3). Not more than 9.9 Endotoxin
Units per mg of ondansetron.
Test solution.Transfer an accurately measured volume of the

injection containing about 2 mg of ondansetron to a 25-ml
volumetric flask, dilute to volume with the mobile phase and mix.
Storage. Store protected from light, at a temperature not

exceeding 30.
equivalent amount of ondansetron.
Ondansetron Orally Disintegrating

Tablets
Ondansetron Orally Disintegrating Tablets contain not less
than 90.0 per cent and not more than 110.0 per cent of the
stated amount of ondansetron, ClsH19N30.
Usual strength. 4 mg; 8 mg.
Identification
Tests
Apparatus No.1,
1816
IP 2010
ONDANSETRON ORALLY DISINTEGRATINGTABLETS

dissolution medium ifnecessary, at the maximum at about 31 0
nm (2.4.7). Calculate the content ofClsH19N30 in the medium
from the absorbance obtained by using a solution of known
concentration of ondansetron RS in same medium.
D. Not less than 80 per cent ofthe stated amount ofC ls H19N30.
(2.4.14).
Test solution. Disperse a quantity of powdered tablets

containing about 40 mg of ondansetron with 100.0 ml of the
mobile phase. Centrifuge a portion ofthis solution at 3000 rpm
for 10 minutes. Use the supernatant.
1,2,3,9-tetrahydro-9-methyl-3-methylene-4H-carbazol-4-one
RS (ondansetron impuirty DRS) in acetonitrile.
2-methylimidazole in acetonitrile.
ondansetron RS in acetonitriile.
Reference solution (d). Dilute 5.0 ml ofreference solution (c)
Reference solution (e). Dilute 10.0 ml ofreference solution (c)
Reference solution (j). Transfer 5.0 ml of each reference
solution (a), (b) and (c) to a 100-ml volumetric flask, dilute to
volume with the mobile phase.
- a stainless steel column 25 cm x 4.6 rom, packed with
porous silica chemically bonded to nitrile groups (5 /-lm),
buffer prepared by dissolving 2.72 g of monobasic
potassium phosphate in 1000 ml of water, adjusted to
pH 5.4 with 1 M sodium hydroxide and 20 volumes of
acetonitrile,
Inject reference solution (t). The test is not valid unless the
resolution between ondansetron and adjacent peak is not less
than 1.5, the theoretical plates is not less than 8000 for
ondansetron and tailing factor is not more than 2.0.
Inject reference solution (e). The signal to noise ratio for the
ondansetron peak is not less than 15. The relative retention
time with reference to ondansetron for 2-methylimidazole is
about 0.16 and for ondansetron impurity D is about 0.45.
Inject reference solution (d) and the test solution. In the

chromatogram obtained with the test solution, the area of the
peak due to 2-methylimidazole is not more than 0.1 5 per cent,
multiply with response factor 0.5, the area of peak due to
ondansetron impurity D is not more than 0.12 per cent, multiply
with response factor 1.2. The area of any other secondary
peak is not more than 0.1 per cent and the sum of all the
secondary peaks is not more than 0.5 per cent.
LOg.
Other tests. Comply with the tests stated under tablets.

of powder containing about 40 mg of ondansetron with
100.0 mlO.01 Mhydrochloricacid.Dilute 1.0 ml ofthe solution
to 10.0 ml with 0.01 Mhydrochloric acid.
ondansetron RS in 0.01 M hydrochloric acid.
3[Dimethylamino methylj-1,2,3,9-tetrahydro-9-methyl-Hcarbozol-4-one-RS (ondansetron impurity A RS) in 0.01 M
hydrochloric acid.
Reference solution (c). Transfer 8.0 ml each of reference
solution (a) and (b) to 50-ml volumetric flask and dilute to
volume with 0.01 M hydrochloric acid.
porOl.jS silica bonded to nitrile groups (5 /-lm),
- mobile phase: a mixture of52 volumes of0.272 per cent
w/v solution of monobasic potassium phosphate,
adjusted to pH 5.4 with 1 M sodium hydroxide and 48
volumes of acetonitrile,
resolution between the peaks due to ondansetron and
ondansetron impurity A is not less than 1.5. The relative
retention time with reference to ondansetron for ondansetron
impurity A is about 1.1. The tailing factoris not more than 2.0
for ondansetron peak.
Calculate the content ofCIsH19N30 in the tablets.
Storage. Store protected form light and moisture, at a
Labelling. The label states the active ingredient in terms of
1817
ONDANSETRON ORAL SOLUTION
IP 2010
Ondansetron Oral Solution

Ondansetron Hydrochloride Oral Solution
Ondansetron Oral Solution is a solution of Ondansetron
Hydrochloride in a suitable vehicle..
Ondansetron Oral Solution contains not less than 95.0 per
ondansetron, ClsH'9N30.
Usual strength. 2 mg per 5 ml; 4 mg per 5 ml.
Identification
the plate with silica gel G.
Mobile phase. A mixture of 90 volumes of chloroform, 50
volumes of ethyl acetate, 40 volumes of methanol and 1volume
of ammonium hydroxide.
Test solution. Dilute an accurately measured volume of the
oral solution containing about 20 mg of ondansetron to 100.0
ml in a mixture of 50 volumes of methanol and 50 volumes of
water.
ondansetron RS in methanol.
Apply 10 /J.I of each solution. Allow the solvent front to rise
15 cm. Dry the plate in air and examine in ultraviolet light at
254 nm. The chro\natogram obtained with the test solution
corresponds to that obtained with the reference solution.
Tests
pH (2.4.24).3.3 to 4.0.
Ondansetron impurity D. Not more than 0.1 per cent.
Test solution. Dilute an accurately measured volume of oral

solution to obtain a solution having a concentration of 0.08
per cent w/v of ondansetron in the mobile phase.
1,2,3,9-tetrahydro-9-methyl-3-methylene-4H-carbazol~4-on
e
RS (ondansetron impurity D RS) in the mobile phase.

Reference solution (b). A solution containing 0.00005 per cent
w/v of ondansetron impurity D RS and 0.0002 per cent w/v of
N-methyl [2 -(2 -methylbenzhydryloxy) ethyl} am ine
hydrochloride RS (ondansetron impurity C RS) in the mobile
phase.
porous silica bonded to nitrile groups (5 /J.m),
monobasic potassium phosphate, previously adjusted
to pH 5.4 and 20 volumes of acetonitrile,
- flow rate. 1.5ml per minute,
injection volume. 20/J.1.
resolution between ondansetron impurity D and ondansetron
impUlity C is not less than 2.0, the tailing factor for ondansetron
impurity D is not more than 2.0 and the relative standard
deviation forreplicate injections is notmore than 4.0 per cent.
Calculate the content of ondansetron impurity D in the oral
solution.
(2.4.14), as described under Assay with the following
modifications.
Test solution. Weigh accurately a quantity of oral solution

containing about 45 mg of Ondansetron Hydrochloride in
50-ml volumetric flask, dissolve and dilute with the mobile
phase and mix. Dilute 5.0 ml ofthis solution to 50 ml with the
mobile phase.
resolution between ondansetron impuirty A and ondansetron
is not less than 1.5, the tailing factor is not less than 2.0 and
more than 1.5 per cent. The relative retention time with
reference to ondansetron for ondansetron impurity A is
about 1.1.
chromatogram obtained with the test solution, the area ofthe
peak due to ondansetron impurity D is not more than
0.1 per cent, the area of each peak due to imidazole, 2-methyl
imidazole, des-(-methy1)ondansetron hydrochloride,
N-Desmethyl ondansetron maleate, ondansetron impurity A
is not more than 0.2 per cent. The area of any other secondary
peak is not more than 0.2 per cent and the sum of all the
secondary peaks is not more than 0.5 per cent.
Test solution. Dilute a volume of the oral solution containing

about 9 mg ofondansetron to 100.0 ml with themobi1e phase.
1818
IP 2010
ONDANSETRON TABLETS

of 3[DimethylaminomethylJ-l, 2, 3, 9-tetrahydro-9-methyl4H-carbozol-4-one RS (ondansetron impurity A RS) in the
mobile phase.
- a stainless steel column 25 cm x 4.6 mrn, packed with
porous silica (5 11m) with chemically bonded nitrile
groups (such as Supelco LC-CN),
ondansetron impurity A is about 1.1. The resolution between
Inject reference solution (a) The tailing factor is not more
Inject tbe test solution and reference solution (a).
Calculate the content ofClsHl9N30, HCl in the oral solution.
Microbial contamination (2.2.9). Complies with the microbial
contamination test. The total aerobic count is not more than
100 cfu per g, Enterobacteriaceae count is not more than 10
cfu per g and the total combined molds and yeasts count is
not more than 50 cfu per g.
Medium. 500 ml ofwatel;

(2.4.7). Calculate the content ofCIsHI9N30 in the medium from
concentration of ondansetron hydrochloride dihydrate RS
in the same medium.
1 mg of CIsHI9N30 is equivalent to 1.25 mg of
C,sHI9N30,HCl,2HzO.
D. Not less than 70 per cent ofthe stated amount ofClsHl9N30.
(2.4.14).
Use the chromatographic system, test solution and reference
solution (b) described under Assay.
is not more than 0.5 times the area of the peak in the
cent).
Tablets.
chromatographic conditions and reference solution (a)
described in the Assay.
Test solution. Disperse one tablet in' the mobile phase and
dilute to obtain a solution containing 0.01 per cent w/v of
Ondanseteron Hydrochloride and filter.
Calculate the content ofCIsHI9N30 in the tablet.
Ondansetron Tablets
Ondansetron Hydrochloride Tablets
Ondansetron Tablets contain not less than 95.0 per cent and
ondansetron, ClsH19N30.

a quantity ofpowder containing about 50 mg of ondansetron,
disperse in 50 ml ofthe mobile phase and filter. Dilute 5.0 ml of
the solution to 50.0 ml with the mobile phase.
Usual strengths. 2 mg; 4 mg; 8 mg

Identification
. chromatogram obtained withthe reference solution (a).
Tests
Dissolution (2.5.2)
Apparatus No.1,

of 3[DimethylaminomethylJ-l, 2, 3, 9-tetrahydro-9-methyl4H-carbozol-4-one RS (ondansetron impurity A RS) in the
mobile phase.
1819
ORAL REHYDRATION SALTS
IF 2010
porous silica (5 /lm) bonded to nitrile groups (such as
Supelco LC-CN),
mobile phase: a mixture of 50 volumes of 0.02 M
Composition ofthe formulation in terms ofthe amount, in g, to

be dissolved in sufficient water to produce 1000 ml.
Sodium Chloride
Dextrose (anhydrous)
or
Dextrose Monohydrate
Potassium Chloride
Sodium Citrate
2.6
13.5
14.85
1.5
2.9
The molar concentrations of sodium, potassium, chloride and

citrate ions in terms ofmillimoles per litre are given below:
ondansetron impurity A is about 1.1. The resolution between
Sodium
Potassium
75
20
Chloride

Citrate
Dextrose
65
10
mmol/l
75
The total osmolar concentration of the solution in terms of

mOsmol per litre is 245.

Calculate the content ofC,sH,9N30 in the tablets.
Oral Rehydration Salts contain not less than 90.0 per cent and
not more than 110.0 per cent ofthe stated amount ofDextrose
(anhydrous) or Dextrose Monohydrate (as appropriate) and
ofthe requisite amQunts ofsodium, Na, potassium, K, chloride,
Cl, and citrate, C6H s0 7, calculated from the stated amounts of
the relevant constituents.
Description. A white to creamy-white, amorphous or
crystalline powder; odourless.
Identification
ORS Powder
Oral Rehydration Salts are dry, homogeneously mixed powders
containing Dextrose, Sodium Chloride, Potassium Chloride
and either Sodium Bicarbonate or Sodium Citrate for use in
oral rehydration therapy after being dissolved in the requisite
amount ofwater.
They may contain suitable flavouring agents and, where
necessary, suitable flow agents in the minimum quantity
required to achieve a satisfactory product but may not contain
artificial sweetening agents like mono- and/or polysaccharides. If saccharin/saccharin sodium or aspartame is
used in preparations meant for paediatric use, the concentration
of saccharin should be such that its daily intake is not more
than 5 mg/kg ofbody weight and that of aspartame should be
such that its daily intake is not more than 40 mg/kg of body
weight.
Category: Replacement solution for diarrhoeal rehydration.
Usual strength. A formulation of reduced osmolarity (given
below) recommended by the World Health Organization
(WHO) for the Diarrhoeal Diseases Control Programme, and
ofthe United Nations Children's Emergency Fund (UNICEF).
A. When heated, melting and charring occurs and an odour of

burnt sugar is produced.
B. Add a few drops ofthe solution prepared as directed in the
label to 5 ml of potassium cupri-tartrate solution; a copious
red precipitate is produced on boiling.
C. Gives reaction A of sodium salts, reaction A of potassium
salts and reaction A of chlorides (2.3.1).
D. A quantity containing about 50 mg of citric acid gives the
reactions ofcitrates (2.3.1).
Tests
Uniformity of weight. Comply with the test for contents of
packaged dosage forms (2.5.6).
Seal test (onlyfor sachets). Loosely bundle 10 sachets with a
rubber band and submerge the bundle under water in a vacuum
desiccator maintained at a pressure not exceeding 18 kPa for
one minute. Examine the bundle for any fine stream ofbubbles.
Re-establish normal pressure and open the bundle. No
penetration of water is observed in any sachet.
1820
IP 2010
ORMELOXIFEN HYDROCHLORIDE
Other tests. Comply with the tests stated under Oral Powders.
Assay. Carry out the following assays on the well-mixed
contents of an individual sachet or on a suitable sample from
the well-mixed contents ofa bulle container. Where the amount
in an individual sachet is insufficient to carry out all the assays,
take a separate sachet for the Assay for citrate and for the
Assay for dextrose. For the Assays for total sodium, for
potassium and for total ch.loride weigh accurately about
8.0 g and dissolve in sufficient water to produce 500.0 ml
(solution A).
For total sodium - Dilute a suitable volume of solution A

with a s1,1fficient volume of a solution of strontium chloride
such thatthe final solution contains a 1500 to 2000-fold excess
of strontium ions and determine by Method A for atomic
absorption spectrophotometry (2.4.2), measuring at 589 nm
and using sodium solution AAS, suitably diluted with the
strontium chloride solution, for the standard solutions or,
alternately by Method A for flame photometry (2.4.4).
1 g ofSodium Chloride, and ofSodium Citrate is equivalent to
0.3934 and 0.2345 g ofNa respectively.
For potassium - Dilute a suitable volume of solution A with

a sufficient volume of solution of strontium chloride such
that the final solution contains a 1500 to 2000-fold excess of
strontium ions and determine by Method A for atomic
and using potassium solution AAS, suitably diluted with the
strontium chloride solution, for the standard solutions or,
alternatively by Method A for flame photometry (2.4.4).
in g ofC6H 120 6and C6H1206,H20 respectively, as appropriate,

in the weight taken for the Assay.
Storage. Store protected from moisture in sachets, preferably
made ofaluminum foil, containing sufficient powder for a single
dose or for a day's treatment or for use in hospitals, in bulle
containers containing sufficient quantity to produce a volume
of solution appropriate to the daily requirements of the
hospital concerned.
Labelling. The label states (l) for sachets, the total weights,
in g, of each constituent; (2) for bulle containers, the weights,
in g, of each constituent in a stated quantity, in g, of the oral
powder; (3) the molar concentration in millimoles per litre of
sodium, potassium, chloride and citrate ions, and of dextrose
as well as the total osmolar concentration in mOsmol per litre
of the solution prepared from the oral powder; (4) the total
weight ofthe contents of the container; (5) the directions for
use; (6) that any portion ofthe solution prepared from the oral
powder that remains unused for 24 hours after preparation
should be discarded; (7) the storage conditions. g, of the oral
powder.
Ormeloxifene Hydrochloride
Centchroman Hydrochloride; Centchroman
H3 CO
1 g ofPotassium Chloride is equivalent to 0.5245 g ofK.

I
For total chloride - Titrate 50 ml of solution A with 0.1 M

silver nitrate using potassium chromate solution as indicator.
1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g ofCI.

1 g of Sodium Chloride, and of Potassium Chloride is
equivalent to 0.6066 and 0.4756 g ofCl respectively.
For citrate - Weigh accurately about 2.0 g and dissolve in

50 ml of anhydrous glacial acetic acid by heating at about
50. Cool, allow to stand for 10 minutes. Titrate with 0.1 M
perchloric acid, using 1-naphtholbenzein solution as
indicator. Carry OJ.lt a blank titration.
CJfS07'
1 g ofSodium Citrate is equivalentto 0.6430 g ofC6Hs0 7
For dextrose - Weigh accurately about 7.5 g, dissolve in

40 ml of water, add 0.5 ml of dilute ammonia solution, and
dilute to 50 ml with water. Mix well, allow to stand for
30 minutes, filter the solution, ifturbid, and measure the optical
rotation in a 2-dm tube (2.4.22). The observed rotation, in
degrees, multiplied by 0.9477 and 1.0424 represents the weight
,CH 3
CH 3
I":
~
HCI
O~O
Mol. Wt. 493.5
Ormeloxifene Hydrochloride is trans-7-methoxy-2,2dimethyl-3-phenyl-4[4-(2pyrrolidinoethoxy)phenyl]chroman hydrochloride.

Ormeloxifene Hydrochloride contains not less than 98.5 per
cent and not more than 101.5 per cent of C30H3sN03,HCl,
Category. Antifertility agent (female oral contraceptive).
Dose. 120 mg twice a week, 3 days apart, for three months and
thereafter once a week.
1821
ORMELOXIFEN HYDROCHLORIDE
IP 2010
Identification .
Compare the spectrum with that obtained with ormeloxifene
hydrochloride RS orwith the reference spectrum oformeloxifene.
B, When examined in the range 230 urn to 360 run (2.4.7), a
maxima at about 278 and 282 urn.
C. Determine by thin-layer chromatography (2.4.17), coating

ormeloxifene hydrochloride RS in chloroform.
dry the plate in air, until the odour ofthe solvents is no longer
detectable and spray with a 0.3 per cent w/v solution of
potassium permanganate. The principal spot in the
D. To 1 ml ofa 2 per centw/v solution in ethanol (95 percent)
add 1 ml of a saturated solution of picric acid in water, stir
well and set aside for 5 minutes. The yellow precipitate obtained
after washing with water and drying at 60 for 4 hours melts at
212to 218(2.4.21).
Tests
Total basic substances. Weigh accurately 0.5 g, dissolve in
25 ml of anhydrous glacial acetic acid, add 10 ml of a 5 per
cent w/v solution of mercuric acetate in anhydrous glacial
acetic acid. Titrate with 0.1 M perchloric acid, using clystal
violet solution as indicator to a bluish green end-point. Carry
C30H3sN03,HCl.
cis~Isomer.
Not more than 1.5 per cent ofthe total content of

hydrochlorides of trans-and cis-isomers determined in the
Assay.
a stainless steel column 25 cm x 4.6 mm,packed with
mobile phase: 80 volumes of acetonitrile and 20 volumes
of water containing 0.04 per cent w/v solution of
tetramethylammonium hydroxide adjusted to pH 7.6
with phosphoric acid,
.
.
spectrophotonieter set at 280 run,
Inject the reference solution and use an attenuation such that
the response for theprincipal peak due to trans-ormeloxifene
hydrochloride is more than 50 per cent of the full-scale
deflection ofthe recorder. Make a total ofsix injections. When
the chromatograms are recorded under the conditions
described above, trans-ormeloxifene hydrochloride is eluted
before cis-ormeloxifene hydrochloride.The test is not valid
unless the relative standard deviation ofsix replicate injections
is not greater than 6 per cent and the resolution between the
peaks due to cis-ormeloxifene hydrochloride and transormeloxifene hydrochloride is greater than 1. If necessary,
adjust the proportions and the flow rate of the mobile phase
to obtain proper resolution. Inject a suitable volume of test
solutiop and record the response. From the average peak areas
of the six replicate analyses calculate the content of transormeloxifene hydrochloride and cis-ormeloxifene
hydrochloride in the substance under examination by
comparing with the peak responses for trans-ormeloxifene
hydrochloride and cis-ormeloxifene hydrochloride respectively
obtained with reference solution.
Ormeloxifene Hydrochloride Tablets

Centchroman Hydrochloride Tablets; Centchroman
Tablets
Ormeloxifene Hydrochloride Tablets contain not less than
amount ofOrmeloxifene hydrochloride, C30H3sN03,HCI.

Test solution.. A 0.004 per cent w/v solution of the substance

of trans-ormeloxifene hydrochloride RS and 0.0006 per cent
w/v of cis-ormeloxifene hydrochloride RS in the mobile phase.
Identification
Ormeloxifene Hydrochloride with! 0 ml of chloroform, filter
and evaporate the filtrate to dryness at a pressure not
exceeding 0.7 kPa. The residue complies with the following
tests.
Compare the spectrum with that obtained with ormeloxifene
1822
IP 2010
ORNlDAZOLE
hydrochloride RS or with the reference spectrum of

ormeloxifene.
maxima at about 278 and 282 nm.


ormeloxifene hydrochloride RS in chloroform.
dry the plate in air, until the odour of the solvents is no longer
detectable and spray with ~ 0.3 per cent w/v solution of
potassium permanganate. The principal spot in the
Tests
Apparatus No.2,
of water containing 0.04 per cent w/v solution of
with phosphoric acid,
Inject the reference solution and use an attenuation such that
the response for the principal peak due to trans-ormeloxifene
hydrochloride is more than 50 per cent offull-scale deflection
of the recorder. Make a total of six injections. When the
chromatograms are recorded under the conditions described
above, trans-ormeloxifene hydrochloride is eluted before cisormeloxifene hydrochloride.The test is not valid unless the
relative standard deviation of six replicate injections is not
greater than 6 per cent and the resolution between the peaks
due to cis-ormeloxifene hydrochloride and trans-ormeloxifene
hydrochloride is greater than 1. If necessary, adjust the
proportions and the flow rate of the mobile phase to obtain
proper resolution. Inject a suitable volume of test solution
and record the response. From the average peak areas of the
six replicate analyses calculate the content of transormeloxifene hydrochloride and cis-ormeloxifene
hydrochloride in the substance under examination.
Withdraw a suitable volume ofthe medium

, and filter. Measure
the absorbance of the filtrate, suitably diluted if necessary, at
C30H3sN03,HCl in the medium from the absorbance obtained
from a solution of known concentration of Ormeloxifene
hydrochloride RS in water.
Ornidazole
OH
~CI

C30H3sN03,HCl.
cis-Isomer. Not more than 1.5 per cent ofthe total content of
ormeloxifene hydrochlorides of trans- and cis-isomers
determined in the Assay.
02N~rCH3
C7HIQClN30 3
Mol. Wt. 219.6
Omidazole is (RS)-I-chloro-3-(2-methyl-5-nitroimidazol-lyl)propan-2-ol.
Omidazole contains not less than 98.0 per cent and not more
than 101.0 p~r cent ofC7H IQClN30 3,calculated on the anhydrous
basis.
Category. Antiamoebic.
Dose. 500 mg twice a day orally for five days.
Description. A white to yellowish white crystalline powder.

ofthe powder containing 0.1 g ofOrmeloxifene Hydrochloride
with three quantities, each of 5 ml, of methanol, centrifuge
each extract, dilute the combined extracts to 25 ml with
methanol and then dilute 250 III ofthe resulting solution to 25
Identification

of trans-ormeloxifene hydrochloride RS and 0.0006 per cent
w/v of cis-ormeloxifene hydrochloride RS in the mobile phase.

Compare the spectrum with that obtained with ornidazole RS
or with the reference spectrum of omidazole.
1823
ORNIDAZOLE
IP 2010

an absorption maximum at about 277 nm; absorbance at
277 nm, 0.580 to 0.630.

Tests
Ornidazole Tablets

(2.4.14).
Ornidazole Tablets contain not less than 95.0 per cent and not
more than 105.0 per cent of the stated amount of ornidazole,
C7H IOCIN30 3


2-methyl-5-nitroimidazole RS in the mobile phase.
ornidazole RS in the mobile phase.
Reference solution (c). Dilute 20.0 ml ofreference solution (a)
and 1.0 ml ofreference solution (b) to 200.0 ml with the mobile
phase.
Chromatographic system .
- a stainless steel column 25 cm x 4.6 mID, packed with
- mobile phase: a mixture of 70 volumes of O.OlM
potassium dihydrogen orthophosphate and 30
volumes of methanol,
resolution between the peaks corresponding to 2-methyl-5nitroimidazole and ornidazole is at least 1.5.
Inject the test solution, reference solutions (a) and (b). In the
peak corresponding to 2-methyl-5-nitroimidazole is not more
than 0.2 times the area of the peak obtained with reference
solution (a) (0.2 per cent) and the sum of areas of all the
secondary peaks is not more than the area of the peak in the
chromatogram obtained with reference solution (b)
(1.0 per cent).
Identification
Extract a quantity ofthe powdered tablets containing 0.1 g of
Ornidazole with 40 ml of chloroform, filter and evaporate the
filtrate to dryness. The residue complies with the following
test.
Compare the spectrum with that obtained with ornidazole RS
or with the reference spectrum of ornidazole.
B. Heat a quantity ofthe powdered tablets containing 0.1 g of
Ornidazole with 10 mg of zinc powder, 1 ml of water and
0.25 m1 hydrochloric acid for 5 minutes, cool in ice, add 0.5 ml
of sodium nitrite solution and remove the excess of nitrite
with sulphuric acid. Add 0.5 ml of 2-naphthol solution and 2
ml of5 M sodium hydroxide solution. An orange-red colour is
produced.
Tests
Dissolution (2.52).
Apparatus No. 1,
Withdraw a suitable volume ofthe medium and filter promptly,
rejecting the first few ml of the filtrate and dilute a suitable
volume ofthe filtrate with 0.1 M hydrochloric acid. Measure
about 277 nm (2.4.7). Calculate the content of ornidazole,
C7H IOCIN30 3 in the medium from the absorbance obtained from
a solution of known concentration of ornidazole RS
C7H IOCIN30 3
(2.4.14).
Test solution. Disperse a quantity of the powdered tablets

containing 25 mg ofornidazole in 25.0 m1 of the mobile phase.


2-methyl-5-nitroimidazole RS in the mobile phase.
1 ml of 0.1 M perchloric acid is equivalent of 0.02196 g of

C7H IOCIN30 3.

ornidazole RS in the mobile phase.
1824
IP 2010
ORPHENADRINE CITRATE

and 1.0 ml ofreference solution (b) to 200.0 ml with the mobile
phase.
Orphenadrine Citrate contains not less than 98.5 per cent and
not more than 101.0 per cent ofC 1sH 23NO, C6H s0 7, calculated
on the dried basis.
potassium dihydrogen orthophosphate and 30 volumes
of methanol,
Category. Skeletal muscle relaxant.
resolution between the peaks corresponding to 2-methyl-5nitroimidazole and omidazole is at least 2.0.
Inject the test solution, reference solution (a) and (b). In the
peak corresponding to 2-methyl-5-nitroimidazole is not more
than 0.2 times the area of the peak obtained with reference
solution (a) (0.2 per cent) and the sum of areas of all the
secondary peaks is not more than the area of the peak in the
chromatogram obtained with the reference solution (b) (1.0
per cent).
quantity ofthe powder containing 0.2 g ofOmidazole, transfer
to a sintered-glass crucible and extract with 10 ml of hot
acetone. Repeat the extraction 6 times with hot acetone. Cool,
add to the combined extracts 50 ml of acetic anhydride. Titrate
with 0.1 M perchloric acid, determining the end point
C7H IOClN30 3
Storage. Store protected from ligh! and moisture, at a
Dose. By intramuscular or by slow intravenous injection (over

5 minutes), 60 mg repeated after 12 hours if necessary.
odourless or almost odouriess.
Identification
out.
A. Determine by infrared absorption spectrophotometry,(2.4.6).

Compare the spectrum with that obtained with orphenadrine
citrate RS.
0.06 per cent w/v solution in ethanol (95 per cent) shows
absorption maxima at 258 nm, 264
and 271 nm; absorbances
at the maxima are about 0.68, 0.72 and 0.47 respectively.
mn
C. Dissolve 50 mg in 10 ml of ethanol (50 per cent), add 10 ml

of picric acid solution and allow to stand. The precipitate
after recrystallisation from ethanol (95 per cent), melts at
about 89 or at about 107 (2.4.21).
D. Dissolve 5 mg in 2 ml of sulphuric acid; an orange-red
colour is produced.
E. To 1 g add 10 ml water and 2 ml of 5 M sodium hydroxide,
shake with two quantities, each of 10 ml, of chloroform and
discard the chloroform. Heat the aqueous solution to boiling
with an excess of mercuric sulphate solution, filter ifnecessary
and boil the resulting solution with 0.2 ml of dilute potassium
permanganate solution; the solution is decolorised and a
white precipitate is produced.
Tests
Quaternary ammonium salt. Determine by thin-layer
Mobile phase. A mixture of 50 volumes of 2-propanol,

30 volumes of butyl acetate. 15 volumes of water and
5 volumes of strong ammonia solution.
HO
eOOH

examination in 10 ml methanol.
Hooe~eooH
Mol. Wt. 461.5
Orphenadrine Citrate is (RS)-dimethyl[2-(2methylbenzhydryloxy)ethyl]amine dihydrogencitrate.
Reference solution. A 0.005 per cent wlv solution of

ethyldimethyl [2-(2-methylbenzhydryloxy)ethyl ]ammonium
chloride RS.
dry the plate in air and spray with dilute potossium
1825
ORPHENADINE CITRATE
IP 2010
iodobismuthate solution. Any spot corresponding to

ethyldimethyl [2-(2-methylbenzhydryloxy) ethyl] ammonium
chloride in the chromatogram obtained with the test solution
is not more intense than the spot in the chromatogram obtained
Secondary amine. Determine by thin-layer chromatography
(2.4.17) coating the plate with silica gel GF254.
Mobile phase. A mixture of 96 volumes of I-butanol and

examination in'l 0 ml methanol.
Reference solution. A 0.02 per cent wlv solution of methyl [2(2-methylbenzhydlyloxy) ethyl]amine hydrochloride R8.
Apply to the plate 10 /!l of each solution. After development,
dry in air and examine in ultra-violet light at 254 nm. Spray the
plate with dilute potassium iodobismuthate solution and
examine again. By each method of visualisation any spot
corresponding to methyl [2-(2-methylbenzhydryloxy)
ethyl]amine hydrochloride in the chromatogram obtained with
heavy metals, Method B (10 ppm). Use 2 ml oflead standard
solution (l0 ppm Pb) to prepare the standard.
C 1sH23NO, C6Hs07.
C 1sH23NO,HCl
Mol. Wt.305.8

Identification
Tests A andF may be omitted iftestsB, C, D andE are carried
out. Tests B, C and D may be omitted iftests A, E and Fare
carried out.
A. Determine by infrared absorption spectrophotometry,(2.4.6).
Compare the spectrum with that obtained with orphenadrine
hydrochloride RS.
B. When examined in the range 230 nm to 360 nmJ2.4.7), a
0.06 per cent wlv solution in ethanol (95 per cent) shows
three absorption maxima at about 258 nm,264nm and 271 nm;
absorbances at the maxima, about 1.07, 1.13 and 0.73
respectively.
C. Dissolve about 5 mg in 2 mlof sulphuric acid; an orangered colour is produced.

D. Dissolve about 50 mg inl0 ml of ethanol (95percent), add
10 ml of picric acid solution and allow to stand. The
precipitate, after recrystallisation from ethanol (95 per cent)
melts at about 89 or at about 107 (2.4.21).
Tests
Quaternary ammonium salt. Determine by thin-layer

30 volumes of butyl acetate, 15 volumes of water and
chloride R8.
dry the plate in air and spray with dilute potassium
iodobismuthate solution. Any spot corresponding to
chloride in the chromatogram obtained with the test solution
Orphenadrine Hydrochloride is (RS)-dimethyl[2(2methylbenzhydryloxy) ethyl]amine hydrochloride.
Secondary amine. Determine by thin-layer chromatography

(2.4.17) coating the plate with silica gel GF254.
Orphenadrine Hydrochloride contains not less than 98.5 per

cent and not more than 101.0 percent of C 1sH 23NO,HCl.

Category. Antiparkinsonian.

Dose. 150 mg daily, in divided doses, gradually increased;

maximum 400 mg daily.
Reference solution. A 0.02 per cent wlv solution of methyl [2(2-methylbenzhydryloxy) ethyl]amine hydrochloride R8.
1826
IP 2010
OSELTAMIVIR PHOSPHATE

dry in air and examine in ultra-violet light at 254 nm. Spray the
plate with dilute potassium iodobismuthate solution and
examine again. By each method of visualisation any spot
corresponding to methyl [2-(2-methylbenzhydryloxy)
ethyl]amine hydrochloride in the chromatogram obtained with
heavy metals, Method B (10 ppm). Use 2 ml oflead standard
solution (10 ppm Pb) to prepare the standard.
anhydrous glacial acetic acid, add 20 ml of mercuric acetate
solution and titrate with 0.1 M perchloric acid using
l-naphtholbenzein solution as indicator. Carry out a blank
titration.

quantity ofthe powder containing about 70 mg Orphenadrine
Hydrochloride and dissolve as completely as possible in a
mixture of 5 ml of water and 5 ml of 2 M hydrochloric acid
Without delay extract with four quantities, each 15 ml, of
chloroform, filter the combined extracts and evaporate to about
20 m1. Add 30 ml of anhydrous glacial acetic and 2 ml of
mercuric acetate solution and titrate with 0.02 M perchloric
acid determining the end point potentiometrically (2.4.25).
I ml of 0.02 Mperchloric acid is equivalent to 0.006117 g of
C 1SH23NO,HCI.
Oseltamivir Phosphate

C 1sH23NO, HCI.
CI6H2SN204, H3P04
Orphenadrine Hydrochloride Tablets
Oseltamivir Phosphate is phosphoric acid salt of ethyl

(3R,4R,5S)-4-(acetylamino)-5-amino-3-(1-ethylpropoxy)-Icyclohexene-l-carboxylate.
Orphenadrine Tablets contain not less than 92.5 per cent and
orphenadrine hydrochloride, C 1SH23NO,HCI.
Mol. Wt. 410.4
Oseltamivir Phosphate contain not less than 98.0 per cent and
not more than 102.0 per cent ofCI6H2sN204 .H3P0 4,calCulated
on the dried basis.
Identification
Category. Antiviral.
Extract a quantity of the powdered tablets containing about

0.15 g ofOrphenadrine Hydrochloride with chloroform, filter
and evaporate the filtrate to dryness. The residue complies
with the following tests.
Description. A white or off-white powder.
A. Dissolve 5 mg in 2 ml of sulphuric acid; an orange-red

colour is produced.
B. Dissolve 50 mg in 10 ml of ethanol (50 per cent), add 10 ml

of picric acid solution and allow to stand. The precipitate
after recrystallisation from ethanol (95 per cent), melts at
about 89 or at about 107(2.4.21).
C. A 5 per cent w/v solution gives reaction A of chlorides
(2.3.1).
Tests
Identification
Compare the spectrum with that obtained with oseltamivir
phosphate RS.
Tests
Specific optical rotation (2.4.22). - 42.0 to - 48.0, determined
(2.4.14).
1827
OSELTAMIVIR PHOSPHATE
IP 2010
NOTE - Prepare the solutions immediately before use.

oseltamivir phosphate RS in the mobile phase.
octylsilanebonded to porous silica (5 flm) (Such as
YMC pack PRO C8),
- mobile phase: a mixture of66 volumes of buffer solution
prepared by dissolving 6.8 g of anhydrous monobasic
potassium phosphate in 1000 ml of water, adjusting the
pH to 6.0 with dilute sodium hydroxide, 24.5 volumes
of methanol and 23.5 volumes of acetonitrile,
octylsilane bonded to porous silica (5 flm) (such as YMC
pack PRO C8),
- column te~perature 50,
- mobile phase: a mixture of66 volumes ofa buffer solution
potassium phosphate in 1000 ml of water and adjusting
the pH to 6.0 with dilute sodium hydroxide, 24.5 volumes
column efficiency is not less than 2000 theoretical plates and

in the chromatogram obtained with the reference solution (b)
(0.5 per cent) and the sum of all the secondary peaks is not
more than the area of the peak in the chromatogram obtained
with the reference solution (b) (1.0 per cent).
Phosphoric acid. 23.4 to 24.4 per cent, calculated on a dried
basis.
Weigh accurately about 0.2 g and dissolve in 40 ml ofdistilled
water. Titrate with 0.1 M sodium hydroxide, determining the
phosphoric acid.
Calculate the content ofC16H28N204,H3P04'
Oseltamivir Phosphate Capsules

Oseltamivir Capsules contain not less than 90.0 per cent and
oseltamivir, C16H28N204.
Identification
Tests
Heavy metals (2.3.13). 1 g complies with the limit test for heavy
metals, Method A (20 ppm).
Apparatus No.1,
Speed and time. 50 rpm and 45 minutes
on 1 g by drying in an oven at 105.
Withdraw a suitable volume ofthe medium and filter. Determine

Test solution. Use the filtrate.

examination in 50.0 ml ofthe mobile phase. Dilute 10.0 ml of
Reference solution. Dissolve 50 mg of oseltamivirphosphate

RS in 20 ml ofthe mobile phase and dilute to 100 ml with the
dissolution medium. Dilute 5 ml ofthe solution to 25 ml with
the dissolution medium.
Reference solution. A 0.02 per cent w/v solution of oseltamivir

phosphate RS in the mobile phase.
1828
IP 2010
OSELTAMIVIR ORAL SUSPENSION

C16H2sN204'

potassium phosphate in 1000 ml of water and adjusting
the pH to 6.0 with dilute sodium hydroxide, 24.5 volumes

(2.4.14).

Test solution. Weigh accurately a quantity of the mixed
contents of20 capsules containing about 50 mg ofoseltamivir,
disperse in 50 ml ofmobile phase and filter.
Reference solution (b). Dilute 1 ml of reference solution to
octylsilane bonded to porous silica (5 /-lm) (such as YMC
pack PRO C8),
potassium phosphate in 1000 ml of water, adjusting the
pH to 6.0 with dilute sodium hydroxide, 24.5 volumes
injection volume. 20 /-ll.
chromatogram obtained with the reference solution (b) (1.0
Calculate the content ofC16H2sN204.
exceeding 30.
equivalent amount of oseltamivir.
Oseltamivir Oral Suspension

Oseltamivir Phosphate Oral Suspension
Oseltamivir Oral Suspension is a mixture consisting of
Oseltamivir phosphate with buffering agents and other
excipients. It contains a suitable flavouring agent. It is filled in
a sealed container.
The suspension is constituted by dispersing the contents of

the sealed container in the specified volume of water just
before issue.
Oseltamivir Oral Suspension contains not less than 90.0 per
cent and not more than 110.0 percent ofthe stated amount of
oseltamivir C16H2sN204.
The contents of the sealed container comply with the

following requirement.

Assay. Determine by liquid chromatography (2.4.14)..
Usual strength. 15 mg per mI.


contents of20 capsules containing about 200 mg ofoseltamivir,
disperse in 100.0 mI ofthe mobile phase and filter. Dilute 5.0 ml
ofthe solution to 50.0 ml with the mobile phase.
Reference solution. A 0.02 per cent w/v solution of oseltamivir
phosphate RS in the mobile phase.
The constituted suspension complies with the requirements

stated under Oral liquids and with the following
requirements.
Identification
B. To 2 ml of the reconstituted suspension, add 2 ml of dilute
nitric acid and 4ml ofa 10 per cent w/v solution of ammonium
1829
IP 2010
OSELTAMIVIR ORAL SUSPENSION
molybdate and wann the solution. A bright yellow precipitate

isfonned.
Tests
pH (2.4.24). 3.0 to 5.0.
(2.4.14).

Test solution. Weigh accurately a quantity of the suspension
containing 25 mg ofoseltamivir, dissolve in 25 ml ofthe mobile
phase and filter.
octylsilane bonded to porous silica (5 Ilm),
- mobile phase: 70 volumes of a buffer solution prepared
by dissolving 6.8 g ofpotassium dihydrogen phosphate
in 1000 ml of water and adjusting the pH to 6.0 with
dilute sodium hydroxide, 15 volumes of methanol and
15 volumes of acetonitrile,
octy1silane bonded to porous silica (5 Ilm),
phosphate in 1000 m1 of water and adjusting the pH to
6.0 with dilute sodium hydroxide, 24.5 volumes of
methanol and 23.5 volumes of acetonitrile,
Calculate the content of CI6HzsNz04'
exceeding 30.
Labelling. The label states (1) the quantity ofactive ingredient
in tenns of the equivalent amount of oseltamivir; (b) the
temperature of storage and the period during which the
constituted suspension may be expected to be satisfactory
for use.
Oxazepam
is not more than 3 times the area of the peak in the
chromatogram obtained with 'the reference solution (b)
(3.0 per cent).
CI
Mol. Wt. 286.7

Oxazepam is 7-chloro-3-hydroxy-5-phenyl-1,3-dihydro-2H1,4-benzodiazepin-2-one.
Assay. Detennine by liquid chromatography{2.4. 14).
Oxazepam contains not less than 98.5 per cent and not more
than 101.0 per cent ofClsHIICINzOz, calculated on the dried
basis.
Category. Anxiolytic.
Test solution. Weigh accurately a quantity of the suspension

containing 60 mg of oseltamivir, dissolve in 250.0 ml of the
mobile phase and filter. Dilute 10.0 ml ofthe solution to 25.0 ml
with the mobile phase and filter.
Dose. 15 to 30 mg, 2 to 3 times daily.


Band D may be omitted if tests A and C are carried out.
Other tests. Complies with the tests stated under Oral liquids.
Description. A white or alrnostwhite, crystalline powder.
Identification
1830
IP 2010
OXAZEPAM TABLETS

Compare the spectrum with that obtained with oxazepam RS.
B. Prepare the solutions immediately before use, protected
./iom light.
Dissolve about 20 mg in sufficient ethanol (95 per cent) to

produce 100 ml. Dilute 10 ml of the solution to 50 ml with
ethanol (95 per cent) (solution A). Dilute 10 ml of solution A
to 100 ml with ethanol (95 per cent) (solution B).
When examined in the range 230 nm to 360 urn (2.4.7), solution
A shows an absorption maxjmum at about 316 nm. When
examined in the range 220 urn to 250 urn, solution B shows an
absorption maximum at about 229 nm; absorbance at about
229 nm, 1.220 to 1.300.
that in the chromatogram obtained with reference solution (b).
D. Dissolve about 20 mg in a mixture of5 ml of hydrochloric
acid and 10 ml of wate/: Heat to boiling for 5 minutes and cool.
Add 2 ml of a 0.1 per cent w/v solution of sodium nitrite and
allow to stand for 1 minute. Add 1 ml of a 0.5 per cent w/v
solution ofsulphamic acid, mix and allow to stand for 1minute.
Add 1 ml of 0.1 per cent w/v solution of a N- (i-naphthyl)
ethylenediamine dihydrochloride; a red colour is produced.
Tests
Related substances. Carry out the test protectedfrom light.
Wash the plate with methanol before use.
Test solution (a). Dissolve 50 mg of the substance under
examination in sufficient acetone to produce 10m!.
Test solution (b). Dilute 2 ml oftest solution (a) to 10 ml with
acetone.

solution (a) is not more intense than the spot in thechromatogram obtained with reference solution (c) and at most
one such spot is more intense than the spot in the
chromatogram obtained with reference solution (d). The test
is not valid unless the chromatogram obtained with reference
solution (b) shows two clearly separated spots.
on 1.0 g by drying in an oven at 105at a pressure not exceeding
0.7kPa.
of 10 m1 of anhydrous glacial acetic acid and 90 ml of acetic
anhydride and titrate with 0.1 M perchloric acid detennining
the end point potentiometrically (2.4.25). Carry out a blank
titration.
1 ml of 0.1 M perchloric acid is eqUivalent to 0.02867 g of
ClsHIICIN202.
Oxazepam Tablets
Oxazepam Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated. amount of oxazepam,
ClsH11ClN202.
Identification
A. Extract a quantity of the powdered tablets containing
20 mg ofOxazepam with 25 ml of chloroform, filter, evaporate
to dryness and dry the residue at 60 at a pressure not exceeding
0.7kPa.
On the residue, determine by infrared absorption
obtained with oxazepam RS or with the reference spectnnn of
oxazepam.
Reference solution (a). Dissolve 10 mg of oxazepam RS in

sufficient acetone to produce 10 ml.
B. When examined in the range 210 nm to 360 urn (2.4.7), the
Reference solution (b). Dissolve 10 mg each of oxazepam RS

and bromazepam RS in sufficient acetone to produce
solution obtained in the Assay shows absorption maxima at

about 230 nm and 316 nm.
lOml.
Tests
Reference solution (c). Dilute 1 ml ofreference solution (a) to

100 ml with acetone.
Related substances. CarlY out the test protectedfrom light.
Reference solution (d). Dilute 5 ml ofsolution (d) to 10 m1 with

acetone.
Detennine by thin-layer chromatography (2.4.17) coating the

plate with silica gel GF 254.
.
Apply to the plate 20 !J.I of each solution. Allow the mobile

phase to rise 17 cm in the same direction as the washing with
methanol. Dry in air and examine in ultraviolet light at 254 urn.
Wash the plate with methanol before use.

1831
IP 2010
OXAZEPAM TABLETS
Test solution. Shake a quantity ofpowdered tablets containing

30 mg ofOxazepam with 6 rnl ofacetone and centrifuge.
Reference solution (b). Dilute 1 volume ofthe test solution to
w/v each of oxazepam RS and bromazepam RS.
Apply to the plate 20 fl1 of each of the solutions. Allow the
mobile phase to rise 17 em in the same direction as in the
washing with methanol. Dry in air and examine in ultraviolet
spot in the chromatogram obtained with reference solution (a)
(1 per cent) and not more than one such spot is more intense
than the spot in the chromatogram obtained with reference
solution (b) (0.2 per cent). The test is not valid unless the
chromatogram obtained with reference solution (c) shows two
clearly separated spots.
Otber tests. Comply with the tests stated under Tablets.
quantity of the powder containing about 25 mg of Oxazepam
and shake with 150 ml ofethanol (95 per cent) for 30 minutes.
Add sufficient ethanol (95 per cent) to produce 250.0 rnl and
centrifuge. Dilute 5.0 ml ofthe supernatant liquid to 100.0 ml
with the same solvent and measure the absorbance of the
Calculate the content of ClsHIIClNzOz taking 1250 as the
specific absorbance at 230 TIm.
Storage. Store protected from light at a temperature not
exceeding 30.
Oxcarbazepine
Identification
Compare the spectrum with that obtained with oxcarbazepine
RS or with the reference spectrum of oxcarbazepine.
Tests
(2.4.14).
Note - Use freshly prepared solutions.
oxcarbazepine RS in the mobile phase.
Reference solution (b). Dilute 1.0 ml ofreference solution (a)
a stainless steel column 25 em x 4.6 rom, packed with
octadecylsilane bonded to porous silica (5 flm) (such as
Inertsil C18),
- column temperature 50 0 ,
- mobile phase: a mixture of22 volumes of methanol, 16
volumes of acetonitrile and 62 volumes of a buffer
solution prepared by dissolving 6.8 g of potassium
dihydrogen orthophosphate in 1000 ml ofwater, adding
2 ml of triethylamine and adjusting the pH to 6.0,
- spectrophotometer set at 215 TIm,
chromatograms for about 60 minutes~ The relative retention
times with reference to oxcarbazepine are 1.7 for
dibenz[b,fJazepine-5-carboxylic acid amide (oxcarbazepine
impurity A), 7.9 for 10-methoxy-5H-dibenz[b,fJazepine
(oxcarbazepine impurity B), and 2.5 for 10-methoxy-5Hdibenz[b,fJazepine-5-carboxylic acid amide (oxcarbazepine
impurity C ).
ClsHIZNzOz,
Mol. Wt. 252.3
Oxcarbazepine is 10,11-dihydro-1O-oxo-5H-dibenz[b,jJazepine5-carboxarnide.
Oxcarbazepine contains not less than 98.0 per cent and not
more than 102.0 per cent ofClsHlzNzOz, calculated on the dried
basis.
Category. Non-opioid analgesics.
Dose. 300 mg twice a day.
Description. An off-white to yellow crystalline powder.
Calculate the contents of the impurities in the test solution

using the correction factors, 0.68 for oxcarbazepine impurity
A, 0.76 for oxcarbazepine impurity Band 0.80 for oxcarbazepine
impurity C. Not more than 1.0 per cent of oxcarbazepine
impurity A and not more than 0.1 per cent each of
oxcatbazepine impurity Band oxcarbazepine impurity C and
not more than 0.1 per cent ofany other impurity is found. The
sum of all the impurities is not more than 1.5 per cent.
1832
IP 2010
OXCARBAZEPINE TABLETS

Assay. Determine by liquid chromatography (2.4.14) as
described under related substances using the following
solution.

oxcarbazepine RS in the mobile phase.

ClsH12N202.
(2.4.14).
Note - Useji-eshly prepared solutions.

Test solution (a). Weigh accurately a quantity ofthe powdered
tablets containing 50 mg of Oxcarbazepine add 15 ml methanol,
mix with the aid of ultrasound for 15 minutes and dilute to
100.0 ml with methanol. Mix and centrifuge.
Test solution (b). Dilute 5.0 ml of test solution (a) to 25.0 ml
Reference solution (a). Dissolve 25.0 mg of oxcarbazepine
RS in methanol with the aid ofultrasound and dilute to 50.0 ml
with methanol.
Calculate the content ofC,sH,2N202'

Reference solution (c). Dilute 1.0 ml of reference solution (a)

Oxcarbazepine Tablets contain not less than 90.0 per cent and
oxcarbazepine, ClsH12N202.
Identification
obtained with the test solution (b) corresponds to the peak in
B. Extract a quantity ofthe powdered tablets containing about
50 mg ofoxcarbazepine with 100 ml with methanol and filter.
Diluted 2.0 ml ofthis solution to 100.0 ml with methanol. When
examined in the range 200 urn to 350 nm (2.4.7), the solution
shows an absorption maximum at about 254 nm.
Tests
Apparatus No. 1
Medium. 900 ml of 0.75 per cent w/v solution ofsodium lauryl
sulphate in water,
the medium if necessary, at the maximum at about 305 urn
(2.4.7). Calculate the content of ClsH12N202 in the medium
concentration of oxcarbazepine RS prepared by dissolving
in minimum volume of methanol and then diluting with the
dissolution medium.
octadecylsilane bonded to porous silica (5 Ilm) (Such
as Hypersil BDS CI8),
column temperature 500 ,
mobile phase: a mixture of 66 volumes of O.05M
potassium dihydrogen phosphate, 14 volumes of
acetonitrile and 20 volumes of methanol, add 0.1 mlof
triethylamine and adjust the pH to 6.0 with.
- spectrophotometer set at 215 hm,
tailing factor is not more than 2.0 and relative standard deviation
Inject test solution (a) and reference solution (c). Run the
chromatograms for three times the retention time of the
principal peak. In the chromatogram obtained with the test
solution, the area of any secondary peak is not more than the
area ofthe peak in the chromatogram obtained with reference
solution (c) (1.0 per cent) and the sum of areas of all the
secondary peaks is not more than twice the area ofthe peak in
the chromatogram obtained with reference solution (c) (2.0
per cent).
described under Related substances.
Inject test solution (b) and reference solution (b).
1833
IP 2010
OXPRENOLOL HYDROCHLORIDE
Calculate the contentofCIsHI2N202 in the tablets.

exceeding 30.

o:
~
OH

substance under examination in a mixture of 90 volumes of
chloroform and 10 volumes of methanol.
O~NyCH3
O~CH2 CH 3
C 1sH23N03,HCl

examination in 10 rnl ofa mixture of90 volumes of chloroform

,HCI
Mol. Wt. 301.8
Oxprenolol Hydrochloride is (RS)-I-(2-allyloxyphenoxy)-3isopropylaminopropan-2-01 hydrochloride.

Oxprenolol Hydrochloride contains not less than 98.5 per cent
and not more than 101.5 per cent ofClsH23N03,HCl, calculated
on the dried basis.
Category. Beta-adrenoceptor antagonist (antihypertensive;
antianginal; antiarrhythmic).
Dose. As antihypertensive, initially 80 mg twice daily, increased
as required at weekly intervals; maximum 480 mg daily. As
antianginal, 40 to 160 mg thrice daily. As antiarrhythmic, initially
20 to 40 mg thrice daily, increased as necessary.

oxprenolol hydrochloride RS in a mixture of 90 volumes of
Apply to the plate 2 f.l.1 of each solution. Allow the mobile
phase to rise 13 em. Dry the plate in warm air for 10 minutes,
allow to cool, spray with anisaldehyde solution, heat at 105
for 10 minutes and examine in daylight. Any secondary spot
in the chromatogram obtained with test solution (a) is not
with reference solution (a) and not more than one such spot is
Description. A white Of almost white, crystalline powder.
Heavy metals (2.3.13). 1.0 g complies with the iimit test for
Identification

at a pressure of 1.5 to 2.5 kPa for 6 hours.

Compare the spectrum with that obtained with oxprenolol RS
or with the reference spectrum of oxprenolol.


solution. Titrate with 0.1 Mperchloric acid, determining the
end-point potentiometrically (2.4.25).Carry out a blank
titration.
C 1SH23N03,HCl.
Tests
Appearance ofsolution. A 10.0 per cent w/v solution in carbon

dioxide-free water is clear (2.4.1), and not more intensely
coloured than reference solution GYS6 (2.4.1).
pH (2.4.24). 4.5 to 6.0, determined in a freshly prepared
10.0 per cent wIv solution.

12 volumes of methanol and 2 volumes of strong ammonia
solution.
Oxprenolol Tablets
Oxprenolol Hydrochloride Tablets
Oxprenolol Hydrochloride Tablets contain not less than
amount of oxprenolol hydrochloride, CISH23N03,HCl. The
tablets are coated.
1834
IP 2010
OXYGEN 93 PER CENT
Oxygen
Identification
A.To a quantity of the powdered tablets containing 50 mg of
Oxprenolol Hydrochloride add 10 ml of water and 2 ml of
dilute sodium hydroxide solution, extract with 10 ml of
chloroform and reserve the aqueous layer for test C. Dry the
chloroform extract over anhydrous sodium sulphate, filter and
evaporate the filtrate to dryness.
obtained with oxprenolol hydrochloride RS or with the
reference spectrum of oxprenolol hydrochloride.

only at about 273 nm.
C. The aqueous layer obtained in test A gives reaction A of
chlorides (2.3.1).
Mol. Wt. 32.0

Oxygen contains not less than 99.0 per cent vlv of Oz.
This monograph applies to oxygen for medicinal use only.
Identification
with the gas.
B. Shake with alkaline pyrogallol solution; the gas under
examination is absorbed and the solution becoming dark
brown.
C. When tested as described under Assay, not more than
1.0 ml ofgas remains.
Tests
D. The residue obtained in test A melts at about 76 (2.4.21).

Tests
Carbon dioxide. Not more than 300 ppm vlv, determined by

using a carbon dioxide detector tube (2.1.1).

Carbon monoxide. Not more than 5 ppm vlv, determined by

using a carbon monoxide detector tube (2.1.1).

solution.
Water vapour. Not more than 67 ppm vlv, determined by using

a water vapour detector tube (2.1.1).

containing 0.25 g of Oxpreno101 Hydrochloride with 5 ml of
water, centrifuge and use the supernatant liquid.
phase to rise 13 cm. Dry the plate in warm air for 10 minutes,
allow to cool, spray with anisaldehyde solution, heat at 105
for 10 minutes and examine in daylight. Any secondary spot
in the chromatogram obtained with the test solution is not
.
quantity ofthe powder containing about 20 mg ofOxprenolol
Hydrochloride, add 25 ml of 0.1 M hydrochloric acid and
sufficient water to produce 250.0 m!. Mix with the aid of
ultrasound for 5 minutes, shake for 15 minutes and filter.
C,sHz3N03,HCl taking 74.5 as the specific absorbance at
273nm.
Assay (2.3.33). Use 100 ml ofthe gas under examination and

place spirals of freshly cleaned copper wire and 125 ml of
ammonia buffer pH 10.9 in the pipette. The volume of the
residual gas in the burette is not more than 1.0 m!.
Storage. Store under pressure in metal cylinders of the type
conforming to the appropriate safety regulations. Valves and
taps should not be lubricated with oil or grease.
Labelling. The shoulder of the metal cylinder should be
painted white and the remainder should be painted black. The
cylinder should carry a label stating "Oxygen". In addition,
"Oxygen" or the symbol "Oz" should be stencilled in paint on
the shoulder of the cylinder.
Oxygen 93 Per Cent

Oxygen 93 per Cent contains not less than 90.0 per cent and
not more than 96.0 per cent, vlv ofOz, the remainder consisting
mostly of argon and nitrogen. It is produced from air by the
molecular sieve process.
Identification
with the gas.
1835
OXYGEN 93 PER CENT
IP 2010
B. Shake with alkaline pyrogallol solution; the gas under

examination is absorbed and the solution becomes dark brown.
C. When tested as described under Assay, not more than
10.0 ml and not less than 4.0 ml ofgas remain.
Tests
Carbon dioxide. Not more than 300 ppm v/v, determined by
using a carbon dioxide detector tube (2.1.1).
Oxytetracycline has a potency not less than 900 flg of
CzzHz4Nz09, per mg, calculated on the anhydrous basis.

Dose. Orally, 250 to 500 mg every 6 hours; by intramuscular
injection, 1 to 2 g daily.
Description. A tan yellow or light yellow (with or without a
greenish tinge), crystalline powder; odourless.
Carbon monoxide. Not more than 5 ppm v/v, determined by

using a carbon monoxide detector tube (2.1.1).
Identification
Assay (2.3.33). Use 100 ml of the gas under examination and

place spirals of freshly cleaned copper wire and 125 ml of
ammonia buffer pH 10.9 in the pipette. The volume of the
residual gas in the burette is not more than 10.0 ml and not
less than 4.0 ml.

the plate with the substance prepared by mixing 25 g of silica
gel Gwith 50 ml ofa mixture of2.5 ml ofglycerin and 47.5 ml of
0.1 M disodium edetate previously adjusted to pH 7.0 with
dilute ammonia solution. After spreading the plate, allow it to
stand at room temperature till it is dry (70 to 90 minutes).
Storage. Store in cylinders or in a low pressure collecting

tank. Containers used for Oxygen 93 Per Cent must not be
treated with any toxic, sleep-inducing or narcosis-producing
compounds and must not be treated with any compound that
will be irritating to the respiratory tract when the Oxygen
93 Per Cent is used.
Mobile phase. The lower layer formed after shaking 200 ml of

a mixture of 2 volumes of ethyl acetate, 2 volumes of
chloroform and 1 volume of acetone with 25 ml of 0.1 M
disodium edetate previously adjusted to pH 7.0 with dilute
ammonia solution.
Labelling. Label each outlet "Oxygen 93 Per Cent", when it is

piped directly from the collecting tanle to the point ofuse. Ifit
is stored in cylinders, reduce the pressure by means ,of a
regulator. Measure the gases with a gas volume meter
downstream from the detector tube in order to minimise
contamination or change of the specimens. The shOlilder of
the cylinder should be painted white and the remainder should
be painted black. The cylinder should carry a label stating
"Oxygen 93 Per Cent" and "For medicinal use".
Oxytetracycline
0
o
NH z
C:1zHz<tNZC?9,2HzO

oxytetracycline RS in methanol.
w/v each of demethylchlortetracycline hydrochloride RS,
oxytetracycline hydrochloride RS and tetracycline
Apply to the plate 1 III of each solution, freshly prepared.
After development, dry the plate in air, expose to the vapours
of ammonia and examine in ultraviolet light at 365 urn. The
with reference solution (a). The test is not valid unless the
chromatogram obtained with reference solution (b) shows
three clearly separated spots.
OH

,2H zG
B. To about 0.5 mg add 2 ml of sulphuric acid; a red colour is

produced. Add the solution to 1 ml of water; the colour
changes to yellow.
Mol. Wt. 496.5
Oxytetracycline is (4S,4aR,5S,5aR,6S, 12aS)-4-dimethyl

amino-1 ,4,4a,5,5a,6,11,12a-octahydro-3,5,6,10, 12,12ahexahydroxy-6-methyl-1,II-dioxonaphthacene-2carboxamide dihydrate, a substance produced by the
growth of certain strains of Streptomyces rimosus or obtained
by any other means. It contains a variable quantity of water.
C. Dissolve about 2 mg in 5 ml of a 1 per cent w/v solution of

sodium carbonate and add 2 ml of diazotised sulphanilic
acid solution; an orange-red to brownish-red colour is
produced.
Tests
suspension in freshly boiled and cooled water.
1836
IP 2010
OXYTETRACYCLINE DIHYDRATE
Specific optical rotation (204.22). -203 to -216, detennined

at 20 in a 1.0 per cent w/v solution in 0.1 M hydrochloric
acid, after allowing the solution to stand protected from light
for 30 minutes before measurement.
Light absorption. Absorbance ofa 0.002 per cent w/v solution
in buffersolution pH2.0 at the maximum at about 353 run, 0.58
to 0.62 (204.7).
Light-absorbing impurities. A. Dissolve 20 mg in sufficient
of a mixture of I volume of 1 M hydrochloric acid and
99 volumes of methanol to produce 10 ml. Absorbance ofthe
resulting solution at about 430 nm, when measured within
1 hour ofpreparing the solution, not more than 0.25, calculated
on the anhydrous basis (204.7).
B. Dissolve 0.1 g in sufficient ofa mixture of 1 volume of 1 M

hydrochloric acid and 99 volumes of methanol to produce
10 ml. Absorbance ofthe resulting solution at about 490 nm,
when measured within 1 hour of preparing the solution, not
more than 0.20, calculated on the anhydrous basis (204.7).
(204.14).

examination in 25 ml of O. DIMhydrochloric acid.
Reference solution (a). Dissolve 20 mg of oxytetracycline RS
in 25 ml of O. DIM hydrochloric acid.
Reference solution (b). Dissolve 20 mg of 4-epioxytetracycline RS in 25 ml of 0.01 M hydrochloric acid.
Reference solution (c). Dissolve 20 mg of tetracycline
hydrochloride RS in 25 ml of 0.01 M hydrochloric acid.
Reference solution (d). Dilute a mixture of 1.5 ml ofreference
solution (a), 1.0 ml of reference solution (b) and 3.0 ml of
reference solution (c) to 25 ml with 0.01 M hydrochloric
acid.
Reference solution (e). Dilute a mixture of 1.0 ml ofreference
solution (b) and 4.0 ml.ofreference solution (c) to 200 rn1 with
spectrophotometer set at 254 run,

injection volume. 20 /ll.
Adjust the sensitivity so that the heights of the peaks in the
chromatogram obtained with reference solution (d) are at least
50 per cent of the full-scale deflection of the recorder.
The test is not valid unless (a) the resolution between the first
peak (4-epioxytetracycline) and the second peak
(oxytetracycline) is at least 4.0 (b) the resolution between the
second peak and the third peak (tetracycline) is at least 5.0
(the content of 2-methyl-2-propanol in the mobile phase may
be adjusted if necessary) and (c) the symmetry factor for the
second peak is at most 1.25.
Inject reference solution (a) six times. The test is not valid
unless the relative standard deviation of the area of the peak
due to oxytetracycline is not greater than 1.0 per cent.
Inject the test solution and reference solution (e). In the
chromatogram obtained with the test solution the area of any
peak corresponding to 4-epioxytetracycline or tetracycline is
not greater than the area of the corresponding peak in the
chromatogram obtained with reference solution (e). In the
peak appearing on the tail of the principal peak is not greater
than 4.0 times that ofthe peak due to 4-epioxytetracycline in
the chromatogram obtained with reference solution (e).
Heavy metals (2.3.13).004 g complies with limit the test for
Water (2.3043). 4.0 to 9.0 per cent, detennined on 0.5 g.
Assay. Detennine by the microbiological assay ofantibiotics,
Method A or B (2.2.10), and express the results in /lg of
oxytetracycline, CzzHz4Nz09' per mg.
Oxytetracycline intended for use in the manufacture of

parenteral preparations without a fUrther procedure for the
removal ofbacterial endotoxins complies with the jrjl!owing
additional requirement.
Bacterial endotoxins (2.2.3). Not more than 004 Endotoxin Unit

.
per
mg ofOxytetracycline.
styrene- divinylbenzene co-polymer (8 to 10 /lm),
Oxytetracycline intended for use in the manufacture of
parenteral preparations without a further sterilisation
- 'mobile phase: add 60 g of 2-methyl-2-propanol to a procedure complies with the following additional
volumetric flask with the aid of 200 ml of water, add requirement.
60 ml of 0.33 M phosphate buffer pH 7.5, 50 ml of a
1.0 per cent w/v solution of tetrabutylammonium
hydrogen sulphate previously adjusted to pH 7.5 with Storage. Store protected from light and moisture. If it is
2 M sodium hydroxide and 10 ml of a 0.04 per cent w/v intended for use in the manufacture ofparenteral preparations,
solution of disodium edetate previously adjusted to the container should be sterile and sealed so as to exclude
pH 7.5 with 2 M sodium hydroxide and dilute to micro-organisms.
1000.0 ml with water,
.Labelling. The label states whether or not the contents are
- flowrate. 1 ml per minute,
1837
IP 2010
OXYTETRACYCLINE INJECTION

per mg ofOxytetracycline.
Oxytetracycline Injection is a sterile solution ofoxytetracycline
with or without one or more suitable buffering agents,
anaesthetics, preservatives, antioxidants, complexing agents
and solvents.
Oxytetracycline Injection contains not less than 90.0 per cent
anhydrous oxytetracycline, CzzHz4Nz09'
Usual strengths. 50 mg per ml; 125 mg per ml.
Description. A clear, yellow to tan yellow solution. It may
have a greenish tinge.

Method A or B (2.2.10), and express the result in mg of
oxytetracycline, CzzHz4Nz09' per ml.
Labelling. The label states (1) the strength in mg ofanhydrous
oxytetra,cycline per ml; (2) that the contents are to be used for
intramuscular use only; (3) the names of any preservatives
used.
Identification
A. Determine by thin-layer chromatography (204.17), coating
gel G with 50 ml ofa mixture of2.5 ml ofglycerin and 47.5 ml of
Mobile phase. The lower layer fonned after shaking 200 ml of

ammonia solution.
Test solution. Shake a quantity containing 10 mg ofanhydrous
oxytetracycline with 20 ml of methanol, centrifuge if necessary
and use the clear supernatant liquid.
oxytetracycline hydrochloride RS in methanol.
Apply to the plate 1 fll of each solution, freshly prepared.
of ammonia and examine in ultraviolet light at 365 nm. The
B. Add 0.1 ml to 2 ml ofsulphuric acid; a red colour is produced.
Add the solution to 1 ml of water; the colour changes to
yellow.
Tests
pH (204.24). 8.0 to 9.0.
Mol. Wt. 496.9
Oxytetracycline Hydrochloride is (4S,4aR,5S,5aR,6S, l2a8)4-dimethylaIDino-l,4,4a,5,5a,6,11,12a-octahydro-3,5,
6,10,12, 12a-hexahydroxy-6-methyl-l ,11-dioxonaphthacene-2carboxamide hydrochloride, a substance produced by
the growth of certain strains of Streptomyces rimosus or
obtainedby any other means.
Dose. Orally, 250 to 500 mg every 6 hours; by intravenous
infusion, in a concentration of 0.1 per cent w/v, 1 to 2 g daily.
Description. A pale yellow, crystalline powder; odour1ess;
hygroscopic.
Oxytetracycline Hydrochloride has a potency not less than

835 flg of CzzHz4Nz09, per mg, calculated on the anhydrous
basis.
Identification
gel G with 50 ml ofa mixture of2.5 mlofglycerin and 47.5 ml of
Mobile phase. The lower layer formed after shaking 200 m1 of
chloroform and 1 volume of acetone with 25 m1 of 0.1 M
ammonia solution.
1838
IP 2010
OXYTETRACYCLINE HYDROCHLORlDE

Apply to the plate 1 J!l of each solution, freshly prepared.

produced. Add the solution to 1 ml of water; the colour
changes to yellow.
C. Dissolve about 2 mg in 5 ml of a 1 per cent w/v solution of

produced.
D. A 5 per cent w/v solution gives the reactions of chlorides
(2.3.1).
Tests
Specific optical rotation (2.4.22). -188 to -200, determined
at 20 in a 1 per cent w/v solution in 0.1 M hydrochloric acid.
Light absorption (2.4.7). Absorbance ofa 0.002 per cent w/v
solution in chloride buffer solution pH 2.0 at the maximum at
about 353 nm, 0.54 to 0.58.
of a mixture of 1 volume of 1 M hydrochloric acid and
resulting solution at about 430 urn, when measured within
1 hour ofpreparing the solution, not more than 0.50, calculated
on the anhydrous basis (2.4.7).
B. Dissolve 0.1 g in sufficient ofa mixture of 1 volume of 1 M

10 ml. Absorbance of the resulting solution at about 490 nm,
more than 0.20, calculated on the anhydrous basis (2.4.7).
(2.4.14).

examination in 25 ml of O. 01 M hydrochloric acid.
Reference solution (a). Dissolve 20 mg of oxytetracycline RS
in 25 ml of O. 01 M hydrochloric acid.
Reference solution (b). Dissolve 20 mg of 4-epioxytetracycline RS in 25 ml of 0.01 M hydrochloric acid.
Reference solution (c). Dissolve 20 mg of tetracycline

hydrochloride RS in 25 ml of 0.01 M hydrochloric acid.
Reference solution (d). Dissolve 20 mg of a..-apooxytetracycline RS in 20 ml of 0.01 M sodium hydroxide and
dilute to 250 ml with 0.01 M hydrochloric acid.
Reference solution (e). Dissolve 20 mg of ~ -apooxytetracycline RS in 20 ml of 0.01 M sodium hydroxide and
dilute to 250 ml with 0.01 M hydrochloric acid.
Reference solution (f), Dilute a mixture of 1.5 ml ofreference
solution (a), 1.0 ml of reference solution (b), 3.0 ml each of
reference solutions (c) (d) and (e) to 25 ml with 0.01 M
hydrochloric acid.
Reference solution (g). Dilute a mixture of 1.0 ml ofreference
solution (b), 4.0 ml of reference solution (c) and 40.0 ml of
reference solution (e) to 200 ml with 0.01 M hydrochloric
acid.
styrene-divinylbenzene co-polymer (8 to 10 J!m),
- mobile phase: transfer separately 30 g (for mobile phase
A) orl 00 g (for mobile phase B) of 2-methyl-2-propanol
to volumetric flasks with the aid of200 ml of water; to
each flask add 60 ml of 0.33 M phosphate buffei'pH 7.5,
50 ml of a 1.0 per cent w/v solution of tetrabutylammonium hydrogen sulphate previously adjusted to
pH 7.5 with 2 M sodium hydroxide and 10 ml of a 0.04
per cent w/v solution of disodium edetate previously
adjusted to pH 7.5 with 2 M sodium hydroxide and
dilute each solution to 1000.0 ml with water.,
- injection volume. 20 J!l.
Carry out a one-step gradient elution in the following manner.
Pump a mixture containing 30 volumes ofmobile phase Band
70 volumes of mobile phase A for 15 minutes, then pump a
mixture containing 30 volumes of mobile phase A and
70 volumes of mobile phase B for 15 minutes and finally
equilibrate with the first mixture. Adjust the sensitivity so that
the heights of the peaks in the chromatogram obtained with
reference solution (f) are at least 50 per cent of full-scale
deflection ofthe recorder.
The test is not valid unless, in the chromi;ltogram obtained
with reference solution (f), (a) the resolution between the first
peak (4-epioxytetracycline) and the second peak
(oxytetracycline) is at least 4.0, (b) the resolution between the
second peak and the third peak (tetracycline) is at least 5.0, (c)
the resolution between the fourth peak (a..-apooxytetracycline) and the fifth peak (~-apo-oxytetracycline)
is at least 3.5, and (d) the symmetry factor ofthe second peak
1839
OXYTETRACYCLINE HYDROCHLORIDE
IP 2010
is at most 1.25. If necessary adjust the proportions of the

mobile phases used to produce the one-step gradient elution.
Adjust the time-programme for the one-step gradient elution
if necessary.
Inject reference solution (a) six times. The test is not valid if
the relative standard deviation of the area of the peak due to
oxytetracycline is greater than 1.0 per cent. Ifnecessary, adjust
the integrator parameters.
Inject the test solution and reference solution (g). In the
peak corresponding to 4-epioxytetracycline or tetracycline
is not greater than the area of the corresponding peak in the
chromatogram obtained with reference solution (g) and the
total area ofthe peaks corresponding to a-apo-oxytetracycline
and to ~ -apo-oxytetracycline and any peak between the latter
two is not greater than the area of the peak due to ~ -apooxytetracycline in the chromatogram obtained with reference
solution (g). In the chromatogram obtained with the test
solution the area of any peak appearing on the tail of the
principal peak is not greater than 4.0 times that ofthe peak due
to 4-epioxytetracycline in the chromatogram obtained with
reference solution (g).
Heavy metals (2.3.13).004 g complies with the limit test for
Oxytetracycline Hydrochloride Capsules
Oxytetracycline Capsules contain not less than 95.0 per cent
oxytetracycline hydrochloride, CzzHz4Nz09,HCl.
Identification
the plate with the substance prepared by mixing 25 g of
silica gel G with 50 ml of a mixture of 2.5 ml of glycerin and
47.5 ml of 0.1 M disodium edetate previously adjusted to pH
7.0 with dilute ammonia solution. After spreading the plate,
allow it to stand at room temperature till it is dry (70 to 90
minutes).

ammonia solution.
Test solution. Extract a quantity ofthe contents ofthe capsules

containing 10 mg ofOxytetracycline Hydrochloride with 20 ml
methanol, centrifuge and use the supernatant liquid.
Water (2.3043). Not more than 2.0 per cent w/w, determined on
0.5 g.


Method A or B (2.2.10), and express the result in Ilg of
oxytetracycline, CzzHz4Nz09, per mg.

Oxytetracycline Hydrochloride intended for use in the

manufacture ofparenteral preparations without a fitrther
procedure for the removal ofbacterial endotoxins complies
with the following additional requirement.
per mg.
Oxytetracycline Hydrochloride intended for use in the

.manufacture ofparenteral preparations without a jitrther
sterilisation procedure complies with the following
Apply to the plate 1 III of each solution, freshly prepared.

Sterility. Complies with test for sterility (2.2.11).
B. To 0.5 mg of the contents of the capsules add 2 ml of

sulphuric acid; a red colour is produced. Add the solution to
1 ml of water; the colour changes to yellow.
Storage. Store protected from light and moisture. If it is

intended for use in the manufacture ofparenteral preparations,
the container should be sterile and sealed so as to exclude
micro-organisms.
C. Dissolve about 2 mg ofthe contents ofthe capsules in 5 ml

ofa 1 per cent w/v solution of sodium carbonate and add 2 ml
of diazotised sulphanilic acid solution; a light brown colour
is produced.

D. The contents ofthe capsules give the reactions ofchlorides

(2.3.1).
1840
IP 2010
OXYTETRACYLINE EYE OINTMENT
Tests
Identification
Light-absorbing impurities. Dissolve a portion ofthe mixed

contents of five capsules as completely as possible in
sufficient of a mixture of 1 volume of 1 M hydrochloric acid
and 99 volumes of methanol to produce two solutions of
Oxytetracycline Hydrochloride containing (1) 0.2 per cent w/v
and (2) 1.0 per cent w/v and filter each solution. Absorbance
ofthe filtrate obtained from solution (1) at about 430 om, when
measured within 1 hour of preparing the solution, not greater
than 0.75 and ofthe filtrate obtained from solution (2) at about
490 om, not more than 0.40 (2.4.7).

plate with the substance prepared by mixing 25 g of silica gel
G with 50 ml of a mixttLre of2.5 ml ofglycerin and 47.5 ml of
Apparatus No.1,
Withdraw a suitable volume ofthe medium and filter through
than 1.0 j.Lm. Measure the absorbance of the filtrate, suitably
diluted ifnecessary, at the maximum at about 353 nm (2.4.7).
Calculate the content ofCzzH24N209,HCI in the medium taldng
282 as the specific absorbance at 353 om.
CnH2~209,HCl.
Loss on drying (2.4.19). Not more than 5.0 percent, determined

on 1.0 g ofthe mixed contents ofthe capsules by drying in an
oven at 60 at a pressure not exceeding 0.7 kPa for 3 hours.
Assay. Weigh accurately a quantity of the mixed contents of
20 capsules containing about 0.25 g of Oxytetracycline
Hydrochloride, add 250.0 ml of water, shake, filter.
A or B (2.2.10), and express the results in mg ofoxytetracycline
hydrochloride per capsule taking each mg of oxytetracycline
to be equivalent to 1.079 mg ofoxytetracycline hydrochloride,
CnH2~209,HCl.
Mobile phase. The lower layer formed after shaldng 200 ml of

ammonia solution.
Test solution. A solution prepared by heating a quantity
containing 20 mg ofOxytetracycline Hydrochloride with 20 ml
of methanol for 20 minutes, cooling in ice, filtering, carefully
evaporating the filtrate to dryness and dissolving the residue
in 20 ml of methanol.
Apply to the plate 1 j.LI of each solution, freshly prepared.
Tests
Ointments.
Assay. Weigh accurately about 1.0 g and transfer to a
separating funnel. Add 25 ml of peroxide-free ether, shake
well and extract with five quantities, each of20 ml, of 0.1 M
hydrochloric acid. Combine the extracts and dilute to
200.0 ml with 0.1 Mhydrochloric acid. Dilute a suitable volume
of the resulting solution with buffer solution No 3 (2.2.10), to
produce a solution containing 1 j.Lg of oxytetracycline per mI.

Oxytetracycline Eye Ointment contains not less than 90.0 per
oxytetracycline hydrochloride, CZ2H24N209,HCI.

B (2.2.10), and express the results as a percentage of
oxytetracycline hydrochloride taking each mg of
oxytetracycline to be equivalent to 1.079 mg ofoxytetracycline
hydrochloride, C22H24Nz09,HCl.
Usual strength. 1 per cent w/w.
Oxytetracycline Hydrochloride Eye Ointment
1841
OXYTETRACYCLINE HYDROCHLORIDE INJECTION
IP 2010
Injection
Oxytetracycline Hydrochloride Injection is a sterile material
consisting of Oxytetracycline Hydrochloride with or without
buffering agents and other excipients. It is filled in a sealed
container.
The injection is constituted by dissolving the contents ofthe
sealed container in the requisite amount of sterile Water for
Injections, immediately before use.
The constituted solution complies with the requirements for

Clarity of solution and Particulate matter stated under
Parenteral Preparations (Injections).
Storage. The constituted solution may be used within three
days of preparation when stored in a refrigerator (20 to 8).
Oxytetracycline Hydrochloride Injection contains not less than
amount ofoxytetracycline, CzzHz4NzOg.

requirements stated under Parenteral Preparations
(Powdersfor Injection) and with thefollowing requirements.
Usual strengths. The equivalent of 250 mg and 500 mg of
oxytetracycline.
Description. A pale yellow, crystalline powder.
Identification
gel G with 50 ml ofa mixture of2.5 ml ofglycerin and 47.5 m1 of

chloroform and I volume of acetone with 25 ml of 0.1 M
ammonia solution.
examination in 100 m1 of methanol.

produced. Add the solution to I ml of water; the colour
changes to yellow.
C. Dissolve about 2 mg in 5 ml of a I per cent w/v solution of
produced.
Tests
Appearance of solution. A 10.0 per centw/v solution is clear
(2.4.1) and yellow.
pH (2.4.24). 2.0 to 3.0, determined in a 1.0 per cent w/v solution.
of a mixture of I volume of 1 M hydrochloric acid and
resulting solution at about 430 nm, when measured within
I hour ofpreparing the solution, not more than 0.50, calculated
on the anhydrous basis (2.4.7).
B. Dissolve 0.1 g in sufficient ofa mixture of I volume of 1 M
10 ml. Absorbance of the resulting solution at about 490 nm,
more than 0.20, calculated on the anhydrous basis (2.4.7).
method A or B (2.2.10), and express the result in )..I.g of
oxytetracycline, CzzHz4NzOg, per mg.
Bacterial endotoxins (2.2.3). Not more than 0.4 Endotoxin Unit
per mg.
Labelling. The label states (I) the quantity ofOxytetracycline
Hydrochloride contained in it in terms of the equivalent
amount of oxytetracycline; (2) that the contents are to be
used for intravenous injection only; (3) the names of the
buffering agents used.
Oxytocin

Apply to the plate I )..1.1 of each solution, freshly prepared.
Cys-Tyr-lle-Gln-Asn-Cys-Pro-Leu-GlyNH z
I
Mol. Wt.1007.2
Oxytocin is a cyclic nonapeptide hormone obtained by a

process offractionation from the posterior lobe ofthe pituitary
1842
IP 2010
OXYTOCIN
gland of healthy oxen or other mammals or by synthesis that

has the property of stimulating contraction of the uterus and
mille ejection in receptive animals. It may be presented as a
solid or as a solution in a solvent containing an appropriate
antimicrobial preservative such as 0.2 per cent w/v solution of
chlorbutol.
Ifit is derived from animal species, Oxytocin contains not less
than 95.0 percent and not more than 105.0 per cent of the
stated number of Units of oxytocic activity. If it is a synthetic
product presented as a solid, it contains not less than 560
Units per mg, calculated with reference to the peptide content
and when presented as a liquid, it contains not less than 150
Units per ml.
Category. Oxytocic.
Description. When presented as a solid, a white or almost

white powder. When presented as a liquid, a clear colourless
liquid.
Identification
Test B may be omitted if tests A and C are carried out. Test C

B. Amino acid analysis.
For hydrolysis-Acid hydrolysis using hydrochloric acid

containing phenol is the most common procedure used for
protein/peptide hydrolysis preceding amino acid analysis. The
addition of phenol to the reaction prevents the halogenation
of tyrosine.
Hydrolysis solution. 6 M hydrochloric acid containing 0.1
per cent to 1.0 per cent of phenol.
Procedure
Liquidphase hydrolysis. Place the protein or peptide sample
in a hydrolysis tube, and dry (the sample is dried so that water
in the sample will not dilute the acid used for the hydrolysis).
Add 200 fl.l of hydrolysis solution per 500fl.g oflyophilised
protein. Freeze the sample tube in a dry ice-acetone bath, and
flame seal in vacuum. Samples are typically hydrolysed at
110 for 24 hours in vacuum or in an inert atmosphere to
prevent oxidation. Longer hydrolysis times (e.g. 48 hours and
72 hours) are investigated ifthere is a concern that the protein
is not completely hydrolysed.
Vapour phase hydrolysis. This is one of the most common
acid hydrolysis procedures, and it is preferred for microanalysis
when only small amounts of the sample are available.
Contamination of the sample from, the acid reagent is also
minimised by using vapour phase hydrolysis. Place vials
containing the dried samples in a vessel that contains an

appropriate amount of hydrolysis solution. The hydrolysis
solution does not come in contact with the test sample. Apply
an inert atmosphere or vacuum (less than 200 fl.m ofmercury
or 26.7 Pa) to the headspace of the vessel, and heat to about
110 for a 24 hours hydrolysis time. Acid vapour hydrolysis
the dried sample. Any condensation ofthe acid in the sample
vials is to be minimised. After hydrolysis, dry the test sample
in vacuum to remove any residual acid.
For analysis-Past-column ninhydrin derivatisation. Ionexchange chromatography with post-column ninhydrin

derivatisation is one ofthe most common methods employed
for quantitative amino acid analysis. As a rule, a lithium-based
cation-exchange system is employed for the analysis of the
more complex physiological samples, and the faster sodiumbased cation-exchange system is used for the more simplistic
amino acid mixtures obtained with protein hydrolysates
(typically containing 17 amino acid components). Separation
ofthe amino acids on an ion-exchange column is accomplished
through a combination of changes in pH and cation strength.
A temperature gradient is often employed to enhance
separation.
When the amino acid reacts with ninhydrin, the reactant has a
characteristic purple or yellow colour. Amino acids, except
imino acid, give a purple colour, and show an absorption
maximum at 570 nm. The imino acids such as proline give a
yellow colour, and show an absorption maximum at 440 urn.
The post-column reaction between ninhydrin and amino acids
eluted from the column is monitored at 440 nm and 570 urn,
and the chromatogram obtained is used for the determination
of amino acid composition.
The detection limit is considered to be 10 pmol for most ofthe
amino acid derivatives, but 50 pmol for the proline derivative.
Response linearity is obtained in the range of 20-500 pmol
with con-elation coefficients exceeding 0.999. To obtain good
composition data, samples larger than 1 fl.g before hydrolysis
are best suited for this amino acid analysis ofprotein/peptide.
Express the content ofeach amino acid in moles. Calculate the
relative proportions of the amino acids, taking 1/6 of the sum
ofthe number ofmoles ofaspartic acid, glutamic acid, proline,
glycine, isoleucine and leucine as equal to 1.
The values fall within the following limits: aspartic acid: 0.90
to 1.10; glutamic acid: 0.90 to 1.10; proline: 0.90 to 1.10; glycine:
0.90 to I.I 0; leucine: 0.90 to I.I 0; isoleucine: 0.90 to I.I 0;
tyrosine: 0.7 to 1.05; half-cystine: 1.4 to 2.1. Not more than
traces of other amino acids are present.
C. For biological response, dissect out the uterus from a virgin
female rat weighing between 120 to 200 g and in diestrus stage.
Immediately before injection confirm the uterine stage ofthe
rat by vaginal smear. Suspend one hom of the uterus in a
1843
OXYTOXIN
IP 2010
mobile phase: appropriate proportions ofa 1.56 per cent

w/v solution of sodium dihydrogen phosphate (mobile
phase A) and a mixture ofequal volumes of acetonitrile
and water (mobile phase B),
injection volume: 20 /ll
organ bath containing 9.0 g of sodium chloride, 0.42 g of

potassium chloride, 0.16 g of calcium chloride, 0.50 g of
sodium bicarbonate, 0.25 g of dextrose, and 0.0053 g of
magnesium chloride per litre of the solution. Maintain the
bath temperature at 32 or any other suitable temperature at
which spontaneous contractions of the uterus are abolished
and the preparations maintain its sensitivity. Oxygenate the
bath solution with a mixture of 95 per cent oxygen and 5 per
cent carbon dioxide. Record the contractions of the uterine
muscle on a recorder, using a suitable instrument producing
linear response (for example an isotonic liver with a load of
not exceeding 2 g or isotonic and linear transducer). Add to
the bath two appropriate dilutions of the oxytocin reference
solution, and record the contraction of the muscle following
each dilution. The appropriate dilutions (doses) are those
dilutions of the reference solution which produce clearly
distinctive submaximal contractions. The required dilution
normally lies between 0.1 to 5 micro units per ml of the bath
solution. When maximal contraction is reached, replace the
bath solution by a fresh solution and wait until the muscle is
relaxed completely and the pointer of the recorder returns to
the base line. The doses of the different reference solutions
should be added at regular intervals depending upon the rate
of the recovery of the uterine muscle. Dissolve or dilute the
preparation to be tested in a suitable diluent (preferably using
bath solution) to obtain responses on the addition of two
dilutions similar to the one used with the oxytocin reference
solution. The two selected dilutions ofthe reference solution
and preparation under examination should be applied
according to a randomised block or Latin square design and
at least three responses to each dilution should be recorded.
The magnitude of contractions obtained with the reference
solution is comparable to the contractions obtained with the
test solution.
Equilibrate the column with a mixture of 70 volumes ofmobile

phase A and 30 volumes of mobile phase B and record the
chromatograms as follows. Operate by gradient elution
increasing continuously and linearly the proportion ofmobile
phase B by 1.0 per cent v/v per minute for 30 minutes. Finally
elute using the same mixture for 15 minutes to re-equilibrate
the column.
Calculate the content ofthe peptide, C43H66NIZ0IZSZ.
Test solution. Dissolve about 25 mg of the substance under

examination in 100 ml ofmobile phase A.
Reference solution. A 0.025 per cent w/v solution of oxytocin
RS in mobile phase A.
a stainless steel column 12.5 cm x 4.6 mm, packed with
mobile phase: A. a 1.6 per cent w/v solution of sodium
dihydrogen phosphate,
B. a mixture of 50 volumes of acetonitrile
below,
Tests
Peptide. 90.0 to 11 0.0 per cent ofthe stated amount ofoxytocin,
C43H66NIZ0IZSZ expressed per mg for the solid, and in mg per
ml for the liquid,
Time
(in min)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
0-30
30-30.1
30.1- 45
70-740
40-770
70
30-760
60-730
30
Test solution. Dissolve 3.5 mg of the substance under

examination in sufficient of a 1.56 per cent w/v solution of
sodium dihydrogen phosphate to produce 10.0 ml or use the
Calculate the content OfC43H66N120IzSz.
liquid preparation as appropriate.
Reference solution. Dissolve 3.5 mg ofoxytocin RS in sufficient

of a 1.56 per cent w/v solution of sodium dihydrogen
phosphate to produce 10.0 ml.
Oxytocin intended for use in the manufacture ofparenteral

preparations without a fUrther procedure for the removal of
bacterial endotoxins complies with the following additional
requirement.
Bacterial endotoxins (2.2.3). Less than 0.5 Endotoxin Units
per Unit ofoxytocin.
Oxytocin intended for use in the manufacture ofparenteral

preparations without a further sterilisation procedure
complies with the following additional requirement.
1844
IP 20ID
OXYTOCIN INJECTION
Sterility (2.2.11). Complies with the test for sterility.
- mobile phase: a mixture of85 volumes ofa 0.2 per cent

v/v solution of orthophosphoric acid and 15 volumes
of acetonitrile,
- injection volume. 200 J.Ll.
Storage. Store protected from moisture. If the substance is

the container should be sterile, tamper-evident and sealed so
as to exclude micro-organisms.
Labelling. The label states (1) the number ofUnits ofoxytocic
activity per mg (for solid) or per ml (for liquid); (2) either the
animal species from which it is obtained or whether it is
synthetic, as appropriate; (3) whether or not the contents are
theoretical plates are not less than 50,000.
Calculate the content ofC43H66NlzOlzSz in the injection.
Oxytocin Injection containing Oxytocin of natural origin

obtained by extraction and purification complies with the
follOWing additional requirement.
Oxytocin Injection
Vasopressin impurity. Not more than 0.5 Unit perml.
Oxytocin Injection is a sterile solution of Oxytocin in Water

for Injections.
Oxytocin Injection contains not less than 95.0 per cent and
not more than 105.0 per cent ofthe stated number ofUnits of
oxytocin activity.
Solvent mixture. Dissolve 5.0 g of chlorobutanol in 5.0 ml of

glacial acetic acid, add 1.1 g of sodium acetate, 5.0 g of
ethanol, and dilute to 1000 ml with water and mix.
Usual strengths. 5 Units per ml; 10 Units per ml.
Test solution. Dilute 2.0 ml of injection under examination to

25 ml with 0.25 per cent w/v of glacial acetic acid and mix.
Description. A clear colourless liquid.
Identification
Tests
pH(2.4.24).3.0t05.0
per Unit ofoxytocin.
Test solution. Use the injection under examination.

Reference solution. Dissolve the contents of one vial of
oxytocin RS in a 1.65 per cent w/v solution of sodium
dihydrogen orthophosphate to produce a solution containing
the same concentration in J.Lg of oxytocin as that stated on the
label ofthe injection.
J.Lm) (such as Nucleosil C18),

vasopressin RS in a lmown volume of solvent mixture. If
necessary dilute the prepared solution to a working
concentration range.
endcapped octadecylsilane bonded to porous silica
(5 J.Lm),
v/v solution of dibasic ammonium phosphate, adjusted
to pH 3.0 with orthophosphoric acid and 13 volumes of
acetonitrile, filter through 0.45 J.Lm nylon membrane,
spectrophometer set at 220 nm,
- injection volume. 20 J.L1.
Equilibrate the column at least for one hour. Run the
chromatogram minimum of60 minutes.
resolution between vasopressin and the adjacent peak is not
less than 1.5 and relative standard deviation for replicate
Labelling. The label states (1) the number ofUnits ofoxytocin
activity per ml; (2) either the animal spieces from which it is
obtained or whether it is synthetic, as appropriate.
1845
OXYTOXIN NASAL SOLUTION
IF 2010
Calculate the content ofC43H66NlzOlzSzin the nasal solution.
Oxytocin Nasal Solution is a solution ofOxytocin in a suitable

solvent containing an appropriate antimicrobial preservative.
Oxytocin Nasal Solution containing Oxytocin of natured

origin obtained by extraction and purification complies with
the follOWing additional requirement.
Oxytocin Nasal Solution contains not less than 85.0 per cent
and not more than 120.0 per cent ofthe stated number ofUnits
ofoxytocic activity.
Usual streugth. 40 Units perml.

Description. A clear, colourless solution.
Identification
Tests
pH (2.4.24). 3.0 to 5.0
Other tests. Complies with the tests stated under Nasal
Preparations.
Test solution. Use the nasal solution under examination.

the same concentration in f-lg ofoxytocin as that stated on the
label of the nasal solution.
f-lm) (such as Nucleosil CI8),
mobile phase: a mixture of85 volumes ofa 0.2 per cent
of acetonitrile,
- injection volume. 200 f-ll.
theoretical plates is not less than 50,000.
Vasopressin impurity. Not more than 0.5 Unit perml.

vasopressin RS in a known volume of solvent mixture. If
/lIn),
mobile phase: a mixture of87 volumes of6.6per cent vi
v solution of dibasic ammonium phosphate, adjusted
acetonitrile, filter through 0.45 f-lm nylon membrane,
spectrophometer set at 220 urn,
injection volume. 20 f-ll.
Storage. Store at a temperature not exceeding 30.
Labelling. The label states (1) the number ofUnits ofoxytocic
activity per ml; (2) either the animal species from which it is
obtained or whether it is synthetic, as appropriate; (3) that the
preparation is intended for intranasal administration only.
1846
MONOGRAPHS
p
1853
1854
1855
1856
1856
1857
1859
1860
1861
1862
. 1862
Paclitaxel
Paclitaxel fujection
Pancreatin
D-Panthenol
Pantoprazole Sodium
Pantoprazole Sustained-release Tablets
Paracetamol
Paracetamol Syrup
Paracetamol Tablets
Hard Paraffin
Liquid Paraffin
Penicillamine Tablets
1863
1863
1863
1864
1865
1865
1866
1868
Diluted Pentaerythritol Tetranitrate
18~68
Pentaerythritol Tetranitrate Tablets
1870
1871
1872
1873
1873
1874
1875
1876
1877
1877
1878
Light Liquid Paraffin

Liquid ParaffinEmulsion
White SoftParaffin
Yellow SoftParaffin
Paraffin Ointment
Paraldehyde
Penicillamine
Pentamidine Isethionate
Pentamidine fujection
Pentazocine
Pentazocine Hydrochloride
Pentazocine Tablets
Pentazocine Lactate
Pentazocine fujection
Pentobarbitone Sodium
Pentobarbitone Tablets
Pepsin
1847
MONOGRAPHS
1879
1881
1882
1883
1884
1885
1886
1887
1888
1889
1889
1890
1891
1891
1892
Peritoneal Dialysis Solutions

Perphenazine
Perphenazine Tablets
Pethidine Hydrochloride
Pethidine Injection
Pethidine Tablets
Phenindione
Phenindione Tablets
Pheniramine Maleate
Pheniramine Injection
Pheniramine Tablets
Phenobarbitone
Phenobarbitone Tablets
Phenobarbitone Sodium
Phenobarbitone Injection
1893
1893
1894
1895
1896
1897
1898
1899
1899
1900
1901
1901
1902
Phenobarbitone Sodium Tablets

Phenol
Phenolphthalein
Phenoxyethanol
PhenoxymethylpenicillinPotassium
PhenoxymethylpenicillinPotassiumTablets
Phentolamine Mesylate
Phentolamine Injection
Phenylephrine Hydrochloride
Phenylephrine Injection
PhenylmercuricAcetate
Phenylmercuric Nitrate
Phenylpropanolamine Hydrochloride
1903
1904
1905
1906
1906
Phenytoin
Phenytoin Sodium
PhenytoinInjection
Phenytoin Capsules
Phenytoin Tablets
1848
MONOGRAPHS
Phenytoin Oral Suspension
1907
Pholcodine
1908
Pholcodine Linctus
1908
Phosphoric Acid
1909
Physostigmine Salicylate
1910
Physostigmine fujection
1911
Pilocarpine Nitrate
1911
Pimozide
1912
Pimozide Tablets
1913
Pindolol
1914
Pindolol Tablets
1915
Pioglitazone HydrocWoride
1916
Pioglitazone Tablets
1917
Piperacillin
1918
Piperacillin futravenous Infusion
1919
PiperazineAdipate
1920
PiperazineAdipate Tablets
1921
Piperazine Citrate
1921
Piperazine Citrate Syrup
1922
Piperazine Hydrate
1923
Piperazine Phosphate
1924
Piperazine Phosphate Tablets
1924
Piracetam
1925
Piroxicam
1926
Piroxicam Capsules
1927
Plaster of Paris
1928
Poloxamer
1928 .
Polyethylene Glycol 1500
1929
1930
1930
Polyoxyl35 Castor Oil
1931
Polyoxyl40 Hydrogenated Castor Oil
1931
Polysorbate 20
1932
1849
:1'~
MONOGRAPHS
Polysorbate 80
...
1932
Potassium CWoride
1933
Potassium Chloride and Dextrose Injection
1934
Potassium CWoride, Sodium Chloride and Dextrose Injection
1935
PotassiumCitrate
1936
Potassium Clavulanate
1937
Potassium Clavulanate Diluted
1938
Potassium Iodide
1939
PotassiumPermanganate
1940
Povidone
1941
Povidone..Iodine
1943
Povidone-Iodine Solution
1943
PralidoximeCWoride
1944
Pralidoxime CWoride Injection
1944
Pravastatin Sodium
1945
Pravastatin Tablets
1946
Praziquantel
1947
Praziquantel Tablets
1948
Prazosin HydrocWoride
1949
Prazosin Tablets
1950
Prednisolone
1951
Prednisolone Tablets
1952
Prednisolone Acetate
1954
Prednisolone Sodium Phosphate
1955
Prednisolone Sodium Phosphate Injection
1956
Prednisone
1957
Prednisone Tablets
1958
Pregabalin
1960
Pregabalin Capsules
1961
Pregelatinised Starch
....
1963
Primaquine Phosphate
1963
PrimaquineTablets
1964
Probenecid
1965
1850
MONOGRAPHS
1966
1966
1967
1968
1968
1969
1970
1971
1972
1972
1973
1974
1974
1975
1976
1977
1977
1978
1979
1979
1980
1981
1982
1982
1983
1984
1985
1986
1987
1988
1989
1990
1990
Probenecid Tablets
Procainamide Hydrochloride
Procainamide fujection
Procainamide Tablets
Procaine Hydrochloride
Procaine andAdrenaline fujection
Procaine Penicillin
Fortified Procaine Penicillin fujection
ProchlOlperazine Maleate
Prochlorperazine Tablets
Prochlorperazine Mesylate
Prochlorperazine fujection
Procyclidine Hydrochloride
Procyclidine Tablets
Progesterone fujectable Suspension
Proguanil Hydrochloride
Proguanil Tablets
Promazine Tablets
Promethazine Hydrochloride
Promethazine fujection
Promethazine Syrup
Promethaiine Tablets
Promethazine Theoclate
Promethazine Theoclate Tablets
Propane
Propionic Acid
Propofol
Propofol fujection
Propranolol Hydrochloride
Propranolol fujection
Propranolol Tablets
Propyl Gallate
Propylene Glycol
1851
MONOGRAPHS
Propylparaben
1991
1992.
. 1993.
Propylthiouracil
Propylthiouracil Tablets
1994
1995
1996
1996
1997
1998
1999
1999
2000
2001
2001
.... 2002
2003
2004
2005
Propyphenazone
Protamine Sulphate
Protamine Sulphate Injection
Prothionamide
Prothionamide Tablets
Protriptylihe HydrocWoride
Protriptyline Tablets
Pseudoephedrine HydrocWoride
Pseudoephedrine Syrup
Pseudoephedrine Tablets
Psoralen
Pyrantel Pamoate
Pyrante1Pamoate Oral Suspension
Pyrazinamide
Pyrazinamide Tablets
Pyridoxine HydrocWoride
Pyrimethamine Tablets
2005
2006
2007
..... 2008
Pyrimethamine and Sulphadoxine Tablets
.... 2009
Pyridoxine Tablets
Pyrimethamine
1852
IP 2010
PACLITAXEL
Paclitaxel
octadecylsilane bonded to porous silica(3/lm),
mobile phase: A. a mixture of 60 volumes of water and
B. acetonitrile
below,
spectrophotometer set at 227nm,
Taxo!
C47HsINOl4
Mol. Wt. 853.9
Paclitaxel is 5~,20-epoxy-l,2a;4,7 f3,lOf3,13a-hexahydroxytaxll-en-9-one 4,1 O-diacetate 2-benzoate 13-ester with(2R,3S)N-benzoyl-3-phenylisoserine.

A taxane derivative first isolated from the bark ofthe Pacific
yew tree, Taxus brevifolia.
Paclitaxel contains not less than 97.0 per cent and not more
than 102.0 per cent ofC47Hs1N014, calculated on the anhydrous
basis.
Category. Anticancer.
CAUTION - Paclitaxel is potentially cytotoxic. Great care

should be taken in handling the powder and preparing
solutions.
Identification
Compare the spectrum with that obtained with paciltaxel RS.

obtained withthe test solution corresponds to the peak inthe
Tests
Specific optical rotation (2.4.22). -49.00 to - 55.00 , determined
in 1.0 per cent w/v solution in methanol.
(2.4.14).

in 10 ml of acetonitrile.
paclitaxel RS in acetonitrile.
w/vof 10 deacetyl-7-epipaclitaxel and 0.1 per cent w/v of
Time
(in min)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
100
O.
20
100
60
10
90
62
100
70
100
o
o
resolution between the peak due to paclitaxel and 10-deacetyl7-epipaclitaxel is not less than 1.2. The relative retention time
for 10-deacetyl-7-epipaclitaxel is about 0.94 in respect to
paclitaxel.
in the chromatogram obtained with the reference solution (a)
is not more thantwi<;:e the area ofthe peak in the chromatogram
obtained with the reference solution (a) (2.0 per cent).
Heavy metals (2.3.13). 1 g complies with the limittest for heavy

metals, Method B (20ppm ).
'
Sulphated ash (2.3.18). Not more than 0.2 per cent. '
Water (2.3.43). Not more than 4.0 percent, determined on 0.1
g by coloumetry method.
per mg ofpaclitaxel.
Microbial contamination (2.2.9). The total viable aerobic count
does not exceed 100 cfu per g. It meets the requirements ofthe
tests for the absence of Staphylococcus aureus, Pseudomonas
aeruginosa, Salmonella species, and Escherichia coli.
Solvent mixture. Dissolve 200 III of glacial acetic acid in

1000 ml of methanol.
examination in 100.0 ml in solvent mixture.
1853
PACLITAXEL INJECTION
IP 2010
Reference solution. A 0.1 per cent w/v solution ofpaclitaxel

a stainless steel column 25 ern x.4;6 mm, packed with
pentafltioro phenyl groups bonded to porous silica (5
J.Ull),
column temperature 35,
mobile phase: a mixture of 11 volumes of water and
flow rate. 1.5 rn1 per minute,
tailing factor is not more than L5. The relativestandard

w/v of 10 deacetyl-7-epipaclitaxel RS and 0.12 per cent w/v
of paclitaxel RS in acetonitrile.
octadecylsilane bonded to porous silica (311m),
mobile phase: A. a mixture of 00 volumes of water and
B. acetonitrile,
flow rate. L2 rn1 per minute,
below,
injection volume. lO Ill.
Time
(in min)
Mobile phase A
(per cent v/v)
Mobile phase B
(percent v/v)
Calculate the content OfC47HsIN014.
100

exceeding 25.
26
100
o
o
66
17
83
67
100
Inject the test solution and reference solution.
Paclitaxel Injection
Paclitaxel Injection is a sterile solution of Paclitaxel suitable
for dilution,for intravenous use.
Paclitaxel Injection contains not less than 90.0 per cent and
not more than 110.0 per centofthestated amount of paclitaxel,
C47HsIN014.
.
Description. A clear colourless to slight yellow viscous
solution.
Identification
with test solution corresponds to the peak in the chromatogram
Inject reference solution (b). The test is not valid uniess the
resolution between the peak due to paclitaxel and 10-deacetyl7-epipaclitaxel is not less than 1.2. The relative retention time
for 1O-deacetyl-7-epipaclitaxe1 is about 0.94 in respect to
paclitaxel.
in the chromatogram obtained with the reference solution (a)
obtained with the reference solution (a) (2.0 per cent).
Tests

Unit per mg ofpaclitaxel.
pH (2.4.24). 3.0to 7.0, determined in a 10percentv/v solution

in water.
Light absorption. Absorbance of the injection at about 425

nm, not more than 0.1.
(2.4.14).
Solvent mixture. Dissolve 200 III of glacial acetic acid in

1000 ml of methanol.
Test. solution. Accurately measure the volume of injection

containing 12 mg of Paclitaxel, dilute to 10 rn1 with acetonitrile.
Test solution. Accurately measure the volume of injection

containing 6 mg ofPaclitaxel and dissolve in 10 ml ofsolvent
mixture.

Reference solution. A 0.06 per cent w/v solution ofpaclitaxel

1854
lP 2010
PANCREATIN
penta fluro phenyl group chemically bonded to porous
silica'(5 jlm),
mobile phase: a mixture of 11 volumes of water and 9
- spectrophotometer set at 227nm,
- injection volume. 10 jll.
and solution (2) to the remainder and maintain the mixtures at

39 1 for 5 minutes. To I ml of each mixture add 10 mlof
iodinated potassium iodide solution. The liquid containing
solution (2) retains the colour of the solution of iodine and
the liquid containing solution (1) acquires an intense blue
colour.
Tests
Fat. Not more than 5.0 per cent, determined by the following
method. Extract I g with light petroleum (40 to 60) for
tailing factor is not more than 1.5. The relative standard
3 hours in an apparatus for the continuous extraction ofdrugs

(2.1.8), evaporate the extract and dry the residue at 105 for
2 hours.
Microbial contamination (2.2.9). 1g is free from Escherichia

coli; 10 g is free from Salmonellae.
Calculate the content ofC47HsINOI4.

exceeding 25.
Pancreatin
Pancreatin is a preparation ofmammalian pancreas containing
protease, lipase and amylase activity. It may contain Sodium
Chloride.
Pancreatin contains not less than the minimum protease
activity, amylase activity and lipase activity determined under
the conditions of the Assay.
Category. Digestive enzyme.
Dose. 500 mg to 1g.
Description. A white or buff-coloured, amorphous powder;'
odour, meaty and not unpleasant.
Identification
A. Triturate 0.5 g with 10 ml of water and adjust to pH 8.0 by
the addition of 1 M sodium hydroxide using cresol red
solution as indicator. Divide the resulting solution into two
equal portions. Boil one portion [solution (1)] and leave the
other untreated [solution (2)]. To each add a few shreds of
congo red fibrin, warm to 39 1 and maintain at this
temperature for 1 hour. Solution (2) is stained red and solution
(1) is colourless or not more than slightly pink.
B. Triturate 0.25 g with 10 ml of water and adjustto pH 8.0 by
the addition of 1 M sodium hydroxide using cresol red
solution as' indicator. Divide the resulting solution into two
equal portions. Boil one portion [solution (1)] and leave the
other untreated [solution (2)]. Dissolve 0.1 g ofsoluble starch
in 100 ml ofboiling water, boil for 2 minutes, cool and dilute to
150 ml with water. Add solution (1) to halfthe starch mucilage
on 0.5 g by dying in an oven at 60 at a pressure not exceeding
Assay. For protease activity - Weigh accurately 4.0 g of
purified casein and dissolve in about 90 ml of water containing
3 ml of1 M sodium hydroxide, adjust the pH ofthe solution to
8.7 and add sufficient water to produce 100.0 ml. Weigh
accurately about 0.5 g of the substance under examination,
triturate with water and add sufficient water to produce
300.0 ml to give the test solution. Dilute 15.0 ml ofthe casein
solution with 30 ml of water, warm to 55 and add 10.0 ml ofthe
unfiltered test solution. Heat rapidly to 55 and keep at this
temperature for 20 minutes. Cool rapidly to room temperature.
Dilute a further portion of 15.0 ml ofthe casein solution with
30 ml of water, add 10.0 ml of the unfiltered test solution,
previously boiled and cooled, heat rapidly to 55 and keep at
this temperature for 20 minutes. Cool to room temperature. To
each solution add 0.75 ml ofphenolphthalein solution and 10
ml ofJormaldehyde solution. Titrate each solution with 0.1 M
sodium hydroxide until the colour of the solution matches
that produced by mixing 10 ml of buffer solution pH 8.7 and
0.15 ml of phenolphthalein solution. The difference between
the two titrations is not less than 4.5 ml.
For lipase activity- To 95 ml of water, add 6.5 mloftriacetin

and 0.2 ml ofa 0.1 per centw/v solution of bromocresolpurple,
neutralise with 0.5 M sodium hydroxide and add sufficient
water to produce 110 mI. Place 50 ml ofthis solution in each of
two large tubes 3 cm x 20 cm A and B contained in a thermostat

at 30. Insert in each tube a rubber stopper having two holes,
onefor the tip ofa burette and the other fora short glass tube
through which passes a thread operating a glass stirring coil.
Stir the contents of the tube until they attain the temperature
of the thermostat. Prepare a solution of 0.1 g ofthe substance
under examination in 10.0 ml of water. To tube A add 1.0 ml of
the solution, to tube B add 1.0 ml of the solution previously
boiled. Adjust and maintain the pH ofthe solutions in the two
1855
IP 2010
PANCREATIN
tubes to 6.2 to 6.4 by the addition of 0.05 M sodium hydroxide

dropwise, stirring frequently. After 30 minutes, the difference
between the volumes of 0.05 M sodium hydroxide added to
the two tubes is not less than 1.0 ml.
For amylase activity- Not less than I00 Units per g. Dissolve
0.1 g or a quantity containing 10 Units, accurately weighed, in
sufficient buffer solution pH 6.8 to produce 1000.0 ml. Filter if
necessary (1 ml of the test solution should be capable of
digesting about 10 mg of dry soluble maize or corn starch).
Into each of six stoppered test~tubes add 5.0 ml of starch
substrate without touching the sides of the test-tube. Place
the test-tubes in a water-bath maintained at 40 0.1. When
the temperature of the solution in the tubes has reached 40,
add 0.35 ml, 0.4 rnl, 0.45 rnl, 0.5 ml, 0.55 ml and 0.6 rnl ofthe test
solution to each of the test-tubes marked 1 to 6 respectively
and record the time of addition. Mix thoroughly and replace
the tubes in the water-bath. After exactly 60 minutes remove
the tubes and cool rapidly in cold water. Add to each tube
0.05 ml of 0.02 M iodine and mix well. Note the tube containing
the lowest volume of test solution, which does not show a
bluish or violet tinge (if there is doubt, warm the solution
slightly, when the colour distinction is prominent). From this
volume calculate the number ofgrams ofdry soluble maize or
corn starch digested by 1.0 g of the substance under
examination. This represents the number ofUnits of amylase
activity per g.
Identification
Boil 50 mg with 5 ml of 0.1 M sodium hydroxide for 1 minute,
cool and add 5 ml of1 M hydrochloric acid and 0.1 mlofjerric
chloride test solution; a deep yellow colour is produced.
Tests
pH (2.4.24). Not more than 10.5, determined in a 5.0 percent
w/v solution in carbon dioxide-free water.
at 20 in a 5.0 per cent w/v solution.
Refractive index (2.4.27). 1.490 to 1.498, determined at 20.
Heavy metals (2.3.13). 1.0 g dissolved in 25 rnl ofwater complies
with the limit test for heavy metals, Method A (20 ppm).
Assay. Weigh accurately about 0.5 g and carry out Method A
for the determination ofnitrogen (2.3 .30).
1 ml of 0.05 Msulphuricacidis equivalentto 0.001401 g ofN.
Pantoprazole Sodium

Labelling. The label states the name of any added substance.
Mol. Wt. 432.4
Pantoprazole Sodium is sodium 5-(difluromethoxy)-2[[(3,4
uimethoxy-pyridin-2-yl)methyl]sulphinyl]benzimidazol-1-ide,
sesquihydrate.
D-Panthenol
Pantothenol; Dextro-pantothenyl Alcohol
Pantoprazole Sodium contains not less than 98.0 per cent and
not more than 102.0 per cent ofCI6HI4F2N3Na04S, calculated
Category. Antiulcer.
C9H I9N04
Mol. Wt. 205.3
Identification
D-Panthenol is (R)-2,4-dihydroxy-N-(3-hydroxypropyl)-3, 3dimethylbutanamide.

D-Panthenol contains not less than 6.60 per cent and not more
than 6.95 per cent of nitrogen, N.

Compare the spectrum with that obtained with pantoprazole
sodium RS or with the reference spectrum of pantoprazole
sodium.
Category. Vitamin B (enzyme co-factor).

Dose. 250 to 500 mg.
Description. A clear, colourless or slightly yellow, viscous
liquid; odourless.

obtained with the test solution corresponds to the principal
peak in the chromatogram obtained with the reference solution.
1856
IP 2010
PANTOPRAZOLE SUSTAINED-RELEASE TABLETS
C. Gives the reaction (a) ofsodium (2.3.1).
Tests
Solvent mixture (a). Dilute 25.0 ml of ammonium hydroxide

to 500.0 ml with water.
Optical rotation (2.4.22). -0.40 to +0.40, determined on 1.0

per cent w/v solution.
Solvent mixture (b). Equal volumes of acetonitrile and water.

(2.4.14).
Solvent mixture. Equal volumes of acetonitrile and 0.001 M

sodium hydroxide.
examination in 50.0 ml ofthe solvent mixture.
pantoprazole sodium RS in the solvent mixture. Dilute 2.5 ml
ofthe solution to 50.0 ml with the solvent mixture.
- a stainless steel column 12.5 cm x 4.0 mm packed with
as Inertsil ODS-3),
- mobile phase: A. dissolve 1.74 g of dibasic potassium
phosphate to 1000 ml with water; adjusted to pH
7.0 with orthophosphoric acid,
B. acetonitrile,
Time
Mobile phase A
Mobile phase B
(per cent v/v)
(in min)
(per cent v/v)
20-780
040
80-720
40-45
20
80
45-55
20-780
80-720

examination 10.0 ml ofsolvent mixture (b) and dilute to 50.0 ml
with solvent mixture (a). Dilute 3.0 ml ofthis solution to 20.0 ml
with solvent mixture (a).
Reference solution. Dissolve 20 mg of pantoprazole sodium
RSin 10.0 ml of solvent mixture (b) and dilute to 50.0 ml with
solvent mixture (a). Dilute 3.0 ml ofthis solution to 20.0 ml with
solvent mixture (a).
as Inertsil ODS-3),
- sample temperature.4,
solution prepared by dissolving 1.32 g of dibasic
ammonium phosphate to 1000 ml with water, adjusted
acetonitrile,
B. a mixture of7 volumes of acetonitrile
and3 volumes of methanol,
Mobile phase B
Time
Mobile phase A
(per cent v/v)
(in min)
(per cent v/v)
than 2.0 per cent.
peak due to impurity C determined at wavelength 305 nm at
relative retention time about 0.6 is not more than 0.77 times the
area of the principal peak at 290 nm in the chromatogram
obtained with reference solution (b) (0.5 per cent), the area of
any secondary peak is not more than 0.077 times the area of
solution (b) (0.5 per cent) and the sum of the areas of all the
secondary peaks is not more than 0.15 times the area of the
solution (b) (1.0 per cent).
Water (2.3.43). Not less than 5.0 per cent and not more than
8.0 per cent, determined on 0.15 g.
0-10
86
35
42
36
86
86
46
14
58
14
14
than 2.0 per cent.
Calculate the content ofC16H14F2N3Na04S,
Storage. Store protected from light and moisture, between 2
to 8.

Pantoprazole Sodium Sustained-release Tablets
Pantoprazole Sustained-release Tablets contain not less than
amount ofpantoprazole, C16HlSF2N304S.
1857
IP 2010
PANTOPRAZOLE SUSTAINED-RELEASE TABLETS

Tablets.
Usual strenth. 40 mg.
Identification
peak: in the chromatogram obtained with the reference solution.
B. When examined in the range 230 urn to 350 nm (2.4.7), a
maxima at about 289 urn.
Determine by liquid chromatography (2.4.14), as described in

the Assay using the following solutions.
Test solution. Disperse 1 intact tablet in 100.0 ml ofthe mobile

phase and filter.
Tests
Reference solution. Dissolve an accurately weighed quantity

of pantoprazole sodium RS in the mobile phase and dilute
with the mobile phase to obtain a solution having a known
concentration similar to the test solution.
NOTE-Prepare the solutions immediately before use.

Protect the solutions from light.
Calculate the content ofCI6HIsF2N304S in the tablet.

A. Apparatus No.1,

Protect the solutions from light.
Test solution. Withdraw the medium completely and disperse

the intact tablet in 100 ml of the mobile phase and filter.
of pantoprazole sodium RS in the mobile phase and dilute
with the mobile phase to obtain a solution having a known
Use chromatographic system as described under Assay.
Calculate the content of CI6HISF2N304S released in the acid
medium by subtracting the content of CI6HISF2N304S in the
test solution from the total content of Pantoprazole,
CI6HISF2N304S determined in the Assay.
D. Not more than 10 per cent of the stated amount of
CI6HISF2N304S.
B. Apparatus No.1,
Medium. 1000 ml of tris-acetate buffer solution pH 8.5,

Run method A on another 6 tablets and discard the medium
completely and fill the empty vessel with the dissolution
medium. Withdraw a suitable volume ofthe medium and filter.
Dilute the filtrate, if necessary, with the dissolution medium.
Measure. the absorbance at the maximum at about 290 nm
(2.4.7). Calculate the content ofC,6H,sF2N304S in the medium
concentration of pantoprazole sodium RS.
CI6HISF2N304S.

a quantity ofpowder containing about 20 mg ofPantoprazole
Sodium, disperse in 100 ml ofthe mobile phase and filter.
pantoprazole sodium RS in the mobile phase.
stainless steel column 25 cm x 4.6 mm, packed with
octadecylsilane bonded to porous silica (5 Ilm) (such as
Inertsil ODS-3),
mobile phase: a mixture of50 volumes ofbuffer solution
orthophosphate and. 1 g of hexane sulphonic acid
sodium salt in 1000 ml of water, adjusted to pH 7.3 with
1 M sodium hydroxide and 50 volumes of acetonitrile,
theoretical plates are not less than 2000, the tailing factor is
not more than 2.0 and the relative standard deviation for
replicate injections is not more than 2.0 per cent.
Calculate the content OfC16HISF2N304S in the tablets.
exceeding 30.
Labelling. The label states the strength in terms ofthe amount
ofPantoprazole.
1858
IP 2010
PARACETAMOL
Paracetamol

(2.4.14).
Acetaminophen
Note-Prepare the solutions immediately before use.

examination in 2.5 ml of methanol containing 0.46 per cent
w/v of tetrabutylammonium hydroxide solution (40 per cent
w/v) and dilute to 10.0 ml with the solution containing equal
volumes of 1.79 per cent w/v ofdisodium hydrogenphosphate
and 0.78 per cent w/v of sodium dihydrogen phosphate.
Mol. Wt. 151.2

Paracetamol is 4-hydroxyacetanilide.
Paracetamol contains not less than 99.0 per cent and not more
than 101.0 per cent ofCsHgN02, calculated on the dried basis.
Category. Analgesic; antipyretic.
Dose. 500 mg to I g every 4 to 6 hours, upto 4 g daily, in
divided doses
Description. White crystals or a white, crystalline powder.
Identification
TestA may be omitted if tests B C andD are carried out. Tests
B, C and D may be omitted if test A is carried out. .
Compare the spectrum with that obtained with paracetamol
RS or with the reference spectrum ofparacetamol.
B. Dissolve 50 tng in sufficient methanol to produce 100 ml.
To 1 ml ofthis solution add 0.5 m1 of0.1 M hydrochloric acid
and dilute to 100 ml with methanol. Protect the resulting
solution from bright light and immediately measure the
absorbance at the maximum at about 249 nm; absorbance at
249 nm, about 0.44 (2.4.7).
C. Boil 0.1 g in 1 ml of hydrochloric acid for 3 minutes, add
10 ml of water and cool; no precipitate is produced. Add
0.05 ml of 0.0167 M potassium dichromate; a violet colour
develops which does not tum red.
D. Gives the reaction of acetyl groups (2.3.1).
Tests
4-Aminophenol. Dissolve 0.5 g in sufficient methanol (50 per
cent) to produce 10 ml. Add 0.2 ml offreshly prepared alkaline
sodium nitroprusside solution, mix and allow to stand for
30 minutes. Any blue colour in the solution is not more intense
than that in 10 ml of a solution prepared at the same time and
in the same manner containing 0.5 g of 4-aminophenol-free
paracetamol and 0.5 ml of a 0.005 per cent w/v solution of
4-aminophenol in methanol (50 per cent) (50 ppm).

Reference solution (b). Dilute 1.0 ml of reference solution (a)
Reference solution (c). Asolution containing 0.025 per cent
w/v each of 4-aminophenol, paracetamol RS and
chloroacetanilide in methanol. Dilute 1.0 ml of this solution
Reference solution (d). Dissolve 20 mg of 4-nitrophenol in
50.0 ml of methanol. Dilute 1.0 ml ofthis solution to.20.0 ml
octylsilane bonded to porous silica (5 /lm),
- mobile phase: a mixture of 37.5 volumes of a 1.79 per
cent w/v solution of disodium hydrogen phosphate,
37.5 volumes of a 0.78 per cent w/v solution ofsodium
dihydrogen phosphate and 25 volumes of methanol
containing 0.46 per cent v/v of tetrabutylammonium
hydroxide solution (40 per cent w/v),
resolution between the peaks due to 4-aminophenol
(paracetamol impurity K) and paracetamol is not less than 4.0
and the signal-to-noise ratio of the peak due to
chloroacetanilide (paracetamol imputity J) is not less than 50.
The relative retention time with reference to paracetamol for
paracetamol impurity K is about 0.8, for 4-nitrophenol
(paracetamol impurity F) is about 3.0 and for paracetamol
impurity J is about 7.0.
Inject the test solution, reference solution (a), (b) and (c). Run
the chromatogram 12 times the retention time ofthe principal
peale In the chromatogram obtained with the test solution the
area ofthe peak due to paracetamol impurity J is not more than
0.2 times the area of the corresponding peak in the
chromatogram obtained with reference solution (c) (10 ppm)
1859
PARACETAMOL SYRUP
IP 2010
and the area ofthe peak due to paracetamol impurity K is not Reference solution. A 0.25 per cent w/v solution of
more than the area of the corresponding peak in the paracetamol RS in methanol
chromatogram obtained with reference solution (c) (50 ppm). Apply to the plate 10 III of each solution. After development,
The area of any other secondary peak is not more than 0.5 dry the plate in a current of warm air, examine in ultraviolet
times the area of the principal peak in the chromatogram light at 254 nm. The principal spot in the chromatogram
obtained with reference solution (a) (0.05 per cent). The sum obtained with the test solution corresponds to that in the
ofareas ofother secondary peaks is not more than the area of chromatogram obtained with the reference solution.
solution (a) (0.1 per cent). Ignore any peak with an area less B. In the Assay, the principal peak in the chromatogram
than 0.5times the area ofthe principal peak in the chromatogram obtained with the test solution corresponds to the peak in the
chromatogram obtained with reference solution.
Tests
4-Aminophenol. Determine by liquid chromatography (2.4.14).

Loss on drying (2.4.19). Not more thanO.5 per cent, determined
Test solution. Shake 5 ml ofthe preparation under examination

with 15 ml ofthe mobile phase, dilute to 25 ml with the mobile
phase and filter if necessary.

10 ml of water and 50 ml of 1 M sulphuric acid. Boil under a
4-aminophenol in the mobile phase.
reflux condenser for 1 hour, cool and dilute to 100.0 ml with
water. To 20.0 ml of the solution add 40 mlofwater, 40 g of Chromatographic system
water in the form of ice, 15 ml of 2 M hydrochloric acid and
0.1 ml of ferro in solution and titrate with 0.1 M eerie
mobile phase: 0.01 M sodium butanesulphonate in a
ammonium sulphate until a yellow colour is produced. Carry
mixture
of85 volumes of water, 15 volumes of methanol
and 0.4 volume offormic acid,
1 ml of 0.1 M eerie ammonium sulphate is equivalent to
0.00756 gofCsHgN02
of any peak corresponding to 4-aminophenol is not greater
than the area of the peak in the chromatogram obtained with
the reference solution. In the chromatogram obtained with the
test solution peaks with a long retention time may occur due
to preservatives in the preparations.
Paracetamol Syrup
Paracetamol Oral Solution; Acetaminophen Syrup
Paracetamol Syrup is a solution ofParacetamol in a suitable
flavoured vehicle.
Paracetamol Syrup contains not less than 95.0 per cent and
not more than 105.0 per cent w/v solution ofthe stated amount
ofparacetamol, CsHgN0 2
Usual strength. 125 mgper 5 ml.
Identification
25 volumes of acetone, 10 volumes of toluene and 0.5 volumes
of glacial acetic acid.
Test solution. Dilute a volume containing 25 mg ofParacetamol
to 10 ml with methanol and filter ifnecessary.
,

Test solution. Mix an accurately weighed quantity of the
preparation under examination containing 25 mg of
Paracetamol in 100 ml ofthe mobile phase, dilute to 200.0 ml
with the mobile phase and filter if necessary.
Reference Solution. A 0.0125 per cent w/v solution of
paracetamol RS in the mobile phase.
- mobile phase: 0.01 M sodium butanesulphonate in a
mixture of85 volumes of water, 15 volumes of methanol
1860
PARACETAMOL TABLETS
IF 2010

injection volume. 20 !-d.
test solution peaks with a long retention times may occur due
to excipients.
Determine the weight per ml (2.4.29) ofthe syrup and calculate

the percentage content ofCsH 9N02, weight in volume.

(2.4.14).

containing about 0.2 g ofParacetamol in 10.0 ml ofthe mobile
phase, filter.
Paracetamol Tablets
Acetaminophen Tablets
Reference solution (a). Dilute 1 ml ofthe test solution to 20 ml

with the mobile phase. Dilute lml ofthis solution to 20 ml with
the mobile phase.
Paracetamol Tablets contain not less than 95.0 per cent and
paracetamol, CsH9N02

w/v each of 4-aminophenol and paracetamol RS in the
mobile phase.
Reference solution (c). ) A 0.00002 per cent w/v solution of 4chloroacetanilide in the mobile phase.
Identification
Extract a quantity ofthe powdered tablets containing 0.5 g of .
Paracetamol with 20 ml of acetone, filter, evaporate the filtrate
to dryness and dry at 105. The residue complies with the
following tests.
Compare the spectrum with that obtained with paracetamol
RSor with the reference spectrum ofparacetamol.
B. Boil 0.1 gin 1 ml of hydrochloric acid for 3 minutes, add
10 ml of water and cool; no precipitate is produced. Add
0.05 ml of 0.0167 M potassium dichromate; a violet colour
develops which does not tum red.
Tests
4-Aminophenol. Determine by liquid chromatography (2.4.14).

containing 1 g ofParacetamol with 15 ml of methanol, dilute to
100 ml with water and filter.
4-aminophenol in methanol (15 per cent).
octadecylsilane bonded to porous silica (10 j..1m),
- mobile phase: 0.01 M sodium butanesulphonate in a
mixture of85 volumes of water, 15 volumes of methanol
- injection volume. 20 J.1l.
of any peak corresponding to 4-aminophenol is not greater
than the area of the peak in the chromatogram obtained with
the reference solution. In the chromatogram obtained with the
octylsilane bonded to porous silica (5 J.1m) (Such as
Zorbax Rx C8),
- column temperature. 35 0,
- mobile phase: a mixture of 25 volumes of methanol
containing 1.15 g of tetrabutylammonium hydroxide
solution (40 per centw/v), with 37.5 volumes of 0.05 M
disodium hydrogen orthophosphate and 37.5 volumes
of 0.05 M sodium dihydrogen orthophosphate,
- injection volume. 20 j..1l.
4.0.
Inject the test solution and reference solution (a), (b) and (c).
Run the chromatogram 12 times the retention time of the
solution the area of peak corresponding to 4-aminophenol is
not more than the area ofthe corresponding peak in reference
solution (b) (0.1 per cent), the area of peak corresponding to
4- chloroacetanilide is not more than the area of the principal
peak in reference solution (c) (10 ppm) and the area of any
other secondary peak is not more than the area ofthe principal
peak obtained with reference solution (a) (0.25 per cent).
Apparatus. No 1
Medium. 900 ml ofphosphate bufferpH 5.8
Withdraw a suitable volume ofthe medium and filter and dilute
a suitable volume ofthe filtrate with the same solvent. Measure
1861
IP 2010
HARD PARAFFIN
about 243 nm (2.4.7). Similarly measure the absorbance of a

solution oflmown concentration ofparacetamol RS. Calculate
the content of CsHgNO z.
D. Not less than 80 per cent ofthe stated amount ofCsHgNO z.
Description. A transparent, colourless, oily liquid~ free from

fluorescence by daylight; odourless or almost odouiless.
Tests
Weight per ml (2.4.29). 0.860 g to 0.904 g.

quantity ofthe powder containing about 0.15 g ofParacetamol,
add 50 mloiO.l M sodium hydroxide, dilute with 100 ml of
water, shake for 15 minutes and add sufficient water to produce
200.0 ml. Mix, filter and dilute 10.0 ml ofthe filtrate to 100.0 ml
with water. To 10.0 ml of the resulting solution add 10 ml of
0.1 M sodium hydroxide, dilute to 100.0 ml with water and
mix. Measure the absorbance of the resulting solution at the
CsHgNO z taking 715 as the specific absorbance at 257 nm.
Dynamic viscosity (2.4.28). 110 mPas to 230 mPas, determined

at20010 bymethodB or Viscosity (2.4.28). 10 cps to 40 cps,
determined at 30 l by method C using LVI spindle at 30
rpm.
Acidity or alkalinity. To 10.0 g add 20 ml ofboiling water, heat
in a water-bath for 5 minutes, shake vigorously for 1 minute,
cool, allow to separate and filter the aqueous layer. To 10 ml of
the filtrate add 0.1 ml of phenolphthalein solution. The"
solution is colourless and not more than 0.1 ml of 0.1 M sodium
hydroxide is required to change the colour of the solution to
pink.
Light absorption. When examined in the range 240 nrn to
280 nm (2.4.7), a 2.0 per cent w/v solution in 2,2,4-trimethylpentane shows an absorption of not more than 0.1.
Hard Paraffin
HardParaffin is a purified mixture of solid hydrocarbons
obtained from petroleum or from shale oil.
Category. Pharmaceutical aid(stiffening agent).
Description. A white or colourless, translucent mass,
frequently showing a crystalline structure; odourless even
when freshly cut; slightly greasy to the touch. Bums with a
luminous flame. When melted, the liquid is free from
fluorescence by daylight.
Tests
in a water-bath for 5 minutes, shake vigorously for I minute,
the filtrate add 0.1 rnl of phenolphthalein solution. The
solution is colourless and not more than 0.1 ml of 0:1 M sodium
pink
Congealing range (2.4.10). 50 to 65.
Liquid Paraffin
White Mineral Oil; Liquid Petrolatum
Liquid Paraffin is a pmified mixture of liquid hydrocarbons
obtained from petroleum to which not more than 10 ppm of
. tocopherol or of butylated hydroxytoluene may be added.
Category. Laxative; faecal softener.
Dose. 10 to 30 ml.
Readily carbonisable substances. Place 5 ml in a dry, heatresistant glass-stoppered test-tube (125 mm x 18 mm)
previously rinsed with chromic acidsolution, then with water
and dried. Add 5 ml of nitrogen-free sulphuric acid
(containing 94.5 per cent to 95.5 per cent w/w ofHzS04), insert
the stopper and shake as vigorously as possible in the
longitudinal direction of the tube for 5 seconds. Loosen the
stopper, immediately place the tube in a bath ofboiling water,
supporting it so as to prevent contact of the tube with the
bottom or side ofthe bath and heat for 10 minutes. At the end
of the second, fourth, sixth, and eighth minutes, remove the
tube from the bath and shake as vigorously as possible in the
longitudinal direction ofthe tube for 5 seconds. At the end of
10 minutes from the time the tube was placed in the bath
remove the tube and allow to stand for 10 minutes. The lower
acid layer is notmore intensely coloured than a mixture oD ml
ofFCS, 1.5 ml ofCCS and 0.5 ml ofCSS (2.4.1), overlaid with
5 ml ofliquid paraffin. lithe sulphuric acid remains dispersed
in the molten paraffin, the colour ofthe emulsion is not darker
than that of the standard mixture when shaken vigorously.
Solid paraffins. Place a suitable quantity, previously dried by
heating at 100 for 2 hours and cooled in a desiccator over
sulphuric acid, in a glass cylindrical vessel having an internal
diameter ofapproximately 25 mm. Close the vessel and immerse
in a mixture of ice and water; after 4 hours the liquid is
sufficiently clear that a black line, 0.5 mm in width, held vertically
behind the vessel is easily seen.
Sulphur compounds. Mix 4 ml with 2 ml of ethanol (95 per
cent), and 2 drops of a clear, saturated solution of lead
monoxide iii sodium hydroxide solution and heat at 70 for
1862
IP 2010
WHITE SOFT PARAFFIN
10 minutes with frequent shaking; the mixture remains

colourless.
Light Liquid Paraffin

Light Mineral Oil; Light Liquid Petrolatum
Light Liquid Paraffin is a purified mixture ofliquid saturated
hydrocarbons obtained from petroleum. It may contain a
suitable stabiliser.
dispersed in the molten paraffin, the colour ofthe emulsion is

not darker than that of the standard mixture when shaken
vigorously.
Solid paraffins. Place a suitable quantity, previously dried by
heating at 100 for 2 hours and cooled in a desiccator over
sulphuric acid, in a glass cylindrical vessel having an internal
diameter ofapproximately 25 mm. Close the vessel and immerse
in a mixture of ice and water; after 4 hours the liquid is
sufficiently clear that a black line, 0.5 mm in width, held vertically
behind the vessel is easily seen.
Description. A transparent, colourless, oily liquid, free from

fluorescence by daylight; almost odourless when cold.
Sulphur compounds. Mix 4 ml with 2 ml of ethanol (95 per

cent), and 2 drops of a clear, saturated solution of lead
monoxide in sodium hydroxide solution and heat at 70 for
10 minutes with frequent shaking; the mixture remains
colourless.
Tests
Category. Pharmaceutical aid (vehicle).

Dynamic viscosity (2.4.28). 25 mPas to 80 mPas, determined at
20 l by method B or Viscosity (2.4.28). 10 cps to 40 cps,
determined at 30 l by method C using LVI spindle at 30
rpm.
the filtrate add 0.1 ml of phenolphthalein solution. The
pink.
Light absorption. When examined in the range 240 nm to 280
nm (2.4.7), a 2.0 per cent w/v solution in 2,2,4-trimethylpentane
shows an absorption of not more than 0.1.
Readily carbonisable substances. Place 5 ml in a dry, heatresistant glass-stoppered test-tube (125 mm x 18 mm)
previously rinsed with chromic acid solution, then with water
and dried. Add 5 ml of nitrogen-free sulphuric acid
(containing 94.5 per cent to 95.5 per cent w/w ofH 2S04), insert
the stopper and shake as vigorously as possible in the
longitudinal direction of the tube for 5 seconds. Loosen the
stopper, immediately place the tube in a bath ofboiling water,
supporting it so as to prevent contact of the tube with the
bottom or side ofthe bath and heat for 10 minutes. At the end
of the second, fourth, sixth, and eighth minutes, remove the
tube from the bath and shake as vigorously ssas possible in
the longitudinal direction of the tube for 5 seconds. At the
end of 10 minutes from the time the tube was placed in the
bath remove the tube and allow to stand for 10 minutes. The
lower acid layer is not more intensely coloured than a mixture
of3 ml ofFCS, 1.5 ml ofCCS and 0.5 ml ofCSS (2.4.1), overlaid
with 5 ml of liquid paraffin. If the sulphuric acid remains
Liquid Paraffin Emulsion

Liquid Paraffm Oral Emulsion
Liquid Paraffin Emulsion is an oral emulsion ofLiquid Paraffin
in Purified Water.
Liquid Paraffin Emulsion contains not less than 44.0 per cent
and not more than 49.0 per cent w/w ofliquid paraffin.
Category. Laxative; faecal softener.
Dose. 10 to 30 m!.
Tests
Assay. Weigh accurately about 5.0 g, add 10 ml ofwater, extract
with two quantities, each of40 ml, ofa mixture of2 volumes of
ethanol (95 per cent), 3 volumes of light petroleum (40 to
60) and 3 volumes of ether and then with 30 ml ofa mixture of
equal volumes of lightpetroleum (40 to 60) and ether. Wash
the combined extracts with 15 ml of 0.5 M sodium hydroxide
and then with 15 ml of water, evaporate the solvent, add 5 ml
of acetone and evaporate again. Repeat the addition and
evaporation of acetone until the residue is free from water,
dry at 105 for 15 minutes and weigh.
White Soft Paraffin

White Petroleum Jelly
White Soft Paraffin is a purified, semi-solid mixture of
hydrocarbons obtained from petroleum and bleached.
1863
WHITE SOFT PARAFFIN
IP 2010
Category. Pharmaceutical aid (ointment base).

Description. A white, translucent, soft unctuous mass,
retaining these characteristics on storage and when melted
. and allowed to cool without stirring; not more than slightly
fluorescent by daylight, even melted; odour1ess when rubbed
on the skin.
Tests
Melting range (2.4.21).38 to 56, determined by Method IV
solution is colourless and not more than 0.1 m1 of 0.1 M sodium
pink.
Light absorption (2.4.7). Absorbance o(a 0.05 per cent w/v
solution in 2,2,4-trimethylpentane at about 290 nm, not more
than 0.5.
Fixed oils, fats and resin. Digest 109 with 50 ml of sodium
-hydroxide solution at 100 for 30 minutes and allow the
aqueous layer to separate. On acidifYing the aqueous layer
with dilute sulphuric acid, no precipitate or oily matter is
produced.
Foreign organic matter. Volatilises when heated, without
emitting an acrid odour.
Consistency. 100 to 300, determined by the following method.
Apparatus. The apparatus is essentially in agreement with IS

4887: 1980 and comprises a penetrometer fitted with a polished
cone-shaped metal plunger weighing 150 g having a detachable
steel tip of the following dimensions. The tip of the cone has
an angle of 30, the point being truncated to a diameter of
0.38 0.08 mID, the base ofthe tip is 8.38 0.13 mID in diameter
and the length ofthe tip is 15 0.25 mID. The remaining portion
ofthe cone has an angle of 90, is 28 to 29 mm in height, and
has a maximum diameter of65.1 mID at the base. The containers
of the test are flat-bottomed metal or glass cylinders that are
102 6 mID in diameter and not less than 60 mm in height.
Procedure. Melt a sufficient quantity at a temperature below
85 and pour into one or more ofthe containers filling to within
6 mID ofthe rim. Cool to 25 2.so over a period ofnot less than
16 hours, protected from drafts. Two hours before the test,
place the containers in a water-bath at 25 0.5. If the room
temperature is below 23.5 or above 26.5, adjust the
temperature of the cone to 25 0.5 by placing it in a waterbath.
Without disturbing the surface of the substance under
examination, place the container on the penetrometer table,
and lower the cone until the tip just touches the top surface of
the test substance at a spot 25 mID to 38 mm from the edge of

the container. Adjust the zero setting and quickly release the
plunger, then hold it free for 5 seconds. Secure the plunger
and read the total penetration from the scale. Make three or
more trials, each so spaced that there is no overlapping ofthe
areas of penetration. Where the penetration exceeds 20 mID,
use a separate container of the test substance for each trial.
Read the penetration to the nearest 0.1 mm. Calculate the
average of the three or more readings and conduct further
trials to a total of 10 if the individual results differ from the
average by more than 3 per cent. The final average of the
trials is not less than 10.0 mID and not more than 30.0 mID
indicating a consistency value between 100 and 300.

Yellow Soft Paraffin

Yellow Petroleum Jelly
Yellow Soft Paraffin is a purified, semi-solid mixture of
hydrocarbons obtained from petroleum.

Description. A pale yellow to yellow, translucent, soft
unctuous mass, retaining these characteristics on storage and
when melted and allowed to cool without stirring; not more
than slightly fluorescent by daylight, even melted; odourless
when rubbed on the skin.
Tests
Melting range (2.4.21).38 to 56, determined by Method IV.
Light absorption (2.4.7). Absorbance of a 0.05 per cent w/v
solution in 2,2,4-trimethylpentane at about 290 nm, not more
than 0.75.
pink.
Fixed oils, fats and resin. Digest 109 with 50 ml of sodium
hydroxide solution at 100 for 30 minutes and allow the
aqueous layer to separate. On acidifYing the aqueous layer
with dilute sulphuric acid, no precipitate or oily matter is
produced.
Foreign organic matter. Volatilises when heated, without
emitting an acrid odour.
Consistency. 100 to 300, determined by the following method.
1864
IP 2010
PARALDEHYDE
Apparatus. The apparatus is essentially in agreement with IS

4887.1980 and comprises a penetrometer fitted with a polished
cone-shaped metal plunger weighing 150 g having a detachable
steel tip of the following dimensions. The tip of the cone has
an angle of 30, the point being truncated to a diameter of
0.38 0.08 rom, the base ofthe tip is 8.38 0.13 rom in diameter
and the length ofthe tip is 15 0.25 rom. The remaining portion
of the cone has an angle of 90, is 28 to 29 mm in height, and
has a maximum diameter of65.1 rom at the base. The containers
of the test are flat-bottomed metal or glass cylinders that are
102 6 mm in diameter and not less than ,60 mm in height.
Procedure. Melt a sufficient quantity at a temperature below
85 and pour into one or more ofthe containers filling to within
6 rom ofthe rim. Cool to 25 2S over a period ofnot less than
16 hours, protected from drafts. Two hours before the test,
place the containers in a water-bath at 25 0.5. If the room
temperature is below 23.5 or above 26.5, adjust the
temperature of the cone to 25 0.5 by placing it in a waterbath.
Without disturbing the surface of the substance under

examination, place the container on the penetrometer table,
and lower the cone until the tip just touches the top surface of
the test substance at a spot 25 mm to 38 mm from the edge of
the container. Adjust the zero setting and quickly release the
plunger, then hold it free for 5 seconds. Secure the plunger
and read the total penetration from the scale. Make three or
more trials, each so spaced that there is no overlapping of the
areas of penetration. Where the penetration exceeds 20 rom,
use a separate container of the test substance for each trial.
Read the penetration to the nearest 0.1 mm. Calculate the
average of the three or more readings and conduct further
trials to a total of 10 if the individual results differ from the
average by more than 3 per cent. The final average of the
trials is not less than 10.0 rom and not more than 30.0 mm
indicating a consistency value between 100 and 300.
Tests
Paraffin Ointment complies with the tests stated under
Ointments.
Paraldehyde
Mol. Wt. 132.7

Paraldehyde is 2,4,6-trimethyl-1 ,3,5-trioxane, the cyclic trimer
of acetaldehyde. It may contain a suitable amount of
antioxidant.
Category. Anticonvulsant in status epilepticus; hypnotic;
sedative.
Dose. By deep intramuscular injection, as a single dose, 5 to
10 ml; by rectal injection, 5 to 10 ml, suitably diluted with
physiological saline.
Description. A colourless or slightly yellow, transparent liquid;
odour, strong and characteristic. Solidifies at low temperature
to form a crystalline mass.
Identification'
A. Heat 5 ml with 0.1 ml of 1 M sulphuric acid; acetaldehyde,
B. To 5 ml ofa 10 per cent v/v solution add 5 ml ofammoniacal

silver nitrate solution in a test-tube and heat on a water-bath;
metallic silver is deposited as a mirror on the sidesofthe tube.
C. A 10 per cent w/v solution in carbon dioxide-free water is
clear (2.4.1), but becomes turbid on warming.
Tests
Congealing range (2.4.1 0). 10 to 13.
Paraffin Ointment
Distillation range (2.4.8). Not more than 10 per cent distils

below 123 and not less than 95 per cent distils below 126.
Category. Pharmaceutical aid (ointment basis).
White Beeswax
20 g
Relative density (2.4.29).0.991 to 0.996.
Hard Paraffin
30 g
Acetaldehyde. Shake 5 ml with a mixture of5 ml of ethanol

(60 per cent), 5 ml of hydroxylamine hydrochloride reagent
in ethanol (60 per cent) and 2 drops of methyl orange solution
and titrate with 0.5 M sodium hydroxide to full yellow colour;
not more than 0.8 ml of 0.5 M sodium hydroxide is required.
Cetostearyl Alcohol
50 g
White Soft Paraffin
900 g
*May be replaced by Yellow Soft Paraffin ifother medicaments

to be incorporated are coloUred.
Mix the ingredients, heat gently with stirring until
homogeneous and stir until cold.
Acidity. Mix 5 ml with 45 ml of carbon dioxide-free water and

titrate with 0.1 M sodium hydroxide using phenolphthalein
solution as indicator; not more than 1.5 ml is required.
1865
IP 2010
PARALDEHYDE
Chlorides. To 5 ml ofa 1 per cent v/v solution add one drop of

nitric acid and three drops of silver nitrate solution; no
opalescence is produced immediately.
Sulphates. To 5 ml ofa 1 per cent v/v solution add one drop of
hydrochloric acid and three drops of barium chloride
solution; no turbidity is produced.
Peroxides. In a stoppered vessel, dissolve 5 ml in sufficient of
recently boiled and cooled water to produce 50 ml, add 5 ml of
dilute sulphuric acid and 10 ml ofpotassium iodide solution.
Close the flask and set aside in the dark for 15 minutes. Titrate
with 0.1 M sodium thiosulphate using starch solution as
indicator; set aside for 5 minutes and, if necessary, complete
the titration. Not more than 2.0 ml of 0.1 M sodium
thiosulphate is required.
Non-volatile matter. Heat 5 ml in a small dish on a water-bath
and dry at 105 for 1 hour; the residue weighs not more than 3
mg (0.06 per cent w/v).
Storage. Store protected from moisture, in complete darkness
and at a temperature of8 to 15. Ifsolidified, the whole ofthe
contents of the container should be liquified by warming
before use.
NOTE - Do not use Paraldehyde if it has a brownish colour

or an odour of acetic acid. Avoid contact with rubber and
plastics.
Labelling. The label states (1) the nature and the proportion
of any antioxidant added; (2) that it may decompose on
standing to form potentially harmful substances.
Identification
Test A may be omitted if test B, C and D carried out. Test D

may be omitted if test A, Band C are carried out.
A. Determine by thin layer chromatography (2.4.17), coating

10 volumes of glacial acetic acid and 10 volumes of water.
Test solution.. Dissolve 0.25 g of the substance under
examination in 100 ml of wate]:
penicillamine RS in water.
Apply to the plate 2 III of each solution. Allow the mobile.
phase to rise 10 cm. Dry the plate at 105 for 5 to 10 minutes
and expose to iodine vapour for 5 to 10 minutes. The principal
reference solution.
B. Dissolve 0.5 g in a mixture of 0.5 ml of hydrochloric acid

and 4 ml ofwarm acetone, cool in ice and scratch the inside of
the tube with a glass rod to initiate crystallisation; a white
precipitate is produced. Filter under vacuum, wash the
precipitate with acetone and dry with suction. A 1 per cent
w/v solution of the dried material is dextrorotatory.
C. To 4 ml of a 1 per cent w/v solution add 2 ml of
phosphotungstic acid solution and heat nearly to boiling; a
blue colour is produced.
D. In the testfor Penicillamine disulphide, the principal peak
in the chromatogram obtained with test solution (b)
corresponds to the peak due to penicillamine in the
Penicillamine
D-Penicillamine
CH s 0
HsC--L - Jl
HS/
Tests
~N~~H
CsHIlNOzS
Mol. Wt. 149.2
Penicillamine is 3-mercapto-o-valine.

dioxide-free water (solution A) is clear (2.4.1), and not more
intensely coloured than degree 6 of the appropriate range of
reference solutions (2.4.1).
Penicillamine contains not less than 98.0 per cent and not
more than 101.0 per cent ofCsH II NOzS, calculated on the dried
basis.
pH (2.4.24). 4.5 to 5.5, determined ina 1.0percentw/v solution.
Category. Chelating agent in copper and lead poisoning;

antirheumatoid arthritic.
Heavy metals (2.3.13).10 ml ofsolution A, complies with the

limit test for heavy metals, Method D (20 ppm). Use lead
standard solution (2 ppm Pb) to prepare the standard.
Dose. In poisoning, 500 mg to 2 g daily, in divided doses or in

accordance with the needs of the patient. In rheumatoid
arthritis, initial dose, 125 to 250 mg daily before food, increased
gradually every 4 to 12 weeks until remission occurs; usual
maintenance dose, 500 to 750 mg daily.
Description. A white or almost white, crystalline powder.

in a 5.0 per cent w/v solution in 1 M sodium hydroxide.
Mercuric salts. Determine by atomic absorption

spectrophotometry (2.4.2), using a solution prepared in the
following manner. To 1.0 g ofthe substance under examination
add 10 ml of water and 0.15 ml of perchloric acid and swirl
until dissolution is complete. Add 1 ml of ammonium
1866
IP 2010
PENICILLAMINE
pyrrolidinedithiocarbamate solution that has been washed

three times immediately before use, each time with an equal
volume of 4-methyl-2-pentanone. Mix, add 2 ml of 4-methyl2-pentanone, shake for 1 minute, dilute to 25 mlwith water,
allow the layers to separate and use the 4-methyl-2-pentanone
layer. Measure the absorbance at 254 nm using a mercury
hollow-cathode lamp and an air-acetylene flame and setting
the zero using a 4-methyl-2-pentanone layer obtained by
repeating the procedure described above but omitting the
substance under examination. For the standard solution
dissolve 0.108 g of yellow mercuric oxide in the minimum
volume of 2 M hydrochloric acid, add sufficient water to
produce 1000.0 ml and treat suitable volumes in the same manner
as the solution ofthe substance under examination (10 ppm).
Penicillamine
disolphide.
Determine by
liquid
chromatography (2.4.14).
examination in 5 ml ofthe mobile phase, add 1 ml ofa 0.0025
per cent w/v solution of sulphanilamide (internal standard)
in the mobile phase and dilute to 10 ml with the mobile phase.
Test solution (b). Dissolve 40 mg of the substance under
examination in the mobile" phase and dilute to 10 ml with the
same solvent.
penicillamine RS in the mobile phase.
Reference solution (b). Add 1 m1 oftest solution (a) to 1 m10f
a 0.04 per cent w/v solution ofpenicillamine disulphide RS in
the mobile phase and dilute to 10 ml with the mobile phase.
- a stainless steel column 25 cm x 5 mm, packed with
octylsilane bonded to porous silica (5 to 10 Ilm),
- mobile phase: an equal volume of 0.2 per cent w/v
solution of methanesulphonic acid and 0.01 per cent
w/v solution of disodium edetate,
In the chromatogram obtained with test solution (a) the ratio
of the area of any peak corresponding to penicillamine
disulphide to the area of the peak due to the internal standard
is not greater than the corresponding ratio in the chromatogram
obtained with reference solution (b).
Penicillin. Carry out the following procedure in a penicillinfree atmosphere and with equipment reserved for the test.
Sterilise the equipment at 180 for 3 hours and the buffer
solutions at 121 for 20 minutes before use.
LiquefY a suitable nutrient medium such as that described
below and inoculate at a suitable temperature with a culture of
Micrococcus flavus (ATCC 9341) to give 5 x 104 microorganisms per ml or a quantity necessary to obtain the required
sensitivity and formation of clearly defined inhibition zones

ofsuitable diameter. Immediately pour the inoculated medium
into five Petri dishes (10 cm in diameter) to give uniform layers
2 to 5 mm in depth. Alternatively, the medium may consist of
two layers, only the upper layer being inoculated. Store the
dishes so that no appreciable growth or death of microorganisms occurs before use and so that the surface of the
medium is dry at the time of use. In each dish, place five
stainless steel hollow cylinders (6 mm in diameter) on the
surface ofthe medium evenly spaced on a circle with a radius
of about 25 mm and concentric with the dish. For each dish,
place in separate cylinders 0.15 ml of each of the following
five solutions.
For solution (1) dissolve LO g of the substance under
examination in 8 ml ofphosphate buffer pH 2.5, add 8 ml of
ether and shake vigorously for 1minute. Repeat the extraction
and combine the ether layers. Add 8 ml ofphosphate buffer
pH 2.5, shake for 1 minute, allow to settle and separate the
ether layer quantitatively, taking care to eliminate the aqueous
phase completely. (Penicillin is unstable at pH 2.5; carry out
the operations at this pH within 6 to 7 minutes). Add 8 mlof
phosphate bufferpH 6.0, shake for 5 minutes, allow to settle,
separate the aqueous layer and check that the pH is 6.0. For
solution (2) add 20 III of pr:micillinase solution to 2 ml of
solution (1) and incubate at 37 for 1 hour. For solution (3)
dissolve 5 mg of benzylpenicillin sodium in 500 ml of
phosphate buffer pH 6.0 and dilute 0.25 mlofthis solution to
200 ml with phosphate bufferpH 2.5. Carry outthe extraction
procedure described under solution (1) using 8 ml of this
solution and beginning at the words "add 8 ml ofether...". For
solution (4) add 20 ml of penicillinase solution to 2 ml of
solution (3) and incubate at 37 for 1 hour. Prepare solution (5)
in the same manner as solution (1) but omitting the substance
under examination.
Maintain the dishes at 30 for at least 24 hours. Measure the
diameters ofthe zones ofinhibition to within 0.1 mm. The test
is not valid unless solution (3) gives a clear zone of inhibition
and solutions (4) and (5) give no zones ofinhibition. Ifsolution
(1) gives a zone ofinhibition it is caused by penicillin provided
solution (2) gives no zone of inhibition. If this is the case, the
average diameter of the zones of inhibition given by solution
(1) for the five Petri dishes is less than that given by solution
(3)(0.1 ppm).
Nutrient medium
Peptone
Yeast extract
Meat extract
Sodium chloride
Agar
Distilled water
Adjust the pH to
1867
5 g
L5g
L5g
3.5g
15 g
1000 ml
6.0
IP 2010
PENICILLAMINE TABLETS
Penillic acid. Absorbance of a 0.2 per cent w/v solution at

about 268 run, not more than 0.07 (2.4.7)(about0.5 per cent).
Loss on drying (2.4.19). Not morethan 0.5 per cent, determined
at a pressure not exceeding 0.7 kPa.
Assay. Dissolve 0.1 gin 30 ml of anhydrous glacial acetic
acid Titrate with 0.1 M perchloric acid, determining the endpoint potentiometrically (2.4.25). Carry out a blank titration.
CsH11NOzS.
D-Penicillamine Tablets
Penicillamine Tablets contain not less than 95.0 per cent and
penicillamine, CsH11NOzS. The tablets are coated.
Penicillamine disulphide.
Determine
by
liquid
Test solution. Shake quantity of the powdered tablets

containing 40 mg of Penicillamine with 10 ml of the mobile
phase, filter and use the filtrate.
penicillamine disulphide RS in the mobile phase.
a stainless steel column 25 cm x 5 mm, packed with
octylsilane bonded to porous silica (5 to 10 /lm),
mobile phase: an equal volume of 0.2 per cent w/v
solution of methanesulphonic acid and 0.01 per cent
w/v solution of disodium edetate,
of any peak corresponding to penicillamine disulphide is not
greater than the area ofthe principal peak in the chromatogram
obtained with the reference solution (1.0 per cent).
Identification
quantity of the powder containing 0.1 g of Penicillamine,

dissolve as completely as possible in 50 ml of water and filter.
Add to the filtrate 5 ml of1 M sodium hydroxide and 0.2 ml of
a 0.1 per cent w/v solution of dithizone in ethanol (95 per
cent) and titrate with 0.02 M mercuric nitrate.
A. Shake a quantity ofthe powdered tablets containing 20 mg

ofPenicillamine with 4 ml of water and filter. Add to the filtrate
2 ml of phosphotungstic acid solution and allow to stand for
5 minutes; a blue colour is produced.
1 ml of 0.02 M mercuric nitrate is equivalentto 0.005968 gof

CsH11NOzS.
B. Dissolve a quantity of the powdered tablets containing

10 mg ofPenicillamine in 5 ml of water and add 0.3 ml of5 M
sodium hydroxide and 20 mg of ninhydrin; an intense blue or
violet-blue colour is produced immediately.
Tests
Mercuric salts. Disperse a quantity of the powdered tablets

containing 1 g ofPenicillamine in 10 ml of water in a stoppered
flask, add 0.2 ml of9 M perchloric acid and swirl to dissolve.
Add 1 ml of ammonium pyrrolidinedithiocarbamate solution,
mix, add 2 ml of 4-methyl-2-pentanone, shake for 1 minute and
add sufficient water to produce 25 ml. Detennine by atomic
absorption spectrophotometry (2.4.2), using a mercury hollowcathode lamp and an air-acetylene flame and setting the zero
using a 4-methyl-2-pentanone layer obtained by repeating
the procedure described above but omitting the substance
under examination, measuring at 254 run. Use mercury solution
AAS, suitably diluted with water, for the standard solutions,
adjusted to contain the same concentrations of9 M perchloric
acid, ammonium pyrrolidinedithiocarbamate solution and
4-methyl-2-pentanone as the solution under examination
(40 ppm).
Diluted Pentaerythritol Tetranitrate is a dry mixture of2,2bis(hydroxymethyl)propane-l ,3-diol tetranitrate with

Lactose or Mannitol or a mixture of Lactose and Starch or
any other suitable inert excipients which permit safe
handling.
Diluted Pentaerythritol Tetranitrate contains not less than
amount ofpentaerythritol tetranitrate, CsHgN40IZ'
1868
IP 2010
DILUTED PENTAERYTHRlTOL TETRANITRATE
Category. Antianginal.
Dose. The equivalent of 20 to 60 mg of pentaerythritol
tetranitrate, three to four times daily.
Description. A white or almost white, powder; odour, faint
and mild.
Identification
A. Transfer a quantity of powder containing 10 mg of
pentaerythritol tetranitrate to a medium porosity sintered-glass
filter, add 5 ml of dry acetone and collect the filtrate. Repeat
with two further quantities, each of 5 ml, of dry acetone and
evaporate the combined filtrate at a temperature not exceeding
60, with the aid of a gentle current of air, and dry the residue
at 60 for 4 hours; the residue melts at 138 to 142(2.4.21).
B. Suspend 10 mg ofthe residue obtained in test A in a mixture
of2 ml of sulphuric acid and 1 ml of water; cool and carefully
overlay with 3 ml offerrous sulphate solution; a reddish brown
colour is produced at the interface of the two liquids.

Mobile phase. A mixture of20 volumes of ethyl acetate and 80

volumes of toluene.
examination containing about 10 mg of Pentaerythritol
Tetranitrate in 10 ml of ethanol (95 per cent), filter.
Reference solution. Shake a quantity of diluted
pentaerythritol tetranitrate RS containing about 10 mg of
Pentaerythritol Tetranitrate with 10 ml of ethanol (95 per
cent).
phase to rise 8 cm. Dry the plate in air and examine at 254 urn,
Tests
Impurity A. Determine by thin-layer chromatography (2.4.17),
coating the plate with silica gel G.
Mobile phase. A mixture of 15 volumes of glacial acetic acid,

30 volumes of acetone and 60 volumes of toluene.
Test solution. Dissolve about 0.1 g of the substance under
examination in 5.0 ml of ethanol (95 per cent), filter.
Reference solution. Dissolve about 10 mg ofpotassium nitrate
in I ml of water and dilute to 100 ml with ethanol (95 per
cent).
phase to rise 8 cm. Dry the plate in air and spray with freshly
prepared potassium iodide and starch solution and examine

at 254 urn. The spot due to nitrate in the chromatogram obtained
cent, calculated as potassium nitrate).
(2.4.14).
Test solution (a). Shake about 25 mg of the substance under

examination in 20 ml of the mobile phase for 15 rp.inutes and
dilute to 25.0 ml with the mobile phase, filter.
Reference solution (a). Dissolve a quantity of diluted
pentaerythritol tetranitrate RS containing about 25 mg of
Pentaerythritol Tetranitrate in 20 ml of the mobile phase,
sonicate for 15 minutes and dilute to 25.0 ml with the mobile
phase.
Reference solution (c). Dilute 0.3 ml ofreference solution (b)
Reference solution (d). Dilute 200 III of glyceryl trinitrate
solution RS to 25.0 ml with the mobile phase.
Reference solution (e). To 1 ml of reference solution (b), add
1 ml of reference solution (d) and dilute to 10.0 ml with the
mobile phase.
Reference solution (f). Dilute 1.0 ml ofreference solution (a)
to 20.0 ml with the mobile phase. Dilute 0.5 ml ofthis solution
octylsilane bonded to porous silica (5 11m),
- mobile phase: a mixture of 35 volumes of water and 65
Inject reference solution (e). The test is not valid unless the
resolution between the peaks due to glyceryl trinitrate and
pentaerythritol tetranitrate is not less than 3.0. The relative
retention time with reference to pentaerythritol tetranitrate for
pentaerythritol tetranitrate impurity B is about 0.7 and for
pentaerythritol tetranitrate impurity C is about 0.3.
Inject test solution (a), reference solution (c) and (t). Run the
In the chromatogram obtained with test solution (a) the area
ofthe peak due to pentaerythritol tetranitrate impurity C is not
1869
PENTAERYTHRITOL TETRANlTRATE TABLETS
IP 2010
obtained with reference solution (c) (0.3 per cent), the area of
any other secondary peak is not more than not more than
obtained with reference solution (t) (0.1 per cent). The sum of
all the secondary peaks is not more than twice the area of the
solution (c) (0.6 per cent). Ignore any peak with an area less
obtained with reference solution (t) (0.05 per cent).
Heavy metals (2.3.13). 2.0 g complies with limit test for heavy
metals, Method B (10 ppm).
Assay. Determine by liquid chromatography (2.4.14), as
described in the test for Related substances.
Inject test solution (b) and reference solution (b).
Calculate the content ofCsHsN4012.
NOTE - Undilutedpentaelythritol tetranitrate is a powerfUl
explosive. It can be exploded with percussion or excessive
heat. Great care and appropriate precautions should be
taken in handling and only exceedingly small amounts should
be isolated.
Labelling. The label states the percentage content of
pentaerythritol tetranitrate.

Pentaerythritol Tetranitrate Tablets contain not less than
amount ofpentaerythritol tetranitrate, CsHsN 40 12
Identification
A. Transfer a quantity of powder containing 10 mg of
pentaerythritol tetranitrate to a medium porosity sintered-glass
filter, add 5 ml of dry acetone and collect the filtrate. Repeat
with two further quantities, each of 5 ml, of dry acetone and
evaporate the combined filtrate at a temperature not exceeding
60, with the aid of a gentle current of air, and dry the residue
at 60 for 4 hours; the residue melts at 138 to 142 (2.4.21).
Tests
Uniformity of content. (For tablets containing 10 mg or less)
- Comply with the test stated under Tablets.
Crush one tablet and transfer to a 50-ml volumetric flask with
the aid of 15 m1 ofacetone. Add sufficient acetone to produce
25 ml, heat the mixture on a water-bath at a temperature not
exceeding 60 and boil gently, with occasional swirling, for
5 minutes. Cool, dilute to volume with acetone and mix.
Transfer a portion of the mixture to a glass-stoppered

centrifuge tube and centrifuge at 1500 rpm for 5 minutes.
Transfer 2.5 ml of the supernatant solution to a 100-ml
volumetric flask and evaporate at 35 with the aid ofa current
of air to dryness. To the residue add 1.0 m1 of glacial acetic
acid and swirl to dissolve. Add 2 ml of phenoldisulphonic
acidsolution, mix and allow to stand for 5 minutes. Add 25 ml
ofwater and 10 ml ofstrong ammonia solution, cool, dilute to
volume with water and mix. Measure the absorbance of the
using water as the blank.
Weigh accurately 0.130 g of potassium nitrate, previously
dried at 105 for 4 hours, dissolve in 3 ml ofwater, dilute with
sufficient glacial acetic acid to produce 200.0 ml and mix
well. Using 1.0 ml of this solution repeat the procedure
beginning at the words "Add 2 ml ofphenoldisulphonic acid
solution,.......".Calculate the content ofCsHsN4012 in the tablet.
1 ml ofthe potassium nitrate solution is equivalent to 0.000503
g ofCsHsN40'2'
pentaerythritol tetranitrate and transfer to a 100-m1 volumetric
flask with the aid of about 30 ml of acetone. Add sufficient
acetone to produce 50 ml and warm on a water-bath at a
temperature not exceeding 60 and boil gently, with occasional
swirling, for 5 minutes. Cool, dilute to volume with acetone
and mix. Transfer a portion ofthe mixture to a glass-stoppered
centrifuge tube and centrifuge at 1500 rpm for 5 minutes.
Transfer 1.0 ml of the supernatant solution to a 100-ml
volumetric flask and evaporate at 35 with the aid ofa current
of air to dryness. To the residue add 1.0 ml of glacial acetic
acid and swirl to dissolve. Add 2 ml of phenoldisulphonic
acidsolution, mix and allow to stand for 5 minutes. Add 25 ml
ofwater and 10 ml ofstrong ammonia solution, cool, dilute to
volume with water and mix. Measure the absorbance of the
resulting solution at the maximum at about 409 urn (2.4.7),
Weigh accurately 0.13 g ofpotassium nitrate, previously dried
at 105 for 4 hours, dissolve in 3 ml of water, dilute. with
sufficient glacial acetic acid to produce 200.0 mland mix
well. Using 1.0 ml of this solution repeat the procedure
beginning at the words "Add 2 ml ofphenoldisulphonic acid
solution,.......".
Calculate the content of CsHsN40 12 from the values of the
absorbances so obtained.
1 ml of the potassium nitrate solution is equivalent to
0.000503 g ofCsHsN40 12
equivalent amount of pentaerythritol tetranitrate.
1870
IP 20ra
PENTAMIDINE ISETHIONATE
Pentamidine Isethionate
and dilute to 50 ml with water. Dilute 1.0 ml ofthe solution to

Mol. Wt. 592.7

Pentamidine Isethionate is 4,4' -[pentane-l ,5-diylbis(oxy)]
bisbenzenecarboximidamide di(2-hydroxyethanesulphonate).
Pentamidine Isethionate contains not less than 98.5 per cent
and not more than 101.0 per cent ofC19H24N402, 2C2H 60 4S,
Category. Antiprotozoal.
Dose. By intramuscular injection, 3 to 4 mg per kg body weight
daily for 10 days, maximum 200 mg.
Description. A white or almost white powder or crystals;
odourless or almost odourless; hygroscopic.
Identification
Compare the spectrum with that obtained with pentamidine
isethionate RS or with the reference spectrum ofpentamidine
isethionate.
octadecylsilane bonded to porous silica (5 ilm),
35 volumes of 3 per cent w/v solution of ammonium
acetate, adjusted to pH 7.5 with triethylamine,
- injection volume. 10 ill.
resolution between the 2 principal peaks is not less than 2.0.
peale In the chromatogram obtained with the test solution,
the area of any secondary peak is not more than the area of
solution (a) (0.2 per cent), the sum of all the secondary peaks
Tests
Ammonium isethionate. To 1.0 g in a test-tube (about 4 cm in

diameter) add 10 ml of water and 20 ml of 1 M sodium
hydroxide. Immediately attach a bung carrying a splash head
and an aspirator tube (about 5 mm in diameter).. Connect the
splash head to two test-tubes in series, each containing 20 ml
of 0.01 M sulphuric acid. Heat the tube containing the
substance under examination in a water-bath at 45 to 50 and,
maintaining this temperature, draw a current ofair, previously
passed through 1 M sulphuric acid, through the liquids in a
series of tubes for 3 hours at such a rate thatthe bubbles are
just too rapid to count. Titrate the combined solutions from
the two absorption tubes with 0.02 M sodium hydroxide using
methyl red-methylene blue solution as indicator; not less than
36.5 ml of 0.02 M sodium hydroxide is required.
pH (2.4.24). 4.5 to 6.5, determined in a 5.0 percentw/v solution.

(2.4.14).

Assay. Dissolve 0.250 g in 50 ml of dimethylformamide. Add

0.25 ml of thymol blue solution and titrate with 0.1 M
tetrabutylammonium hydroxide, under a current of nitrogen,
until the colour of the indicator changes to blue. Carry out a
blank titration.
B. When examined in the range 230 om to 360 nm (2.4.7), a

0.001 per cent w/v solution in 0.01 Mhydrochloric acid shows
an absorption maximum only at about 262 om; absorbance at
about 262 om, about 0.46).
C. To 10 ml ofa 0.05 per cent w/v solution add 1 ml ofa 0.1 per
cent w/v solution of glyoxal sodium bisulphite and 1 ml of a
solution prepared by dissolving 4 g of boric acid in a mixture
of 27 ml of 1 M sodium hydroxide and sufficient water to
produce 100 ml. Heat on a water-bath for 10 minutes; a magenta
colour is produced.

Reference solution (b). To 0.1 g in a conical flask, add 40 ml of
water and glass beads, adjusted to pH 10.5 with dilute sodium
hydroxide solution and boil under reflux for 20 minutes. Cool

0.02963 g ofC23H36N401OS2.
1871
PENTAMIDINE INJECTION
IP 2010
Reference solution (b). Dissolve 0.1 g of substance under

examination in 40 ml of water, adjusted to pH 10.5 with 2 M
Pentamidine Isethionate Injection
sodium hydroxide, heat under a reflux condenser for 20
Pentamidine Injection is a sterile material consisting of minutes, cool and dilute to 50 ml with water. Dilute 1.0 m1 of
Pentamidine Isethionate with or without buffering agents and this solution to 50.0 ml with the mobile phase.
other excipients. It is filled in a sealed container.
Pentamidine Injection

octadecylsilane bonded to porous silica (5 f!m) (Such
as Spherisorb ODS 1), .
mobile phase: a mixture of 130 volumes of methanol and
70 volumes of3 per cent w/v solution of ammonium
acetate, adjusted to pH 7.5 with triethylamine,
injection volume. 10 f!l.
The injection is constituted by dissolving the contents of the


Storage. The constituted solution should be used immediately
after preparation but, in any case, within the period
recommended by the manufacturer.
Pentamidine Injection contains not less than 95.0 per cent and
pentamidine isethionate, C19Hz4N40z, 2CzH 60 4S.

(Powders for Injection) and with thefollowing requirements.
Identification
Compare the spectrum with that obtained with pentamidine
isethionate RS or with the reference spectrum ofpentamidine
isethionate.
2.0.
area of any secondary peak is not more 0.2 times the area of
solution (a) (0.2 per cent) and the sum of the areas of all the
principal peak in the chrotnatogram obtained with reference
solution (a) (0.4 per cent).
Ammonium isethionate. To 1.0 g in a test-tube (about 4 cm in

diameter) add 10 ml of water and 20 ml of 1 M sodium
B. When examined in the range 230 nm to 360 nm (2.4.7), a hydroxide. Immediately attach a bung carrying a splash head
0.001 per cent w/v solution in 0.01 M hydrochloric acid shows and an aspirator tube (about 5 mm in diameter). Connect the
an absorption maximum only at about 262 nm; absorbance at splash head to two test-tubes in series, each containing 20 ml
of 0.01 M sulphuric acid. Heat the tube containing the
C. To 10ml ofaO.05 per cent w/v solution add 1 m1 ofaO.1 per substance under examination in a water-bath at 45 to 50 and,
cent w/v solution of glyoxal sodium bisulphite and 1 ml of a maintaining this temperature, draw a current ofair, previously
solution prepared by dissolving 4 g of boric acid in a mixture passed through 1 M sulphuric acid, through the liquids in a
of 27 ml of 1 M sodium hydroxide and sufficient water to series of tubes for 3 hours at such a rate that the bubbles are
produce 100 ml. Heat on a water-bath for 10 minutes; a magenta just too rapid to count. Titrate the combined solutions from
the two absorption tubes with 0.02 M sodium hydroxide using
colour is produced.
methyl red-methylene blue solution as indicator; not less than
36.5 ml of 0.02 M sodium hydroxide is required.
Tests
pH (2.4.24). 4.5 to 6.5, determined in a5.0 per centw/v solution.
(2.4.14).
Test solution. Dissolve 0.1 g of substance under examination

in 100.0 ml ofthe mobile phase.
Reference solution (a). Dilute 1 ml ofthe test solution to 100
Assay. Dissolve 0.25 g ofthe mixed contents of 10 containers

in 50 ml of dimethylformamide. Titrate with 0.1 M
tetrabutylammonium hydroxide, determining the end-point
0.02963 g ofC,9Hz4N40z, 2CzH 60 4S.
Storage. Store in single dose containers.
1872
IP 2010
PENTAZOCINE HYDROCHLORIDE
Pentazocine

substance 'under examination in chloroform.
Reference solution (c). A 0.005 per cent w/v solution of the
Mol. Wt. 285.4

Pentazocine is (2RS, 6RS, llRS)-6, II-dimethyl-3-(3-methylbut2-enyl)-1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocin-8-01.
Pentazocine contains not less than 98.0 per cent and not more
than 101.0 per cent ofC I9H27NO, calculated on the dried basis.

Heat the plate at 105 for 15 minutes, allow to cool, expose to
iodine vapour and re-examine in ultraviolet light at 254 nm.
chromatogram obtained with reference solution (a); not more
than one such spot is more intense than the spot in the
chromatogram obtained with reference solution (b) and not
more than four such spots are more intense than the spot in
the chromatogram obtained with reference solution (c).
Category. Narcotic analgesic.
Dose. By subcutaneous, intramuscular or intravenous

injection, 30 to 60 mg every 3 to 4 hours.
Description. A white or pale cream powder.
Identification
A Determine by infrared absorption spectrophotometry (2.4.6).
Compare the spectrum with that obtained with pentazocine
RS or with the reference spectrum ofpentazocine.
B.To 1 mg in a porcelain crucible add 0.5 ml of a solution of
sulphuric acid containing 1 per cent w/v solution of
ammonium molybdate; an intense blue colour is produced
which changes to bluish green, green and finally, on standing,
yellow.
C. Dissolve 5 mg in 5 ml ofsulphuric acid, add 0.05 mlofferric
chloride solution and mix; a yellow colour is produced which
deepens slightly in intensity on warming. On the addition of
0.05 ml ofnitric acid the yellow colour is unchanged.

anhydrous glacial acetic acid and titrate with 0.1 M perchlpric
blank titration.
C I9H27NO.
Tests
Light absorption (2.4.7). To 0.1 g add 20 ml ofwater and 10 ml
of 1 M hydrochloric acid, shake to dissolve and add sufficient
water to produce 100 ml. Dilute 10 ml to 100 ml with water. The
278 nm, 0.67 to 0.71.
3 volumes of 2-propylamine and 3 volumes of methanol.
Referencesolution (a). A 0.02 per cent w/v solution of the
C I9H27NO,HCl
Mol. Wt. 321.9
Pentazocine Hydrochloride is (2RS,6RS, llRS)-6, 11dimethyl-3-(3-methylbut-2-enyl)-1,2,3,4,5,6-hexahydro-2,6methano-3-benzazocin-8-01 hydrochloride.

Pentazocine Hydrochloride contains not less than 98.0 per
cent and not more than 101.0 per cent of C I9 H27NO,HCl,
Dose. 25 to 100 mg, every 3 to 4 hours after food.
1873
IP 2010
PENTAZOCINE HYDROCHLORIDE
Description. A white or pale cream-coloured, crystalline

powder; odourless. The material exhibits polymorphism.
0.7kPa.
Identification

blank titration.

pentflzocine hydrochloride.
B. To 1 mg in a porcelain crucible add 0.5 ml ofa solution of
yellow.

C I9H27NO,HCl.
Pentazocine Tablets
Pentazocine Hydrochloride Tablets

Tests
pH (2.4.24). 4.0 to 6.0, determined ina 1.0 per centw/v solution.
Light absorption (2.4.7). Dissolve 0.1 g in 10 ml of 1 M
hydrochloric acid and add sufficient water to produce
100 ml; dilute 10 ml to 100 ml with water. Absorbance ofthe
resulting solution at the maximum at about 278 urn, 0.59 to
0.63.
Mobile phase. A mixture of 94 volumes of chlorofor.m,

iodine vapour and re-examine in ultraviolet light at 254 urn.
Pentazocine Tablets contain not less than 92.5 per cent and
pentazocine hydrochloride, C I9H 27NO,HCl.
Identification
A. Shake a quantity of the powdered tablets containing 0.1 g
of Pentazocine Hydrochloride with 10 ml of water, filter, add
1 ml of1 M sodium hydroxide and shake the resulting solution
with 20 ml of chloroform. Wash the chloroform extract with
5 ml of water, dry over anhydrous sodium sulphate and filter.
Evaporate the chloroform using a rotary evaporator and dry
the oily residue at a temperature not exceeding 25 at a pressure
of2 kPa for 1 hour.
obtained with pentazocine RS or with the reference spectrum
of pentazocine.
B. To a quantity of the powdered tablets containing 50 mg of
Pentazocine Hydrochloride add 70 ml of water, shake for
15 minutes, add sufficient water to produce 100 ml and filter.
To 10 ml ofthe filtrate add 10 ml of1 M sodium hydroxide and
sufficient water to produce 100 ml. When examined in the
range 230 nm to 360 urn (2.4.7), the resulting solution shows
C. Shake a quantity ofthe powdered tablets containing 25 mg

ofPentazocine Hydrochloride with 5 ml of water and 0.5 ml of
2 M nitric acid for 1 minute and filter. The filtrate gives reaction
A ofchlorides (2.3.1).
Tests
1874
IP 2010
PENTAZOCINE LACTATE

containing 0.2 g ofPentazocine Hydrochloride with 10 ml of
0;1 M methanolic ammonia for 10 minutes, centrifuge and
use the supernatant liquid.
Reference solution (a). Dilute 1 volume of test solution to
100 volumes with the same solvent.
Reference solution (b). Dilute 1 volume of test solution to
Reference solution (c). Dilute 1 volume of test solution to
Pentazocine Lactate contains not less than 98.5 per cent and
not more than 101.0 per cent ofCI9H27NO,C3H603, calculated
on the dried basis.
Dose. By subcutaneous, intramuscular or intravenous
injection, the equivalent ono to 60 mg ofpentazocine every 3
to 4 hours.
Description. A white or pale cream-coloured, crystalline
powder; odourless.
Identification
lactate RS or with the reference spectrum of pentazocine
lactate.

chromatogram obtained with reference solution (a), not more
more than four such spots are mo~e intense than the spot in
B. To 1 mg in a porcelain crucible add 0.5 ml of a solution of

yellow.
pH (2.4.24). 5.5 to 6.5, determined in a 1.0percentw/v solution.

quantity ofthe powder containing about 25 mg ofPentazocine
Hydrochloride, shake with 100 ml ofwater for 15 minutes, add
2.5 ml of 1 M hydrochloric acid and sufficient water to
produce 250.0 ml and filter. Measure the absorbance of the
filtrate at the maximum at about 278 nm (2.4.7). Calculate the
content ofC I9H27NO,HCl taking 61.2 as the specific absorbance
at278nm.
Light absorption (2.4.7). Dissolve 0.1 g in 10 ml of 1 M

hydrochloric acid and add sufficient water to produce
100 ml; dilute 10 ml to 100 ml with water. Absorbance ofthe
resulting solution at the maximum at about 278 nm, 0.50 to
0.54.

HO



H,C"'. CH N-"'=t,
3
Tests

Pentazocine Lactate
C. Gives reaction A oflactates (2.3.1).

CH 3
Mol. Wt. 375.4

Pentazocine Lactate is (2RS, 6RS, llRS)-6, ll-dimethyl-3-(3methylbut-2-enyl)-1,2,3,4,5,6-hexahydro-2,6-methano-3benzazocin-8-ol1actate.

1875
PENTAZOCINE INJECTION
IP 2010

Su.phated ash (2.3.18). Not more than 0.1 per cent.
Assay. Weigh accurately about 0.75 g of the substance under
examination, dissolve in 50 ml of anhydrous glacial acetic
acid. Titrate with 0.1 M perchloric acid, using crystal violet
CI9H27NO,C3H603.
C. To a volume containing 30 mg ofpentazocine add 2 ml of

0.1 M sodium hydroxide, extract with 2 ml of chloroform and
evaporate 0.1 ml of the chloroform extract to dryness in a
porcelain crucible. Add to the residue 0.5 ml of a 1 per cent
w/v solution of ammonium molybdate in sulphuric acid; an
intense blue colour is produced which changes to bluish green,
green and finally, on standing, yellow.
Tests
pH (2.4.24). 4.0 to 5.0.

Test solution. Dilute a volume ofthe injection with sufficient

ethanol (95 per cent) to produce a solution containing
2.0 per cent w/v solution ofpentazocine.
Pentazocine Injection

100 volumes with ethanol (95 per cent).
Pentazocine Lactate Injection

Pentazocine Injection is a sterile solution in Water for
Injections ofeither Pentazocine Lactate or pentazocine lactate
prepared by the interaction of Pentazocine and Lactic Acid.
Pentazocine Injection contains not less than 95.0 per cent and
pentazocine, C I9 H27NO.
Usual strength. The equivalent of30 mg of pentazocine per
ml; the equivalent of60 mg ofpentazocine per ml.
Description. A clear, colourless or almost colourless liquid.
Identification
A. To a volume containing 90 mg ofpentazocine add 5 ml of
0.1 M sodium hydroxide and shake the resulting solution
with 5 ml of chloroform. Wash the chloroform extract with 2 ml
of water, dry over anhydrous sodium sulphate and filter.
Evaporate the chloroform without applying heat and dry the
oily residue at a temperature not exceeding 25 and at a pressure
of2 kPa for 1 hour.

obtained with pentazocine RS or with the reference spectrum
ofpentazocine (form B).

.
Reference solution (c). Dilute 1 volume ofthe test solution to
iodine vapour and re-examine in ultraviolet light at 254 urn.
chromatogram obtained with reference solution (a), not more
0.15 g of pentazocine add sufficient water to produce
100.0 ml. To 5.0 ml add 1 ml of 1 M hydrochloric acid and
sufficient water to produce 100.0 ml. Measure the absorbance
ofthe resulting solution atthe maximum at about 278 urn (2.4.7).
Calculate the content of C I9H27NO, taking 69 as the specific_
absorbance at 278 urn.
B. Dilute a volume containing 30 mg ofpentazocine to 100 ml

with water. To 10 ml add 10 ml of 1 M sodium hydroxide and Storage. Store protected from light.
sufficient water to produce 100 ml. When examined in the
range 230 urn to 360 urn (2.4.7), the solution shows absorption. Labelling. The label states the strength in terms of the
equivalent amount ofpentazocine in a suitable dose-volume.
maxima, at about 238 urn and 298 nm.
1876
PENTOBARBITONE TABLETS
IP 2010

under examination.
Soluble Pentobarbitone; Pentobarbital Sodium

substance under examination.
Do not ignore any spot remaining on the line of application.
Mol. Wt. 248.3
Free pentobarbitone. Not more than 3.5 per cent, determined

by the following method. Weigh accurately about 2.0 g and
dissolve in 75 ml of dimethylformamide, heating gently if
necessary. Add 0.25 ml of a 1 per cent w/v solution of thymol
blue in dimethylformamide and titrate with 0.1 M sodium
methoxide to a blue end-point. Carry out a blank titration.
Pentobarbitone Sodium is sodium 5-ethyl-5-[(IRS)-Imethylbutyl]barbiturate.
1 ml of 0.1 M sodium methoxide is equivalent to 0.02263 g of

pentobarbitone.
Pentobarbitone Sodium contains not less than 99.0 per cent

and not more than 101.5 per cent ofC\\HI7N2Na03, calculated
on the dried basis.
Isomer. Dissolve 0.3 gin 5 ml of a 5 per cent w/v solution of

anhydrous sodium carbonate and add 0.3 g of 4-nitrobenzyl
bromide dissolved in 10 ml of ethanol (95 per cent). Heat
under a reflux condenser for 30 minutes, cool to 25 scratch
the side of the vessel with a glass rod if necessary to induce
crystallisation and filter. Wash the residue with five quantities,
each of 5 ml, of water. Transfer the residue as completely as
possible to a small flask, add 25 ml of ethanol (95 per cent)
and heat under a reflux condenser for 10 minutes; the solid
dissolves completely. Cool to 25 and scratch the side of the
flask with a glass rod to induce crystallisation. Filter, wash the
residue with two quantities, each of 5 ml, of water and dry at
105 for 30 minutes. The dried residue melts at 136 to 148
(2.4.21).
Category. Hypnotic.,
Description. A white, crystalline powder or granules;
hygroscopic.
Identification
A. To 10 mlofa 10 per cent w/v solution add 5 mlof2 M acetic
acid; a white, crystalline precipitate is produced. Filter, wash
the precipitate with water and dry at 105. Determine the
melting point ofthe dried precipitate (2.4.21). Mix equal parts
ofthe dried precipitate and phenobarbitone RS and determine
the melting point (2.4.21). The difference between the melting
points (which are about 131) is not greater than 2.
B. Complies with the test for identification of barbiturates

(2.3.1), using the dried precipitate obtained in test A for
preparing the test solution.
.C. To 10 mg add 10 mg of vanillin and 2 ml of sulphuric acid,
mix and heat on a water-bath for 2 minutes; a reddish-brown
colour is produced. Cool and add 5 ml of ethanol; the colour
changes to violet and then blue.
D. Ignite 1 g; the residue gives reaction A of sodium salts
(2.3.1).
12.75 per cent w/v solution of silver nitrate in pyridine and
titrate with 0.1 M ethanolic sodium hydroxide using 0.5 mlof
thymolphthalein solution as indicator, until a pure blue colour
is obtained. Carry out a blank titration.
1 ml of 0.1 Methanolic sodium hydrOXide is equivalent to
0.02483 g ofC\\HI7N2Na03'
Tests
Appearance of solution. Prepare freshly a 10.0 per cent w/v
solution in carbon dioxide-free water (solution A). Solution
A is clear (2.4.1).
pH (2.4.24). 9.6 to 11.0, determined in solution A.
Pentobarbitone Sodium Tablets; Pentobarbital Sodium

Tablets
Related substances. Complies with the test for related

substances in barbiturates (2.3.4), but applying 10 III of each
of the following solutions.
Pentobarbitone Tablets contain not less than 92.5 per cent

pentobarbitone sodium, C\\HI7N2Na03.
1877
PENTOBARBITONE TABLETS
IP 2010
Pepsin
Identification
Pepsin is obtained from the gastric mucosa of pigs, cattle or

sheep. It contains gastric proteinases that are active in an
acid medium, pH 1 to 5. It may contain a suitable diluent such
as Lactose.

of Pentobarbitone Sodium with 10 ml of a 10 per cent w/v
solution ofpyridine and filter. Add to the filtrate 1 ml ofcupric
sulphate with pyridine solution and set aside for 10 minutes;
a reddish violet precipitate is produced.
B. Shake a quantity of the powdered tablets containing 0.1 g

of Pentobarbitone Sodium with 10 ml of water and filter. To
the filtrate add 2 ml of hydrochloric acid; a white precipitate
is produced (distinction from pentobarbitone).
Pepsin has an activity equivalent to its ability to digest not

less than 3000 times its weight of coagulated egg albumin
when determined by the method given under Assay.
Category. Proteolytic enzyme.
Dose. 300 mg to 1 g.
C. The residue obtained in the Assay melts at 127 to

130(2.4.21).
Description. A white or light buff-coloured, crystalline or

amorphous powder or translucent scales; odour, faint and
meaty but not rancid; hygroscopic.
D. The powdered tablets, when moistened with hydrochloric

acid and introduced on a platinum wire into a flame, impart a
yellow colour to the flame.
Identification
Tests
Isomer. Dissolve a quantity ofthe powdered tablets containing
0.3 g of Pentobarbitone Sodium in 5 ml of a 5 per cent w/v
solution of anhydrous sodium carbonate and add 0.3 g of
4-nitrobenzyl bromide dissolved in 10 ml of ethanol (95 per
cent). Heat under a reflux condenser for 30 minutes, cool to
25 scratch the side of the vessel with a glass rod ifnecessary
to induce crystallisation and filter. Wash the residue with five
quantities, each of 5 ml, of water. Transfer the residue as
completely as possible to a small flask, add 25 ml of ethanol
(95 per cent) and heat under areflux condenser for 10 minutes;
filter the hot solution. Cool to 25 and scratch the side of the
flask with a glass rod to induce crystallisation. Filter, wash the
residue with two quantities, each of 5 ml, of water and dry at
105 for 30 minutes. The dried residue melts at 136 to 148
(2.4.21).
B. The proteolytic activity of a solution in water is destroyed

at once by boiling. It is destroyed by warming for 10 minutes
at 40 at a pH of8.0.
Tests

quantity of the powder containing about 0.3 g of
Pentobarbitone Sodium, dissolve as completely as possible
in 10 ml of a 2 per cent w/v solution of sodium hydroxide,
saturate with sodium chloride, acidify with hydrochloric acid
and extract with successive quantities, each of 15 ml, of ether
until complete extraction is effected. Wash the combined
extracts with two quantities, each of2 ml, ofwater and extract
the combined washings with 10 ml of ether. Add the ether to
the main ether layer, filter and wash the filter with ether.
Evaporate the solvent and dry the residue to constant weight
atl05.
1 g ofresidue is equivalent to 1.097 g ofCIIHI7N2Na03'
A. Place 1 ml of congo redfibrin on a filter paper and wash

until a colourless filtrate is obtained with a solution prepared
by diluting 30 ml of 1 M hydrochloric acid to 1000 ml with
water and adjusting the pH 1.5 to 1.7. Perforate the filter paper
and wash the congo red fibrin through it with 20ml of the
same hydrochloric acid solution. Shake this suspension before
use. Dissolve about 10 mg ofthe substance under examination
in 2 ml ofthe hydrochloric acid solution and adjust the pH 1.5
to 1.7. Place 4 ml ofthe congo red fibrin suspension in each of
two tubes. To one of the tubes add 1 m1 ofthe solution of the
substance under examination and to the other tube add 1 mlof
water (control solution). Mix the contents of each tube and
place ina water~bathat 25 with gentle shaking for 15 minutes;
the control solution is eoi()urless and the solution of the
substance under examination is violet blue.
Microbial contamination (2.2.9). 1 g is free from Escherichia

coli and 109 is free from Salmonellae.
Assay. Weigh accurately 0.25 g, triturate with 1.0 g ofsodium
chloride, add slowly acidified water prepared by diluting
65 ml of1 M hydrochloric acidto 1000 ml with water, continue
the trituration, dilute to 1000.0 ml with the acidified water and
shake for 15 minutes. Prepare coagulated egg albumin by
boiling fresh hen-eggs in water for 15minutes, cooling rapidly
to room temperature by immersion in cold water, separating
1878
IP 2010
PERlTONEAL DIALYSIS SOLUTIONS
the whites and rubbing through a no. 44 sieve. Reject the first
portion that passes through the sieve and triturate 15.0 g of
freshly prepared coagulated egg albumin with 50 ml of the
acidified water ensuring that the particles of egg albumin are
thoroughly disintegrated, add a further 50 ml of the acidified
water and keep in a water-bath at 51 0 10 for 15 minutes. Add
20.0 ml of the prepared solution of the substance under
examination and digest at 51 0 10 for 4 hours, shaking at
intervals of 15 minutes. Centrifuge and decant offmost ofthe
clear supernatant liquid, wash the remainder into a 10-ml
graduated cylinder and allow to stand for 30 minutes. The
volume of the undissolved albumin is not more than 2 ml.
Description. Clear, colourless or faintly straw-coloured

solutions.
Identification
A. To 5 ml of the solution under examination, add 2 ml of
dilute sodium hydroxide solution and 0.05 ml of potassium
cupri-tartrate solution; the solution remains blue and clear.
Heat to boiling; a copious red precipitate is formed.
B. 20 ml gives reactions of chlorides, sodium salts, potassium
salts and calcium salts (2.3.1).
C. To 5 ml add 1 ml of hydrochloric acid in a test-tube fitted

with a stopper and a bent tube, heat and collect a few ml ofthe
distillate. The distillate gives reaction C of acetates (2.3.1).

Intraperitoneal Dialysis Fluids
Peritoneal Dialysis Solutions are sterile preparations for
intraperitoneal use containing electrolytes with a concentration
close to the electrolytic composition ofplasma. They contain
dextrose in varying concentrations and/or other suitable
osmotic agents. They do not contain antioxidants such as
metabisulphite salts.
Peritoneal Dialysis Solutions contain not less than 97.5 per
sodium, Na, not less than 95.0 per cent and not more than
105.0 per cent ofthe stated amounts ofpotassium, K, calcium,
Ca, magnesium, Mg, chloride, Cl, acetate, C2H3 0 2 , lactate,
C3H;03, sodium bicarbonate, NaHC03, bicarbonate, HC0 3and
dextrose,C6H 120 6
Usual strengths: Several formulations are used. The
concentrations of the components per litre of solution are
usually in the following range.
Concentration in
mmolllitre
Sodium
Potassium
Calcium
Magnesium
Acetate and/or
Lactate and/or
Bicarbonate
CWoride
Dextrose
Unless otherwise justified and authorised, antioxidants such

as metabisulphite salts are not added to the solutions.
125-150
0-4.5
0-2.5
0.25- 1.5
30-60
90-120
25-250
When bicarbonate is present, the solution of sodium

bicarbonate is supplied in a separate container or a separate
compartment and is added to the electrolyte solution
immediately before use.
D. ToO.l ml of titan yellow solution add lOmlofwater,2mlof

the solution under examination and 1 mt of 1 M sodium
hydroxide; a pink colour is produced if magnesium salts are
present.
E. Lactates and bicarbonates are identified together with ,the
Assay for lactate and bicarbonate.
Tests
Appearance of solution. The solution under examination is
clear (2.4.1), and not more intensely coloured than reference
solution YS4 (2.4.1).
pH (2.4.24). 4.5 to 6.5. Ifthe solution contains bicarbonate, 6.5
to 8.0.
5-Hydroxymethylfurfural and Related substances. Dilute a
volume containing 1.0 g of Dextrose to 250.0 ml with water
and measure the absorbance of the resulting solution (2.4.7)
at the maximum at about 284 urn; absorbance at'about 284 urn,
not more than 0.25.
Aluminium. Adjust the pH of 400 ml of the solution under
examination to pH 6.0 and add 10 ml of acetate bufferpH 6.0.
Extract the resulting solution with successive quantities of
20, 20 and 10 ml of a 0.5 per cent w/v solution of
8-hydroxyquinoline in chloroform and dilute the combined
extracts to 50.0 ml with chloroform. Use as the blank a mixture
of lO ml of acetate bufferpH 6. 0 and 100 ml of water treated in
the same manner and as the standard solution a mixture of
2.0 ml of aluminium standard solution (2 ppm AI), 10 ml of
acetate buffer pH 6.0 and 90 ml of water treated in the same
manner. Measure the fluorescence of the test solution (II), of
the standard solution (12) and ofthe blank (13), (2.4.5), using an
excitation wavelength of392 nm and a secondary ftlterwith a
transmission band centred at 518 urn, or a monochromator set
to transmit at this wavelength. The fluorescence of the teSt
solution (I, - 13) is not greater than that ofthe standard solution
(I2 - 13),
1879
PERITONEAL DIALYSIS SOLUTIONS
IP 2010
Particulate contamination (2.5.9). Carry out the test using

50 ml ofthe solution under examination.
solution as indicator until a reddish yellow colour is produced.

The preparation meets the requirements ofthe test ifit contains

particles within the maximum limits shown below.
1 ml of 0.1 M silver nitrateisequivalent to 0.003545 g oftota1

chloride, calculated as Cl.
Particle size in /lm

(Equal to or larger than)
For acetate (if present) chromatography (2.4.14).
Maxirnumnumber
ofparticles per ml
10
25
25
Determine by liquid

preparation under examination quantitatively with water to
obtain a solution containing about 1.0 mg of acetate per ml.

Unitperml.
Pyrogens (2.2.8). Solutions for which a validated test for
bacterial endotoxins cannot be carried out, comply with the
test for pyrogens, injecting 10 ml of the solution per kg ofthe
rabbit's body weight.
Assay. For sodium - Dilute suitably with water and determine
by Method A for flame photometry (2.4.4), or by Method A for
atomic absorption spectrophotometry (2.4.2), measuring at
589 Dill and using sodium solution FP, or sodium solution
AAS respectively, suitably diluted with water for the standard
solutions.
For potassium - Dilute suitably with water and determine

by MethodA for flame photometry (2.4.4), or by Method A for
767 Dill and using potassium solution FP orpotassimn solution
solutions.
For calcium - Dilute suitably with water and determine by
Method A for flame photometry (2.4.4), or by Method A for
422.7 Dill and using calcium solution FP or calcium solution
solutions.
For magnesium - To 50.0 ml add 50 ml of water and 5 ml of
strong ammonia"ammonium chloride solution and titrate with
0.005 M disodium edetate using 50 mg of eriochrome black
T mixture as indicator.
1 ml of O. 005 M disodium edetate is equivalent to 0.1215 mg
of Mg.
For total chloride - Dilute an accurately measured volume

containing about 60 mg ofchloride to 50.0 ml with water. Add
25.0 ml of 0.1 M silver nitrate and 2 ml of nitric acid. Filter,
wash the precipitate with water slightly acidified with nitric
acid and titrate the excess of silver nitrate with 0.1 M
ammonium thiocyanate using ferric ammonium sulphate

of sodium acetate in water to obtain a solution having a known
concentration of about 0.12 per cent w/v of sodium acetate.
a stainless steel column 30 cm x 7.8 mm, packed with a
strong cation-exchange resin consisting of sulphonated
cross-linked styrene-divinylbenzene copolymer in the
hydrogen form (7 /lm),
mobile phase: filtered and degassed 0.1 M sulphuric
acid,
spectrophotometer set at 210 Dill,
Inject the reference solution and record the chromatograms.
The test is not valid unless the tailing factor is not more than
2.0 and the relative standard deviation for replicate injections
is not more than 2.0 per cent.
Iriject separately the test and standard solutions and record
the chromatograms. Measure the responses forthe major peak
and calculate the content of acetate in the preparation under
examination.
For lactate (ifpresent) - Determine by liquid chromatography

(2.4.14).
Test solution. Use the preparation under examination.
Reference solution(a). Dissolve an accurately weighed
quantity of sodium lactate RS in water to obtain a solution
having a lmown concentration of about 2 mg per ml.
Reference solution (b). Prepare a solution in water containing
about 3 mg of anhydrous sodium acetate and 3 mg of sodium
lactate RS per ml.
octadecylsi1ane chemically bonded to porous silica or
ceramic microparticles 3 to 10 /lm,
mobile phase: a filtered and degassed solution in water
containing about I ml of formic acid and 1 ml of
dicyclohexylamine per litre,
1880
PERPHENAZINE
IP 2010
Titrate with 0.1 M sodium thiosulphate using starch solution

as indicator. Carry out a blank titration using 25 ml of water.

Inject separately reference solutions (a) and (b), and record

the chromatograms. The test is not valid unless the resolution
between the peaks due to acetate and lactate is not less than
2.0, the tailing factor for the analyte peak is not more than 2.0
and the, relative standard deviation for replicate injections is
Calculate the content of anhydrous dextrose, C 6H 120 6, from

the following Table.
Volume of
0.1 M sodium thiosulphate
consumed (ml)
Anhydrous dextrose
8
9
10
19.8
22.4
25.0
27.6
30.3
33.0
35.7
38.5
41.3
Inject separately the test solution and reference solution (a),

and record the chromatograms. Measure the responses for
the major peak and calculate the content of lactate in the
preparation under examination.
11
12
For sodium bicarbonate - Titrate with 0.1 M hydrochloric

acid a volume ofthe preparation under examination containing
about 0.1 g of sodium bicarbonate, determining the end-point
potentiometrically (2.4.25).
1 ml of 0.1 M hydrochloric acid is equivalent to 8.40 mg of
NaHC03
For lactate and bicarbonate chromatography (2.4.14).
Determine by liquid
Test solution. Use the preparation under examination.

Reference solutions. Dissolve accurately weighed quantities
oflactates and bicarbonates in order to obtain solutions having
concentrations of about 90 per cent, 100 per cent and 110 per
cent ofthe claim in 100 ml of water.
cation-exchange resin (9 /lm),
mobile phase: filtered and degassed 0.005 M sulphuric
acid,
- ,flow rate. 0.6 ml per minute,
differential refractometer detector,
Inject separately the test solution and the reference solutions
in duplicate, and record the chromatograms in the prescribed
conditions. The peaks elute in the following order, lactates,
then bicarbonates. Determine the concentration of lactates
and bicarbonates in the test solution by interpolating the peak
area for lactate and the peak height for bicarbonate from the
linear regression curve obtained with the solutions prepared
as reference solutions.
For dextrose - Transfer a volume of the preparation under

examination containing about 25 mg ofDextrose to a 250-ml
conical flask with a ground-glass neck and add 25.0 ml of
cupri-citric solution. Add a few grains of pumice, fit a reflux
condenser, heat so that boiling occurs within 2 minutes and
boil for exactly 10 minutes. Cool and add 3 g of potassium
iodide dissolved in 3 ml of water. Carefully add, in small
amounts, 25 rnl ofa 25 per cent w/w solution ofsulphuric acid.
13
14
15
16
(mg)
Storage. Peritoneal Dialysis Solutions are supplied in rigid or

semi-rigid plastic containers, in flexible plastic containers fitted
with a special connecting device (these are generally pIled to
a volume below their nominal capacity and presented in clo:>ed
protective envelopes) or in glass containers. Store at a
CAUTION - Exposure to temperatures below 4 may cause

crystallisation and separation of solid particles rendering
the preparation unsuitable for use.
Labelling. The label states (1) the formula ofthe solution for
peritoneal dialysis, expressed in grams per litre and in millimoles
per litre; (2) the total osmolar concentration in mOsmol per
litre; (3) the nominal volume ofthe solution in the container;
(4) that the solution is free from bacterial endotoxins, or where
applicable, that it is apyrogenic; (5) that the solution is not to
be used for intravenous infusion; (6) that any unused portion
of the solution is to be discarded; (7) that the solution
containing visible particles should not be used; (8) the storage
conditions.
Perphenazine
C:z 1H26ClN30S
Mol. Wt. 404.0
Perphenazine is 2-[4-[3-(2-chloro-l OH-phenothiazin-l0yl)propyl]piperazin-l-yl]ethanol.
1881
IP 2010
PERPHENAZINE
Perphenazine contains not less than 99.0 per cent and not
more than 101.0 per cent OfCZIHz6ClN30S, calculated on the
dried basis.
D. Melting range (2.4.21). 96 to 100.
Category. Dopamine receptor antagonist; neuroleptic.

methanol is clear (2.4.1).
Description. A white or yellowish-white, crystalline powder.
Tests

Identification
Compare the spectrum with that obtained with perphenazine
RS or with the reference spectrum of perphenazine.

Mobile phase. A mixture of 1 volume of concentrated
ammonia, 14 volumes of water and 85 volumes of butanol.

the plate with kieselguhr G.
Reference solution. Dilute 0.5 ml ofthe test solution to 100 m1

with methanol.
Mobile phase. A mixture of 2 volumes of diethylamine and

100 volumes of light petroleum, saturated with
phenoxyethanol (add 6 volumes phenoxyethanol to 8 volumes
ofthe above mixture until there is a persistent cloudiness after
shaking, decant, and use the supernatant liquid, even if it is
cloudy).

phase to rise 15 cm. Dry the plate in air and examine under
ultraviolet light at 254 nm. Any secondary spot in the
intense than the spot in the chromatogram obtained with the
reference solution.


perphenazine RS in chloroform.
Impregnate the plate by placing it in a closed tank containing
the necessary quantity ofthe impregnation mixture containing
2.5 per cent v/v of phenoxyethanol and 7.5 per cent v/v of
formamide in acetone so that the plate dips about 5 mm beneath
the surface of the liquid. When the impregnation mixture has
risen at least 17 cm from the lower edge of the plate, remove
the plate and use immediately. Carry out the chromatography
in the same direction as the impregnation.
Apply to the plate 2 III of each solution. Develop in the dark
and allow the mobile phase to rise 15 cm. Expose the plate to
ultraviolet light at 365 nm and examine after a few minutes.
with the reference solution. Dry the plate at 120 for 20 minutes,
allow to cool and spray with a 10 per cent v/v solution of
sulphuric acid in ethanol (95 per cent). The principal spot in
the chromatogram obtained with the test solution corresponds
to that in the chromatogram obtained with the reference
solution.
C. When examined in the range 230 Dill to 350 Dill (2.4.7), a
maxima at 257 Dill and 313 Dill. The ratio of the absorbance
measured at the maximum at 313 Dill to that measured at the
maximum at 257 nmis 0.120 to 0.128.
Loss on drying (2.4.19). Not more than 0.5 percent, determined

on 1.0 g by drying in vacuum at 65 for 4 hours.
Assay. Weigh accurately about 0.15 g and dissolve in25 mlof
1 ml of 0,1 M perchloric acid is equivalent to 0.0202 g of
CZIHz6CIN30S.
Calculate the content ofCzlHz6ClN30S.
Perphenazine Tablets contain not less than 92.5 per cent and
not more tha.n 107.5 per cent of the stated amount of
perphenazine, CZIHz6CIN30S.
Identification
A. To a quantity of the powdered tablets containing 40 mg of
Perphenazine, add 10 ml of water and 2 .ml of 1 M sodium
hydroXide, shake and extract with 15 ml of ether. Wash the
ether layer with 5 ml of water, dry with anhydrous sodium
sulphate and evaporate the ether to dryness. On the residue,
determine by infrared absorption spectrophotometry (2.4:6).
1882
IP 2010
PETHIDINE HYDROCHLORIDE
Compare the spectrum with that obtained with pelphenazine

RS or with the reference spectrum of perphenazine.
B. Extract a quantity ofthe powdered tablets containing 20 mg

ofPerphenazine with 10 ml of chloroform, filter and evaporate
the filtrate to dryness. Dissolve the residue in 2 ml of methanol,
pour into a 4.0 per cent w/v solution ofpicric acid in methanol
at 50 0 , cool and allow to stand for 4 hours. After recrystallisation
from methanol, the precipitate melts at about 248 0 (2.4.21)
with decomposition.
Perphenazine, add 5 ml of sulphuric acid and allow to stand
for 5 minutes. A red colour is produced.
disintegrated, heat on a water-bath for 3 minutes, swirling

continuously, cool, add 400 ml of ethanol (95 per cent), mix
with the aid ofultrasound for 2 minutes, shake for 5 minutes,
dilute to 500 ml with ethanol (95 per cent) and filter through a
glass microfibre filter paper. Dilute the filtrate with ethanol
(95 per cent) to produce a solution containing 0.001 per cent
w/v of Perphenazine. Record second-derivative ultraviolet
absorption spectra ofthe solutions in the range 210 to 290 nm
(2.4.7). Measure the amplitude from the peak at 265 nm to the
trough at 255 nm.
Calculate the content of C21H26N30S in the tablet from the
absorbance obtained by repeating the operation using
pelphenazine RS in place ofthe substance under examination.
Tests
For tablets containing 10 mg or less.
Related substances. Comply with the test for Related

substances in Phenothiazines (2.3.5) with the following
modifications.
.
Use the average of the 10 individual results obtained in the

test for Uniformity of content.
Use mobile phase (c) and applying to the plate 50 III ofeach of
the following freshly prepared solutions.

containing 20 mg ofPerphenazine with 10 ml of ethanol (95
per cent) and filter.
Meperidine Hydrochloride
CH 3

with the ethanol (95 per cent).
Uniformity of content. (For tablets containing 10 mg or

less). Comply with the test stated under Tablets.
NOTE-Carry out the test protectedfrom light.

Transfer one tablet to a 1OO-rnl volumetric flask and add 5 ml of
water, mix with the aid ofultrasound for 15 minutes or until the
tablet has completely disintegrated, heat on a water-bath for 3
minutes, swirling continuously and cool. Add 50 ml of ethanol
(95 per cent), mix with the aid of ultrasound for 2 minutes,
shake for 5 minutes, dilute to 100 ml with ethanol (95 per cent)
and filter through a glass microfibre filter paper. Dilute the
filtrate with ethanol (95 per cent) to produce a solution
containing 0.001 per cent w/v of Perphenazine. Record the
second-derivative ultraviolet absorption spectra of the
resulting solution inthe range 210 to 290nm (2.4.7). Measure
the amplitude from the peak at 265 nm to the trough at 255 nm.
ClsH2IN02,HCI
Mol. Wt. 283.8
Pethidine Hydrochloride is ethyll-methyl-4phenylpiperidine-4-carboxylate hydrochloride.

Pethidine Hydrochloride contains not less than 99.0 per cent
and not more than 10 1.0 per cent ofCIsH21N0 2,HCl, calculated
on the dried basis.
Dose. Orally, 50 to 150 mg every 4 hours; by subcutaneous or
intramuscular injection, 25 to 100 mg every 4 hours; by slow
intravenous injection, 25 to 50 mg every 4 hours.
Calculate the content of C21H26N30S in the tablet from the

perphenazine RS in place ofthe substance under examination.
Description. Colourless crystals or a white, crystalline powder.
Identification
Assay. For tablets containing more than 10 mg.
NOTE-Carry out the test protectedfrom light.

Shake 10 whole tablets with 50 ml of water and mix with the aid
ofultrasound for 15 minutes or until the tablets have completely

Compare the spectrum with that obtained with pethidine
1883
IP 2010
PETHIDINE HYDROCHLORIDE
hydrochloride RS or with the reference spectrum ofpethidine

hydrochloride.
Time
(min.)
0-15
15-31
31-40
40-41
41-50
B. To 5 ml of a 1 per cent w/v solution add a few drops of

potassium men::uri":iodide solution; a cream-coloured
C. Dissolve 5 mg in 0.5 ml of water and add 0.1 ml of

formaldehyde solution and 2 ml of sulphuric acid; an orangered colour is produced.
D. Gives reaction A ofchlorides (2.3.1).
Tests
Appearance ofsolution. A2.0 per centw/v solution in carbon
dioxide-free water is clear (2.4.1), and colourless (2.4.1).
Acidity or alkalinity. To 10 ml ofa 2.0 per cent wIv solution in
carbon dioxide-free water add 0.2 ml of methyl red solution
and 0.2 ml of 0.01 Msodium hydroxide; the solution is yellow.
Add 0.3 ml of 0.01 M hydrochloric acid; the solution is red.
(2.4.14).
Solvent mixture. 20 volumes of acetonitrile and 80 volumes

of water.
Test solution (a). Dissolve about 0.1 g ofthe substance under
Test solution (b). Dissolve about 0.125 g of the substance
under examination in 10.0 ml ofthe solvent mixture.
Reference solution (a). Dilute 0.5 ml of test solution (a) to
Reference solution (b). Dissolve 12.5 mg of 1-methyl-4phenyl-1,2,3,6- tetrahydropyridine in 10.0 ml of the solvent
mixture. Dilute 1.0 ml of this solution to 100.0 ml with the
solvent mixture:
and 1.0 ml ofreference solution (c) to 100.0 ml with the solvent
mixture.
/lm) (Such as Inertsil ODS2),
mobile phase: A. a mixture of equal volumes of 4.2 per
cent w/v solution of sodium perchlorate and 1.2 per
cent v/v solution of orthophosphoric acid, adjusted to
pH 2.0 with triethylamine,
B. acetonitrile,
below,
injectiOn volume. 50 Ill.
Mobile phase A
(per cent v/v)
80 -'775
75 -'755
55
55 -'780
80
Mobile phase B
(per cent v/v)
20 -'725
25 -745
45
45 -'720
20
signal-to-noise ratio for the first peak is not less than 10 and
peak-to-valley ratio where H p is height above the baseline,
and H v is height above the baseline ofthe lowest point of the
curve separating this peak from the peak due to impurity A is
not less than 4.0. The relative retention time with reference to
pethidine for pethidine impurity B is about 0.66 and for
pethidine impurity A is about 0.68.
Inject test solution (a), (b), reference solution (a) and (c). In
the chromatogram obtained with test solution (b), the area of
the peale due to I-methyl-4-phenyl-l ,2,3,6-tetrahydropyridine
(pethidine impurity B) is not more than the area of the
corresponding peak in the chromatogram obtained with
reference solution (c) (10 ppm). In the chromatogram obtained
with test solution (a), the area of any secondary peak is not
obtained with reference solution (a) (0.5 per cent) and the sum
of all the secondary peaks is not more than twice the area of
solution (a) (1.0 per cent). Ignore any peak with an area less
than 0.1 times the area ofthe principal peak in the chromatogram
solution. Titrate with 0.1 M perchloric acid, using crystal
violet solution as indicator. Carry out a blank titration.
ClsH21N02,HCI.
Pethidine Injection
Pethidine Hydrochloride Injection; Meperidine
Hydrochloride Injection
Pethidine Injection is a sterile solution of Pethidine
Pethidine Injection contains not less than 95.0 per cent and
not more than 105.0 per cent of pethidine hydrochloride,
ClsH21N02,HCI.
1884
PETHIDINE TA-\3LETS
IP 2010
Identification
A. To a volume containing 50 mg ofPethidine Hydrochloride
add sufficient 1. M sodium hydroxide to make strongly alkaline
to litmus paper and extract with two quantities, each of 10 ml,
of chloroform. Wash the combined extracts with 5 ml of water,
dry over anhydrous sodium sulphate, filter and evaporate the
filtrate to dryness. Remove the last traces of chloroform by
drying the residual oil at 60 ata pressure not exceeding
0.7kPa.
On the oily residue, determine by infrared absorption
obtained with pethidine hydrochloride RS or with the
reference spectrum of pethidine.
B. To 0.5 ml add 0.1 ml offormaldehyde solution and 2 ml of

sulphuric acid; an orange-red colour is produced.
C. Gives the reactions ofchlorides (2.3.1).
Tests
(2.4.14).
of water.
Test solution. Dilute a volume ofthe injection containing 0.1 g
ofPethidine HydrocWoride with 25.0 ml ofthe solvent mixture.
Reference solution. Dilute 0.5 ml of the test solution to 100.0
ml with the solvent mixture.
a stainless steel column 25 cm x 4.0 rom packed with
(5 Ilm) (Such as Kromasil C18),
- mobile phase: A. a mixture of equal volumes of 4.2 per
cent w/v solution of sodium perchlorate and 1.2 per
cent v/v solution of orthophosphoric acid, adjusted to
pH 2.0 with triethylamine,
B. acetonitrile,
below,
Time
Mobile phase A
Mobile phase B
(min.)
(per cent v/v)
(per cent v/v)

in the chromatogram obtained with the reference solution (0.5
per cent) and the sum ofthe areas of all the secondary peaks

Test solution. Dilute a volume ofthe injection containing about
0.1 g of Pethidine Hydrochloride with 100.0 ml of the water.
Dilute 3.0 ml ofthis solution to 25.0 ml with the mobile phase.
Reference solution. A 0.1 per cent w/v solution of pethidine
hydrochlorideRS. Dilute 3.0 ml ofthis solution to 25.0 ml with
the mobile phase.
- a stainless steel column 25 cm x 4.0 rom packed with
(5Ilm) (Such as Spherisorb ODSl),
and 90 volumes of a solution prepared by dissolving
6.8 g ofpotassium dihydrogen orthophosphate in 1000
ml of water, add 10 ml triethylamine, adjusted to pH 7.0
- injection volume. 20 ,.d.
theoritical plates is not less than 8000.
Injet the test solution and the reference solution.
Calculate the content ofClsHzlNOz,HCl in the injection.
Pethidine Tablets
20 -725
Pethidine Hydrochloride Tablets; Meperidine

Hydrochloride Tablets
55 -780
25 -745
45
45 -720
Pethidine Tablets contain not less than 92.5 per cent and not
more than 107.5 per cent of the stated amount of pethidine
hydrochloride, ClsHzlNOz,HCI.
80
20
0-15
15-31
31-40
80 -775
75 -755
55
40-41
41-50

1885
PETHIDINE TABLETS
IP 2010
Identification
"

of Pethidine Hydrochloride with 20 ml of chloroform, filter,
evaporate the filtrate to dryness and dry the residue at a
pressure of 2 kPa.
obtained with pethidine hydrochloride RS or with the
reference spectrum of pethidine hydrochloride.
B. Shake a quantity ofthe powdered tablets containing 0.2 g
of Pethidine Hydrochloride with 20 ml of water and filter. To
5 ml ofthe filtrate add 10 ml ofpicric acid solution. The crystals
so obtained, after washing with water and drying, melt at
about 190(2.4.21).
Tests

reference solution.
quantity of the powder containing about 0.3 g of Pethidine
Hydrochloride in 40 ml of water,add 2 rnl of 5 M sodium
hydroxide and extract immediately with successive quantities
of25, 10 and 10 ml of chloroform. Wash each extract with the
same 15 ml of water and filter into a dry flask. Combine the
extracts (which should be clear and free from droplets of water).
Titrate with 0.05 M perchloric acid, using 0.15 ml of
1-naphtholbenzein solution as indicator. Carry out a blank
titration.
ClsH21N02,HCl.

(2.4.17), coating the plate with laeselguhr G.
Mobile phase. The upper layer obtained by shaking together

100 volumes of light petroleum (50 to 70), 8 volumes of
2-phenoxyethanol and 1 volume of diethylamine.
Phenindione
Test solution. The upper layer obtained by shaking a quantity

of the powdered tablets containing 0.1 g of Pethidine
Hydrochloride with 5 ml of water, filtering, shaking the filtrate
with 0.5 ml of 5 M sodium hydroxide and 2 ml of ether and
allowing the layers to separate.
Reference solution. Dilute 0.5 mlofthe test solution to 50 ml
with ether.
Impregnate the dry plate by placing it in a closed tank
containing a mixture of90 volumes of acetone and 10 volumes
of 2-phenoxyethanol so that the plate dips about 5 mm beneath
the surface ofthe liquid, allowing the impregnating solvent to
ascend at least 15 cm, removing the plate from the tank and
drying in a current ofair. Use immediately, with the flow ofthe
mobile phase in the same direction as the impregnation.
Apply to the plate 5 )11 of each solution. Allow the mobile
phase to rise 12 cm. Dry the plate in air for 10 minutes, return
the plate to the tanle and repeat the development. Remove the
plate, allow it to dry in air for 10 minutes and spray with a
0.2 per cent w/v solution of 2,7-dichlorofluorescein in
methanol. Allow to stand for 5 minutes and spray with water
until the background is white to pale yellow. Examine in
daylight. The chromatograms show red or orange spots. Any
secondary spot in the chromatogram obtained with the test
solution is not more intense than the spot in the chromatogram
obtained with the reference solution. Examine without delay
in ultraviolet light at 365 nm. The chromatograms show spots
with intense yellow fluorescence. Any secondary spot in the
o}o
o
Mol. Wt. 222.2

Phenil1dione is 2-phenylindane-l ,3-dione.
Phenindione contains not less than 98.0 per cent and not
more than 100.5 per cent ofC 1sHIQ02, calculated on the dried
basis.
Dose. Initially, 200 to 300 mg; subsequently 25 to 100 mg daily,
in accordance with the needs of the patient.
Description. Soft, white or creamy-white crystals; almost
odourless.
Identification
Compare the spectrum with that obtained with phenindione
RS.
B. Dissolve 0.1 gin 30 ml of ethanol (95 per cent) with the aid
of heat, cool and add sufficient ethanol (95 per cent) to
produce 50 ml. Dilute 10 ml of this solution to 250 ml with
0.1 M sodium hydroxide and ftuther dilute 5 ml to 100 ml with
0.1 M sodium hydroxide. When examined in the range 230 nm
and 360 nm (2.4.7), the solution shows absorption maxima at
1886
IP 2010
PHENINDIONE TABLETS
about 278 nm and at about 330 nm; absorbance at about

278 nm, about 0.55 and at about 330 nm, about 0.16.
C. To 1 g add 50 ml of ethanol (95 per cent) and 0.5 mlof
aniline, heat gently under a reflux condenser for 3 hours, cool
in ice and filter. The residue, after washing with 2 ml ofethanol
(95 per cent) and recrystallisation from chloroform, melts at
about 225(2.4.21).
Phenindione Tablets
Phenindione Tablets contain not less than 92.5 per cent and
phenindione, ClsHIOO Z
Identification
Tests
Related substances. Determme by thin-layer chromatography
Mobile phase. A 0.02 per cent w/v solution of butylated
hydroxytoluene in a mixture of 80 volumes of toluene,
20 volumes of ethyl acetate and 4 volumes of glacial acetic
acid.
examination in 10 m1 ofdichloromethane.
phase to rise 4 cm. Dry the plate in warm air and examine in
reference solution (a) and not more than one such spot is
more intense than the spot in the chromatogram 9btained
on 1.0 g by drying in oven at 105 for 2 hours.
Assay. Weigh accurately about 0.3 g, add 50 ml of ethanol
(95 per cent) and warm until solution is effected. Cool to
room temperature, add 10 ml ofa 10 per cent v/v solution of
bromine in ethanol (95 per cent) and allow to stand for
10 minutes, shaking occasionally. Add 1 g of 2-naphthol and
shake until the colour ofthe bromine is discharged. Remove
any vapour of bromine in the flask with a current of air, add
50 m1 of water and 10 ml of dilute potassium iodide solution
and titrate the liberated iodine with 0.1 M sodium thiosulphate
using starch solution as indicator.
1 ml of 0.1 M sodium thiosulphate is equivalent to 0.01111 g
ofC1SHIOOz.

Phenindione with 50 ml ofchloroform, filter and evaporate the
filtrate to dryness. Recrystallise the residue from ethanol
(95 per cent). The crystals complies with the following tests.
A. Determine by infrared absorption spectrophotometry (2.4.6)..
Compare the spectrum with that obtained with phenindione
RS.
B. Dissolve 0.1 gin 30 ml ofethanol (95 per cent) with the aid
of heat, cool and add sufficient. ethanol (95 per cent) to
produce 50 ml. Dilute 10 ml of this solution to 250 ml with
0.1 M sodium hydroxide and further dilute 5 ml to 100 ml with
0.1 M sodium hydroxide. When examined in the range 230nm
to 360 nm(2.4.7), the solution shows absorption maxima at
about 278 nm and 330 nm; absorbance at about 278. about
0.55 and at about 330 nm, about 0.16.
C. To 50 mg add 1 ml ofsulphuric acid; a deep blue to violet

solution is produced. On dilution with water the solution
becomes colourless and a white precipitate is produced.
Tests
Mobile phase. A 0.02 per cent w/v solution of butylated
hydroxytoluene in a mixture of 80 volumes of toluene,
20 volumes of ethyl acetate and 4 volumes of glacial acetic
acid.
containing 50 mg of P4enindione with 15 m1 of
dichloromethane, filter, evaporate the filtrate to dryness and
dissolve the residue in 5 ml of dichloromethane.
50 volumes with dichloromethane.
Reference solution (b). Dilute 1 volume of the test solution to
200 volumes with dichloromethane.
phase to rise 4 cm. Dry the plate in warm air and examine in
1887
IP 2010
PHENlRAMINE MALEATE

Uniformity ofcontent. (For tablets containing 50 mg or less)
Place one tablet in 50 ml of0.1 M sodium hydroxide, dissolve
completely by shaldng gently, add a further 100 ml of 0.1 M
sodium hydroxide and shake for 1 hour. Dilute to 250.0 ml with
0.1 M sodium hydroxide, filter and dilute a portion of the
filtrate with sufficient 0.1 M sodium hydroxide to produce a
solution containing 4 Ilg ofPhenindione per ml. Measure the
278 nm (2.4.7). Calculate the content ofC 1SH lO0 2taking 1310
as the specific absorbance at 278 nm.
Assay. Weigh and powder 20 Tablets. Weigh accurately a
quantity ofthe powder containing about 50 mg ofPhenindione
and shake with 150 ml of 0.1 M sodium hydroxide for 1 hour,
add sufficient 0.1 M sodium hydroxide to produce 250.0 ml,
filter and dilute 5.0 ml of the filtrate to 250.0 ml with 0.1 M
sodium hydroxide. Measure the absorbance of the resulting
content ofC 1SH lO0 2taking 1310 as the specific absorbance at
278nm.
Identification

Compare the spectrum with that obtained with pheniramine
maleate RS or with the reference spectrum of pheniramine
maleate.
an inflection at about 262 nm; absorbance at about 265 nm,
about 0.42.
C. Dissolve 0.25 gin 5 ml ofwater, add 2 ml ofstrong ammonia
solution and extract with three quantities, each of 5 ml, of
chloroform. Evaporate the aqueous extract to dryness, add
0.2 ml of 1 M sulphuric acid and 5 ml of water, extract with
four quantities, each of 25 ml, of ether and evaporate the
combined ether extracts to dryness in a current ofwarm air. To
the residue add 50 mg of resorcinol and 1 ml of sulphuric
acid, heat in a water-bath.for 2 minutes, shake well, heat in a
water-bath for a further 30 minutes and cool in ice. Carefully
add 5 ml ofwater; a yellow colour is produced. To 2 ml ofthe
solution add 3 ml of a 50 per cent w/v solution of ammonium
acetate, previously cooled in ice; a pink colour is produced
which persists for at least 10 minutes in the cooled solution.
Tests
Pheniramine Maleate

40 volumes of chloroform and 10 volumes of diethylamine.
examination in 10 ml ofmethanol.
"
(COOH

substance under examination in methanol.
COOH

Mol. Wt. 356.4

Pheniramine Maleate is (3RS)-N,N-dimethyl-3-phenyl-3(pyridin-2-yl)propan-l-amine hydrogen maleate.
Pheniramine Maleate contains not less than 99.0 per cent and
not more than 101.0 per cent ofC16H2oN2,C4H404, calculated
on the dried basis.
Category. Antihistaminic.
Dose. Orally, 25 to 50 mg daily, in divided doses; by
intramuscular or slow intravenous injection, 22.5 to 45 mg
daily, in divided doses.

iodobismuthate solution. Any secondary spot in the
Heavy metals (2.3.13). Dissolve 1.0 g in 10 ml ofwater and add
2 ml ofacetic acid and sufficient water to produce 25 ml. The
resulting solution complies with the limit test for heavy metals,
Method A (20 ppm).
1888
IP 2010
PHENIRAMINE TABLETS
0.7kPa.
acid, using 1- naphtholbenzein solution as indicator. Carry
CI6H20N2,C4~04.
Related substances. Determine by the method described under

th'e Identification test using as the reference solution a
solution prepared by diluting 1 volume of the test solution to
500 volumes with chloroform. Any secondary spot in the
reference solution.
Pheniramine Injection contains not less than 90.0 per cent and
pheniramine maleate, C16H20N2, C4H40 4.

0.11 g ofPheniramine Maleate add sufficient water to produce
50.0 ml and mix well. To 20.0 ml add sufficient 1 M sodium
hydroxide to make the solution just alkaline to litmus paper,
add 2 ml in excess and extract with two quantities, each of
50 ml, ofether. Wash each ether extract in succession with 20,
20 and 5 ml of 0.1 M hydrochloric acid, dilute the combined
extracts to 100.0 ml with 0.1 M hydrochloric acid and mix.
Dilute 5.0 ml to 100.0 ml with 0.1 M hydrochloric acid and
measure the absorbance of the resulting solution at the
maximum at about 265 nrn (2.4.7), using 0.1 M hydrochloric
acid as the blank. Calculate the content OfCI6H2oN2, C4H40 4
Usual strength. 22.75 mg per mI.
Pheniramine Maleate Injection
Pheniramine Injection is a sterile solution of Pheniramine
Maleate in Water for Injections.
Identification
Test solution. Evaporate an appropriate volume ofthe injection
to dryness in a current ofnitrogen using the minimum amount
of heat, dissolve the residue in sufficient chloroform to
produce a solution containing 2.0 per cent w/v solution of
Pheniramine Maleate and centrifuge.
Reference solution. A2.0 per cent w/v solution ofpheniramine
maleate RS in chloroform.
Apply to the plate 10 ).11 of each solution. After development,
dry the plate in air and examine in ultraviolet light at 254 nrn.
The two principal spots in the chromatogram obtained with
the test solution correspond to those in the chromatogram
obtained with the reference solution. Spray the plate with
dilute potassium iodobismuthate solution. The principal spot
reference solution.
Tests
pH (2.4.24). 4.5 to 5.5.
Pheniramine Tablets
Pheniramine Maleate Tablets
Pheniramine Tablets contain not less than 92.5 per cent and
pheniramine maleate, C16H20N2, C4~04'
Usual strengths. 12.5 mg; 25 mg; 50 mg.
Identification
Boil a quantity ofthe powdered tablets containing about 0.5 g
ofPheniramine Maleate with 150 ml ofacetone under a reflux
condenser for about 45 minutes. Filter and evaporate the filtrate
to dryness on a water-bath. The residue complies with the
following tests.

Compare the spectrum with that obtained with pheniramine
maleate RS or with the reference spectrum of pheniramine
maleate.
B. When examined in the range 230 nrn to 360 nrn (2.4.7), a

an inflection at about 262 nm; absorbance at about 265 nrn,
about 0.42.
1889
PHENIRAMINE TABLETS
IP 2010
C. Dissolve 0.25 gin 5 ml of water, add 2 ml ofstrong ammonia

solution and extract with three quantities, each of 5 ml, of
chloroform. Evaporate the aqueous extract to dryness, add
0.2 ml of 1 M sulphuric acid and 5 ml of water, extract with
four quantities, each of 25 ml, of ether and evaporate the
combined ether extracts to dryness in a current ofwarm air. To
the residue add 50 mg of resorcinol and 1 ml of sulphuric
acid, heat in a water-bath for 2 minutes, shake well, heat in a
water-bath for a further 30 minutes and cool in ice. Carefully
add 5 ml of water; a yellow colour is produced. To 2 ml ofthe
solution add 3 ml of a 50 per cent w/v solution of ammonium
acetate, previously cooled in ice; a pink colour is produced
which persists for at least 10 minutes in the cooled solution.
Tests

containing 20 mg of Pheniramine Maleate with 10 ml of
100 volumes with methanol.
Reference solution (b). Dilute 1 volume of reference solution
(a) to 20 volumes with methanol.
Apply to the plate 10 J.Ll of each solution. After development,
dry the plate in air, spray with . dilute potassium
Phenobarbital
Mol. Wt. 232.2

Phenobarbitone is 5-ethyl-5-phenylbarbituric acid.
Phenobarbitone contains not less than 99.0 per cent and not
more than 101.0 per cent of ClzHlZNz03, calculated on the
dried basis.
Category. Sedative; anticonvulsant.
Dose. 60 to 300 mg at night.
odourless.
Identification
Tests C, D and E may be omitted if tests A and B are carried
out.
Compare the spectrum with that obtained with phenobarbitone
RS or with the reference spectrum of phenobarbitone.
B. Determine the melting point (2.4.21) ofthe substance under

examination and of a mixture of equal quantities of the
substance under examination and phenobarbitone RS. The
difference between the melting points, which are about 175,
is not greater than 2.
C. Complies with the test for identification of barbiturates
(2.3.2).

quantity ofthe powder containing about 45 mg ofPheniramine
Maleate, shake with 20 l11l of 0.1 Mhydrochloric acid, centrifuge
and transfer the supernatant liquid to a 1OO-ml volumetric flask.
Repeat the extraction with three further quantities, each of20
ml, of 0.1 M hydrochloric acid. Combine the extracts and add
sufficient 0.1 M hydrochloric acid to produce 100.0 ml. Mix
and dilute 5.0 ml to 100.0 ml with 0.1 M hydrochloric acid;
maximum at about 265 nm (2.4.7), using 0.1 M hydrochloric
acid as the blank. Calculate the content of C16HzoNz,C4H404
taking 210 as the specific absorbance at 265 urn.
Phenobarbitone
D. Dissolve about 20 mg in 5 ml of eth.anol, add a drop of

cobalt chloride solution and a drop of dilute ammonia
solution; a violet colour is produced.
E. Gives the reaction ofnon-nitrogen substituted barbiturates
(2.3.1).
Tests
Appearance of solution. A 10.0 per cent w/v solution in a
mixture of20 volumes of2 M sodium hydroxide and 30 volumes
of water is clear (2.4.1), and not more intensely coloured than
reference solution YS6 (2.4.1).
Acidity. Mix 1.0 g with 50 ml ofwater, boil for 2 minutes, allow
to cool, filter and adjust the volume to 50 ml. To 10 m1 of the
1890
IP 2010
PHENOBARBITONE SODIUM
filtrate add 0.1 5 ml of methyl redsolution; not more than 0.1 ml

of 0.1 M sodium hydroxide is required to change the colour of
the solution from orange-yellow to pure yellow.
anhydrous sodium sulphate and evaporate the ether to

dryness on a water-bath. The residue complies with the
following tests.


Mobile phase. A mixture of 5 volumes of concentrated

ammonia, 15 volumes of ethanol (95 per cent) and 80 volumes
of chloroform.
Reference solution. Dilute 0.5 ml ofthe test solution to 100 ml
with ethanol (95 per cent).
Apply 20 JlI of each solution. Allow the mobile phase to rise
15 em. Dry the plate in air and examine in ultraviolet light at 254
om. Spray with diphenylcarbazone mercuric reagent. Allow
the plate to dry in air and spray with freshly prepared
alcoholic potassium hydroxide solution ( 20 per cent w/v).
Heat at 105 for 5 minutes. Any secondary spot in the
chromatogram obtained with the test solution, is not more
B. Dissolve 50 mg in 2 ml of a 0.2 per cent w/v solution of

cobaltous acetate in methanol, warm, add 50 mg ofpowdered
borax and heat to boiling; a bluish violet colour is produced.
Tests
Disintegration (2.5.1). 30 minutes
Phenobarbitone and extract in a continuous extraction
apparatus (2.1.8) with ether until complete extraction is
effected. Remove the ether and dry the residue, which is
C12H12N203, to constant weight at 105,

Loss on drying (2.4.19). Not more than 1.0 per cent w/w,
determined on 1.0 g by drying in an oven at 105 for 2 hours.
pyridine, add 0.25 ml of thymolphthalein solution and 10 ml
of silver nitrate-pyridine reagent and titrate with 0.1 M
ethanolic sodium hydroxide until a pure blue colour is
obtained. Repeat the operation without the substance under
the amount of sodium hydroxide required.
Phenobarbital Sodium; Soluble Phenobarbitone; Soluble
Phenobarbital
H
NyONa
&
H3C

0.01161 g OfC12H12N203.
~ ~ 0

C l2H ll N 2Na03
Mol. Wt. 254.2
Phenobarbitone Sodium is sodium 5-ethyl-5-phenylbarbiturate.
Phenobarbital Tablets
Phenobarbitone Sodium contains not less than 99.0 per cent

and not more than 101.0 per cent OfC12HllN2Na03, calculated
on the dried basis.
Phenobarbitone Tablets contain not less than 92.5 per cent

phenobarbitone, CI2H12N203.
Category. Sedative; anticonvulsant.
Identification
Dose. Orally, 60 to 300 mg at night; by intramuscular or

intravenous injection, 50 to 200 mg, repeated after 6 hours if
necessary; maximum 600 mg daily.

0.5 g of Phenobarbitone with 50 rn1 of ether, filter through
Description. A white powder or crystalline granules or flaky

crystals; hygroscopic.
1891
PHENOBARBITONE SODIUM
IP 2010
Identification
Test A may be omitted iftests B, C, D, E andF are carried out.
Tests C, D andE may be omitted iftests A, BandF are carried
out.
A. Dissolve 0.2 gin 20 ml of ethanol (50 per cent), acidify

with dilute hydrochloric acid and extract with 50 ml of ether.
Wash the ether layer with 10 ml of water, dry over anhydrous
sodium sulphate and filter. Evaporate the filtrate to dryness
and dry the residue at 105.
obtained with phenobarbitone RS or with the reference
spectrum of phenobarbitone.
Apply 20 I!l ofeach solution. Allow the mobilephase to rise 15

em. Dry the plate in air and examine in ultraviolet light at 254
nm. Spray with diphenylcarbazone mercuric reagent. Allow
the plate to dry in air and spray, with freshly prepared
alcoholic potassium hydroxide solution ( ,20 per ,cent w/v).
Heat at 105 0 for 5 minutes. Any secondary spot in the
chromatogram' obtained with the test solution, is not more

(2.3.2), but using the following solutions.

water and add 8 ml of 0.05 M sulphuric acid. Heat to boiling
and cool. Add 30 ml of methanol and shake until dissolution
is complete. Titrate with 0.1 M sodium hydroxide, determining
the end-point potentiometrically (2.4.25). After the first
inflection, stop the addition of the sodium hydroxide, add
10 ml of pyridine, mix and continue the titration until the
second inflection is reached. The difference between the
volumes represents the amount of sodium hydroxide
required.

under examination in ethanol (50 per cent).

C12HIlNzNa03'

phenobarbitone RS in ethanol (50 per cent).
B. Determine the melting point (2.4.21), ofthe residue obtained

in test A and ofa mixture ofequal quantities ofthe residue and
phenobarbitone RS. The difference between the melting
points, which are about 175, is not greater than 2.
D. 1 g dissolves completely in 20 ml of ethanol (90 per cent)

(distinction from barbitone sodium).
Phenobarbitone Injection
E. Gives the reaction of non-nitrogen substituted barbiturates

(2.3.1).
Phenobarbital Sodium Injection; Phenobarbitone Sodium

InjeCtion; Soluble PhenobarbitoneInjeetion
F. Ignite about 0.1 g; the residue gives the reactions ofsodium
Phenobarbitone Injection is a sterile solution of

Phenobarbitone Sodium in a mixture of nine volumes of
Propylene Glycol and one volume of Water for Injections.
salts (2.3.1).
Tests
Appearance ofsolution. Al 0.0 per cent w/v solution in ethanol
(50 per cent) is clear (2.4.1), and not more intensely coloured
than reference solution YS7 (2.4.1).
pH (2.4.24). Not more than 10.2, determined in a 10.0 per cent
w/v solution.
Related substances. Deterrriine by thin-layer chromatography

ammonia, 15 volumes of ethanol (95 per cent) and 80 volumes
of chloroform.
Test solution Dissolve about 1.0 g of the substance under
Phenobarbitone Injection contains not less than 95.0 per cent

phenobarbitone sodium, ClzHIINzNa03'
Usual strengths. 30 mg, 60 mg, 65 mg and 130 mg per ml.
Identification
To a volume containing 1 g of Phenobarbitone Sodium add
15 ml of water if necessary, make slightly acidic with 1 M
sulphuric acid and filter. The residue, after washing with water
and drying at 105, complies with the following tests.

B. Dissolve 50 mg in 2 ml of a 0.2 per cent w/v solution of
cobalt acetate in methanol, warm, add 50 mg of powdered
borax and heat to boiling; a bluish violet colour is produced.
1892
PHENOL
Tests
the filtrate is alkaline to litmus solution and yields a white

precipitate on the addition of dilute hydrochloric acid.
pH (2.4.24).10.0 to 11.0.
Assay. Weigh accurately about 2.0 g, add 30 ml of water and
3 g of sodium carbonate, stir to dissolve and titrate with
0.1 M silver nitrate until a distinct turbidity is observed when
viewed against a black background, the solution being stirred
vigorously throughout the titration.
1 ml of 0.1 M silver nitrate is equivalent to 0.02542 g of
Ct2HIIN2Na03'
Determine the weight per ml of the injection (2.4.29) and
calculate the percentage weight in volume ofC12H11N2Na03.

Labelling. The label states that the injection should not be
used ifthe solution is discoloured or ifit contains a precipitate.
E. The powdered tablets, when moistened with hydrochloric

Tests
Phenobarbitone Sodium, dissolve as completely as possible
in 10 ml of a 2 per cent w/v solution of sodium hydroxide,
saturate with sodium chloride, acidify with hydrochloric acid
and extract with successive quantities, each of 15 ml, of ether
extracts with two quantities, each of2 ml, of water and extract
the main ether layer and dry the residue to constant weight at
105.
1 g ofthe residue is equivalent to 1.095 g ofC12HlIN2Na03.

Phenobarbital Sodium Tablets; Soluble Phenobarbitone
Tablets; Soluble Phenobarbital Tablets
Phenobarbitone Sodium Tablets contain not less than
amount ofphenobarbitone sodium, C12HllN2Na03'
Phenol
Carbolic acid
OH
Identification
Mol. Wt. 94.1
A. Heat 0.1 g ofthe residue obtained in Assay on a water-bath

with 15 ml of ethanol (25 per cent) until dissolved, filter while
hot and allow to cool. Filter through a sintered-glass crucible,
wash with a small quantity of ethanol (25 per cent) and dry at
105. Heat in a sealed tube at 105 for 1 hour.
obtained with phenobarbitone RS or with the reference
spectrum of phenobarbitone.
B. The residue obtained in testA melts at about 175 (2.4.21).
Phenol contains not less than 99.0 per cent and not more than
100.5 per cent ofC 6H 60.
Category. Antiseptic; antipruritic; pharmaceutical aid

(antimicrobial preservative).
Description. Colourless or faintly pink or faintly yellowish
crystals or crystalline masses; odour, characteristic;
deliquescent.
.
Identification
C. Dissolve 50 mg ofthe residue obtained in Assay in 2 ml of

a 0.2 per cent w/v solution of cobaltous acetate in methanol,
warm, add 50 mg of powdered borax and heat to boiling; a
bluish violet colour is produced.
A. Dissolve 0.5 gin 2 ml of strong ammonia solution. Dilute

this solution to about 100 ml with water and to 2 ml of the
resulting solution, add 0.05 ml of 3 per cent w/v solution of
sodium hypochlorite; a blue colour develops which becomes
progressively more intense.
D. Triturate a quantity of the powdered tablets containing

0.2 g ofPhenobarbitone Sodium with 5 ml of water and filter;
B. Dissolve 1.0 g in sufficient water to produce 15 ml (solution

A) and to 1 ml,add 10 ml of water and 0.1 mlofJerricchloride
1893
PHENOL
IP 2010
solution; a violet colour is produced which disappears on the

addition of 5 ml of 2-propanol.
C. To 1 ml ofsolutionA add 10 ml of water and 1 m1 of bromine
solution; a pale yellow precipitate is produced.
Dose. 30 to 200 mg.

Description. A white or yellowish white, crystallimi or
amorphous powder; odourless or almost odourless.
Identification
Tests
Appearance of solution. Solution A is clear (2.4.1), and not
more intensely coloured than reference solution BS6 (2.4.1).
Acidity. To 2 ml of solution A add 0.05 ml of methyl orange
solution; the solution is yellow.
Freezing point (2.4.11). Not less than 39.so.
Non-volatile matter. Not more than 0.05 per cent, when 5.0 g is
volatilised on a water-bath and dried to constant weight at
105.
Assay. Weigh accurately about 0.5 g and dissolve in sufficient
water to produce 250.0 ml. Transfer 25.0 ml to a ground-glassstoppered flask, add 50.0 ml of 0.05 M bromine and 5 ml of
hydrochloric acid, stopper, allow to stand for 30 minutes,
swirling occasionally, and allow to stand for a further
15 minutes. Add 5 ml ofa 20 per cent w/v solution ofpotassium
iodide, shake and titrate with 0.1 M sodium thiosulphate until
a faint yellow colour remains. Add 0.5 ml of starch solution
and 10 ml of chloroform and continue the titration with
vigorous shaking. Repeat the operation without the substance
under examination. The difference between the titrations
represents the amount of bromine required.
1 ml of 0.05 Mbromine is equivalentto 0.001569 g ofC6H 60.
A. Dissolves in dilute solutions of alkali hydroxides and in

hot solutions ofalkali carbonates forming a red solution which
is decolorised by dilute acids.
B. Dissolve 25 mg in 100 ml of ethanol (95 per cent) (solution
A). To 2.0 ml ofsolutionAadd 5.0 ml of 1 Mhydrochloric acid
and dilute to 50.0 ml with ethanol (95 per cent) (solution AI)'
To 10.0 ml of solution A add 5.0 ml ofl M hydrochloric acid
and dilute to 50.0 ml with ethanol (95 per cent) (solution A 2).
To 2.0 ml of solution A add 5.0 ml of 1 M sodium hydroxide
and dilute to 50.0 ml with ethanol (95 per cent) (solution B).
Examined between 220 urn and 250 urn (2.4.7), solution AI
shows an absorption maximum at 229 nm. The specific
absorbance at the maximum at 229 urn is 922 to 1018. Examined
between 250 urn and 300 urn, solutionA2 shows an absorption
maximum at 276 urn. The specific absorbance at the maximum
at 276 urn is 142 to 158. Examined between 230 urn and 270 urn,
solution B shows an absorption maximum at 249 urn. The
specific absorbance at the maximum at 249 urn is 744 to 822.
Tests
Solution A. To 2.0 g add 40 ml of distilled water, boil, cool

and filter.
Appearance of solution. A 4 per cent w/v solution in ethanol
(95 per cent) is clear (2.4.1) and not more intensely coloured
Acidity or alkalinity. To 10 ml of solution A, add 0.15 ml of
bromothymol blue solution. Add 0.05 ml of 0.01 M
hydrochloric acid, the solution is yellow. Add 0.1 ml of 0.01
M sodium hydroxide, the solution is blue.
Phenolphthalein

(2.4.17), coating the plate with silica gel F254.
Mobile phase. A mixture of 50 volumes of acetone and 50

volumes of dichloromethane.
examination in 10 ml ethanol (95 per cent).
OH
HO
Mol. Wt. 318.3
Phenolphthalein is 3,3-bis(4-hydroxyphenyl)phthalide.
Phenolphthalein contains not less than 98.0 per cent and not
more than 102.0 per cent of C2oH1404, calculated on the dried
basis.
Category. Laxative.

with ethanol (95 percent). Dilute 5 ml ofthis solution to 100
ml with ethanol (95 per cent).
Reference solution (b). Dissolve 25 mg ofjluorene in ethanol
(95 per cent), add 0.5 ml ofthe test solution and dilute to 10ml
phase to rise 15 em. Dry the plate in air and examine at 254 urn
1894
IP 2010
PHENOXYETHANOL
and re-examine after exposure to ammonia vapour. Any

obtained with reference solution (a) (0.5 per cent). The test is
not valid unless the chromatogram obtained with reference
solution (b) shows 2 clearly separated spots.
Chlorides (2.3.12). Dilute 10 ml of solution A to15 ml with
water. The solution complies with the limit test for chlorides
(lOOppm).
Heavy metals (2.3.13). Heat 5 g with 50 ml of dilute
hydrochloric acid on a water-batl). for 5 min and filter.
Evaporate the filtrate almost to dryness and dissolve the
residue in 50 ml of water. 12 ml ofthis solution complies with
the limit test for heavy metals, Method D (20 ppm). Use 10 ml
of lead standard solution (2 ppm Pb) to prepare the standard.
Identification
Shake 2 ml of Phenoxyethanol with a mixture of 4.0 g of
potassium permanganate, 5.4 g of sodium carbonate and 75
ml of water for 30 minutes. Add 25 g of sodium chloride and
stir continuously for 60 minutes, filter and adjusted to pH 1.7
with hydrochloric acid. The precipitate, after recrystallisation
from water complies with the following tests.
Compare the spectrum with that obtained with phenoxyethanol
RS or with the reference spectrum of phenoxyethanol.

0.008 per cent w/v solution in water, shows two absorption
maxima at 269 nm and 275 nm and specific absorbance at 269
nm is 95 to 105 and at 275 nm is 75 to 85.
Sulphates (2.3.17). 15 ml ofsolution A complies with the limit

test for sulphates (200 ppm).
Tests
Refractive index (2.4.27). 1.537 to 1.539
Relative density (2.4.29). 1.105 to 1.110
Assay. Dissolve 0.1 gin 5 ml of dimethylformamide. Add 5 ml

of sodium carbonate solution, 10 ml of sodium hydrogen
carbonate solution, 35 ml of water and 50.0 ml of 0.05 M
iodine. Add 10 ml of dichloromethane and 20 ml of dilute
sulphuric acid. Titrate the excess ofiodine with 0.1 M sodium
thiosulphate; using 0.3 ml of starch solution added towards
the end ofthe titration, as indicator. Carry out a blank titration.
1 mlof 0.05 M iodine is equivalent to 0.003979 g ofCzoHl404.
Related substances. Determine by Gas chromatography

(2.4.13).
Internal standard solution. Dissolve 1.25 g of methyllaurate

in 25.0 ml of dichloromethane.
Test solution. Dissolve 5 g ofthe substance under examination
in 10.0 ml of dichloromethane and add 1.0 ml ofthe internal
standard solution.
Reference solution. Add 10.0 ml of the internal standard
solution to 1.0 ml of the test solution and dilute to 100.0 ml
with dichloromethane.
- a glass column 1.5 m x 4 mm, packed with silanised
diatomaceous earth support (150 to 180 mesh) coated
with 3 per cent w/w solution of polymethylphenylsiloxane,
temperature:
column. 130,
inlet port and detector at 200,
flame ionization detector,
flow rate. 30 ml per minute using nitrogen as a carrier
gas.
Phenoxyethanol
2-Phenoxyethanol
Mol. Wt.138.2
Phenoxyethanol is l-hydroxy-2-phenoxyethane.
Phenoxyethanol contains not less than 99.0 per cent and not
more than 100.5 per cent ofC~HIOOz, calculated on the dried
basis.
Category. Pharmaceutical aid.
Description. A colourless, slightly viscous liquid.
Inject 1 f!1 ofthe reference solution. The test is not valid unless
the resolution between the peaks due to phenoxyethanol and
methyllaurate is not less than 12.
Inject 1 f!1 of the test solution and the reference solution. In
the chromatogram obtained with the test solution, the ratio of
the area due to peak corresponding tophenoxyethanol to the
area of the peak due to the internal standard from the
chromatogram obtained with the reference solution. from the
1895
PHENOXYMETHYLPENICILLIN POTASSIUM
IP 2010
chromatogram obtained with test solution, the ratio of the

area ofthe sum ofany secondary peale and the peak due to the
internal standard, 'is not more than 1.0 per cent.
Phenol. Not more than 0.1 per cent.
Shake about 1 g ofpropoxyphenol in 50 ml of dichloromethane
with 1 ml of dilute sodium hydroxide solution and 10 ml of
water. Wash the upper layer with 2 quantities, each of20 ml of
dichloromethane and dilute to 100.0 ml with water. The
absorbance at the maxima at 287 urn (2.4.7) is not more than
0.27.
Assay. Weigh accurately 2 g, dissolve in 10.0 ml of freshly
prepared acetic anhydride solution, heat in a water bath for
45 minutes. Cool, add 10.0 ml of water. further heat for 2
minutes, cool and add 10.0 ml of butanol, shake vigorously
and titrate excess of acetic acid with 1 M sodium hydroxide
using 0.2 ml of phenolphthalein solution as indicator. Carry
1 ml of 1 M sodium hydroxide is equivalent to 0.1382 g of
C8H IOOZ
Identification

Compare the spectrum with that obtained with
phenoxymethylpenicillin potassium RS.
B. Gives reaction B ofpenicillins and cephalosporins (2.3.1).

C. Gives reaction A ofpotassium salts (2.3.1).
Tests
pH (2.4.24). 5.5 to 7.5, determined in a 0.5 per centw/v solution.
in a 1.0 per cent w/v solution in carbon dioxide-free water.
solution in 0.1 M sodium hydroxide at the maximum at about
306 nm, not more than 0.33. Absorbance of a 0.02 per cent
w/v solution in O.lM sodium hydroxide at the maximum at
about 274 nm, not less than 0.50.
Phenoxyacetic acid. Not more than 0.5 per cent.
Phenoxymethylpenicillin Potassium
Test solution. Weigh accurately a suitable quantity of the

substance under examination, dissolve in phosphate buffer
pH 6.6 and dilute to obtain a solution having a known
concentration of about 20 mg per ml.
Penicillin V Potassium
Reference solution. Weigh accurately a suitable quantity of

phelloxyacetlc acid, dissolve in the phosphate buffer pH 6.6
and dilute to obtain a solution having a known concentration
of about 0.1 mg per ml.
Mol. Wt. 388.5
(5Ilm),
Phenoxymethylpenicillin Potassium is potassium (6R)-6-(2phenoxyacetamido)penicillinate, produced by the growth of

certain strains of Penicillium notatum or related organisms
on a culture medium containing an appropriate precursor, or
obtained by any other means.
Phenoxymethylpenicillin Potassium contains not less than
86.0 per cent of total penicillins CI6HI7NzOsS, calculated on
the anhydrous basis.
Dose. For an adult, the equivalent of 250 to 500 mg of
phenoxymethylpenicillin every 6 hours, at least 30 minutes
before food. For a child every 6 hours, upto 1 year, 62.5 mg; 1
to 5 years, 125 mg; and 6 to 12 years, 250 mg.
Description. A white, crystalline powder.
- mobile phase: a mixture of 65 volumes of water,

35 volumes of acetonitrile and 1 volume ofglacial acetic
acid,
Inject the reference solution. The tailing factor is not more
Inject alternately the test solution and the reference solution.
Calculate the content of phenoxyacetic acid.
Limit ofp-hydroxyphenoxymethylpenicillin. Not more than
5.0 per cent.
1896
IP 2010
PHENOXYMETHYLPENICILLIN POTASSIUM TABLETS
Using the chromatograms obtained with the test solution in Phenoxymethylpenicillin Potassium
the Assay, calculate the content ofp-hydroxyphenoxymethylpenicillin from the peak response of p-hydroxyphenoxy~ Tablets
methylpenicillin and the sum of the peak responses of Phenoxymethylpenicillin Tablets; Penicillin V Potassium
p-hydroxyphenoxymethylpenicillin and phenoxymethylTablets; Penicillin V Tablets
penicillin.
Phenoxymethylpenicillin Potassium Tablets contain not less
Water (2.3.43). Not more than 1.0 per cent, determined on than 92.5 per cent and not more than 107.5 per cent of the
1.0 g.
stated amount ofphenoxymethylpenicillin, CI6HISNzOsS.

examination in the mobile phase and dilute to 50.0 ml with the
mobile phase.
quantity of phenoxymethylpenicillin potassium RS in the
mobile phase and dilute to obtain a solution having a known
concentration of about 2.5 mg per ml.
Reference solution (b). A solution in the mobile phase

containing 0.25 per cent w/v each ofbenzylpenicillin potassium
and phenoxymethylpenicillin potassium.
- a stainless steel column 30 cm x 4 rom, packed with
(3 to 10 Jlm),
35 volumes of acetonitrile and 5.75 volumes of glacial
acetic acid,
Identification
ofphenoxymethylpenicillin with water, dilute to 250 ml with
water and filter. When examined between 230 and 360 nm
(2.4.7), the filtrate shows absorption maxima at about 268 nm
and 274 nm and a minimum at about 272 urn.
B. Shake a quantity ofthe powdered tablets containing 10 mg

ofphenoxymethylpenicillin with I 0 ml of water, filter and add
0.5 ml of neutral red solution. Add sufficient 0;01 M sodium
hydroxide to produce a permanent orange colour and then
add 1.0 ml of penicillinase solution; the solution changes
rapidly to red.
C. Ignite 0.5 g of the powdered tablets, add 5 ml of 2 M
hydrochloric acid, boil, cool and filter. The filtrate gives
reaction B ofpotassium salts (2.3.1).
Tests

Inject reference solution (b).The relative retention times are

about 0.8 for benzylpenicillin and 1.0 for phenoxymethylpenicillin. The column efficiency determined from the
phenoxymethylpenicillin peak is not less than
1800 theoretical plates and the resolution between benzylpenicillin and phenoxymethylpenicillin is not less than 3.0.
Inject reference solution (a). The relative standard deviation
Inject the test solution and reference solution (a). Record the
chromatograms and measure the responses for the
phenoxymethylpenicillin peak and any p-hydroxyphenoxymethylpenicillin peak with a retention time ofabout 0.4 relative
to that ofthe main phenoxymethylpenicillin peak.
Calculate the content of CI6HI7NzOsS, from the sum of the
peak responses ofthep-hydroxyphenoxymethylpenicillin and
phenoxymethylpenicillin peaks in the chromatograms obtained
with the test solution and reference solution (a).
Usual strengths. The equivalent of62.5 mg, 125 mg, 250 mg

and 500 mg ofphenoxymethylpenicillin.
Apparatus No.1,
Medium. 900 m1 of watel;
the absorbance ofthe filtrate, suitably diluted if necessary, at
the maximum at about 268 urn (2.4.7). At the same time measure
the absorbance of a solution of known concentration of
phenoxymethylpenicillin potassium RS at the maximum at
about 268 urn. Calculate the content of CI6HlsNzOsS, in the
medium.
CI6HlsNzOsS.
Test solution. Weigh and finely powder 20 tablets. Dissolve

an accurately weighed quantity ofthe powder containing about
0.25 g of phenoxymethy1penicillin in the mobile phase by
shaking for 5 minutes and dilute to 100.0 ml with the mobile
phase. Filter through a 0.5 Jlm or finer filter and use the filtrate.
1897
PHENOXYMETHYLPENICILLIN POTASSIUM TABLETS
IP 2010

quantity of phenoxymethylpenicillin potassium RS in the
mobile phase and dilute to obtain a solution having a known
concentration ofabout 2.5 mg per ml.
Reference solution (b). A solution in the. mobile phase
containing 0.25 per cent w/v each ofbenzylpenicillin potassium
and phenoxyinethylpenicillin potassium.
octadecylsilane chemically bonded to porous silica,
350 volumes of acetonitrile and 5.75 volumes ofglacial
acetic acid,
Inject reference solution (b). The relative retention times are
about 0.8 for benzylpenicillin and 1.0 for phenoxymethylpenicillin. The column efficiency determined from the
phenoxymethylpenicillin peak is not less than 1800 theoretical
plates and the resolution between the benzylpenicillin and
phenoxymethylpenicillin peaks is not less than 3.0.
Inject reference solution (b). The relative standard deviation
chromatograms and measure the responses for the
phenoxymethylpenicillin peak and any p-hydroxyphenoxymethylpenicillin peak with a retention time ofabout 004 relative
to that ofthe main phenoxymethylpenicillin peak.
Calculate the content of C16HI7NzOsS, from the sum of the
peak responses ofthe p-hydroxyphenoxymethylpenicillin and
phenoxymethylpenicillin peaks in the chromatograms obtained
with the test solution and reference solution (a)
equivalent amount ofphenoxymethylpenicillin.
Phentolamine Mesylate is 3-[[4,5-dihydro-1H-imidazol-2yl)methy1](4-methylphenyl)aminophenol

methanesulphonate.
Phentolamine Mesylate contains not less than 99.0 per cent
and not more than 100.5 per cent of C 17H I9N 30, CH4 0 3 S,
Category. Alpha-adrenoceptor antagonist.
Dose. By intravenous injection, 5 to 10 mg.
Identification

A. Determine by infrared absorption spectrophotometry(2A.6).
Compare the spectrum with that obtained with phentolamine
mesylate RS or with the reference spectrum of phentolamine
mesylate.
B. When examined in the range 230 nm to 360 nm (204.7), a
0.002 per cent w/v solution shows an absorption maximum
C. Dissolve 0.5 gin 5 ml of ethanol (95 per cent) and 5 mlof
0.1 M hydrochloric acid and add 2 ml of a 0.5 per cent w/v
solution of ammonium metavanadate; a light green precipitate
is produced.
D. Mix 50 mg with 0.2 g ofpowdered sodium hydroxide, heat
to fusion and continue the heating for a few seconds longer.
Cool, add 0.5 ml of water and a slight excess of 2 M
hydrochloric acid amI warm; szdphur dioxide is evolved,
which turns moistened starch iodate paper blue.
Tests
Acidity or alkalinity. Dissolve 0.1 gin 10 m1 of carbon dioxidefree water and add 0.1 ml of methyl red solution. The solution
is not red and not more than 0.05 ml of 0.1 Msodium hydroxide
is required to change the colour of the solution.
(204.17), coating the plate with silica gel G.
Phentolamine Mesylate
Mobile phase. A mixture of 85 volumes of 2-butanone,

15 volumes of acetone and 5 volumes of strong ammonia
solution.
Mol. Wt. 377.5

1898
IP 2010
PHENYLEPHRINE HYDROCHLORlDE

reference solution.
Phenylephrine Hydrochloride
H,,,OH H
HO~N'CH3

anhydrous 2-propanol with the aid ofultrasound ifnecessary.
Titrate with 0.1 M tetrabutylammonium hydroxide in
2-propanol. Determine the end-point potentiometrically
(2.4.25), using a glass electrode and a calomel electrode
containing a saturated solution of tetramethylammonium
chloride in 2-propanol. Carry out a blank titration.
0.03775 g ofC 17H 19N 30, CH40 3S.
~H13N02,HCI
,HCI
Mol. Wt. 203.7
Phenylephrine Hydrochloride is (R)-I-(3-hydroxyphenyl)-2methylaminoethanol hydrochloride.

Phenylephrine Hydrochloride contains not less than 98.5 per
cent and not more than 101.0 per cent of C9H 13N02, HCI,
Dose. By subcutaneous or intramuscular injection, 2 to 5 mg;
by slow intravenous injection, 100 to 500 IJ.g; by intravenous
infusion 5 to 20 mg in 500 ml, adjusted according to response.
Identification
Phentolamine Injection

Band Cmay be omitted if tests A and D are carried out.
Phentolamine Mesylate Injection

Phentolamine Injection is a sterile solution of Phentolamine
Mesylate in Water for Injections containing Dextrose.
Phentolamine Injection contains not less than 95.0 per cent
and not more than 105.0per cent of the stated amount of
phentolamine mesylate, C 17H 19N 30, CH40 3S.
The residue obtained in the Assay melts at about 138 (2.4.21 ).
Tests
B. Dissolve about 10 mgin I ml of water and add 0.05 mlof

cupric sulphate solution and I ml of 5 M sodium hydroxide;
a violet colour is produced. Add I ml of ether and shake; the
C. Dissolve 0.3 gin 3 ml of water, add I ml of 6 M ammonia
and initiate crystallisation by scratching the side of the tube
with a glass rod. The melting range of the crystals, after
washing with iced water and drying at 105 for 2 hours, is 171
to 176(2.4.21).
D. Gives reaction A ofchlorides (2.3.1).
pH (2.4.24). 3.5 to 5.0.

Assay. Dilute an accurately measured volume containing about
0.1 g ofPhentolamine Mesylate to 40 ml with water, add 20 ml
of a 20 per cent w/v solution of trichloroacetic acid, allow to
stand for 3 hours, filter, wash the residue with two quantities,
each of 5 ml, of water and dry to constant weight at 105.
I g of the residue is equivalent to 0.8487 g of C 17H 19N 30,
phenylephrine hydrochloride.
etherlayer remains colourless.
Identification
C~03S,

Compare the spectrum with that obtained with phenylephrine
Tests
Appearance of solution. Dissolve 2.0 g in 100 ml of carbon
dioxide-free water prepared from distilled water (solution
A). Solution A is clear (2.4.1), and colourless (2.4.1).
methyl red solution and 0.2 ml of 0.01 M sodium hydroxide.
The solution is yellow and not more than 0.4 ml of 0.01 M
solution to red.
Specific optical rotation (2.4.22). -43.0 to -47.0. determined
in solution A.
1899
PHENYLEPHRINE HYDROCHLORIDE
IP 2010

(2.4.17), coating the plate with silica gel H.

15 volumes of 10Mammonia and 5 volumes of chloroform.
Apply to the plate 5 !J.I of each solution. After development,
dry the plate in cold air, spray with ninhydrin solution, heat at
105 for 10 minutes and examine in daylight. Any secondary
spot in the chromatogram obtained with the test solution is
not moreintense than the spot in the chromatogram obtained
with reference solution (a) and not more than two such spots
are more intense than the spot in the chromatogram obtained
Phenones. To 10 ml of solution A add sufficient 0.01 M
hydrochloric acid to produce 50 ml. Absorbance of the
resulting solution at the maximum at about 310 om, not more
than 0.20 (2.4.7).
Losson drying{2.4.19). Not more than 1.0 per cent,deterrnined
0.5 ml of 0.1 M hydrochloric acid and 80 ml of ethanol
(95 per cent) and titrate with 0.1 M ethanolic sodium
hydroxide determining the end-point potentiometrically
(2.4.25). Record the volume added between the two inflections.
0.02037 gofCgH 13NOz,HCl.

Identification
A. In the test for Related substances, the principal spot in the

chromatogram obtained with test solution (b) c9rresponds to
B. To a volume containing 10 mg of Phenylephrine
Hydrochloride add, if necessary, sufficient water to produce
1 ml and then add 0.05 ml of cupric sulphate solution and I ml
of 5 M sodium hydroxide; a violet colour is produced. Add
1 ml of ether and shake; the ether layer remains colourless.
Tests
pH (2.4.24). 4.5 to 6.5.

15 volumes of 10Mammonia and 5 volumes of chloroform.
Test solution (a) Evaporate a volume ofthe injection containing
20 mg ofPhenylephrine Hydrochloride to dryness and dissolve
the residue in 1 rol of methanol.
Test solution (b). Dilute 1 volume of test solution (a) to
Reference solution (a). Dilute 1 volume of test solution (b) to
2.5 volumes with methanol.
phenylephrine hydrochloride RS in methanol.
Apply to the plate 5 !J.I of each solution. After development,
dry the plate in cold air, spray with ninhydrin solution, heat at
105 for 10 minutes and examine in daylight. Any secondary
spot in the chromatogram obtained with test solution (a) is
not more intense than the spot in the chromatogram obtained
with test solution (b) and not more than two such spots are
with reference solution (a).
Phenylephrine Injection
Phenylephrine Hydrochloride Injection
Phenylephrine Injection is a sterile solution of Phenylephrine
Phenylephrine Injection contains not less than 95.0 per cent
and not more than 105.0 per cent of the stated amount.of
phenylephrine hydrochloride, CgH 13NOz,HCl.

50 mg of Phenylephrine Hydrochloride add sufficient 0.5 M
sulphuric acid to produce 100.0 ml. Dilute 10.0 ml of this
solution to 100.0 ml with 0.5 M sulphuric acid and measure
about 273 om (2.4.7). Calculate the content ofC gH I3NO z,HCl
1900
IP 2010
PHENYLMERCURIC NITRATE
Phenylmercuric Acetate
thiocyanate. Repeat the operation without the substance under

the amount of ammonium thiocyanate required.
1 ml of 0.1 M ammonium thiocyanate is equivalent to
0.01684 g ofC gHgHg02
Mol. Wt. 336.7
Phenylmercuric Acetate is (acetato)phenylmercury.

Phenylmercuric Acetate contains not less than 98.0 per cent
and not more than 100.5 per cent ofC gH gHg02
Category. Pharmaceutical aid (antimicrobial preservative).
Description. A white to creamy white, crystalline powder, or
small white prisms or leaflets.
Identification
A. To 100 mg add 0.5 mlofnitricacid, warm gently until a dark
brown colour is produced and dilute with water to 10 ml; the
characteristic odour of nitrobenzene is produced.
B. To 100 mg add 0.5 ml of sulphuric acid and I ml of ethanol
(95 per cent) and warm; the characteristic odour of ethyl
acetate is produced.
C. To 5 ml of a saturated solution in water, add a few drops of
a freshly prepared 10 per cent w/v solution ofsodium sulphide;
a white precipitate is formed which darkens slowly on boiling
and allowing to stand.
Tests
Mercuric salts and heavy metals. Heat about 100 mg with
15 rn1 of water, cool and filter. To the filtrate add a few drops of
a freshly prepared 10 per cent w/v solution ofsodium sulphide;
the precipitate formed shows no immediate colour.
Polymercurated benzene compounds. Shake 2.0 g with 100 rn1
of acetone. Filter, wash the residue with successive portions
of acetone until a total of 50 ml is used, dry the residue at 105
for 1 hour and weigh. The weight of the residue does not
exceed 30 mg (1.5 per cent).
Phenylmercuric Nitrate
Phenylmercuric Nitrate is a mixture ofphenylmercuric nitrate,
C6H sHgN03 and phenylmercuric hydroxide, C 6H sHgOH.
Phenylmercuric Nitrate contains not less than 62.5 per cent
and not more than 63.5 per cent ofmercury, Hg, calculated on
the dried basis.
Category. Antiseptic; pharmaceutical aid (antimicrobial
preservative).
Description. A white or pale yellow powder.
Identification
A. To 0.1 g add 3 ml of sulphuric acid; the mixture becomes
yellow and the characteristic odour of nitrobenzene is
produced.
B. To 0.1 g add 45 ml of water and heat to boiling with shaking.
Cool, filter and add sufficient water to produce 50 ml (solution
A). To 1 ml ofsolution A add 1 mlof2 Mhydrochloricacid; a
white, flocculent precipitate is produced.
C. To 5 ml of solution A add 8 ml of water and 0.1 ml of a
freshly prepared 10 per cent w/v solution of sodium sulphide;
a white precipitate is formed which darkens slowly on boiling
and allowing to stand.
D. To 5 ml of solution A add 1 ml of 2 M hydrochloric acid,
2 m1 of dichloromethane and 0.2 ml of dithizone solution and
shake; the lower layer is orange-yellow.
E. Solution A gives reaction ofnitrates (2.3.1).
Tests
Appearance ofsolution. Solution A is colourless (2.4.1).
Assay. Weigh accurately about 0.4 g, transfer to a 100-ml flask,

add 15 ml of water, 5 ml ofjormic acid and 1 g ofzinc dust and
reflux for 30 minutes. Cool, filter and wash the filter paper and
the amalgam with water until the washings are no longer acidic
to litmus. Dissolve the amalgam in 40 ml of 8 M nitric acid.
Heat on a water-bath for 3 minutes and add 0.5 g urea and
sufficient 0.02 M potassium permanganate to produce a
permanent pink colour. Cool and add hydrogen peroxide
. solution to decolorise the solution. Add 1 ml of jerrie
ammonium sulphate solution and titrate with 0.1 M ammonium
Inorganic mercuric compounds. To a 10 ml ofsolution A add

2 ml ofpotassium iodide solution and 3 ml of2 M hydrochloric
acid and filter; the filtrate is colourless. Wash the precipitate
with 2 ml of water, combine the filtrate and washings and add
2 ml of2 M sodium hydroxide and sufficient water to produce
20 m!. 12 ml of the solution complies with the limit test for
heavy metals, Method A (2.3.13). Use lead standard solution
(1 ppm Pb) to prepare the standard (0.1 per cent).
1901
IP 2010
PHENYLPROPANOLAMINE HYDROCHLORIDE
on 1.0 g by drying overphosphoruspentoxide at a pressure
of 1.5 to 2.5 kPa for 24 hours.
D. Melting range (2.4.21). 194 to 197

of 90 ml of water and 10 ml of nitric acid. Add 2 ml offerric
ammonium sulphate solution and titrate with 0.1 M ammonium
thiocyanate until a persistent reddish yellow colour is
obtained. Repeat the operation without the substance under
the amount of ammonium thiocyanate required.
Tests
1 ml of 0.1 A1ammonium thiocyanate is equivalent to 0.02006

gofHg.
Storage. Store protected from light and moi~ture.
Appearance ofsolution. AS.0 per cent w/v solution in water is

clear (2.4.1) and colourless (2.4.1).
Acidity or alkalinity. To 10 ml of 5.0 per cent w/v solution in
water, add 0.1 ml of methyl red solution and 0.2 ml of 0.01 M
sodium hydroxide. The solution turns yellow. Add 0.4 ml of
0.01 M hydrochloric acid. The solution turns red.
Mobile phase. Amixture of6 volumes of 13.5 M ammonia, 24

volumes of ethanol (95 per cent) and 70 volumes of butanol.
Phenylpropanolamine Hydrochloride

, Hel
~HI3NO,HCl
Mol. Wt.187.7
Reference solution (a). Dissolve 20. mg of

phenylpropanolamine hydrochloride RS in 10 ml of ethanol
(95 per cent).
Phenylpropanolamine Hydrochloride is (IRS,2SR)-2-aminoI-phenylpropan-l-01 hydrochloride.

to 10.0 ml with ethanol (95 per cent).
Phenylpropanolamine Hydrochloride contains not less than

99.0 percent and not more than 101.5 per cent ofCcjH I3NO,HCl,
Reference solution (c). Dissolve 20 mg of

norpseudoephedrine hydrochloride RS in ethanol (95 per
cent), add 1 ml of test solution (a) and dilute to 10 mlwith
ethanol (95 per cent).
Category. Adrenoreceptor antagonist.
Reference solution (d). Dissolve 60 mg of ammonium chloride

in 10 ml ofthe methanol.
Identification
Tests A and E may be omitted if tests B, C and D are carried
out. Tests B, C and D may be omitted if tests A and E are
carried out.
phenylpropanolamine hydrochloride RSor with the reference
spectrum of phenylpropanolamine hydrochloride.
C. To a 1.0 per cent w/v solution of the substance under
examination in the water, add 0.2 ml of copper sulphate
solution and 0.3 ml of dilute sodium hydroxide solution. A
violet colour develops. Add 2 ml of ether and shake. A violet
precipitate is formed between the two layers.
Before use, spray the plate with a 2 per cent w/v solution of
disodium tetraborate, using 8 ml for a plate 100 mm by 200 mm
and dry in a stream of cold air for 30 minutes. Apply to the
plate 10 f.tl of each solution as bands about 10 mm by 3 mm.
Allow the mobile phase to rise 10 cm. Dry the plate in a current
ofwarm air, allow to cool, spray with a 0.2 per cent w/v solution
of ninhydrin in ethanol (95 per cent) and heat at 110 for 15
minutes. Any spot in the chromatogram obtained with test
solution (a) other than the principal spot and the spot
corresponding to ammonium chloride is not more intense than
the spot in the chromatogram obtained with reference solution
(b) (1.0 per cent). The test is not validunless the chromatogram
obtained with reference solution (c) shows two clearly
separated spots.
Phenylpropanonamine. Absorbance of a 2.0 per cent w/v
solution in 0.01 M hydrochloric acid at the maximum at about
283 urn (2.4.7) is not more than 0.1.
1902
IP 2010
PHENYTOIN
Heavy metals (2.3.13).12 ml of5.0 per cent w/v solution in

water complies with the limit test for heavy metals, Method D
(20 ppm).
of5 ml of 0.01 Mhydrochloric acid and 50 m1 of ethanol (95
per cent). Titrate with 0.1 M sodium hydroxide. Determine
the end-point potentiometrically (2.4.25). Carry out a blank
titration.
~HI4CINO.
Tests
Appearance of solution. Dissolve 1.0 g in a mixture of5 m1 of
1 M sodium hydroxide and 20 ml of water. The solution is
clear (2.4.1) and not more intensely coloured than reference
solution BYS5 (2.4.1).
Acidity or alkalinity. To 1.0 g, add 45 ml of water and boil for
2 minutes. Allow to cool and filter. Wash the filter with carbon
dioxide-free water and dilute the combined filtrate and
washings to 50 ml with the same solvent. To 10 ml of the
solution add 0.15 ml of methyl red solution; not more than 0.5
ml of 0.01 M hydrochloric acid is required to change the
colour ofthe solution to red. To 10 ml ofthe solution add 0.15
ml of bromothymol blue solution; not more than 0.5 ml of O. 01
M sodium hydroxide is required to change the colour of the
solution to blue.
Phenytoin

Diphenylhydantoin
Solvent mixture. Equal volumes of acetone and meth,anol.

Mobile phase. A mixture of 30 volumes of dioxan and 75
volumes of hexane.
Mol.Wt. 252.3
Phenytoin is 5,5-diphenylimidazolidine-2,4-dione.
Phenytoin contains not less than 99.0 per cent and not more
than 101.0 per cent of ClsHI2NzOz, calculated on the dried
basis.
Category. Anticonvulsant.
Reference solution (a). Dissolve 20 mg ofphenytoin RSin 10

ml ofthe solvent mixture.
Reference solution (b). Dissolve 8 mg of benzophenone in
100 ml ofthe solvent mixture.
Reference solution (c). Dissolve 8 mg of benzil in 100 ml of
Reference solution (d). Dilute 1 ml oftest solution (a) to 100
Reference solution (e). Mix 1 ml ofreference solution (b) and

1 ml ofreference solution (c).
Identification
Compare the spectrum with that obtained with phenytoin RS
or with the reference spectrum of phenytoin.

that in the chromatogram obtained with reference solution
(a).
C. To about 10mg, add 1 mlofwaterand 0.05 mlofammonia.
Heat until boiling begins. Add 0.05 ml of a 5 per cent w/v
solution of copper sulphate in dilute ammonia and shake, a
pink crystalline precipitate is formed.
Before use, wash the plate with a mixture of 30 volumes of

dioxan and 75 volumes of hexane. Allow the plate to dry in air.
Apply to the plate 10 ,...1 of each solution. Dry the plate in a
current ofcold air for 2 minutes. Allow the mobile phase to rise
15 cm. Dry the plate in air and examine under ultraviolet light
at 254 nm. In the chromatogram obtained with test solution (a)
any spot corresponding to benzophenone is not more intense
than the spot in the chromatogram obtained with the reference
solution (b) (0.2 per cent); any spot corresponding to benzil
with reference solution (c) (0.2 per cent) and any spot, apart
from the principal spot and any spot corresponding to benzophenone and benzil, is not more intense than the spot in the
chromatogram obtained with reference solution (d) (1 per
1903
. PHENYTOIN SODIUM
IP 2010
cent). The test is not valid unless the chromatogram obtained

with reference solution (e) shows two clearly separated spots.
B. Dissolve 0.25 gin 5 ml of water and acidifY with dilute

hydrochloric acid; a white precipitate is produced.
heavy metals, Method D (10 ppm).
C. Dissolve 0.1 g in 10 ml of a 10 per cent w/v solution of

pyridine, add 1 ml of cupric sulphate with pyridine solution
and allow to stand. for 10 minutes; a blue precipitate is
produced.
.
Sulphated ash (2.3.18); Not more than 0.1 per cent.

Loss ondrying (2.4.19). Not more than 0.5 per cent, detenhined
on LOg by drying in an oven at 105.
dimethylformamide. Titrate with 0.1 M sodium methoxide,
1 ml of 0.1 M sodium methoxide is equivalent to 0.02523 gof
ClsHlzNzOz.
D. Incinerate 0.1 g; the residue after neutralisation with

hydrochloric acid and addition of 2 ml of water gives the
reactions ofsodium salts (2.3.1).
Tests
Appearance of solution. Suspend 1.0 g in 5 ml of water and
dilute to 20 ml with 0.1 M sodium hydroxide; the solution is
Free phenytoin. Dissolve 0.3 gin 10 ml of a mixture of equal
volumes of pyridine and water and add 0.5 ml of dilute
phenolphthalein solution and 3 ml of silver nitrate-pyridine
reagent. Not more than 1.0 ml of 0.1 M sodium hydroxide is
required to change the colour of the solution to pink.
Phenytoin Sodium
Diphenylhydantoin Sodium
Related substances. Detenhine by thin-layer chromatography


45 volumes of 2-propanol and 10 volumes ofstrong ammonia
solution.
Mol. Wt. 274.3
Phenytoin Sodium is 4-oxo-5,5-diphenyl-2-imidazolidin-2-01il.te

Phenytoin Sodium contains not less than 98.0 per cent and Reference solution (a). A 0.04 per cent w/v solution of the
not more than 101.0 per cent ofClsHIINzNaOz, calculated on substance under examination in methanol.
. Reference solution (b). A 0.02 per cent w/v solution of
benzophenone in methanol.
Category. Anticonvulsant; antiarrhythmic.
Dose. As anticonvulsant, orally, 150 to 300 mg daily, increasing
gradually to 600 mg in accordance with the needs of patient.
In status epileptieus, by slow intravenous injection, 10 to 15
mg per kg at a rate not exceeding 50 mg per minute as a loading
dose; maintenance doses of 100 mg thereafter every 6 hours.
In arrhythmias, by intravenous injection, 3.5 to 5 mg per kg at
a rate not exceeding 50 mg per minute and repeated once if
necessary.
Apply to the plate 10 !J.l of each solution. After development,

and any spot corresponding to benzophenone is not more
intense than the spotin the chromatogram obtailled with
reference solution (b).
Description. A white powder; odourless; somewhat

hygroscopic.
Identification

A. Detenhine by infrared absorption spectrophotometry (2.4.6).

Compare the spectrum with that obtained with phenytoin
sodium RS or with the reference spectrum ofphenytoin sodium.
Assay. Weigh accurately about 0.18 g, suspend in 2 ml of

water, add 8 ml of 0.05 M sulphuric acid and heat gently for
1 minute. Add 30 ml of methanol, cool. Titrate with 0.1 M
potentiometrically (2.4.25). After the fIrst inflection, stop the
addition of sodium hydroxide, add 5 ml of silver nitrate
1904
IP 2010
PHENYTOIN INJECTION
solution in pyridine, mix and continue the titration until a

second inflection is reached. Record the volume of 0.1 M
sodium hydroxide added between the two inflections.
D. Incinerate 0.1 g; the residue after neutralisation with

hydrochloric acid and addition of 2 ml of water gives the
reactions of sodium salts (2.3.1).

CjsHjjNzNaOz.
Tests
Appearance of solution. Suspend 1.0 g in 5 ml of water and

dilute to 20 ml with 0.1 M sodium hydroxide; the solution is
Phenytoin Injection
Completeness of solution. The contents dissolve in the

quantity of the solvent recommended on the label and give a
clear solution.
Phenytoin Sodium Injection; Diphenylhydantoin Sodium

injection
Phenytoin Injection is a sterile material consisting ofPhenytoin
Sodium with or without buffering agents and other excipients.
It is filled in a sealed container.
Injection or other suitable solvent, immediately before use.

Phenytoin Injection contains not less than 90.0 per cent and
not more than 110.0 per centofthe stated amount ofphenytoin
sodium, ClsHjjNzNaOz.
Description. A white powder; odourless; somewhat
hygroscopic.

stated under Parenteral Preparations
requir~ments

solution in the stated solvent.
Mobile phase. A mixture of 45 volumes of chloroform, .

45 volumes of 2-propanol and 10 volumes of strong ammonia
solution.
Apply to the plate 10 p.l of each solution. After development,
and any spot corresponding to benzophenone is not more
Identification


l.Og.

Assay. Weigh accurately about 0.18 g ofthe mixed contents of

10 containers, suspend in 2 ml of water, add 8 ml of 0.05 M
sulphuric acid and heat gently for 1 minute. Add 30 ml of
methanol, cool. Titrate with 0.1 M sodium hydroxide,
determining the end-point potentiometrically (2.425). After
the first inflection, stop the addition of sodium hydroxide,
add 5 ml ofsilver nitrate solution inpyridine, mix and continue
the titration until a second inflection is reached. Record the
volume of 0.1 M sodium hydroxide added between the two
inflections.
B. Dissolve 0.25 g in 5 ml of water and acidify with dilute

hydrochloric acid; a white precipitate is produced.

pyridine, add 1 ml of cupric sulphate with pyridine solution
and allow to stand for 10 minutes; a blue precipitate is
produced.
1905
PHENYTOIN CAPSULES
IP 2010
1 m1 of 0.1 M sodium hydroxide is equivalent to 0.02743 g of

ClsHIINzNaOz.
exceeding 30.
Labelling. The label states (1) the quantity of Phenytoin
Sodium contained in it; (2) the directions for preparing the
Injection.
Apply to the plate 10 l!1 of each solution. After development,

dry at 80 for 5 minutes and examine in ultraviolet light at 254
nm. Any spot in the chromatogram obtained with the test
solution corresponding to benzophenone is not more intense
solution (b) (0.5 per cent) and any other secondary spot is not
with reference solution (a) (0.5 per cent).
Phenytoin Capsules
Phenytoin Sodium Capsules; Diphenylhydantoin Sodium
Capsules
Phenytoin Capsules contain not less than 92.7 per cent and
not more than 107.5 per cent ofthe stated amount ofphenytoin
sodium, CISHIINzNaOz.
Assay. Shake a quantity ofthe mixed contents of20 capsules

containing about 0.25 g of Phenytoin Sodium with 50 ml of
0.01 M sodium hydroxide. Centrifuge, acidify 25 ml of the
clear liquid with 10 ml of 0.1 M hydrochloric acid and extract
with successive quantities of50, 40 and 25 ml of ether. Wash
the combined extracts with 10 ml of water, evaporate to
dryness and dry the residue at 105. Dissolve in 50 ml of
anhydrous pyridine. Titrate with 0.1 M tetrabutylammonium
hydroxide using 0.3 per cent w/v solution of thymol blue in
anhydrous pyridine as indicator.
0.02743 g ofClsHIINzNaOz.
Identification
A. Centrifuge the precipitated mixture obtained in test B,
dissolve the residue in methanol, evaporate and dry at 105
for 30 minutes. The residue complies with the following test.
B. Shake a quantity of the content of capsules containing

about 0.5 g ofPhenytoin Sodium with 10 ml ofwater and filter.
The filtrate yields a white precipitate on the addition of 2 M
hydrochloric acid.
C. To 0.1 g ofthe residue obtained in testA, add 0.5 ml of 1 M
sodium hydroxide, 10 ml of a 10per cent w/vsolution of
pyridine and 1 ml of copper sulphate-pyridine reagent and
allow to stand for 10 minutes. A blue precipitate is produced.
Tests
Mobile phase. A mixture of 10 volumes of 13.5 M ammonia,

45 volumes of chloroform and 45 volumes ofpropan-2-ol.
Test solution. Shake a quantity of the content of capsules
containing about 0.2 g of Phenytoin Sodium with 5 m1 of
methanol, warm on a water-bath with shaking and filter.
ml with methanol.
Phenytoin Tablets
Phenytoin Sodium Tablets; Diphenylhydantoin Sodium
Tablets
Phenytoin Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of phenytoin
sodium, ClsHIINzNaOz. The tablets are coated.
Identification
of Phenytoin Sodium with 20 ml of water, filter, acidify with
dilute hydrochloric acid and extract with 10 ml of chloroform.
Wash the chlorofonn extract with water, dry with anhydrous
sodium sulphate and evaporate to dryness and dry the residue
at 105. The residue complies with the following test.
DetermIne by infrared absorption spectrophotometry (2.4.6).
sodium sodium RS treated in the same manner or with the
reference spectrum of phenytoin sodium.
B. Triturate a quantity ofthepowdered tablets containing 0.5

g ofPhenytoin Sodium with 10 ml of water and filter. Acidify
with dilute hydrochloric acid; a white precipitate is produced.
C. The powdered tablets, when moistened with hydrochloric
1906
IP 2010
PHENYTOIN ORAL SUSPENSION
Tests
When stored at the temperature and for the period stated on

the label during which the constituted suspension may be
expected to be satisfactory for use, it contains not less than
80.0 per cent of the stated amount of phenytoin, ClsH12N202.
Related substances. Detennine bythin-layer chromatography

containing 0.1 g ofPhenytoin Sodium with 5 ml of methanol,
wann on a water-bath with shaking and filter.

light at 254 nm. In the chromatogram obtained with the test
solution any spot corresponding tobenzophenone is not more
reference solution.
Usual strength. 25 mg per m!.

exceeding 30.
The constituted suspension complies with the tests stated
under Oral Liquids and with the following tests.
Identificatio~

Compare the spectrum with that obtain with phenytoin RS or
with the reference spectrum of phenytoin.
Tests
pH (2.4.24).4.5 to 5.5 determined on 1.0 g.

quantity of the powder containing about 0.25 g of Phenytoin
Sodium, shake with 40 ml of 0.01 M sodium hydroxide for
5 minutes and add sufficient 0.01 M sodium hydroxide to
produce 50.0 m!. Centrifuge, aciditY 25.0 rnl ofthe clear liquid
with 10 ml of 0.1 M hydrochloric acid and extract successively
with 50, 40, 25 and 25 ml of ether. Wash the combined extracts
with 10 ml of water, evaporate to dryness and dry the residue
at 105. Dissolve in 50 m1 of anhydrous pyridine and titrate
with 0.1 Mtetrabutylammonium hydroxide, using 0.3 per cent
w/v solution of thymol blue in pyridine as indicator and taking
care to prevent absorption of carbon dioxide from the
atmosphere. Carry out a blank titration.
1 m1 of 0.1 M tetrabutylammonium hydroxide is equivalent to
0.02743 g ofClsHIIN2Na02.
Benzil and benzophenone. Determine by thin-layer

chromatography (2.4.17), coating the plate with silica gel
GF254.
Mobile phase. A mixture 000 volumes of 1, 4-dioxan and 75
volumes of hexane.
Test solution. Disperse a quantity of suspension containing
about 30 mg of Phenytoin in 5 ml of water add 2 ml of 2 M
hydrochloric acid, mix well and extract with five 20 ml quantities
of ether. Combine the ether extracts, wash with three 10 ml
quantities of water, evaporate to dryness and dissolve the
residue in 1.5 ml ofa mixture on volume ofglacial acetic acid
and 9 volumes of acetone.
benzophenone in ethanol (95 per cent).
Reference solution (b). A 0.004 per cent w/v solution of benzil
in ethanol (95 per cent).

Diphenylhydantoin Oral Suspension
Phenytoin Oral Suspension is a mixture of consisting of
Phenytoin with buffering agents and other excipients. It
contains a suitable flavouring agent. It is filled in a sealed
container.
The suspension is constituted by dispersing the contents of
the sealedcontainer in the specific volume ofWaterjust before
use.
Phenytoin Oral Suspension contains not less than 90.0 per

phenytoin, ClsHI2N202.

phase to.rise 12 cm. Dry the plate in air and examine in ultraviolet
spot in the chromatogram obtain with reference solution (a)
(0.2 per cent) and not more than one such spot is more intense
Assay. Disperse a quantity ofthe suspension containing about
0.2 g of Phenytoin, add 10 ml of 2 M hydrochloric acid and
15 ml of water, extract with three 50 ml quantities ofa mixture
00 volumes of chloroform and 1volume ofpropan-2-01 and
evaporate the combined extract to dryness. Dry the residue at
105 for 1 hour, cool and dissolve in a mixture 00 ml of 1 M
sodium hydroxide and 50 ml of water with the aid of gentle
1907
PHOLCODINE
IP 2010
heat. Cool and pass a current of carbon dioxide through the

solution until precipitation of phenytoin is complete, filter
and wash the residue with two 10 ml quantities of water and
dry at 105 for 2 hours.
Determine the weight per ml (2.4.29) ofthe oral suspension.
Calculate the content OfClSHI2N202, weight in volume.
sodium nitrite, allow to stand for 15 minutes and add 3 ml of

6 M ammonia. The solution is not more intensely coloured
than reference solution BS4 (2.4.1).

cent), 70 volumes of toluene, 65 volumes of acetone and
Pbolcodine

Referencesolution (b). A 0.0125 per cent w/v solutioriofthe
Pholcodine contains not less than 98.0 per cent and not more
than 101.0 per cent of C23H30N204, calculated on the dried
basis.

reference solution (a) and not more than one such spot of
higher Rf value than the principal spot is more intense than
the spot in the chromatogram obtained with reference solution
(b).
Category. Cough suppressant.
C23H30N204,H20
Mol. Wt. 416.5
Pholcodine is 7,8-didehydro-4,5a-epoxy-17-methyl-3-[2(morpholin-4-yl)ethoxy]morphinan-6a-ol monohydrate.
Dos~. Upto 40 mg daily, in divided doses.
Loss on drying (2.4.19). 3.9 to 4.5 per cent, determined on 0.5

g by drying in an oven at 105.
Identification
Compare the spectrum with that obtained with pholcodine RS
or with the reference spectrum of pholcodine.
B. To 10 ml ofa 0.1 per cent w/v solution add 75 ml of water
and 10 ml of 1 M sodium hydroxide and dilute to 100 ml with
water. When examined in the range 230 nm and 360 nm (2.4.7),
the resulting solution shows an absorption maximum at about
284 nm; absorbance at about 284 nm, 0.36 to 0.39.
C. Dissolve 50 mgin 1 ml of sulphuric acid and add 0.05 ml of
a 10 per cent w/v solution of ammonium molybdate; a pale
blue colour is produced. Warm gently; the colour changes to
deep blue. Add 0.05 ml of2 M nitric acid; the colour changes
to brownish red.
Tests
at 20 in a 2.0 per cent w/v solution in ethanol (95 per cent).
Morphine. Dissolve 0.1 g in sufficient of 0.1 M hydrochloric
acid to produce 5 ml, add 2 ml of a 1 per cent w/v solution of

anhydrous glacial acetic acid, warming gently. Titrate with
0.1 M perchloric acid, determining the end-point
potentiometrically at the second inflection (2.4.25). Carry out
a blank titration.
C23H30N204.
Pbolcodine Linctus
Pholcodine Linctus is a solution containing 0.1 per cent w/v
solution of Pholcodine and 1 per cent w/v solution of Citric
Acid Monohydrate in a suitable flavoured vehicle.
Pholcodine Linctus contains not less than 0.090 per cent and
not more than 0.110 per cent w/v of pholcodine,
C23H30N204.H20.
Category. Opioid cough suppressant.
1908
PHOSPHORIC ACID
IP 2010
Identification
Category. Pharmaceutical aid (acidifYing agent).
To 20 ml add 20 ml of water, make alkaline to litmus paper with

5 M ammonia, extract with two quantities, each of 20 ml, of
chloroform, washing each extract with 5 ml of water, dry the
combined extracts with anhydrous sodium sulphate, filter and
evaporate to dryness. If necessary, add 0.1 ml of ether and
scratch the sides of the vessel with a glass rod to induce
crystallisation. The crystals, dried at a pressure not exceeding
2 kPa, comply with the following tests.
Description. A clear, colourless, syrupy liquid; corrosive.

When kept at a low temperature it may solidifY, producing a
mass of colourless crystals which do not melt until the
temperature reaches 28.

Compare the spectrum with that obtained with pholcodine RS
or with the reference spectrum of pholcodine.
B. When examined in the range 230 urn and 360 nm (2.4.7), a
0.01 per cent w/v solution in 0.01 Msodium hydroxide shows
an absorption maximum only at about 284 nm.
C. To a portion ofthe crystals add 0.05 ml of nitric acid and
mix; a yellow colour is produced.
D. Dissolve the remainder ofthe crystals in 1 ml of sulphuric
acid and add 0.05 ml of ammonium molybdate-sulphuric acid
solution; a pale blue colour is produced. Warm gently; the
colour changes to deep blue. Add 0.05 ml of 2 M nitric acid;
the colour changes to brownish red.
Tests
Assay. Weigh accurately about 50 g and add sufficient 5 M
ammonia to make the solution alkaline to litmus paper, extract
with four quantities, each of 25 ml, of chloroform, washing
each extract with the same 5 ml of water. Combine the extracts
and evaporate until the volume is reduced to 15 m!. Titrate
with 0.02 M perchloric acid, using quinaldine red solution
as indicator. Carry out a blank titration.
~3H30Nz04,HzO.
Determine the weight perm! ofthe linctus (2.4.29), and calculate

the content ofCz3H30Nz04,HzO, weight in volume.
Identification
A. Dilute with water; the solution is strongly acidic.
B. Dilute 10.0 g to 150 ml with water (solution A). Neutralise

with 2 M sodium hydroxide; the resulting solution gives the
reactions of phosphates (2.3.1).
Tests
Appearance of solution. Solution A is clear (2.4.1), and
colourless (2.4.1).
Arsenic (2.3.1 0). Dissolve 5.0 g in 50 ml of water and add 10 ml
of stannated hydrochloric acid; the resulting solution
complies with the limit test for arsenic (2 ppm).
Heavy metals (2.3.13). Dilute 1.2 ml with 10 ml of water,
neutralise with dilute ammonia solution, add sufficient dilute
acetic acid to render the solution acidic and then.dilute to
25 ml with water. The resulting solution complies with the
limit test for heavy metals, Method A (I 0 ppm).
Iron (2.3.14). 10 ml ofsolutionA complies with the limit testfor
iron (60 ppm).
Chlorides (2.3.12). 3 ml ofsolution A complies with the limit
test for chlorides (50 ppm).
Alkali phosphates. To 1.7 g in a graduated cylinder add 6 ml of
ether and 2 ml of ethanol (95 per cent); no turbidity is
produced.
Aluminium and calcium. To 1.7 g add 10 ml of water and 8 ml
of dilute ammonia solution; the solution remains clear.

equivalent amount of pholcodine.
Assay. Weigh accurately about 1.0 g, add a solution oflO g of

sodium chloride in 30 ml of water and titrate with1 Msodium
hydroxide using dilute phenolphthalein solution as indicator.
Phosphoric Acid
Orthophosphoric Acid; Concentrated Phosphoric Acid
H3P04
Mol. Wt. 98.0
Phosphoric acid contains not less than 84.0 per cent w/w
and not more than 90.0 per cent w/w ofH3P04.
Hypophosphorus acid and phosphorous acid. To 5 m! ofsolution

A add 2 ml of a 1.7 per cent w/v solution of silver nitrate and
heat on a water-bath for 5 minutes; the appearance of the
solution does not change.

H 3P04
Storage. Store protected from moisture, in glass containers.
1909
PHYSOSTIGMINE SALICYLATE
IP 2010
water, and dilute to 100 ml with the same solvent (solutionA).

The solution, examined immediately after preparation, is clear
Eserine Salicylate
pH (2.4.24). 5.1 to 5.9, determined in solution A immediately

after preparation.
COO~H
&
Specific opticalrotation (2.4.22). -90.0 to -94.0, determined

in solution A immediately after preparation.
I"::
o
Eseridine. To 5 ml of solution A, examined immediately after

preparation, add a few crystals of potassium iodate and a
drop of 2 M hydrochloric acid and 2 ml of chloroform and
shake; the cWoroform layer does not tum violet within 1minute.
Mol. Wt. 413.5

Physostigmine Salicylate is (3aS,8aR)-1 ,2,3,3a,8,8ahexahydro-l ,3a,8-trimethylpyrrolo[2,3-b]indol-5-yl
methylcarbamate salicylate.

solution.
Physostigmine Salicylate contains not less than 98.5 per cent

and not more than 101.0 per cent of ClsH21N302, C 7H 60 3,


physostigmine salicylate RS in ethanol (95 per cent).
Dose. By subcutaneous or intramuscular injection, 600 flg to

1.2 mg.
Description. A colourless or faintly yellow crystals, turning
red gradually under the action of air and light and rapidly in
presence of moisture; odourless.
Identification
Test A may be omitted if tests B, C and D are carried out. Test
C may be omitted if tests A, Band D are carried out.
Compare the spectrum with that obtained with physostigmine
salicylate RS.
C. To a 1 per cent w/v solution add 1 M sodium hydroxide; a
white precipitate which turns pink is produced. The precipitate
dissolves in an excess ofthe reagent, producing a red solution.

physostigmine salicylate RS in ethanol (95 per cent).
dry the plate in cold air, carry out a second chromatographic
development in same direCtion, dry the plate in air and spray
with freshly prepared acetic potassium iodobismuthate
solution and then with hydrogen peroxide solution (IO vol).
Examine the plate within 2 minutes. Any secondary spot in the
chromatogram obtained with test solution (a) is not more
test for sulphates (0.1 per cent).
on the residue obtained in the test for Loss on drying.
Tests

mixture ofequal volumes of chloroform and anhydrous glacial
acetic acid. Titrate with 0.1 M perchloric acid, determining
titration.
Appearance of solution. Dissolve 0.9 g without heating in

95 ml of carbon dioxide-free water prepared from distilled
ClsH2IN302,C7~03'
D. A 0.9 per cent w/v solution in carbon dioxide-free water

gives reaction A of salicylates (2.3.1).
1910
IP 2010
PILOCARPINE NITRATE
Pilocarpine Nitrate
NlJ+JO
f'l
Physostigmine Injection
H3 C
Physostigmine Salicylate Injection; Eserine Salicylate

Injection
Physostigmine Injection is a sterile solution ofPhysostigmine
Salicylate in Water for Injections.
Physostigmine Injection contains not less than 90.0 per cent
physostigmine salicylate, ClsH21N302, C7H 60 3.
Usual strength. 600 flg per ml.
CH 3
\.
HN0 3
HH
CIIHI6N202, RN03
Mol. Wt. 271.3
Pilocarpine Nitrate is (3S,4R)-3-ethyl-4-[(I-methyl-lHimidazol-5-yl)methyl]dihydrofuran-2(3H)-one nitrate..

Pilocarpine Nitrate contains not less than 98.5 per cent and
not more than 101.0 per cent ofCIIHI6N202, RN0 3, calculated
on the dried basis.
Category. Cholinergic (ophthalmic) in glaucoma.
odourless; sensitive to light.
Identification
Identification
A. Warm a volume containing 3 mg of Physostigmine
Salicylate with 0.3 ml of5 M ammonia; a yellowish red solution
is produced which on evaporation gives a bluish residue.
B. The residue obtained in testA dissolves in ethanol (95 per
cent) producing a blue solution which, on the addition of
6 M acetic acid, appears blue by transmitted light and exhibits
a red fluorescence which intensifies on dilution with water.
C. The residue obtained in test A dissolves in sulphuric acid
producing a green solution which, on the gradual addition of
ethanol (95 per cent), changes to red but reverts to green
when the ethanol is evaporated.
Tests

Band C may be omitted if tests A andD are carried out.

Compare the spectrum with that obtained with pilocarpine
nitrate RS.
C. Dissolve about 10 mg in 2 ml of water, add 2 drops ofa 5 per
cent w/v solution ofpotassiumdichromate, 1 ml of hydrogen
peroxide solution (10 vol) and 2 ml of chloroform and shake;
the chlorofonn layer turns violet.
D. Gives reaction A ofnitrates (2.3.1).
pH (2.4.24).4.0 to 6.0.
Tests


coloured than reference solution YS6 (2.4.1).
Assay. Transfer an accurately measured volume containing

30 mg of Physostigmine Salicylate to a separator, add about
0.25 g of sodium bicarbonate and extract with six quantities,
each of 15 ml, of chloroform. Filter the combined chloroform
extracts through about 109 of anhydrous sodium sulphate.
Add 25 ml of anhydrous glacial acetic acid to the filtrate.
ClsH21N302, C7H 60 3
The injection should not be used if it is more than slightly
discoloured.
pH (2.4.24).3.5 to 4.5, determined in a 5.0 per cent w/v solution

prepared immediately before use in carbon dioxide-free water.
in a 5.0 per cent w/v solution in carbon dioxide-free water,
prepared immediately before use.

solution.
1911
IP 2010
PILOCARPINE NITRATE

examination in 100 m1 ofwater.
pilocarpine nitrate RS in water.
pilocarpine nitrate RS in water.
dry the plate at 105 for 10 minutes, cool and spray with
potassium iodobismuthate solution. Any secondary spot in
the chromatogram obtained with test solution (a) is not more
Other alkaloids. To a 1 per cent wlv solution add dilute
ammonia solution; no turbidity is produced. To a 1 per cent
w/v solution add a few drops of potassium dichromate
solution; no turbidity is produced.
Chlorides (2.3.12). 25 m1 ofa 10.0 per cent w/v solution complies
with the limit test for chlorides (100 ppm).
Pimozide contains not less than 99.0 per cent and not more
than 101.0 per cent ofCzsHz9FzN30, calculated on the dried
basis.
Description. A white to almost white powder.
Identification

Compare the spectrum with that obtained with pimozide RS or
with the reference spectrum ofpimozide.
Mobile phase. A mixture of 10 volumes of acetone and 90

volumes of methanol.
Iron (2.3.14). 20 ml ofa 10.0 per cent w/v solution complies

with the limit test for iron (20 ppm).
Reference solution (a). A 0.3 per cent w/v solution ofpimozide

on 0.5 g.
Reference solution (b). A solution containing 0.3 per cent wi

veach ofpimozide RS and benperidol RS in the mobile phase.

phase to rise 15 cm. Dry the plate in a current ofwarm air for 15
minutes and expose it to iodine vapour until the spots appear.
chromatogram obtained with reference solution (b) shows two

CIIHI6NzOz, RN03.
Pimozide
F
C. Mix about 5 mg ofthe substance under examination with 45

mg ofheavy magnesium oxide and ignite in a crucible until an
almost white residue is obtained. Allow to cool, add 1 ml of
water, 0.05 ml ofphenolphthalein solution and about 1 mlof
dilute hydrochloric acid to render the solution colourless.
Filter. To a freshly prepared mixture of 0.1 ml of alizarin S
solution and 0.1 ml ofzirconyl nitrate solution, add 1.0 ml of
the filtrate. Mix allow to stand for 5 minutes and compare the
colour of the solution with that of a blank prepared in the
same manner. The test solution is yellow and the blank is red.
D. Melting point (2.4.21).216 to 220.
Tests
F
Mo1.Wt. 461.6
Pimozide is 1-[I-[4,4-bis(4-fluorophenyl)butyl]piperidin-4yl]-l ,3-dihydro-2H-benzimidazol-2-one.

reference solution YS7 (2.4.1).
(2.4.14).
1912
IP 2010
PIMOZIDE TABLETS

Reference solution (a). Dissolve 5 mg of pimozide RS and 2
mg of mebendazole RS in 100 ml of methanol.
Reference solution (b). Dilute 5.0 ml ofthe test solution to 100
ml with methanol. Dilute 1.0 ml ofthis solution to 10 ml with
methanol.
mobile phase: A. a solution containing 0.25 per cent
w/v of ammonium acetate and 0.85 per cent w/v of
tetrabutylammonium hydrogen sulphate,
B. acetonitrile,
flow rate. 2ml per minute,
below,
Time
Mobile phase A
Mobile phase B
(per cent v/v)
(per cent v/v)
(in min.)
0-10
80~70
20~30
10-15
70
30
15-20
80
20
resolution between the peaks due to mebendazole and pimozide
.
is not less than 5.0.
peak due to (1-(piperidin-4-yl)-1 ,3-dihydro-2H-benzimidazol2-one) (pimozide impurityA) , (1-[1-[(4RS)-4-(4-fluorophenyl)4-phenylbutyl]piperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2one) (pimozide impurity B), (1-[1-[(4RS)-4-(2-fluorophenyl)-4(4-fluorophenyl)butyl]piperidin-4-yl]-1 ,3-dihydro-2Hbenzimidazol-2-one) (pimozide impurity C), (1-[ 1-[4,4-bis(4fluorophenyl)butyl]-1 ,2,3,6-tetrahydropyridin-4-yl]-1 ,3dihydro-2H-benzimidazol-2-one) (pimozide impurity D), (1-[1[4,4-bis(4-fluorophenyl)butyl]piperidin-4-yl I-oxide]-1,3dihydro-2H-benzimidazol-2-one) (pimozide impurity E), is not
obtained with reference solution (b) (0.5 per cent). The sum of
the areas of all the secondary peaks is not more than 1.5 times
the area of the principal peak in the chromatogram obtained
with reference solution (b) (0.75 per cent). Ignore any peak
with an area less than 0.1 times the area ofthe principal peale in
per cent).
Assay. Dissolve 0.3 g in 50 ml of a mixture of 1 volume of

anhydrous glacial acetic acid and 7 volumes of methyl ethy(
ketone and titrate with 0.1 M perchloric acid, using 0.2 ml of
naphtholbenzein solution as indicator. Carry out a blank
titration.
I ml 0.1 M perchloric acid is equivalent to 0.04616 g of
C2sH29F2N30.
Pimozide Tablets
Pimozide Tablets contain not less than 95.0 per cent and not
more than 105.0 per cent of the stated amount ofpimozide,
C2sH29F2N30.
Identification
20 mg ofPimozide with 20 rnl of dichloromethane for 5 minutes,
filter and evaporate the filtrate to dryness under reduced
pressure. On the residue, determine by infrared absorption
obtained with pimozide RS or with the reference spectrum of
pimozide.

Tests
Apparatus No.1,
Medium. 900 ml of 0.01 M hydrochlorIc acid,
Test solution. Dilute the filtrate, if necessary with the

dissolution medium.
Reference solution. A 0.0002 per cent w/v solution ofpimozide
RS in dissolution medium.
Use chromatographic system as described under Related
substances.
Calculate the content ofpimozide, C2sH29F2N30.

C2sH29F2N30.

(2.4.14).
1913
PIMOZIDE TABLETS
IP 2010

about 40 mg ofPimozide with 20 ml ofmethanol for 30 minutes,
mix with the aid ofultrasound for 10 minutes, centrifuge and
filter the supernatant liquid.
Reference solution (a). Dilute 1 ml of the test solution to 200
ml with methanol.
w/v of pimozide RS and 0.002 per cent w/v of mebendazole
RS in methanol.
/lm) (Such as Hypersil ODS),
mobile phase: A. a solution containing 0.25 per cent
w/v of ammonium acetate and 0.85 per cent w/vof
tetrabutylammonium hydrogen sulphate,
B. acetonitrile,
below,

RS in methanol.
substances.
resolution between the peaks due to pimozide and mebendazole
Calculate the content ofCzsHz9FzN30 in each tablet.

about 20 mg ofPimozide with 35 ml of methanol for 30 minutes,
dilute to 50 ml with methanol, mix with the aid of ultrasound
for 10 minutes, centrifuge and filter the supernatant liquid.
pimozide RS in methanol.
RS in methanol.
Time
(min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
80
20
10
70
30
15
70
30
16
80
20
resolution between the peaks due to pimozide and mebendazole
30
80
20

substances.
reSolution between the peaks obtained with mebendazole and
pimozide is not less than 5.0.
per cent) and the sum of the areas of all secondary peaks is
not more than 1.5 times the area of the principal peak in the
cent).
Calculate the content ofCzsHz9FzN30 in the tablets.

Pindolol
Uniformity of content (For tablets containing 10 mg or less).

Test solution. Shake one tablet with 7 ml of methanol for 30

minutes, dilute with methanol to produce a solution containing
0.01 per cent w/v ofPimozide, mix with the aid ofultrasound
for 10 minutes, centrifuge and filter the supernatant liquid.
pimozide RS in methanol.
Mol. wt. 248.3

Pindolol is (RS)-1-indol-4-yloxy-3-isopropylaminopropan-2-
01.
Pindolol contains not less than 99.0 per cent and not more
than 101.0 per cent of CIJIzoNzOz, calculated on the dried
basis.
Category. Beta-adrenoceptor antagonist.
1914
IP 2010
PINDOLOL TABLETS
heavy metals, Method C (20 ppm).
Description. A white or almost white, crystalline powder..
Identification
Compare the spectrum with that obtained with pindolol RS.
0.002 per cent w/v solution in a 0.085 per cent v/v solution of
hydrochloric acid in methanol shows absorption maxima at
about 264 nm and at about 287 nm, and a shoulder at about
275 nm, absorbances at about 264 nm, 0.66 to 0.70 and at
about 287 nm, 0.34 to 0.38.
methanol. Titrate with 0.1 M hydrochloric acid, determining
the end-point potentiometrically (2.4.25).
1 ml of 0.1 M hydrochloric acid is equivalent to 0.02483
C1Ji20N20 2.
of
e. In the test for Related substances, the principal spot in the Pindolol Tablets
Tests
Pindolol Tablets contains not less than 90.0 per cent and not
more than 11 0.0 per cent of the stated amount of pindolol,
C1Ji20N 20 2'
Appearance of solution. A 5.0 per cent w/v solution in dilute

acetic acid is clear (2.4.1) and not more intensely coloured
than reference solution BYS4 (2.4.1).
NOTE- Prepare the solutions immediately before use and

protected from light.
Solvent mixture. 99 volumes of methanol and 1 volume of
anhydrous glacial acetic acid.
Mobile phase. A freshly prepared mixture of 50 volumes of
ethyl acetate, 50 volumes of methanol and 4 volumes ofstrong
ammonia solution.
Reference solution (a). A 0.2 per cent w/v solution ofpindolo I
RS in the solvent mixture.
pindolol RS in the solvent mixture.
Apply to the plate 5 J!l of each solution. Allow the mobile
phase to rise 10 cm. Dry the plate immediately in a current of
cold air and examine in ultraviolet at 254 nm without delay.
cent).
Identification
ofPindolol with 80 m1 of ether for 30 minutes, filter and dry the
extract with anhydrous sodium sulphate. Filter the extract,
remove the ether using a rotary evaporator and dry the residue
over phosphoruspentoxide at 110 at a pressure not exceeding
2 kPa for 1 hour. The residue complies with the following test.
Compare the spectrum with that obtained with pindolol RS.
e. Shake a quantity ofthe powdered tablets containing 20 mg

of Pindolo1with 5 m1 ofa mixture of99 volumes of methanol
and 1 volume ofglacial acetic acid for 45 minutes. Centrifuge
and dilute 1 m1 of the supernatant liquid to 50 ml with the
acetic acid-methanol mixture. To 2 ml ofthis solution add 1 ml
of dimethylaminobenzaldehyde solution; a violet-blue colour
is produced.
Tests

protectedfrom light.
Solvent mixture. 99 volumes of methanol and 1 volume of
anhydrous glacial acetic acid.
1915
PINDOLOL TABLETS
IP 2010

ethyl acetate, 50 volumes of methanol and 4 volumes of strong
ammonia solution.
Pioglitazone Hydrochloride contains not less than 98.0 per

cent and not more than 102.0 per cent of C I9 Hzo N z 0 3S,HCl,

containing 20 mg ofPindolo1with 5 mI ofthe solvent mixture
for 15 minutes, centrifuge and apply the supernatant liquid to
the plate as the last solution.
Category. Antidiabetic.

with the solvent mixture and further dilute 7 ml ofthe solution
to 100 ml with the solvent mixture.
Reference solution (b). Dilute 1 ml ofthe test solution to 10 ml
with the solvent mixture and further dilute 3 ml ofthis solution
phase to rise 10 cm. Dry the plate in air, spray immediately with
dimethylaminobenzaldehyde solution and warm at 50 for
20 minutes. Any spot with R r value of about 0.1 in the
reference solution (a) (0.7 per cent). Any other secondary
with reference solution (b) (0.3 per cent). Ignore any spot
remaining on the line of application.
quantity of the powdered tablets containing about 90 mg of
Pindolol and shake with 100.0 ml of methanol for 45 minutes.
Centrifuge and dilute 15.0 ml of the supernatant liquid to
100.0 mI with methanol and measure the absorbance of the
Calculate the content ofC'4HzoNzOz taking 338 as the specific
Identification
Compare the spectrum with that obtained with pioglitazone
pioglitazone hydrochloride.
Tests
Related Substances. Detennine by liquid chromatography
(2.4.14)

examination in 100.0 mI of methanol.
pioglitazone hydrochloride RS in methanol.
Reference solution (b). Dilute 1.0 mI ofreference solution (a)
to 100.0 ml with methanol.
octadecylsilane bonded to porous silica ( 5 Ilm),
potassium dihydrogen phosphate and 50 volumes of
acetonitrile,
tailingfactQr is not more than 2.0 and the relative standard
Pioglitazone Hydrochloride
Mol.Wt. 392.9
Inject the blank, the test solution and reference solution (b).
of any secondary peak is not more than 0.5 times the area of
the peak in the chromatogram obtained with reference solution
(b) (0.5 per cent) and the sum ofthe areas ofall the secondary
peaks is not more than the area ofthe peak in the chromatogram
obtained with reference solution (b) (1.0 per cent ).
Pioglitazone Hydrochloride is (RS)-5-{4-[2-(5-ethyl-2pyridinyl)ethoxy]benzyl}thiazolidine-2,4-dione hydrocWoride.
heavy metals, Method B (20 ppm ).
, Hel
CI9HzoNz03S,HCl
1916
IP 2010
PIOGLITAZONE TABLETS

on 1.0 g by dying in an oven at 105.
described under the test for Related substances.
Test solution: Weigh and powder 20 tablets. Transfer a quantity

of the powder equivalent to 30 mg of pioglitazone and add
about 150 ml of methanol. Mix with the aid ofultrasound at a
temperature not exceeding 30 for 15 minutes and shake for 20
minutes. Dilute to 250 ml with methanol and mix. Filter through
a membrane filter and discard the first 5 ml. Use the filtrate as
the test solution.
Reference solution. A solution of pioglitazone hydrochloride
RS in methanol containing 0.00012 per cent w/v of
Calculate the content ofC l9 H zo N z0 3S,HCl.
pioglitazone.
Pioglitazone Hydrochloride Tablets
Pioglitazone Tablets contain not less than 90.0 per cent and
pioglitazone, CI9HzoNz03 S.
octadecylsilane bonded to porous silica (5 I!m),
potassium dihydrogen phosphate and 50 volumes of
acetonitrile,
-
Identification
of pioglitazone with 40 ml of methanol, filter and evaporate
the filtrate to dryness. The residue complies with the following
test.
Compare the spectrum with that obtained with pioglitazone
pioglitazone hydrochloride.
Tests
Apparatus No. 1

injection volume. 20 I!l.

Solvent mixture. A mixture of 35 volumes of water and 65

volumes of acetonitrile.
a quantity ofthe powder containing about 30 mg ofpioglitazone,
add about 20 ml of water and mix with the aid of ultrasound.
Add about 150 ml ofthe solvent mixture and mix with the aid
ofultrasound for 15 minutes. Dilute to 250.0 ml with the solvent
mixture.
pioglitazone hydrochloride RS in the solvent mixture.

(2.4.7). Calculate the content ofC19HzoNz03S in the medium
concentration ofpioglitazone hydrochloride RS in the same
medium.
CI9HzoNz03S,
(2.4.14).
octadecylsilane bonded to porous silica ( 5 I!m) ,
orthophosphate and 1.15 g of diammonium hydrogen
phosphate in 1000 ml of water, adding 1.0 ml of
triethylamine and adjusting the pH to 5.0 with
orthophosphoric acid, and 65 volumes of acetonitrile,
1917
PIPERACILLIN
IP 2010
- injectionvolume.20.gl.
deviation forreplicate injections is not more than 2.0 per cent.
Inject the test solutioIl and the. reference solution.

on .12 per cent w/v solution ofsodium dihydrogen phosphate.
examination in 50 m1 ofthe solvent mixture.
Calculate the content ofCI9HzoNz03 S in the tablets.

Labelling, The label state.s the strength in terms of the

equivalent amount ofpioglitazone.

piperacillin RS in the solvent mixture.

Reference solution (c). Dissolve 10 mg of piperacillin RS
and 10 mg of anhydrous ampicillin RS (piperacillin impurity
A RS) in 50 ml solvent mixture.
Piperacillin
Reference solution (d). Dilute 1.0 ml ofreference solution (a)

to 100 m1 with the solvent mixture. Dilute 1.0 ml ofthis solution
. H2 0
Mol.Wt. 535.6
Piperacillin is (6R)-6-[R-2-(4-ethyl-2,3-dioxopiperazine-lcarboxamido)-2-phenylacetamido]penicillanic acid
monohydrate.
Piperacillin contains not less than 96.0 per cent and not more
than 102.0 per cent of CZ3Hz7Ns07S, calculated on the
anhydrous basis.
Category. Penicillin antibacterial.
Description. A white oralmost white powder.
Identification
Compare the spectrum with that obtained with piperacillin
RS or with the reference spectrum ofpiperacillin.
a stainless steel column 25 em x 4.6 mm packed with
octadecylsilane bonded to porous silica (5 gm),
mobile phase: A. a mixture of576 volumes of water, 200
volumes of 3.12 per cent w/v solution of sodium
dihydrogen phosphate and 24 volumes of an 8.0 per
cent w/v solution of tetrabutylammonium hydroxide,
adjusted to pH 5.5 with dilute phosphoric acid or dilute
sodium hydroxide solution; add 200 volumes of
acetonitrile,
B. a mixture 126 volumes of water, 200
volumes of a 3.12 per cent w/v solution of sodium
dihydrogen phosphate and 24 volumes of an 8.0 per
cent w/v solution of tetrabutylammonium hydroxide,
adjusted to pH 5.5 with dilute phosphoric acici or dilute
sodium hydroxide solution; add 650 volumes of
acetonitrile, .
below,
injection volume. 20 gl.
Tests
Appearance of solution. A 10 per cent w/v solution in carbon
dioxide-free water is clear (2.4.1) and not more intensely
coloured than the reference solution BS8 (2.4,1).
Specific optical rotation (2.4.22). + 165 to + 175 , determined
in a 1.0 per centw/v solution in methanol.
0
Mobile phase A
(per cent v/v)
Mobile phaseB
(per cent v/v)
O-tg
88
12
tg -(tg +30)
88~0
12~100
(tg+30 )-( tg-45)
0~88
100~12

(2.4.14).
NOTE-Use freshly prepared solutions.
Time
(in min.)
where, tg
retention time ofpiperacillin determined with

reference solution (b)
Inject reference solution (b), (c) and (d). The test is not valid
unless the. resolution between the peaks due to piperacillin
impurity A and piperacillin in the chromatogram obtained with
1918
IP 2010
PIPERACILLIN INTRAVENOUS INFUSION
reference solution (c) is not less than 10; signal-to-noise ratio

is not less than 3 for the principal peak in the chromatogram
obtained with reference solution (d); mass distribution ratio
is (2:3) for the peak due to piperacillin in the chromatogram
obtained with reference solution (c). The area ofany secondary
peak is not more than twice the area of the principal peak in
per cent).
N,N-Dimethylaniline (2.3 .21). Not more than 20 ppm.
Water. (2.3.43). 2.0 per cent to 4.0 per cent, determined on
0.5g.
than 1.0 per cent.
Usual strengths. 1 g per vial; 2 g per vial; 4 g per vial.

requirements stated under Parental Preparations (Powder
for Injections) and with the following requirements.
Identification
A. Dissolve a quantity ofthe powder containing about 0.25 g
ofanhydrous piperacillin in 20 ml of water, add 0.5 ml of dilute
hydrochloric acid, mix well, allow to stand until a precipitate
appears and filter. Determine by infrared absorption
obtained with piperacillin RS or with the reference spectrum
ofpiperacillin.
C. Gives the reaction ofsodium (2.3.1).
Tests
Inject test solution (a) and reference solution (a).
pH (2.4.24).4.8 to 6.8, detennined in a 10.0 percent w/v solution.
Calculate the content of C23H27N507S,

(2.4.14).
Test solution. Disperse a quantity ofpowder containing about

39 mg ofanhydrous piperacillin in 100 ml ofthe mobile phase.
Piperacillin Intravenous Infusion

Piperacillin Intravenous Infusion is a sterile solution of
Piperacillin Sodium in Water for Injections. It is prepared by
dissolving Piperacillin for Intravenous Infusion in the requisite
amount of Water for Injections before use.
The intravenous infils ion complies with the requirements

stated under Parenteral Preparations (Injections).
Piperacillin for Intravenous Infusion

Piperacillin for Intravenous Infusion is a sterile material
consisting of Piperacillin Sodium or Piperacillin Sodium
prepared by the interaction of piperacillin with sodium
bicarbonate, with or without excipients. It is filled in a sealed
container.
.

Parental Preparations. (Injections).
Piperacillin for Intravenous Infusion contains not less than
amount ofpiperacillin, C23 H 27N507S.

piperacillin RS in the mobile phase.
octadecylsilane bonded to porous silica (5 to 10 Ilm),
(Such as Beckman Ultrasphere ODS),
mobile phase: a mixture of 0.3 volume of 0.4 M
tetrabutylammonium hydroxide" 10 volumes of 0.2 M
sodium dihydrogen orthophosphate monohydrate, 44.7
volumes of water and 45 volumes of methanol, cool
and adjust to pH 5.5 with orthophosphoric acid,
peak with a relative retention time of about 0.6 is not more
obtained with reference solution (b) (3.5 per cent). The area of
solution (a) (2 per cent).
1919
PIPERACILLIN INTRAVENOUS INFUSION
IP 2010

Bacterial endotoxins (2.2.3). Not more than 2.5 Endotoxin Units
per ml ofa solution prepared in the following manner. Dissolve
a quantity in water BETto obtain a solution containing 50 mg
ofpiperacillin per ml. Carry out the test using Maximum Valid
dilution ofthis solution calculated from the declared sensitivity
of the lysate used in the test.
Test solution. Disperse a quantity ofpowder containing about

39 mg ofanhydrous piperacillin in 100 ml ofthe mobile phase.
Reference solution (a). Dissolve 40 mg ofpiperacillin RS in
methanol and dilute to 100 ml with the mobile phase.
w/vof anhydrous ampicillin RS and 0.02 per cent w/v of
octadecylsilane bonded to porous silica (5 f..l.m), (Such
as Beckman Ultrasphere ODS),
mobile phase: a mixture of 0.3 volume of 0.4 M
tetrabutylammonium hydroxide, 10 volumes of 0.2 M
sodium dihydrogen orthophosphate monohydrate, 44.7
volumes of water and 45 volumes of methanol, cool
and adjust to pH 5.5 with orthophosphoric acid,
- injection volume. 10 f..l.l.
Inject reference solution (b). The resolution between the peaks
due to anhydrous ampicillin and piperacillin is not less than
16.
Inject the test solution and the reference solution (a).
Piperazine Adipate contains not less than 98.0 per cent and
not more than 101.0 per cent ofC4H ION 2, C 6H IO0 4, calculated
Identification
Test A may be omitted iftests B, C andD are carried out. Tests .

Compare the spectrum with that obtained with piperazine
adipate RS or with the reference spectrum of piperazine
adipate.
B. In the test for Related substances, examine the plate after
spraying with both the ninhydrin and iodine solutions. The
principal spot in the chromatogram obtained with test solution
(b) corresponds to that in the chromatogram obtained with
reference solution (a).
C. Dissolve 0.1 g in 5 ml of water, add 0.5 g of sodium
bicarbonate, 0.5 ml of a freshly prepared 5.0 per cent w/v
solution of potassium ferricyanide and 0.1 ml of mercury,
shake vigorously for 1 minute and allow to stand for
20 minutes; a reddish colour slowly develops.
Tests
(2.4.1), and not more intensely coloured than reference solutio!1
BS8 (2.4.1).
(2.4.17), coating the plate with silica gel G..
Solvent mixture. 60 volumes of strong ammonia solution and
40 volumes of ethanol.
Calculate the content ofC23H27Ns07S,

Labelling. The label ofthe sealed container states the quantity
of active ingredient contained in it in terms ofthe equivalent
amount of anhydrous piperacillin.

acetone and 20 volumes of strong ammonia solution.
Test solution (a). Dissolve I g of the substance under
Test solution (b). Dissolve 1 g of the substance under
Piperazine Adipate
Reference solution (a). A I per cent w/v solution ofpiperazine

adipate RS in the solvent mixture.
ethylenediamine in the solvent mixture.
triethylenediamine in the solvent mixture.
Reference solution (d). A solution containing 0.025 pei cent
w/v of triethylenediamine and 1 per cent w/v ofthe substance
under examination in the solvent mixture.
1920
IP 2010
PIPERAZINE CITRATE

dry the plate at 105, spray with a 0.3 per cent w/v solution of
ninhydrin in a mixture on volumes of anhydrous acetic acid
and 100 volumes of 1-butanol and then with a 0.15 per cent
w/v solution of ninhydrin in ethanol and dry at 105 for 10
minutes. Any secondary spot in the chromatogram obtained
with test solution (a) is not more intense than the spot in the
chromatogram obtained with reference solution (b). Spray the
plate with 0.05 M iodine and allow to stand for about 10
minutes. Any spot corresponding to triethylenediamine in
reference solution (c). The test is not valid unless the
chromatogram obtained with reference solution (d) shows two
separated spots. Ignore any spot remaining on the line of
application.
Heavy metals (2.3.13). Dissolve l.0 g in 20 ml ofwater, 0.5 ml of
0.1 M hydrochloric acid and add sufficient water to produce
25 ml. The resulting solution complies with the limit test for
heavy metals, Method A (20 ppm).
Sulphated ash(2.3.18). Not more than 0.1 per cent.
l.Og.
anhydrous glacial acetic acid with gentle heating and dilute
to 70 ml with the same solvent. Titrate with 0.1 Mperchloric
acid, using 0.25 ml of 1-naphtholbenzein solution as indicator
and titrating until the colour of the solution changes from
brownish yellow to green. Carry out a blank titration.
C4HIQN2, C6HIQ 0 4.
vigorously for 1 minute and allow to stand for 20 minutes; a

reddish colour slowly develops.
B. To 10 ml of the filtrate obtained in test A add 5 ml
hydrochloric acid and extract with three quantities, each
10 ml, of ether, evaporate the combined ether extracts
dryness; the residue, after washing with a small volume
water and drying at 105, melts at about 152 (2.4.21).
of
of
to
of
Tests
quantity of the powder containing about 0.2 g of Piperazine
Hydrate, shake with 10 ml of water for 1 hour, filter and wash
the residue with two quantities, each of 10 ml, of water. To the
combined extract and washings add 5 ml of 1 M sulphuric
acid and 50 ml of picric acid solution, bring to boil, allow to
stand for 1 hour and filter through a sintered-glass crucible
(porosity No.4) and wash the residue with successive
quantities, each of 10 ml, of a mixture of equal volumes of a
saturated solution ofpicric acid and water until the washings
are free from sulphate. Wash with five quantities, each of
10 ml, of ethanol and dry to constant weight at 105.
1 g ofthe residue is equivalent to 0.3567 g ofC4HIQN2 , 6H20.
equivalent amount of piperazine hydrate.
Piperazine Citrate
HO
Piperazine Adipate Tablets
Piperazine Adipate Tablets contain not less than 92.5 per cent
piperazine hydrate, C4HIQNz, 6H20.
Usual strength. The equivalent of 300 mg of piperazine
hydrate. (144 mg of Piperazine Adipate is approximately
equivalent to 120 mg ofPiperazine Hydrate).
GOOH
HOOG~GOOH
2

Piperazine Citrate is a salt of piperazine with citric acid
containing a variable amount ofwater of crystallisation.
Piperazine Citrate contains not less than 98.0 per cent and not
more than 101.0 per cent of(C4HIQN2)3, 2C6Hg0 7, calculated on
Identification
A. Extract a quantity ofthe powdered tablets containing 1 g of

Piperazine Hydrate with 20 ml of water and filter. Dilute 1mlof
the filtrate to 5 ml with water, add 0.5 g ofsodium bicarbonate,
0.5 ml of a freshly prepared 5.0 per cent w/v solution of
potassium ferricyanide and 0.1 ml of mercury, shake
Dose. For an adult, in the treatment ofthreadworm infestation,

equivalent of 1 to 2 g of Piperazine Hydrate daily, in divided
doses for 7 days; in the treatment of roundworm infestation,
equivalent of4.5 g ofPiperazine Hydrate as a single dose. For
a child, in the treatment of threadworm infestation, the
1921
PIPERAZINE CITRATE
IP 2010
equivalent of 40 mg of Piperazine Hydrate per kg of body

weight daily, in divided doses for 7 days; inthe treatment of
roundworm infestation, as a sillgle dose, the equivalent of 120
mg of Piperazine Hydrate per kg of body weight, up to a
maximum dose of4 g.
(150 mg ofPiperazine Citrate or 132 mg ofanhydrous piperazine
citrate is approximately equivalent to 120 mg of Piperazine
Hydrate).
Description. A white, granular powder; almost odourless.
Identification
citrate RS or with the reference spectrum ofpiperazine citrate.
spraying with both the ninhydrin solutions. The principal
spot in the chromatogram obtained with test solution (b)
C. A 10 per cent w/v solution gives the reactions of citrates

(2.3.1).
Tests
(2.4.1), and not more intensely coloured than reference solution
BS8 (2.4.1).

dry the plate at 105, spray with a 0.3 per cent w/v solution of
ninhydrin in a mixture on volumes of anhydrous acetic acid
and 100 volumes of 1-butanol and then with a 0.15 per cent
w/v solution of ninhydrin in ethanol and dry at 105 for
with test solution (a) is not more intense than the spot in the
chromatogram obtained with reference solution (b). Spray the
plate with 0.05 M iodine and allow to stand for about
10 minutes. Any spot corresponding to triethylenediamine in
reference solution (c). The test is not valid unless the
chromatogram obtained with reference solution (d) shows two
separated spots. Ignore any spot remaining on the line of
application.
to 70 ml with the same solvent. Titrate with 0.1 Mperchloric
brownish yellow to green. Carry out a blank titration.
(C4HIONz)3,2C6H807'

(2.4.17), coating the plate with silica gel G.

Piperazine Citrate Oral Solution; Piperazine Citrate Elixir

Piperazine Citrate Syrup is a solution ofPiperazine Citrate in a

suitable flavoured Vehicle.
Test solution (a). Dissolve 1 g of the substance under

piperazine citrate RS in the solvent mixture.
Piperazine Citrate Syrup contains the equivalent of not less

stated amount ofpiperazine hydrate, C4H ION z, 6H zO.
Usual strength. The equivale.nt of 750 rug of piperazine
hydrate in 5 ml.

(125 mg ofPiperazine Citrate or 110 mg ofanhydrous piperazine

citrate is approximately equivalent to 100 mg of Piperazine
Hydrate).

Identification
Reference solution (d). A solution containing 0.025 per cent

w/v of triethylenediamine and 1 per cent w/v ofthe substance
A. To 1 ml add 5 ml of2 M hydrochloric acid and, with stirring,

1 ml of a freshly prepared 50 per cent w/v solution of sodium
nitrite, cool in ice for 15 minutes, induce crystallisation, wash
1922
IP 2010
PIPERAZINE HYDRATE
the crystalline precipitate with water and dry at 105. The

crystals melt at about 159 (2.4.21).
B. Warm 10 ml with activated charcoal and filter. Boil a portion
of the filtrate with an excess of mercuric sulphate solution,
filter, boil the filtrate and add 0.25 ml of dilute potassium
permanganate solution; the permanganate solution is
decolorised and a white precipitate is produced.
C. AcidifY a portion of the filtrate obtained in test B with 1 M
sulphuric acid, add 0.25 ml of dilute potassium permanganate
solution, warm until the colour is discharged and add an excess
of bromine water; a white precipitate is produced either
immediately or on cooling.
Tests
Assay. Weigh accurately a quantity containing about 0.2 g of
Piperazine Hydrate, add 3.5 ml of 0.5 M sulphuric acid and
10 ml of water, add 100 ml ofpicric acid solution, heat on a
water-bath for 15 minutes, allow to stand for I hour and filter
through a sintered-glass crucible (porosity No.4). Wash the
residue with successive quantities, each of I 0 ml, ofa mixture
of equal volumes of a saturated solution of picric acid and
water until the washings are free from sulphate. Wash the
residue with five quantities, each of 10 ml, of ethanol and dry
to constant weight at 105.
1 g ofthe residue is equivalent to 0.3567 g ofC4HIQNz, 6H zO;
Determine the weight per ml ofthe syrup (2.4.29), and calculate
the content ofC4HIQNz, 6H zO, weight in volume.
equivalent amount of piperazine hydrate.
Description. Colourless, glassy deliquescent crystals.
Identification
A. Detennine by infrared absorption spectrophotome1:Iy (2.4.6).
hydrate RS.
spraying with both the ninhydrin solutions. The principal spot
in the chromatogram obtained with test solution (b)
C. Dissolve 0.2 g in 5 ml of dilute hydrochloric acid, add 0.5 g

of sodium nitrite and heat to boiling. Cool in ice for 15 minutes,
scratching the walls of the container with a glass rod and
filter. The crystals, after washing with 10 ml ofice-cold water
and drying at 105, melt at about 159 (2.4.21).
Tests
coloured than reference solution BS8 (2.4.1).
solution in carbon dioxide:free water.

Piperazine Hydrate

eN)

N
H
C4HIQNz,6HzO
treatment of threadworm infestation, 40 mg per kg of body

weight daily, in divided doses for 7 days. In the treatment of
roundworm infestation, as a single dose, 120 mg per kg of
body weight, up to a maximum dose of4 g.
Reference solution (a). A 1 per cent w/v solution ofpiperazine

hydrate RS in the solvent mixture.
Mol. Wt.194.2
Piperazine Hydrate contains not less than 98.0 per cent and
not more than 10 I. 0 per cent ofC4H IQNz, 6H zO.



1 to 2 g daily, in divided doses for 7 days; in the treatment of
roundworm infestation, 4 g as a single dose. For a child, in the

w/v of triethylenediamine and 1 per cent w/v of the substance
1923
PIPERAZINE HYDRATE
IF 2010
Apply to the plate 5 III of each solution. After development, Description. A white, crystalline powder; odourless or almost
dry the plate at 105, spray with a 0.3 per cent w/v solution of odourless.
ninhydrin in a rriixture oD volumes ofanhydrous acetic acid
and 100 volumes off-butanol and then with a 0.15 per cent Identification
w/v solution of ninhydrin in ethanol and dry at 105 for
10 minutes. Any secondary spot in the chromatogram obtained A. Dissolve 0.2 g in 5 ml of dilute hydrochloric acid, add
with test solution (a) is not more intense than the spot in the 0.5 g of sodium nitrite and heat to boiling. Cool in ice for
chromatogram obtained with reference solution (b). Spray the 15 minutes, scratching the walls ofthe container with a glass
plate with 0.05 M iodine and allow to stand for about rod and filter. The crystals, after washing with 10 ml of ice10 minutes. Any spot corresponding to triethylenediamine in cold water and drying at 105, melt at about 159 (2.4.21).
the chromatogram obtained with test solution (a) is not more B. Dissolve 0.1 g in 5 ml of water, add 0.5 g of sodium
intense than the spot in the chromatogram obtained with bicarbonate, 0.5 ml of a freshly prepared 5.0 per cent w/v
reference solution (c). The test is not valid unless the solution of potassium ferricyanide and 0.1 ml of mercury,
chromatogram obtained with reference solution (d) shows two shake vigorously for 1 minute and allow to stand for
separated spots. Ignore any spot remaining on the line of 20 minutes; a reddish colour slowly develops.
application.
C. Gives the reactions of phosphates (2.3.1).
Tests

to 70 ml with the same solvent. Titrate with 0.1 M perchloric
brownish yellow to green. Canoy out a blank titration.
Heavy metals (2.3.13). Dissolve 1.0 g in 20 ml of2 M acetic

acid and add sufficient water to produce 25 ml. The solution
complies with the limit test for heavy metals, Method A
(20 ppm).

C4H ION2,6H20.

3.5 ml of0.5 M sulphuric acid and 10 ml ofwater. Add 100 ml
of picric acid solution, heat on a water-bath for 15 minutes
and allow to stand for 1 hour. Filter through a sintered-glass
crucible (porosity No.4) and wash the residue with successive
quantities, each of 10 ml, of a mixture of equal volumes of a
saturated solution ofpicric acid and water until the washings
are free from sulphate. Wash the residue with five quantities,
each of 10 ml, of ethanol and dry to constant weight at 105.
C4H ION2, H3P04 , H20
Mol. Wt. 202.2
Piperazine Phosphate contains not less than 98.5 per cent

and not more than 100.5 per cent ofC 4H ION 2 , H3P0 4 ,
Water (2.3.43). 8.0 to 9.5 percent, determined on 0.25 g.
1 g ofthe residue is equivalent to 0.3382 g ofC 4H ION 2 , H3P04

the equivalent of 1 to 2 g ofPiperazine Hydrate daily, in divided
doses for 7 days; in the treatment of roundworm infestation,
the equivalent of4.5 g ofPiperazine Hydrate as a single dose.
For a child, in the treatment of threadworm infestation, the
equivalent of 40 mg of Piperazine Hydrate per kg of body
weight daily, in divided doses for 7 days; in the treatment of
roundworm infestation, as a single dose, the equivalent of 120
mg of Piperazine Hydrate per kg of body weight, up to a
maximum dose of4 g.
(125 mg ofpiperazine phosphate is approximately equivalent
to 120 mg ofPiperazine Hydrate).

If the tablets are intended to be chewed before swallowing
they may contain suitable flavouring agents.
Piperazine Phosphate Tablets contain not less than 92.5 per
piperazine phosphate, C4H ION 2, H3P04, H 20.
Usual strengths. The equivalent of260 mg; 520 mg Piperazine
Hydrate (125 mg of piperazine phosphate is approximately
equivalent to 120 mg ofPiperazine Hydrate).
1924
IP 2010
PlRACETAM
Identification
Extract a quantity of the powdered tablets containing 1 g of
Piperazine Phosphate with 20 rnl ofwater and filter. The filtrate
A. To 4 ml of the filtrate, add 1 ml of hydrochloric acid, 0.5 g
ofsodium nitrite and heat to boiling. Cool in ice for 15 minutes,
scratching the walls of the container with a glass rod and
filter. The crystals, after washing with 10 ml ofice-cold water
and drying at 105, melt at about 159 (2.4.21).
B. Dilute 1 ml of the filtrate to 5 ml with water, add 0.5 g of

sodium bicarbonate, 0.5 ml of a freshly prepared 5.0 per cent
w/v solution ofpotassiumferricyanide and 0.1 ml ofmercwy,
shake vigorously for 1 minute and allow to stand for
20 minutes; a reddish colour slowly develops.
Identification
Compare the spectrum with that obtained with piracetam RS
or with the reference spectrum ofpiracetam.
Tests
Appearance of solution. A20.0 per cent w/v solution in water
is clear (2.4.1) and colourless (2.4.1).
(2.4.14).
Solvent mixture. A mixture of 10 volumes of acetonitrile and
90 volumes ofwater.
C. Gives the reactions of phosphates (2.3.1).
Tests
Disintegration. The test does not apply to Piperazine
Phosphate Tablets intended to be chewed before swallowing.
quantity of the powder containing about 0.15 g of Piperazine
Phosphate, shake with 10 ml of water for 1 hour, filter and
wash the residue with two quantities, each of 10 ml, ofwater.
To the combined extract and washings add 5 ml of 1 M
sulphuric acid and 50 ml ofpicric acid solution, boil, allow
the mixture to stand for several hours and filter through a
sintered glass crucible (porosity No.4). Wash the residue
with successive quantities, each of 10 ml, ofa mixture ofequal
volumes of a saturated solution ofpicric acid and water until
the washings are free from sulphate. Wash the residue with
five quantities, each of 10 ml,. ofethanol (95 per cent) and dry
to constant weight at 105.
1 g ofthe residue is equivalent to 0.3714 g ofC 4HIQN2 , H 3P0 4,
H20.
Labelling. The label states, where applicable, that the tablets
are to be chewed before swallowing.
Test solution (a). Dissolve 50.0 mg of the substance under

Test solution (b). Dilute 10.0 ml oftest solution (a) to 50.0 ml
with the solvent mixture.
Reference solution (a). Dissolve 5 mg ofthe substance under
examination and 10 III of 2-pyrrolidone in sufficient solvent
mixture to produce 100.0 ml.
Reference solution (b). Dilute 1.0 ml of test solution (a) to
100.0 ml with the solvent mixture. Dilute 5.0 ml ofthis solution
to 50.0 ml with the solvent mixture.
Reference solution (c). Dissolve 50.0 mg ofpiracetam RS in
100.0 ml ofthe solvent mixture. Dilute 10.0 ml ofthis solution
- a stainless steel column 25 cm x 4.6 mm, packed with end
capped octadecylsilane bonded to silica (5 Ilm),
dipotassium hydrogen phosphate, adjusted to pH 6.0
resolution between the peaks due to 2-pyrrolidone and
piracetam is not less than 3.0 and the symmetry factor is not
more than 2.0 for the peak due to piracetam.
Piracetam
C6HIQN20 2
Piracetam contains not less than 98.0 per cent and not more
than 102.0 per cent ofC 6HIQN20 2, calculated on the dried basis.
Mol.Wt.142.2
Piracetam is 2-(2-oxopyrrolidin-l-yl)acetarnide .
Inject test solution (a) and reference solution (b). Run the
chromatogram for 8 times the retention time ofpiracetam. In
the chromatogram obtained with the test solution (a), the area
ofany secondary peak is not more than the area ofthe principal
(b) (0.1 per cent) and the sum ofareas of all the secondary
1925
PIROXICAM
IP 2010
per cent). Ignore any peak with an area less than 05 times the
reference solution (b) (0.05 per cent).
acid shows absorption maxima at about 242 urn and 334 urn
and a minimum at about 270 urn; absorbance at about 334 urn,
aboutO.8T
Heavy Metals (2.3.13). 2.0 g complies with limit test for heavy
Mobile phase. A mixtureof95 volumes of toluene and

5 volumes of acetic acid.

examination in 100 ml of a mixture of equal volumes of
chloroform and methanol.

Reference solution. A 0.1 per cent w/v solution of piroxicam

RS in a mixture ofequal volumes of chloroform and methanol.
than 2.0 per cent.

Inject test solution (b) and reference solution (c).

Calculate the content ofC 6H ION 20 2
Tests

(2.4.14).
Piroxicam

examination in 50.0 ml of acetonitrile.
Reference solution. Dilute 1.0 ml ofthe test solution to 10.0 ml
with acetonitrile. Dilute 1.0 ml ofthis solution to 50.0 ml with
acetonitrile.
Mol. Wt. 331.4

Piroxicam is 4-hydroxy-2-methyl-N-(2-pyridyl)-2H-l,2benzothiazine-3-carboxamide-l, I-dioxide.
Piroxicam contains not less than 97.0 per cent and not more
than 103.0 per cent of ClsH13N304S, calculated on the
anhydrous basis.
Category. Analgesic; antiinflammatory; antipyretic.
Description. An off-white to light tan or light yellow powder;
odourless.
Identification
Compare the spectrum with that obtained with piroxicam RS
or with the reference spectrum ofpiroxicam.
0.001 per cent w/v solution in 0.01 M methanolic hydrochloric
/lill),
potassium dihydrogen phosphate, adjusted to pH 3.0
symmetry factor ofthe peak due to piroxicam impurity B is not
more than 5.0. The relative retention time with reference to
piroxicam for piroxicam impurity B is about 0.85.
chromatogram 5 times the retention time ofthe principal peale.
(b) (0.2 per cent), the sum of all the secondary peaks is not
1926
IP 2010
PIROXICAM CAPSULES
more than twice the area of the principal peak in the

Test solution. Dissolve a portion of the contents of the

capsules in sufficient of a mixture of equal volumes of
chloroform and methanol to obtain a solution containing
about 0.1 per cent w/v ofPiroxicam. Shake for 10 minutes, filter
and use the filtrate.
Reference solution. A 0.1 per cent w/v solution of piroxicam

RS in a mixture ofequal volumes of chloroform and methanol.


2.0g.

under examination in 0.1 M methanolic hydrochloric acid.
Tests
Reference solution. A 0.005 per cent w/v solution ofpiroxicam

RS in 0.1 M methanolic hydrochloric acid.
Apparatus No.2,
mobile phase: a mixture of45 volumes of methanol and
55 volumes of a buffer solution prepared by diluting a
mixture of 7.72 g of anhydrous citric acid in 400 ml of
water and 5.35 g ofsodiumphosphate in 100 ml of water
to 1000 ml with water;
Uniformity of content. (For capsules containing 10 mg or

lessJ- Comply with the test stated under Capsules.
than2.0 per cent.
Test solution. Dissolve the contents ofa capsule in 100.0 ml of

0.1 M methanolic hydrochloric acid and filter. Dilute further
if necessary.
Determine by liquid chromatography (2.4.14) using the

chromatographic system and the reference solution described
under Assay.
Calculate the content OfC1SH13N304S.

Withdraw 10 ml of the medium and filter. Measure the

absorbance ofthe filtrate (2.4.7), suitably diluted ifnecessary,
at the maximum at about 242 nm. Calculate the content of
ClsH13N304S, in the medium taking 352 as the specific
absorbance at 242 run.
ClsH13N304S.
Calculate the content ofCIsHI3N304S in the capsule.

0.25 g.
Piroxicam Capsules

Piroxicam Capsules contain not less than 92.5 per cent and
not more than 107.5 per cent ofthe stated amount ofpiroxicam, Test solution. Dissolve an accurately weighed quantity of the
mixed contents of the capsules containing about 50 mg of
ClsH13N304S.
Piroxicam in 100.0 ml of 0.1 Mmethanolic hydrochloric acid.
Further dilute 1.0 ml ofthe solution to 10.0 ml with the same
solvent.
Identification


5 volumes of acetic acid.
Reference solution. A 0.005 per cent w/v solution ofpiroxicam

RS in 0.1 M methanolic hydrochloric acid.
1927
PLASTER OF PARIS
IP 2010

55 volumes of a buffer solution prepared by diluting a
mixture of7.72 g of anhydrous citric acid in 400 ml of
water and 5.35 g ofsodium phosphate in 100 ml of water
to 1000 ml with water,
than 2.0 per cent.
retain their sharpness of outline and do not crumble under

pressure.
Loss on ignition (2.4.20). 4.5 to 8.0 per cent, detennined by
igniting to constant weight at red heat.
Poloxamers
Inject altemately the test solution and the reference solution.

b is atleast 15 and (CHzCHzO)a+c is varied from 20 to 90% by weight
Calculate the content ofCIsH13N304S in the capsules.

Mol. Wt. ranges from 1000 to > 16000
Poloxamers are polyethyleneprQpylpropyleneglycols.

Synthetic block copolymer of ethylene oxide and propylene
oxide represented by the following general fonnula:
Plaster of Paris
Dried Calcium Sulphate; Exsiccated Calcium Sulphate
CaS04, YzHzO
Mol. Wt. 145.1
Plaster of Paris is prepared by heating powdered gypsum,

CaS04,YzHzO, at about 1500 in a controlled manner such that it
is substantially converted into the hemihydrate, CaS04,YzHzO,
with minimum production ofthe anhydrous phases ofcalcium
sulphate. It may contain suitable accelerators or decelerators.
Poloxamer Ethylene Propylene Content of Average

oxide oxyethylene relative
Type
oxide
molecular
units
(per
units
mass
(b)
cent)
(a)
Category. Surgical aid.

Description. A white or almost white powder; odourless or
almost odourless; hygroscopic.
124
10-15
18-23
44.8-48.6
2090-2360
188
75-85
25-30
79.9-83.7
7680-9510
237
60-68
35-40
70.5-74.3
6840-8830
338
137-146
42-47
81.4-84.9
12700-17400
407
95-105
54.60
71.5-74.9
9840-14600
Identification
A suitable antioxidant may be added.
Gives the reactions of calcium salts and of sulphates (2.3.1).
Description. Poloxamer 124 is a colourless or ahnost colourless

liquid and poloxamers 188,237,338,407 are a white or ahnost
white, waxy powder, microbeads or flakes.
Tests
pH (2.4.24). 6.5 to 9.0, detennined in a20.0 percentw/v slurry
in water.
Acid insoluble Dlatter: Dissolve 0.5 gin 30 ml ofa mixtureof
1 volume of hydrochloric acid and 2 volumes of water and
evaporate to dryness in a dish on a water-bath. Heat for
2 hours at 1200 and again add 20 ml ofthe acid mixture. Wann
for a few minutes and filter. Wash the residue with wann water
until free from chlorides, dry, ignite and weigh. The residue
weighs not more than 5 mg (1.0 per cent).
Setting properties. 20 g mixed with 10 ml of wate,; at 150 to 200
in a cylindrical mould about 2.4 cm in diameter sets in not less
than 4 minutes and not more than 6 minutes. The mass thus
formed, after standing for 3 hours, possesses sufficient
hardness to resist pressure of the fingers at the edges, which
Identification
Compare the spectrum with that obtained with poloxamer RS
or with the reference spectrum ofpoloxamer.
B. Average relative molecular mass.
C. Oxypropylene: oxyethylene ratio.
Tests
Melting point (2.4.21). About 500 for poloxamers 188, 237, 338
and 407.
1928
IP 2010
POLYETHYLENE GLYCOL 1500

dioxide-ji-ee water (solution A) is not more intensely coloured
pH (2.4.24).5.0 to 7.5, determined in solution A.'
Ethylene oxide, propylene oxide and dioxan. Determine by gas
Ethylene oxide stock solution. Disperse about 0.5 g of
ethylene oxide RS with 50.0 ml of dimethyl sulphoxide.
Ethylene oxide solution. Dilute 1.0 ml of the ethylene oxide
stock solution to 250.0 ml with dimethyl sulphoxide.
Propylene oxide stock solution. Introduce about 7 ml of
dichloromethane into a volumetric flask, add 0.5 g ofpropylene
oxide and dilute to 10.0 ml with dichloromethane. Dilute 0.5
mlofthis solution to 50.0 ml with dimethyl sulphoxide.
Propylene oxide solution. Dilute 1.0 ml ofthe propylene oxide
stock solution to 50.0 ml with dimethyl sulphoxide.
Dioxan solution. Introduce 0.1 g of dioxan into a flask and
dilute to 50.0 ml with dimethyl sulphoxide. Dilute 2.5 ml ofthis
solution to 100.0 ml with dimethyl sulphoxide.
Mixture solution. Dilute a mixture of 6.0 ml of the ethylene
oxide solution, 6.0 ml ofthe propylene oxide solution and 2.5
ml ofthe dioxan solution to 25.0 ml with dimethyl sulphoxide.
Test solution. To 1.0 g ofthe substance under examination in
a head-space vial, add 4.0 ml ofdimethyl sulphoxide and close
the vial immediately.
Reference solution. To 1.0 g ofthe substance under examination
in a head-space vial, add 2.0 ml of dimethyl sulphoxide and
2.0 ml ofthe mixture solution. Close the vial immediately.
- a fused silica column 50 m x 032 mm, packed with
poly(dimethyl)(diphenyl)siloxane (film thiclmess 5 J.l.m),
- temperature:
column 70 0 to 240,
inlet port and detector at 250 0 ,
- a flame ionisation detector,
- flow rate. 1.4 ml per minute using nitrogen as the carrier
gas,
Head-space conditions
- equilibration temperature 1100 ,
- equilibration time 30 minutes,
- transfer line temperature 140 0 ,
- pressurisation time 1 minute,
- injection time 0.05 minute.
Inject 1 J.l.I of the gaseous phase of the test solution and the
reference solution. The relative retention time with reference
to ethylene oxide is about 6 minutes, for propylene oxide is
about 1.3, for dichloromethane is about 1.6, for dioxan is about
3.0, for dimethyl sulphoxide is about 3.7.
The ethylene oxide is not more than 0.5 times the area of the
corresponding peak in the chromatogram obtained with the
reference solution (1 ppm); propylene oxide is not more than
0.5 times the area of the corresponding peak in the
chromatogram obtained with the reference solution (5 ppm)
and dioxan is not more than 0.5 times the area of the
corresponding peak in the chromatogram obtained with the
reference solution (10 ppm).
Average relative molecular mass. Weigh 15 g ofthe substance
under examination into a 250-ml ground glass stoppered flask,
add 25.0 ml of phthalic anhydride solution and a few glass
beads and swirl to dissolve. Boil gently under a reflux
condenser for 60 minutes, cool and add 2 quantities, each of
10 ml, ofpyridine, through the condenser. Add 10 ml ofwater,
mix and allow to stand for 10 minutes. Add 40.0 ml of 0.5 M
sodium hydroxide and 0.5 ml of a 1.0 per cent w/v solution of
phenolphthalein in pyridine. Titrate with 0.5 M sodium
hydrOXide to a light pink endpoint that persists for 15 second
and record the volume ofsodium hydroxide used (S). Prepare
a blank Record the volume of sodium hydroxide used (B).
Calculate the average relative molecular mass using the
following expression:
Oxypropylene:oxyethylene ratio. Nuclear magnetic resonance
spectrometry (2.434).
Use a 10.0 per cent w/v solution of the substance under
examination in deuterated chloroform. Record the average
area of the doublet appearing at about 1.08 ppm due to the
methyl groups ofthe oxypropylene units (A I) and the average
area of the composite band from 3.2 ppm to 3.8 ppm due to
CH20 groups ofboth the oxyethylene and oxypropylene units
and the CHO groups of the oxypropylene units (A 2) with
reference to the internal standard. Calculate the percentage
of oxyethylene, by weight, in the sample under examination
using the following expression:
Total ash (23.19). Not more than 0.4 percent.
LOg.
Labelling. The label states the type ofpoloxamer.

Macrogol1500
Polyethylene Glycol 1500 is a mixture ofthe polycondensation
products of ethylene oxide and water obtained under
controlled conditions. It is represented by the formula
HOCH2[CH20CH2]nCH20H, where n is between 28 and 36.
1929
POLYETHYLENE GLYCOL 1500
IP 2010
Description. A white or creamy white, soft, wax-like solid;

Tests
Appearance of solution (2.4.1). A25.0 per centw/v solution is
not more intensely coloured than reference solution BYS6.
pH (2.4.24). 4.0 to 7.0, determined ina5.0 percentw/v solution.
Freezing point (2.4.11).42 to 46.
Hydroxyl value (2.3.27).70 to 86, determined on 5.0 g.
Viscosity (2.4.28). 25 mm2s- 1 to 32 mm2s-1, determined at 100
by Method A using a V-tube viscometer (size D).
Arsenic (2.3.10). Mix 3.3 g with 3 g of anhydrous sodium

carbonate, add 10 ml of bromine solution and mix thoroughly.
Evaporate to dryness on a water-bath, gently ignite and
dissolve the cooled residue in 16 ml of brominated
hydrochloric acid and 45 ml of water. Remove the excess of
bromine with 2 ml of stannous chloride solution AsT. The
resulting solution complies with the limit test for arsenic
(3 ppm).
Heavy metals (2.3.13). Dissolve 4.0 g in 5 ml ofa 1.0 per cent
w/v solution of hydrochloric acid and sufficient water to
produce 25 ml. The solution complies with the limit test for

(3 ppm).
Macrogo16000
Heavy metals (2.3.13). Dissolve 4.0 g in5 ml ofa 1.0 percent

produce 25 m!. The solution complies with the limit test for
heavy metals, MethodA(5 ppm).

HOCH2[CH20CH2]nCH20H, where n is between 112 and 158.
Category. Pharmaceutical aid (ointment base and tablet

excipient).
Description. A white to creamy white, wax-like solid or flakes;

Tests
Macrogo14000
Appearance ofsolution (2.4.1). A 15.0 per cent w/v solution is


HOCH2[CH20CH2]IICH20H, where n is between 69 and 84.
pH (2.4.24). 4.5 to 7.5, determined in a5.0percentw/v solution.

Freezing point (2.4.11). 56 to 60.
Viscosity (2.4.28). 250 mm2s- 1 to 390 mm 2s- l , determined at
100 by Method A using a V-tube viscometer (size F).
Category. Phannaceutical aid (ointment base).

Description. A creamy white, hard, wax-like solid, powder or
flakes; odour, faint llndcharacteristic.
Tests
Appearance of solution (2.4.1). A20.0 per cent w/v solution is
Freezing point (2.4.11). 53 to 56.
Hydroxyl value (2.3.27). 30 to 36, detennined on 20.0 g.
Viscosity (2.4.28).76 mm2s-1 to 110 mm2s- l , determined at 100
by Method A using a V-tube viscometer (size E).
Arsenic (2.3.10). Mix 3.3 g with 3 g of anhydroussodium

(3 ppm).
Heavy metals (2.3.13). Dissolve 4.0 g in 5 ml ofa 1.0 percent
produce 25 ml. The resulting solution complies with the limit
test for heavy metals, Method A (5 ppm).
1930
IP 2010
POLYOXYL 40 HYDROGENATED CASTOR OIL
filtrate and rinsing in a 50-ml Nessler cylinder, dilute with water

to 40 ml, and mix.
Polyoxyl 35 Castor Oil

Polyoxy135 Castor Oil contains mainly the tri-ricinoleate ester
ofethoxylated glycerol, with smaller amounts ofpolyethylene
glycol ricinoleate and the corresponding free glycols. It results
from the reaction of glycerol ricinoleate with about 35 moles
of ethylene oxide.
To each of the cylinder containing the standard solution and

the test solution, add 2 ml of pH 3.5 acetate buffer, then add
1.2 ml of thioacetamide- glycerine base, dilute with water to
50 ml, mix, allow to stand for 2 minutes, and view downward
over a white surface: the colour ofthe solution obtained from
the test solution is not more intense than that of the solution
obtained from the standard solution, produced by treating 2
mlof lead standard solution (l ppm) in a similar manner.

Water (2.3.43). Not more than 3.0 per cent, determined on 1 g.
Identification

Compare the spectrum with that obtained with polyoxyl 35
castor oil RS or with the reference spectrum of polyoxyl 35
castor oil.
B. Dissolve about 0.1 g in 10 ml of ethanolic potassium
hydroxide solution, boil for about 3 minutes and evaporate to
dryness. The residue dissolve with 5 ml of water, yielding a
clear solution. Add a few drops of glacial acetic acid; a white
precipitate is formed.
C. To a 5 .0 per cent w/v solution, add bromine solution,
dropwise, the bromine is decolorized.
Polyoxyl 40 Hydrogenated Castor Oil

Polyoxy140 Hydrogenated Castor Oil contains mainly the trihydroxystearate ester of ethoxylated glycerol, with smaller
amounts of polyethylene glycol tri-hydroxystearate and of
the corresponding free glycols. It results from the reaction of
glycerol tri-hydroxystearate with about 40 to 45 moles of
ethylene oxide.
Identification
Tests
Weight per ml (2.4.29). 1.05 to 1.06.
Viscosity (2.4.28). 600 to 850 centipoises at 25, a capillary
viscometer being used.
A. Dissolve 0.1 gin 1 ml of water, add 9 ml of5.0percentw/v

solution of sodium chloride and heat on a water-bath, the
solution becomes turbid at a temperature between 700 and 85.
Saponification value (2.3.37).60 to 75.
B. Dissolve about 0.1 g in 10 ml of ethanolic potassium

hydroxide solution, boil for about 3 minutes, and evaporate
to dryness. Dissolve the residue with 5 ml of water, yielding a
clear solution. Add a few drops of glacial acetic acid; a white
precipitate is fonned.
Iodine value (2.3.28).25 to 35.
Tests
Heavy metals (2.3 .13). Weigh about 2 g ofthe substance under

examination in a crucible and add sufficient sulphuric acid to
Congealing temperature (2.4.10). 16 to 26.
Acid value (2.3.23) Not more than 2.0.

Hydroxyl value (2.3.27). 65 to 80.
wet the substance, carefully ignite at a low temperature until

thoroughly charred. Add to the carbonized mass 2 ml of nitric
acid and 5 drops of sulphuric acid, and heat cautiously until
white fumes no longer are evolved. Ignite, preferably in a
muffle furnace, at 500 to 600 0 , until the carbon is completely
burned off. Cool, add 4 ml of dilute hydrochloric acid, cover,
digest on a steam-bath for 15 minutes, and slowly evaporate
on a steam-bath to dryness. Moisten the residue with 1 drop
of hydrochloric acid, add 10 ml ofhot water, and digest for 2
minutes. Add dilute ammonium hydroxide dropwise until the
solution is just allcaline to litmus paper, dilute with water to 25
ml, and adjust at pH 3.0 with dilute acetic acid, filter. Rinse
the crucible and the filter with 10 ml of water, combine the
Acid value (2.3.23). Not more than 2.0.

Hydroxyl value (2.3.27).60 to 80.
Saponification value (2.3 .37).45 to 69.
Iodine value (2.3.28). Not more than 2. O.
Heavy metals (2.3.13). Weigh 2 g of the substance under
examination in a crucible and add sufficient sulphuric acid to
wet the substance, carefully ignite at a low temperature until
thoroughly charred. Add to the carbonized mass 2 ml of nitric
acid and 5 drops of sulphuric acid, and heat cautiously until
white fumes no longer are evolved. Ignite, preferably in a
muffle furnace, at 5000 to 600 0 , until the carbon is completely
burned off. Cool, add 4 ml of dilute hydrochloric acid, cover,
1931
POLYSORBATE 20
IP 2010
digest on a stearn bath for 15 minutes, and slowly evaporate

on a stearn bath to dryness. Moisten the residue with 1 drop
ofhydrochloric acid, add 10 ml ofhot water, and digest for 2
minutes. Add dilute ammonium hydroxide dropwise until the
solution is just alkaline to litmus paper, dilute with water to 25
ml, and adjust with diluteacetic acid to a pH between 3.0 and
4.0, using short-range pH indicator paper as an external
indicator. Filter if necessary, rinse the crucible and the filter
with 10 ml ofwater, combine the filtrate and rinsing in a 50-rnl
Nessler cylinder, dilute with water to 40 ml, and mix.
To each of the cylinder containing the standard solution and
the test solution, add 2 ml of acetate buffer pH 3.5, then add
1.2 ml of thioacetamide- glycerine base, dilute with water to
50 ml, mix, allow to stand for 2 minutes, and view downward
over a white surface: the colour ofthe solution obtained from
the test solution is not more intense than that of the solution
obtained from the standard solution, produced by treating 2
mlof lead standard solution (l ppm) in a similar manner.
Polysorbate 20
B. Dissolve 0.1 ginS mlofchlorojorm, add 0.1 gofpotassium

thiocyanate and 0.1 g of cobalt nitrate and stir with a glass
rod; a blue colour is produced.
Tests
pH (2.4.24). 5.0 to 7.0, determined in a 5.0 per cent w/v solution
in carbon dioxide-free water.
Acid value (2.3.23). Not more than 2.0, determined on 5.0 g.
Hydroxyl value (2.3.27). 96 to 108, determined on 2.0 g.
Saponification value (2.3.37). 40 to 50, using 15 ml of0.5 M
ethanolic potassium hydroxide and diluting with 50 ml of
water before carrying out the titration.
Iodine value (2.3.28). Not more than 5.0, determined by the
iodine bromide method.
Reducing impurities. Dissolve 2.0 g in 25 ml ofhot water, add
25 ml of 1 M sulphuric acid and 0.1 ml ofjerroin solution and
titrate with 0.01 M eerie ammonium sulphate shaking
continuously until the colour changes from red to greenish
blue persists for 30 seconds. Carry out a blank titration. Not
more than 2.0 ml of 0.01 M eerie ammonium sulphate is
required.
Sulphated ash. Not more than 0.2 per cent, determined by the
following method. Weigh accurately about 2.0 g in a silica or
platinum crucible, add 0.5 ml ofsulphuric acid and heat on a
water-bath for 2 hours. Carefully ignite at a low temperature
until thoroughly chaned. Add to the carbonised mass 2 ml of
nitric acid and 0.25 ml ofsulphuric acid, cautiously heat until
white fumes are evolved and then ignite at 6000 until the. carbon
is completely burnt off. Allow to cool, weigh and repeat the
operation for periods of 15 minutes until two successive
weighings do not differ by more than 0.5 mg.
Polyoxyethylene 20 Sorbitan Monolaurate

Polysorbate 20 is a mixture ofpartial lauric esters of D-glucitol
and its anhydridescopolyrnerised with approximately 20 moles
ofethylene oxide for each mole of D-glucitol and its anhydrides.
The lauric acid used for the esterification may contain other
fatty acids.
Polysorbafe80
Polyoxyethylene 80 Sorbitan Monooleate
Category. Pharmaceutical aid (non-ionic surfactant).

Description. A clear or slightly opalescent, oily, yellowish or
brownish yellow liquid; odour, faint and characteristic.
Identification
Compare the spectrum with that obtained with polysorbate
RS or with the reference spectrum of polysorbate.
1932
w+x+y+z =20
IP 2010
POTASSIUM CHLORIDE
Polysorbate 80 is a mixture ofpartial oleic esters of D-glucitol

and its anhydrides copolymerised with approximately 20 moles
ofethylene oxide for each mole ofD-glucitol and its anhydrides.
Potassium Chloride
Category. Pharmaceutical aid (non-ionic surfactant).
Potassium Chloride contains not less than 99.0 per cent and
not more than 100.5 per cent of KC1, calculated on the dried
basis.
Description. Aclear or almost clear, oily, yellowish or brownish
yellow liquid; odour, faint and characteristic.
KCl
Mol. Wt. 74.6
Category. Electrolyte replenisher.
Identification
Compare the spectrum with that obtained with polysorbate
RS or with the reference spectrum of polysorbate.
Dose. Prophylactic; 2 to 4 g (approximately 25 to 50 mmol)

daily; therapeutic, in established potassium depletion, 10 to
15 g (approximately 135 to 200 mmol) daily.
B. Dissolve 0.1 gin 5 ml of chloroform, add 0.1 g ofpotassium

thiocyanate and 0.1 g of cobalt nitrate and stir with a glass
rod; a blue colour is produced.
odourless.
C. To 2 ml of a 5 per cent w/v solution add 0.5 ml of bromine

water; the bromine is decolourised.
Dissolve 109 in 100 ml of carbon dioxide-fi-ee water prepared

from distilled water (solution A). The solution gives the
reactions of potassium salts and of chlorides (2.3.1).
Tests
Identification

Tests
Acid value (2.3.23). Not more than 2.0, determined on 5 g.
colourless (2.4.1).
Hydroxyl value (2.3.27). 65 to 80, determined on 2.0 g.
Acidity or alkalinity. 5.0 g dissolved in 50 rnl ofcarbon dioxideSaponification value (2.3.37). 45 to 55, using 15 ml of 0.5 M free water requires not more than 0.5 ml of 0.01 M sodium
ethanolic potassium hydroxide and diluting with 50 ml of hydroxide or 0.01 M hydrochloric acid for neutralisation to
water before carrying out the titration.
bromothymol blue solution.
Iodine value (2.3.28). 18 to 24, determined by the iodine bromide
Arsenic (2.3.10). Dissolve 10.0 g in 50 ml of water and add
method.
10 ml of stannated hydrochloric acid AsT. The resulting
Reducing impurities. Dissolve 2.0 g in 25 ml ofhot water, add solution complies with the limit test for arsenic (1 ppm).
25 ml of 1 M sulphuric acid and 0.1 ml offerroin solution and
Barium. To 5 ml of solution A add 5 ml of water and 1 ml of
titrate with 0.01 M eerie ammonium sulphate shaking
1 M sulphuric acid; the solution, after not less than 15 minutes,
continuously until the colour changes from red to greenish

blue persists for 30 seconds. Carry out a blank titration. Not
more than 5.0 ml of 0.01 M eerie ammonium sulphate is
required.

Sulphated ash. Not more than 0.2 per cent, determined by the
is not more opalescent than a mixture of5 ml ofsolution A and

6 ml of water.
Heavy metals (2.3.13). Dissolve 2.0 g in 10 ml of water to
which are added 2 ml of dilute acetic acid and 13 ml of water.
The solution complies with the limit test for heavy metals,
Method A (10 ppm).
following method. Weigh accurately about 2.0 g in a silica or Calcium and magnesium. Dissolve 1gin 20 ml of water, add
platinum crucible, add 0.5 ml of sulphuric acid and heat on a 1 ml of dilute ammonia solution and 1ml ofsodium phosphate
water-bath for 2 hours. Carefully ignite at a low temperature solution; the solution remains clear.
until thoroughly charred. Add to the carbonised mass 2 ml of Iron (2.3.14). 20 ml ofsolutionA complies with the limit test for
nitric acid and 0.25 ml of sulphuric acid, cautiously heat until iron (20 ppm).
white fumes are evolved and then ignite at 6000 until the carbon
Bromides. Dilute 1.0 ml ofsolution Ato 50.0 mlwith water. To
is completely burnt off. Allow to cool, weigh and repeat the
5.0 ml of the solution add 2.0 ml of phenol red reagent and
operation for periods of 15 minutes until two successive
. 1.0 ml of a 0.0 I per cent solution of chloramine T and mix
weighings do not differ by more than 0.5 mg.
immediately. After exactly 2 minutes, add 0.15 ml of 0.1 M
Water (2.3.43). Not more than 3.0 percent, determined on 1.0 g. sodium thiosulphate, mix and dilute to 10.0 ml with water. The
absorbance ofthe solution measured at about 590 nm (2.4.7),
1933
POTASSIUM CHLORIDE
IP 2010
using water as the blank, is not more than that of a standard

prepared at the same time and in the same manner, using 5.0 ml
of a 0.0003 per cent w/v solution of potassium bromide
(0.1 percent).
Iodides. Moisten 5 g by adding dropwi~e, a solution freshly
prepared by mixing 25 ml of iodide-free starch solution, 2 ml
of 0.5 M sulphuric acid, 0.15 ml of sodium nitrite solution
and 25 ml of water and examine the mixture in daylight; the
substance shows no blue colour after 5 minutes.
water and titrate with 0.1 M silver nitrate using potassium

chromate solution as indicator.
Potassium Chloride and Dextrose

Injection
Potassium Chloride in Dextrose Injection; Potassium CWoride
and Dextrose Intravenous Infusion; Potassium Chloride and
Glucose Intravenous Infusion
Potassium Chloride and Dextrose Injection is a sterile solution
of Potassium Chloride and Dextrose in Water for Injections.
Potassium Chloride and Dextrose Injection contains not less
stated amount of potassium chloride, KCl, and not less than
amount ofdextrose, C 6H 1Z0 6 It contains no antimicrobial agent.
Usual strengths: Injections containing the following amounts
ofpotassium chloride, KCl, and dextrose, C 6H IZ06.
per cent w/v of
Potassium Chloride
(KCl)
1 ml of 0.1 M silver nitrate is equivalent to 0.007455 g ofKCl.
Potassium Chloride intended for use in the manufacture of

dialysis solutions complies with the following additional
requirement.
Aluminium. Dissolve 4.0 g in 100 ml of water and add 10 ml of
acetate buffer pH 6.0. Extract with successive quantities of
20, 20 and 10 ml of a 0.5 per cent w/v solution of
8-hydroxyquinoline in chloroform and dilute the combined
extracts to 50 ml with chloroform. Use as the standard solution
a mixture of2 ml of aluminium standard solution (2 ppm AI),
10 ml of acetate bufferpH6.0 and 98 ml of water treated in the
same manner and as the blank solution a mixture of 10 ml of
acetate bufferpH 6.0 and 100 ml of water treated in the same
manner. Measure the fluorescence of the test and standard
solutions (2.4.5), using an excitation wavelength of about
392 urn and emission wavelength ofabout 518 urn and setting
the instrument to zero with the blank solution in each case.
The fluorescence of the test is not greater than that of the
standard solution (1 ppm).
per cent w/v of'

Dextrose
(C6H 1Z0 6)
0.Q75
0.15
0.225
0.30
Description. A clear, colourless or faintly straw-coloured

solution.
Identification
A. To I ml add 0.05 mlofpotassium cupri-tartrate solution;
the solution remains blue and clear. Heat to boiling, a copious
red precipitate is formed.
B. Gives reaction B of potassium salts and reaction A of
chlorides (2.3.1).
Tests
Potassium chloride intended for use in the manufacture of pH (2.4.24).3.5 to 6.5.

Parenteral Preparations or for the preparation of
liaemlJdzalyszsslJlution complies wliliiliejolllJwing 5-Hydroxymethylfurfural and Related substances. Dilute a
Sodium. Not more than 0.1 per cent, determined by atomic
absorption spectrophotometry (2.4.2), using a 1.0 per cent
w/v solution and measuring at 589 urn. Use sodium solution
AAS, suitably diluted with water, for the standard solutions.
Labelling. The label states whether or not the material is
suitable for use in the manufacture ofParenteral Preparations
or for the preparation ofhaemodialysis or dialysis solutions.

maximum at about 284 urn, absorbance at about 284 urn (2.4.7);
not more than 0.25.
Heavy metals (2.3 .13). Evaporate a volume containing 4.0 g of

Dextrose to 10 ml and add 2 ml of dilute acetic acid and
sufficient water to produce 25 ml. The solution complies with
the limit test for heavy metals, Method A (5 ppm).
perrnI.
1934
IP 2010
POTASSIUM CHLORIDE, SODIUM CHLORIDE AND DEXTROSE INJECTION
per cent w/v of

Potassium
Chloride
(KCI)

Assay. For potassium chloride - Titrate an accurately
measured volume containing 0.1 g ofPotassium Chloride with
0.1 M silver nitrate using potassium chromate solution as
indicator.
0.075
0.075
0.075
1 ml of 0.1 Msilver nitrate is equivalent to 0.007455 g ofKCl.
0.150
0.150
0.150
For dextrose To an accurately measured volume

containing 2 g to 5 g of Dextrose, add 0.2 ml of 5 M ammonia
and sufficient water to produce 100.0 ml. Mix well, allow to
stand for 30 minutes and measure the optical rotation in a
2-dm tube (2.4.22). The observed rotation in degrees multiplied
by 0.9477 represents the weight, in g, ofdextrose, C 6H 1Z0 6, in
the volume taken for assay.
0.150
0.150
0.225
0.225
0.225

Labelling. The label states (1) the strength as the percentages
w/v ofPotassium Chloride and Dextrose; (2) the total osmolar
concentration in mOsmol per litre; (3) where the contents are
less than 100 ml, or where the label states that the Injection is
not for direct use but is to be diluted before use, the label
alternatively may state the total osmolar concentration in
mOsmol per ml; (4) the content ofpotassium in millirnoles; (5)
that the injection containing visible particles in the solution
should not be used.
per cent w/v of

Sodium
Chloride
(NaCl)
per cent w/v of

Dextrose
(C6H12 06J
0.250
0.330
0.450
0.225
5
5
5
10
0.225
0.330
0.450
0.900
0.225
0.225
0.225
0.300
0.330
0.450
0.225
0.300
0.300
0.330
0.450
0.300
0.900
5
5
5
5
10
5
5
5
5
5
5
Identification
A. To 1 ml add 0.05 ml ofpotassium cupri-tartrate solution;
the solution remains blue and clear. Heat to boilIng, a copious
B. Gives reaction A of potassium salts, reaction B of sodium

salts and reaction A of chlorides (2.3.1)
Potassium Chloride, Sodium Chloride

and Dextrose Injection
Tests
Potassium Chloride in Dextrose and Sodium Chloride

Injection; Potassium Chloride, Sodium Chloride
and Dextrose Intravenous Infusion; Potassium
Chloride, Sodium Chloride and Glucose Intravenous
Infusion
Potassium Chloride, Sodium Chloride and Dextrose Injection
is a sterile solution of Potassium Chloride, Sodium Chloride
and Dextrose in Water for Injections.
Potassium Chloride, Sodium Chloride and Dextrose Injection
contains not less than 95.0 per cent and not more than
110.0 per cent ofthe stated amounts ofsodium, Na, potassium,
K, and chloride, Cl, and not less than 95.0 per cent and not
more than 105.0 per cent of the stated amount of dextrose,
C 6H 1Z 0 6 It contains no antimicrobial agent.
Usual strengths: Injections containing the following amounts
of potassium chloride, KCl, sodium chloride, NaCl, and
dextrose, C 6H 1Z0 6
pH (2.4.24).3.5 to 6.5, determined on a portion diluted with

water, ifnecessary, to a concentration ofnot more than 5.0 per
cent of dextrose.
maximum at about 284 urn, absorbance at about 284 urn (2.4.7);
not more than 0.25.
Heavy metals (2.3.13). Evaporate a volume containing 4.0g of
Dextrose to 10 ml and add 2 ml of dilute acetic acid and
perml.
Assay. For sodium - Dilute appropriately with water and
determine by Method A for flame 'photometry (2.4.4), or by
1935
POTASSIUM CHLORIDE, SODIUM CHLORIDE AND DEXTROSE INJECTION
Method A for atomic absorption spectrophotometry (2.4.2),

measuring at 589 nm and using sodium solution FP or sodium
solution AAS respectively, suitably diluted with water for the
standard solutions.
For potassium Dilute appropriately with water and

determine by Method A for flame photometry (2.4.4), or by
measuring at 767 nm and using potassium solution FP or
potassium solution AAS respectively, suitably diluted with
water for the standard solutions.
For total chlorides - To 20.0 ml add 30 ml of water, 50.0 ml of
0.1 M silver nitrate and 2 ml of nitric acid. Filter, wash the
precipitate with water slightly acidified with nitric acid and
titrate the excess of silver nitrate with 0.1 M ammonium
thiocyanate using ferric ammonium sulphate solution as
indicator until a reddish yellow colour is produced. Carry out
a blank titration.
1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g oftotal
For dextrose- To an accurately measured volume containing

2 g to 5 g ofDextrose, add 0.2 ml of5 M ammonia and sufficient
water to produce 100.0 ml. Mix well, allow to stand for
30 minutes and measure the optical rotation in a 2-dm tube
(2.4.22). The observed rotationin degrees multiplied by 0.9477
represents the weight, in g, ofdextrose, C6H 1Z0 6, in the volume
taken for assay.
w/v ofPotassium Chloride, Sodium Chloride and Dextrose; (2)
the total osmolar concentration in mOsmol per litre; (3) where
the contents are less than 100 ml, or where the label states that
the Injection is not for direct use but is to be diluted before
use, the label alternatively may state the total osmolar
concentration in mOsmol per ml; (4) the content ofpotassium,
sodium and chloride in millimoles; (5) that the injection
containing visible particles in the solution should not be used.
IP 2010
Category. Systemic alkaliniser.

Dose. 4 to 109.
Description. White, granular crystals or a crystalline powder;
odourless; hygroscopic,
Identification
A. Dissolve 10 g in 100 ml of carbon dioxide-free water
prepared from distilled water (solution A). 0.5 ml of solution
A gives the reactions of potassium salts (2.3.1).
B. To 1 ml of solution A add 4 ml of water; the solution gives
the reactions of citrates (2.3.1).
Tests
colourless (2.4.1).
Acidity or alkalinity. To 10 ml of solution A add 0.1 mlof
dilute phenolphthalein solution; not more than 0.2 ml of
0.1 M hydrochloric acid or 0.1 M sodium hydroxide is
required to change the colour ofthesolution.
Arsenic (2.3.10). Dissolve 5.0 gin 50 ml of water and add 15 ml
of stannated hydrochloric acid. The resulting solution
heavy metals, Method A (1 0 ppm).
Sodium. Not more than 0.3 per cent, determined by atomic
absorption spectrophotometry (2.4.2), Method II, measuring
at 589 nm. Prepare the test solution by addition of 1 ml of2 M
hydrochloric acid to 10.0 ml of solution A and diluting to
100.0 ml with distilled water. Use sodium solution AAS,
suitably diluted with distilled water, for the standard
solutions.
Chlorides (2.3.12). Dilute 25.0 ml ofsolution A to 35 ml with
water. The resulting solution complies with the limit test for
chlorides (l 00 ppm).
Potassium citrate is tripotassium 2-hydroxypropane-l ,2,3tricarboxylate monohydrate.
Oxalate. Dissolve 0.5 gin 4 ml ofwater, add 3 ml ofhydrochloric

acid and 1 g of granulated zinc and heat on a water-bath for
1 minute. Allow to stand for 2 minutes, decant into 0.25 ml ofa
1 per cent w/v solution of phenylhydrazine hydrochloride,
heat to boiling, cool rapidly, add an equal volume of
hydrochloric acid and 0.25 ml of potassium ferricyanide
solution, shake and allow to stand for 30 minutes. Any pink
colour produced is not more intense than that obtained by
treating 4.0 ml of a 0.005 per cent w/v solution of oxalic acid
at the same time and in the same manner (300 ppm, calculated
as anhydrous oxalic acid).
Potassium Citrate contains notless than 99.0 per cent and not
more than 101.0 per cent of C 6H sK30 7, calculated on the
anhydrous basis.
Sulphates (2.3.17). To 10.0 ml ofsolution A add 2 ml of 7 M

hydrochloric acid and dilute to 15 ml with distilled water; the
solution complies with the limit test forsulphates (150 ppm).
Potassium Citrate
HO
COOK
._~-KOOC-/ ~COOK
C6HsK307,HzO
,H 0
2
Mol. Wt. 324.4
1936
IF 2010
POTASSIUM CLAVULANATE
Readily carbonisable substances. Heat 0.20 g, in powder, with

10 ml of sulphuric acid (96 per cent w/w) in a water-bath at
90 1 for 60 minutes and cool rapidly. The solution is not
more intensely coloured than reference solution YS2 or GYS2
(2.4.1).

examination in mobile phase A and dilute to 25 ml with mobile
phase A.
Water (2.3.43). 4.0 to 7.0 per cent, determined on 0.5 g, titration

being done after stirring for 15 minutes subsequent to addition
of the substance under examination.

veach of lithium clavulanate RS and amoxycillin trihydrate

anhydrous glacial acetic acid, heat to about 50, allow to
cool. Titrate with 0.1 M perchloric acid, using 0.25 ml of
l-naphtholbenzein solution as indicator and titrating until a
green colour is obtained. Carry out a blank titration.

ml with mobile phaseA.

C6HsK 30 7
O~K
o~rr -~
Lj--o
OH
CsHsKNOs
Mol. Wt. 237.3
Potassium Clavulanate is potassium (Z)-(2R,5R)-3-(2hydroxyethylidene)-7-oxo-4-oxa-l-azabicyclo[3.2.O]heptane2-carboxylate.

Potassium Clavulanate contains not less than 96.5 per cent
and not more than 102.0 per cent of potassium clavulanate,
CsHsKNOs, calculated on the anhydrous basis.
Description. A white to off white, crystalline hygroscopic
powder.
Identification
Inject reference solution (b). The resolution between

clavulanate (first peak) and amoxycillin (second peak) is not
less than 13.
Inject the test solution and reference solution (b). The area of
any individual impurity peak obtained is not more than the
reference solution (a) (1.0 per cent). The sum of all impurity
peaks obtained is not more than twice the area ofthe principal
(a) (2.0 per cent). Ignore any peaks with an area 0.05 times the
Water (2.3.43). Not more than 1.5 percent,determinedonO.5 g.
B. Gives reaction A ofpotassium salts (2.3.1).
Tests
NOTE -Prepare the solutions immediately before use.
pH (2.4.24).5.5 to 8.0, determined in 1.0 per cent w/v solution.

(2.4.14).

octadecylsilane bonded to porous silica (5 f.lm),
mobile phase: A. a 0.78 per cent w/v solution ofsodium
dihydrogen phosphate adjusted to pH 4.0 with
phosphoric acid and filtered through a 0.5 f.lm filter,
B. a mixture of equal volumes ofmobile
phase A and methanol,
below,
spectrophotometer set at 230 om,
injection volume. 20 f.ll.
Time
Mobile phase A
Mobile phase B
(in min.)
(per cent v/v)
(per cent v/v)
0-4
100
o
4-15
100--750
050
15 -18
50
50
18-24
50--7100
50--7 0
24- 39
100
o

examination in a 0.41 per cent w/v solution of sodium acetate
previously adjusted to pH 6.0 with glacial acetic acid, and
dilute to 50.0 ml with the same solution.
1937
POTASSIUM CLAVULANATE
IP 2010
Reference solution (a). AO.1 per cent w/v solution of lithium

clavulanate RS in a 0.41 per cent w/v solution of sodium
acetate previously adjusted to pH 6.0 with glacial acetic
acid.
veach of lithium clavulanate RS and amoxycillin trihydrate
RS in a 0.41 per cent w/v solution ofsodium acetate previously
adjusted to pH 6.0 with glacial acetic acid.
octadecy1silane bonded to porous silica (5 /lm),
- mobile phase: a mixture of 5 volumes of methanol and
95 volumes of a 1.5 per cent w/v solution of sodium
dihydrogen phosphate previously adjusted to pH 4.0
with dilute phosphoric acid,
less than 3.5.
Potassium Clavulanate Diluted contains not less than 91.2 per

potassium clavulanate, CsHsKNOs.
Description. A white or almost white powder, hygroscopic.
Identification
B. Gives reaction A ofpotassium salts (2.3.1).

C. Depending on the diluent used, carry out one ofthe relevant
identification tests given below.
(i) On a watch-glass place a quantity of the substance under
examination corresponding to 20 mg ofcellulose and disperse
in 4 ml of iodinated zinc chloride solution. The powder
becomes violet-blue in colour.
(ii) It gives the reaction ofsilicates (2.3.1).
Inject alternately the test solution and reference solution (a).
Tests
Calculate the content ofCsHsKNOs.
pH (2.4.24) 4.8 to 8.0, determined on a quantity ofthe substance

under examination containing 0.2 g ofpotassium clavulanate
dissolved in 20 ml of carbon dioxide-free water.
1 mg of clavulanate (CsHgNO s) is equivalent to 1.191 mg of

CsHsKNOs.
Potassium Clavulanate intendedfor use in the manufacture

ofParenteral Preparations without a further procedure for
the removal of bacterial endotoxins complies with the
folloWing additional requirement.
Light absorption. When examined in the range 230 nm to 360

nm (2.4.7), a 0.1 per cent w/v solution in 0.1 M phosphate
buffer solution pH 7.0 shows an absorption maximum 0.40
measured immediately at 278 nm.

Unit per mg of potassium clavulanate.

(2.4.14).
Potassium Clavulanate intendedfor use in the manufacture

of Parenteral Preparations without a further sterilisation
procedure complies with the folloWing additional
requirement.

examination containing 0.25 g of potassium clavulanate in 5
ml ofmobile phase A, dilute to 25.0 ml with mobile phase A and
filter.

100.0 ml with mobile phase A.

Labelling. The label states whether it is intended for use in
the manufacture of parenteral preparations.
Potassium Clavulanate Diluted

Mol. Wt. 237.3
Potassium Clavulanate Diluted is a dry mixture of Potassium
Clavulanate and Microcrystalline Cellulose, or Silica, colloidal
anhydrous or Silica, colloidal hydrated.
Reference solution (b). Dissolve 10 mg of amoxycillin

trihydrate RS in 1 ml of the test solution and dilute to 100 ml
with mobile phase A.
- mobile phase: A. a 0.78 per cent w/v solution of sodium
dihydrogen phosphate with the pH adjusted to 4.0 with
dilute phosphoric acid,
B. a mixture of equal volumes of mobile
phase A and methanol,
1938
IP 2010
POTASSIUM IODIDE

below,
Inject reference solution (b). The resolution between the

clavulanate peak and amoxycillin peak is not less than 3.5.
Inject alternately the test solution and reference solution (a)..
Calculate the content ofCsHsKNOs .
1 mg of clavulanate (CsHgNOs) is equivalent to 1.191 mg of
CsHsKNOs.
Time
Mobile
Mobile
Comment
phase A
phase B
(in min.) (per cent vIv) (per cent v/v)
100
100
o
o
15
50
50
18
50
50
end chromatogram,
return to 100A
24
100
end equilibration,
begin'next
chromatogram
Isocratic
Labelling. The label states the percentage contents of

potassium clavulanate and the diluent used to prepare the
mixture.
begin linear
gradient
Potassium Iodide
K.I
Inject reference solution (b). The resolution between the

less than 13.
Inject alternatively the test solution and reference solution
(a). Any individual impurity is not more than 1.0 per cent and
the sum of all impurities found is not more than 2.0 per cent.
LOg.

examination containing about 50.0
mg of potassium
clavulanate in a 0.4 per cent w/v solution of sodium acetate
with the pH previously adjusted to 6.0 with glacial acetic
acid, dilute to 50.0 ml with the same solution and filter.
Refei-ence solution (a). Dissolve 50.0 mg of lithium
clavulanate RS in a 0.4 per cent w/v solution ofsodium acetate
with the pH previously adjusted to 6.0 with glacial acetic
acid and dilute to 50.0 ml with the same solution.
Reference solution (b). Dissolve 10 mg of amoxycillin
trihydrate RS in 10 ml ofreference solution (a).
octadecylsilane bonded to porous silica (10 jJ.m),
95 volumes of a 1.5 per cent w/v solution of sodium
dihydrogen phosphate with the pH previously
adjusted to 4.0 with dilute phosphoric acid,
Mol. Wt. 166.0
Potassium Iodide contains not less than 99.0 per cent and
not more than 100.5 per cent ofKI, calculated on the dried
basis.
Category. Antithyroid; antifungal; expectorant.
Dose. In preoperative treatment of thyrotoxicosis, 30 to 60
mg; as expectorant, 250 to 500 mg.
Description. Colourless crystals or a white powder; odourless.
Identification
Dissolve 109 in 100 ml of carbon dioxide-ji-ee water (solution
A). The solution gives the reactions of potassium salts, and
ofiodides (2.3.1).
Tests
colourless (2.4.1).
Alkalinity. To IOmlofsolutionAaddO.2rnlofO.Ol Msulphuric
acid; no colour is produced on addition of a drop of
phenolphthalein solution.
Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and 12 ml of
stannated hydrochloric acid AsT. The resulting solution
Iron (2.3.14). 20 rnl ofsolutionA complies with the limit test for
iron (20 ppm).
Bar~um. Dissolve 0.5 g in 10 ml of water and add 1 mlofdilute
sulphuric acid; no turbidity develops within one minute.
Cyanide. Warm 5 ml of Solution A, add one drop offerrous

sulphate solution and 0.5 ml of sodium hydroxide solution
1939
POTASSIUM IODIDE
IP 2010
and acidify with hydrochloric acid; no blue colour is

produced.
Iodate. Dissolve 0.5 g in 10 ml of carbon dioxide-free water
and add 0.15 ml of dilute sulphuric acid and a drop of iodidefree starch solution; no blue colour is produced within
2 minutes.
Sulphates (2.3.17). 1.0 g dissolved in 15 ml of water complies
with the limit test for sulphates (150 ppm).
Thiosulphate. Dissolve 1 gin 10 ml of water, add 0.1 mlof
starch solution and 0.1 ml of 0.005 M iodine; a blue colour is
produced.
on 1.0 g of the powdered substance by drying in an oven at
105 for 3 hours.
Tests
Appearance of solution. Dissolve 1.5 g in 50 m1 of distilled
water, heat on a water-bath and add gradually 6 ml of ethanol
(95 per cent), cool, dilute to 60 ml with distilled water and
filter. The filtrate (solution A) is colourless (2.4.1).
Water-insoluble matter. Dissolve 0.5 gin 50 ml of water, heat
to boiling, filter through a tared, sintered-glass filter (porosity
No.4) and wash with water until the filtrate is colourless. The
weight of the residue, dried at 105 to constant weight, is not
more than 5 mg (1.0 per cent).
Assay. Weigh accurately about 0.35 g, dissolve in about 10 ml

of water, add 35 ml of hydrochloric acid and 5 ml of chloroform.
Titrate with 0.05 M potassium iodate until the purple colour
ofiodine disappears from the chloroform. Add the last portion
of the iodate solution dropwise and agitate vigorously and
continuously. Allow to stand for 5 minutes. If any colour
develops in the chloroform layer continue the titration until
the chloroform is decolorised.

water to produce lOO.Omi. To 20.0 ml add 20 m1 of water, 1 gof
potassium iodide and 10 ml of 2 M hydrochloric acid and
titrate the liberated iodine with 0.1 M sodium thiosulphate
using starch solution, added towards the end of the titration,
as indicator.
1 ml of 0.05 M potassium iodate is equivalent to 0.0166 g of

KI.

ofKMn04'
CA UTION - Great care should be taken in handling

potassium permanganate as dangerous explosions are liable
to occur if it is brought into contact with organic or other
readily oxidisable substances, either in solution or in the
dry .condition.
Potassium Permanganate
KMn0 4
Mol. Wt. 158.0
Potassium Sorbate
Potassium Permanganate contains not less than 99.0 per cent

and not more than 100.5 per cent ofKMn04.
CH 3 CH=CHCH=CHCOOK
Category. Antiseptic.
Mol. Wt.150.2
Description. A dark purple or brownish black, granular powder

or dark purple or almost black slender, prismatic crystals, having
a, metallic Illstre;odQ.urless. It decomposes on contact with
celtain organic substances.
Potassium Sorbate is potassium 2,4-hexadienoate.
Identification
Category. Food preservativ.
A. A solution in water acidified with sulphuric acid and heated

to 70 is decolorised by hydrogen peroxide solution (20 vol).
Description. A white or almost white powder or granules.
B. Heated to redness, it decrepitates, evolves oxygen and

leaves a black residue which with water forms potassium
hydroxide solution; the resulting solution when neutralised
with dilute hydrochloric acid gives the reactions ofpotassiurn
salts (2.3.1).

Potassium Sorbate contains not less than 99.0 per cent and
not more than 101.0 per cent of C6H7KO z, calculated on the
dried basis.
Identification

Compare the spectrum with that obtained with potassium
1940
IP 2010
POVIDONE
sorbate RS or with the reference spectrum of potassium

sorbate.
B. When examined in the range 230 nm to 350 nm (2.4.7), a 0.02
per cent w/v solution in water, dilute 1.0 ml ofthis solution to
100.0 ml with 0.1 M hydrochloric acid shows an absorption
maximum at about 264 nm and the specific absorbance at 264
nm is 1650 to 1900.
using 0.1 ml of crystal violet solution as indicator until the

colour changes from violet to bluish-green. Carry out a blank
titration.
C6H7KO z.
C. Dissolve 0.2 g in 2 ml of water and add 2 ml of dilute acetic

acid, filter. The solution gives reaction B ofpotassium (2.3.1).
Povidone
Tests
Polyvinylpyrrolidone; Polyvidone

dioxide-free water (solution A) is clear (2.4.1) and not more
Acidity or alkalinity. To 20 ml of solution A, add 0.1 ml of
phenolphthalein solution. Titrate with 0.1 M sodium
hydroxide or 0.1 M hydrochloric acid. Not more than 0.25 ml
is required to change th~ colour of the indicator.
Aldehydes. Not more than 0.15 per cent, determined by
dissolving 1.0 g in a mixture of 50 ml of 2-propanol and 30 ml
of water, adjust to pH 4.0 with 1 M hydrochloric acid. and
dilute to 100 ml with water. To 10 ml ofthe solution add 1 ml of
decolorisedfuchsin solution and allow to stand for 30 minutes.
Any colour produced is not more intense than that obtained
in a solution prepared simultaneously by adding by 1 ml of
decolorised filChsin solution to a mixture of 1.5 ml of
acetaldehyde standard solution (l00 ppm C2H 4 0j, 4 ml of 2propanol and 4.5 ml of water.
Heavy metals (2.3.13). Weigh in a silica crucible 2 g of the
substance under examination, mix with 0.5 g of magnesium
oxide. Ignite to dull redness until a homogeneous white or
greyish-white mass is obtained. After 30 minutes of ignition if
mixture remains coloured, allow to cool, mix using a fine glass
rod and repeat the ignition. If necessary repeat the operation.
Heat at 8000 for about 1 hour. Dissolve the residue in 5 ml
ofa mixture ofequal volumes of hydrochloric acid and water.
Add 0.1 ml of phenolphthalein solution and then
concentrated ammonia until a pink colour is obtained. Cool,
add glacial acetic acid until the solution is decolourised and
add 0.5 ml in excess. Filter if necessary and wash the filter.
Dilute to 20 ml with water, 12 ml of the resulting solution
complies with the limit test for heavy metals, Method D (10
ppm). Use lead standard solution (10 ppm pb), diluting 1 ml
standard solution to 10 ml with a mixture ofequal volumes of
hydrochloric acid and water.
anhydrous acetic acid. Titrate with 0.1 M perchloric acid
n
Mol. Wt. (111.2)11
Povidone is poly(2-oxopyrrolidin-I-ylethylene) and consists
oflinear polymers of 1-vinylpyrrolidin-2-one. The different
types of Povidone are characterised by their viscosity in
solution, expressed as K-value, in the range 10 to 95.
Povidone with a nominal K-value of 15 or less has a K-value
ofnot less than 85.0 per cent and not more than 115.0 per cent
ofthe declared value. The K-value ofpovidone with a nominal
K-value ofmore than 15, or a nominal K-value range with an
average ofmore than 15, is not less than 90.0 percent and not
more than 107.0 per cent ofthe declared value or ofthe average
of the declared range. It contains not less than 11.5 per cent
and not more than 12.8 per cent of nitrogen, N, calculated on
Category. Pharmaceutical aid (tablet binder, coating agent,
dispersing and suspending agent).
Description. A white or yellowish white powder or flakes;
odourless or almost odourless; hygroscopic.
Identification
Compare the spectrum with that obtained with povidone R8.
B. Add 2.5 g in small portions to a suitable volume of carbon
dioxide-fi-ee water, stirring with a magnetic stirrer, and dilute
to 25 ml with the same solvent (solution A). To 0.4 ml ofsolution
A add 10 ml of water, 5 ml of2 M hydrochloric acid and 2 ml
1941
IP 2010
POVIDONE
of potassium dichromate solution; an orange-yellow

C. To 1 ml of solution A add 0.2 ml of dimethylaminobenzaldehyde reagent and 0.1 ml of sulphuric acid; a pink
colour is produced.
D. To 0.1 ml of solution A add 5 ml of water and 0.2 ml of
0.05 M iodine; a red colour is produced.
Tests
more intensely coloured than reference solution BS6 or BYS6
(2.4.1).
Heavy metals. Mix 2.0 g with 0.5 g of magnesium oxide in a
silica crucible. Ignite to dull red heat until a homogeneous
white or greyish white mass is produced. Heat at 800 for
about 1 hour, dissolve the residue using two quantities, each
of 5 ml, of 5 M hydrochloric acid, add 0.1 ml of
phenolphthalein solution and strong ammonia solution until
a pink colour is produced. Cool, add glacial acetic acid until
the solution is decolorised and add a further 0.5 ml. Filter if
necessary and dilute the solution to 20 ml with water. To 12 ml
ofthe resulting solution add 2 ml of acetate bufferpH 3.5, mix,
add 1.2 ml of thioacetamide reagent, mix immediately and
allow to stand for 2 minutes. Any brown colour produced is
not more intense than that obtained by treating in the same
manner a mixture of 2 ml ofthe test solution obtained above
and 10 ml of the 20 ml of solution obtained by repeating the
procedure using 2 ml of lead standard solution (10 ppm Pb)
in place of the substance under examination, adding 0.5 g of
magnesium oxide in a silica crucible and continuing as
described above beginning at the words "Ignite to dull red
heaL." (10 ppm).
Aldehydes. Boil 20.0 g in 180 rnI ofa 25 per cent v/v solution of
sulphuric acid in a round-glass-stoppered flask under a reflux
condenser for 45 minutes and allow to cool. Fit a distillation
head, distil and collect 60 rnI ofthe distillate in 20 rnI ofa 7.0 per
cent w/v solution of hydroxylamine hydrochloride, previously
adjusted to pH 3.1 using 1 M sodium hydroxide, and cooled
in ice. Titrate with 0.1 M sodium hydroxide to pH 3.1. Carry
out a blank titration. Not more than 9.1 ml of 0.1 M sodium
hydroxide is required (0.2 per cent, calculated as acetaldehyde,
CzILO).
l-vinylpyrrolidin-2-one. Determine by liquid chromatography
(2.4.14).
Reference solution (a). A 0.05 per cent w/v solution of 1vinylpyrrolidin-2-one in methanol. Dilute 1.0 ml of this
solution to 100.0 ml with methanol. Further dilute 5.0 ml of
this solution to 100.0 ml with the mobile phase.
Reference solution (b). Dissolve 10 mg of I-vinylpyrrolidin2-one and 0.5 g of vinyl acetate in 100.0 ml of methanol.
Dilute 1.0 rnI ofthis solution to 100.0 ml with the mobile phase.
Inject reference solution (a) and (b). The test is not valid unless
the resolution between the peaks due to I-vinylpyrrolidin-2one and vinyl acetate is not less than 2.0 in the chromatogram
obtained with reference solution (b) and the relative standard
deviation for replicate injections is not more than 2.0 in the
chromatogramobtained with the test solution the area of the
peak due to I-vinylpyrrolidin-2-one is not more than the area
reference solution (a) (10 ppm).
Water (2.3.43). Not more than 5.0 percent determined on 0.5 g.
K-value. For Povidone with a stated K-value of 18 or less,
prepare a 5.0 per cent w/v solution. For Povidone with a
declared K-value of more than 18, prepare a 1.0 per cent w/v
solution. Allow the solution to stand for 1 hour and carry out
Method B for the determination ofviscosity (2.4.28), at 25
0.2 using a size no. 1 viscometer with a minimum flow time of
100 seconds. Calculate the K-value (1<.0) from the expression
_ 1.5logz-1
K 00.15+0.003c
~300C logz + (c + 1.5c 10gz)2

0.15c + 0.003c 2
where c is the percentage concentration w/v of the substance

urider examiIlatioIl, calculated Oil the arihymous basts, aridi
is the viscosity of the solution relative to that of water.
Nitrogen (2.3.30). Follow Method C, using 0.3 g, accurately
weighed and 11 ml of nitrogen-free sulphuric acid. For
complete destruction of organic matter repeat the addition of
hydrogen peroxide (10 vol) (usually 3 to 6 times) until a clear,
light-green solution is obtained, then heat for a further 4 hours.
Labelling. The label states the viscosity in terms ofa K-value
orK-range.
1942
IP 2010
POVIDONE-IODINE SOLUTION
Povidone-Iodine
CH-CH z
Assay. Weigh accurately about 3 g, transfer to a beaker and

add 200 m1 of water. Cover the beaker and stir with a mechanical
stirrer at room temperature for not more than 1hour to dissolve
as completely as possible. Titrate immediately thereafter with
0.1 M sodium thiosulphate using 3 mlof starch solution,
added towards the end of the titration, as indicator.
,x I
(yo

ofl.
Povidone-Iodine is a complex produced by interaction

between iodine and poly(2-oxopyrrolidin-l-ylethylene).
Povidone-Iodine contains not less than 9.0 per cent and not
more than 12.0 per cent ofavailable iodine, I, calculated on the
dried basis.
Category. Topical anti-infective; antiseptic.
Description. A yellowish brown amorphous powder; odour,
slight and characteristic of iodine.
Identification
A Add 0.05 ml ofa 10 per cent w/v solution to a mixture ofl ml
of starch solution and 9 ml of water; a deep blue colour is
produced.
B. Spread 1 ml of a 10 per cent w/v solution over an area of
about 20 cm x 20 cm on a glass plate and allow it to dry in air at
room temperature and in an atmosphere of low humidity
overnight; a brown, dry, non-smearing film is formed which
dissolves readily in water.
Tests
Nitrogen (2.3.30).9.5 to 11.5 per cent, calculated on the dried
basis, determined on about 0.3 g, by Method A
Iodide. Not more than 6.6 per cent, calculated on the dried
basis, determined by the following method. Weigh accurately
about 0.5 g and dissolve in 100 ml of water. Add sufficient
sodium bisulphite solution until the colour of iodine is
discharged. Add 25.0 ml of 0.1 M silver nitrate and 10 ml of
nitric acid and mix. Titrate with 0.1 M ammonium thiocyanate,
usingferric ammonium sulphate solution as indicator. Repeat
the procedure without the substance under examination. The
silvernitrate required. 1 ml of 0.1 M silver nitrate is equivalent
to 0.01269 g ofl. From the percentage oftotal iodine, calculated
on the dried basis, subtract the percentage of available iodine
determined in the Assay and obtain the percentage of iodide.
Povidone-Iodine Solution is a solution of Povidone-Iodine in
Purified Water. It may contain a small amount ofEthanoL
Povidone-Iodine Solution contains not less than 85.0 per cent
iodine, 1.
Usual strengths. 5 per cent w/v; 7.5 percent w/v; 10 per cent
w/v.
Description. A deep brown liquid; odour, characteristic of
iodine.
Identification
A Dilute 1 m1 to 20 rnl with water and add 1 ml ofthe resulting
solution to a mixture of 1 ml of starch solution and 9 ml of
water; a deep blue colour is produced.
B. Transfer 10 ml to a small flask and cover the mouth ofthe
flask with a filter paper soaked in starch solution; no blue
colour is produced on the paper within 60 seconds.
C. Dilute 20 ml to 100 ml with water. To 10 ml add dropwise
0.1 M sodium thiosulphate until the colour is just discharged.
To 5 ml ofthe resulting solution add 5 ml of 2 M hydrochloric
acid and 2 ml of potassium dichromate solution; a red
Tests
pH (2.4.24).3.0 to 6.5.
Ethanol (ifpresent) (2.3.45). 90.0 to 11 0.0 per cent ofthe stated
amount ofCzHsOH, determined by MethodA after decolorising
the solution with just sufficient quantity of a 10 per cent w/v
solution of sodium thiosulphate and treating with a few drops
of dilute sodium hydroxide solution.
50 mg of iodine add sufficient water to produce not less than
30 ml and titrate immediately with 0.02 Msodium thiosulphate,
1943
PRALIDOXIME CHLORIDE
IP 2010
using 3 ml of starch solution, added towards the end of the

titration, as indicator. Carry out a blank titration.
1 ml of 0.02 M sodium thiosulphate is equivalent to 0.002538
gofI.
Labelling. The label states the quantities ofiodine and ethanol
(if present) as percentages w/v.
1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g ofCl.

Pralidoxime Chloride
C7~CINZO
Chloride content. 20.2 to 20.8 percent, calculated on the dried

about 0.3 g, dissolve in about 150 ml of water, add 20 ml of
glacial acetic acid and 10 drops of (4-tert-octylphenoxy)
nonaethoxyethanol and titrate with 0.1 M silver nitrate,
determining the end-point potentiometrically (2.4.25).
Mol. Wt. 172.6
Pralidoxime Chloride is 2-hydroxyiminomethyl-lmethylpyridinium chloride.

Pralidoxime Chloride contains not less than 97.0 per cent and
not more than 103.0 per cent ofC7H gClNzO, calculated on the
dried basis.

water to produce 250.0 ml and mix. Dilute 5.0 ml ofthis solution
to 100.0 ml with water. Transfer 5.0 ml to a 50-ml volumetric
flask, dilute to about 40 ml with water, add 5.0 ml of 1 M
sodium hydroxide and dilute to volume with water. Within 10
minutes ofthe addition of the alkali, measure the absorbance
ofthe solution at the maximum at about 332 nm (2.4.7), using a
solution of5.0 ml of 1 M sodium hydroxide diluted with water
to 50.0 ml as the blank.
Calculate the content of C 7H gC1NzO from the absorbance
obtained by carrYing out the assay simultaneously using about
0.5 g, accurately weighed, of pralidoxime chloride RS.
Category. Antidote for cholinesterase inhibitors.

Dose. By intravenous injection, 1 to 2 g as a 5 per cent w/v
solution, administered over not less than 5 minutes period; by
intramuscular injection, 1 g initially, followed by 1 to 2 further
doses ifnecessary, upto a maximum of 12 g in 24 hours.
Description. A white to pale yellow, crystalline powder;
odourless.
Identification

Compare the spectrum with that obtained with pralidoxime
chloride RS or with the reference spectrum of pralidoxime
chloride.
Pralidoxime Chloride Injection

Pralidoxime Chloride Injection is a sterile material consisting
ofPralidoxime Chloride with or without buffering agents and
other excipients. It is filled in a sealed container.
sealed container in the requisite amount Of sterile Water for


absorption maxima at about 242 nm and 292 nm and in
0.1 M sodium hydroxide, a maximum at about 332 nm.

C. To 0.1 ml of a 20 per cent w/v solution add 1 ml of a 0.6 per

cent w/v solution offerric chloride; an amber-brown colour
is produced.
Pralidoxime Chloride Injection contains not less than 90.0 per

pralidoxime chloride, C7HgClNzO.
Usual strength. 1g.
D. Gives the reactions of chlorides (2.3.1).
Description. A white to pale yellow powder.

Tests

requirements stated under Parenteral Preparations (powders
for Injection) and with the following requirements:
1944
IP 2010
PRAVASTATIN SODIUM
Identification
Compare the spectrum with that obtained with pralidoxime
chloride R8 or with the reference spectrum of pralidoxime
chloride.

absorption maxima at about 242 nm and 292 nm and in
0.1 M sodium hydroxide, a maximum at about 332 nm.
C. To 0.1 ml ofa 20 per cent w/v solution add 1 ml ofa 0.6 per
cent w/v solution offerric chloride; an amber-brown colour
is produced.
Pravastatin Sodium is sodium (3R,5R)-3,5-dihydroxy-7[(IS,2S,6S,8S,8aR)-6-hydroxy-2-methyl-8-[[(2S)-2methylbutanoyl]oxy]-1 ,2,6,7,8,8a-hexahydronaphthalen-lyl]heptanoate

Pravastatin Sodium contains not less than 97.0 per cent and
not more than 102.0 per cent ofCz3H3sNa07, calculated on the
anhydrous and ethanol free basis.
Category. Antihyperlipidaemic.
Description. A white to yellowish white powder or crystalline
powder, hygroscopic.
Identification
A. Determine by infi:ared absorption spectrophotometry (2.4.6).

Compare the spectrum with that obtained with pravastatin
sodium RS or with the reference spectrum of pravastatin
sodium.
B. 1 ml of a 5 per cent w/v solution in carbon dioxide-free

water (Solution A) gives reaction A of sodium (2.3.1).

per mg ofpralidoxime chloride.
Tests
Tests

water to produce 250.0 rnl and mix. Dilute 5.0 ml ofthis solution
to 100.0 ml with water. Transfer 5.0 ml to a 50-ml volumetric
flask, dilute to about 40 ml with water, add 5.0 ml of
1 M sodium hydroxide and dilute to volume with water. Within
10 minutes ofthe addition ofthe alkali, measure the absorbance
ofthe solution at the maximum at about 332 nm (2.4.7), using a
solution of5.0 ml of 1 M sodium hydroxide diluted with water
to 50.0 ml as the blank.
Appearance of solution. Dilute 2.0 ml ofsolutionAto 10 ml

with water. The solution is clear (2.4.1) and not more intensely
coloured than the reference solution BYS6 (2.4.1).
Specific optical rotation (2.4.22). + 153 0 to+159 0 , detelmined
(2.4.14).
Calculate the content of C 7H 9CINzO from the absorbance

obtained by carrying out the assay simultaneously using about
0.5 g, accurately weighed, of pI'alidoxime chloride RS.
Solvent mixture. 9 volumes of methanol and 11 volumes of

water.
Labelling. The label states the period within which the

constituted injection should be used.

Test solution (b). Dilute 10 ml of test solution (a) to 100 ml
Pravastatin Sodium
Reference solution (a). Dissolve the contents of a vial of

pravastatin impurity A RS in 1 ml of test solution (b).
Reference solution (b). Dilute 2 ml oftest solution (a) to 100
ml with the solvent mixture. Dilute 1 ml ofthis solution to 10 ml
H3
CYO
CH
Reference solution (c). Dissolve 12.4 mg of pravastatin

1,1,3,3- tetramethylbutylamine RS in 100 ml of thesolvent
mixture.
Mol. Wt. 446.5
octadecylsilane bonded to porous silica (5 J.l.m),
1945
PRAVASTATIN SODIUM
IP 2010
- mobile phase: a mixture of 1 volume of glacial acetic

acid, 1 volume of triethylamine, 450 volumes of
methanol and 550 volumes of watel;
- injection volume. 10 )..Ll.
resolution between the peaks due to pravastatin impurity A
and pravastatin is not less than 7.0.
The relative retention times with reference to pravastatin for
(3R,5R)-3 ,5-dihydroxy-7-[(1 S,2S,6S,8S,8aR)-6-hydroxy-8[[(2S, 3R)-3 - hydroxy-2-methylbutanoy1] oxy]-2-methy11,2,6,7,8,8a-hexahydronaphthalen-1-y1] heptanoic acid (3"hydroxypravastatin) (pravastatin impurity B), (3R,5R)-3,5dihydroxy-7-[(1 S,2S,6S,8S,8aR)-6-hydroxy-8-[[(2S,3S)-3hydroxy-2-methylbutanoyl]oxy]-2-methyl-1 ,2,6,7 ,8,8ahexahydronapthalen-1-yl] heptanoic acid (3" -(S)hydroxypravastatin) (pravastatin impurity E), (3R,5R)-3,5dihydroxy-7-[(lS,2S,6R,8S,8aR)-6-hydroxy-2-methyl-8-[[(2S)2-methylbutanoyl]oxy]-1,2,6,7,8,8a-hexahydronaphthalen-1yl]heptanoic acid (6'-epipravastatin) (pravastatin impurity
A), (lS,3S,7S,8S,8aR)-3-hydroxy-8-[2-[(2R,4R)-4-hydroxy-6oxotetrahydro-2H-pyran-2-yl]ethyl]-7-methyl-1 ,2,3,7,8,8ahexahydronaphthalen-l-yl (2S)-2-methylbutanoate
(pravastatin lactone) (pravastatin impurity D ) and (3R,5R) 3,5-dihydroxy-7-[(lS,2S,6S,8S,8aR)-6-hydroxy-2-methyl-8[[(2S)-2-methylpentanoyl]oxy]-1 ,2,6,7 ,8,8ahexahydronaphthalen-l-yl] heptanoic acid) (pravastatin
impurity C) and (3R, 5R)-7-[(1S,2S,6S,8S,8aR)-6,8-dihydroxy2-methyl-l ,2,6,7,8,8a-hexahydronaphthalen-l-yl]-3,5dihydroxyheptanoic acid (pravastatin impurity F) are about
0.2,0.3,0.6, 1.9 and 2.1 respectively.
Inject test solution (a) and reference solution (b). Run the
peak of pravastatin. In the chromatogram obtained with test
solution (a), the area of peak due to pravastatin impurity A is
not more than 1.5 times the area ofthe principal peak obtained
with reference solution (b) (0.3 per cent), the area ofpeaks due
to pravastatin impurities B, C, D and E is not more than the
area ofthe principal peak obtained with reference solution (b)
(0.2 per cent), the area ofpeak due to pravastatin impurity F is
not more than 0.75 times the area ofthe principal peak obtained
with reference solution (b) (0.15 per cent) the area of other
principal peak obtained with reference solution (b) (0.1 per
cent) and sum of the areas of all the secondary peaks is not
more than 3 times the area ofthe principal peale obtained with
reference solution (b) (0.6 per cent). Ignore any peak with an
cent).
Heavy metals (2.3.13). Dissolve 2.0 g in a mixture orI5 volumes

of water and 85 volumes of methanol and dilute to 20 ml with
the same solvent mixture and use 12ml of the solution. The
Method B (20 ppm). Prepare the reference solution using lead
standard solution (2 ppm Pb) obtained by diluting lead
standardsolution (l 00 ppm Pb) with a mixture of 15 volumes
of water and 85 volumes of methanol.
~
Ethanol (2.3.45). Not more than 3.0 per cent v/v (determined
by method I).
0.5 g.
described in the test for Related substanceswith the following
modification.
Inject the test solution (b) and reference solution (c).
Calculate the content ofCZ3H3sNa07 from the declared content
of pravastatin in pravastatin 1,1,3,3-tetramethylbutylamine
RS.
1 mg ofpravastatin is equivalent to 1.052 mg ofpravastatin
sodium.
Pravastatin Tablets
Pravastatin Sodium Tablets
Pravastatin Tablets contain not less than 92.5 per cent and
pravastatin sodium, C23H3sNa07.
Identification
10 mg ofPravastatin Sodium with 80 rn1 ofwater for 10 minutes,
dilute to 100 ml with water, filter. Dilute 1 ml to 10 ml with
water. When examined in the range 220 to 350 nm (2.4.7),
exhibits a maximum only at 238 um.
Tests
(2.4.14).
Solution A. Dissolve 4.1 g of anhydrous sodium acetate in

400 ml of water, adjust the pH to 5.6 with glacial acetic acid
and add sufficient water to produce 500 ml.
1946
IP 2010
PRAZIQUANTEL
Solution B. Mix 20 volumes of methanol with 80 volumes of

solution A.
Test solution. Add 20 ml of solution A to a quantity of the
powdered tablets containing about 10 mg of Pravastatin
Sodium, mix using a vortex mixer and then with the aid of
ultrasound for 15 minutes, centrifuge. Dilute 1 ml ofthe clear
supernatant liquid to 5 ml with solution B.
Reference solution (a). Dilute 1 ml of the test solutionto 100
ml with solution B.
5 ml with solution B.
Test solution. Add 20 ml of solution A to a quantity of the

powdered tablets containing about 10 mg of Pravastatin
Sodium, mix using a vortex mixer and then with the aid of
ultrasound for 15 minutes, centrifuge. Dilute 1 ml ofthe clear
supernatant liquid to 5 ml with solution B.
Reference solution (a). Dissolve 12.4 mg of pravastatin
1,1,3,3,-tetramethylbutylamine RS in 20 ml of solution A.
Dilute 1 ml ofthis solution to 5 ml with solution B.
Reference solution (b). Dissolve 2 mg of pravastatin impurity
A RS in the minimum volume of methanol and dilute to 20 ml
with the test solution.
Reference solution (c). Dilute 1 ml ofreference solution (b) to

4 ml with solution B.
Use the chromatographic system, solution A and B as

Reference solution (d). Dissolve 2 mg of (3R,5R)-3,5dihydroxy-7-[(lS,2S,6R,8S,8aR)-6-hydroxy-2-methyl-8[[(2S) -2-methyl bu tanoyIJoxyJ-1, 2,6,7,8, 8ahexahydronaphthalen-1-yUheptanoic acid RS (pravastatin
impurity A RS) in the minimum volume of methanol and dilute
to 20 ml with the test solution.
octadecylsilane bonded to porous silica (5 f..lm) (such as
Ultrasphere ODS), .
acid, 1 volume of triethylamine, 450 volumes of
methanol and 550 volumes ofwater,
injection volume. 20 f..ll.

Calculate the content of C23H3SNa07 in the tablets using the
declared conterit of pravastatin in pravastatin 1,1,3,3tetramethylbutylamine RS.
1 mg ofC23H3607 is equivalent to 1.052 mg ofC23H3sNa07'
Praziquantel
Inject reference solution (d). The test is not valid unless the
Inject the test solution, reference solution (a), (b) and (c). In
the chromatogram obtained with the test solution the area of
any secondary peak is not more than twice the area of the
solution (a) (2 per cent), the area of one such peak is not more
obtained with reference solution (a) (1 per cent), not more
than a further one such peak has an area more than the area of
secondary peaks is not more than three times the area of the
solution (a) (3 per cent). Ignore any peak with an area less
obtained with reference solution (c) (0.05 per cent).
Mol.Wt. 312.4
Praziquantel is (11bRS)-2-(cydohexylcarbonyl)-l ,2,3,6,7,11 bhexahydro-4H-pyrazino[2, I-a ]isoquinolin-4-one.
Praziquantel contains not less than97.5 per cent and not more
than 102.0 per cent of C19H24N202, calculated on the dried
basis.
Category. Antihelminthic.
Identification
Compare the spectrum with that obtained with praziquantel
RS or with the reference spectrum ofpraziquantel.
1947
PRAZIQUANTEL
IP 20ID
Tests
(2.4.14).

Test solution (b). Dilute 1.0 ml oftest solution (a) to 20 ml with
the mobile phase.
Reference solution (a). Dissolve 40 mg bfpraziquantel RS in
10 ml ofthe mobile phase. Dilute 1.0 ml to 20 ml with the mobile
phase.
Reference solution (b). Dissolve 5.0 mg of (11 bRS)-2-benzoyl1,2,3,6,7,11b-hexahydro-4H-pyrazino[2, 1-a]isoquinolin-4one RS (praziquantel impurity A RS) in 25 ml of reference
solution (a). Dilute 2.0 ml of this solution to 20 ml with the
mobile phase.
Reference solution (c). Dilute 1.0 ml oftest solution (a) to 20
ml with the mobile phase. Dilute 5.0 ml ofthis solution to 50 ml
Inject reference solutions (b). The test is not valid unless the
resolution between the peaks corresponding to praziquantel
impurity A and praziquantel is not less than 3.0.
Inject test solution (a), reference solution (b) and (c). Run the
chromatogram 5 times the retention time ofthe principal peale.
In the chromatogram obtained with test solution (a), the area
(c) (0.5 per cent). The area of one such peak is not more than
obtained with reference solution (c) (0.2 per cent). The sum of
the areas of all other secondary peaks is not more than the
area 0.1 times the area ofthe principal peale in the chromatogram
on 1.0 g by drying in an oven at 50 over diphosphorus
pentoxide at a pressure not exceeding 0.7 kPa for 2 hours.

than 1.0 per cent.
Inject the test solution (b) and reference solution (a).
Calculate the content of CI9Hz4NzOz.
Praziquantel Tablets contain not less than 90.0 per cent and
Praziquantel, CI9Hz4NzOz.
Identificaton
Compare the spectrum with that obtained with praziquantel
RS or with the reference spectrum of praziquantel.
the plate with silica gelG.
Mobile phase. Ethyl acetate.

containing 30 mg of praziquantel in 5 ml of methanol for 5
minutes, centrifuge and use the clear supernatant liquid.
Reference solution. A 0.6 per cent w/v solution ofpraziquantel
RS in methanol.
urn. The principal spot in the chromatogram obtained with the
test solution corresponds to that in the chromatogram obtained
Tests
Apparatus No.1,
Medium. 900 ml of 0.1 M hydrochloric acid containing 2.0 mg
per ml of sodium lauryl sulphate,
the absorbance ofthe filtrate at the maximum at about 263 urn
(2.4.7). Calculate the content of CI9Hz4NzOz in the medium
concentration of praziquantel RS in the same medium.
1948
IP 2010
PRAZOSlN HYDROCHLORIDE

C19H24N202'

almost odourless.
Identification

a quantity of the powder containing 150 mg ofPraziquantel,
add 70 ml ofmobile phase, sonicate for 5 minutes, dilute with
mobile phase to 100 ml, mix and filter. Dilute 3.0 ml of the
solution to 25 ml with the mobile phase and mix.
praziquentel RS in the mobile phase.
Chromatographic System
- a stainless steel colurnn25 cm x 4.0 mID, packed with
Inject the reference solutIon. The test is not valid unless the

Compare the spectrum with that obtained with prazosin
hydrochloride RS.
B. Prepare a 0.05 per cent w/v solution ofthe substance under
examination in a 0.1 per cent v/v solution of hydrochloric
acid in methanol. Dilute separately 1 ml and 5 ml ofthis solution
to 100 ml with the same acid solution (solutions A and B
respectively). When examined in the range 220 nm to 280 nm
(2.4.7), solution A shows an absorption maximum at about
247 nm; absorbance at the maximum, 0.66 to 0.70. When
examined in the range 280 nm to 400 nm, solution B exhibits
two maxima at about 330 nm and 343 nm; absorbances at the
two maxima, 0.65 to 0.70 and 0.60 to 0.66 respectively.

Solvent mixture. A mixture of 1 volume of diethylamine, 10

volumes of methanol and 10 volumes of methylene chloride.
Mobile phase. A mixture of 5 volumes of diethylamine and 95
volumes of ethyl acetate.
Calculate the content OfC19H24N202. in the tablets.


examination in 10 ml ofsolvent mixture.
Reference solution. A 0.1 percent w/v solution of prazosin
hydrochloride RS in solvent mixture.
Prazosin Hydrochloride
Apply to the plate 10 fll of each solution. Allow the mobile

phase to rise 8 em. Dry the plate in warm air and examine under
ultra-violet light at 254 nm. The principal spot in the chroma
togram obtained with the test solution corresponds to that in
the chromatogram obtained with the reference solution.
D. A 0.1 per cent w/v solution gives the reactions of chlorides
(2.3.1).
Tests
Mol. Wt.4l9.9
Prazosin Hydrochloride is 2-[4-(2-furoyl)piperazin-l-yl]-6,7dimethoxyquinazolin-4-ylaminehydrochloride.
Prazosin Hydrochloride contains not less than 98.5 per cent
and not more than 101.0percentofC19H21Ns04,HCl,calculated
Category. Antihyperten,sive.
Dose. 500 flg to 1 mg two to three times daily.
Iron.To 1.0 g add dropwise about 1.5 ml of nitric aCid,heat

cautiously on a water-bath until fumes are no longer evolved.
Ignite by slowly raising the temperature from 150 to 1000,
maintaining the final temperature for 1 hour. Cool, dissolve
the residue in 20 ml of 2 M hydrochloric acid, evaporate to
about 5 ml, dilute to 25 ml with 2 M hydrochloric acid and
examine the resulting solution by atomic absorption
spectrophotometry (2.4.2), measuring at 248 nm using an iron
hollow-cathode light as a source ofradiation, an air-acetylene
flame and iron standard solution (8 ppm Fe), diluted as
1949
PRAZOSIN HYDROCHLORIDE
IP 2010
necessary with water to prepare. the. standard solutions

(100 ppm).
acid to produce 50 rril. The resulting solution complies with

the limit test for heavy metals, Method B (50 ppm).
Reserve about 10 ml of the solution for the Nickel test.
Nickel. Examine the final solution prepared in the test for Iron
by atorriiC absorption spectrophotometry (2.4.2), measuring
at 232 urn using a nickel hollow-cathode light as a source of
radiation, an air-acetylene flame and using nickel standard
solution (10 ppm Ni},diluted as necessary with water to
prepare the standard solutions (50 ppm).
Water (2.3.43). Not more than 0.5 per cent w/w, using 2.0 g
dissolved in a mixture of equal volumes of dichloromethane
and methanol.

(2.4.14).

examination in the mobile phase and dilute to 50 ml with the
mobile phase.
Reference solution (a). Dilute 1 ml of the test solution to
100 ml with the mobile phase. Further dilute 1 rril ofthe solution
Reference solution (b). Dissolve 8 mg of metoclopramide
hydrochloride RS in 1 ml of the test solution and dilute to
25 rril with the mobile phase. Further dilute 1 ml ofthe solution
octadecylsilane bonded to porous silica (5 ~m),
50 volumes of a solution containing 3.484 g per litre of
sodium pentanesulphonate and 3.p4 g per litre of
with glacial acetic acid,
- injection volume. 20 ~L
InjeCt reference solution (b). The retention times are: prazocine
about 9 minutes and metoclopramide abOut 5 minutes.The
test is not valid unless the resolution between the peaks
corresponding to prazocine and metoclopramide is at least 8.
Inject the test solution and reference solution (a). Continue
the chromatography of the test solution for 6 times the
retention time of the principal peak. Any secondary peak
obtained with the test solution is not more than twice the area
reference solution (a) (0.2 per cent).The sum ofall the impurities
is not more than five times the area ofthe principal peale in the
cent). Ignore any peale with an area 0.5 times the area of the
solution (a) (0.05 per cent).
Heavy metals (2.3.13). Determine on the residue obtained in
the test for Sulphated ash. Dissolve in sufficient 2 M nitric

anhydrous formic acid and 30 ml of acetic anhydride. Titrate
C19H21Ns04, HCL
Storage. Store proteCted from light.
Prazosin Tablets
Prazosin Hydrochloride Tablets
Prazosin Tablets contain not less than 90.0 per cent and not
more than 11 0.0 per cent of the stated amount of prazosin,
C19H21Ns04.
Usual strengths. The equivalent of500 ~g; 1 mg; 2 mg; 5 mg
ofprazosin.
Identification
Shake a quantity of the powdered tablets containing about
10 mg ofprazosin with a mixture of 10 ml of dichloromethane
and 10 ml of 0.05 M potassium hydroxide, filter the organic
layer through cotton, evaporate to dryness and drythe residue
at 60 at a pressure not exceeding 2 kPa at 60 for 2 hours.
obtained with prazosin hydrochloride RS or with the reference
spectrum of prazosin.
Tests
Mobile phase. A mixture of 95 volumes of ethyl acetate and

5 volumes of diethylamine.
containing 5 mg ofprazosin with 2 rril ofa mixture ofl 0 volumes
of dichloromethane, 10 volumes of methanol and 1 volume of
diethylamine, centrifuge a~d pass the supernatant liquid
through a 0.5 ~m PTFE (Polytetrafluoroethylene) filter.
200 volumes with the same solvent mixture
1950
IF 2010
PREDNISOLONE
Reference solution (b). Dilute 2 volumes ofreference solution

(a) to 5 volumes with the same solvent mixture.
Prednisolone
Apply to the plate 20 III of each solution. Any secondary spot

with reference solution (a) and not more than two such spots
are more intense than the spot in the chromatogram obtained
OH
-OH
Uniformity of content. Comply with the tests stated under

Tablets.
Mol. Wt. 360.4

Prednisolone is 1113,17a,21-trihydroxypregna-l ,4-diene-3,20dione.
Test solution. Shake one tablet for 1 hour in a suitable volume

of a mixture of96 volumes of methanol, 2 volumes of glacial
acetic acid and 2 volumes of water to produce a solution
containing 0.002 per cent w/v solution ofprazosin, centrifuge
and use the supernatant liquid.
Prednisolone contains not less than 96.0 per cent and not
more than 104.0 per cent of C Z1 HZ 80 S, calculated on the dried
basis.
Reference solution. A 0.0022 per cent w/v solution ofprazosin

hydrochloride RS in the same solvent mixture.
Dose. Upto 30 mg daily, in divided doses.
a stainless steel column 20 cm x 4 rom, packed with porous
silica particles, (5 Ilm),
mobile phase: 0.01 per cent w/v solution of diethylamine
in a mixture of 96 volumes of methanol, 2 volumes of
glacial acetic acid and 2 volumes of water,
Category. Adrenocortical steroid.

hygroscopic.
Identification
Compare the spectrum with that obtained with prednisolone
RS or with the reference spectrum of prednisolone.
Calculate the content OfC19HzINs04 in the tablet.


10 volumes offormamide.

of the powdered tablets containing about 2. mg of prazosin
with 100 ml in a mixture of96 volumes of methanol, 2 volumes
of glacial acetic acid and 2 volumes of water for 30 minutes,
centrifuge and use the supernatant liquid:
Reference solution. A 0.0022 per cent w/v solution ofprazosin
hydrochloride RS in a mixture of 96 volumes of methanol,
Mobile phase. Chloroform.

Reference solution (a). Dissolve 25 mg ofprednisolone RS in

was done.

equivalent amount of prazosin.

The chromatographic conditions as described under

Uniformity of content may be used.
Calculate the content OfC19HzINs04 in the tablets.
1951
IP 2010
PREDNISOLONE

in daylight and in ultraviolet light at 365 nrn. The principal
compact spot. A.
C. Dissolve 2 mg in 2 ml of sulphuric acid by shaking and
allow to stand for 5 minutes; an intense red colour is produced
with a reddish brown fluorescence when examined in ultraviolet
light at 365 nrn. Pour the solution into 10 ml of water and mix;
the colour fades and there is a yellow fluorescence under
ultra-violet light (365 nrn).
Tests
Specific optical rotation (2.4.22). +96.0 to +102, determined
solution in ethanol (95 per cent) at the maximum at about
240 nrn, 0.40 to 0.43.
(2.4.14).

examination in 2 ml of tetrahydrofilran and dilute to 10m1 with
water.
Reference solution (a). Dissolve 2 mg of prednisolone RS
and 2 mg of hydrocortisone RS in the mobile phase and dilute
Reference solution (b). Dilute 1 ml of the test solution to
base deactivated end-capped octadecylsilane bonded
to porous silica (5 I!m),
- mobile phase: a mixture of 220 ml of tetrahydrofitran
and 700 ml of water, allowed to equilibrate, diluted to
1000 ml with water and mixed again,
- spectrophotometer set at 254 nrn,
- injection volume. 20 I!l.
Equilibrate the column with the mobile phase for about 30
minutes.
Adjust the sensitivity of the system so that the height of the
solution (b) is not less than 50 per cent of the full scale of the
recorder.
Inject reference solution (a). The retention times are:

prednisolone, about 14 minutes and hydrocortisone about
15.5 minutes. The test is not valid unless the resolution between
the peaks corresponding to prednisolone and hydrocortisone
is at least 2.2. If necessary, adjust the concentration of
tetrahydrofitran in the mobile phase.
Inject separately the solvent mixture ofthe test solution as a
blank, the test solution and reference solution (b). Continue
the chromatography of the test solution for 4.5 times the
than the principal peak, is not more than the area ofthe principal
(b) (1.0 per cent) and not more than one such peak has an area
more than half the area of the principal peak in the
cent); the sum of the areas of all the peaks other than the
principal peak is not more than 2.0 times the area ofthe principal
(b) (2 per cent). Ignore any peak obtained with the blank run
and any peak with an area less than 0.05 times that of the
solution (b).
ethanol to produce 100.0 ml. Dilute 2.0 ml of this solution to
100.0 ml with ethanol. Measure the absorbance ofthe resulting
solution at the maximum at about 243.5 nrn. Calculate the
content of CZIHzsOs taking 415 as the specific absorbance at
243.5nrn.
Prednisolone Tablets contain not less than 90.0 per cent and
prednisolone, CZIHzsOs.
Identification
Extract a quantity ofthe powdered tablets containing 30 mg of
Prednisolone with 10 ml of chloroform, filter and evaporate
tests.

RS or with the reference spectrum of prednisolone.
1952
IP 2010
PREDNISOLONE TABLETS

mobile phase: a mixtl.J[e of 220 ml of tetrahydrofilran

and 700 ml of water, allowed to equilibrate, diluted to
1000 ml with water and mixed again,
injection volume. 20 !-tl.

Test solution. Dissolve 25 mg of the residue in 10 ml of the
solvent mixture.
Equilibrate the column with the mobile phase for about

30 minutes.
Reference solution (a). Dissolve 25 mg ofprednisolone RS in


recorder.

top, remove the plate from the tanle and allow the solvent to
was done.

prednisolone, about 14 minutes and hydrocortisone about
15.5 minutes. The test is not valid unless the resolution between
the peaks corresponding to prednisolone and hydrocortisone
is not less than 2.2. If necessary, adjust the concentration of
tetrahydrojitran in the mobile phase.
Apply to the plate 2 !-tl of each solution. Allow the mobile

the hot plate with ethanolic sulphuric acid (20 per' cent v/v).
in daylight and in ultraviolet light at 365'nm. The principal
compact spot.
Inject separately the solvent mixture of the test solution as a

blank, the test solution and reference solution (b). Continue
the chromatography of the test solution for 4.5 times the
retention time of the principal peale In the chromatogram
obtained with the test solution, the area of any secondary
peak is not more than the area of the principal peak in the
cent) and the sum of the areas of all the peaks other than the
principal peak is not greater than three times the area of the
solution (b) (3.0 per cent). Ignore any peak obtained with the
blanle run and any peak with an area less than 0.05 times that
reference solution (b) (0.05 per cent) and any peak with a
retention time of3 minutes or less.
Tests
(2.4.14).

containing 10 mg ofPrednisolone with 25 ml of methanol for
10 minutes and mix with the aid of ultrasound for 2 minutes;
filter the extract (Whatman GF/F is suitable), washthe filter
with two 10-ml quantities of methanol, combine the filtrate
and washings and evaporate to dryness using a rotary
evaporator and a warm water-bath, dissolve the residue in
10 ml of tetrahydrofilran and dilute to 20 ml with water.
Reference solution (a). Dissolve 2 mg of prednisolone RS
and 2 mg of hydrocortisone RS in the mobile phase and dilute
100 ml with a 50 per cent v/v solution of tetrahydrofitran.

Powder one tablet, add 50 ml of ethanol (95 per cent), shake
for 30 minutes, add sufficient ethanol (95 per cent) to produce
100.0 ml. Centrifuge and pipette a suitable volume ofthe
supernatant liquid containing 0.5 mg of Prednisolone and
dilute to 50.0 ml with ethanol (95 per cent). Measure the
240 nm (2.4.7). Calculate the content of CZIHzsOs taking
415 specific absorbance at 240 nm.
Apparatus. No.1,
Withdraw a suitable volume ofthe medium, filter and dilute a
suitable volume ofthe filtrate with the same solvent. Measure
the absorbance ofthe filtrate at the maximum at about240 nm
1953
PREDNISOLONE TABLETS
IP 2010
(2.4.7). Calculate thecontentof~21H2s0sin the medium from

concentration of prednisolone RS.
Prednisolone Acetate contains not less than 97.0 per cent and
not more than 103.0 per cent of C23H3006, calculated on the
dried basis.
D. Not less than 70 per cent ofthe stated amount ofC21H2s0s.
Category. Antiinflammtory; immunosuppressant.

a quantity of the powder containing about 5 mg of
Prednisolone, add 58 ml of methanol, shake for 10 minutes
and add sufficient water to produce 100.0 ml. Mix well and
filter.
Reference solution (a). A solution containing 0.005 per cent

w/v of prednisolone RS and 0.0075 per cent w/v of
dexamethasone (internal standard) in a mixture of58 volumes
of methanol and 42 volumes of water.
Reference solution (b). Prepare in the same manner as the test
solution but adding 10 ml ofa 0.075 per cent w/v solution of
dexamethasone in methanol and 48 ml of methanol in place of
58 mlofmethanol.
octadecylsilyl silica gel (5 ~m),
- mobile phase: a mixture of 42 volumes of water and
58 volumes of methanol,
- injection volume. 20 ~l.
The assay is not valid unless the resolution factor between
the peaks due to prednisolone and dexamethasone is greater
than 2.5 and the column efficiency, determined using the peak
due to prednisolone in the chromatogram obtained with the
test solution is greater than 15,000 theoretical plates per metre.
Calculate the content ofC21H2s0s in the tablets.
Identification
acetate RS or with the reference spectrum of prednisolone
acetate.

the plate with silica gel F254.
Solvent muture. 10 volumes of methanol and 90 volumes of

dichloromethane.
Mobile phase. Add a mixture of 1.2 volumes of water and 8
volumes of methanol to a mixture of 15 volumes of ether and
77 volumes of dichloromethane.
prednisolone acetate RS in the solvent mixture.
Reference solution (b). Dissolve 10 mg of prednisolone
pivalate RS in 10.0 ml ofreference solution (a).
Apply to the plate 5 ~l of each solution. Allow the mobile
light at 254 nm. Spray with alcoholic solution ofsulphuric
acid. Heat at 105 for 10 minutes or until the spots appear.
Allow to cool and examine under ultraviolet light at 365 urn.
C. Add about 2 mg ofthesubstance under examination in 2 ml
of sulphuric acid and shake to dissolve. Within 5 minutes, an'
intense red colour develops. When examined under ultraviolet
light at 365 urn, a reddish-brown fluorescence is seen. Add the
solution to 10 ml of water and mix. The colour fades and there
is greenish-yellow fluorescence in ultraviolet light at 365 urn.
D. It gives the reaction ofacetyl (2.3.1).
~3H3006
Mol.Wt. 402.5
Prednisolone Acetate is 11~, 17a-dihydroxy-3,20dioxopregna-l ,4~dien-2l-yl acetate.
Tests
Specific optical rotation (2.4.22). + 128 to + 137, determined
1954
IP 2010
PREDNISOLONE SODIUM PHOSPHATE

(2.4.14).

Solvent mixture. Equal volumes of acetonitrile and buffer
solution pH 4 prepared by mixing 1 volume of dilute
hydrochloric acid, 5 volumes of a 6.81 per cent w/v solution
of sodium acetate, 15 volumes ofa 3.73 percent w/v solution
of potassium chloride and 79 volumes of water.
Reference solution (a). Dissolve 2 mg each of prednisolone
acetate RS and hydrocortisone acetate RS (prednisolone
impurity A RS) in 100 ml ofthe solvent mixture.
Reference solution(b). Dilute 1.0 ml ofthe test solution to 100
ml with the solvent mixture. Dilute 2.0 ml ofthis solution to 10
ml with the solvent mi;lture.
j.ll11),
The relative retention time with reference to prednisolone
acetate for prednisolone acetate impurity B is about 0.4, for
prednisolone acetate impurity A is about 1.1, for prednisolone
acetate impurity C is about 2.
resolution between the peaks due to prednisolone acetate
and prednisolone acetate impurity A is not less than 2.0.
area of secondary peak corresponding to prednisolone acetate
impurity A and B is not more than 5 times the area of the
solution (b) (1.0 per cent), the area of secondary peak
corresponding to prednisolone acetate impurity C is not more
any other secondary peak is not more than 0.5 times the area
reference solution (b) (0.1 per cent) and the sum of the areas
ofall secondary peaks is not more than 10 times the area of,the
solution (b) (2.0 per cent). Ignore any peak with an area less
than 0.25 times the area of the principal peak in the

cent).
Assay. Dissolve 0.1 gin 100.0 ml of ethanol (95 per cent).
Dilute 2 ml of this solution to 100 ml with the same solvent.
Measure the absorbance at the maximum at about 243 urn
(2.4.7). Calculate the content of CZ3H3006 taking 370 as the
specific absorbance at 243 nm.

oII
O-P-ONa
I
-OH ONa
o
CzIHz7Naz08P
Mol. Wt. 484.4
Predisolone Sodium Phosphate is 11~,17a, 21trihydroxypregna-l,4-diene-3,20-dione-2l(dihydrogenphosphate) disodium salt.

Prednisolone Sodium Phosphate contains not less than 96.0
per cent and not more than 102.0 per cent of CZIHz7Naz08P,
Category. Antiinflammtory; immunosuppressant.
Identification

sodium phosphate RS. If the spectra obtained in the solid
state show differences, dissolve the substance under
examination and the reference substance separately in the
minimum volume of ethanol (95 per cent) evaporate to dryness
on a water-bath and record the spectra again using the residues.
B. To about 40 mg add 2 ml of sulphuric acid and heat gently
until white fumes are evolved. Add nitric acid dropwise,
continue the heating until the solution is almost colourless
and cool. Add 2 ml of water, heat until white fumes are again
evolved, cool, add 10 ml of water and neutralise to red litmus
paper with dilute ammonia solution. The solution complies
with reaction A of sodium salts and reaction B ofphosphates
(2.3.1).
1955
PREDNISOLONE SODillM PHOSPHATE
IP 2010
Tests
pH (2.4.24). 7.5 to 10.5 determined in 1.0 per cent w/v solution.

in a 1.0 per cent w/v solution in a mixture of 9 volumes of
phosphate bufferpH 7.0 and 1 volume of carbon dioxide-free
water.
than half the area of the principal peak in the chromatogram

obtained with reference solution (b) (1 per cent); the sum of
the areas of all the peaks other than the principal peak is not
greater than 1.5 times the area of the principal peak in the
chromatogram obtained with reference solution (b) (3 per
cent). Ignore any peak due to the solvent and any peak with
an area less than 0.025 times the area of the principal peak in
Inorganic phosphate. Not more than 1.0 per cent ofphosphate,
(2.4.14).

mobile phase.
Reference solution (a). Dissolve 25 mg of prednisolone
sodium phosphate RS and 25 mg of prednisolone RS in the
mobile phase and dilute to 25.0 ml with the same solvent.
Dilute 1.0 ml ofthe solution to 25.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of the test solution to
- a stainless steel column 15 cm x 4.6 IDm, packed with
microparticles (5 /lm),
- mobile phase: mix 1.36 g of potassium dihydrogen
phosphate and 0.6 g of hexylamine and allow to stand
for 10 minutes and dissolve in 185 ml of water, add 65 ml
of acetonitrile,
Inject reference solution (b). Adjust the sensitivity of the
system so that the height of the principal peak in the
chromatogram obtained with reference solution (b) is 70 per
cent to 90 per cent ofthe full scale of the recorder.
Equilibrate the column with the mobile phase for about 30
minutes.
prednisolone sodium phosphate, about 6.5 minutes and
prednisolone, about 8.5 minutes. The test is not valid unless
the resolution between the peaks due to prednisolone sodium
phosphate and prednisolone is at least 4.5; ifthis resolution is
not achieved, increase the concentration. of acetonitrile or
increase the concentration of water in the mobile phase.
Inject separately the test solution and reference solution (b).
Continue the chromatography for three times the retention
time ofthe principal peak. III the chromatogram obtained with
the test solution, the area of any peak other than that from the
principal peak is not greater than the area ofthe principal peak
in the chromatogram obtained with reference solution (b) (2
per cent) and not more than one such peak has an area greater
P04
Dissolve 50 mg in sufficient water to produce 100 ml. To 10 ml
ofthe resulting solution add 5 ml of molybdovanadic reagent,
mix and allow to stand for 5 minutes. Any yellow colour in the
solution is not more intense than that produced in a standard
prepared at the same time in the same manner using 10 ml of
phosphate standard solution (5 ppm PO,J.

1.0 g.

water to produce 100.0 ml and mix. Dilute 5.0 ml ofthe resulting
solution to 250.0 ml with water. Measure the absorbance at
CZJHz7NazOsP taking 312 as the specific absorbance at 247 nm.

Injection
Prednisolone Sodium Phosphate Injection is a sterile solution
of Prednisolone Sodium Phosphate in Water for Injections.
Prednisolone Sodium Phosphate Injection contains not less
stated amount of prednisolone phosphate, CZIHz90SP.

Description. A clear, colourless liquid.
Identification
A. In the Assay, the chromatogram obtained with the test
solution corresponds to the peak due to prednisolone sodium
phosphate in the chromatogram obtained with reference
solution (a).
B. To a volume containing 0.2 mg of Prednisolone Sodium
Phosphate slowly add 1 ml of sulphuric acid. and allow to
stand for 2 minutes. A deep red colour is produced.
Tests
pH (2.4.24).7.0 to 8.0.
1956
IP 2010
PREDNISONE

per mg of prednisolone phosphate.
Category. Adrenocortical steroid.

Dose. 5 to 60 mg daily, in divided doses.
odourless.

injection to obtain a solution containing 0.001 per cent w/v of
prednisolone phosphate.
Reference solution (a). Weigh accurately about 10 mg of
prednisolone sodium phosphate RS, dissolve in sufficient
water to produce 100.0 ml (solution A) and dilute 10.0 ml of
the solution to 100.0 ml with watel:
Reference solution (b). Add 10 ml of a 0.01 per cent w/v
solution of betamethasone sodium phosphate RS in water to
10 ml of solution A and dilute to 100 ml with water.
microparticles (l0 /lm) (such as Spherisorb ODS 1),
55 volumes of citro-phosphate buffer pH 5.0,
resolution between the peaks due to betamethasone sodium
phosphate and prednisolone sodium phosphate is at least 2.5.
Inject alternatively the test solution and reference solution (a).
Calculate the content ofCzIHz90sP in the injection.
Storage. Store protected from light, in a single-dose or in multidose containers.
Prednisone
Identification
Tests A and B may be omitted iftests C and D are carried out.
Tests C and D may be omitted if tests A and B are carried out.

Compare the spectrum with that obtained with prednisone RS
or with the reference spectrum of prednisone.

Reference solution (a). Dissolve 25 mg of prednisone RS in
Place the dry plate in a tame containing a shallow layer of the
top, remove the plate from the tame and allow the solvent to
was done.
compact spot.
Prednisone is 17a.,21-dihydroxypregna-I,4-diene-3,11,20trione.
C. Dissolve 2 mg in 2 ml of sulphuric acid and allow to stand

for 5 minutes; an orange colour is produced within 5 minutes,
which exhibits a blue fluorescence in ultraviolet light at 365
om. Pour the solution into 10 ml of water; the colour changes
first to yellow and then fades gradually but the blue
fluorescence in ultraviolet light remains.
Prednisone contains not less than 96.0 per cent and not more
than 104.0 per cent ofCzlHz60s, calculated on the dried basis.
D. Dissolve 1 mg in 1 ml of ethanol (95 per cent), evaporate to

dryness at a pressure not exceeding 0.7 kPa, add 5 ml of 1 M
o
CZIHz60S
Mol. Wt. 358.4
1957
PREDNISONE
IP 2010
sodium hydroxide and heat at 70 for 30 minutes; not more

than a slight yellow colour is produced (distinction from
cortisone acetate).
Tests
Specific optical rotation (204.22). +167 to +175, determined
Light absorption (204.7). Absorbance ofa 0.001 per cent w/v
solution in methanol at the maximum at about 240 om, 0040 to
0043; the ratio of the absorbance at the maximum at about
240 nm to that at about 263 om, 1.85 to 2.05.
(2.4.14).

examination in methanol and dilute to 10 ml with the same
solvent.
Reference solution (a). Dissolve 2.0 mg of prednisolone RS
and 2.0 mg ofprednisone RS in methanol and dilute to 100 ml
with the same solvent.
octadecylsilyl silica gel (5 /-lm),
mobile phase: A. a mixture of 100 ml of acetonitrile,
200 m1 of methanol and 650 ml of water, allowed to
equilibrate, diluted to 1000 m1 with water and mixed again,
B. acetonitrile,
-flow rate. 2.5 ml per minute,
below,
injection volume. 20 /-l1.
Time
Mobile
Mobile
Comment
Phase A
PhaseB
(min) (per cent v/v) (per cent v/v)
Isocratic
o
100
o
begin linear
o
25
100
gradient
40
end chromatogram,
60
40
change to 100B
being treatment
41
o
100
withB
100
end treatment,
46
o
return to 100A
begin equilibration
47
100
o
with A
end equilibration,
100
o
52
begin next
chromatogram
Equilibrate the column with the mobile phaseB for at least

30 minutes and then with mobile phase A foTS minutes: For
subsequent chromatograms, use the conditions described from
40.0 to 52.0 minutes.
solution (b) is not less than 50 per centofthe full scale of the
recorder.
prednisone, about 19 minutes and prednisolone about
23 minutes. The test is not valid unless the resolution between
the peaks corresponding to prednisone and prednisolone is
at least 2.7. Ifnecessary, adjust the concentration ofacetonitrile
in mobile phase A.
Inject separately methanol as a blank, the test solution and
reference solution (b). In the chromatogramobtained with the
test solution: the area of any peak other than the principal
peak is not greater than 0.25 times the area of the principal
(b) (0.25 per cent); the sum ofthe areas of all the peaks, apart
from the principal peak, is not greater than 0.75 times the area
reference solution (b) (0.75 per cent). Ignore any peak due to
the blank run and any peak with an area less than 0.05 times
the area ofthe principal peak in the chromatogram obtained
Loss on drying (2,4.19). Not more than 1.0 percent, determined
ethanol to produce 100.0 m1. Dilute 2.0 ml of this solution to
100.0 ml with ethanol. Measure the absorbance ofthe resulting
solution at the maximum at about 238 om. Calculate the content
of C Z1 HZ60 Staking 425 as the specific absorbance at 238 om.
Prednisone Tablets
Prednisone Tablets contain not less than 90.0 per cent and
prednisone, CZIHz60S'
Identification
Shake a quantity ofthe powdered tablets containing 30 mg of
Prednisone with 10 m1 of chloroform, filter and evaporate the
1958
IP 2010
PREDNISONE TABLETS

tests.
Compare the spectrum with that obtained with prednisone RS
or with the reference spectrum of prednisone.
octadecylsilane bonded to poroussilica(5 Ilm),
mobile phase: A. a mixture of 100 ml of acetonitrile,
200 ml of methanol and 650 ml of water, allowed to
equilibrate, diluted to 1000 ml with water and mixed again,
B. acetonitrile,


below,

solvent mixture.

Comment
Mobile
Mobile
Phase A
Phase B
(min) (per cent v/v) (per cent v/v)
Time
Reference solution (a). Dissolve 25 mg of prednisone RS in

was done.
compact spot.
Tests
(2.4.14).
containing 25 mg of Prednisone with 10 ml of methanol for
10 minutes, mix with the aid of ultrasound for 2 minutes and
filter the extract (Whatman GFIF is suitable).
Reference solution (a). Dissolve 2.0 mg of prednisolone RS
and 2.0 mg ofprednisone RS in methanol and dilute to 100 ml
0
25
100
100
0
0
Isocratic,
begin linear
gradient
40
40
60
end chromatogram,
change to 100B
41
100
begin treatment
withB
46
100
end treatment,
return to 100A
47
100
begin equilibration
with A
52
100
end equilibration,
begin next
chromatogram
Equilibrate the column with the mobile phase B for at least

30 minutes and then with mobile phase A for 5 minutes. For
subsequent chromatograms use the conditions described from
40.0 to 52.0 minutes.
recorder.
prednisone, about 19 minutes and prednisolone about
23 minutes. The test is not valid unless the resolution between
the peaks corresponding to prednisone and prednisolone is
at least 2.7. Ifnecessary, adjust the concentration ofacetonitrile
in mobile phase A.
Inject separately methanol as a blank, the test solution and
reference solution (b). In the chromatogram obtained with the
test solution: the area of any peak other than the principal
1959
PREDNISONE TABLETS
IP 2010
peak is not greater than 0.25 times the area of the principal
(b) (0.25 per cent); the sum ofthe areas of all the peaks, apart
from the principal peak, is not greater than 0.75 times the area
reference solution (b) (0.75 per cent). Ignore any peak due to
the blank run and any peak with an area less than 0.05 times
Tablets.
Powder one tablet, add 50 ml of ethanol (95 per cent), shake
for 30 minutes, add sufficient ethanol (95 per cent) to produce
100.0 ml. Centrifuge and pipette a suitable volume of the
supernatant liquid equivalent to 0.5 mg of Prednisone and
dilute to 50.0 ml with ethanol (95 per cent). Measure the
240 nm (2.4.7). Calculate the content ofCzIHz60s taking 415 as
specific absorbance at 240 om.
standard solution and dilute to 50.0 ml with a 50 per cent v/v

solutIon of methanol.
octadecylsilane bonded to porous silica (3 to 10 J..Lm),
- mobile phase: a suitable filtered mixture of688 volumes
of water, 250 volumes ofperoxide-free tetrahydrofuran
and 62 volumes of methanol such that at a flow rate of
1 ml per minute the retention times of prednisone and
acetanilide are about 8 and 6 minutes respectively,
- injection volume. 10 J..Ll.
Inject the reference solution. Adjust the operating parameters
such that the peak obtained is about 50 per cent of the full
scale. The relative standard deviation for replicate injections
is not more than 2.0 per cent and the resolution between
prednisone and the internal standard is not less than 3.0.
Inject the test solution and the reference solution. Calculate
the content of CZIHz60S in the tablets.
Apparatus No.1,
Pregabalin

the absorbance ofthe filtrate at the maximum at about 240 nm
(2.4.7). Calculate the content ofCzIHz60s in the medium from
concentration of prednisone RS.
D. Not less than 70 per cent ofthe stated amount OfCZIHz60s.
CH
H 3C
NHz
3 ~
~COOH
CgH 17NOz
Mol. Wt. 159.2
Pregabalin is (S)-4-amino-3-(2-methylpropyl)butyric acid.

Internal standard solution. Dissolve an accurately weighed

quantity of acetanilide in a 50 per cent v/v solution of
methanol to obtain a solution having a known concentration
of0.1 1 mg per ml.
Test solution. Weigh and powder 20 tablets. To an accurately
weighed quantity of the powder containing about 20 mg of
Prednisone, aM 5mlofwater, mixWltl1 the aId ofuitras()und
for one minute, add 50 ml of methanol and mix with the aid of
ultrasound for I minute. Dilute with water to 100.0 rnl and mix.
To 5.0 m1 ofthis solution add 5.0 ml ofinternal standard solution
and dilute to 50.0 ml with a 50 per cent v/v solution of methanol
and mix. Filter through a 5 J..Lm filter and discard the fIrst 20 ml
ofthe filtrate.
Pregabalin contains not less than 98.0 per cent and not more
than 102.0 per cent ofC gH 17NOz, calculated on the dried basis.
Identification
Compare the spectrum with that obtained with pregabalinR8
or with the reference spectrum of pregabalin.
Tests
Specific optical rotation (2.4.22). +10 to +12.0, determined
on 1.0 per cent w/v solution.
--:::--:;;----'---'--;-~~~;--;----'---'--__,____--'-______;___.__,..___._--'-___;--'-......,.-Enantiomeric-purity.,...Determine-by~liquidc0hr0mat0graphy--'---'-
Reference solution. Weigh accurately a suitable quantity of

prednisone RS and dissolve in a 50 per cent v/v solution of
methanol to obtain a solution having a concentration of about
0.2 mg per ml. To 5.0 rnl ofthis solution add 5.0 ml ofinternal
(2.4.14).
Merfey's reagent. Dissolve about 0.1502 g ofmerfey's reagent
in 50 ml of acetone.
1960
PREGABALIN CAPSULES
IP 2010
NOTE--8tore the reagent at 2 to 8.

Test solution (aJ. Dissolve about 100 mg of the substance
under examination in 100.0 ml of water.
Test solution (b). To 2.0 ml of test solution (a), add 0.5 ml of
merfey's reagent and 0.5 ml of 1 M sodium bicarbonate in the
test tube. Heat at 40 for 60 minutes. Allow to equilibrate to
room temperature.
as Hypersil BDS C18),
prepared by diluting about 7.18 ml of triethylamine to
1000 ml water, adjusted to pH 3.0 with orthophosphoric
acid and 38 volumes of acetonitrile,
The retention time for S- Pregabalin is about 9 minutes and RPregabalin is about 12 minutes.
Inject test solution (b). The area of peak due to R-pregabalin
is not more than 0.5 per cent the area of the S-pregabalin.
(2.4.14).
acetonitrile.
with the solvent mixture. Further dilute 5.0 ml ofthis solution

relative standard deviation of replicate injections is not more
than 5.0 per cent.
secondary peak is not more than the area ofprincipal peak in
the chromatogram obtained with reference solution (0.5 per
more than twice the area of the principal peak in the
chromatogram obtained with reference solution (1.0 per cent).
Heavy Metals (2.3.13). 1 g complies with the limit test for
Loss on drying (2.4.19). Not more than 0.5 per cent determined
Reference solution. A 0.2 per cent w/v solution ofpregabalin
as Inertsil ODS-3),
deviation ofreplicate injections is not more than 2.0 per cent.
Calculate the content of CgH 17N02
Pregabalin Capsules
Pregabalin Capsules contain not less than 90.0 per cent and
pregabalin, CgH 17N0 2
Usual strengths. 25 mg; 50 mg; 75 mg; 100 mg; l50mg; 225
mg;300mg.
Identification
with the test solution corresponds to the principal peak in the
Tests
Dissolution (2.5.2);
Apparatus No.1,
Medium. 900 ml of O. 05 M hydrochloric acid,
Test solution. Use the filtrate and dilute, if necessary, with the
dissolution medium.
1961
IP 2010
PREGABALIN CAPSULES

pregabalin RS in the dissolution medium.
Use chromatographic system as described under Assay using
injection volume 100 Ill.
theoretical plates of the principal peak is not less than 2000,
Calculate the content ofCsH 17NO z.
D. Not less than 70 per cent ofthe stated amount ofCsH 17NO z.
(2.4.14).
Solvent mixture. Dissolve 1.2 g of potassium dihydrogen

phosphate in 1000 ml of water, adjust the pH to 6.9 with dilute
potassium hydroxide.
Test solution. Mix the content of 10 capsules. Weigh and
disperse a quantity containing about 15 mg of Pregabalin, in
about 25 ml ofthe solvent mixture, sonicate for 30 minutes and
dilute to 100.0 ml with the solvent mixture.
pregabalin RS in the solvent mixture.
Reference solution (b). Dissolve 4.5 mg of lactam RS in 10.0
m1 ofthe solvent mixture.
Reference solution (c). Dissolve 750 mg ofpregabalin RS in
30 m1 ofsolvent mixture. Add about 5 ml ofreference solution
(b) and dilute to 50.0 ml with the solvent mixture.
octadecylsilane bonded to porous silica (5 Ilin) (Such
as Inertsil ODS-3V),
mobile phase: A. a mixture of90 volumes ofthe solvent
mixture and 10 volumes of acetonitrile,
B. acetonitrile,
below,
Time
(in min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
100
100
o
o
25
35
65
30
100
40
100
o
o
theoretical plates is not less than 3000, the tailing factor is not
more than 2.0 and the relative standard deviation of replicate
Inject reference solution (a) and the test solution. The relative
retention time with reference to lactam peak for lactose
conjugate impurity is about 0.78. In the chromatogram
obtained with the test solution, the area of the peak due to
lactam multiplied with relative response factor, 0.07 is not more
obtained with reference solution (a) (0.5 per cent), the area of
lactose conjugate impurity peak multiplied with relative
response factor, 0.05 is not more than 1.5 times the area ofthe
solution (a) (1.5 per cent). The area of any other secondary
chromatogram obtained with reference solution (a)'(1.0 per
more than 2.5 times the area of the principal peak in the
cent). Ignore any peak with an area less than 0.05 times ofthe
area of principal peak in the chromatogram obtained with
Test solution. Mix the contents of 20 capsules. Disperse a

quantity of powder containing about 200 mg of Pregabalin
with 35 ml of the mobile phase, sonicate for 10 minutes and
dilute to 50 ml with the mobile phase.
Reference solution. A 0.4 per cent w/v solution ofpregabalin
as Inertsil ODS-3),
mobile phase: a mixture of 92 volumes of solution
containing 2.72 g of potassium dihydrogen
orthophosphate in 900 ml of water, add 2 ml of
triethylamine and adjusted to pH 6.0 with
orthophosphoric acid, 5 volumes of methanol and 3
theoretical plates are not less than 2000, tailing factor is not
more than 2.0 and the relative standard deviation of replicate
1962
IP 2010
PRIMAQUINE PHOSPHATE
Calculate the content of CgH 17NO z in the capsules.

exceeding 30.
equivalent amount ofPregabalin.
Foreign matter. Examined under a microscope using a mixture

of equal volumes ofglycerol and water, not more than traces
of matter other than starch granules are present.
Loss on drying (2.4.19). NOhum'ethan 15.0 percent, determined
on 1.0 g by drying in an oven at 130 for 90 minutes.
Microbial contamination (2.2.9). Total viable aerobic count is
not more than 103bacteria and 10z fungi per gram, determined
by plate count. It complies with the test for Escherichia coli.
Pregelatinised Starch is prepared from maize starch, potato
starch or rice starch by mechanical processing in the presence
of water, with or without heat, to rupture all or part of the
starch granules and subsequent drying. It contains no added
substances but it may be modified to render it compressible
and to improve its flow characteristics.
Labelling. The label states the type of starch used as starting

material.
Description. A white or yellowish-white powder.
Identification
A. Microscopic- In a mixture of equal volumes of glycerol
and water, it presents irregular, translucent, white or yellowishwhite flakes or pieces with an uneven surface. Under polarised
light (between crossed nicol prisms), starch granules with a
distinct black cross intersecting at the hilum may be seen.
E. Disperse 0.5 g in2 ml ofwater without heating, add 0.05 ml

of iodine solution; a reddish-violet to blue colour is produced.
Tests
pH (2.4.24). 4.5 to 7.0, determined in 3.0 percent w/v solution
Oxidising substances. Transfer 4.0 g to a glass-stoppered,
125 ml conical flask and add 50.0 ml of a mixture of equal
volumes of water and methanol. Insert the stopper and swirl
for 5 minutes. Transfer to a glass-stoppered 50 ml centrifuge
tube and centrifuge. Transfer 30.0 ml ofthe clear supernatant
liquid to a glass-stoppered 125-ml conical flask. Add I mlof
glacial acetic acid and 0.5 g to 1.0 g of potassium iodide.
Insert the stopper, swirl, and allow to stand for 30 minutes in
the dark. Add I ml ofstarch solution and titrate with 0.002 M
sodium thiosulphate until the starch-iodine colour disappears.
Carry out a blank titration. Not more than 1.4 ml of O. 002 M
sodium thiosulphate is required (0.002 per cent, calculated as
HzOz).
I ml of0.002 M sodium thiosulphate is equivalent to 0.034 mg
of oxidising substances, calculated as HzO z.
Sulphur dioxide (2.3.40). Not more than 50 ppm.
Iron (2.3.14). Dissolve the residue obtained in the test for
Sulphated ash in 20 ml ofdilute hydrochloric acid, filter. 10 ml
ofthe filtrate complies with the limit test for iron (20 ppm).
ClsHzIN30,2H3P04
Mol. Wt. 455.3
Primaquine Phosphate is (RS)-8-(4-amino-lmethylbutylamino)-6-methoxyquinoline diphosphate.

Primaquine Phosphate contains not less than 98.5 per cent
and not more than 101.5 per cent of ClsHzIN30,2H3P04,
Category. Antimalarial.
Dose. The equivalent of 15 mg ofprimaquine, once a day for
14 days.
Description. An orange, crystalline powder; odourless or
almost odourless.
Identification
Compare the spectrum with that obtained with primaquine
phosphate RS or with the reference spectrum of primaquine
phosphate.
E. When examined in the range 300 nm to 450 nm (2.4.7), a

0.015 per cent w/v solution in 0.01 Mhydrochloricacidshows
absorption maxima at about 332 nm and 415 nm; absorbance
at about 332 nm,aboutO.68 to 0.78 and at about 41 5 nm, 0.41 to
0.53. Dilute 5 ml of the solution to 50 ml with 0.01 M
hydrochloric acid. When examined in the range 215 urn to
1963
PRIMAQUINE PHOSPHATE
IP 2010
310 nm, the resulting solution shows absorption maxima at

about 225 urn, 265 urn and 282 urn; absorbances at the maxima
are 0.74 to 0.77, 0.50 to 0.53 and 0.50 to 0.52, respectively.
the plate with silica gel F254.
solution.
Test solution. DissolveO.2 g in 5 m1 of water, dilute to 10 ml
with methanol and dilute 1 volume ofthe resulting solution to
10 volumes with methanol (50 per cent).
Reference solution. Dissolve 20 mg ofprimaquinephosphate
RS in 5 ml of water, dilute to 10 ml with methanol.
Apply to the plate 5 j..Ll of each solution. After development,
D. Dissolve 50 mg in 5 ml of water, add 2 ml of 2 M sodium
hydroxide and shake with two quantities, each of 5 ml, of
chloroform. The aqueous layer, acidified by the addition of
nitric acid, gives reaction B of phosphates (2.3.1).
The test is not valid unless in the chromatogram obtained

with reference solution (c) there is a peak just before the
principal peak with an area of about 6 per cent of that of the
principal peak and the resolution between these peaks is not
less than 2.0.
Inject each solution and record the chromatograms for at least
twice the retention time ofprimaquine. The sum ofthe areas of
any secondary peaks in the chromatogram ob~ained with the
test solution is not greater than the area of the principal peak
in the chromatogram obtained with reference solution (a).
Ignore any peak the area of which is less than that of the
solution (b).
anhydrous glacial acetic acid with gentle heating. Titrate
ClsHzIN30,2H3P04.
Tests

(2.4.14).
Primaquine Tablets
Primaquine Phosphate Tablets
Test solution. Add 0.2 ml ofstrong ammonia solution to 1 ml

of a 1.0 per cent w/v solution of the substance under
examination, shake with 10 ml ofthe mobile phase and use the
clear, lower layer.
Primaquine Tablets contain not less than 90.0 per centand not
more than 110.0 per cent ofthe stated amount ofprimaquine,
ClsHz1N30. The tablets are coated.

Usual strength. The equivalent of2.5 mg of primaquine (13

mg of Primaquine Phosphate is approximately equivalent to
7.5 mg ofprimaquine).
Reference solution (b). Dilute 1 m1 ofthe test solution to 10m1

with the mobile phase and further dilute 1 ml ofthe resulting
solution to 50 ml with the same solvent.
Identification
Reference solution (c). Add 0.2 ml ofstrong ammonia solution

to 1 m1 of a 1.0 per cent w/v solution of the primaquine
phosphate RS, shake with 10 ml of the mobile phase and use
the clear, lower layer.
porous silica particles (10 j..Lm),
- mobile phase: a mixture of 45 volumes of chloroform,
45 volumes of hexane, 10 volumes of methanol and
0.1 volume of strong ammonia solution,
- injection volume. 20 j..L1.
A. Dissolve a quantity of the powdered tablets containing

60 mg ofprimaquine in a mixture of 10 m1 ofwater and 2 m1 of
2 M sodillll1 hydroxide and extractwith two quantities, each
of 20 ml, of chloroform. Wash the chloroform extracts with
water, dry over anhydrous sodium sulphate, evaporate to
dryness and dissolve the residue in 2 ml of chloroform. The
residue complies with the following test.
Compare the spectrum with that obtained with primaquine
phosphate RS or with the reference spectrum of primaquine.
of primaquine with 10 ml of water and filter. To 2 m1 of the
filtrate add 3 ml ofwater and 1 ml ofa 5 percentw/v solution
1964
IP 2010
PROBENECID
of eerie ammonium sulphate in 2 M nitric acid; a deep violet

colour is produced immediately.
Tests
Tablets.
Transfer one tablet into a suitable container, add 5 ml of
hydrochloric acid, disperse the tabletin about 25 g ofcrushed
ice and add sufficient water to produce 50.0 ml. Carry out the
nitrite titration (2.3 .31), using 0.01 M sodium nitrite as titrant.
1 ml of 0.01 M sodium nitrite is equivalent to 0.002594 g of
ClsH21N30.
quantity ofthe powder containing about 0.15 g ofprimaquine,
dissolve in 20 ml of water, add 5 ml of2 M sodium hydroxide
and extract with four quantities, each of25 ml of chloroform.
Combine the chloroform extracts and evaporate to a volume
of about 10 ml. Add 40 ml of anhydrous glacial acetic acid,
Titrate with 0.1 M perchloric acid, determining the end point
ClsH21N30.
equivalent amount ofprimaquine.
Identification
Compare the spectrum with that obtained with probenecidRS
or with the reference spectrum of probenecid.
0.001 per cent w/v solution in a mixture of9 volumes ofethanol
(95 per cent) and 1 volume of 0.1 M hydrochloric acid shows
absorption maxima at about 223 urn and 248 nm; absorbance
at about 248 nm is about 0.33.
C. Dissolve 0.2 g in about 0.6 ml of2 M ammonia and add 3 ml
of 1.7 per cent w/v solution of silver nitrate; a white precipitate
is produced which is soluble in an excess of dilute ammonia
solution.
Tests
Appearance of solution. A 10.0 per cent w/v solution i.n 2 M
sodium hydroxide is clear (2.4.1), and not more intensely
Acidity. Add 2.0 g to 100 ml of water, heat on a water-bath for
30 minutes, cool, filter and dilute with water to 100.0 mI. Titrate
50.0 ml of the solution with 0.1 M sodium hydroxide using
phenolphthalein solution as indicator. Not more than 0.5 ml
of 0.1 M sodium hydroxide is required to change the colour of
the solution.
Mobile phase. A mixture of55 volumes of toluene, 20 volumes

of di-isopropyl ether, 15 volumes of chloroform and
10 volumes of glacial acetic acid.
Probenecid

Cl3Hl9N04S
Mol. Wt. 285.4
Probenecid is 4-(dipropylsulphamoyl)benzoic acid.

Probenecid contains not less than 99.0 per cent and not more
than 101.0 per cent of C 13H I9N04S, calculated on the dried
basis.
Category. Uricosuric agent and for delaying renal excretion of
penicillins and cephalosporins.
Dose. 250 mg to 500 mg twice daily.

ethanol (95 per cent), shaking well and heating gently if
1965
PROBENECID TABLETS
IP 2010
necessary. Cool and titrate with 0.1 M sodium hydroxide,

using bromothymol blue solution as indicator. Carry out a
blank titration.
CI3HI9N04S.

Withdraw a suitable volume of the medium and filter, Dilute
4.0 ml ofthe filtrate to 100.0 ml with 0.1 M sodium hydroxide.
maximum at about 244 nm (2.4.7). Similarly measure the
absorbance of a solution of a known concentration of
probenecid RS. Calculate the content of C 13H I90 4S.
Probenecid Tablets

CI3HI9N03S,
Probenecid Tablets contain not less than 95.0 per cent and
probenecid, CI3HI9N04S.
Identification
A.Triturate a quantity of the powdered tablets containing 0.5
g of Probenecid with ethanol (95 per cent), filter and
concentrate the filtrate by evaporation on a water-bath. Cool,
filter and recrystallise the residue from ethanol (50 per cent).
Compare the spectrum with that obtained with probenecidRS
or with the reference spectrum of probenecid.

quantity of the powder containing about 0.2 g ofProbencid,
add 200 ml of ethanol (95 per cent) and 5 ml of 1 M
hydrochloric acid, heat on a water-bath at 70 0 for 30 minutes,
shaking occasionally. Cool, add sufficient ethanol (95 per
cent) to produce 250.0 ml and filter. To 5.0 ml ofthe filtrate add
5 ml of 0.1 M hydrochloric acid, dilute to 250.0 ml with ethanol
(95 per cent) and measure the absorbance of the resulting
content of C 13H I9N04S taking 332 as specific absorbance at
248nm.
C. The residue obtained in test A melts at about 1990 (2.4.21).

Tests
H2N
(CH
~JlN~N,--/,CH3
~ H
C 13Hz1 N30,HCl
.HCI
Mol. Wt. 271.8

of di-isopropyl ether, 15 volumes of chloroform and
Procainamide Hydrochloride is 4-amino-N-[2-(diethylamino)

ethyl)benzamide hydrochloride.

containing 0.2 g ofProbenecid with 20 ml of acetone, filter and
use the filtrate.
Procainamide Hydrochloride contains not less than 98.0 per

cent and not more than 101.0 per cent of C 13 Hz1 N 30,HC1,

with acetone.
Category. Antiarrhythmic.

Apparatus. No.1,
Dose. Orally, 500 mg to 1.5 g daily, in divided doses; by slow

intravenous injection
Description. A white or very slightly yellow, crystalline
powder; hygroscopic.
Identification
1966
IP 2010
PROCAINAMIDE INJECTION

Compare the spectrum with that obtained with procainamide
hyc/rochloride RS or with the reference spectrum of
procainamide hydrochloride.

273 nm, 0.58 to 0.61.
C. Dilute I ml of a solution, prepared by dissolving 2.5 g in

sufficient carbon dioxide-free water to produce 25 ml, to 2 ml
with water. 1 ml ofthis solution gives the reaction ofprimary
aromatic amines (2.3.1).
D. A 2 per cent w/v solution gives reaction A of chlorides
(2.3.1).
Tests
pH (2.4.24). 5.6to 6.3, deterrnined in a 10.0 per cent w/v solution.


30 volumes of water and 15 volumes of glacial acetic acid.
Procainamide Injection
Procainamide Hydrochloride Injection
Procainamide Injection is a sterile solution of Procainamide
Hydrochloride in Water for Injections. It may contain Sodium
Metabisulphite as a stabilising agent.
Procainamide Injection contains not less than 95.0 per cent
procainamide hydrochloride, C 13Hz1 N 30, HC1.
Usual strength. 100 mg per m1.
Identification
A. Dilute a volume containing 0.25 g of Procainamide

Hydrochloride to 25 ml with water, make alkaline with 5 M
sodium hydroxide and extract with two quantities, each of
5 ml, of chloroform. Filter the combined extracts through
anhydrous sodium sulphate, evaporate the filtrate to dryness
using a rotatory evaporator and dissolve the residue in 5 ml of
chloroform.
procainamide.
B. Dilute a suitable volume of the injection with water to
produce a solution containing 0.0005 per cent w/v of
Procainamide Hydrochloride. Absorbance of the resulting
solution at about 280 nm, about 0.30 (2.4.7).
Tests
pH (2.4.24). 4.0 to 5.5.

Test solution. Dilute a volume ofthe injection containing 0.1 g

ofProcainamide Hydrochloride to 5 ml with methanol.


water and 10 ml of hydrochloric acid and carry out the nitrite
titration (2.3.31).

dry the plate in air and examine in ultraviolet light at 254nm.
I ml of 0.1 M sodium nitrite is equivalent to 0.02718 g of

C13Hz1N30, HCL

1967
PROCAINAMIDE TABLETS
IP 2010
Assay. To a volume containing about 0.25 g of Procainamide

Hydrochloride add 45 ml of 6 M hydrochloric acid and boil
for 1 minute. Carry out the nitrite titration (2.3.31).

C13H21N30, HCl.

quantity ofthe powder containing about 0.25 g ofProcainamide
Hydrochloride, add 100 ml of 6 M hydrochloric acid, shake
for 15 minutes and boil for 1 minute. Carry out the nitrite
titration (2.3.31).

C13H21N30, HCl.
Procainamide Hydrochloride Tablets
Procainamide Tablets contain not less than 95.0 per cent and
procainamide hydrochloride, C 13H2J N 30,HCl.
, Hel
Identification
A. Shake a quantity ofthe powdered tablets containing 0.25 g
of Procainamide Hydrochloride with 25 ml of water, make
alkaline with 5 M sodium hydroxide and extract with two
quantities, each of 5 ml, of chloroform. Filter the combined
extracts through anhydrous sodium sulphate, evaporate the
filtrate to dryness using a rotatory evaporator and dissolve
procainamide.
B. Triturate a quantity ofthe powdered tablets containing 1 g
ofProcainamide Hydrochloride with 10 ml of water and filter.
The filtrate gives the reactions of chlorides (2.3.1).
C13H20N202,HCl
Mol. Wt. 272.8
Procaine Hydrochloride is 2-(diethylamino)ethy14aminobenzoate hydrochloride.

Procaine Hydrochloride contains not less than 99.0 per cent
and not more than 101.0 per cent ofC13H2oN202,HCl, calculated
on the dried basis.
Category. Local anaesthetic.
Dose. To be adjusted according to the site of operation and
response of the patient.
odourless.
Identification
Tests
Mobile phase. A mixture of 60 volumes of I-butanol,

containing 0.4 g ofProcainamide Hydrochloride with 20 ml of
methanol (90 per cent) for 15 minutes and filter.
dry the plate in air and examine in ultraviolet light at 254 nrn.

out.
Compare the spectrum with that obtained with procaine
hydrochloride RS or with the reference spectrum of procaine
hydrochloride.
B. To 0.2 ml ofa 5 per cent w/v solution add 2 ml of water and
0.5 ml of 1 M sulphuric acid, shake and add 1 ml of a 0.1 per
cent w/v solution of potassium permanganate; the colour is
immediately discharged.
C. To about5 mg add 0.5 ml ofjUming nitric acid, evaporate to
dryness on a water-bath, cool, dissolve the residue in 5 ml of
acetone and add 1 ml of 0.1 M ethanolic potassium
hydroxide; only a brownish red colour develops.
1968
IP 2010
PROCAINE AND ADRENALINE INJECTION
D. Dilute 1 mlofa5 percentw/v solution to 100ml with water;

2 ml of the solution gives the reaction of primary aromatic
arnines (2.3.1).
Procaine and Adrenaline Injection contains not less than

1.9 per cent and not more than 2.1 per cent w/v of procaine
hydrochloride, C13HzoNzOz, HCI and the equivalent ofnot less
than 0.00175 percent and not more than 0.00225 per cent w/v
ofadrenaline, C9H I3N0 3.
Tests

Identification
pH (2.4.24). 5.0to 6.5, determined in a 2.0 per cent w/v solution.

Mobile phase. A mixture of 80 volumes of dibutyl ether,

16 volumes of n-hexane and 4 volumes of glacial acetic acid.
Test solution. A 10 per cent w/v solution of the substance
under examination in water.
4-aminobenzoic acid in water.
light at 254 nm. Any secbndary spot in the chromatogram
The principal spot remains on the line of application.
Assay. Weigh accurately about 0.3 g, dissolve in 50 ml of 2 M
hydrochloric acid, add 3 g ofpotassium bromide, cool in ice
and titrate slowly with 0.1 M sodium nitrite, stirring constantly.
Determine the end point potentiometrically (2.4.25).
C13HzoNzOz, HCl.
A. To 5 ml add 5 ml of water and 10 ml ofpicric acidsolution,

shake gently and set aside for 1 hour; the crystalline precipitate,
after washing with water and drying at 100, melts at about
134 (2.4.21).
B. To 5 ml add 1 m1 of hydrochloric acid, cool to 0, add 5 ml of

sodium nitrite solution and pour the mixture into 2 ml of
2-naphthol solution containing 1 g of sodium acetate; an
orange-red colour is produced.
C. To 10 ml add 4 ml of disodium hydrogen phosphate solution
and sufficient 0.1 M iodine to produce a distinct brown colour.
Add 0.1 M sodium thiosulphate to remove the excess of
iodine; a pink colour is produced.
Tests
pH (2.4.24). 3.0 to 5.5.
Assay. For procaine hydrochloride - To 10.0 ml of the
injection add 0.5 g ofsodium carbonate and extract with three
quantities, each of20 ml, ofa mixture of 1 volume of2-propanol
and 3 volumes of chloroform until complete extraction of
procaine is effected. Shake the combined extracts with 5 ml of
water, wash the water with the solvent mixture and add the
washing to the combined extracts. Shake the combined extracts
and washings with 10.0 ml of 0.1 M hydrochloric acid,
separate the acid layer, wash the combined extracts and
washings with 5 ml of water, add the aqueous extract to the
separated acid layer and titrate with 0.1 M sodium hydroxide,
using methyl red-methylene blue solution as indicator.
1 m1 of 0.1 M hydrochloric acid is equivalent to 0.02728 g of
CI3HzoNzOz,HCl.
Procaine and Adrenaline Injection

Procaine Hydrochloride and Adrenaline Bitartrate
Injection; Procaine Hydrochloride and Epinephrine
Bitartrate Injection
Procaine and Adrenaline Injection is a sterile solution of
Procaine Hydrochloride and Adrenaline Bitartrate in Water
for Injections.
For adrenaline
To 10.0 m1 of the injection add 20 mg of
sodium metabisulphite, 0.1 ml of ferrous sulphate-citrate
solution, 1 ml of glycine buffer solution and mix. Allow to
stand for 10 minutes, extract with 10 ml of ether, allow to
separate, reject the ether and 'measure- the absorbance of a
4-cm layer of the solution at about 540 nm (2.4.7). Calculate
the content of adrenaline, C9H 13N0 3, from a reference curve
prepared by treating suitable aliquots of a solution of
adrenaline bitartrate RS in the same manner.
1969
PROCAlNE PENICILLIN
IP 2010
1 mg of adrenaline bitartrate is equivalent to 0.0005497 g of

C9H I3N03.
Labelling. The label states the strength as "Procaine
Hydrochloride, 2 per cent w/v; Adrenaline, 0.002 per cent
w/v".
Assay. Determine the contents of benzyl penicillin and

procaine by liquid chromatography (2.4.14).
Test solution. Weigh accurately 70 mg ofthe substance under

examination and dissolve in 30 ml ofthe mobile phase with the
aid of ultrasound for about 2 minutes. Dilute to 50.0 ml with
the mobile phase.
benzyl penicillin potassium RS and 40 mg of procaine
hydrochloride RS and dissolve in 25 ml of the mobile phase
with the aid of ultrasound for about 2 minutes. Dilute to
Procaine Penicillin
CI3H20N202,CI6HlSN204S,H20
Mol. Wt. 588.7
Procaine Penicillin is 2-diethylaminoethyI4-aminobenzoate

(6R)-6-(2-phenylacetamido)penicillanate monohydrate.
Procaine Penicillin contains not less than 51 per cent and not
more than 59.6 per cent of penicillin G, calculated as
CI6HISN204S, and not less than 37.5 per cent and not more
than 43.0 per cent ofprocaine, C13H20N202, both calculated on
Dose. By intramuscular injection, 300 to 900 mg daily (300 mg
ofProcaine Penicillin is approximately equivalent to 200 mg of
benzylpenicillin).
Identification
Compare the spectrum with that obtained with procaine
penicillin RS or with the reference spectrum of procaine
penicillin.
chromatogram obtained with the reference solution (a).
C. A turbid solution of 0.1 gin 2 ml of 2 M hydrochloric acid
gives the reaction ofprimary aromatic amines (2.3.1).
Tests
pH (2.4.24).5.0 to 7.5, determined in a solution prepared by
shaking 50 mg in sufficient carbon dioxide-free water to
produce 15 ml until dissolution is complete.
Specific optical rotation (2.4.22). +165 to +180, determined
in a solution prepared by dissolving 0.25 g in sufficient of a
mixture of 3 volumes of acetone and 2 volumes of water to
produce 25 ml.
Reference solution (b). Mix 1 volume of a 0.24 per cent w/v

solution of phenoxymethylpenicillin potassium RS in the
mobile phase with 3 volumes of reference solution (a).
octadecylsilane bonded to porous silica (l0 /lm),
mobile phase: a mixture of 50 volumes of a solution
prepared by mixing 14 g of potassium dihydrogen
phosphate in 6.5 g of tetrabutylammonium hydroxide
(40 per cent) and adjusting the pH to 7.0 with 1 M
potassium hydroxide or dilute phosphoric acid,
25 volumes of acetonitrile and 25 volumes of water,
spectrophotometer set at ~35 om,
Inject reference solution (b). The resolution between benzyl
penicillin potassium and phenoxymethylpenicillin potassium
is not less than 2. The relative retention time ofprocaine with
reference to benzyl penicillin is about 2.2.
peak responses of the main peaks of benzyl penicillin and
procaine. Calculate the content of benzyl penicillin,
Cl6HlSN204S and the content of procaine, Cl3H20N202 using
the factor of 0.866 for converting the content of procaine
hydrochloride to that of procaine.
Procaine Penicillin intended for use in the manufacture of

Parenteral Preparations without afitrther procedure for the
removal ofbacterial endotoxins complies with the following
Bacterial endotoxins (2.2.3). Not more than 0.1 0 Endotoxin
Unit per mg.
Procaine Penicillin intended for use in the manufacture of

Parenteral Preparations without a further sterilisation
procedure complies with the follOWing additional
requirement.
1970
IP 2010
FORTIFIED PROCAINE PENICILLIN INJECTION
Storage. Store protected from moisture. If the material is

intended for use in the manufaGture ofparenteral preparations,
micro-organisms.
Labelling. The label states (1) where applicable, that the
contents are free from bacterial endotoxins; (2) where
applicable, that the contents are sterile.

C. Give the reaction of primary aromatic amines (2.3.1),
producing a bright, orange-red precipitate.
Tests
0.5g.
Fortified Procaine Penicillin Injection

Procaine Penicillin with Benzylpenicillin Injection
Fortified Procaine Penicillin Injection is a sterile mixture offive
parts of Procaine Penicillin and one part of Benzylpenicillin
Potassium or Benzylpenicillin Sodium, together with suitable
dispersing and buffering agents. It is filled in a sealed container.
The injection is constituted by suspending the contents of
the sealed container in the requisite amount of sterile Water
for Injections, immediately before use.
Storage. The constituted injection should be used within
24 hours (4 days ifa buffering agent is present) ofpreparation
when stored at a temperature not exceeding 20 or within
7 days (14 days if a buffering agent is present) when stored at
a temperature between 2 and 8.
Fortified Procaine Penicillin Injection contains a quantity of
total penicillins calculated as CI6H17NzNa04S and equivalent
to not less than 60.0 per cent and not more than 74.0 per cent
ofthe stated amounts ofprocaine penicillin and benzylpenicillin
potassium or benzylpenicillin sodium and a quantity of
procaine, CI3HzoNzOz, equivalent to not less than 36.0 per cent
procaine penicillin.
Usual strengths. Procaine Penicillin, 300 mg (300,000 Units)
and Benzylpenicillin Potassium or Benzylpenicillin Sodium 60
mg (100,000 Units), Procaine Penicillin 3 g (3,000,000 Units)
and Benzylpenicillin Potassium or Benzylpenicillin Sodium,
600 mg (1,000,000 Units).

contents of 10 containers equivalent to about 70 mg of
procaine penicillin and dissolve in 30 ml of the mobile phase
benzylpenicillin potassium RS and 40 mg of procaine
hydrochloride RS and dissolve in 25 ml of the mobile phase
Reference solution (b). Mix 1 volume of a 0.24 per cent w/v
solution of phenoxymethylpenicillin potassium RS in the
mobile phase with 3 volumes ofreference solution (a).
containing 1.4 per cent w/v of potassium dihydrogen
orthophosphate and 0.65 per cent w/v tetrabutyl
ammonium hydroxide adjusted to pH 7.0 with 1 M
potassium hydroxide or orthophosphoric acid,
benzylpenicillin potassium and phenoxymethylpenicillin
potassium is not less than 2. The relative retention time of
procaine with reference to benzylpenicillin is about 2.2.
Identification
peak responses of the main peaks of benzyl penicillin and
procaine. Calculate the total penicillin content as
benzylpenicillin, CI6HISNz04S and the content of procaine,
CI3HzoNzOz using the factor of0.866 for converting the content
of procaine hydrochloride to that of procaine.
A. Dissolve 10 mg in 10 ml of water and add 0.5 ml of neutral

red solution. Add sufficient 0.01 M sodium hydroxide to give
a permanent orange colour and then add 1 mlofpenicillinase
solution; a red colour is produced rapidly.
Labelling. The label states (1) the quantities of Procaine

Penicillin and Benzylpenicillin Potassium or Benzylpenicillin
Sodium contained in it; (2) the names ofany added dispersing
and buffering agents; (3) "for intramuscular injection only".
The contents of the sealed container comply with the tests

stated under Parenteral Preparations (Powders for
Injection) and with the following requirements.
1971
PROCHLORPERAZINE MALEATE
IP 2010
Prochlorperazine Maleate
Apply 4 f.!l of each solution.
C2oH24CIN3S,2CJ!404
D. Triturate 0.2 g with a mixture oB ml of water and 1 m1 of10

M sodium hydroxide and shake with three quantities, each of
5 ml, of ether. To 0.1 ml ofthe aqueous layer add a solution of
10 mg of resorcinol in 3 ml of sulphuric acid and heat on a
water-bath for 15 minutes; no colour develops. To the
remainder ofthe aqueous layer add 2 ml of bromine solution,
heat on a water-bath for 15 minutes, then heat to boiling and
cool. To 0.1 ml of the solution add a solution of 10 mg of
resorcinol in 3 ml of sulphuric acid and heat on a water-bath
for 15 minutes; a blue colour develops.
,[(COOH]
COOH
2
Mol. Wt. 606.1
Prochlorperazine Maleate is 2-chloro-1 0-[3-(4methylpiperazin-I-yl)propyl]phenothiazine di(hydrogen

maleate).
Tests
pH (2.4.24). 3.0 to 4.0, determined in a freshly prepared saturated
solution.
Prochlorperazine Maleate contains not less than 98.0 per cent

and not more than 101.0 per cent of C2oH24CIN3S, 2C 4H40 4,
Related substances. Complies with the test for Related

substances in Phenothiazines (2.3.5), using mobile phase A.
Category. Antipsychotic; antiemetic.
NOTE- Prepare the test solution immediately before use.
Dose. As antipsychotic, 15 to 100 mg daily, in divided doses;

as antiemetic, 10 to 30 mg daily, in divided doses.
Description. A white or pale yellow, crystalline powder;

Identification
Assay. Weigh accurately about 0.2 g, in powder, dissolve in
50 ml of anhydrous glacial acetic acid, heating gently on a
water-bath, allow to cool. Titrate with 0.1 M perchloric acid,
determining the end-point potentiometrically (2.4.25). Carry .
A.To 20 mg add 5 ml of water and I m1 of 1 M sodium hydoxide,

shake and extract with 10 ml of ether. Wash the ether extract
with 5 ml of water, dry with anhydrous sodium sulphate,
evaporate to dryness and dissolve the residue in 0.2 ml of
chloroform.

~OH24CIN3S,2CJ!404.

prochlOlperazine maleate RS or with the reference spectrum
ofprochlorperazine.

0.001 per cent w/v solution in ethanol (95 per cent) containing
0.01 per cent v/vof strong ammonia solution shows an
absorption maximum at about 258 nm and a less well-defined
maximum at about 313 nm; absorbance at about 258 nm, about
0.6.
Prochlorperazine Maleate Tablets

Prochlorperazine Tablets contain not less than 92.5 per cent
prochlorperazine maleate, C2oH24CIN3S, 2C4~04'
Identification
Test solution. A solution containing 0.1 per cent w/v of the

substance under examination in a mixture ofequal volumes of
methanol and chloroform.

Prochlorperazine Maleate add 10 ml of water and 2 ml of 1 M
sodium hydroxide, shake and extract with 15 ml of ether. Wash
the ether with 5 ml of water, dry with anhydrous sodium
sulphate, evaporate to dryness and dissolve the residue in
0.4 ml of chloroform.

prochlorperazine maleate RS in the same solvent mixture.
Detelmine by infrared absorption spectrophotometry (2.4.6).

C. Complies with the test for identification ofphenothiazines

(2.3.3).
1972
PROCHLORPERAZlNE MESYLATE
IP 2010
prochlorperazine maleate RS or with the reference spectrum

ofprochlorperazine.
B. To a quantity of the powdered tablets containing 5 mg of
Prochlorperazine Maleate add 5 ml ofsulphuric acid and allow
to stand for 5 minutes; a red colour is produced.
C. Shake a quantity of the powdered tablets containing 0.2 g
ofProchlorperazine Maleate with 2 ml of water and 1 ml of5 M
sodium hydroxide, mix and extract with three quantities, each
of 10 ml, of ether. Dry the combined extracts with anhydrous
sodium sulphate, filter, evaporate the filtrate to dryness and
dissolve the residue in 10 ml of methanol and add a solution
of 0.15 g ofpicric acid in 10 ml of methanol. The precipitate,
after washing with a small quantity of methanol, melts at about
255, with decomposition (2.4.21).
ethanol, dilute 10.0 ml ofthis solution to 50.0 ml with ethanol

CZOHZ4ClN3S, 2C4H40 4 taking 620 as the specific absorbance at
258nm.
Tests
Related substances. Comply with the test for Related,
Mol. Wt. 566.2
NOTE- Prepare the following solutions freshly.

containing 0.1 g of Prochl.orperazine Maleate with 10 ml of
methanol containing 0.5 per cent v/v of strong ammonia
solution and filter.
Prochlorperazine Mesylate is 2-chloro-1 0-[3-(4methylpiperazin-1-yl)propyl]phenothiazine

di(methanesulphonate).
Reference solution. Dilute 1.0 ml of the test solution to

200.0 ml with the same solvent.
Prochlorperazine Mesylate contains not less than 98.0 per

cent and not more than 101.0 per cent ofCzoHz4ClN3S,2CH4S03,
Apply to the plate 20 JlI of each solution.
Uniformity ofcontent. (For tablets containing 10 mg or less)

Dose. As antipsychotic, by intramuscular injection, 12.5 to 25

mg; twice or thrice daily; as antiemetic, by intramuscular
injection, 12.5 mg.
NOTE- Protect the solutions from light throughout the

Assay.
Crush one tablet and extract with three quantities, each of
10 ml, of ethanol containing 1 per cent v/v of strong ammonia
solution. Filter the extracts and to the combined filtrates add
sufficient ethanol to produce 50.0 ml. Dilute 10.0 ml of this
solution to 100.0 ml with ethanol. Dilute further with ethanol,
if necessary, to give a final solution containing 10 Jlg of
Prochlorperazine Maleate per ml and measure the absorbance
ofthe resulting solution at the maximum at about 258 nm (2.4.7).
Calculate the content of CZOHZ4ClN3S, 2C4H40 4 taking 620 as
Assay. Protect the solutions from light throughout the Assay.
the powder containing about 25 mg of Prochlorperazine
Maleate and extract with three quantities, each of 10 ml, of
ethanol containing 1 per cent v/v of strong ammonia solution.
Filter the extracts and to the combined filtrates add sufficient
ethanol to produce 100.0 ml. Dilute 10.0 ml to 50.0 ml with
Description. A white or almost white powder; odourless.
Identification
prochlOlperazine mesylate RS or with the reference spectrum
ofprochlorperazine mesylate.
B. When examined in the range 230 run to 360 nm (2.4.7), a
0.001 per cent w/v solution in ethanol containing 0.01 per
cent v/v of strong ammonia solution shows an absorption
maximum at about258 nm and a less well-defined maximum at
about 313 nm; absorbance at about 258 nm, about 0.6.
for 5 minutes; a red colour is produced.
D. Mix 50 mg with 0.2 g ofpowdered sodium hydroxide, heat
to fusion and continue the heating for a few seconds longer.
Cool, add 0.5 ml of water and a slight excess of 2 M
hydrochloric acid and warm; sulphur dioxide is evolved which
turns moistened starch iodate paper blue.
1973
IP 2010
PROCHLORPERAZINE MESYLATE
Tests
B. To a volume containing 5 mg ofProchlorperazine Mesylate

add carefully 2 ml of sulphuric acid and allow to stand for
5 minutes; a red colour is produced.
Tests
pH (2.4.24).5.5 to 6.5.
Test solution. Dissolve the substance under examination in

methanol containing 0.5 per cent v/v of strong ammonia
solution.
Related substances. Carry out the test for Related substances

in Phenothiazines (2.3.5), using mobile phase A.
0.7kPa.

40 ml with methanol containing 0.5 per cent v/v of strong
ammonia solution immediately before use.
Assay. Weigh accurately about 0.5 g, dissolve in 10 ml of Reference solution (b). Dilute 1.0 ml of the test solution to
water, add 5 ml of 1 M sodium hydroxide and extract with 200.0 ml with the methanol-ammonia mixture immediately
successive quantities of 50, 25, 25 and 25 ml of ether. Wash before use.
the combined ether extracts with 5 ml of water, shake the "Any secondary spot in the chromatogram obtained with the
washings with 5 ml of ether, add the ether to the combined test solution is not more intense than the spot in the
ether extracts and evaporate to dryness. Add 2 ml of ethanol, chromatogram obtained with reference solution (a) and not
evaporate to dryness. Titrate with 0.1 M perchloric acid, more than one such spot is more intense than the spot in the
using 1-naphtholbenzein solution as indicator. Carry out a chromatogram obtained with reference solution (b).
blank tihation.
I ml of 0.1 M perchloric acid is equivalent to 0.02831 g of Preparations (Injections).
C2oH24ClN3S,2CH4S03.
Assay. Protect the solutions from light throughout the Assay.
To an accurately measured volume containing about 25 mg of
Prochlorperazine Mesylate add sufficient ethanol containing
0.01 per cent v/v of strong ammonia solution to produce
Prochlorperazine Injection
200.0 ml. Dilute 5.0 ml of this solution to 100.0 ml with the
ammoniacal ethanol and measure the absorbance of the
Prochlorperazine Mesylate Injection
Prochlorperazine Injection is a sterile solution of Calculate the content ofC2oH24ClN3S, 2CH4S03taking 635 as
Prochlorperazine Mesylate in Water for Injections free from the specific absorbance at 258 nm.
dissolved air.
Prochlorperazine Injection contains not less than 95.0 per cent
prochlorperazine mesylate, C2oH24ClN3S,2CH4S03'
Usual strength. 12.5 mg perml.
Identification.
A. To a volume containing 0.1 g ofProchlorperazine Mesylate
add 20 ml of water and 2 ml of 10Msodium hydroxide. Shake
and extract with 25 ml of ether. Wash the ether layer with two
quantities, each of5 ml, of water, dry with anhydrous sodium
sulphate and evaporate to dryness. l)issolve the residue in
1 ml of chloroform.
prochlorperazine mesylate RS h'eated in the same manner or
with the reference spectrum ofprochlorperazine.
o
C I9H29NO,HCI
, Hel
Mol. wt. 323.9
Procyclidine Hydrochloride is (RS)-I-cyclohexy1-1-phenyl-3pyrrolidin-1-ylpropan-1-01 hydrochloride.
1974
PROCYCLIDINE TABLETS
IP 2010
Procyclidine Hydrochloride contains not less than 99.0 per

cent and not more than 101.0 per cent ofC I9 H29NO,HCI,
Category. Antiparkinsonian.
Dose. Initially, 2.5 mg thrice daily, after meals increasing at
intervals oftwo to three days by 2.5 to 5 mg daily; maximum,
30 mg daily.
Description. A white, crystalline powder, odourless or almost

odourless.
Identification

Compare the spectrum with that obtained with procyclidine
hydrochloride RS.
B. Dissolve 0.25 gin 10 ml of water, make allcaline with 5 M
ammonia and extract with three quantities, each of 10 ml, of
ether. Dry the combined ethereal extracts over anhydrous
sodium sulphate, filter, remove the ether and scratch the
residue with a glass rod to induce solidification. The residue
melts at about 85 (2.4.21).
Tests
Related substances. A. Determine by thin-layer
GF254.
Mobile phase. A mixture oflOO volumes of ether and 1volume
Test solution. Add 5 ml of 1.25 M sodium hydroxide to 20 ml

examination and mix. Extract with two quantities, each of
20 ml, of ether, add to the combined extracts 5 ml ofa 0.06 per
cent w/v solution of triphenylethylene (internal standard) in
ether, shake with anhydrous sodium sulphate and filter.
Evaporate the filtrate and dissolve the residue in 1 ml of ether.
Reference solution (a). Prepare in the same manner as the test
solution but using 20 ml of a 0.5 per cent w/v solution of the
substance under examination and omitting the addition ofthe
internal standard solution.
Reference solution (b). Prepare in the same manner as the test
solution but using 20 ml of a 0.5 per cent w/v solution of the
a glass column 1.5 m x 4 mm, packed with acid-washed,
silanised diatomaceous support (such as HP
chromosorb W) coated with 10 per cent w/w ofmodified
polyethylene glycol 20M (such as SP-1000) and 2 per
cent w/w of potassium hydroxide,
temperature:
column. 240,
- flow rate. 30 ml per minute ofthe carrier gas.
The ratio of the sum of the areas of any secondary peaks to
the area of the peak due to the internal standard in the
chromatogram obtaihed with reference solution (b) is not more
than the ratio ofthe area ofthe principal peak to the area ofthe
internal standard peak in the chromatogram obtained with the
test solution.


1-phenyl-3-pyrrolidinopropan-1-one hydrochloride RS in
chloroform.

anhydrous glacial acetic acid, warm if necessary to effect
solution and cool. Add 10 ml of mercuric acetate solution.
Titrate with 0.1 M perchloric acid, using crystal violet

light at 254 nm. Any spot corresponding to 1-phenyl-3pyrrolidinopropan-l-hydrochloride-l-one in the chromatogram
(a). Spray the plate with dilute potassium iodobismuthate
B. Determine by gas chromatography (2A.13).

C I9H29NO,HCI.
Procyc1idine Hydrochloride Tablets
Procyclidine Tablets contain not less than 90.0 per cent and
procyclidine hydrochloride, C I9H 29NO,HCl.
1975
PROCYCLIDINE TABLETS
IP 2010
Usual strengths. 2.5 mg; 5 mg.
Identification
about 25 mg ofProcyclidine Hydrochloride in 10 ml of water,
shake with 20 ml of ether and discard the ether layer. Make the
aqueous layer alkaline with 2 M sodium hydroxide and extract
with two quantities, each of20 ml, ofether. Wash the combined
ether extracts with two quantities, each of 10 ml, of water, dry
by shaking with anhydrous sodium sulphate, filter and
evaporate the filtrate to dryness. If necessary, induce
crystallization by scratching with a glass rod.
to settle. Transfer 5.0 ml of the supernatant solution to a

separating funnel, extract with 20.0 ml of chloroform and filter
the extract, discarding the first 5 mlofthe filtrate. Measure the
absorbance of the filtrate at the maximum at about 405 nm
(2.4.7), using as the blank a solution prepared by treating
0.5 ml of water and 4.5 ml ofa 0.025 per cent w/v solution of
bromocresol pUiple in the same manner beginning at the
words "extract with 20 ml of chloroform
".
Calculate the content of C 19Hz9NO.HCI from the absorbance
obtained by repeating the operation using procyclidine
hydrochloride RS in place of the powdered tablets.

obtained with procyclidine hydrochloride RS.
B. The powdered tablets give the reactions ofchlorides (2.3.1).
Progesterone Injectable Suspension
Tests
Progesterone Injectable Suspension is a sterile suspension of

Progesterone in Water for Injections.

Mobile phase. A mixture ofl 00 volumes of ether and 1volume

containing 25 mg ofProcyclidine Hydrochloride with 5 ml of
chloroform and filter.
I-phenyl-3-pyrrolidinopropan-l-one hydrochloride RS in
chloroform.
Reference solution (b). Dilute 1 volume of test solution to
light at 254 nm. Any spot corresponding to I-phenyl-3pyrrolidinopropan-l-one in the chromatogram obtained with
cent). Spray the plate with dilute potassium iodobismuthate
cent). Ignore any spot due to excipients on the line of
application.
Progesterone Injectable Suspension contains not less than

amount of progesterone, CZIH300Z'
Usual Strengths. 25 mg per ml; 50 mg per ml; 100 mg per ml;
200 mg per m!.
Identification
A. Filter a volume of suspension containing about 100 mg of
progesterone, through a medium-porosity, sintered-glass
crucible, filter again ifthe fluid is not clear. Wash with several
5.0 ml portions of water, evaporate on a steam bath, dry the
residue at 105. On the residue, determine by infrared absorption
obtained with progesterone RS or with the reference spectrum
of progesterone.
B. Melting point (2.4.21). 126 to 131.
Tests
pH (2.4.24). 4.0 to 7.5.
Solvent mixture. 85 volumes of ethanol (95 per cent) and 15

volumes of water.

quantity ofthe powder containing about 2.5 mg ofProcyclidine
Hydrochloride, transfer to a 100-ml volumetric: flask, add
10.0 ml of water and mix, and dilute to volume with a 0.025 per
cent w/v solution of bromocresol purple in 2 per cent v/v
solution ofglacial acetic acid. Allow the undissolved particles
Internal standard solution. A 0.66 per cent w/v solution of

methyltestosterone RS in the solvent mixture.
Test solutiO/i. Shake aVolume Ofsl.lspellsioll cOlltairiingabollt
25 mg ofprogesterone with 16 ml ofthe solvent mixture into a
polytef-lined, screw-capped, 25 ml test tube until the solution
1976
PROGUANIL TABLETS
IP 2010
is clear. Add 2.0 ml ofthe internal standard solution and dilute

A. Detelmine by infrared absorption spectrophotometry (2.4.6).

Compare the spectrum with that obtained with proguanil
hydrochloride RS or with the reference spectrum ofproguanil
hydrochloride.

progesterone RS in the solvent mixture. To 4.0 ml of this
solution, add 2.0 ml ofthe internal standard solution and dilute B. To 10 ml of a saturated solution add 0.25 ml of potassium
ferrocyanide solution; a white precipitate is produced which
dissolves on addition of a few drops of dilute nitric acid.
-

octadecylsilane chemically bonded to porous silica (3050llm),
- mobile phase: a mixture ofn volumes of water and 28
volumes of isopropyl alcohol,
resolution between the peaks due to progesterone and
methyltestosterone is not less than 3.5 and the relative
standard deviation for replicate injections is not more than 1.5
per cent.
Calculate the content of CZIH300z in the suspension.
Storage. Store in single dose or multiple dose containers,

preferably of Type 1 glass.
Chloroguanide Hydrochloride
C. Dissolve 5 mg in 5 ml ofa wann 1.0 percentw/v solution of

cetrimide and add 1 ml of 5 M sodium hydroxide and 1 ml of
bromine solution; a deep red colour is produced.
Tests
Acidity or alkalinity. To 35 ml of water maintained at 60 to
65 add 0.2 ml of methyl red solution, neutralise with 0.01 M
sodium hydroxide or 0.01 M hydrochloric acid, add 0.4 g of
the substance under examination and stir until dissolved. The
resulting solution is not acidic and requires for neutralisation
not more than 0.2 ml of 0.01 M hydrochloric acid.
4-Chloroaniline. Dissolve 0.1 g in 1 ml of 2 M hydrochloric
acid, add sufficient water to produce 20 ml, cool to 5, add 1
ml of 0.05 M sodium nitrite, allow to stand at 5for 5 minutes,
add 2 ml ofa 5 per cent w/v solution of ammonium sulphamate
and allow to stand for 10 minutes. Add 2 ml of a 0.1 per cent
w/v solution of N-(l-naphthyl)ethylenediamine
dihydrochloride, dilute to 50 ml with water and allow to stand
for 30 minutes. Any magenta colour produced is not more
intense than that obtained by treating in the same manner and
at the same time 20 ml of a solution containing 1.25 Ilg of
4-chloroaniline.
CjlHj6CINs, HCl
Mol. Wt. 290.2
Proguanil Hydrochloride is 1-(4-chlorophenyl)-5isopropylbiguanide hydrochloride.

Proguanil Hydrochloride contains not less than 99.0 per cent
and not more than 101.0 per cent ofCjjH16ClNs, HCl, calculated
on the dried basis.

anhydrous glacial acetic acid and 10 ml of mercuric acetate
solution. Titrate with 0.1 M perchloric acid, determining the
CjlH16ClNS, HCI.
Proguanil Tablets
Dose. Suppressive, 100 to 300 mg daily.
Proguanil Hydrochloride Tablets; Chloroguanide

Description. A white, crystalline powder; odourless.

Identification

Proguanil Tablets contain not less than 95.0 per cent and not
more than 105.0 per cent of the stated amountofproguanil
hydrochloride, CjjHj6ClNs, HCI.
1977
IP 2010
PROGUANIL TABLETS
Identification
A. Boil a quantity ofthe powdered tablets containing 0.5 g of
Proguanil Hydrochloride with 5 ml of dilute hydrochloric acid,
cool and filter. To the filtrate add a slight excess of sodium
hydroxide solution, extract with 30 ml of ether and evaporate
the ethereal extract; the residue, after drying at 105, melts at
about 131 (2.4.21).
Dissolve the residue obtained in testA in the minimum quantity

of dilute hydrochloric acid; the solution diluted with water
to about 40 ml and neutralised if necessary, with cautious
a4dition of dilute ammonia solution, complies with the
following tests.
B. To 10 ml add 0.25 ml ofpotassiumferrocyanide solution; a
white precipitate is produced which dissolves on addition of
a few drops of dilute nitric acid.
Calculate the content ofCIIHI6ClNs,HCl from the absorbance

obtained by repeating the operation using 10 ml ofa 0.01 per
cent w/v solution of proguanil hydrochloride RS beginning
at ~he words "add 70 ml ofwater,..,.".
Promazine Tablets
Promazine Hydrochloride Tablets
Promazine Tablets contain not less than 92.5 per cent and not
more than 107.5 per cent of ~he stated amount of promazine
hydrochloride, CI7HzoNzS,HCl.
Identification
C. To 0.5 ml add 5 ml of a warm 1.0 per cent w/v solution of

cetrimide and add 1 ml of5 M sodium hydroxide and 1 ml of
bromine solution; a deep red colour is produced.
A. To a quantity ofthe powdered tablets containing 40 mg of

Promazine Hydrochloride, add 10 ml of water and 2 ml of1 M
the extract with 5 ml of water, dry with anhydrous sodium
Tests
4-Chloroaniline. To a quantity of the powdered tablets 0.4 ml of chloroform. On the residue, determine by infrared
containing about 0.1 g ofProguanil Hydrochloride add 5 ml of absorption spectrophotometry (2.4.6). Compare the spectrum
ethanol (95 per cent) and shake for 10 minutes. Add 2.5 mlof with that obtained with promazine hydrochloride RS or with
2 M hydrochloric acid and 15 ml of water, mix and filter the reference spectrum ofpromazine hydrochloride.
through a wetted filter paper, washing the filter with 5 ml of B. To a quantity of the powdered tablets containing 5 mg of
water. Cool to 5, add 1 ml of 0.05 M sodium nitrite, allow to Promazine Hydrochloride, add 5 ml ofsulphuric acid and allow
stand at 5 for 5 minutes, add 2 ml ofa 5 per cent w/v solution to stand for 5 minutes. An orange colour is produced.
of ammonium sulphamate and allow to stand for 10 minutes.
Add 2 ml of a 0.1 per cent w/v solution of C. Triturate a quantity of the powdered tablets containing 0.2
N-(l-naphthyl)ethylenediamine dihydrochloride, dilute to g ofPromazine Hydrochloride with 4 rnl of methanol, centrifuge,
50 ml with water and allow to stand for 30 minutes. Any filter the supernatant liquid, heat almost to boiling, immediately
magenta colour produced is not more intense than that add 2 ml ofa boiling 3.5 per cent w/v solution ofpicric acid in
obtained by treating in the same manner and at the same time methanol and boil for 2 minutes. Cool in ice, filter, wash the
crystals with three quantities of methanol, dissolve in 10 ml of
20 ml of a solution containing 1.25 Ilg of 4-chloroaniline.
hot methanol and repeat the crystallisation and washing. The
rust red crystals so obtained, after drying at 105 for 1 hour,
Assay. Weigh and powder 20 tablets. Weigh accurately a melts at about 144 (2.4.21).
quantity of the powder containing about 0.1 g of Proguanil
Hydrochloride, add 5 ml of water and warm on a water-bath Tests
with stirring until a smooth paste is obtained. Add 50 ml of
water, continue warming for 10 minutes, cool, add sufficient Related substances. Comply with the test for Related
water to produce 100.0 rnl and filter. Dilute 10.0 ml ofthe filtrate substances in Phenothiazines (2.3.5), using mobile phase (a)
to 100.0 ml with water and to 10.0 ml ofthe resulting solution and the following freshly prepared solutions.
add 70 ml of water, 5 ml of a 20 per cent w/v solution of Test solution. Extract a quantity of the powdered tablets
cetrimide and 1 ml of 2-propanol. Adjust the temperature of containing 0.1 g of Promazine Hydrochloride with 10 ml of
the solution to 20 and add 2 ml of alkaline sodium methanol and filter.
hypobromite solution and sufficient water to produce
Reference solution. Dilute 1 ml oftestsolutiont0200ml with
100.0 rnl. Allow to stand at 20 for 25 minutes and measure the
methanol.
480 nm (2.4.7).
1978
IP 2010
PROMETHAZINE INJECTION
Assay.
Heat the solution to boiling; it becomes orange and an orangered precipitate is produced.
NOTE-earlY out the test protectedfi-om light.

the powder containing about 50 mg of Promazine
Hydrochloride and triturate with 10 ml of 2 M hydrochloric
acid and add 200 ml of water. Shake for 15 minutes, add
sufficient water to produce 500 ml and centrifuge about 50 ml
ofthe mixture. To 5 ml ofthe clear, supernatant liquid add 10 m1
of 0.1 M hydrochloric acid and sufficient water to produce
100 mi. Measure the absorbance (2.4.7) ofthe resulting solution
at the maximum at about 251 urn.
Calculate the content of C 17H zoN zS,HCI taking 935 as the
specific absorbance at 251 urn.
D. Gives reaction B ofchlorides (2.3.1).
Tests
.
prepared immediately before use.
in Phenothiazines (2.3.5), protected from bright light using
mobile phase B.

examination in 10 ml ofa mixture of95 volumes of methanol
and 5 volumes of diethylamine.
Reference solution (aj. A 0.01 per cent w/v solution of the
Reference solution (bj. A 0.02 per cent w/v solution of

isopromethazine hydrochloride RS in the same solvent
mixture.
, Hel
Apply to the plate 10 )11 of each solution.
Mol. Wt. 320.9

Promethazine Hydrochloride is (RS)-dimethyl(2phenothiazin-1 O-ylpropyl)amine hydrochloride.
Promethazine Hydrochloride contains not less than 98.5 per
cent and not more than 101.0 per cent of C 17H zoN zS, HCI,
Category. Antiemetic.
Description. A white or faintly yellowish, crystalline powder.
Identification

Compare the spectrum with that obtained with promethazine
promethazine hydrochloride.
B. Complies with the test for Identification ofPhenothiazines
Any spot corresponding to isopromethazine in the

reference solution (b) and any other secondary spot is not
5 ml of 0.01 M hydrochloric acid and 50 ml of ethanol (95 per
cent) and titrate with 0.1 M sodium hydroxide, determining
the end-point potentiometrically (2.4.25). Record the volume
added between the two inflections.
C 17HzoNzS, HCI.
Promethazine Injection
(2.3.3).
Promethazine Hydrochloride Injection
C. Dissolve 0.1 gin 3 ml of water and add 1 ml of nitric acid

dropwise; a precipitate is produced which dissolves rapidly
to give a red solution which becomes orange and then yellow.
Promethazine Injection is a sterile solution of Promethazine

Hydrochloride in Water for Injections free from dissolved air.
It may contain suitable stabilising agents.
1979
PROMETHAZINE INJECTION
IP 2010
Promethazine Injection contains not less than 95.0 per cent

promethazine hydrochloride, C 17H2oN2S, HCl.
Usual strengths. 25 mg in 1 ml; 50 mg in 2 ml.
Identification
A. To a volume containing 0.1 g ofPromethazine Hydrochloride
and extract the mixture with 25 ml of ether. Wash the ether
layer with two quantities, each of 5 ml, of water, dry with
anhydrous sodium sulphate and evaporate to dryness.
Dissolve the residue in 1 ml of chloroform.
hydrochloride RS treated in the same manner or with the
reference spectrum ofpromethazine.
B. To a volume containing 0.2 g ofPromethazine Hydrochloride
add sufficient potassium carbonate to saturate the solution,
extract with two quantities, each of 10 ml, of ether and
evaporate the combined extracts to dryness. Dissolve the
residue in 2 ml of methanol and pour into a solution of0.4 g of
picric acid in 10 ml of methanol, previously warmed to about
50. Cool, scratch the sides ofthe tube to induce crystallisation,
allow to stand for 3 to 4 hours and filter. The residue, after
washing with methanol and drying, melts at about 160 (2.4.21).
C. To a volume containing 5 mg ofPromethazine HydrocWoride

5 minutes; a red colour is produced.
Tests

intense than the spot in the chromatogramI obtained with
Assay. Carry out the following procedure protected from
light.
Promethazine Hydrochloride add sufficient 0.01 M
hydrochloric acid to produce 100.0 ml. Dilute 10.0 ml to
100.0 ml with 0.01 Mhydrochloric acid, dilute 1O.0ml ofthis
solution to 50.0 ml with 0.01 M hydrochloric acid and measure
about 249 nm (2.4.7). Calculate the content ofC 17H 2oN 2S, HCl
taking 910 as the specific absorbance at 249 om.
Promethazine Syrup
Promethazine Hydrochloride Syrup; Promethazine
Hydrochloride Oral Solution; Promethazine Oral Solution
Promethazine Syrup is a solution containing Promethazine
Hydrochloride in a suitable flavoured vehicle.
Promethazine Syrup contains not less than 90.0 per cent and
promethazine hydrochloride, C 17H 2oN 2S, HCl.
pH (2.4.24).5.0 to 6.0.

in Phenothiazines (2.3.5), using mobile phase B.
Identification
Test solution. Dilute a volume ofthe injection with a mixture of

95 volumes of methanol and 5 volumes of diethylamine to
contain 1 per cent w/v of Promethazine Hydrochloride.
Reference solution (a). Dilute 1 volume of the test solution to
40 volumes with the same solvent mixture.
Reference solution (b). Dilute 1 volume of the test solution to
mixture.
Apply separately to the plate 10 f.1l of each solution.
Any spot corresponding to isopromethazine in the
reference solution (c). Any other secondary spot in the
Carry out the method for identification of phenothiazines

(2.3.3).
Test solution. A solution prepared by diluting a suitable volume

of the syrup with water to contain 0.08 per cent w/v of
Promethazine Hydrochloride. Apply 5 f.11 to the plate.
Tests
Assay. Carry out the following procedure protected from
light.
Weigh accurately a quantity containing about 10 mg of
Promethazine Hydrochloride, add 25 ml of water and 5 ml ofa
5 per cent w/v solution of sodium hydroxide. Extract the
mixture with two quantities, each of 50 ml, of chloroform,
shaking vigorously for 1 minute each time, evaporate the
combined extracts to dryness at .about 30 at a pressure of
1980
IP 2010
PROMETHAZINE TABLETS
2 kPa and dissolve the residue in sufficient 0.1 M hydrochloric

acidto produce 50.0 ml (solution A). Dilute 10 ml ofsolution A
to 50.0 ml with water (solution B). To a further 10 ml ofsolution
A add 5 ml ofperoxyacetic acid solution, allow to stand for
10 minutes and add sufficient water to produce 50.0 m1 (solution
C). Measure the absorbance of solution C at the maximum at
about 336 run (2.4.7), using solution B as the blank and measure
the absorbance of solution B at the same wavelength using
water as the blank. Repeat the procedure using a 0.02 per cent
w/v solution of promethazine hydrochloride RS in 0.1 M
hydrochloric acid in place of solution A and beginning at the
words "Dilute 10 ml of......".
Determine the weight per ml ofthe symp (2.4.29), and calculate
the content ofC 17HzoN zS, HCI, weight in volume. The result is
not valid unless the absorbance of solution B is less than
0.10.

Promethazine Hydrochloride add 5 ml of sulphuric acid and
allow to stand for 5 minutes; a red colour is produced.
Tests
in Phenothiazines (2.3.5), using mobile phase B.
Test solution. A solution freshly prepared by extracting a
Promethazine Hydrochloride with 10 ml of a mixture of
95 volumes of methanol and 5 volumes of diethylamine and
filtering.
40 ml with the same solvent.
200.0 mlwith the same solvent.
isopromethazine hydrochloride RS in the same solvent.
Promethazine Tablets
Promethazine Hydrochloride Tablets
Promethazine Tablets contain not less than 92.5 per cent and
promethazine hydrochloride, C 17HzoN zS, HCl. The tablets are
coated.
Apply to the plate 10 III of each solution. Any spot

corresponding to isopromethazine in the chromatogram
(c). Any other secondary spot in the chromatogram obtained
Identification
Crush one tablet; add 1 ml of dilute hydrochloric acid and 30

A. To a quantity of the powdered tablets containing 40 mg of ml ofwater and shake for 15 minutes. Add sufficient water to
Promethazine Hydrochloride add 10 ml of water and 2 ml of produce 50.0 ml and centrifuge. Complete the procedure
1 M sodium hydroxide, shake and extract with 15 ml of ether. described in the Assay beginning at the words "To 5.0 ml of
Wash the ether extract with 5 ml ofwater, dry with anhydrous . the clear supernatant liquid....".
sodium sulphate, evaporate to dryness and dissolve the
residue in 0.4 ml ofchloroform.
Determine by infrared absorption spectrophotometry (2.4.6). Assay. earlY out the following procedure protected froin
Compare the spectrum with that obtained with promethazine light.
hydrochloride RS treated in. the same manner or with the
reference spectrum ofpromethazine.
0.2 g of Promethazine Hydrochloride in 2 ml of water, filter,
add sufficient potassium carbonate to saturate the solution,
extract with two quantities, each of 10 ml, of ether and
evaporate the combined extracts to dryness. Dissolve the
residue in 2 ml ofmethanol and pour into a solution of0.4 g of
picric acid in 10 ml of methanol, previously warmed to about
50. Cool, scratch the sides ofthe tube to induce crystallisation,
allow to stand for 3 to 4 hours and filter. The residue, after
washing with methanol and drying, melts at about 160 (2.4.21).

the powder containing about 50 mg of Promethazine
Hydrochloride with 10 ml of 2 M hydrochloric acid and add
200 ml ofwater. Shake for 15 minutes, add sufficient water to
produce 500.0 ml and centrifuge about 50 ml ofthe mixture. To
5.0 ml of the clear supernatant liquid add 10 ml of 0.1 M
hydrochloric acid and sufficient water to produce 100.0 ml.
C 17HzoN zS, HCI taking 910 as the specific absorbance at
249 run.
1981
PROMETHAZINE THEOCLATE
IP 2010
E. Fuse 50 mg ofthe residue obtained in test D with 0.5 g of

anhydrous sodium carbonate, boil the residue with 5 ml of
water, acidify to litmus paper with nitric acid and filter. The
filtrate gives reaction A ofchlorides (2.3.1).
Tests
CWorides (2.3.12). Shake 1.5 g with 50 ml ofwater for 2 minutes
and filter. 25 ml of the filtrate complies with the limit test for
Mol. Wt. 499.0
Promethazine Theoclate is the (RS)-dimethyl(2phenothiazin-10-ylpropyl)amine salt of8chlorotheophylline.
Promethazine Theoclate contains not less than 98.0 per cent
and not more than 101.0 per cent of C 17H zoN zS, C7H 7C1N40 z,
Category. Antiemetic.
almost odourless.
Identification
out.
A. Shake 0.15 g with 2.5 ml of water, add 1 ml of5 M ammonia
and extract with 30 ml of ether. Wash the ether extract with
10 ml of water, dry with anhydrous sodium sulphate and
evaporate to dryness. Dissolve the residue in 1 ml of
chloroform.
RS or with the reference spectrum ofpromethazine.
0.0007 per cent w/v solution in ethanol containing 0.01 per
cent v/v of strong ammonia solution shows an absorption
maximum at about 255 nm; absorbance is a.bout 0.53.
for 5 minutes; a red colour is produced.
D. Shake 0.4 g with 10 ml of water, add 4 rlil of5 M ammonia,
shake with two quantities, each oBO ml, of ether and add 4 ml
of hydrochloric acid to the aqueous solution; a white
precipitate is produced. Filter, wash with water and dry at
105. Dissolve 10 mg of the residue in 1 ml of hydrochloric
acid, add 0.1 g of potassium chlorate and evaporate to
dryness; a reddish residue remains which becomes purple on
exposure to the vapour of ammonia.

in Phenothiazines (2.3.5), protected from bright light, using
mobile phase B.
NOTE-Prepare the following solutions immediately before

use.
examination in 10 ml of a mixture of95 volumes of methanol
and 5 volumes of diethylamine.
mixture.
Apply to the plate 10 III of each solution. Any spot
acetone. Titrate with 0.1 M perchloric acid, using 3 ml of a
saturated solution of methyl orange in acetone as indicator.
C17HzoNzS,C7H7ClN40Z.

Promethazine Theoclate Tablets contain not less than 92.5 per
promethazine theoclate, C 17H zoN zS, C7H7ClN40 Z
1982
PROPANE
IP 2010
Identification
Promethazine Theoclate add 10 ml of water and 2 ml of 1 M
the ether extract with 5 ml of water, dry with anhydrous sodium
0.4 ml of chloroform.

mixture.

theoclate RS treated in the same manner or with the reference
spectrum ofpromethazine.
Apply to the plate 10 /ll of each solution. Any spot

(c). Any other secondary spot in the chromatogram obtained
0.2 g of Promethazine Theoclate in 2 ml of water, filter, add

sufficient potassium carbonate to saturate the solution, extract
with two quantities, each of 10 ml, of ether and evaporate the
combined extracts to dryness. Dissolve the residue in 2 ml of
methanol and pour into a solution of 0.4 g of picric acid in
10 ml of methanol, previously warmed to about 50. Cool,
scratch the sides ofthe tube to induce crystallisation, allow to
stand for 3 to 4 hours and filter. The residue, after washing
with methanol and drying, melts at about 160 (2.4.21).
Promethazine Theoclate add 5 m1 of sulphuric acid and allow
to stand for 5 minutes; a red colour is produced.
D. Extract a quantity ofthe powdered tablets containing 0.2 g
of Promethazine Theoclate with chloroform, filter and
evaporate the filtrate to dryness. Shake the residue with 10 ml
of water, add 4 ml of 5 M ammonia and extract with two
quantities, each of30 ml, of ether. Wash the combined extracts
with 10 ml of water. Combine the aqueous layer and washings,
add 4 ml of hydrochloric acid; a white precipitate is produced.
Filter, wash the residue with water and dry at 105. Dissolve
10 mg ofthe residue in 1 ml of hydrochloric acid, add 0.1 g of
potassium chlorate and evaporate to dryness; a reddish
residue remains which becomes purple on exposure to the
vapour ofammonia.
Assay. CarlY out the following procedure protected from

light.
the powder containing about 50 mg ofPromethazine Theoclate,
add 5 ml of water and 1 m1 of strong ammonia solution and
allow to stand for 5 minutes. Add 50 ml of ethanol, shake for
5 minutes and filter, washing the residue with five quantities,
each of 5 ml, of ethanol. Add sufficient ethanol to the filtrate
to produce 100.0 ml. Dilute 10.0 ml to 100.0 ml with ethanol
and dilute 10.0 ml of this solution to 100.0 ml with ethanol.
maximum at about 255 om (2.4.7). Calculate the content of
C 17H zoN zS, C7H 7ClN40 z taking 755 as the specific absorbance
at255 om.
Propane
H3C
/"'--..
C3Hg
CH3
Mol. Wt. 44.1
Propane is dimethylmethane.
Tests
Propane contains not less than 98.0 per cent of C3H g
Related substances. Carry out the test for related substances

in phenothiazines (2.3.5), using mobilephase B.
CAUTION-Propane is highly flammable and explosive.
Test solution. A solution freshly prepared by extracting a

Promethazine Theoclate with 10 ml ofa mixture of95 volumes
of methanol and 5 volumes of diethylamine and filtering.
Identification
Determine by infrared absorption spectrophotometry (2.4.6),
the solution give maxima at the wavelength of3.4 /lm, 6.8 /lm
and7.2/lm.
Tests
Water(2.3.43). Not more than 0.001 per cent with the following
modifications (a) provide the closed-system titrating vessel
with an opening through which passes a coarse-porosity gas
1983
PROPANE
IP 2010
dispersion tube connected to a sample cylinder; (b) dilute the

reagent with anhydrous methanol to give a water equivalence
factor ofbetween 0.2 and 1.0 mg per ml, age this diluted solution
for not less than 16 hours before sanitation; (c) obtain a 100 g
sample as directed under inhalation preparation, and introduce
the sample into the titration vessel through the gas dispersion
tube at a rate of about 100 ml of gas per minute.
High-boiling residues. Not more than 5 flg per ml, determined
by the following method. Prepare a cooling coil from copper
tubing (about 6 mm outside diameter x about 6.1 m long) to fit
into a vacuum-jacketed flask. Immerse the cooling coil in a
mixture ofdry ice and acetone in a vacuum-jacketed flask, and
connect one end of the tubing to the propellant sample
cylinder. Carefully open the sample cylinder valve, flush the
cooling coil with about 50 ml of the propellant, and discard
this portion of liquefied propellant. Continue delivering
liquefied propellant from the cooling coil, and collect it in a
previously chilled 1000-ml sedimentation cone until the cone
is filled to the 1OOO-mi mark. Allow the propellant to evaporate,
using a warm water bath maintained at about 400 to reduce
evaporating time. When all ofthe liquid has evaporated, rinse
the sedimentation cone with two 50-ml portions of pentane,
and combine the rinsings in a tared 150-ml evaporating dish.
Transfer 100 ml ofthe pentane solvent to a second tared 150ml evaporating dish, place both evaporating dishes on a water
bath, evaporate to dryness, and heat the dishes in an oven at
1000 for 60 minutes. Cool the dishes in a desiccator, and weigh.
Repeat the heating for IS-minute periods until successive
weighings are within 0.1 mg, and calculate the weight of the
residue obtained from the propellant as the difference between
the weights of the residues in the two evaporating dishes.
prepared from crushed firebrick and calcined or burned

with a clay binder ab~ve 900 0 with subsequent acidwash. It may be silanized),
a thermal-conductivity detector,
- flow rate. 50 ml per minute using nitrogen as carrier gas.
Inject the test solution. The test is not valid unless"the relative
standard deviation for replicate injections is not more than
1.0.
Inject 2 fll of test solution.
Calculate the percentage purity by dividing 100 times the
propane response by the sum of all of the responses in the
chromatogram.
Storage. Store protected from moisture and prevent exposure
to excessive heat.
Propionic Acid
H3C~OH
o
C3H60 z
Mol. Wt. 74.1
Propionic Acid contains not less than 99.5 per cent and not
more than I00.5 per cent ofC3H60 z.
Tests
Weight per ml (2.4.29). 0.988 to 0.993.
Distilling range (2.4.8). 138,SO to 142'so.
Acidity of residue. Add 10 ml of water to the residue obtained

in the test for High boiling residues, mix by swirling for about
30 seconds, add 2 drops of methyl orange solution, insert the
stopper in the tube, and shake vigorously; no pink or red
color appears in the aqueous layer.
Heavy metals (2.3 .13). To the residue obtained in the test for
Nonvolatile residue, .add 8 ml of 0.1 M hydrochloric acid,
warm gently until solution is complete, dilute with water to
100 ml. 10 ml ofthe solution complies with the limit test for
Sulphur. Carefully open the container valve to produce a

moderate flow of gas. Do not direct the gas stream toward the
face, but deflect a portion of the stream toward the nose, the
odour free from the characteristic odour of sulphur
compounds.
Limit of nonvolatile residue. Evaporate 20 g in a tared dish,

and dry at 105 0 for 1 hour, the weight ofthe residue does not
exceed 2 mg.
Assay. Detennine by Gas chromatography (2.4.13).
Test solution. Connect 1 Propane cylinder to the chromatograph

through a suitable sampling valve and a flow control valve
downstream from the sampling valve. Flush the liquid sample
through the sampling valve, taking care to avoid entrapment
of gas or air in the sampling valve.
an aluminum column 6 m x 3 mm, packed with 10 per cent
of liquid phase G30 ( Tetraethylene glycol dimethyle
ether) on non-acid-washed support SIC ( A support
Readily oxidizable substances. Dissolve 15 g of sodium

hydroxide in 50 ml of water, cool, add 6 ml of bromine, stirring
to effect complete solution, and dilute with water to 2000 ml.
Transfer 25.0 ml ofthis solution to a glass-stoppered, 250-ml
conical flask containing 100 ml of water, and add 10 ml of20.0
percentw/vsolution sodium acetate and 10.0mlofPropionic
Acid. Allow to stand for 15 minutes, add 5 ml of25.0 per cent
w/v solution of potassium iodide and 10 ml of hydrochloric
acid, and titrate with 0.1M sodium thiosulphate just to the
disappearance ofthe brown color. Carry out a blank titration.
The difference between the volume of 0.1 M sodium
thiosulphate required for the blank and that required for the
test solution is not more than 2.2 ml.
1984
PROPOFOL
IP 2010
Aldehydes. Transfer 10.0 ml to a glass-stoppered, 250-ml

conical flask containing 50 ml of water and 10.0 ml of 1.25 per
cent w/v solution of sodium bisulphite, insert the stopper,
and shake vigorously. Allow the mixture to stand for 30
minutes, then titrate with 0.1 M iodine to the same brownish
yellow endpoint obtained with a blank treated with the same
quantities of the same reagents. The difference between the
volume of 0.1 M iodine required for the blank and that required
for the test solution is not more than 1.75 ml.
Assay. Weigh accurately about 1.5 g and dissolve with 100 ml
ofrecently boiled and cooled water in a 250-ml conical flask,
and titrate with 0.5 M sodium hydroxide using dilute
phenolphthalein solution as indicator ,
C3H60 Z
Propofol
Mol. Wt. 178.3

Propofol is 2,6-diisopropylphenol.
Propofol contains not less than 98.0 per cent and not more
than 102.0 per cent ofC1zHlsO.
Description. A colourless or very light yellow, clear liquid.
Identification
Compare the spectrum with that obtained with propofol RS or
with the reference spectrum ofpropofol.
Tests
(2.4.14).

examination in 10 ml of hexane.
examination in 100 ml of hexane.
Reference solution (a). A solution containing 5 mg of the
substance under examination and 15 III containing 15 mg of
2, 6-bis(l-methylethyl) benzene-1, 4-dione RS (propofol

impurity J RS) in 50 ml of hexane.
Reference solution (b). Dilute 0.1 ml of propofol for peak
identification RS (containing 3,3' ,5,5' -tetrakis(1methylethyl)biphenyl-4,4'-diol (propofol impurities E) and 2(1-methylethoxy)-1,3-bis(1-methylethyl)benzene (propofol
impurities G) to 1.0 ml with hexane.
Reference solution (c). Dilute 1.0 ml oftest solution (a) to 100
ml with hexane. Dilute 1.0 ml of this solution to 10 ml with
hexane.
Reference solution (d). A 0.24 per cent w/v solution of
propofol RS in hexane.
silica gel (5Ilm),
- mobile phase: a mixture of 1 volume of anhydrous
ethanol, 7.5 volumes of acetonitrile and 990 volwnes
of hexane,
resolution between the peaks due to propofol impurity J and
propofol is not less than 4.0. The relative retention times with
reference to propofol for propofol impurity G is about 0.5, for
oxydibenzene (propofol impurity G) is about 0.6, for 2-(1methylethenyl)-6-(1-methylethyl)phenol (propofol impurity B)
is about 0.7, for 4-hydroxy-3,5-bis(1-methylethyl)benzoic acid
(propofol impurity F) is about 2.3, for 2,5-bis(1methylethyl)phenol (propofol impurity D) is about 2.5 , for 1methylethy14-hydroxy-3,5-bis(1-methylethyl)benzoate
(propofol impurity P) is about 2.9, for 2,4-bis(1methylethyl)phenol (propofol impurity A) is about 3.0, for 2(l-methylethyl)phenol (propofol impurity C) is about 3.4, for
propofol impurity E is about 4.0, for 3-(1-methylethyl)phenol
(propofol impurity F) is about 5.8 and for 4-(1methylethyl)phenol (propofol impurity H) is about 6.4.
I
Inject test solution (a), reference solution (a), (b) and (c). Run
the chromatogram 7 times the retention time of the principal
peak. In the chromatogram obtained with test solution (a), the
area ofpeak due to propofol impurity G is not more than twice
with reference solution (c) (0.2 per cent); the area ofthe peak
due to propofol impurity E is not more than 0.1 times the area
reference solution (c) (0.01 per cent). The area of any
solution (c) (0.05 per cent). The sum of the areas of all the
secondary peaks is not more than 3 times the area of the
1985
PROPOFOL
IP 2010

solution (c) (0.3 per cent). Ignore any peak other than propofol
impurity E with an area less than 0.3 times the area of the
solution (c) (0.03 per cent)~
Impurities J, K, Land O. Determine by gas chromatography
(2.4.13).

ml with dichloromethane. Further dilute 1.0 ml ofthis solution
to 10 ml with dichloromethane.
Reference solution (b). Dilute 5 III containing 5 mg ofpropofol
impurity J RS to 100 ml with dichloromethane. Dilute 1.0 ml of
this solution to 25 ml with dichloromethane.
Reference solution (c). Dissolve 4 mg ofpropofol RS in 1.0 ml
of reference solution (b).
a fused-silica capillary column 30 m x 0.32 mm, coated
with polymethylphenylsiloxane (0.5 Ilm),
- temperature:
Column 80 from 0-3 minutes, 80-120 from 3-25 minutes
and 210 from 25-40 minutes,
Injection port at 100 and detector at 270,
- flow rate. 1.7 ml per minute using nitrogen as carrier gas.
Inject 1 III ofreference solution (c). The test is not valid unless
the peak- to- valley ratio isnot lessthan3.0 where Hp is the
height above the baseline ofthe peak due to propofol impurity
J and HI' is the height above the baseline ofthe lowest point of
the curve separating this peak from the peak due to propofol.
The relative retention time with reference to propofol for 1-(1methylethoxy)-2-( l-methylethyl)benzene (propofol impurity K)
is about 0.76, for 2,2-dimethyl-4-(l-methylethyl)-1,3benzodioxole (propofol impurity L) is about 0.81 ,forpropofol
impurity J is about 1.01 and for 2-(1-methylethyl)-6propylphenol (propofol impurity 0) is about 1.03.
Inject 1 III ofthe test solution, reference solution (a) and (c). In
the chromatogram obtained with the test solutioIl the
of
the peak due to propofol impurities J, K, Land 0, for each
impurity, is not more than 0.5 times the area of the principal
(a) (0.05 percent).
area

Inject test solution (b) and reference solution (d).
Calculate the content ofC 12H 1sO.
Storage. Store protected from light under an inert gas.
Propofol Injection
Propofol Injection is a sterile emulsion ofPropofol in a suitable
base.
Propofol Injection contains not less than 95.0 per cent and
not more than 105.0 per cent ofthe stated amount ofpropofol,
C1zH1sO.
UsmH strength. 10 mg per ml.
Identification
A. Extract a volume ofthe injection containjng 0.1 g ofPropofol
with three 25 ml quantities of hexane and filter the combined
extracts using a glass fibre filter (such as Whatrnan GF/C).
Extract the filtrate with two 20 ml quantities of methanol (90
per cent) and evaporate the combined extracts under reduced
pressure. Dissolve the residue obtained in 2 ml of ethanol and
evaporate to dryness under reduced pressure. Dry the residue
at 50 over phosphorus pentoxide for 1 hour. On the residue,
determine by infrared absorption spectrophotometry (2.4.6).
Compare the spectrum with that obtained with propofol RS or
with the reference spectrum of propofol.
Tests
pH (2.4.24).6.0 to 8.5.
Propofol quinone and propofol dimer. Determine by liquid
Test solution. Dilute a volume ofinjection containing 80 mg of

Propofol in 100 ml ofpropan-2-ol.
ofpropofol RS, 0.00008 per cent w/v of2, 6-bis(l-methylethyl)1,4-benzoquinone RS (propofol impurity J RS) and 0.0002
per cent w/v of 3,3',5,5'-tetrakis (l-methylethyl)biphenyl4,4'-diol RS (propofol dimer RS) in propan-2-o1 containing
6.8 per cent v/v of water.
a stainless steel column 10 cm x 4.6 mrn, packed with
HypersilODS),
mobile phase: a mixture of 40 volumes of
tetrahydrojUran and 60 volumes of water,
Inject the reference solution. The relative retention time with
reference to propofol for propofol impurity J is about 0.8 and
for propofol dimer is about 2.5.
1986
IP 2010
PROPRANOLOL HYDROCHLORIDE
chromatogram three times the retention time of the principal
peale. In the chromatogram obtained with the test solution the
area ofthe peak due to propofol quinone (propofol impurity 1)
is not more than the areaof'the corresponding peak in the
cent) and the area of the peak due to propofol dimer is not
more than the area of the corresponding peak in the
cent).
Globule size. Not more than 5000 globules more than or equal
to 2 Jlm per ml ofa 0.01 per cent vIv dilution in a filtered 0.9 per
cent w/v solution of sodium chloride. By means of suitable
equipment, pass the solution through a 50 Jlm aperture and
count the number of globules by monitoring the change in
electrical current between two electrodes immersed on either
side of the aperture (such as Coulter Counter). Repeat the
procedure using the 0.9 per cent w/v solution of sodium
chloride only to determine the background count.
Free fatty acid. Not more than 7 millimoles per litre, when
determined by the following method.
To 5 ml ofa well shaken injection add 25 ml ofa mixture of40
volumes ofpropan-2-ol, 10 volumes ofn-heptane and 1 volume
of 0.5 sulphuric acid and shake for 1 minute. Allow to stand
for 10 minutes, add 15 ml ofn-heptane and 15 ml ofwater. Mix
by inverting the container 10 times and allow to stand for 15
minutes. To 15 ml of the upper phase add 5 ml of nile blue A
solution and pass a stream of nitrogen, previously passed
through 0.1 M sodium hydroxide, through the solution. Titrate
with 0.02 M sodium hydroxide, using a microburette. Calculate
the content offree fatty acid from a calibration curve prepared
from quantities of 1, 2, 4, 6 and 8 ml of a 1.282 per cent w/v
solution ofpalmitic acid in n-heptane, each diluted to 50 ml
with n- heptane. Carry out the method described above, using
5 ml ofeach solution and beginning at the words 'add 25 mlof
a mixture'.


Calculate the content of lysolecithin.
Units per ml.
Other tests. Complies. with the tests stated under Parenteral
Test solution. Dilute a volume of injection containing 80 mg of
Propofol to 100 ml with the propan-2-ol.
Reference solution (a). A 0.08 per cent wIv solution ofpropofol
RS in in propan-2-o1 containing 6.8 per cent v/v of water.
w/v of propofol RS and 0.00008 per cent w/v of propofol
impurity J RS in propan-2-o1 containing 6.8 per cent v/v of
water.
The chromatographic system described under Propofol
quinone and propofol dimer may be used but using a detection
wavelength of275 run and injecting 20 Jll of each solution.
resolution between the peaks due to propofol and propofol
impurity J is not less than 2.5.
Inject the test solution and reference solution (b).
Calculate the content ofC1zHIsO in the injection.
Storage. Store at a temperature not exceeding 30. It should
not be allowed to freeze.
Lysolecithin. Not more than 0.2 per cent w/v.

Test solution. Dilute a volume of injection containing 0.1 g of
Propofol in 100 ml ofthe mobile phase.
Reference solution. A 0.01 per cent w/v solution oflysolecithin
in the mobile phase.
- a stainless steelcolurnn 15 cm X 4.6 mm, packed with
octadecylsilane bonded to porous silica (5 Jlm) (Such
as Nucleosil C 18),
- mobile phase: a mixture of 1.4 volumes of
orthophosphoric acid, 40 volumes of water and 60
CI6HzINOz,HCl
Mol. Wt. 295.8
Propranolol Hydrochloride is (RS)-I-[( I-methylethyl)amino]3-(naphthalen-l-yloxy)propan-2-01 hydrochloride.

Propranolol Hydrochloride contains not less than 99.0 per
cent and not more than 101.0 per cent of CI6HzINOz, HCl,
Category. Antihypertensive, antianginal and antiarrhythmic
agent.
1987
IP 2010
PROPRANOLOL HYDROCHLORIDE
Dose. Orally, 20 mg to 2 g daily, in divided doses; the initial

dose should not exceed 40 mg; by slow intravenous injection,
3 to 10mg.
Identification
Compare the spectrum with that obtained with propranolol
propranolol hydrochloride.
maxima at about 290 nm, 306 nm and 319 nm.
Assay. Weigh accurately about 0.25 g, dissolve in 25 mlof

ethanol (95 percent) and titrate with 0.1 Msodium hydroxide,
CI6HzINOz, HCl.
Propranolol Injection
Tests
Propranolol Hydrochloride Injection

methanol is clear (2.4.1), and not more intensely coloured
than degree 60fthe appropriate range of reference solutions
(2.4.1).
Propranolol Injection is a sterile solution of Propranolol

Hydrochloride in Water for Injections containing Citric Acid.
pH (2.4.24).5.0 to 6.0, determined in a 1.0 per centw/v solution.

Specific optical rotation (2.4.22). -1.0 to +1.0, determined in
a 4.0 per cent w/v solution.
.
(2.4.14).

ml with the mobile phase. Dilute 1.0 ml ofthis solution to 10.0
- mobile phase: dissolve 1.6 g of sodium laurylsulphate
and 0.31 g of tetrabutylammonium dihydrogen
phosphate in a mixture of 1 ml of sulphuric acid, 450 ml
of water and 550 ml of acetonitrile, adjust to pH 3.3
using dilute sodium hydroxide solution,
- spectrophotometer set at292 nm,
injection volume. 20 ~I.
solution (0.1 per cent) and the sum ofall the secondary peaks
Propranolol Injection contains not less than 90.0 per cent and
propranolol hydrochloride, CJ6HzJNOz, HCI.
Usual strength. 1 mgpermI.
Identification
A. Make alkaline with 1 M sodium hydroxide a volume
containing 10 mg of Propranolol Hydrochloride and extract
with three quantities, each of5 ml, of ether. Wash the combined
extracts with water until the washings are freefrorrialkali, dry
with anhydrous sodium sulphate, filter, evaporate the filtrate
to dryness and dry the residue at 50 at a pressure of2 kPa for
1 hour.
obtained with propranolol hydrochloride RS treated in the
same manner or with the reference spectrum of propranolol.
B. When examined in the range 230 nm to 360 nm (2.4.7),the
solution obtained in the Assay shows absorption maxima at
about 290 nm, 306 nm and 319 nm.
Tests
pH (2.4.24).3.0 to 3.5.
(2.4.14).
Test solution. Dilute a volume of injection containing about

100 mg ofPropranolol HydrocWoride in 100 ml of acetonitrile.
1988
IP 2010
PROPRANOLOL TABLETS
(5 /lm) (Such as Hypersil ODS),
- mobile phase: a mixture of 1.15 g of sodium dodecyl
sulphate, 10 ml of a mixture of 1 volume of sulphuric
acid and 9 volumes of water, 20 ml of a 1.7 percent
w/v solution of tetrabutylammonium dihydrogen
orthophosphate, 370 ml of water and 600 ml of
acetonitrile, adjusted to pH 3.3 with 2 M sodium
hydroxide,
- injection volume. 10 ~Ll.
peaks is not more than four times the area ofthe principal peak
per cent).
2 mg ofPropranolol Hydrochloride add sufficient methanol to
produce 100.0 ml and measure the absorbance ofthe resulting
solution at the maximum at about 290 urn (2.4.7). Calculate the
content of CI6HzINOz, HCI taking 206 as the specific
to dryness and dry the residue at 500 at a pressure of2 kPa for
1 hour.
obtained with propranolol hydrochloride RS treated in t he
same manner or with the reference spectrum of propranolol.

at about 290 nm, 306 nm and 319 nm.
Tests
Transfer one tablet to a 100-ml volumetric flask, add 5 ml of
dilute hydrochloric acid and allow to stand, swirling
occasionally, until it is disintegrated. Add about 70 ml of
methanol and shake well for about 1 minute. Dilute to volume
with methanol, mix and centrifuge an aliquot of the solution.
Dilute a suitable volume of the clear solution with methanol
to produce a solution containing 20 /lg of Propranolol
Hydrochloride per ml. Measure the absorbance ofthe resulting
solution at the maximum at about 290 nm (2.4.7), using
methanol as the blank. Calculate the content of CI6HzINOz,
HCI taking 206 as the specific absorbance at 290 nm.
Apparatus No.1,
Medium. 900 mltf 0.1 M hydrochloric acid,
Storage. Store protected from light, in a single dose containers.
Withdraw a suitable volume ofthe medium and filter. Dilute a

suitable volume ofthe filtrate with the same solvent. Measure
about 290 nm (2.4.7). Calculate the content ofC,6Hz,NOz,HCI
in the medium taking 206 as the specific absorbance at 290 urn.
Propranolol Tablets
D. Not less than 75 per cent ofthe stated amount ofC,6HzINOz,

HCI.
Propranolol Hydrochloride Tablets

(2.4.14).
Propranolol Tablets contain not less than 92.5 per cent and
propranolol hydrochloride, CI6HzlNOz, HCI.

containing about 100 mg ofPropranolol HydrochlOlide in 100.0
ml of methanol and filter.
Identification

A.Suspend a quantity of the powdered tablets containing

0.1 g of Propranolol Hydrochloride in 20 ml of water, filter,
make the filtrate alkaline with 1 M sodium hydroxide and extract
with three quantities, each of 10 ml, ofether. Wash the combined
extracts with water until the washings are free from alkali, dry
with anhydrous sodium sulphate, filter, evaporate the filtrate
(5 /lm) (Such as Hypersil ODS),
mobile phase: a mixture of 1.15 g of sodium dodecyl
sulphate, 10 ml of a mixture of 1 volume of sulphuric
1989
IP 2010
PROPYL GALLATE
acid and 9 volumes of water, 20 ml of a 1.7 per cent

w/v solution of tetrabutylammonium dihydrogen
orthophosphate, 370 ml of water and 600 ml of
acetonitrile, adjusted to pH 3.3 with 2 M sodium
hydroxide,
peaks is not more than four times the area ofthe principal peak
per cent).
quantity ofthe powder containing about 20 mg ofPropranolol
Hydrochloride and shake with 20 ml of water for 10 minutes.
Add 50 ml of methanol, shake for a further 10 minutes, add
sufficient methanol to produce 100.0 ml and filter. Dilute
10.0 ml ofthe filtrate to 50.0 ml with methanol and measure the
290 nm (2.4.7). Calculate the content ofC16HzlNOz, HCI taking
206 as the specific absorbance at 290 nm.
B. Dissolve 10 mg in 50 ml of water, cool and add 5 ml of dilute

ammonia solution; a red colour is produced which becomes
brown on standing and is restored on shaking.
C. Dissolve 5 mg in 50 ml of water and add 0.05 mlofjerric
chloride test solution; a bluish black colour is produced.
Tests
Chlorides (2.3.12). Shake 1.0 gwith 50 ml ofwater for 5 minutes
Sulphates (2.3.17). Shake 0.8 g with 100 ml of water for
5 minutes and filter. 15 ml ofthe filtrate complies with the limit
Storage. Store protected from light and moisture, in nonmetallic containers.
Propylene Glycol
1,2-Propanediol
'ii',
Storage. Store protected from light and mOIsture.
Propyl Gallate
C3H sOz
Mol. Wt. 76.1
Propylene Glycol is (RS)-propane-l ,2-diol.
HO~O~CH3
Category. Pharmaceutical aid (humectant; solvent).

Description. A clear, colourless, viscous liquid; practically
HO~
OH
Identification
Mol. Wt. 212.2
Propyl Qallate is propy13,4,5-trihydroxybenzoate.

Category. Pharmaceutical aid (antioxidant).
Description. A white or creamy white, crystalline powder;
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), a
about 0.49.
A. To 0.5 ml ofa 0.01 percentw/v solution, cooled in ice, add

5 ml ofa cooled mixture of 10 ml ofwater and 90 ml ofsulphuric
acid. Heat for 10 minutes on a water-bath at 70, cool and add
0.2 ml ofa 3 per cent w/v solution of ninhydrin in a 2.5 per cent
w/v solution of sodium metabisulphite; a violet colour slowly
appears.
B. Heat 0.15 ml with 0.1 g of boric acid; a pleasant odour

develops.
C. Add 1 ml to 0.5 g ofpotassiumbisulphate and heat gently;
a fruity odour develops and when the solution is heated to
dryness, no sharp, acrid smell of acrolein is perceptible.
1990
IP 2010
PROPYLPARABEN
Tests
diethylene glycol. The test is not valid unless in the

chromatogram obtained with the reference solution, the
resolution between the peaks due to propylene glycol (first
peak) and ethylene glycol (second peak) is not less than 1.0.
Appearance of solution. The substance under examination is

clear (2.4.1), and colourless (2.4.1).
Acidity. Mix 10 ml with 40 m1 of water and add 0.1 mlof
bromothymol blue solution. The solution is greenish yellow
and not more than 0.05 ml of 0.1 M sodium hydroxide is
required to change the colour to blue.
No peaks corresponding to ethylene glycol and diethylene

glycol are obtained in the chromatogram obtained with the
test solution.
Sulphated ash (2.3.18). Not more than 0.01 per cent w/w,
detennined by the following method. Heat 50 g until it burns,
and ignite. Allow to cool, moisten the residue with sulphuric
acid and ignite; repeat the operations.
Boiling range (2.4.8). 184 to 189.

Relative density (2.4.29). 1.035 to 1.040.
Heavy metals (2.3.13). Dilute 3 ml to 12 ml with water. The
Method D (5 ppm). Use lead standard solution (l ppm Pb) to
prepare the standard.
Oxidising substances. To 10 ml add 5 ml of water, 2 m1 of
potassium iodide solution and 2 m1 of 1 M sulphuric acid and
allow to stand in a ground-glass-stoppered flask protected
from light for 15 minutes. Titrate the liberated iodine with
0.05 M sodium thiosulphate using 1 ml of starch solution,
added towards the end of the titration, as indicator. Not more
than 0.2 ml of 0.05 M sodium thiosulphate is required.

5.0g.
Propylparaben
Propyl Hydroxybenzoate
Reducing substances. Mix 1 rnl with 1ml of6 M ammonia and

heat on a water-bath at 60 for 5 minutes; the solution is not
yellow. Immediately add 0.15 ml of 0.1 M silver nitrate; the
solution does not change its appearance within 5 minutes.
Ethylene glycol and diethylene glycol. Determine by gas
in sufficient ethanol (95 per cent) to produce 100 ml.
Reference solution. Dissolve 2 g of the substance under
examination, 0.02 g ofethylene glycol and 0.02 g ofdiethylene
glycol in ethanol (95 per cent) and dilute to 100 ml with the
same solvent.
- a glass column 1.5 m x 3 mm, packed with 12 per cent
Sorbitol ~)ll untreated siliceous earth (Such as
Chromosorb W-NAW (SINS),
temperature:
column: 165,
inlet port and detector at 260,
Inject 3 ).11 or other suitable volume ofthe test solution. Record
the chromatograms adjusting the sensitivity so that the height
ofthe peak due to propylene glycol is more than 50 per cent of
the full-scale deflection in the chromatograms. Inject the same
volume of reference solution and record the chromatograms.
The order of elution is propylene glycol, ethylene glycol and
Mol. Wt. 180.2

Propylparaben is propyI4-hydroxybenzoate.
Propylparaben contains not less than 99.0 per cent and not
more than 101.0 per cent ofC IOH I20 3, calculated on the dried
basis.
Identification
A. When examined in the range 230 nm to 360 urn (2.4.7), a
an absorbance maximum at about 258 nm; absorption at
258 urn, 0.44 to 0.47.
B. To about 10 mg in a test-tube add 1 ml ofsodium carbonate
solution, heat to boiling for 30 seconds and cool (solution A).
To a further 10 mg in a test-tube add 1 rnl ofsodium carbonate
solution; the substance partly dissolves (solution B). Add at
the same time to each of the solutions A and B 5 m1 of
aminophenazone solution and 1 ml ofpotassiumferricyanide
solution and mix. Solution B is yellow to orange-brown.
Solution A is orange to red and the colour is clearly more
intense than any similar colour that may be obtained with
solution B.
1991
PROPYLPARABEN
IP 2010
Tests
Appearance ofsolupon. Al 0.0 per cent w/v solution in ethanol

(95 Pier cent) is clear (2.4.1), and not more intensely coloured
Propylthiouracil
Acidity. Dissolve 1.0 g in sufficient ethanol (95 per cent) to

produce 10 ml. To 2 ml of the solution add 3 ml of ethanol
(95 per cent), 5 ml of carbon dioxide-ji-ee water and 0.1 mlof
bromocresol green solution. Not more than 0.1 ml of Od M
. solution.
H3C~NyS
. . lrr
NH
Related substances. Detelmine by thin-layer chromatography

C7H ION 20S
Mobile phase. A mixture of 1 volume ofglacial acetic acid, 30

volumes of water and 70 volumes of methanol.
Propylthiouracil is 2,3-dibydro-6-propyl-2-thioxopyrimidin4(1H)-one.

examination in 10.0 ml of acetone.
Propylthiouracil contains not less than 98.0 per cent and not
more than 100.5 per cent ofC 7H ION 20S, calculated on the dried
basis.

100.Oml with acetone.
Reference solution (b). Dissolve 10 mg of ethyl
parahydroxybenzoate RS in 1.0 ml of the test solution and
dilute to 10.0 ml with acetone.
Apply 2 !!l of each solution. Allow the mobile phase to rise 15
cm. Dry the plate in air and examine in ultraviolet light at 254
nm. In the chromatogram obtained with the test solution, any
secondary spot is not more intense than the cOlTesponding
(0.5 per cent) The test is not valid unless the chromatogram
obtained with reference solution (b) shows two Clearly
separated principal spots.
Chlorides (2.3.12). Heat 2.0 gwith 100 ml of water, cool, add
sufficient water to restore the original volume, and filter. 25 ml
of the filtrate complies with the limit test for chlorides
(500 ppm).
Sulphates.To 10 ml of the filtrate obtained in the test for
Chlorides add 0.15 ml of dilute hydrochloric acid and 0.1 m1
of barium chloride solution; no turbidity is produced within
10 minutes.
on 1.0 g by drying over silica gel for 5 hours.
Assay. To 1.0 g add 20.0 ml of 1 M sodium hydroxide. Heat at
about 70 for 1 hour. Cool rapidly in an ice bath. Titrate the
excess sodium hydroxide with 0.5 M sulphuric acid,
continuing the titration until the second point ofinflexion and
detelmining the end-point potentiometrically (2.4.25).
CIOH I20 3
Mol. Wt. 170.2
Category. Antithyroid.
Description. A white or pale cream-coloured crystals or
crystalline powder; odourless.
Identification
A. Detenmne by infrared absorption spectrophotometry (2.4.6).
propylthiouracil RS or with the reference spectrum of
propylthiouracil.
B. Examine the chromatograms obtained in the test for Related

substances in ultraviolet light at 254 nm before exposure of
the plate to iodine vapour. The principal spot in the
chromatogram obtained with test solution (b) cOlTesponds to
(b).
C. To about 20 mg add 8 ml of bromine water and shake for a
few minutes. Boil until the mixture is decolorised, allow to cool

and filter. Add 2 ml of barium chloride solution; a white
precipitate is produced. Add 5 ml of 2 M sodium hydroxide;
the precipitate does not become violet.
Tests

12 volumes of 2-propanol and 0.2 volume of glacial acetic
acid.
1992
IP 2010
PROPYLTHIOURACIL TABLETS

Reference solution (a). A 0.01 per cent w/v,solution of the
propylthiouracil RS in methanol.
thiourea in methanol.
Expose the plate to iodine vapour for 10 minutes. By both
methods of visualisation, any spot corresponding to thiourea
in the chromatogram obtained with test solution (a) is not
with reference solution (c) and any other secondary spot is
not more intense than the spot in the. chromatogram obtained
Arsenic (2.3.1 0). Dissolve 2.0 g in 50 ml ofwater and add 10 rnl
of stannated hydrochloric acid AsT. The resulting solution
heavy metals, Method C (20 ppm).
on 1.0 g by drying in oven at 105.
30.0 ml (n] ml) of0.1 M sodium hydroxide, boil and shake until
solution is complete. Add 50 ml of 0.1 M silver nitrate with
stirring, boil gently for 5 minutes, cool and titrate with 0.1 M
potentiometrically (2.4.25) (n2 ml). Record the total volume
(n] + n2 ml) of 0.1 M sodium hydroxide added.
1 ml of0.1 M sodium hydroxide is equivalent to 0.008511 g of
C7H ION20S.
Identification
of Propylthiouracil with 20 ml of methanol for 10 minutes,
filter and evaporate to dryness.
obtained with propylthiouracil RS or with the reference
spectrum ofpropylthiouracil.
of Propylthiouracil with 60 ml of methanol for 20 minutes,
dilute to 100 ml with methanol and filter. Dilute 5 ml of the
filtrate to 250 ml with methanol. When examined in the range
230 nm to 360 nm (2.4.7), the resulting solution shows an
absorption maximum only at about 274 nm.
C. Extract a quantity of the powdered tablets in a continuous

extraction apparatus (2.1.8) with ether and evaporate the
solution to dryness. The residue, after drying at 105, melts at
about 219 (2.4.21).
Tests
12 volumes of 2-propanol and 0.2 volume of glacial acetic
acid.
containing 50 mg ofPropylthiouracil with 5 ml ofmethanol for
15 minutes, filter and use the filtrate.
thiourea in methanol.
dry the plate in air and examine in ultraviolet light at 254 mTI.
Expose the plate to iodine vapour for 10 minutes. By both
methods of visualisation, any spot corresponding to thiourea
with reference solution (b) and any other secondary spot is
Other tests. Comply with the tests stated under Tablets
Propylthiouracil Tablets contain not less than 92.5 per cent
propylthiouracil, C7H ION 20S.

Propylthiouracil, dissolve in a mixture of20 ml of0.1 M sodium
hydroxide and 75 ml ofwater with the aid ofgentle heat. Cool,
add 4 g of sodium acetate, just acidity the solution to litmus
paper with 6 M acetic acid, add 0.5 ml of a freshly prepared
1993
PROPYPHENAZONE
IP 2010
0.5 per cent w/v solution of 1,5-diphenylcarbazone in ethanol

(95 per cent) and titrate with 0.02 M mercuric nitrate until a
pinkish violet colour persists for 2 to 3 minutes.
I ml of 0.02 M mercuric nitrate is equivalent to 0.006808 g of
C7H ION zOS.
Propyphenazone
Tests
colourless (2.4.1).
phenolphthalein solution; the solution is colourless. Add
0.2 ml of 0.01 M sodium hydroxide; the solution is pink. Add
0.4 ml of 0.01 M hydrochloric acid; the solution becomes
colourless. Add 0.2 ml of methyl red solution; the solution is
orange or red.

45 volumes of ethyl acetate and 10 volumes of I-butanol.
Test solution (a). An 8 per cent w/v solution ofthe substance
Test solution (b). A 1.6 per cent w/v solution of the substance
Mol. Wt. 230.3

Propyphenazone is 4-isopropyl-2,3-dimethyl-l-phenyl-3pyrazolin-5-one.

propyphenazone RS in methanol.
Propyphenazone contains not less than 99.0 per cent and not
more than 101.0 per cent ofC 14H IsNzO, calculated on the dried
basis.

dry the plate in a current ofhot air for 15 minutes and examine
in ultraviolet light at 254 nm. Spray the plate with a mixture of
equal volumes of potassium ferricyanide solution and ferric
chloride solution. By both methods of visualisation, any
secondary spot in the chromatogram obtained with test
Category. Analgesic; antipyretic.

Dose. 1.5 to 3 g daily, in divided doses.
Description. A white or slightly yellowish, crystalline powder;
odourless.
Identification
propyphenazone RS or with the reference spectrum of
propyphenazone.
(b).
C. Dissolve 2 g in sufficient of a mixture of equal volumes of

ethanol (95 per cent) and carbon dioxide-free water to
produce 50 ml (solutionA). To 1 ml ofsolutionA add O.lml of
ferric chloride solution; a brownish red colour is produced
which becomes yellow on addition ofl ml of2 M hydrochloric
acid.
Arsenic (2.3.10). Mix 1.0 g with 10 ml ofa 2 per cent w/v

solution of magnesium nitrate in ethanol in a silica or platinum
dish, evaporate on a water-bath and heat gradually in order to
incinerate. If the material remains incompletely carbonised,
moisten with a small quantity of nitric acid and ignite again.
Cool, add 3 ml of hydrochloric acid and heat on a water-bath
to dissolve the residue. The resulting solution complies with
the limit test for arsenic (10 ppm).
at a pressure of 1.5 to 2.5 kPa for 4 hours.
Assay. Weigh accurately about 0.2 g of the dried material,
dlssoive in 3()11110f anhjid,;ous glacial acetic acid. titraTe
with 0.1 M perchloric acid, determining the end-point
1994
IP 2010
PROTAMINE SULPHATE
1 m1 of 0.1 M perchloric acid is equivalent to 0.02303 g of

C1JflSN20.
Protamine Sulphate
Protamine Sulphate is a purified mixture of the su1phates of
basic peptides prepared from the sperm or mature testes of
suitable species of fish. It binds with heparin in solution,
inhibiting its anticoagulant activity. It is prepared in conditions
designed to minimise the degree of Microbial contamination.
Each mg of Protamine Sulphate precipitates not less than
100 Units of heparin sodium RS, calculated on the dried basis.
Category. Heparin antidote.
Dose. By slow intravenous injectiCln over a 1O-minute period,
1 mg for every 100 Units ofheparin remaining in the patient if
given within 15 minutes; maximum 50 mg.
Iron (2.3.14). Dissolve 2.0 g in water with the aid ofheat and
dilute to 20 ml with water. The resulting solution complies
with the limit test for iron (20 ppm).
Mercury. Add 20 ml of a mixture of equal volumes of nitric
acid and sulphuric acid to 2.0 g in a 250-ml flask fitted with a
ground-glass stopper, boil under a reflux condenser for 1 hour,
cool and carefully dilute with water. Boil until nitrous fumes
are no longer evolved. Cool, carefully dilute the solution to
200 ml with water, mix and filter. Transfer 50 ml ofthe filtrate to
a separating funnel. Shake with successive small quantities of
chloroform until the chloroform layer remains colourless. To
the aqueous layer add 25 ml of 1 M sulphuric acid, 115 ml of
water and 10 ml ofa 20 per cent w/v solution of hydroxylamine
hydrochloride. Titrate with dithizone solution; after each
addition, shake the mixture 20 times and towards the endpoint of the titration allow to separate and discard the
chloroform layer. Titrate until a greenish blue colour is
produced. Calculate the content of mercury using the
equivalent ofmercury in Ilg per ml oftitrant determined in the
standardisation ofthe dithizone solution (10 ppm).
Description. A white or almost white powder; hygroscopic.
Nitrogen (2.3.30). 21.0 to 26.0 per cent, calculated on the dried

basis, determined by Method C.
Identification
Sulphates. 16 to 24 per cent, determined by the following

method. Dissolve 0.15 gin 15 ml of water in a beaker, add 5 ml
of 2 M hydrochloric acid and heat to boiling. Slowly addto
the boiling solution 10 ml of barium chloride solution. Cover
and heat on a water-bath for 1 hour. Filter and wash the
precipitate several times with small quantities of hot water.
Dry and ignite the residue to constant weight at 600.
A. Produces a precipitate under the conditions of the Assay.

B. Dissolve 0.2 g in 5 ml of water and dilute to 10 ml with the
same solvent (solution A). To 0.5 ml ofsolution A add 4.5 ml of
water, 1 ml of a 10 per cent w/v solution of sodium hydroxide
and 1 m1 ofa 0.02 per cent w/v solution of I-naphthol and mix.
. Cool to 5 and add 0.5ml of alkaline sodium hypobromite
solution; an intense red colour is produced.
C. Heat 2 ml ofso1utionAon a water-bath at 60, add 0.1 ml of
mercuric sulphate solution and mix; no precipitate is produced.
Cool the mixture in ice; a white precipitate is produced.
D. Gives reactionAofsulphates (2.3.1).
Tests
Appearance ofsolution. To 2.5 ml ofsolution A add 7.5 mlof
water. The resulting solution is not more opalescent than
opalescence standard OS2 (2.4.1), and not more intensely
.coloured than reference solution BYS6 (2.4.1).
acid.
Light absorption (2.4.7). Dilute 2.5 ml ofsolution A to 5.0 ml
with water. Absorbance of the resulting solution at 260 to
280 nm is not more than 0.1.
1 g ofthe residue is equivalent to 0.4117 g ofS04

toxicity, using 0.5 mg dissolved in 0.5 ml ofwater for injections.
Assay. Prepare solutions of the substance under examination
in water containing (1) 0.015 per cent w/v (2) 0.01 per cent
w/v and (3) 0.005 per cent w/v. Titrate each ofthe solutions in
duplicate with a 174 ill per ml or a suitable dilution of heparin
sodium RS using the following procedure. Introduce an
accurately measured volume ofthe solution to be titrated, for
example 1.5 m1, into the cell of a suitable spectrophotometer,
set the instrument at a suitable wavelength (none is critical) in
the visible range and add the titrant in small volumes until
there is a sharp increase in the absorbance. Note the volume
of titrant added.
Carry out three independent assays. For each individual
titration, calculate the number ofUnits of heparin titrated per
mg of the substance under examination. Calculate the result
ofthe assay as the average ofthe 18 values. Test the linearity
of the response by standard statistical methods. The assay is
1995
IP 2010
PROTAMINE SULPHATE INJECTION
not valid unless the standard deviations calculated for the

results obtained with each test solution are less than 5 per
cent of the average result.
Light absorption. Dilute the injection, ifnecessary, with water

to produce a solution containing 1 per cent w/v solution of
Protamine Sulphate. Absorbance of the resulting solution at
260 to 280 urn, not more than 0.1 (2.4.7).
Protamine Sulphate intended for use in the manufacture of

Parenteral Preparations without a further appropriate Bacterial endotoxins (2.2.3). Not more than 7.0 Endotoxin Units
procedure for the removal of bacterial endotoxins complies per mg of protamine sulphate.
Abnormal toxicity. Complies with the test for abnormal toxicity
Bacterial endotoxins (2.2.3). Not more than 7.0 Endotoxin Units (2.2.1), using a volume containing 0.5 mg of Protamine
per mg ofprotamine sulphate~
Sulphate~
Protamine Sulphate intended for use in the mr;lnufacture of Other tests. Complies with the tests stated under Parenteral
Parenteral Preparations without a fitrther appropriate Preparations (Injections).
Assay. Prepare solutionsofthe substance under examination
in water containing (1) 0.015 per cent w/v (2) 0.01 per cent
w/v and (3) 0.005 per cent w/v. Titrate each ofthe solutions in
Storage. Store protected from moisture. If it is intended for duplicate with a 174 IV per ml or a suitable dilution of heparin
use in the manufactUre ofParenteral Preparations, the container sodium RS using the following procedure. Introduce an
should be sterile and sealed so as to exclude micro-organisms. accurately measured volume of the solution to be titrated, for
Labelling. The label states whether or not the contents are example 1.5 ml, into the cell of a suitable spectrophotometer,
intended for use in the manufacture ofParenteral Preparations. set the instrument at a suitable wavelength (none is critical) in
the visible range and add the titrant in small volumes until
there is a sharp increase in the absorbance. Note the volume
of titrant added.
Identification
Carry out three independent assays. For each individual

titration, calculate the number ofUnits of heparin titrated per
mg of the substance under examination. Calculate the result
ofthe assay as the average ofthe 18 values. Test the linearity
of the response by standard statistical methods. The assay is
not valid unless the standard deviations calculated for the
results obtained with each test solution are less than 5 per
cent of the average result.
A. Produces a precipitate under the conditions of the Assay.
B. Dilute a suitable volume with water to give a solution

containing 0.2 per cent w/v solution of Protamine Sulphate.
To 5 ml of this solution add 1 ml of a 10 per cent w/v solution
of sodium hydroxide and 1 ml of a 0.02 per cent w/v solution
of I-naphthol and mix. Cool to 5 and add 0.5 ml of alkaline
sodium hypobromite solution; an intense red colour is
produced.
Labelling. The label states (1) that the dose is calculated from
the results of determinations of the amount required to
produce an acceptable blood-clotting time in the patient; (2)
the approximate number of Units of heparin activity 1 ml is
capable of neutralising.
C. Heat 2 ml on a water-bath at 60, add 0.1 ml of mercuric

sulphate solution and mix; no precipitate is produced. Cool
the mixture in ice; a white precipitate is produced.
Prothionamide
Protamine Sulphate Injection is a sterile solution ofProtamine

Sulphate in Water for Injections.
Protamine Sulphate Injection contains not less than 80.0 per
cent of the stated amount of protamine sulphate.
D. Gives reaction A ofsulphates (2.3.1).
Tests
pH (2.4.24).2.5 to 3.5.
Optical rotation (2.4.22). -0.52to -0.68, determined in a
solution prepared by diluting the injection with 0.5 M
hydrochloric acid so as to contain 0.8 per cent w/v ofProtamine
Sulphate.
C9H 1zNzS
Mol. wt.180.3
Prothionamide is 2-propyl-4-pyridinecarbothioamide.
1996
IP 2010
PROTHIONAMIDE TABLETS
Prothionamide contains not less than 99.0 per cent and not
more than 101.0 per cent ofCsHIQN2 S ,calculated on the dried
basis.
Category. Antitubercular.
reference solution (0.5 per cent) and the sum ofthe areas ofall
such peaks is not more than twice the area of the principal
(1.0 percent).
Description. A yellow, crystalline powder.
Identification

Compare the spectrum with that obtained with prothionamide
RS.

described under Related substances using the following
solutions.

absorption maximum only at about 291 nm. The absorbance at
291 nm is about 0.78.
Tests

examination in the mobile phase and dilute to 100.0 m1 with the
mobile phase. Dilute 5.0 ml ofthis solution to a 50.0 ml with the
mobile phase.
Acidity. Dissolve 2.0 g in 20 ml of methanol, heating to about Reference solution. A 0.05 per cent w/v solution of
50, and add 20 ml of water. Cool slightly, shake until prothionamide RS in the mobile phase. Dilute 5.0 ml of the
crystallisation occurs, if any and allow to cool to room solution to 50.0 ml with the mobile phase.
temperature. Add 60 ml of water and titrate with 0.1 M sodium
. Inject the reference solution. The test is not valid unless the
hydroxide using 0.2 ml of cresol redsolution as indicator. Not
more than 0.2 ml of 0.1 M sodium hydroxide is required to
change the colour of the indicator to red.
(2.4.14).
Calculate the content ofC 9H I2N 2S.
examination in the mobile phase and dilute to 100 ml with the Storage. Store protected from light and moisture.
mobile phase.

prothionamide RS in the mobile phase. Dilute 1 ml of the
solution to 100 ml with the mobile phase.
mobile phase: 60 volumes of a buffer solution prepared
by mixing 2.0 ml of triethylamine with 1000 ml of water,
adjusting the pH to 6.0 with dilute orthophosphoric
- injection volume. 20 !-tl.
Inject the reference solution.The test is not valid unless the
than 2.0 per cent.
Inject alternatively the test solution and the reference solution.
of any individual impurity peak is not more than the area of
the principal peak in the chromatogram obtained with the
Prothionamide Tablets contain not less than 90.0 per cent not
more than 110.0 per cent ofthe stated amount ofprothionamide,
C9H I2N2S.
Identification
A. Extract a quantity of the powdered tablets containing 25
mg ofProthionamide with 5 rnl of methanol, filter and evaporate
test.
Compare the spectrum with that obtained with prothionamide
RS.
1997
IP 2010
PROTHIONAMIDE TABLETS
Tests
of water and adjusting the pH to 6.0 with dilute

orthophosphoric acid, and 40 volumes of acetonitrile,
- flow rate. 1 ml perminute,
- injection volume. 20 jll.
Apparatus No.2,
Medium. 900 ml 0.1 M hydrochloric acid,
the dissolution medium ifnecessary, at the maximum at about
290 nm (2.4.7). Calculate the content ofC9H 1zNzS in the medium
concentration of prothionamide RS in the same medium.
D. Not less than 75 per cent ofthe stated amount ofC 9H 12N zS.
(2.4.14) as described under Assay using the following
solutions.
tailing factor is not more than 2.0 the column efficiency is not
Calculate the content ofC9H 1zN zS in the tablets.
Protriptyline Hydrochloride

tablets containing 50 mg of Prothionamide disperse in the
mobile phase, shake, dilute to 100 ml with the mobile phase
and filter.
, Hel

ofprothionamide RS in the mobile phase. Dilute 1.0 ml ofthe
solution to 100.0 ml with the mobile phase.
than 2.0 per cent.
of any individual impurity peak is not more than the area of
the peak in the chromatogram obtainxd with the reference
solution (0.5 per cent) and the sum of the areas of all such
impurities is not more than twice the area of the peak in the
cent).

Prothionamide, disperse in the mobile phase, shake and dilute
to 100.0 ml with the mobile phase. Dilute 5.0 ml ofthe resulting
prothionamide RS in the mobile phase. Dilute 5.0 ml of the
- a stainless steel Golumn 25 cm x 4.6 mID, packed with
octadecylsilane bonded to porous silica (5 jlm),
prepared by mixing 2.0 ml of triethylamine with 1000 ml
Mol. Wt. 299.8

Protriptyline Hydrochloride is 3-(5H-dibenzo[a,d]cyc1ohept5-yl)propyl(methyl)amine hydrochloride.
Protriptyline Hydrochloride contains not less than 99.0 per
cent and not more than 101.0 per cent ofC I9Hz1 N,HCl, calculated
on the dried basis.
Category. Tricyclic antidepressant.
Description. A white to yellowish white powder.
Identification
A. Dissolve 0.1 gin 10 ml of water, make alkaline with 1M
sodium hydroxide, extract with 5 ml of chloroform, dry with
anhydrous sodium sulphate and evaporate the solvent using
a current of nitrogen. On the oily residue, determine by the
infrared absorption spectrophotometry (2.4:6). Compare the
spectrum with that obtained with protriptyline hydrochloride
RS or with the reference spectrum of protriptyline
hydrochloride.
Tests
pH (2.4.24). 5.0to 6.5,determinedina 1.0percentw/v solution.
0.7kPa.
1998
IP 2010
PSEUDOEPHEDRINE HYDROCHLORIDE
Pseudoephedrine Hydrochloride

Assay. Weigh accurately about 0.7 g, dissolve in 0.5 ml of
acetic anhydride and 20 ml of anhydrous glacial acetic acid,
add 10 m1 of mercuric acetate solution. Titrate with 0.1 M
perchloric acid, using clystal violet solution as indicator.
H pH
~
I
.0
CH3
' NHCH
HCI
1 ml of O.lM perchloric acid is equivalent to 0.02998 g of

C I9H21 N,HCl.
CIOH1sNO,HCl
Pseudoephedrine Hydrochloride contains not less than

99.0 per cent and not more than 101.0 per cent ofCIOHlsNO,
HCl, calculated on the dried basis.
Protriptyline Hydrochloride Tablets

Protriptyline Tablets contain not less than 90.0 per cent and
protriptyline hydrochloride, C 19H 21 N, HCl.
Mol. Wt. 201.7
Pseudoephedrine Hydrochloride is (lS,2S)-2-methylaminoI-phenylpropan-1-01 hydrochloride.
Dose. 60 mg three to four times daily.
Description. A white, crystalline powder; odourless or almost
odourless.
Identification
Identification
A. When examined in the range 230 to 350 nm (2.4.7), a solution

obtained in the Assay exhibits a maximum only at 292 nm.
B. Shake a quantity of the powdered tablets containing about
5 mg ofProtriptyline Hydrochloride with 20 m1 of methanol
and filter. To 1 ml ofthe filtrate add 1 ml of a 2.5 per cent w/v
solution of sodium hydrogen carbonate, 1 ml of a 2 per cent
w/v solution of sodiumperiodate and 1 ml ofa 0.3 per cent wi
v solution ofpotassium permanganate. Allow to stand for 15
minutes, acidify with 1 M sulphuric acid and extract with 10
ml of 2,2,4-trimethylpentane, washing the extract with 10 ml
of 0.25 M sulphuric acid. The absorbance oftrimethyIpentane
.solution does not exhibit a maxima at 265 nm (2.4.7) (distinction
from amitriptyline and nortriptyline).

pseudoephedrine hydrochloride RS or with the reference
spectrum of pseudoephedrine hydrochloride.
0.1 per cent w/v solution shows absorption maxima at about
251 nm, 257 nm and 263 nm; absorbances at the maxima, about
0.75, about 0.98 and about 0.78, respectively.
C. A 5 per cent w/v solution gives the reactions of chlorides
(2.3.1).
Tests
C. Triturate a quantity of the powdered tablets containing

about 0.1 g of Protriptyline Hydrochloride with 10 ml of
chloroform, filter and evaporate the filtrate to dryness.
Dissolve part ofthe residue in 3 ml of water and add 0.05 ml of
a 2.5 per cent w/v solution of quinhydrone in methanol. A red
colour develops.
Tests

(2.4.14).

Assay. Triturate 10 tablets for 15 minutes with 100 ml of a
solution prepared by mixing 1 volume of 1 M hydrochloric
acid and 9 volumes of methanol , transfer to a graduated
flask using sufficient ofthe solvent mixture to produce 250 ml,
mix and filter. Dilute a volume ofthe filtrate containing about 1
mg ofProtriptyline Hydrochloride to 100 ml with the solvent
mixture and measure the absorbance at the maximmn at 292 nm
(2.4.7). Calculate the content ofC I 9H2 IN, HCl taking 465 as the
absorbance.
Appearance of solution. A 5.0 per cent w/v solution is not

more than very faintly opalescent and colourless (2.4.1).
Specific optical rotation (2.4.22). +61.0 to +62.so, determined

in a 5.0 per cent w/v solution using a 2-dm tube.

mobile phase.
Reference solution (a). Dissolve 20 mg of ephedrine
hydrochloride RS (pseudoephedrine impurity A RS) in 20.0
ml ofthe mobile phase. Dilute 1.0 ml ofthis solution to 50.0 ml
1999
PSEUDOEPHEDRINE SYRUP
IP 2010
Reference solulion (c). Dissolve 10. mg of ephedrine

hydrochloride RS (pseudoephedrine impurity A RS) in 5 ml
ofthe test solution and dilute to 100 ml with the mobile phase.
Pseudoephedrine Syrup contains not less than 90.0 percent

and not more than 110.0 per cent of the. stated amount of
pseudoephedrine hydrochloride, CIOH1SNO, HCI.
phenylsilane bonded to porous silica (5 /lm),
94 volumes of 1.2 per cent w/v solution of ammonium
acetate, adjusted to pH 4.0 with glacial acetic acid,
Inject the test solution, reference solution (a), (b) and (c). Run
the chromatogram 1.5 times the retention time of
pseudoephedrine. The relative retention time with reference
to pseudoephedrine for pseudoephedrine impurity A is about
0.89. The test is not validunless the resolution between the
peaks due to pseudoephedrine impurity A and
pseudoephedrine is not less than 2.0. In the chromatogram
obtained with the test solution the area of the peak due to
pseudoephedrine impurity A is not more than the area of the
solution (a) (1.0 per cent), the area of any other secondary
cent). The sum of all the secondary peaks other than
pseudoephedrine impurity A is not more than twice the area of
the principal peale in the chromatogram obtained with reference
than 0.1 times the area oftheprincipal peale in the chromatogram
50 ml of anhydrous glacial acetic acid and 10 ml of mercuric
acetate solution. Titrate with 0.1 M perchloric acid, using
clystal violet solution as indicator. Carry out a blank titration.
1 ml of 0.1 M perchloric acid is equivalenttoO.020l7g of
CIOH1SNO, HCI.
Pseudoephedrine Hydrochloride Syrup
Pseudoephedrine Syrup is a solution of Pseudoephedrine
Hydrochloride in a suitable flavoured vehicle.
Identification
A. Shake a quantity of the syrup containing 120 mg of
Pseudoephedrine Hydrochloride with two quantifies, each of
30 ml, of ether, and discard the ether layer. Add 4 ml of
1 M sodium hydroxide to the aqueous layer and extract with
two quantities, each of 10 ml, of ether. Dry the combined ether
extracts with anhydrous sodium sulphate, filter and evaporate
the filtrate to dryness.
obtained with pseudoephedrine hydrochloride RS treated in
the saame manner or with the reference spectrum of
pseudoephedrine.
B. The residue obtained in testA melts at about U8 (2.4.21).
C. Dissolve 50 mg ofthe residue obtained in testA in 10 ml of
0.1 M hydrochloric acid; it is dextro-rotatory.
Tests
Test solution. To an accurately measured volume of the syrup

containing about 0.12 g of Pseudoephedrine Hydrochloride,
add 50 ml of 0.1 M hydrochloric acid mix, add sufficient
0.1 M hydrochloric acid to produce 100.0 ml, filter and use
the filtrate.
pseudoephedrine hydrochloride RS in 0.1 M hydrochloric
acid.
mobile phase: a mixture of 85 volumes of ethanoland
15 volumes ofa 0.4 per cent w/v solution of ammonium
acetate,
injection volume. 20 /li.
Inject the reference solution and record the chromatogram.
The test is not valid unless the relative standard deviation is
not more than 2.0 per cent and the tailing factor is not more
than 1.5.
Inject altemately the test solution and the reference solution..
2000
IP 2010
PSORALEN
Determine the weight per ml ofthe syrup (2.4.29), and calculate

the content ofCIOHlsNO, HCl weight in volume.
Pseudoephedrine Hydrochloride Tablets
Apply to the plate 10 /-Ll of each solution. After development,

dry the plate in a current of warm air, spray with a solution
containing 0.3 g of ninhydrin in a mixture ofl 00 mIl-butanol
and 3 ml ofglacial acetic acid and heat at 1200 for 20 minutes.
any yellow spot near the line of application.
Pseudoephedrine Tablets contain not less than 95.0 per cent

pseudoephedrine hydrochloride, CIOH1SNO, HCI.
Identification
of Pseudoephedrine Hydrochloride with 10 ml of water and
filter. Shake the filtrate with 10 ml of ether, discard the ether
layer. Add 1 ml of 1 M sodium hydroxide to the aqueous layer
and extract with two quantities, each of 10 ml, of ether. Dry the
combined ether extracts with anhydrous sodium sulphate,
filter and evaporate the filtrate to dryness.
obtained with pseudoephedrine hydrochloride RS treated in
the same manner or with the reference spectrum of
pseudoephedrine.
Test solution. Weigh and powder 20 tablets. To a quantity of

the powdered tablets containing about 0.12 g of
Pseudoephedrine Hydrochloride, add 50 ml of 0.1 M
hydrochloric acid mix with the aid ofultrasound for 15 minutes,
add sufficient methanol to produce 100.0 ml, filter and use the
filtrate.
pseudoephedrine hydrochloride RS in methanol (50 per
cent).
octadecylsilane bonded to porous silica (10 /-Lm),
- mobile phase: 0.005 M dioctyl sodium sulphosuccinate
in a mixture of 65 volumes of methanol, 35 volumes of
water and 1 volume of glacial acetic acid,
- flow rate; 1.5 ml per minute,
- spectrophotometer set at 258 om,
- injection volume. 20 /-Ll.

Tests
Calculate the content ofC IOH 1sNO,HCI in the tablets.

Mobile phase. A mixture of 40 volumes of butyl acetate,

20 volumes of acetone, 20 volumes of I-butanol, 10 volumes
of 5 M ammonia and 10 volumes of methanol.
Test solution (a). Add 25 ml of methanol to a quantity ofthe
powdered tablets containing 0.5 g of Pseudoephedrine
Hydrochloride, shake for 5 minutes, filter, wash the filter with
methanol and evaporate the combined filtrate and washings
to dryness. Dissolve the residue as completely as possible in
5 ml of methanol, centrifuge and use the supernatant liquid.
Psoralen
Mol. Wt. 186.2
Test solution (b). Dilute 1 volume of the test solution to

Psoralen is 7H- furo[3 ,2-g][ 1]benzopyran-7-one, obtained

from the fruits of Psoralea carylifolia Linn. (Fam. Leguminosae) and from the leaves of Ficus carica (Fam.
Urticaceae) or prepared by synthesis.

Psoralen contains not less than 95.0 per cent and not more
than 101.0 per cent ofC Il H 60 3 , calculated on the dried basis.

pseudoephedrine hydrochloride RS in methanol.
Category. Topical pigmenting agent.

Description. Colourless needles; odourless.
2001
IP 2010
PSORALEN
Identification
Pyrantel Pamoate
A. Dissolve 1 mg in 5 ml of ethanol (95 per cent) and add

15 ml ofamixture containing 43 volumes of water, 5 volumes
of acetic acid and 3 volumes of propylene glycol; a blue
fluorescence is visible in ultraviolet light at 365 nm.
Pyrantel Embonate
COOH
OH
B. Dissolve 1 mg in 2 ml of ethanol (95 per cent) and add

0.1 ml of 0.1 M sodium hydroxide; a yellow fluorescence is
visible in ultraviolet light at 365 nm.
OH
Tests
COOH

Mobile phase. A mixture of 90 volumes of benzene and 10

Reference solution. Dilute 1 volume ofthe test solution to 100
volumes with chloroform.
Apply to the plate 5 /-ll of each solution. After development,
methanol to produce 100.0 ml. Dilute 2.0 ml ofthis solution to
100.0 ml with methanol and measure the absorbance of the
resulting solution at the maximum at about 247 nm(2.4.7).
Calculate the content ofC 11 H60 3from the absorbance obtained
by repeating the operation using a final solution of 20 /-lg per
ml ofpsoralen RS in methanol in place ofthe substance under
examination.
CIIHI4NzS, CZ3HI606
Mol. Wt. 594.7
Pyrantel Pamoate is 1,4,5,6-tetrahydro-1-methyl-2-[(E)-2-(2thienyl)vinyl]pyrimidine hydrogen; 4,4'-methylene bis(3hydroxynaphthalene-2-carboxylate).

Pyrantel Pamoate contains not less than 98.0 per cent and not
more than 102.0 per cent of C34H30Nz06S, calculated on the
dried basis.
Dose. 10 mg per kg
Description. A pale yellow or yellow powder.
Identification
Compare the spectrum with that obtained with pyrantel
pamoate RS or with the reference spectrum of pyrantel
pamoate.
Tests
(2.4.14).

protectedii'om light.
Solvent mixture. Mix 25 volumes of glacial acetic acid, 25
volumes of water and 10 volumes of diethylamine with cooling.
examination in 7 ml ofthe solvent mixture and dilute to 100.0
ml with mobile phase. .
Reference solution (a). Dissolve 10.0 mg of 1-methyl-2-[(Z)2-(thiophen-2-yl)-1,4,5,6-tetrahydropyrimidine RS (pyrantel
impurity A RS) in the solvent mixture, add 2.5 ml of the test
solution and dilute to 50.0 ml with the mobile phase. Dilute 2.0
ml ofthis solution to 100.0 ml with the mobile phase.
silica gel (5 /-lm),
2002
PYRANTEL PAMOATE ORAL SUSPENSION
IP 2010
- mobile phase: 7.2 volumes of the solvent mixture and

92.8 volumes of acetonitrile,
- flow rate. I ml per minute,
- spectrophotometer set at 288 mn,
Inject reference solution (a). Run the chromatogram for 4 times
the retention time of pyrantel. The test is not valid unless the
resolution between the peaks due to pyrantel and pyrantel
impurity A is at least 4.0. The relative retention times with
reference to pyrantel pamoate are about 0.5 for pamoic acid,
about 1.3 for pyrantel impurity A and about 1.8 for ((E)-N{methylamino)propyl]-3-(thiophen-2-yl)prop-2-enamide)
pyrantel impurity B.
Inject the test solution, reference solutions (a) and (b). For the
calculation of content, multiply the peak area of pyrantel
impurity B by 0.4. In the chromatogram obtained with the test
solution, the area ofthe peak due to pyrantel impurity A is not
more than the corresponding peak in the chromatogram
obtained with reference solution (a) (0.5 per cent); the area of
the peak due to pyrantel impurity B is not more than the area
reference solution (b) (0.2 per cent); the area any other
principal peak in the chromatogram obtained with the reference
secondary peaks other than those of pyrantel impurities A
and B is not more than 0.6 times the area ofthe principal peak
(0.3 per cent). Ignore any peak with an area less than 0.1 times
with reference solution (b) (0.05 per cent).
Chlorides (2.3.12). To 0.7 g, add 10 ml of dilute nitric acid
and 30 ml of water. Heat on water-bath for 5 minutes and cool.
The resulting solution complies with the limit test for chlorides
(360 ppm).
Sulphates (2.3.17). To 0.5g, add 2.5 ml of dilute nitric acid
and dilute to 50 ml with water. Heat on water bath for 5 minutes.
15 ml complies with the limit test for sulphates (0.1 per cent).
Iron (2.3.14). Ignite 0.54 g at 8000 for 2 hours. Dissolve the
residue in 2.5 ml of dilute hydrochloric acid with gentle heating
for 10 minutes and cool. The resulting solution complies with
the limittest for iron (75 ppm).
for 10 minutes. Allow to cool (a clear solution is not obtained).

potentiometrically (2.4.25). Carry out a blank titration. .
C34H30Nz06S,
Pyrantel Pamoate Oral Suspension

Pyrantel Pamoate Oral Suspension is a suspension ofPyrantel
Pamoate in a suitable flavoured aqueous vehiCle.
Pyrantel Pamoate Oral Suspension contains not less than 90.0
ofpyrantel, CIIHI4NzS.
Usual strength. 50 mg per ml
Identification
the plate with silica gel H.
Mobile phase. The upper layer ofa mixture obtained by shaking

20 volumes of methyl isobutyl ketone, 10 volumesofJormic
acid and 10 volumes of water.
Test solution. Dilute a known volume of the oral suspension
with sufficient 0.05 M methanolic ammonia to produce a
solution containing about 8 mg ofpyrantel per ml. Shake the
mixture by mechanical means and centrifuge. Use the clear
supernatant liquid.
ReJerencesolution. A solution of pyrantel pamoate RS in
0.05 M methanolic ammonia containing 0.008 per cent w/v of
pyrantel.
phase to rise to about 17 cm. Dry the plate in air and examine
in ultraviolet light at 365 nm. The principal spot in the
B. In the Assay, the major peaks in the chromatogram obtained
with the test solution correspond to the peaks in the
Tests
pH (2.4.24). 4.5 to 6.0
Loss on drying (2.4.1 9). Not more than 1.0 per cent, determined
on 1.0 g by drying in vacuum at 60 0 for 3 hours.
NOTE- Carry out the test protectedfi-om light.
Assay. Weigh accurately about 0.5 g, add 10 ml of acetic

anhydride and 50 ml glacial acetic acid. Heat at 500 and stir
Test solution. Weigh accurately a quantity of the oral

suspension containing about 60 mg of pyrantel, disperse in
2003
IP 2010
PYRANTELPAMOATE ORAL SUSPENSION
60 ml of water and add sufficient water to produce 100.0 m!.

Dilute 1.0 ml ofthe well-stirred susptmsion to 25.0 ml with the
mobile phase to obtain a clear solution. Mix and filter.
Reference solution. Weigh accurately about 20 mg ofpyrantel

pamoate RS and dissolve in sufficient mobile phase to produce
25.0 ml. Dilute 10.0 ml ofthe resulting solution to 100.0 ml with
the mobile phase and mix.
- a stainless stefllcolumn 25 cm x4.6 mill, packed with
porous siiica particles (5 to 10 Ilm),
mobile phase: a mixture of 92.8 volumes of acetonitrile,
3 volumes of acetic acid, 3 volumes of water and 1.2
volumes of diethylamine,
- flow rate. 1 mlper minute,
Inject the reference solution. The resolution between pyrantel
and pamoic acid is not less than 10.0, the column efficiency
for the pyrantel peak is not less than 8000 theoretical plates,
the tailing factor for the pyrantel peak is not greater than 1.3
Inject the test solution and t4e reference solution. Continue
the chromatography for a period not less than 2.5 times the
retention times of pyrantel base. The relative retention times
for pamoic acid and pyrantel base are about 0.6 and 1.0
respectively.
Determine the weight per ml (2.4.29) of the suspension and
calculate the content OfCIIHI4NzS, weight in volume.
exceeding 30.
equivalent amount of pyrantel.
Dose. Up to 35 mg per kg of body weight daily, in divided

doses.
Identification
Compare the spectrum with that obtained with pyrazinamide
RS or with the reference spectrum ofpyrazinamide.
B. Dissolve 50 mg in water and dilute to 100 ml with the same

solvent (solution A). Dilute 1 ml ofsolution A to 10 m!. When
examined in the range 290 om to 360 om (2.4.7), the resulting
solution shows an absorption maximum at about 310 om. Dilute
2 ml ofsolution A to 100 ml with water. When examined in the
range 230 om to 290 om, the solution shows an absorption
maximum at about 268 om; absorbance at about 268 om,
between 0.64 and 0.68.
C. Boil 20 mg with 5 ml ofsodium hydroxide solution; ammonia,
Tests
dioxide-free water (solution B) is clear (2.4.1), and colourless
(2.4.1).
Acidity or alkalinity. To 25 ml of solution B add 0.05 mlof
phenolphthalein solution and 0.2 ml of 0.01 M sodium
hydroxide; the solution is red. Add 1 ml of 0.01 Mhydrochloric
acid; the solution is colourless. Add 0.15 ml of methyl red
solution; the solution is red.

Pyrazinamide

Mol. Wt. 123.1
Pyrazinamide is pyrazine-2-carboxamide.
Pyrazinamide contains not less than 99.0 per cent and not
more than 100.5 per cent of CsHsN'lO; calculated on the
anhydrous basis.

dry the plate in air and examine in ultraviolet light at 254 om.
2004
IP 2010
PYRIDOXINE HYDROCHLORIDE

5.0g.
Assay. Weigh accurately about 0.3 g and transfer to the flask
ofan ammonia distillation apparatus. Add 200 ml of water and
75 ml ofsodium hydroxide solution. Boil gently for 20 minutes,
collecting the distillate in 50.0 ml of 0.05 M sulphuric acid.
Boil vigorously to complete the distillation of the ammonia
and titrate the excess of acid with 0.1 M sodium hydroxide,
using methyl red solution as indicator. Repeat the operation
between the titrations represents the amount of acid required
to neutralise the ammonia formed.
Mobile phase. A mixture of 60 volumes of l-butanol,

containing 0.1 g ofPyrazinamide with 50 mr ofa mixture of90
volumes of chloroform and 10 volumes of methanol, filter,
evaporate to dryness and dissolve the residue in sufficient of
the same solvent mixture to produce 10 ml.
Reference solution. Dilute I volume of the test solution to
500 volumes with a mixture of90 volumes of chloroform and
I ml of 0.05 M sulphuric acid is equivalent to 0.01231 g of

CsHsN30.

Pyrazinamide Tablets contain not less than 95.0 per cent and
pyrazinamide, CsH sN 30.
Identification

quantity ofthe powder containing about 0.1 g ofPyrazinamide,
add 200 ml of water, allow to stand for 10 minutes, swirling
occasionally, mix with the aid ofultrasound for 10 minutes and
dilute to 500.0 ml with water. Filter and discard the first 20 ml
ofthe filtrate. Dilute 5.0 ml ofthe filtrate to 100.0 ml with water
CsH sN 30 taking 650 as the specific absorbance at 268 nm.

of Pyrazinarriide with 20 ml of ethanol, filter, evaporate the
filtrate to dryness and my the residue at 105 for 30 minutes.
obtained with pyrazinamide RS or with the reference spectrum
ofpyrazinamide.
.

of Pyrazinamide with 50 ml of watel; dilute to 100 ml with
water and filter (solution A). Dilute I ml ofsolution A to 10 ml
with water. When examined in the range 290 nm to 360 nm
(2.4.7), the resulting solution shows an absorption maximum
at about 310 nm. Dilute 2 rnIofsolutionAto 100mi with water.
When examined in the range 230 nm to 290 nm, the resulting
solution shows an absorption maximum at about 268 nm;
absorbance at 268 nm, between 0.64 and 0.68.
C. Boil a quantity ofthe powdered tablets containing 20 mg of
Pyrazinamide with 5 ml of sodium hydroxide solution;
ammonia, recognisable by its odour, is evolved.
Pyridoxine Hydrochloride
VitaminB 6
, Hel
CgH lI N03,HCI
Mol. Wt. 205.6
Pyridoxine Hydrochloride is 5-hydroxy-6-methylpyridine3,4-dimethanol hydrochloride.

Pyridoxine Hydrochloride contains not less than 99.0 per cent
and not more than 101.0 per cent ofCgH II N0 3,HCI, calculated
on the dried basis.
Tests
Category. B group vitamin; sideroblastic anaemia therapy;

anti-isoniazid neuropathy.

Dose. In deficiency states, prophylactic, upto 2 mg daily;

therapeutic, 20 to 50 mg upto three times daily. In multivitamin
2005
IF 2010
PYRIDOXINE HYDROCHLORIDE
preparations for oral use, prophylactic, 500 J.tg to 1.5 mg daily;

therapeutic, 1.5 to 3 mg daily. In sideroblastic anaemia, 100 to
400 mg daily, in divided doses. In isoniazid neuropathy,
prophylactic, 10 mg daily; therapeutic, 50 mg three times daily.
odourless.
Identification
B may be omitted if tests A, C and D are carried out.
Compare the spectrum with that obtained with pyridoxine
hydrochloride RS or with the reference spectrum ofpyridoxine
hydrochloride.

an absorption maximum at 288 nm to 296 nm; absorbance at
the maximum, 0.420 to 0.445. A solution prepared by diluting
1 ml of a 0.1 per cent w/v solution in 0.1 M hydrochloric acid
to 100 ml with 0.025 M standard phosphate buffer shows
absorption maxima at 248 nm to 256 nm and at 320 nm to
327 nm; absorbances at the maxima, 0.175 to 0.195 and 0.345 to
0.365, respectively.
Apply to the plate 2 J.tl of each solution. After development,

dry the plate in air and spray with a 5 per cent w/v solution of
sodium carbonate in a mixture of 70 volumes of water and
30 volumes of ethanol (95 per cent). Dry in a current of air,
spray with a 0.1 per cent w/v solution of 2,6-dichloroquinone4-chloroimide in ethanol (95 per cent) and examine
obtained with test solution (a) is not more intense than the
(a); Ignore any spots remaining on the line of application.
Heavy metals (2.3.13). 12 ml of a 5.0 per cent w/v solution
complies with the limit test for heavy metals, Method D
(20 ppm). Use lead standard solution (1 ppm Pb) to prepare
the standard.
5 ml of anhydrous glacial acetic acid and 6 ml of mercuric
acetate solution. Titrate with 0.1 M perchloric acid, using
crystal violet solution as indicator, until a green colour is
produced. Carry out a blank titration.
CsH II N03,HCI.
(b).
D. A 5 per cent w/v solution gives reaction A of chlorides
(2.3.1).
Pyridoxine Tablets
Tests
Pyridoxine Hydrochloride Tablets

YS7 (2.4.1).
Pyridoxine Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent ofthe stated amount of pyridoxine
hydrochloride, C SH II N03, HCI.

Identification

ofdichloroinethane, 13 volumes of tetrahydrofuran and
Test solution (a). A 10 per cent w/v solution of the substance
Test solution (b). A 1 per cent w/v solution of the substance
under examination water.
Reference solution (b). A 1 per cent w/v solution ofpyridoxine

ofPyridoxine Hydrochloride with 50 ml of 0.025 M standard
jJhosjJhcie
for i 5 m.inutes and dllute to 16b m.l with the
same solvent. Mix, filter and dilute 5 ml ofthe filtrate to 100 ml
with the same solvent. When examined in the range 230 nm to
360 nm (2.4.7), the resulting solution exhibits two maxima, at
about 254 nm and 324 nm.
lJUffer

20 mg of Pyridoxine Hydrochloride with 50 ml of water and
allow to stand for 20 minutes. To 1 ml ofthe supernatant liquid
add 10 ml ofa 5 per cent w/v solution of sodium acetate,lilll
of water and 1 ml of a 0.5 per cent w/v solution of 2,6-dichloroquinone-4-chloroimide in ethanol (95 per cent) and
2006
PYRIMETHAMINE
IP 2010
shake; a blue colour is produced which fades rapidly and

becomes brown. Repeat the operation but adding 1 ml of a
0.3 per cent w/v solution of boric acid in place of 1 ml of
water; no blue colour is produced.
Pyrimethamine
Tests
of dichloromethane, 13 volumes of tetrahydrofuran and
CI
NH 2
Mol. Wt. 248.7

containing 40 mg of Pyridoxine Hydrochloride with 10 ml of
water for 15 minutes, filter and use the filtrate.
Pyrimethamine is 5-(4-cWorophenyl)-6-ethylpyrimidine-2,4diamine.
Reference solution. Dilute 1 ml oftest solution to 200 ml with

water.
Pyrimethamine contains not less than 99.0 per cent and not
more than 101.0 per cent OfCI2HI3CIN4, calculated onthe dried
basis.
Apply to the plate 10 J.11 of each solution. After development,

sodium carbonate in a mixture of 70 volumes of water and
30 volumes of ethanol (95 per cent). Dry it in a current of air,
spray with a 0.1 per cent w/v solution of 2, 6-dichloroquinone4-chloroimide in ethanol (95 per cent) and examine
Dose. Used only in combination with sulphadoxine (25 mg
pyrimethamine and 500 mg sulphadoxine) or dapsone (12.5
mg pyrimethamine and 100 mg dapsone).
Description. Colourless crystals or an almost white, crystalline
powder; odourless.
Identification

Tablets.

Powder one tablet, add 50 ml of 0.1 M hydrochloric acid and

heat on a water-bath for 15 minutes, swirling occasionally.
Cool, dilute to 100.0 ml with 0.1 M hydrochloric acid, filter,
discarding the first 20 ml of the filtrate. If necessary, dilute
quantitatively and stepwise with 0.1 M hydrochloric acid to
produce a solution containing 10 J.1g of the pyridoxine
hydrochloride per ml and measure the absorbance of the
Calculate the content of C SH 11 N03, HCl taking 430 as the

Compare the spectrum with that obtained with pyrimethamine
RS or with the reference spectrum ofpyrimethamine.

quantity ofthe powder containing about 20 mg ofPyridoxine
Hydrochloride, add 50 ml of 0.1 M hydrochloric ac,id and
Cool, dilute to 100.0 ml with 0.1 M hydrochloric acid and
filter, discarding the first 20 ml ofthe filtrate. Dilute 5.0 ml of
the filtrate to 100.0 ml with 0.1 M hydrochloric acid and
maximum at about 290 ~m (2.4.7). Calculate the content of
C SH 11 N0 3, HCl taking 430 as the specific absorbance at
290nm.

C. Dissolve 0.14 g in sufficient ethanol to produce 100 ml,
dilute 10 ml ofthis solution to 100 ml with 0.1 Mhydrochloric
acid and dilute 10 ml ofthe solution to 100 ml with the same
solvent. When examined in the range 230 nm to 360 nm (2.4.7),
the resulting solution shows an absorption maximum at about
272 nm; absorbance at about 272 nm, 0.43 to 0.46.
Tests
Appearance ofsolution. Dissolve 0.25 g in sufficient ofa mixture
of 3 volumes of dichloromethane and 1 volume of methanol
to produce 10 ml. The resulting solution is clear (2.4.1), and
not more intensely coloured than reference solution BYS6
(2.4.1).
Acidity or alkalinity. Shake 1.0 g with 50 ml of water for
2 minutes and filter (solution A). To 10 ml of solution A add
2007
PYRIMETHAMINE
IP 2010
0.05 ml ofphenolphthalein solution; the solution is colourless

and not more than 0.2 ml of 0.01 M sodium hydroxide is
required to change the colour to pink. Add 0.4 ml of 0.01 M
hydrochloric acid and 0.05 ml of methyl red solution; the
solution is red or orange.
.

of glacial acetic acid, 8 volumes of I-propanol and 4 volumes
of chloroform.
Identification
of Pyrimethamine with 50 ml of ethanol (95 per cent) for 20
minutes, filter and evaporate the filtrate to dryness. On the
residue, determine by infrared absorption spectrophotometry
(2.4.6). Compare the spectrum with that obtained with
pyrimethamine RS or with the reference spectrum of
pyrimethamine.
B. Extract the powdered tablets with 1 M sulphuric acid; filter

and add potassium tetraiodomercurate solution to the filtrate.
A creamy white precipitate is produced.

C. Extract a quantity ofthe powdered tablets containing 50 mg

ofPyrimethamine with two 10 ml quantities of chloroform and
evaporate the combined extracts to dryness. The residue melts
at about 240 0 (2.4.21).

examination in 100 ml ofthe same solvent mixture.
pyrimethamine RS in the same solvent mixture.
Any secondary spot in the chromatogram o15tained with test
Sulphates (2.3.17). 25 ml ofSolution A complies with the limit
test for sulphates. Use a mixture of5.0 ml ofsulphate standard
solution (10 ppm SO4) and 10 ml of distilled water to prepare
the standard (100 ppm).
anhydrous glacial acetic acid, heating gently, cool. Titrate
LlTIlofO.lA1perchloric a9id is equivalent to 0.02487g of

C I2H 13ClN4.
Pyrimethamine Tablets contain not less than 92.5 per cent and
pyrimethamine, C I2H 13 ClN4.
Tests
Solvent mixture. 90 volumes of chloroform and 10 volumes of

methanol.
Mobilephase. A mixture of4 volumes of chloroform, 8 volumes
of I-propanol, 12 volumes of glacial acetic acid and 76
volumes of toluene.
containing 50 mg Pyrimethamine with 5 ml ofsolvent mixture
and filter.
Reference solution. Dilute 1 ml ofthe test solution to 400 ml
Apply to the plate 20 !J.l of each solution. Allow the mobile
phase to rise 10 cm. After development, dry the plate in air and
examine in ultraviolet light at 254 urn. Any secondary spot in
the chromatogram obtained with the test solution is not more
quantity of the powder containing about 0.025g of
Pyrimethamine, add 50 ml ofhot 0.1 M hydrochloric acid and
Mix with theaid of ultrasound for 30 minutes, remove and
cool to room temperature. Add sufficient 0.1 M hydrochloric
acid to produce 100 ml, filter and dilute 5 ml of the filtrate to
100 ml with 0.1 M hydrochloric acid and measure the
absorbance 6ftheresulting solution at the maxiIllUlTI at about
272 urn (2.4.7). Calculate the content OfC12HI3ClN4 taking 316
as the specific absorbance at 272 urn.
2008
IP 2010
PYRIMETHAMINE AND SULPHADOXINE TABLETS
Pyrimethamine and Sulphadoxine

Tablets
Pyrimethamine and Sulphadoxine Tablets contain not less than
amounts ofpyrimethamine, C I2H 13 CIN4, and ofsulphadoxine,
CI2H14N404S.
Usual strength. Pyrimethamine 25 mg and sulphadoxine
500 mg.
Identification


4 volumes of n-heptane, 1 volume of glacial acetic acid and
4 volumes ofa mixture of! volume of methanol and 19 volumes
of ethanol.
containing 25 mg ofPyrimethamine with 50 ml ofa 2 per cent
w/v solution of strong ammonia solution in methanol.
pyrimethamine RS in methanol.
sulphadoxine RS in methanol.
.Apply to the plate 10 III of each solution. After development,
dry the plate in air and examine in ultraviolet light at 254 llill.
One ofthe principal spots in the chromatogram obtained with
the test solution corresponds to the spot in the chromatogram
obtained with reference solution (a) and the other corresponds
to that in the chromatogram obtained with reference
solution (b).

tablets containing about 25 mg ofPyrimethamine and 500 mg
of Sulphadoxine and shake with 35 ml of acetonitrile for
30 minutes in a 1OO-ml volumetric flask. Dilute to volume with
the mobile phase, mix and filter. To 25.0 ml ofthe filtrate add
2.0 ml ofsolution A prepared by dissolving 0.1 g ofphenacetin
(internal standard) in 100 ml of acetonitrile and sufficient of
the mobile phase to produce 50.0 ml.
pyrimethamine RS and 500 mg of sulphadoxine RS, add 35 ml
of acetonitrile and sufficient of the mobile phase to produce
100.0 ml and mix. To 25.0 ml add 2.0 ml ofsolution Aand add
sufficient ofthe mobile phase to produce 50.0 ml and mix well.
mobile phase: a mixture of 4 volumes ofa 1 per cent v/v
solution of glacial acetic acid and 1 volume of
acetonitrile,
spectrophotometer set at 254
nm,
than 2.0 per cent. The relative retention times with reference
to phenacetin for sulphadoxine is about 0.7 and for
pyrimethamine is about 1.3.
Injectthe test solution and the reference solution.
Tests
Calculate the content of CI2HI3ClN4 and the content of

C12HI4N404S in the tablets.
2009
MONOGRAPHS
Q
Quetiapine Fumarate
2013
Quetiapine Tablets
2014
2014
2015
2016
2017
2019
2020
Quinalbarbitone Sodium
Quinalbarbitone Tablets
Quinidine Sulphate
Quinidine Tablets
Quinine Bisulphate
Quinine Bisulphate Tablets
2021
2023
2025
2026
QuinineDihydrochloride
Quinine Dihydrochloride Injection
Quinine Sulphate
Quinine Tablets
2027
2028
2029
2030
2031
2032
Quiniodochlor
Quiniodochlor Cream
Quiniodochlor Ointment
QuiniodochlorTablets
Quiniodochlor and Hydrocortisone Cream
Quiniodochlor and Hydrocortisone Ointment
2011
IP 2010
QUETIAPINE FUMARATE
Reference solution (b). A 0.05 per cent w Iv solution offumariC

acid in the mobile phase.
Quetiapine Fumarate
HXCOOH
HOOC
(C2IH2SN302S)2,C4~04
Mol. Wt. 883.1
Quetiapirte Fumarate is 2-[2-(4-dibenzo[bj] [1,4]thiazepin-llyl-l-piperazinyl)ethoxy]ethanol fumarate

Quetiapine Fumarate contains not less than 98.0 per cent and
not more than 102.0 per cent of C42HsoN604S2,C4H404
Dose. 300 to 450 mg per day.
Identification
Compare the spectrum with that obtained with quetiapine
ji/marate RS. Ifthe spectra obtained show differences, dissolve
the substance under examination and the reference substance
in methanol with the aid ofultrasound. ifnecessary. Evaporate
the solvent under nitrogen at 50 for 2 hours and determine on
the residues.
Reference solution (c). Dissolve 1 mg of quetiapinefumarate

RS and 1.5 mg of 2-(4-Dibenzo[b,f}[1,4] thiazepine-ll-ylPiperazin-1-yl)-ethanol RS (quetiapine impurity A RS) in
100 ml ofthe mobile phase. Dilute 5 m1 ofthe solution to 50 ml
octylsilane bonded to porous silica (5Ilm) (Such as C8Novapak),
- mobile phase: a mixture of 500 volumes of methanol,
400 volumes of water, 100 volumes of acetonitrile and
0.4 volume of triethylamine, adjusted to pH 7.0 with
resolution between the peaks corresponding to quetiapine
impurity A and quetiapine in the chromatogram obtained with
the reference solution (c) is not less than 1.5.
chromatograms for three times the retention time of the
solution, the area ofany secondary peak is not more than 0.5
times the area ofthe peak in the chromatogram obtained with
the reference solution (a) (0.5 per cent) and the sum of the
areas of all the secondary peaks is not more than the area of
the peak in the chromatogram obtained with the reference
solution (a) (1.0 per cent). Ignore the peak due to fumaric acid.

Tests
Fumaricacid.12.6to 13.6 percent.
Weigh accurately about 0.35 g, dissolve in 80 ml of

dimethylformamide. Titrate with 0.1 M tetra butyl ammonium
(2.4.25). Carry out a blank titration.
1 ml of 0.1 M tetra butyl ammonium hydroxide is equivalent
to 0.0058 g offumaric acid.
(2.4.14).
Reference solution (a). A 0.001 per cent wlv solution of
quetiapine jillnarate RS in the mobile phase.

Reference solution. A 0.1 per cent wlv solution of quetiapine
ji/marate RS in the mobile phase.

Related substances.
2013
IP 2010
QUETIAPINE TABLETS
Calculate the content of(CzlHzsN30zS)z,C4Ht04'

exceeding 30.
Quetiapine Tablets
Quetiapine Fumarate Tablets
Quetiapine Tablets contain Quetiapine Fumarate equivalent
of the stated amount of quetiapine, CZIHzsN30zS.
the mobile phase. Dilute 5.0 ml ofthis solution to 50.0 ml with

the mobile phase.
- mobile phase: a mixture of40 volumes of water and 60
volumes of methanol. Add 0.4 ml of triethylamine and
adjust pH to 6.8 with orthophosphoric acid,
than 2.0 per cent.
Dose. 25 mg; 50 mg; 100 mg.
Identification
In the Assay, the principal peak in the chromatogram of the
test solution corresponds to the peak in the chromatogram

Calculate the content ofCzIHzsN30zS in the tablets.
equivalent amount of quetiapine.
Tests
Apparatus No.1,
Test solution. Withdraw about 10 ml of the medium and

centrifuge. Dilute the supernatant liquid suitably with 0.1 M
hydrochloric acid to obtain a solution containing about
10 Ilg per ml, ofquetiapine.
Quinalbarbital Sodium; Secobarbital Sodium;

Secobarbitone Sodium; Soluble Quinalbarbitone
Reference solution. Dissolve about 58 mg of quetiapine

fUmarate RS in 15 ml ofthe dissolution medium and dilute to
100.0 ml with the medium. Dilute 2.0 ml of the solution to
100.0 rnl with the same medium.
Measure the absorbance of the reference solution and test
solution at 248 nm (2.4.7) against 0.1 M hydrochloric acid as
the blank. Calculate the content of quetiapine in the medium
concentration of quetiapinefUmarate RS in the same medium.
D. Not less than 70 per cent ofthe stated amount ofquetiapine.
Assay. Determine by liquid chromatography (2.4.14)
Mol.Wt.260.3
Quinalbarbitone Sodium is sodium (RS)-5-allyl-5-(Imethylbutyl)barbiturate.
Quinalbarbitone Sodium contains not less than 98.5 per cent
a.nd n6t1liore than 102.0 per cent ofClzHI7NzNa03, ca.lclllated
on the dried basis.
Category. Hypnotic.

a quantity of the powder containing 250 mg of quetiapine,
disperse in 100.0 ml of the mobile phase, with the aid of
ultrasound and dilute to 250.0 ml with the mobile phase. Dilute
5 rnl ofthe solution to 50 ml with the mobile phase.
Identification
iiejerencesolution. Weigh accura.tely about 58 mg of

quetiapine fumarate RS, add 30 ml of the mobile phase and
dissolve with aid ofultrasound. Cool and dilute to 50.0 ml with

out.

Description. A white powder; odourless; hygroscopic.
2014
QUINALBARBITONE TABLETS
IP 2010
A. To 10ml ofa 10.0 per cent w/v solution in ethanol (95 per
cent) add 120 ml of water and 5 ml of 2 M acetic acid, stir
vigorously, add 200 ml of water and boil until the precipitate
dissolves and no oily particles remain on the surface of the
liquid. Allow to cool until a haziness begins to appear in the
solution, induce crystallisation, if necessary and allow the
solution to stand for 12 hours. Wash the crystals with three
quantities, each of 10 ml, of water and dry the residue at 80.
obtained with quinalbarbitone RStreated in the same manner.
B. To 0.5 g, add 5 ml of sodium carbonate solution and 10 ml
of nitrobenzyl chloride solution. Heat for 30 minutes on a
water-bath under reflux and allow to stand for I hour. Filter,
wash the precipitate successively with 10 ml of dilute sodium
hydroxide solution and 50 ml of water; recrystallise from a
mixture of equal volumes of chloroform and ethanol (95 per
cent) and dry at 105. The crystals melt atabout 156 (2.4.21)
(2.3.2).
D. Gives the reaction ofnon-nitrogen substituted barbiturates
(2.3.1).
E. Ignite 1 g; the residue gives the reactions of sodium salts
(2.3.1).
Tests
Appearance of solution. A freshly prepared 10.0 per cent w/v
solution in ethanol (95 per cent) is clear (2.4.1), and not more
pH (2.4.24). Not more than 11.0, determined on a 10.0 per cent
w/v solution.
Heavy metals (2.3.13). 0.67 g dissolved in a mixture of5 ml of
1 M sodium hydroxide and 20 ml of water complies with the
limit test for heavy metals, Method C (30 ppm).
substances in barbiturates (2.3.4).
.
ethanol, add 10 ml ofsilver nitrate-pyridine reagent and titrate
with 0.1 M ethanolic sodium hydroxide using 0.5 mlof
thymolphthalein solution as indicator, until a pure blue colour
is obtained. Carry out a blank titration.
0.02603 g ofCI2H17N2Na03'
Quinalbarbital Sodium Tablets; Quinalbarbitone
Sodium Tablets; Secobarbital Sodium Tablets;
Secobarbitone Sodium Tablets
Quinalbarbitone Tablets contain not less than 92.5 per cent
quinalbarbitone sodium, C12H17N2Na03. The tablets are coated.
Identification
A.Shake a quantity of the powdered tablets containing about
0.5 g ofQuinalbarbitone Sodium with 10 ml of water and filter.
To 10 ml ofthe filtrate add 5 ml of sodium carbonate solution
and 10 ml of nitrobenzyl chloride solution. Heat for 30 minutes
on a water-bath under reflux and allow to stand for 1 hour.
Filter, wash the precipitate successively with 10 ml of dilute
sodium hydroxide solution and 50 ml of water; recrystallise
from a mixture of equal volumes of chloroform and ethanol
(95 per cent) and dry at 105. The crystals melt at about
156 (2.4.21)
,
0.5 g of Quinalbarbitone Sodium with 10 ml of water, filter,
acidify the filtrate with acetic acid; oily drops are fonned
which may eventually clystallise.
.
C. The powdered tablets give the reactions of sodium salts
(2.3.1).
Tests
Assay. Weigh and digest 20 tablets with 50 ml of water until
completely disintegrated and not more than a small residue
remains. Add 5 ml of 1 M sodium hydroxide, filter and wash
the residue with sufficient water to produce 100.0 ml. Extract a
volume of the solution containing 0.5 g of Quinalbarbitone
Sodium with two quantities, each of 10 ml, of ether, washing
each ether extract with the same 3 ml of water. Add the water
to the aqueous liquid, acidify witn hydrochloric acid and
extract with successive quantities, each of 15 ml, of ether until
complete extraction is effected. Wash the combined extracts
with two quantities, each of 2 ml, of water and extract
the main ether layer, filter and wash the filter with ether.
Evaporate the solvent and dry the residue to constant weight
at 50.
1 g ofresidue is equivalent to 1.092 g ofC12H17N2Na03.
2015
IP 2010
QUINIDINE SULPHATE
B. To 5 ml ofa 0.1 percentw/v solution add 0.2 ml of bromine

solution and 1 ml of dilute ammonia solution; an emeraldgreen colour is produced.
Quinidine Sulphate
Quinidine Bisulphate
C. To a 0.5 per cent w/v solution add an equal volume of dilute

sulphuric acid; a strong blue fluorescence is produced.
D. To 5 ml ofa 1percentw/v solution add 1 mlofsilvernitrate
solution and stir with a glass rod; after a short interval, a
white precipitate soluble in nitric acid is produced (distinction
from many other allmloids).
E. A 1 per cent w/v solution gives the reactions of sulphates

(2.3.1).
2
(CzoHz~zOz)z,HzS04,2HzO
Mol. Wt. 783.0
Quinidine Sulphate is (8R,9S)-6' -methoxycinchonan-9-01

sulphate dihydrate. The alkaloid is obtained from the bark of
various species of Cinchona and from Remijia pedunculata
Fluckiger (Fam. Rubiaceae) or prepared from quinine.
Quinidine Sulphate contains not less than 99.0 per cent and
not more than 101.5 per cent of alkaloid monosulphates,
calculated as (CzoHz4NzOz)2,H2S04 on the dried basis.
Category. Antiarrhythmic.
Dose. In the treatment ofcardiac arrhythmias, 200 mg daily in
three or four divided doses. In the treatment ofatrial fibrillation,
200 to 400 mg every 2 to 4 hours, upto 3 g daily.
Description. A white or almost white, crystalline powder or
needle-like crystals; odourless.
Identification

of ether and 15 volumes of diethylamine.
Reference solution (a). A 1 per cent w/v solution of quinidine
sulphate RS in methanol.
Reference solution (b). A 1 per cent w/v solution of each of
quinidine sulphate RS and quinine sulphate RS in methanol.
dry the plate in air for 15 minutes and repeat the development.
Dry the plate at 105 for 30 minutes, allow to cool and spray
with potassium iodoplatinate solution. The principal spot in
to the spot in the chromatogram obtained with reference
solution (a). The test is not valid unless the chromatogram
. obtained with reference solution (b) shows two clearly
separated spots.
Tests
Appearance of solution. A2.0 per cent w/v solution in 0.1 M
hydrochloric acid is clear (2..4.1), and not more intensely

in a 2.0 per cent w/v solution in 0.1 M hydrochloric acid.
Dihydroquinidine sulphate. Not more than 15.0 per cent,
calculated on the dried basis and determined by the following
method. Dissolve 0.2 gin 20 ml of water, add 0.5 g ofpotassium
bromide and 15 ml of 2 M hydrochloric acid. Titrate slowly
with 0.0167 M potassium bromate using methyl red solution
as indicator until a yellow colour is obtained. Add a solution
of0.5 g ofpotassium iodide in 200 ml of water and stopper the
flask immediately. Allow to stand in the dark for 5 minutes and
titrate with 0.1 M sodium thiosulphate using starch solution,
added towards the end of the titration, as indicator. Repeat
the operation without the substance under examination.
I ml of 0.0167 M potassium bromate is equivalent to
0.01867 gof(C2oHz4N20z)z, H2S04.
Calculate the content of dihydroquinidine sulphate by

subtracting the result from the assay result.
Other cinchona alkaloids. Determine by liquid

examination in 5 mlof the mobile phase. Heat gently, if
necessary to dissolve the powder as completely as possible,
cool, dilute to 10 ml with the mobile phase and mix.
Reference solution (a). Dissolve 20 mg of quinine sulphate
RS, with gentle heating if necessary, in 5 ml of the mobile
phase and dilute to 10 ml with the mobile phase.
Reference solution (b). Prepare in the same manner as
reference solution (a) but using quinidine sulphate RS in
place of quinine sulphate RS.
Reference solution (c). Mix equal volumes of reference
solutions (a) and (b).
2016
IP 2010
QUININE TABLETS
Reference solution (d). Dilute 1 volume of reference solution

(a) to 10 volumes with the mobile phase and dilute 1 volume of
the resulting solution to 50 volumes with the mobile phase.
Reference solution (e). A solution containing 0.1 per cent
w/v of thiourea in the mobile phase.
octadecylsilane bonded to porous silica (5 11m) (Such
as Hypersil ODS 5 11m),
- mobile phase: a solution prepared by dissolving 6.8 g of
potassium dihydrogen orthophosphate and 3.0 g of
hexylamine in 700 ml of water, adjusting the pH to 2.8
with 1 M orthophosphoric acid, adding 60 ml of
acetonitrile and diluting to 1000 ml with water,
spectrophotometer set at 250 urn for reference solution
(e) and 316 nm for the other solutions,
Inject separately reference solutions (b) and (e). Ifnecessary,
adjust the concentration of acetonitrile in the mobile phase so
that in the chromatogram obtained with reference solution (b)
the capacity factor ofthe peak due to quinidine is 3.5 to 4.5, Va
(the distance along the baseline between the point ofinjection .
and the perpendicular dropped from the maximum ofthe peak
of an unretained component) being calculated from the peak
due to thiourea in the chromatogram obtained with reference
solution (e).
normalisation, ignoring any peaks the areas ofwhich are less

than that of the peak in the chromatogram obtained with
reference solution (d) (0.2 per cent). The content of
dihydroquinidine is not greater than 15 per cent, the content
ofany related substance eluting before quinidine is not greater
than 5 per cent and the content of any other related substance
is not greater than 2.5 per cent.
Loss on drying (2.4.19). 3.0 per centto 5.0 per cent, determined
10 ml of chloroform and 20 ml of acetic anhydride. Titrate
(C2oH24N202)2,H2S04'
Quin.idine Tablets
Quinidine Sulphate Tablets
Quinidine Tablets contain not less than 95.0 per cent and not
more than 105.0 per cent of the stated amount of quinidine
sulphate, (C2oH24N202)2,H2S04,2H20;
Inject reference solutions (a), (b), (c) and (d). The

chromatogram obtained with reference solution (a) shows a
principal peak due to quinine and a peak due to dihydroquinine
with a retention time relative to quinine of about 1.4. The
chromatogram obtained with reference solution (b) shows a
principal peak due to quinidine and a peak due to
dihydroquinidine, with a retention time relative to quinidine
of about 1.2. The chromatogram obtained with reference
solution (c) shows four peaks due to quinine, dihydroquinine,
quinidine and dihydroquinidine which are identified by
comparison of their retention times with those of the
corresponding peaks in the chromatograms obtained with
reference solutions (a) and (b).
Identification
the plate'with silica gel G.

of acetone and 10 volumes of diethylamine.
containing 0.1 g ofQuinidine Sulphate with 10 ml ofa mixture
of2 volumes of chloroform and 1 volume of ethanol (95 per
cent) and filter.
Reference solution. 1.0 per cent w/v solution of quinidine
sulphate RS in a mixture of 2 volumes of chloroform and
1 volume of ethanol (95 per cent).
The test is not valid unless (a) in the chromatogram obtained

with reference solution (c) the resolution between the peaks
due to quinine and quinidine is at least 1.5 and the resolution
between the peaks due to dihydroquinidine and quinine is at
least 1.0 and (b) the signal-to-noise ratio ofthe principal peak
in the chromatogram obtained with reference solution (d) is at
least 5.

dry the plate in air and spray with 0.05 M ethanolic sulphuric
acid and then with dilute potassium iodobismuthate solution.
Inject the test solution and allow the chromatography to

proceed for 2.5 times the retention time ofthe principal peale
Calculate the percentage content of related substances by
B. Extract a quantity ofthe powdered tablets containing 0.1 g

ofQuinidine Sulphate with 20 ml of water and filter. The filtrate
(solution A) is dextro-rotatory.
2017
IP 2010
QUINIDINE TABLETS
flow rate. 1.5 rnl per minute,

spectrophotometer set at 250 nm for reference solution
C. To 1 ml of solution A add 4 ml of water, 2 or 3 drops of

bromine solution and 1 ml of dilute ammonia solution; an
emerald green colour is produced.
D. Solution A gives the reactions ofsulphates (2.3.1).
Tests
Apparatus No.2,
(CzoHz4NzOz)z,HzS04,2HzO in the medium from the absorbance
obtained from a solution ofknown concentration of quinidine
sulphate RS.
(CzoHz4NzOz)z,HzS04,2HzO.

Test solution. Mix a quantity of the powdered tablets

containing 50 mg of Quinidine Sulphate with 20 ml of the
mobile phase. Heat gently to dissolve the powder as completely
as possible, cool, dilute to 25 ml with the mobile phase and
filter, discarding the first few ml offiltrate.
theresulting solution to 50 volumes with the mobile phase.

the capacity factor ofthe peak due to quinidine is 3.5 to 4.5, Vo
(the distance along the baseline between the point of injection
solution (e).
least 5.
proceed for 2.5 times the retention time ofthe principal peak.
normalisation, ignoring any peaks the areas of which are less
dihydroquinidine is not greater than 15 per cent, the content
ofany related substance eluting before quinidine,is not greater
octadecylsilane bonded to porous silica (5 flm) (Such
as Hypersil ODS 5 flm),
mobile phase: a solution prepared by dissolving 6.8 g of quantity of the powder containing about 0.4 g of Quinidine
potassium dihydrogen orthophosphate and 3.0 g of Sulphate, dissolve as completely as possible in 40 ml of acetic
hexylamine in 700 ml of water, adjusting the pH to 2.8 anhydride with the aid of heat and cool. Titrate with 0.1 M
with 1 M orthophosphoric acid, adding 60 ml of perchloric acid, using clystal violet solution as indicator.
2018
IP 2010
QUININE BISULPHATE

(C2oH24N202)2,H2S04,2H20.
C. A 5 per cent w/v solution gives the reactions of sulphates
(2.3.1).
B. A 5 per cent w/v solution has a blue fluorescence.
Tests
Quinine Bisulphate

acid.
Quinine Acid Sulphate

OCH 3
~CH2

C2oH24N202,H2S04,7H20
Mol. Wt. 548.6

examination in 5 ml of the mobile phase. Heat gently, if
Quinine Bisulphate is (8S,9R)-6' -methoxycinchonan-9-01

hydrogen sulphate heptahydrate. The alkaloid is obtained
from the bark of various species of Cinchona.

Quinine Bisulphate contains not less than 98.5 per cent and
not more than 101.5 per cent of alkaloid hydrogen sulphates,
calculated as C2oH24N202,H2S04 on the anhydrous basis.


Dose. Suppressive, 300 to 600 mg daily; therapeutic, 1.2 to 2 g

Description. Colourless or faintly yellow crystals or a white
or faintly yellow, crystalline powder; efflorescent in dry air.
Reference solution (d). Dilute 1 volume ofreference solution

Identification


octadecylsilane bonded to porous silica (5 /lm) (Such
as Hypersil ODS 5 /lm),
hexylamine in 700 ml ofwatel; adjusting the pH to 2.8
- spectrophotometer set at 250 tim for reference solution
(e) and 316 nm for the other solutions, .

in 100 ml methanol.
Reference solution (a). A 1 per cent w/v solution of quinine
qUinidine sulphate RS and quinine sulphate RS in methanol.
obtained with reference solution (b) shows two clearly
separated spots.
Inject separately reference solutions (b) and (e). If necessary,

2019
QUININE BISULPHATE
IP 2010

solution (e).
least 5.
dihydroquinine is not greater than 10 per cent, the content of
any related substance eluting before quinine is not greater
Titratable cation. 75.3 to 79.6 per cent, calculated on the
anhydrous basis, determined by the following method. Titrate
the combined aqueous solutions reserved in the Assay with
0.1 M hydrochloric acid using 0.1 ml of phenolphthalein
solution as indicator.
[CzoHz6NzOzJ2+.

CzoHz~zOz,HzS04'

Quinine Acid Sulphate Tablets
Quinine Bisulphate Tablets contain not less than 95.0 per cent
quinine bisulphate, CzoHz4NzOz,HzS04,7HzO. The tablets are
coated.
Identification
Mobile phase. A mixture of80 volumes oftoluene, 20 volumes

containing 0.1 g ofQuinine Bisulphate with 10 m1 ofa mixture
of2 volumes of chloroform and 1 volume of ethanol (95 per
cent) and filter.
Reference solution. A 1.0 per cent wlv solution of quinine
sulphate in the same solvent mixture.
Apply to the plate 2 111 of each solution. After development,
B. Extract a quantity ofthe powdered tablets containing 0.1 g

ofQuinine Bisulphate with 20 ml ofwater and filter (solution
A). To 5 ml of solution A add 0.2 ml of bromine solution and
1 ml of dilute ammonia solution; an emerald-green colour is
produced.
C. Solution A is leva-rotatory.
D. Solution A gives the reactions of sulphates (2.3.1).

water, add 25 ml of 0.1 M sodium hydroxide and extract with
three quantities, each of 25 ml, of chloroform. Wash each
chloroform extract with the same 20 ml ofwater. Combine the
aqueous solutions and reserve for the test for Titratable cation.
Dry the chloroform extracts with anhydrous sodium sulphate,
evaporate to dryness at a pressure of 2 kPa and dissolve the
residue in 50 ml ofanhydrous glacial acetic acid. Titrate with
0.1 M perchloric acid, using crystal violet solution as
indicator. Carry out a blank titration.
Tests
Apparatus No.2,
2020
QUININE DIHYDROCHLORIDE
IP 2010
C2oH24N202,H2S04,7H20 in the medium taking 99 as the specific

CzOH24N202,H2S04,7H20.
Test solution. Remove any coating from the tablets and mix a
quantity of the powdered tablet cores containing 50 'mg of
Quinine Bisulphate with 20 ml ofthe mobile phase. Heat gently
to dissolve the powder as completely as possible, cool, dilute
to 25 ml with the mobile phase and filter, discarding the first
few ml ofthe filtrate.
Refel'ence solution (c). Mix equal volumes of reference
mobile phase: a solution prepared by dissolving 6.8 g of
with 1 M orthophosphoric acid, add.ing 60 ml of
(the distance along the baseline between the point ofinjection
solution (e).
principal peak due to quinine and a peale due to dihydroquinine

The testis not valid unless (a) in the chromatogram obtained
least 5.
than 5 per cent and the content of any other related substance .
quantity of the powder containing about 0.6 g of Quinine
Bisulphate, dissolve as completely as possible in 40 ml of
acetic anhydride with the aid of heat and cool. Titrate with
C2oH24N202,H2S04,7HzO.
Quinine Dihydrochloride
Quinine Acid Hydrochloride

2021
OCH 3
~CH2
,2HCI
Mol. Wt. 397.3
IP 2010
QUININE DIHYDROCHLORIDE
Quinine Dihydrochloride is the (8S,9R)-6'methoxycinchonan-9-01 dihydrochloride. The alkaloid is

obtained from the bark of various species of Cinchona.
Quinine Dihydrochloride contains not less than 99.0 per cent
and not more than 101.5 per centofalkaloid dihydrochlorides,
calculated as CzoHz4NzOz,2HCl on the dried basis.
Dose. Suppressive, 300 to 600 mg daily; therapeutic, 1.2 to 2 g
daily, in divided doses. By slow intravenous injection, 300 to
600 mg.
Description. A white or almost white powder; odourless.
Identification

quinidine sulphate RS and quinine sulphate RS in methanol.
with potassium iodopldtinate solution. The principal spot in
obtained with reference solution (b) shows two clearly
separated spots.
B. To a 0.5 per cent w/v solution add an equal volume of dilute

(2.3.1 ).
Tests
pH (2.4.24).2.0 to 3.0, determined in a 3.0percentw/v solution.
Barium. To 15 ml of a 2 per cent w/v solution add 1 mlof
dilute sulphuric acid; the solution remains clear for at least
15 minutes.
Dihydroquinine dihydrochloride. Not more than 10.0 per cent,
calculated on the dried basis and determined by the following
method. Dissolve 0.2 g in 20 rnl of water, add 0.5 g ofpotassium

bromide and 15 ml of 2 M hydrochloric acid. Titrate slowly
with 0.0167 M potassium bromate using methyl red solution
as indicator until a yellow colour is obtained. Add a solution
of0.5 g ofpotassium iodide in 200 ml of water and stopper the
flask irnmediat~ly. Allow to stand in the dark for 5 minutes and
titrate with 0.1 M sodium thiosulphate using starch solution,
added towards the end of the titration, as indicator. Repeat
the operation without the substance under examination.
1 m1 of 0.0167 M potassium bromate is equivalent to
0.01987 g of CzoHz4NzOz,2HCl. Calculate the content of
dihydroquinine dihydroch10ride by subtracting the result from
the assay result.

cool, dilute to 10 m1 with the mobile phase and mix.
phase and dilute to 10 m1 with the mobile phase.
octadecylsilane bonded to porous silica (5 !J.m) (Such
as Hypersil ODS 5 !J.m),
hexylamine in 700 ml of water; adjusting the pH to 2.8
acetonitrile and diluting to 1000 ml with water
- spectrophotometer set at 250 nm for reference solution
injection volume. 10 !J.l.
adjust the concentration of acetonitrile in the mobile
2022
phase so
QUININE DIHYDROCHLORIDE INJECTION
IP 2010

solution (e).
.principal peak due to quinine and a peak due to dihydroquinine
due to quinine and quinidine is at least 1S and the resolution
least 5.
anhydride and 10 ml of mercuric acetate solution. Titrate

with 0.1 M perchloric acid, using clystal violet solution as
indicator. Cany out a blank titration.
1 ml of 0.1 M perchloric acid is equivalent of 0.01987 g of
C2oH24N202,2HCl.
Qu.inine Dihydrochloride Injection

Quinine Acid Hydrochloride Injection
Quinine Dihydrochloride Injection is a sterile solution of
Quinine Dihydrochloride in Water for Injections.
Quinine Dihydrochloride Injection contains not less than
amount ofquinine dihydrochloride, C2oH24N202,2HCl.
Description. A clear, almost colourless to light yellow solution.
Identification

Solvent mixture. 2 volumes of chloroform and 1 volume of
Test solution. Extract a volume of the injection containing
0.1 g ofQuinine Dihydrochloride Bisulphate with 10 ml ofthe
solvent mixture and filter.
Titratable cation. 79.7 to 84.2 per cent, calculated on the dried

about 0.4 g, dissolve in 10 ml of water, add 40 ml of methanol
and titrate with 0.1 M sodium hydroxide using
phenolphthalein solution as indicator.

sulphate RS in the solvent mixture.
1 ml of 0.1 Msodium hydroxide is equivalentto 0.01632 g of

[C2oH26N202F+.

with the reference solution (a). The test is not valid unless the

anhydrous glacial acetic acid and add 20 ml of acetic
B. Dilute 1 ml to 20 ml with water, add 0.1 M sodium hydroxide

dropwise until the solution becomed turbid. Add 1 or 2 drops

sulphates ( 0.12 per cent).
Reference solution (b). A solution of 1 per centw/v of each of

quinidine sulphate RS and quinine sulphate RS in the solvent
mixture.
2023
IP 2010
QUININE DffiYDROCHLORIDE INJECTION
of 0.05 M sulphuric acid to obtain a clear solution. To this ' Inject reference solutions (a), (b), (c) and (d). The chromatogram
solution add an equal volume of dilute sulphuric acid; a obtained with reference solution (a) shows a principal peak
strong blue fluorescence is produced.
due to quinine and a peak due to dihydroquinine with a
retention time relative to quinine of about 104. The
C. It gives the reactions of chlorides (2.3.1).
Tests
pH (2.4.24).1.5 to 3.0.
Other cinchona alkaloids. Determine by liquid quinidine and dihydroquinidine which are identified by
Test solution. Dilute, if necessary a suitable volume of the
injection with the mobile phase to produce a solution
containing 0.2 per cent w/v of Quinine Dihydrochloride.
Reference solution (a). Dissolve 20 mg of quinine sulphate with reference solution (c) the resolution between the peaks
RS, with gentle heating if necessary, in 5 ml of the mobile due to quinine and quinidine is at least 1.5 and the resolution
factor between the peaks due to dihydroquinidine and quinine
is at least 1.0 and (b) the signal-to-noise ratio of the principal
Reference solution (b). Prepare in the same manner as peak in the chromatogram obtained with reference solution
reference solution (a) but using quinidine sulphate RS in (d) is at least 5.
Reference solution (c). Mix equal volumes of reference proceed for2.5 times theretention time of the principal peak.
normalisation,
ignoring any peaks the areas of which are less
than
that
of
the
peak in the chromatogram obtained with
reference
solution
(d) (0.2' per cent). The content of
Reference solution (e). A solution containing 0.1 per cent any related substance eluting before quinine is not greater
as Hypersil ODS 5 Ilm),
acetonitrile and diluting to 1000 ml with watel;
(the distance along the baseline betWeen the point ofinjection
of an unretained component) being cakrihitedfrom the peak
solution (e).

0.5 g ofQuinine Dihydrochloride add 20 ml of water and 5 ml
of sodium hydroxide solution. Extract with successive
quantities, each of 10 ml, of chloroform until complete extraction
of the alkaloid is effected, washing each extract with the same
two quantities, each of5 ml, of water. Remove the chloroform
from the combined extracts, dissolve the residue in 50 ml of
anhydrous glacial acetic acid and add 20 ml of acetic
anhydride. Titrate with 0.1 M perchloric acid, using crystal
C2oH24N202,2HCl.
Labelling. The label states that the solution mustbe diluted
to a strength not exceeding 30 mgperml before adhiinistration
and that care should be taken to ensure slow intravenous
injection.
2024
QUININE SULPHATE
IP 2010
D. To 5 ml ofa 1 per centw/v solution add 1 ml of silver nitrate

solution and stir with a glass rod; after a short interval, a
white precipitate soluble in nitric acid is produced (distinction
from many other alkaloids).
Quinine Sulphate
OCH s
E. A 1 per cent w/v solution gives the reactions of sulphates

.
(2.3.1).
Dy
CH 2
Tests
Appearance of solution. A 2.0 per cent w/v solution in 0.1 M

hydrochloric acid is clear (2.4.1), and not more intensely
(C2oH24Nz02hH2S04,2H20
Mol. Wt.783.0
Quinine Sulphate is (8S,9R)-6' -methoxycinchonan-9-ol

sulphate dihydrate. The alkaloid is obtained from the bark of
various species of Cinchona.
Quinine Sulphate contains not less than 99.0 per cent and not
more than 101.0 per cent ofalkaloid monosulphates, calculated
as (C2oHz4N202)Z,H2S04 on the dried basis.
Dose. Suppressive, 300 to 600 mg daily. therapeutic, 1.2 to 2 g
Description. White or almost white, needle-like crystals or a
crystalline powder.
Identification


suspension in water.




Reference solution. A 1 per cent w/v solution of quinine



to the spot in the chromatogram obtained with the reference
solution.
as Hypersil ODS 5 Ilm),
hexylamine in 700 ml of water, adjusting the pH to 2.8
- spectrophotometer set at 250 urn for reference solution
(e) and 316 urn for the other solutions,
B. To 5 ml ofa O.1percentw/v solution add 0.2 ml of bromine

solution and 1 ml of dilute ammonia solution; an emeraldgreen colour is produced.
C. To a 0.5 per cent w/v solution add an equal volume of dilute

2025
IP 2010
QUININE SULPHATE

solution (e).
Quinine Tablets

principal peak due to quinine and a peak due to rlihydroquinine
chromatogram obtained .with reference solution (b) shows a
Identification

least 5.
Loss on drying (2.4.19).3.0 to 5.0 per cent, determined on
10 ml bf chloroform and add 20 ml of acetic anhydride. Titrate
(C2oH24N202)2,H2S04'
Quinine Sulphate Tablets

Quinine Sulphate Tablets contain not less than 95.0 per cent
quinine sulphate, (C2oH24N202)2,H2S04,2Hp. The tablets are
coated.


containing 0.1 g ofQuinine Sulphate with 10 ml ofa mixture of
2 volumes of chloroform and 1 volume of ethanol (95 per
cent) and filter.
Reference solution. A 1 per cent w/v solution of quinine
sillphate in the same solvent mixture.
Apply to the plate 2 f.!l of each solution. After development,
B'. Extract a quantity ofthe powdered tablets containing 0.1 g
ofQuinine Sulphate with 20 ml of water and filter (solution A).
To 5 ml of solution A add 0.2 ml of bromine solution and 1 ml
of dilute ammonia solution; an emerald-green colour is
produced.
C. Solution A is leva-rotatory.
D. Solution A gives the reactions ofsulphates (2.3.1).
Tests
Apparatus No.2,
the maximum at about 348 urn (2.4.7). Calculate the content of
C2oH24N202, H 2S04, 2H20 in the medium from a solution of
known concentration of quinine sulphate RS.
C2oH24Nz02, H2S04, 2H20.
2026
QUINIODOCHLOR
IP 2010
Test solution. Remove any coating from the tablets and mix a
quantity of the powdered tablet cores containing 50 mg of
Quinine Sulphate with 20 ml ofthe mobile phase. Heat gently
to dissolve the powder as completely as possible, cool, dilute
to 25 ml with the mobile phase and filter, discarding the first
few ml ofthe filtrate.
.
spectrophotometer set at 250 urn for reference solution
(e) and 316 urn for the other solutions,
Injectseparately reference solutions (b) and (e). Ifnecessary,
solution (e).
with a retention time relative to quinine of about 104. The

least 5.
proceed for 2.5 times the retention time of the principal peak.
any related. substance eluting before quinine is not greater

quantity of the powder containing about 004 g of Quinine
Sulphate, dissolve as completely as possible in 40 ml of acetic
anhydride with the aid of heat and cool. Filter, if necessary
through Whatrnan No.1 filter paper and rinse with an additional
40 ml of acetic anhydride in small volumes. Titrate with 0.1 M
perchloric acid, using crystal violet solution as indicator.
(C2oH2~202)2,H2S04,2H20.
Quiniodochlor
Clioquinol; Iodochlorhydroxyquinoline;
Iodochlorhydroxyquin
OH
'hN~
~
CI
~HsCI1NO
Mol. Wt.305.5
Quiniodochlor is 5-chloro-7-iodoquinolin-8-ol.
Quiniodochlor contains not less than 97.0 per cent and not
more than 103.0 per cent ofCgHsClINO, calculated on the dried
basis.
2027
QUINIODOCHLOR
IP 2010
Category. Antiamoebic; topical and intestinal antiseptic.
under examination and 0.5 ml ofpyridine, mix, allow to stand

for 15 minutes and add 5 ml of hexane.
Dose. 750 mg to 1.5 g daily, in divided doses.

Description. A yellowish white to brownish yellow powder;
odoUr, faint and characteristic.
Identification

Compare the spectrum with that obtained with quiniodochlor
RS or with the reference spectrum of quiniodochlor.
B. When examined in the range 230 Dill to 360 Dill (2.4.7), a
0.0005 per cent w/v solution in 3 M hydrochloric acid shows
an absorption maximum at about 267 Dill.
C. Bum 20 mg by the oxygen-flask method (2.3.34), using 5 ml
of 2 M sodium hydroxide as the absorbing liquid and dilute to
25 ml with water. To 5 ml add 1 ml of silver nitrate solution; a
yellow precipitate is produced. Add 5 ml of 5 M ammonia,
shake, filter and acidifY the filtrate with nitric acid; a white
Tests
Acidity or alkalinity. Shake 0.5 g with 10 ml ofwater previously
neutralised to phenolphthalein solution. The solution is
colourless and not more than 0.05 ml of 0.1 M sodium hydroxide
is required to change the colour of the solution to pink.
Free iodine. Shake 1.0 g with a solution of 1 g of potassium
iodide in 20 ml of water for 30 seconds, allow to stand for
5 minutes and filter. To 10 ml of the filtrate add 1 ml of 1 M
sulphuric acid and 2 ml of chloroform and shake. Any colour
in the chloroform layer is discharged on the addition of0.1 ml
of 0.005 M sodium thiosulphate.
Reference solution (b). Treat a mixture of0.1 g ofthe substance

under examination and 0.5 ml ofpyridine as described for the
test solution.
- a glass column 1.5 m x 4 mm, packed with silanised
diatomaceous support (100 to 120 mesh) coated with 3
per cent w/w ofmethyl silicone gum,
- temperature:
column.190,
inlet port and detector. 240,
- flame ionisation detector,
- nitrogen as carrier gas.
In the chromatogram obtained with the test solution the peaks
following the solvent peak, in order of emergence, are due to
(a) 5-chloro-8-hydroxyquinoline, (b) 5,7-dichloro-8-hydroxyquinoline, (c) the internal standard, (d) quiniodochlor and (e)
5,7-diiodo-8-hydroxyquino1ine.
In the chromatogram obtained with reference solution (b)
calculate the content of 5-chloro-8-hydroxy-quinoline, 5,7~
dichloro-8-hydroxyquinoline and 5,7-diiodo-8-hydroxyquinoline by reference to the corresponding peaks in the
chromatogram obtained with the test solution.
The total content of the named impurities does not exceed
3.0 per cent w/w, the content of any other impurity does not
exceed 0.2 per cent w/w and the sum of the contents is not
more than 4.0 per cent w/w.
Halide ions. Shake 0.5 g with 25 ml of water for 1 minute and

filter. To the filtrate add 0.5 ml of2 M nitric acid and 0.5 ml of
0.1 M silver nitrate and allow to stand for 5 minutes. Any
opalescence produced is not more intense than that obtained
by adding 0.5 ml of 0.1 M silver nitrate to 25 ml of water
containing 0.5 ml of 2 M nitric acid and 0.2 ml of 0.01 M
hydrochloric acid and allowing to stand for 5 minutes.

anhydrous pyridine. Titrate with 0.1 M tetrabutylammonium
Related substances. Determine by gas chromatography

(2.4.13).
Test solution. Add 0.5 ml ofN,O-bis (trimethylsilyl)acetamide

to 0.5 ml ofa solution in pyridine containing 0.4 per cent w/v
of each of 5-chloro-8- hydroxyquinoline, 5,7-dichloro-8hydroxy- quinoline and 5-chloro-7-iodo-8-hydroxyquinoline
and 0.04 per cent w/v of the substance under examination,
mix, allow to stand for 15 minutes and add 5 ml ofa 0.05 per
cent w/v solution of dibutylphthalate (internal standard) in
hexane.
Reference solution (a). Add 0.5 m1 of N,O-bis
(trimethylsilyl)acetamide to a mixture of 0.1 g ofthe substance

0.03055 gofCgHsClINO.
Quiniodochlor Cream
Clioquinol Cream; Iodochlorhydroxyquinoline Cream;
Iodochlorhydroxyquin Cream
Quiniodochlor Cream contains Quiniodochlor in a suitable
base.
Quiniodochlor Cream contains not less than 90.0 per cent and
quiniodochlor, CgHsClINO.
2028
QUINIODOCHLOR OINTMENT
IP 2010
Usual strength. 4 per cent w/v.
temperature:
column. 165,
inlet port 170 and detector 250,
- flame ionization detector,
flow rate. 30 inl per minute ofhelium as the carrier gas.
Identification
chromatogram obtained with the reference solution. The test
solution and the reference solution prepared by using 1.0 ml
of pyridine instead of the internal standard solution.
B. Weigh accurately a quantity containing about 25 mg of
quiniodochlor, in a 100 ml volumetric flask, add 75 ml of 25 per
cent v/v solution of hydrochloric acid, heat on a steam bath
to melt the cream, shake vigorously to extract the quiniodochlor.
Cool under running water, and dilute to the volume with the
same solvent. Dilute 3.0 ml ofthe filtrate to 100.0 ml with the
same solvent.
When examined in the range 200 nm to 400 run (2.4.7), the

solution shows an absorption maximum at about 267 run.
resolution between the quiniodochlor and the intemal standard
peaks is not less than 3.0. The relative retention time with
reference to pyrene for quiniodochlor is about 0.6.
Calculate the content ofCgHsClINO in the Cream.
Clioquinol Ointment; Iodochlorhydroxyquinoline
Ointment; Iodochlorhydroxyquin Ointment
Tests
Quiniodochlor Ointment contains Quiniodochlor in a suitable

base.
Other tests. Complies with the tests stated under Creams.

Assay. Determine by gas chromatography (2.4.13).

pyrene in pyridine.
Solvent mixture. 80 volumes ofpyridine and 20 volumes of nhexane.
Test solution. Weigh accurately a quantity containing about
150 mg of quiniodochlor, in a 60-ml separator. Place the
separator on its side in a vacuum oven at a pressure of about
10 mm of mercury at 45 for 4 hours. Remove the separator
from the oven, allow to cool, add 15 ml ofthe solvent mixture,
insert a polytefstopper, and mix. Transfer the mixture to a 50ml volumetric flask, and rinse the separator with two 15 ml
portions of the solvent mixture, shaking each time for 30
seconds. Transfer both rinsings to the volumetric flask, dilute
with the solvent mixture to volume, andmix. Transfer 1.0 ml to
a screw-capped glass vial fitted with a septum, add 1.0 ml Qf
bis(trimethylsilyl)acetamide and 1.0 ml of internal standard
solution, attach the cap, and mix. Heat in a water-bath at 50
for 15 minutes, and coo 1.
quiniodochlor RS in the solvent mixture. Transfer 1.0 ml to a
screw-capped glass vial fitted with a septum, add 1.0 ml of
for 15 minutes, and cool.
a glass column 1.83 m x 2 mill, packed with 3 per cent
liquid phase G3 on 80 to 100-mesh support SlAB,
Quiniodochlor Ointment contains not less than 90.0 per cent

quiniodochlor, CgHsCIINO.
Identification
chromatogram obtained with the reference solution. The test
solution and the reference solution prepared by using 1.0 ml
of pyridine instead of the internal standard solution.
B. Weigh accurately a quantity containing about 25 mg of

quiniodochlorin a 100-ml volumetric flask, add 75 ml of 25 per
cent v/v solution of hydrochloric acid, heat on steam bath to
melt the ointment, shake vigorously to extract the
quiniodochlor. Cool under running water, dilute to volume
with the same solvent. Filter and dilute 3.0 ml ofthis solution
When examined in the range 200 run to 400 nm (2.4.7), the
solution shows an absorption maximum at about 267 nm.
Tests

pyrene in pyridine.
2029
IP 2010
QUINIODOCHLOR OINTMENT
Solvent mixture. 80 volumes ofpyridine and 20 volumes of nhexane.

Test solution. Transfer an accurately weighed quantity of the
ointment, containing about 150 mg of Quiniodochlor, to a
suitable conical flask, add 75 ml of n-hexane, and mix. Add 15
ml of dimethylformamide, and mix for 1 minute. Allow the
layers to separate, and transfer the lower layer to a 50-ml
volumetric flask, repeat the extraction with separate 15 ml and
10 ml portions of dimethylformamide, and transfer the lower
layers to the 50-ml volumetric flask. Dilute with
dimethylformamide to volume, and mix. Transfer 1.0 ml ofthis
solution to a screw-capped glass vial fitted with a septum,
and evaporate at about 60 under a stream of nitrogen to
dryness. Add 1.0 ml of a mixture of internal standard solution
to the residue, add 1.0 ml of bis(trimethylsilyl)acetamide and
1.0 ml of internal standard solution, attach the cap, and mix.
Heat in a water-bath at 50 for 15 minutes and cool.
qUiniodochlor RS in the solvent mixture. Transfer 1.0 ml to a
screw-capped glass vial fitted with a septum, add 1.0 ml of
for 15 minutes, and cool.
liquid phase G3 on 80 to 1OO-mesh support SlAB,
- temperature:
column. 165,
- flame ionization detector,
- flow rate. 30 ml per minute ofhelium as the carrier gas.
resolution between the quiniodochlor and the internal standard
peaks is not less than 3. The relative retention time with
reference to pyrene for quiniodochlor is about 0.6.
Calculate the content ofC 9H sCIINO in the Ointment.
Quiniodochlor Tablets
Clioquinol Tablets; Iodochlorhydroxyquinoline Tablets;
Iodochlorhydroxyquin Tablets
90.0per
Quiniodochlorta1:>lets contaiiiii6t less thaii

cent and
quiniodochlor, C9HsClINO.
Identification
250 mg ofQuiniodochlor with 20 ml of acetone, filter and add
20 ml of water to the filtrate. Collect the precipitate formed on
a filter and dry at 105. The residue complies with the following
tests.
Compare the spectrum with that obtained with quiniodochlor
RS or with the reference spectrum of quiniodochlor.
0.0005 per cent w/v solution in 3 M hydrochloric acid shows
an absorption maximum at about 267 nm.
Tests
Disintegration (2.5.1). Maximum time, 30 minutes.
Assay. Weigh and finely powder 20 tablets. Weigh accurately
a quantity ofthe powder containing 0.125 g ofQuiniodochlor,
shake with 20 ml of hot 2-methoxyethanol, decant the hot
supernatant liquid through a fine filter. Repeat the extraction
with two further quantities, of 20 ml and 10 ml, of
2-methoxyethanol, combine the filtered extracts and dilute to
50.0 ml with 2-methoxyethanol. To 5.0 ml ofthis solution add
1 ml of water and sufficient of a mixture of 24 volumes of
2-methoxyethanol and 6 volumes of water to produce 50.0 ml.
To 10.0 ml ofthe solution add 10 ml of 2-methoxyethanol and
2 ml ofa solution prepared by dissolving 0.5 g offerric chloride
hexahydrate in 80 ml of 2-methoxyethanol and adding 0.1 ml
of hydrochloric acid and sufficient 2-methoxyethanol to
produce 100 ml. Dilute the solution to 25.0 ml with
2-methoxyethanol and measure the absorbance of the
resulting solution at the maximum at about 650 nm (2.4:7),
using as blank a solution prepared by treating 10 ml of the
aqueous 2-methoxyethanol in the same manner beginning at
the words "add 10 ml of 2-methoxyethanol... ..".
Calculate the content of C 9H sCIINO from the absorbance
obtained using 10.0 ml ofa solution prepared in the following
manner. Dissolve 0.125 g of quiniodochlor RS in sufficient 2methoxyethanol to produce 50.0 ml, warming to effect solution;
add 1 ml of water to 5.0 ml ofthe solution and add sufficient of
the mixture of24 volumes of 2-methoxyethanol and 6 volumes
of water to produce 50.0 ml. Using 10.0 ml of this solution
repeat the operation beginning at the words "add 10 ml of
2-methoxyethanol.. .."
2030
QUINI0DOCHLOR AND HYDROCORTISONE CREAM
IP 2010
Quiniodocblor and Hydrocortisone

Cream
the filtrate with 2 M nitric acid; a white precipitate is produced

which darkens on exposure to light.
Clioquinol and Hydrocortisone Cream;

Iodochlorhydroxyquinoline and Hydrocortisone
Cream; Iodochlorhydroxyquin and Hydrocortisone
Cream
Tests
Assay. For hydrocortisone chromatography (2.4.14).
Quiniodochlor and Hydrocortisone Cream contains

Hydrocortisone and Quiniodochlor in a suitable base.
Quiniodochlor and Hydrocortisone Cream contains not less
stated amount of quiniodochlor, CgHsCIINO and not less than
amount of hydrocortisone, CZIH300s.
Usual Strength. Quiniodochlor 4 per cent w/w and
Hydrocortisone 1 per cent w/w.
Identification
the plate with silanised silica gel G.
Mobile phase. A mixture of77 volumes of dichloromethane,

15 volumes of ether, 8 volumes of methanol and 1.2 volumes
of water.
Test solution. Disperse by warming and shaking, a quantity of
the cream containing 2.5 g of Hydrocortisone in 10 ml of
ethanol (95 percent), cool, allow to stand at 0 for 30 minutes,
filter and use the filtrate.
hydrocortisone RS in ethanol (95 per cent).
Reference solution (b). Dissolve 12.5 mg of hydrocortisone
RS in 5 ml of test solution.
dry the plate in air and spray with alkaline tefrazoliurn blue
solution. The principal spot in the chromatogram obtained
with test solution corresponds to that in the ~hromatogram
obtained with reference solution (a). The principal spot in the
as a single, compact spot.
B. In the Assay, the principal peak in the chromatogram due to
hydrocortisone obtained with test solution (b) corresponds
to the peak due to hydrocortisone in the chromatogram
C. Fuse a quantity of the cream containing 0.1 g of
quiniodochlor with anhydrous sodium carbonate, dissolve
the fused mass in water and acidify with 2 M nitric acid. Add
silver nitrate solution; a pale yellow precipitate is produced
which is insoluble in 5 M ammonia. Add 5 M ammonia until
the solution becomes alkaline, boil gently, filter and acidify
Determine by liquid
Test solution (a). Add 30 ml of 2,2,4-trimethylpentane to a

quantity of the cream containing 10 mg of Hydrocortisone
and warm on a water-bath until the preparation has melted.
Extract the warm mixture with successive quantities ono, 20
and 20 ml of methanol (80 per cent), combine the aqueous
methanolic layers, cool to about 20 and dilute to 100 ml with
the same solvent.
Test solution (b). Prepare in the same manner as test solution
(a) but adding)O ml of a 4 per cent v/v solution of
bromobenzene in methanol to the cooled methanolic extract
and dilute to 100.0 ml with methanol (80 per cent).
Reference solution. Dissolve 5 mg of hydrocortisone RS in 5
ml of a 4 per cent v/v solution of bromobenzene (internal
standard) in methanol and dilute to 50 ml with methanol (80
per cent).
.
a stainless steel column 25 em x 5 mm, packed with
- mobile phase: methanol (65 per cent),
Calculate the content ofCzIH300s in the cream.
For quiniodochlor - Weigh accurately a quantity of the

cream containing about 25 mg ofquiniodochlor, add 80 ml of
a hot mixture of 6 volumes of water and 24 volumes of
2-methoxyethanol and heat on a water-bath for 5 minutes.
Cool in ice for 10 minutes, allow to warm to room temperature,
dilute to 100 ml with a mixture of 6 volumes of water and
24 volumes of 2-methoxyethanol, mix and filter. To 10 ml ofthe
filtrate, add 10 ml of 2-methoxyethanol and 2 ml ofa solution
prepared by dissolving 0.5 g of iron(III) chloride hexahydrate
in 80 ml of 2-methoxyeth~nol and adding 0.1 ml of
hydrochloric acid and sufficient 2-methoxyethanol to
produce 100 m!. Dilute the solution to 25 ml with
2-methoxyethanol and measure the absorbance of the
using in the reference cell a solution prepared by treating
10 ml ofthe mixture of6 volumes of water and 24 volumes of
2-methoxyethanol in the same manner beginning at the words
"add 10 ml of 2-methoxyethanol...... ".
2031
QUINIODOCHLOR AND HYDROCORTISONE OINTMENT
IP 2010
Repeat the operation beginning at the words 'add 10 ml of

2-methoxyethanol using 10 ml ofa solution prepared in the
following manner. Dissolve 0.125 g of clioquinol RS in
sufficient 2-methoxyethanol to produce 50 ml; warming to
effect solution; add 1 ml of water to 5 ml of the solution and
add sufficient of a mixture of 6 volumes of water and
24 volumes of 2-methoxyethanol to produce 50 ml.
Calculate the content ofC 9HsCIINO in the cream.
Quiniodochlor and Hydrocortisone

Ointment
B. In the Assay, the principal peak in the chromatogram due to

hydrocortisone obtained with test solution (b) corresponds
to the peak due to hydrocortisone in the chromatogram
C. Fuse a quantity of the ointment containing 0.1 g of
Quiniodochlor with anhydrous sodium carbonate, dissolve
the fused mass in water and acidify with 2 M nitric acid. Add
silver nitrate solution; a pale yellow precipitate is produced
which is insoluble in 5 M ammonia. Add 5 M ammonia until
the solution becomes alkaline, boil gently, filter and acidify
the filtrate with 2 M nitric acid; a white precipitate is produced
which darkens on exposure to light.
Tests
Clioquinol and Hydrocortisone Ointment;

Ointment; Iodochlorhydroxyquin and Hydrocortisone
Ointment
Assay. For hydrocortisone

chromatography (2A.14).
Quiniodochlor and Hydrocortisone Ointment contains

Quiniodochlor and Hydrocortisone in a suitable base.
Quiniodochlor and Hydrocortisone Ointment contains not less
stated amount of quiniodochlor, C9HsClINO and not less than
amount of hydrocortisone, CZIH300S'
Identification
the plate with silanised silica gel G.
15 volumes of ether, 8 volumes of methanol and 1.2 volumes
of water.
Test solution. Disperse by warming and shaking, a quantity of
the ointment containing 2.5 g of Hydrocortisone in 10 ml of
ethanol (95 per cent), cool, and allow to stand at 0 0 for 30
minutes, filter and use the filtrate.
hydrocortisone RS in ethanol (95 per cent).
Referencesolution (b). Dissolve 12.5 mg of hydrocortisone
RS in 5 ml of test solution.

dry the plate in air and spray with alkaline tetrazolium blue
with test solution corresponds to that in the chromatogram
Determine by liquid
Test solution (a). Add 30 ml of 2,2,4-trimethylpentane to a

quantity of the ointment containing 10 mg ofHydrocortisone
and warm on a water-bath until the preparation has 'melted.
Extract the warm mixture with successive quantities ono, 20
and 20 ml of methanol (80 per cent), combine the aqueous
methanolic layers, cool to about 20 0 and dilute to 100 ml with
the same solvent.
Test solution (b). Prepare at the same manner as test solution
(a) but adding 10 ml of a 4 per cent v/v solution of
bromobenzene in methanol to the cooled methanolic extract
and dilute to 100.0 ml with methanol (80 per cent).
Reference solution. Dissolve 5 mg of hydrocortisone RS in 5
ml of a 4 per cent v/v solution of bromobenzene (internal
standard) in methanol and dilute to 50 ml with methanol (80
per cent).
mobile phase: methanol (65 per cent),
iI1jection volume. 10 Ill.
Calculate the content ofCzIH300s in the ointment.
For Quiniodochlor- Weigh accurately a quantity of the
ointment containing 25 mg of Clioquinol, add 80 mlof a hot
mixture of24 volumes of 2-methoxy-ethanol and 6 volumes
of water and heat on a water-bath for 5 minutes. Cool in ice for
10 minutes, allow to warm to room temperature, dilute to 100
ml with a mixture of24 volumes of 2-methoxy-ethanol and 6
volumes of water, mix and filter. To 10 rn1 ofthe filtrate, add 10
2032
QUINIODOCHLOR AND HYDROCORTISONE OINTMENT
IP 2010
ml of 2-methoxyethanol and 2 ml of a solution prepared by

dissolving 0.5 g of iron(III) chloride hexahydrate in 80 ml of
2-methoxyethanol and adding 0.1 ml of hydrochloric acid
and sufficient 2-methoxyethanol to produce 100 m!. Dilute
the solution to 25 ml with 2-methoxyethanol and measure the
absorbance of the resulting solution at the maximum at 650
urn (2.4.7), using in the reference cell a solution prepared by
treating 10 m1 ofa mixture of24 volumes of 2~methoxy-ethanol
and 6 volumes of water in the same manner beginning at the
words "add 10 ml of 2-methoxyethanol..... n.
Repeat the operation beginning at the words 'add 10 ml of 2methoxyethanol using 10 ml of a solution prepared in the
following manner. Dissolve 0.125 g of clioquinol RS in
sufficient 2-methoxyethanol to produce 50 ml, warming to
effect solution; add 1 ml of water to 5 ml of the solution and
add sufficient of a mixture of 6 volumes of water and 24
volumes of 2-methoxyethanol to produce 50 m!.
Calculate the content ofC 9HsCllNO in the ointment.
2033
MONOGRAPHS
R
Rabeprazole Sodium
2037
Rabeprazole Tablets
2037
Ramipril
2038
Ramipril Capsules
2039
Ramipril Tablets
2040
Ramipril and Hydrochlorothiazide Tablets

Ranitidine Hydrochloride
2041
2043
Ranitidine Injection
2044
Ranitidine Tablets
2045
Purified Rayon
2046
Reserpine
2047
Reserpine Injection
2048
Reserpine Tablets
2049
Ribavirin
2050
Ribavirin Inhalation Solution

Riboflavin
2051
2052
Riboflavin SodiumPhosphate
2053
Riboflavin Tablets
2054
Rifumpicin
2054
Rifampicin Capsules
Rifampicin Oral Suspension
2055
2056
Rifampicin Tablets
2058
Rifampicin and Isoniazid Tablets

Rifampicin, Isoniazid and Ethambutol Tablets
2059
2061
Rifampicin, Isoniazid and Pyrazinamide Tablets
2063
Rifampicin, Isoniazid, Pyrazinamide and Ethambutol Tablets
2065
Ritonavir
2066
Ritonavir Capsules
2067
Ritonavir Tablets
2068
2070
Rosiglitazone Maleate
2035
MONOGRAPHS
Rosiglitazone Tablets
2070
Rosuvastatin Calcium
2071
Rosuvastatin Tablets
2072
Roxithromycin
2073
Roxithromycin Tablets
2075
2036
IP 2010
RABEPRAZOLE TABLETS
Rabeprazole Sodium
Water (2.3.43). Not more than 7.0 per cent, determined on 0.3 g,
Solvent mixture. 80 volumes of methanol, 20 volumes of water

and 0.1 volume of diethylamine.
Mol. Wt. 381.4
Rabeprazole sodium is 2-( {[4-(3-methoxypropoxy)-3-methyl2-pyridinyl]methyl} sulphinyl)-IH-benzimidazole sodium.
Rabeprazole sodium contains not less than 98.0 per cent and
not more than 102.0 per cent ofClsHzoN303S,Na, calculated on
Category. Antiulcer.
Description. A white to light yellow, crystalline powder,
hygroscopic.
Identification
Compare the spectrum with that obtained with rabeprazole
sodium RS.
B. A 10 per cent w/v solution in carbon dioxide-free water

gives reaction ofsodium (2.3.1).
Tests

examination in 100.0 ml ofsolvent mixture. Dilute 5.0 ml ofthe
solution to 100.0 ml with the same solvent.
rabeprazole sodium RS in the solvent mixture.
octylsilane bonded to porous silica (5 !!m) (Such as
Hypersil keystone betabasic C s),
mobile phase: a mixture of 72 volumes of 0.1 M phosphate buffer pH 7.0 and 28 volumes of acetonitrile,
- injection volume. 10 !!l.

(2.4.14).

examination in 100 ml with the solvent mixture.
Chromatographic system as described under Assay.
chromatogram three times of the principal peale In the
is not more than 1.5 times the area ofthe peak in the chromatogram obtained with the reference solution (b) (1.5 per cent).
Calculate the percentage content ofClsHzoN303S,Na.

Rabeprazole Tablets
Rabeprazole Sodium Tablets
Rabeprazole Tablets contain not less than 90.0 per cent and
rabeprazole sodium, ClsHzoN303SNa. The tablets are enteric
coated.
Identification
Tests
Apparatus No.1,
2037
IP2010
RABEPRAZOLE TABLETS
Replace the 0.1 M hydrochloric acid with phosphate buffer
pH 7.4. Run the apparatus at 75 ipm for 45 minutes. Withdraw
a suitable volume of the medium and filter. Measure the
absorbance ofthe filtered solution immediately, suitably diluted
with the dissolution medium, ifnecessary, at the maximum at
about 291 nm (2.4.7). Calculate the content ofCIsHzoN303SNa
in the medium from the absorbance obtained from a solution
of known concentration of rabeprazole sodium RS, prepared
by dissolving in minimum quantity ofa mixture of?5 volumes
of acetonitrile and 25 volumes of methanol and suitably
diluted with the dissolution medium.
ClsHzoN303SNa.
(2.4.14).

tablet containing 50 mg of Rabeprazole Sodium, disperse in
100 ml ofsolvent mixture and filter.
secondary peak is not more than twice the area of the peak in
the chromatogram obtained with reference solution (b)
is not more than 6 times the area ofthe peak in the chromatogram obtained with the reference solution (b) (6.0 per cent).
Comply with the tests stated under Tablets.
Disperse 1 tablet in sufficient 0.1 M sodium hydroxide to
produce 0.0015 per cent w/v solution. Measure the absorbance
Calculate the content ofCIsHzoN303SNafrom the absorbance
obtained from same concentration of rabeprazole sodium RS
in the same medium.

a quantity of the powdered tablet containing 50 mg of
Rabeprazole Sodium, disperse in 20 ml of 0.1 M sodium
hydroxide and dilute to 100.0 ml with solvent mixture, filter.
rabeprazole sodium RS, dissolve in 10 ml of 0.1 M sodium
hydroxide and dilute to 50.0 ml with solvent mixture.
a stainless steel column 25 cm ' 4.6 mm packed with
mobile phase: a mixture of65 volumes of 0.15 per cent
w/v solution of potassium dihydrogen phosphate
previously adjusted pH to 6.0 with orthophosphoric
acid or sodium hydroxide solution and 35 volumes of
acetonitrile,
Calculate the content ofClsHzoN303SNa.

Ramipril
CZ3H3ZNzOs
Mol. Wt. 416.5
Ramipril is (2S,3aS,6aS)-1-[(S)-2-[[(S)-I-(ethoxycarbonyl)3-phenylpropyl]amino]propanoyl]
octahydrocyclopenta[b]pyrrole-2-carboxylic acid.
Ramipril contains not less than 98.0 per cent and not more
than 101.0 per cent ofCz3H32NzOs. calculated on the dried basis.
Identification
Compare the spectrum with that obtained with ramipril RS.
2038
IP 2010
RAMIPRIL CAPSULES
Tests
is not more than the area of the peak in the chromatogram
Appearance ofsolution. A 1.0 per cent w/v solution in methanol

Specific optical rotation (2.4.22). + 32.0 to + 38.0, detennined
in 1.0 per cent w/v solution in 0.1 M methanolic hydrochloric
acid.
(2.4.14).

examination in 25 ml ofmobile phase B.
Reference solution (a). A 0.1 per cent w/v solution of ramipril
RS in the mobile phase B.
100 ml with mobile phase B.
- mobile phase: A. dissolve 2.0 g of sodium perchlorate
in a mixture of 0.5 ml of triethylamine and 800 ml of
water; adjust pH to 3.6 with orthophosphoric acid and
add 200 ml of acetonitrile,
B. dissolve 2.0 g of sodium perchlorate
in a mixture of 0.5 ml of triethylamine and 300 ml of
water; adjust pH to 2.6 with orthophosphoric acid and
add 700 ml of acetonitrile,
below,
Time
(in min.)
Mobile phase A
(per cent v/v)
90
Mobile phase B
(per cent v/v)
,10
90
10
Sulphated ash (2.3.18). Not more than O.1percent.

on 1.0 g by drying in an oven at 60, under vacuum, for
4 hours.
Assay. Weigh accurately about 0.3 gm, dissolve in 25 ml of
methanol and add 25 ml of water. Titrate with 0.1 M sodium
hydroxide. Detennine the end-point potentiometrically (2.4.25).
1 ml of 0.1 M sodium hydroxide is equivalent to 0.04165 gm of
CZ3H32NzOs.
Ramipril Capsules
Ramipril Capsules contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of ramipnl,
CZ3H32NzOs
Usual strengths. 1.25 mg; 2.5 mg; 5 mg; 10 mg.
Identification
Shake a quantity of the content of the capsules containing
25 mg of Ramipril with 50 ml of acetone, centrifuge for
10 minutes, filter. Evaporate the filtrate to dryness at 60 for
3 hours. The residue complies with the following test.
Tests
75
25
Apparatus No.1,
20
65
35
30
25
75
40
25
75
45
90
10
Test solution. Dilute the filtrate to get 0.00025 per cent w/v
solution of Ramipril with 0.1 M hydrochloric acid.
55
90
10
chromatogram obtained with the te,st solution, the area of any
Reference solution. A 0.00025 per cent w/v solution oframipril

Calculate'the content of CZ3H32NzOs.
Cz3H32NZOS'
'
2039
RAMIPRlL CAPSULES
IP 2010
Uniformity of content (For capsules containing 10 mg or

less). Comply with the test stated under Capsules.
Evaporate the filtrate to dryness at 60 for 3 hours. The residue

complies with the following test.

Test solution. Disperse one capsule in 100 ml of 0.1 M hydrochloric acid, sonicate for 15 minutes. Dilute if necessary, to
produce 0.00025 per cent w/v solution of Ramipril in 0.1 M
hydrochloric acid.
Tests
Apparatus No.1,
Calculate the content ofCz3H3zNzOs.
Test solution. Dilute the filtrate to get 0.00025 per cent w/v
solution of Ramipril with 0.1 M hydrochloric acid.

ramipril RS in 0.1 M hydrochloric acid.
Test solution. Weigh a quantity of the content of capsules

containing 25 mg of Ramipril, disperse in 100.0 ml of
0.1 M hydrochloric acid, mix and centrifuge. Dilute 1.0 ml of
the resulting solution to 100.0 ml with 0.1 M hydrochloric
acid.
Reference solution. A 0.00025 per cent w/v solution of ramipril
- a stainless steel column 12.5 cm X 4.6 mm packed with
and 58 volumes of a solution containing 1.4 per cent
w/v solution of sodium perchlorate and 0.58 per cent
w/v solution of orthophosphoric acid adjusted to
pH 2.5 with triethylamine, adjust the pH ofthe mixture
to 2.1 with orthophosphoric acid,
than 2.0 per cent.
Calculate the content of CZ3H32NzOs.
.Ramipril Tablets
Reference solution. A 0.00025 per cent wIv solution of ramipril

Calculate the content ofCz3H32NzOs.
CZ3H3ZNzOs.
under Assay.
Test solution. Take one tablet, add 5 ml of 0.1 M hydrochloric

acid, sonicate for 10 minutes, dilute, if necessary, with
sufficient 0.1 M hydrochloric acid to produce a solution
containing 0.00025 per cent w/v of Ramipril, centrifuge and
Test solution. Weigh and powder 20 tablets. Weigh a quantity

ofpowdered tablets containing 25 mg ofRamipril, disperse in
100.0 ml of 0.1 M hydrochloric acid and centrifuge. Dilute 1.0
ml ofthe resulting solution to 100.0 ml with 0.1 Mhydrochloric
acid.
Reference solution. A 0.00025 per cent wIv solution of ramipril
Ramipril Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of ramipril,
Cz3H32NZOS'
Usual strengths. 1.25 mg; 2.5 mg; 5 lUg; 10 mg.
Identification
Shake a quantity ofthe powdered tablets containing 25 mg of
. Ramipril with 50 ml of acetone, centrifuge for 10 minutes, filter.
- a stainless steel column 12.5 cm X 4.6 mm packed with
octadecylsilane bonded to porous silica (5/lm),
mobile phase: a mixture of 42 volumys of acetonitrile
w/v solution of sodium perchlorate and 0.58 per cent
w/v solution of orthophosphoric acid adjusted to pH
2.5 with triethylamine, adjust the pH of the mixture to
2040
IP 2010
RAMIPRIL AND HYDROCHLOROTHIAZIDE TABLETS

- mobile phase: a mixture of 55 volumes of water, 45

volumes of acetonitrile, and 0.1 volume of
triethylamine, adjusted to pH 3.0 with orthophoshoric
acid,
than 2.0 per cent.
Inject reference solution (a) and (b). The relative standard

deviation for replicate injections for each peak is not more
than 2.0 per cent.
Inject the test solution, reference solution (a) and (b).
Calculate the content ofCz3H32NzOs and C7HgClN304Sz.

D. Not less than 75 per cent ofthe stated amount ofCz3H32NzOs
and C7HgClN304SZ.
Ramipril and Hydrochlorothiazide

Tablets
Ramipril and Hydrochlorothiazide Tablets contain not less

stated amounts of ramipril, CZ3H32NzOs and
hydrochlorothiazide, C7HgClN304SZ.
Usual strength. Ramipril 2.5 mg and Hydrochlorothiazide

12.5 mg.
Identification
In the Assay, the principal peaks in the chromatogram obtained
with the test solution corresponds to the principal peaks in
Tests
Apparatus No.1,
Medium.750 ml of 0.1 M hydrochloric acid,
Test solution. Dilute the filtrate, if necessary, with the

dissolution medium.
Reference solution (a). Dissolve a quantity of ramipril RS in
the mobile phase and dilute with dissolution medium to obtain
a solution having a known concentration similar to the test
solution.
Reference solution (b). Dissolve a quantity of
hydrochlorothiazide RS in mobile phase and dilute with
dissolution medium to obtain a solution having a known
Chromatographic. system
as Thermo quest Hypersil),
(2.4.14).

containing about 10 mg ofRamipril in mobile phase A, sonicate
for 15 minutes and dilute to 10.0 ml with the same solvent.
w/v of ramipril RS and 1.0 per cent w/v of hydrochlorothiazide
to 50.0 rn1 with mobile phase A. Dilute 5.0 rol ofthis solution to
solution prepared by dissolving 4 g of sodium
perchlorate in 600 ml of water, add 1.0 rol of
triethylamine, adjusted to pH 2.6 with orthophosphoric
B. a mixture of 85 volumes of buffer
solution and 15 volumes of acetonitrile,
below,
Time
Mobile phase A
Mobile phase B
(per cent v/v)
(per cent v/v)
(in min)
0-13
0
100
13 - 17
0 ~50
100 ~50
17-20
50~100
50 ~O
20 - 80
100
o
o ~100
80 - 82
100 ~
82 -87
0
100
2041
IP 2010
RAMIPRIL AND HYDROCHLOROTHIAZIDE TABLETS
relative standard deviation for replicate injections for each
peak is not more than 5.0 per cent. The relative retention time
with reference to ramipril forramipril impurity A is about 0.9,
for ramipril impurity B is about 1.2, for ramipril impurity C is
about 1.5 and for ramipril impurity D is about 1.7.
Inject the test solution and reference solution (b). The retention
time oframipril is about 30 minutes and ofhydrochlorothiazide
is about 10 minutes. In the chromatogram obtained with the
test solution the area of each peak due to ramipril impurity A,
Band C is not be more than the area of the principal peak in
per cent); the area of peak due to ramipril impurity D is not
cent); the area of any other secondary peak is not more than
Tablets.
disperse in water and dilute to 250 ml with the mobile phase,

filter.
Reference solution. A 0.01 per cent w/v solution of ramipril

ChromatographiC system
octadecylsilane bonded to porous silica (5 j.tm), (Such
as Thermo quest Hypersil),
mobile phase: a mixture of 55 volumes of water, 45
volumes of acetonitrile and 0.1 volume of triethylamine,
injection volume. 10 j.tl.
theoretical plates of the principal peak is not less than 2000
theoretical plates and the tailing factor is not more than 2.0.
The relative standard deviation for replicate injections is not
For ramipril- Determine by liquid chromatography (2.4.14).
Calculate the content of C23H32N20S in the tablet.
Test solution. Disperse 1 intact tablet in water and dilute to 50

For hydrochlorothiazide-
Reference solution. Dissolve a quantity of ramipril RS in the

mobile phase and dilute with the mobile phase to obtain a
solution having a known concentration similar to the test
solution.
Reference solution. Dissolve 12.5 mg of hydrochlorothiazide

RS with 10 ml of acetonitrile, sonicate and dilute to 100 ml

Calculate the content of C23H32N20S in the tablet.
For hydrochlorothiazide-Determine
by

containing about 125 mg ofHydrochlorothiazide with 50 ml of
acetonitrile, sonicate for 15 minutes and dilute to 250 ml with
the mobile phase. Dilute 5.0 ml ofthis solution to 20 ml with
the mobile phase.
liquid
Test solution. Disperse 1 tablet with sufficient amount ofthe

mobile phase, sonicate for 15 minutes and dilute to 100 ml with
the mobile phase.
Reference solution. Dissolve a quantity of
hydrochlorothiazide RS in the mobile phase and dilute with
the mobile phase to obtain a solution having a known
octadecylsilane bonded to porous silica (5 j.tm),
mobile phase: a mixture of 9 volumes of 0.1 M sodium
dihydrogen orthophosphate and 1 volume of
acetonitrile, adjusted to pH 3.0 with orthophosphoric
acid,
injection volume. 10 j.tl.
theoretical plates ofhydrochlorothiazide peak is not less than
2000 and the tailing factor is not more than 2.0. The relative
standard deviation for replicate injections is not more than 2.0
per cent.

Calculate the content of C7HsClN304S2 in the tablet.
Assay. For ramipril-Determine by liquid chromatography
(2.4.14).
Test solution. Weigh and powder 20 tablets: Weigh accurately

a quantity of powder containing about 25 mg of Ramipril,
Storage. Store protected from moisture, at. a temperature not

exceeding 30.
Calculate the content ofC7HsCIN304S2 in thetilblet.
2042
IP 2010
RANITIDINE HYDROCHLORIDE

equivalent amount oframipril and hydrochlorothiazide.

methanol.
Ranitidine Hydrochloride
H
~V"S~N~CHN02
H3C--N
CH
C13H22N403S,HCl
I
NHCH 3
, HCI
Reference solution (a). Weigh accurately a quantity of

ranitidine hydrochloride RS in methanol, and dilute with
methanol to obtain a solution containing a known
concentration of about 0.022 per cent w/v.
to 20 ml with methanol.
Mol. Wt. 350.9
Ranitidine Hydrochloride is N-[2-[[[ 5-[(dimethylamino)

methyl]furan-2-yl]methyl]thio]ethyl]-N-methyl-2nitroethene-l, 1-diamine hydrochloride.

Reference solution (d). Dilute 5.0 ml of reference solution (a)
Ranitidine Hydrochloride contains not less than 97.5 per cent

and not more than 102.0 per cent of C13H22N403S, HCl,
Category. Antiulcer (Histamine H2-receptor antagonist).
Dose. Orally, the equivalent of 300 to 600 mg of ranitidine
daily, in divided doses; by intramuscular or slow intravenous
injection, the equivalent of 50 mg of ranitidine every 6 to 8
hours. (1.12 g of ranitidine hydrochloride is approximately
equivalent to 1 g ofranitidine).
Description. A white to pale yellow, crystalline powder.
Identification

Compare the spectrum with that obtained with ranitidine
hydrochloride RS or with the reference spectrum ofranitidine
hydrochloride.
.B. In the test for Related substances, the principal spot in the
(2.3.1).
Tests
BYS5 (2.4.1).
.
Mobile phase. A mixture of 25 volumes of ethyl acetate,

15 volumes of 2-propanol, 4 volumes of strong ammonia
solution and 2 volumes of water.
Reference solution (e). Weigh accurately a quantity of (N,N'bis[2-[[[5-(dimethylamino) methyljfilran-2-yl]methylj

sulphanyl]ethyl]-2-nitroethene-l, l-diamine RS (ranitidine
impurity A RS) in methanol, and dilute with methanol to obtain
a solution containing a known concentration of about 0.127
per cent w/v.
Reference solution (f). Weigh accurately a quantity of 2-[[[5[(dimethyl am ino) me thyl]furan-2-yl]methyl]
sulphanyljethanamine RS (ranitidine impurity B RS) in
methanol, and dilute with methanol to obtain a solution
containing a known concentration of about 0.1 per cent w/v.
Apply to the plate 10 III of each solution except reference
solution (e). Apply separately an additional 10 III of the test
solution and on top'ofthis application, apply 10 III ofreference
solution (e). After development, dry the plate in air and expose
it to iodine vapours in a closed chamber until the spots are
revealed. Any spot in the chromatogram obtained with the
test solution (a) corresponding to the principal spot in the
chromatogram obtained with reference solution (f) is not more
intense than that of the principal spot in the chromatogram
obtained with reference solution (b) (0.5 per cent) and no
other spot in the chromatogram obtained with the test solution
is more intense than the principal spot in the chromatogram
obtained with reference solution (c) (0.3 per cent). The sum of
the intensities of all the secondary spots in the chromatogram
obtained with the test solution does not exceed 1.0 per cent.
The test is not valid unless the chromatogram obtained with
the combined test solution and reference solution (e) shows
two clearly separated principal spots and the chromatogram
obtained with reference solution (d) shows a clearly visible
spot.
Sulphated asb(2.3 .18). Not more than 0.1 per cent.
Loss on drying (2.4.19). Not more than 0.75 percent,
determined on 1.0 g by drying in an oven at 60 at a pressure
2043
RANITIDINE INJECTION
IP 2010
Test solution. A 0.01l2 per cent w/v of the substance under

examination in the mobile phase.
Reference solution. 0.0112 per cent w/v of ranitidine
hydrochloride RS in the mobile phase.
octadecylsilane bonded to porous silica (5 )lm),
15 volumes of 0.1 M ammonium acetate,
-spectrophotometer set at 322 nm,
- injection volume. 20 )ll.
than 2.0 per cent.

Tests
pH (2.4.24).6.7 to 7.3, ifthe preparation is buffered; 4.5 to 7.0,
ifthe preparation is unbuffered.

15 volumes 'of 2-propanol, 4 volumes of strong ammonia
Test solution. Dilute suitably a volume of the injection with
water to produce a solution containing the equivalent of
2.5 per cent w/v ofranitidine in water.
ranitidine hydrochloride RS in water, and dilute with water
to obtain a solution containing a known concentration of
about 0.056 per cent w/v.

Calculate the content ofCI3H22N403S, HCI.

to 20 ml with water.
Ranitidine Hydrochloride Injection
Reference solution (d). Dilute 6.0 ml ofreference solution (c)

Ranitidine Injection is a sterile solution of Ranitidine

Hydrochloride in Water for Injections and may be suitably
buffered.
Reference solution (e). Dilute 5.0 ml ofreference solution (b)

Ranitidine Hydrochloride Injection contains not less than

amount ofranitidine, C13H22N403S.
Usual strength. The equivalent of50 mg ofranitidine in 2 ml
(1.12 g ofranitidine hydrochloride is approximately equivalent
to 1 g ofranitidine).
Identification
A. To a volume ofthe injection containing 25 mg ofranitidine

add 20 ml of methanol, mix and evaporate to dryness. Add
1 ml of light petroleum (6(J' to 80) to the resulting residue,
scratch the side of the vessel with a glass rod to induce
crystallisation, evaporate to dryness and dry the residue at
60 for 10 minutes.The residue complies with the following
test.
hydrochloride.
Reference solution (f). Dilute 5.0 ml of reference solution (e)

Reference solution (g). A 0.127 per cent w/v of solution of
(N,N'-bis[2-[[[5-[(dimethylamino)methyl}furan-2yl]methyl]sulphanyl] ethyl]-2-nitroethene-1, 1-diamine RS
(ranitidine impurity A RS) in methanol.
Apply to the plate 10 )ll of each solution. Apply separately an
additional 10 )ll of the test solution and on top of this
application, apply 10 )ll of reference solution (g). After
development, dry the plate in air and expose it to iodine vapours
in a closed chamber until the spots are revealed. The major
solution is not more intense than that of the principal spot in
the chromatogram obtained with reference solution (a)
(2.0 per cent) and no other secondary spot in the chromatogram
obtained with the test solution is more intense than the
solution (b) (1.0 per cent). The sum ofthe intensities of all the
. secondary spots in the chromatogram obtained with the test
solution does not exceed 5.0 per cent.
2044
IP 2010
RANlTIDINE TABLETS

the combined test solution and reference solution (g) shows
obtained with reference solution (f) shows a clearly visible
spot.
Other tests. Comply with the tests stated under Parenteral
Test solution. Dilute a volume of the injection containing
10.0 mg ofranitidine to 100.0 ml with the mobile phase.
ranitidine hydrochloride RS in the mobile phase.
octadecylsilane bonded to porous silica (5 fJ.m),
- injection volume. 20 fJ.1.
than 2.0 per cent.
dryness and dry the residue at 60 for 10 minutes. The residue

hydrochloride.
Tests
15 volumes of 2-propanol, 4 volumes of strong ammonia
containing 0.45 g of ranitidine with 20 ml of methanol and
filter.
ranitidine hydrochloride RS in methanol, and dilute with
methanol to obtain a solution containing a known
concentration of about 0.022 per cent w/v.
Calculate the content ofCI3H22N403S in the injection.

Reference solution (c). Dilute 30 ml of reference solution (a)

Labelling. The label states (1) the strength in terms of the

equivalent amount of ranitidine; (2) where appropriate, that
the injection is buffered.
Reference solution (d). Dilute 5 ml ofreference solution (a) to

Reference solution (e). Dilute 5 ml ofreference solution (a) to
Ranitidine Tablets
Ranitidine Hydrochloride Tablets
Ranitidine Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent ofthe stated amount ofthe ranitidine,
CI3H22N403S, The tablets are coated.
Usual strengths. The equivalent of 150 mg, 300 mg ofranitidine
(1.12 g ofranitidine hydrochloride is approximately equivalent
to 1 g ofranitidine).
Identification
of ranitidine with 5 ml of methanol for 5 minutes, filter and
evaporate the filtrate to dryness. Add 1 ml of light petroleum
(60 to 80) to the resulting residue, scratch the side of the
vessel with a glass rod to induce crystallisation, evaporate to
Reference solution (f). Weigh accurately a quantity of (N,N'bis[2-[[[5-[(dimethylamino)methyl]furan-2-yl]methyl]

sulphanyl]ethyl]-2-nitroethene-1,1-diamine RS (ranitidine
impurity A RS) in methanol, and dilute with methanol to obtain
a solution containing a lmown concentration of about 0.127
per cent w/v.
Apply to the plate 10 fJ.l of each solution. Apply separately an

additional 10 fJ.l of the test solution and on top of this
application, apply 10 fJ.l of reference solution (f). After
development, dry the plate in air and expose it to iodine vapours
in a closed chamber until the spots are revealed. Any spot in
the chromatogram obtained with the test solution
corresponding to the principal spot in the chromatogram
obtained with reference solution (f) is not more intense than
that of the principal spot in the chromatogram obtained with
reference solution (b) (0.5 per cent) and no other spot in the
chromatogram obtained with the test solution is more intense
2045
RANITIDINE TABLETS
IP 2010
than the principal spot in the chromatogram obtained with

reference solution (c) (0.3 per cent). The sum bfthe intensities
of all the secondary spots in the chromatogram obtained with
the test solution does not exceed 2.0 per cent.
the combined test solution and reference solution (f) shows
obtained with reference solution (e) shows a clearly visible
spot.
Test solution. Weigh and powder 20 tablets. Shake 1.5 g bfthe

powder with 400 ml ofthe mobile phase, dilute to 500.0 ml with
the mobile phase, filter and dilute the filtrate with the mobile
phase to obtain a solution containing the equivalent of
0.01 per cent w/v ofranitidine.
ranitidine hydrochloride RS in the mobile phase.
than 2.0 per cent.
Calculate the content ofC13H22N403S in the tablets.
equivalent amount ofranitidine.
Identification
A. When examined under a microscope in the dry state, or
when mounted in ethanol (95 per cent) and water, the
following characteristics are observed. They are usually of
more or less uniform width. Many longitudinal parallel lines
are distributed unequally over the width in the case ofstandard
viscose fibres, but such lines are absent or very few in fibres
produced through the zinc-free process. The ends are cut
more or less straight. Matt fibres contain numerous granular
particles ofapproximately 1Jl average diameter.
B. Treat with iodinated zinc chloride solution; the fibres
become violet.
C. To 0.1 g add 10 ml ofzinc chloride-jormic acid solution,
heat to 40 and allow to stand for 2 hours 30 minutes, shaking
occasionally. The .fibres dissolve completely except for the
matt variety where titanium dioxide particles remain.
D. Dissolve the residue obtained in the test for Sulphated ash
in 5 ml of sulphuric acid with slight warming, allow to cool,
and carefully add 0.2 ml of hydrogen peroxide solution
(10 volumes). The solution does not undergo colour change
in case of lustrous variety of fibre, but for matt variety an
orange-yellow colour is obtained, the intensity of which
depends on the quantity of titanium dioxide present.
Tests
Colour ofextract. Take 15 g ofmaterial under examination in a
suitable vessel, add 150 ml of water, close the vessel and
allow to macerate for 2 hours. Decant the solution, squeeze
the residual liquid carefully from the sample with a glass rod,
mix and filter. The filtered extract is colourless. Compare the
colour of the extract with water using identical tubes of
colourless, transparent, neutral glass 12 mm in diameter
measuring 2 ml. Compare the colours in diffused daylight,
viewing horizontally against a white background.
Acidity or alkalinity. To 25 ml offiltered extract obtained, add
0.1 ml of dilute phenolphthalein solution; to another 25 ml
add 0.05 ml of methyl orange solution. Neither solution shows
a pink colour.
Foreign fibres. When examined under a microscope, it is seen
to consist exclusively of viscose fibres, except that
occasionally a few isolated foreign fibres may be present.
Purified Rayon
Viscose Fibre; absorbent viscose
Category. Regenerated cellulose used in surgical dressings.
Description. White or very slightly yellow, purified rayon is a
fibrous form ofbleached regenerated cellulose, which can be
produced with a lustrous or matt appearance, and is soft to
the touch. The fibres can be produced with average
staple length between 32 mm to 80 mm, and are practically
odourless.
Fluorescence. Examine a layer about 5 mm in thickness under

ultraviolet light at 365 nm. It displays only a slight, brownishviolet fluorescence and a few yellow particles. Not more than
a few isolated fibres show an intense blue fluorescence.
Absorbancy
A. Sinking time. Not more than 10 seconds, determined by
the following method.
2046
IP20lO
RESERPINE
Apparatus
A dry, cylindrical copper wire basket, 80 mrn high and 50 mrn in

diameter, fabricated from wire ofdiameter 004 mrn and having a
mesh aperture of 15 to 20 mm; the basketweighs 204 to 3.0 g.
Method
Weigh the basket to the nearest 10 mg. Take five samples,

each ofapproximately 1g, from different places in the material
under examination, place loosely in the basket and weigh the
packed basket to the nearest 10 mg. Hold the basket with its
long axis in the horizontal position and drop it from a height of
about 10 mm into water at 25 contained in a beaker at least
12 cm in diameter and filled to a depth of 10 cm. Measure with
a stopwatch the time taken by the basket to sink below the
surface of the water. Repeat the procedure on two further
samples and calculate the average value.
solution prepared at the same time using 0.15 ml of dilute

acetic acid, 1.2 m1 of thioacetamide reagent, 1.7 ml of lead
standard solution (10 ppm Pb) and 10 ml of filtered extract.
Loss on drying (204.19). Not more than 13.0 per cent, determined
Reserpine
B. Water-holding capacity. Not less than 18.0 g per g,

After the sinking time has been recorded in test A, remove the
basket from the water, allow it to drain for 30 seconds with its
long axis in the horizontal position over the beaker, transfer it
to a tared beaker and weigh to the nearest 10 mg. Calculate the
weight of water retained bythe sample. Repeat the procedure
on two further samples and calculate the average value.
Colouring matter. Slowly extract 109 in a narrow percolator
with ethanol (95 per cent) until 50 ml of extract is obtained.
The extract is not more intensely coloured than reference

solution YS5 or GYS6, (204.1) or a solution prepared in the
following manner. To 3.0 ml ofCSS add 7.0 ml ofa solution of
hydrochloric acid containing 1 per cent w/v of hydrochloric
acid and dilute 0.5 ml of the resulting solution to 10 ml with
the same solution of hydrochloric acid.
C33H4oN209
Mol. Wt. 608.7
Reserpine is methyl11,17a.-dimethoxy-18~-[(3,4,5-tri
methoxybenzoyl)oxy]-3~,20a.-yohimbane-16~-carboxy1ate.
Reserpine contains not less than 99.0 per cent and not more
than 101.0 per cent oftotal alkaloids and not less than 98.0 per
cent and not more than 102.0 per cent ofreserpine, C33H4oN209'
both calculated on the dried basis.
Dose. 500 flg daily.
Ether-soluble substances. Not more than 0.5 per cent,

determined by the following method. Extract 5 g with ether in
Description. White to slightly yellow small crystals or a
a continuous extraction apparatus such as a Soxhlet apparatus,

for 4 hours in such a way that the rate is at least four extractions
per hour. Evaporate the ether and dry the residue to constant
weight at 105.
Identification
Water-soluble substances. Not more than 0.7 per cent,

determined by the following method. BoilS g with 500 ml of
water for 30 minutes, stirring frequently and replacing the
water lost by evaporation. Decant the liquid into a beaker,
squeeze the residual liquid from the material carefully with a
glass rod, mix the liquids and filter the extract whilst hot.
Evaporate 400 ml ofthe filtrate and dry the residue to constant
weight at 105.
Hydrogen sulphide. To 10 ml of the filtered extract obtained
in the Colour ofextract, add 1.9 ml ofwater,0.15 ml of dilute
acetic acid and 1 ml of lead acetate solution. After 2 minutes,
the solution is not more intensely coloured than a reference
crystalline powder which darkens slowly on exposure to light.

TestsB, C, D and E may be omitted if test A is carried out.
A. Determine by infrared absorption spectrophotometry (204.6).

Compare the spectrum with that obtained with reserpine RS
or with the reference spectrum of reserpine.
B. Dilute 1 ml of a 0.2 per cent w/v solution in chloroform to
100.0 m1 with ethanol (95 per cent). When examined
immediately after preparation, in the range 230 nm to 360 nm
(204.7), the resulting solution shows an absorption maximum
at about 268 nm; absorbance at about 268 nm, about 0.55.
Over the range 288 nm to 295 nm, the spectrum exhibits a
slight minimum and then a shoulder or a slight maximum;
absorbance over this range, about 0.34.
2047
RESERPINE
IP 2010
C. To about 1 mg add 0.1 ml of a 0.1 per cent w/v solution of

sodium molybdate in sulphuric acid; a yellow colour is
produced which changes to blue within 2 minutes.
D. To 1 mg add 0.2 ml of a freshly prepared 1 per cent w/v
solution of vanillin in hydrochloric acid; a pink colour
develops within 2 minutes.
E. Mix about 0.5 mg with 5 mg of 4-dimethylaminobenzaldehyde and 0.2 ml of glacial acetic acid and add 0.2 ml of
sulphuric acid; a green colour is produced. Add 1 ml of
glacial acetic acid; the colour changes to red.
Reserpine Injection
Reserpine Injection is a sterile solution of Reserpine in Water
for Injections prepared with the aid of a suitable acid. It may
contain suitable antioxidants.
Reserpine Injection contains not less than 90.0 per cent and
not more than 110.0 per cent ofthe stated amount ofreserpine,
C33~oNz09'
Usual strengths. 1 mg per ml; 2.5 mg per ml.
Identification
Tests
in a solution prepared immediately before use by dissolving
0.25 g in sufficient chloroform to produce 25 ml.
Extract a suitable volume ofthe injection containing 10 mg of

Reserpine with 10 ml of chloroform and evaporate the
chloroform layer to dryness. The residue complies with the
following tests.
Oxidation products. Absorbance ofa 0.02 per cent wIv solution

in glacial acetic acid at about 388 nm, measured immediately
after preparation, not more than 0.10 (2.4.7).

Tests B, C, D and E may be omitted if test A is carried out.

on 0.5 g by drying in an oven over phosphorus pentoxide at
60 at a pressure not exceeding 0.7 kPa for 3 hours.
Assay. For total alkaloids - Weigh accurately about 0.5 g
and dissolve in a mixture of40 ml of anhydrous glacial acetic
acid and 6 ml of acetic anhydride. Titrate with 0.1 Mperchloric
total alkaloids.
For reserpine, C33H40N209 - Carry out the following

procedure protected from light. Weigh accurately about
25.0 mg, moisten with 2 ml of ethanol (95 per cent), add 2 ml
of 0.25 M sulphuric acid and 10 ml of ethanol (95 per cent)
and warm gently to dissolve. Cool, dilute to 100.0 ml with
ethanol (95 per cent) and dilute 5.0 ml to 50.0 ml with the
same solvent (solution A). Transfer 10.0 ml to a boiling tube,
add 2 ml of 0.25 M sulphuric acid and 2 ml ofa freshly prepared
0.3 per cent w/v solution of sodium nitrite, mix and heat in a
water-bath at 55 for 35 minutes. Cool, add 1 ml of a freshly
prepared 5 per cent w/v solution of sulphamic acid and dilute
to 25.0 ml with ethanol (95 per cent). Measure the absorbance
ofthe resulting solution atthe maximum at about 388 urn (2.4.7),
using as the blank a solution prepared by treating a further
10.0 ml of solution A in the same manner and at the same time
but omitting the sodium nitrite solution.
Calculate the content of C33H4oNz09 from the absorbance
obtained by repeating the operation using reserpine RS in
place of the substance under examination.

Compare the spectrum with that obtained with reserpine RS
or with the reference spectrum of reserpine.
B. Dilute 1 ml of a 0.2 per cent w/v solution in chloroform to
100 ml with ethanol (95 per cent). When examined immediately
after preparation, in the range 230 nm to 360 nm (2.4.7), the
resulting solution shows an absorption maximum at about
268 urn; absorbance at about 268 nin, about 0.55. Over the
range 288 urn to 295 urn, the spectrum exhibits a slight minimum
and then a shoulder or a slight maximum; absorbance over
this range, about 0.34.
C. To about 1 mg add 0.1 ml of a 0.1 per cent w/v solution of

sodium molybdate in sulphuric acid; a yellow colour is
D. To 1 mg add 0.2 ml of a freshly prepared 1 per cent w/v
solution of vanillin in hydrochloric acid; a pink colour
develops within 2 minutes.
E. Mix about 0.5 mg with 5 mg of 4-dimethylaminobenzaldehyde and 0.2 ml of glacial acetic acid and add 0.2 ml of
sulphuric acid; a green colour is produced. Add 1 ml of
glacial acetic acid; the colour changes to red.
Tests
pH (2.4.24). 3.0t04.0.
Other tests. Comply with the tests stated under Parenteral
Assay. Protect the solutions from light throughout the assay.
Measure accurately a volume ofthe injection containing about
10 mg ofReserpine and dilute with a 2 per cent w/v solution of
citric acid to 100.0 ml. Extract 10.0 ml of this solution for
2048
IP 2010
RESERPINE TABLETS
2 minutes with three quantities, each of 15 ml, of chloroform.

Wash the combined extracts with 10 ml of a 1 per cent w/v
solution of sodium bicarbonate, add sufficient chloroform to
produce 50.0 ml, mix and evaporate to.O ml to dryness on a
water-bath. Dissolve the residue in 10 ml of ethanol (95 per
cent), add 2 ml of 0.25 M sulphuric acid and 10 ml of ethanol
(95 per cent) and warm gently to dissolve. Cool, dilute to
100.0 ml with ethanol (95 per cent) and dilute 5.0 ml to 50.0 ml
with the same solvent (solution A). Transfer to.O ml to a boiling
tube, add 2 ml of 0.25 M sulphuric acid and 2 ml of a freshly
prepared 0.3 per cent w/v solution of sodium nitrite, mix and
heat in a water-bath at 55 for 35 minutes. Cool, add 1 mlofa
freshly prepared 5 per cent w/v solution of sulphamic acid
and dilute to 25.0 ml with ethanol (95 per cent). Measure the
388 nm (2.4.7), using as the blank a solution prepared by
treating a further to. 0 ml ofsolution A in the same manner and
at the same time but omitting the sodium nitrite solution.
Calculate the content of C33H40Nz09 from the absorbance
Storage. Store protected from light in single dose (or if
stabilising agents are present, in multiple dose) containers.
each of 5 ml, of chloroform, filter the extracts through a plug

of cotton moistened with chloroform. Wash the chloroform
extracts with to ml of a 1 per cent w/v solution of sodium
bicarbonate and evaporate the chloroform extracts to dryness
on a water-bath. For tablets containing upto 250 flg of
Reserpine per tablet, dissolve the residue in 10.0 ml of ethanol
(95 per cent). For tablets containing more than 250 flg of
Reserpine per tablet, dissolve the residue in a suitable volume
of ethanol (95 per cent) to give a concentration of250 flg of
Reserpine per 10.0 ml; add 2 ml of 0.25 M sulphuric acid and
2 ml of a freshly prepared 0.3 per cent w/v solution of sodium
nitrite, mix and heat in a water-bath at 55 for 35 minutes. Cool,
add 1 ml of a freshly prepared 5 per cent w/v solution of
sulphamic acid and dilute to 25.0 ml with ethanol (95 per
cent). Measure the absorbance of the resulting solution at
the maximum at about 388 nm (2.4.7), using as the blank a
solution prepared by treating a further 10.0 ml ofsolution A in
the same manner and at the same time but omitting the sodium
nitrite solution.
Calculate the content of C33H40Nz09 in the tablet from the
reserpine RS in place of the substance under examination.
Reserpine Tablets
Reserpine Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of reserpine,
C33~oNz09'
Usual strengths. 100 flg; 250 flg; 500 flg; 1 mg.
Identification
A. Powder a few tablets and extract with chloroform. Evaporate
the extract to dryness, add 0.1 ml ofa 0.1 per cent w/v solution
of sodium molybdate in sulphuric acid; a yellow colour is
B. Powder a few tablets and extract with chloroform. Evaporate
the extract to dryness, add 0.2 ml of a freshly prepared I per
cent w/v solution of vanillin in hydrochloric acid; a pink
colour develops within 2 minutes.
Tests
Uniformity of content. Protect the solutions from light
throughout the test.
Powder one tablet, disperse in 10 ml ofa 2 per cent w/v solution
of citric acid and extract for 2 minutes with three quantities,
Weigh accurately a. quantity of the powdered tablets

containing about 1 mg ofReserpine, add 10 ml ofa 2 per cent
w/v solution of citric acid and extract for 2 minutes with three
quantities, each of 15 ml, of chloroform. Wash the combined
extracts with to m1 of a 1 per cent w/v solution of sodium
bicarbonate, add sufficient chloroform to produce 50.0 ml
and evaporate to.O m1 to dryness on a water-bath. Dissolve
the residue in 10 m1 of ethanol (95 per cent) and add 2 m1 of
0.25 M sulphuric acid and 10 m1 of ethanol (9Jper cent) and
warm gently to dissolve. Cool, dilute to 100.0 m1 with ethanol
(95 percent) and dilute 5.0 ml to 50.0 ml with the same solvent
(solution A). Transfer 10.0 m1 to a boiling tube, add 2 ml of
0.25 M sulphuric acid and 2 ml of a freshly prepared 0.3 per
cent w/v solution of sodium nitrite, mix and heat in a waterbath at 55 for 35 minutes. Cool, add 1 m1 ofa freshly prepared
5 per cent w/v solution of sulphamic acid and dilute to 25.0 ml
with ethanol (95 per cent). Measure the absorbance of the
using as the blank a solution prepared by treating a further
to.O m1 ofsolution A in the same manner and at the same time
but omitting the sodium nitrite solution.
Calculate the content of C33H40Nz09 from the absorbance
2049
RlBAVIRlN
IP 2010
strong cation-exchange resin (9 Ilm),
mobile phase. water, adjusted to pH 2.5 with sulphuric
acid,
- flow rate. ImlpermiIiute,
- spectrophotometer set &t 207 nm,
Ribavirin
Mol.Wt. 244.2
Ribavirin is 1-~-D-ribofuranosyl-lH-l ,2,4-triazole-3carboxamide.
Ribavirin contains not less than 98.0 per cent and not more
than 102.0 per cent ofCsHJ2N40s, calculated on the dried basis.
Identification
Compare the spectrum with that obtained with ribavirin RS or
with the reference spectrum ofribavirin.
Tes'ts
Specific optical rotation (2.4.22). - 33 to - 37, determined in
a 1.0 per cent w/v solution, use freshly prepared solution.
(2.4.14).

peak-to-valley ratio is not less than 1.2, where H p is the height
above the baseline ofthe peak due to ribavirin impurity F and
H v is the height above the baseline of the lowest point of the
curve separating this peak from the peak due to ribavirin.
Inject test solution (a), reference solution (a) and (b). Record
the chromatogram eleven times the retention time of the
principal peak. The relative retention time with reference to
ribavirin for 1-(5-0-acetyl-~-D-ribofuranosyl)-IH-l,2,4triazole-3-carboxarnide (5'-O-acetylribavirin) (ribavirin impurity
F ) is about 1.2. The peak area corresponding to ribavirin
impurity F is not more than the area ofthe principal peak in the
cent). The area of each secondary peak corresponding to 1-~
D-ribofuranosyl-lH-l ,2,4-triazole-3-carboxylic acid (ribavirin
impurity A), l-a-D-ribofuranosyl-lH-l,2,4-triazole-3carboxamide (ribavirin impurity B), IH-l,2,4-triazole-3carboxylic acid (ribavirin impurity C), H-l,2,4-triazole-3carboxamide (ribavirin impurity D), 1-(5-0-benZoyl~a -Dribofuranosyl)-IH-l,2,4-triazole-3-carboxamide (5' -0benzoylribavirin) (ribavirin impurity E), 1- ~ -D-ribofuranosylIH-l ,2,4-triazole-5-carboxamide (N-isomer) (ribavirin impurity
G) is not more than the area of the principal peak in the
cent). The sum of areas ofall the secondary peaks is not more
than twice the area ofthe principal peak in the chromatogram
peak with an area less than 0.5 times the area ofthe principal

examination in100 ml ofthe mobile phase. Dilute 10 ml ofthe
Heavy metals (2.3.13). Dissolve 4.0 g in 20 ml of water with

heating ifnecessary and use 12 ml ofthe solution. The resulting
solution complies with the limit test for heavy metals, Method
B(10ppm).
Reference solution (a). Dilute 1 ml of test solution (a) to 100

ml with the mobile phase. Dilute 1 ml ofthis solution to 10 ml
Reference solution (b). Dissolve the contents of a vial of

ribavirinfor system suitability RS in 2 ml Of the mobile phase.
Reference solution (c). Dissolve 25 mg of ribavirin RS in 100
ml pfthe mobile phase. Dilute 10 ml ofthe solution to 100 ml
Assay. Determine by liquid chromatography (2.4.14) same as
described under Related substances with the following
modification.
2050
IP 2010
RlBAVIRlN INHALATION SOLUTION

similar in position, colour and size to that in the chromatogram
Calculate the content ofCsHl2N40s.

B. In the Assay, the retention time ofthe principal peak in the

that of the principal peak in the chromatogram obtained with
the reference solution.

Ribavirin Solution for Inhalation
Catogery. Antiviral.
Tests
Ribavirin Inhalation Solution is a sterile solution ofRibavirin

in Water for Injections. It is prepared by dissolving Ribavirin
for Inhalation in the requisite amount ofWater for Injections.

(2.4.14).
Ribavirin for Inhalation

Ribavirin for Inhalation is a sterile ribavirin consisting of
Ribavirin with or without excipients. It is filled in a sealed
container.
The inhalation solution is constituted by dissolving the
contents of the sealed container in the requisite amount of
sterile water for injection, immediately before use.

Clarity of solution and particulate matter stated under
Inhalation Preparations.
Ribavirin Inhalation Solution contains not less than 95.0 per
CSH 12N40 s.
The content of the sealed container complies with the

requirements stated under Inhalation Preparations and with
the following requirements.
Identification
Mobile phase. A mixture of 20 volumes of 0.1 M ammonium

chloride and 90 volumes of acetonitrile.
Test solution. Dissolve ribavirin inhalation solution containing
about 0.1 g ofRibavirin in 10 ml of water.
Reference solution. A 1.0 per cent w/v solution of ribavirin
RS in water.
dry the plate in air for 15 minutes, and spray the plate with the
mixture of0.5 ml of anisaldehyde, 0.5 ml ofsulphuric acid, 0.1
ml of glacial acetic acid and 9 ml of ethanol (95 per cent) and
heat at 105 for 40 minutes and allow to cool.The principal
Test solution. A 0.05 per cent w/v solution of ribavirin in the

mobile phase.
ribavirin in the mobile phase.
ribavirin RS in the mobile phase.
strong cation-exchange resin of sulphonated, crosslinked styrene-divinylbenzene co-polymer in the
hydrogen form (7 to 11 Ilm)(Such asArninexHPAH),
mobile phase: water, adjusted to pH 2.5 with sulphuric
acid,
tailing factor for the principal peak is not less than 0.7 and not
more than 1.5.
secondary peak is not more than the area of principal peak in
the chromatogram obtained with reference solution (a) (0.25
cent).
Test solution. Reconstitute the contents of one container as

instructed on the label using an accurately measured volume
of diluent, dilute a suitable volume of the solution to 200
volumes with the mobile phase to produce a solution
containing 0.1 per cent w/v of Ribavirin, mix and dilute 5
volumes of the resulting solution to 20 volumes with the
mobile phase.
2051
RlBOFLAVINE
IP 2010
Reference solution. A 0.025 per cent w/v solution of ribavirin

Dose. Prophylactic, 1 to 4 mg daily; therapeutic, 5 to 10 mg

daily.
strong cation-exchange resin of sulphonated, crosslinked styrene-divinylbenzene co-polymer in the
hydrogen form (7 to 11 J.Lm) (Such as Aminex HPAH),
- mobile phase: water, adjusted to pH 2.5 with sulphuric
acid,
- injection volume. 10 J.Ll.
Description. A yellow to orange-yellow, crystalline powder;

odour, slight.
tailing factor is not less than 0.7 and not more than 1.5.
Identification
Compare the spectrum with that obtained with riboflavine RS
or with the reference spectrum ofriboflavine.
B. Dissolve about 1 mg in 100 ml of water. The solution has a
pale greenish yellow colour by transmitted light and an intense
yellowish green fluorescence by reflected light, which
disappears on addition of mineral acids or allcalis.
Tests
pH (2.4.24).5.5 to 7.2, determined in a saturated solution.
Calculate the content of CgHI2N40s in inhalation solution.

Labelling. The label of the sealed container states (1) the
procedure for preparing the solution, (2) the delivery system
to be used for the constituted solution (3) the conditions
under which the constituted solution should be stored.

in a 0.5 percentw/v solution in carbonate-free 0.05 Msodium
hydroxide. Measure the angle ofrotation within 30 minutes of
preparing the solution.
Light absorption (2.4.7). Dilute a suitable volume ofthe final
solution obtained in the Assay with an equal volume of water.
When examined in the range 210 urn to 460 urn, the resulting
solution exhibits maxima at about 223 urn, 267 urn, 373 nm and
444 nm; the ratio of the absorbance at the maximum at about
373 urn to that at about 267 urn, 0.31 to 0.33 and the ratio ofthe
absorbance at the maximum at about 444 nm to that at about
267 urn, 0.36 to 0.39.
Riboflavin
Lactoflavin; Vitamin B 2
Lumiflavine. Shake 25 mg with 10 ml ofchlorofOrm for 5 minutes

and filter. The filtrate is not more intensely coloured than
Assay. Carry out the procedure in subdued light.
Mol. Wt. 376.4

Riboflavin is 3,1O-dihydro-7,8-dimethyl-l0-[(2S,3S,4R)2,3,4,5-tetrahydroxypentyl]benzo[g]pteridine-2,4-dione.
Riboflavin contains not less than98.0 per cellt and not more
thali 101.0 per celit of CI7H2oN406, calCulated on the dried
basis.
Weigh accurately about 65 mg and transfer to an amber-glass

500-ml volumetric flask, suspend in 5 ml of water, ensuring
that it is completely wetted. Dissolve in 5 ml of 2 M sodium
hydroxide. As soon as dissolution is complete add 100 ml of
water and 2.5 ml of glacial acetic acid and dilute to 500.0 ml
with water. To 20.0 ml of this solution add 3.5 ml ofa 1.4 per
cent w/v solution of sodium acetate and dilute to 200.0 ml
with water. Measure the absorbance ofthe resulting solution
atthe maximum at about 444 nm (2.4.7).
Calculate thec()ntetlt ()fC17H2oN406 taking 328 as the specific
2052
IP 2010
RIBOFLAVINE SODIUM PHOSPHATE
Riboflavin Sodium Phosphate
Tests
Riboflavine-5-phosphate (Sodium Salt); Vitamin B2

Sodium Phosphate

solution.
in a 1.5 per cent w/v solution in 5 M hydrochloric acid.
Heavy metals (2.3.13). To 2.0 g in a silica crucible add 2 ml of
nitric acid dropwise followed by 0.25 ml of sulphuric acid.
Heat cautiously until white fumes are evolved and ignite. Extract
the cooled residue with two quantities, each of 2 ml, of
hydrochloric acid and evaporate the extracts to dryness.
Dissolve the residue in 2 ml of 2 M acetic acid and dilute to
20 ml with water. 12 ml of the solution complies with the
limit test for heavy metals, Method D (10 ppm). Use 1.0 ml
of lead standard solution (10 ppm Pb) to prepare the
standard.
,2H zO
Mol. Wt. 514.4

Riboflavin Sodium Phosphate is monosodium 3, lO-dihydro7,8-dimethyl-1 0-[(2S,3S,4R)-2,3,4-trihydroxypentyl]
benzo[g]pteridine-2,4-dione 5-phosphate dihydrate.
Lumiflavine. Shake 35 mg with 10 ml of dichloromethane for

5 minutes and filter. The filtrate is not more intensely coloured
Inorganic phosphate. Not more than 1.5 per cent, determined

by the following method. Dissolve 0.1 g in sufficient water to
produce
100 mI. Dilute 5 rnl with 10 rnl ofwater and add 5 mI of
Riboflavin Sodium Phosphate contains the equivalent of not
buffered
cupric sulphate solution pH 4.0,2 ml of a 3 per cent
less than 73.0 per cent and not more than 79.0 per cent of
w/v
solution
of ammonium molybdate, 1 ml of a freshly
CI7H2oN406, calculated on the dried basis.
prepared solution containing 2 per cent w/v of 4-methylCategory. B-group vitamin.
aminophenol sulphate and 5 per cent w/v of sodium metabisulphite
and 1 ml of a 3 per cent v/v solution ofperchloric
Dose. Prophylactic, the equivalent of 1 to 4 mg ofriboflavine
acid.
Add
sufficient
water to produce 25 ml, mix and measure
daily; therapeutic, the equivalent of 5 to 10 mg ofriboflavine
of
the
resulting solution at the maximum at
the
absorbance
daily. (1.37 g ofriboflavine sodium phosphate is approximately
about
800
nm
(2.4.7),
within 15 minutes of its preparation,
equivalent to1 g ofriboflavine).
using as the blank a solution prepared in the same manner but
Description. A yellow to orange-yellow, crystalline powder;
omitting the substance under examination. The absorbance is
hygroscopic.
not greater than that produced by repeating the operation
using a solution prepared in the same manner using 15 ml of
Identification
phosphate standard solution (5 ppm PO,J and beginning at
A. When examined in the range 230 nm to 360 nm (2.4.7), a the words "add 5 ml of buffered cupric sulphate solution
0.001 per cent w/v solution in phosphate bufferpH 7.0 shows pH 4.0,"
an absorption maximum at about 267 nm; absorbance at about Loss on drying (2.4.19). Not more than 8.0 per cent, determined
267 nm, 0.58 to 0.64.
B. Dissolve about 10 mg in sufficient 2 M sodium hydroxide

to produce 100 rnl, expose 1 ml to ultraviolet light at 254 nm for
5 minutes, add sufficient 5 M acetic acid to make the solution
acidic to litmus paper and shake the mixture with 2 ml of
dichloromethane; the lower layer exhibits a yellow
fluorescence.
C. To 0.5 g add 10 ml of nitric acid, evaporate the mixture to
dryness on a water-bath, ignite the residue until the carbon is
removed, dissolve the final residue in 5 ml of water and filter.
The filtrate gives the reactions of sodium salts and reaction B
of phosphates (2.4.1).

Assay. Carry out the procedure protectedfrom light.
Weigh accurately about 0.1 g, dissolve in 150 ml of water, add
2 ml of glacial acetic acid and dilute to 1000.0 ml with water.
To 10.0 m1 add 3.5 ml ofa 1.4 per cent w/v solution ofsodium
acetate, dilute to 50.0 ml with water and measure the
444 nm (2.4.7). Calculate the percentage content ofC17H2oN406
2053
RIBOFLAVINE TABLETS
IP 2010
Riboflavin Tablets
Rifampicin
Lactoflavin Tablets; Vitamin B z Tablets
Rifampin
Riboflavin Tablets contain not less than 90.0 per cent and not
more than 115.0 per cent ofthe stated amount of riboflavine,
C17HzoN406.
CH
3 CH 3
I
HO,
~
I
H3C
Identification
OH
CH 3
0
H3CO-
Shake a quantity of the powdered tablets containing 1 mg

of Riboflavine with 100 ml of water and filter; the filtrate
has a pale greenish yellow colour by transmitted light
and an intense yellowish green fluorescence by reflected
light, which disappears on addition of mineral acids or
alkalis.
'CH 3 OH
H3C
CH 3
NH
OH
~
//
OH
,;;N'Nl
~N'CH3
Tests
C43HssN40JZ
Tablets.
Powder one tablet, add a mixture of 2.5 ml of glacial acetic
acid and 50 ml of water and heat on a water-bath for
1 hour with occasional stirring. Dilute with 50 ml of water,
add 15 ml of 1 M sodium hydroxide with continuous
stirring and then sufficient water to produce a solution
containing 10 llg of Riboflavine per ml. Filter and discard
the first few ml of the filtrate. On the clear filtrate carry out
the Assay beginning at the words "Measure the
absorbance....".
Other tests. Compiy with the tests stated under Tablets.
Rifampicin contains not less than 97.0 per cent and not more
than 102.0 per cent of C43HssN40JZ, calculated on the dried
basis.
Dose. For an adult, 450 to 600 mg (about 10 mg per kg) daily
preferably before breakfast. For a child, upto 20 mg per kg
daily to a maximum of600 mg.
Description. A brick-red to reddish brown, crystalline powder;
Assay. earlY out the procedure in subdued light.

the powder containing about 10 mg of Riboflavine, add a
mixture of5 ml ofglacial acetic acid and 100 ml ofwater and
heat on a water-bath for 1 hour with occasional shaking. Dilute
with 50 ml ofwater, cool, add 30 ml of 1 M sodium hydroxide
with continuous stirring. Add sufficient water to produce
1000.0 mI, mix and filter, discarding the first few mI ofthe filtrate.
Measure the absorbance ofthe filtrate at the maximum at about
444 nm (2.4.7).
Calculate the content ofC17HzoN406 taking 328 as the specific
Mol. Wt. 823.0
Rifampicin is (12Z, 14E,24E)-(28,168,178,18R,19R,20R,

218,22R,23S)-1,2-dihydro-5,6,9,17,19-pentahydroxy23-methoxy-2,4,12,16,18,20,22-heptamethyl-8-(4-methylpiperazin-l-yliminomethyl)-l, ll-dioxo-2,7-(epoxypentadeca1,11, l3-trienimino)naphtho[2, I-b ]furan-21-yl acetate.
Identification
Compare the spectrum with that obtained with rifampicin R8
or with the reference spectrum ofrifampicin.
Tests
suspension.
(2.4.14).
2054
IP 2010
RIFAMPICIN CAPSULES
Solvent mixture. A mixture of 10 volumes of a 21.01 per cent

w/v solution of citric acid, 23 volumes of a 13.61 per cent
w/v solution ofpotassium dihydrogen phosphate, 77 volumes
of a 17.42 per cent w/v solution of dipotassium hydrogen
phosphate, 640 volumes of water and 250 volumes of
acetonitrile.
Test solution. Weigh accurately a quantity of powder
containing 20 mg of Rifampicin, add 10 ml of acetonitrile,
shake and filter. Dilute 5 ml of the filtrate to 50 ml with the
solvent mixture.
Reference solution. A solution containing 0.02 per cent w/v of
rifampicin quinone RS in acetonitrile. To 1 ml ofthe solution,
add 1 ml of the test solution and dilute to 100 ml with the
solvent mixture.
- mobile phase: a mixture of 65 volumes of a solution
containing 0.1 per cent v/v of orthophosphoric acid,
0.19 per cent w/v of sodium perchlorate, 0.59 per cent
w/v of citric acid and 2.09 per cent w/v of potassium
dihydrogen phosphate and 35 volumes of acetonitrile,
spectrophotometer set at 254 mn,
resolution between rifampicin and rifampicin quinone is not
less than 4.
of any peak due to rifampicin quinone is not more than
1.5 times the area ofthe peak due to rifampicin quinone in the
cent); the area ofany peak, other than the peak due to rifampicin
and rifampicin quinone, is not more than the area ofthe peak
due to rifampicin in the chromatogram obtained with the
reference solution (1.0 per cent) and the sum of the areas of
any such peaks is not more than 3.5 times the area ofthe peak
due to rifampicin in the chromatogram obtained with the

acetonitrile.

examination in 10.0 ml of acetonitrile and filter. Dilute 5.0 ml of
the filtrate to 100.0 ml with the solvent mixture.
rifampicin RS in acetonitrile. Dilute 5.0 ml of this solution to
Use the chromatographic system and the system suitability
parameters described under the test for Related substances.
than 2.0 per cent.
Calculate the content ofC43H58N4012.
Storage. Store protected from light, in an atmosphere of

nitrogen.
Rifampicin Capsules
Rifampin Capsules
Rifampicin Capsules contain not less than 92.5 per cent and
not more than 107.5 per cent ofthe stated amount ofrifampicin,
C43H58N4012.
Identification
A. Shake a quantity ofthe contents ofthe capsules containing
0.15 g ofRifampicin with 5 ml of chloroform, filter and evaporate
test.
Determine by infrared absorption spectrophotometry (2.4.6)..
Compare the spectrum with that obtained with rifampicin RS
on 1.0 g by drying in an oven at 80 0 at a pressure not exceeding
Tests

(2.4.14).
NOTE -
Prepare the solutions immediately before use.


Solvent mixture. A mixture of 10 volumes ofa 21.01 per cent
2055
IP 2010
RIFAMPICIN CAPSULES

acetonitrile.
Test solution. Shake a quantity ofthe contents ofthe capsules

containing 200 mg of Rifampicin, with 100 ml of acetonitrile
and filter. Dilute 5 ml ofthe filtrate to 50 ml with the solvent
mixture.
w/v of rifampicin RS in acetonitrile. Dilute 1 ml ofthis solution
w/v each of rifampicin RS and rifampicin quinone RS in
acetonitrile. Dilute 5 ml of this solution to 50 ml with the
solvent mixture.
Apparatus No.2,
through a membrane filter disc having an average pore diameter
not greater than 1.0 f.Lm, rejecting the first 1 ml ofthe filtrate.
Dilute the filtrate, ifnecessary, with the same solvent. Measure
the absorbance (2.4.7) ofthe resulting solution at the maximum
at about 475 nm. Calculate the content ofC43HssN4012 in the
medium from the absorbance obtained from a solution of
Imown concentration of rifampicin RS.
C43HssN4012'
octylsilane bonded to porous silica (5 f.Lm),
injection volume. 20 f.Ll.

rifampicin RS in acetonitrile. Dilute 10.0 ml ofthis solution to
50.0 ml with acetonitrile. Dilute 5.0 ml ofthe resulting solution
less than 4, the tailing factor is not more than 2.0 and the
column efficiency is not less than 2000 theoretical plates.

acetonitrile. Dilute 5ml of this solution to 50 ml with the
solvent mixture.
Inject the test solution and reference solution (a). The relative
retention times are 1.0, 0.55, 1.25 and 2.61 for rifampicin,
rifampicin quinone, rifampicin N-oxide and 3-formylrifamycin
SV respectively. The response factors are 1.00, 1.19, 1.03 and
1.25 for rifampicin, rifampicin quinone, rifampicin N-oxide and
3-formylrifamycin SV respectively.
Use the chromatographic system and system suitability

parameters, as described under the test for Related substances.

of any peak due to rifampicin quinone should not be more
than 4 times the area ofthe principal peak in the chromatogram
obtained with the reference solution, the area of any peak due
to rifampicin N-oxide should not be more than 1.5 times the
. area ofthe principal peak in the chromatogram obtained with
the reference solution and the area of any peak due to
3-formylrifamycin SV should not be more than the area ofthe
solution. In the chromatogram obtained with the test solution
the area of allY unknowllpealc shoulclllot
more than the
Test solution. Shake a quantity ofthe contents of the capsules

containing about 300.0 mg of Rifampicin, with 200.0 ml of
acetonitrile, filter. Dilute 10.0 ml ofthe filtrate to 50.0 ml with
acetonitrile. Dilute 5.0 ml of this solution to 50.0 ml with the
solvent mixture.
than 2.0 per cent.
Calculate the content ofC43HssN4012 in the capsules.

Rifampicin oral suspension contains not less than 90.0 per
rifampicin, C43HssN4012.
2056
IP 2010
RIFAMPICIN ORAL SUSPENSION
Identification
A. To a quantity containing 0.1 g of Rifampicin add 30 m1 of
water and shake with two quantities, each of 50 ml, of
chloroform. Dry the combined extracts with anhydrous sodium
sulphate, filter and evaporate the filtrate to dryness at a
temperature not exceeding 70. Wash the residue with 1 ml of
ether and illy at 70. The residue complies with the following
test.
obtained with the test solution con'esponds to the peak in the
Tests
pH (2.4.24). 4.2 to 4.8.
(2.4.14).

acetonitrile.
Test solution. Add 5 m1 of water to a quantity of the oral
suspension containing 20 mg of Rifampicin and extract with
four quantities, each of 10 ml, of dichloromethane, filter the
combined extracts and evaporate to dryness at a temperature
not exceeding 40. Dissolve the residue in 10 ml of acetonitrile.
Dilute 5 ml of the resulting solution to 50 ml with the solvent
mixture.
to a 100 ml with the solvent mixture.
solvent mixture.

less than 4; the tailing factor is not more than 2.0 and the
rifampicin quinone, rifampicin N-oxide and 3-formylrifamycin
SV respectively. Multiply the areas of each Imown impurity
by their response factor. The response factors are 1.00, 1.19,
1.03, 1.22 and 1.25 for lifampicin, rifampicin quinone, rifampicin
N-oxide, Isonicotinyl hydrazone and 3-formylrifamycin SV
respectively.
In the clu'omatogram obtained with the test solution the area
obtained with the reference solution, the area of any peak due
to rifampicin N-oxide should not be more than the area ofthe
solution and the area ofany peak due to 3-fonnylrifamycin SV
should not be more than 5.0 times the area of the principal
of any unlmown peak should not be more than the area ofthe
solution.
Other tests. Comply with the tests stated under Oral
Suspensions.
Test solution. Weigh accurately a quantity of the oral

suspension containing about 150 mg of rifampicin and dilute
to 100.0 ml with acetonitrile. Dilute 10.0 ml ofthis solution to
rifampicin RS in acetonitrile. Dilute 10.0 ml ofthe solution to
50.0 m1 with acetonitrile. Dilute 10.0 ml ofthe resulting solution
w/v of each of rifampicin RS and rifampicin quinone RS in
solvent mixture.
Use the chromatographic system and the system suitability
parameters as described under the test for Related substances.
than 2.0 per cent.
2057
RIFAMPICIN TABLETS
IP 2010

Determine the weight per ml of the suspension (2.4.29) and
calculate the content ofC43HssN4012 weight in volume.
Rifampicin Tablets
Rifampin Tablets
Rifampicin Tablets contain not less than 92.5 per cent and not
more than 107.5 per cent of the stated amount of rifampicin,
C43HssN4012.
Usualstrength.150mg.
Identification
ofRifampicin with 5 ml of chloroform, filter and evaporate the
test.
Tests
(2.4.14).
NOTE

solution w/v ofpotassiurn dihydrogen phosphate, 77 volumes
acetonitrile.
tablets containing 200 mg ofRifampicin, dissolve in 100 ml of
acetonitrile, filter. Dilute 5 ml ofthe filtrate to 50 ml with the
solvent mixture.
w/v of each of rifampicin RS and rifampicin quinone RS in
solvent mixture.
column. temperature. 30,
rifampicin quinone, rifampicin N-oxide, and 3-formylrifamycin
SV respectively. The response factors are 1.00, 1.19, 1.03 and
1.25 for rifampicin, rifampicin quinone, rifampicin N-oxide and
obtained with the reference solution (4 per cent), the area of
any peak due to rifampicin N-oxide should not be more than
obtained with the reference solution (l.5 per cent) and the
area of any peak due to 3-formyl rifamycin SV should not be
more than the area of the principal peak in the chromatogram
obtained with the reference solution (l per cent). In the
unknown peak should not be more than the area of the
solution.
Apparatus No.2,
not greater than 1.0 j.lm, rejecting the first Iml afthe filtrate.
at about 475 nm. Calculate the content ofC43HssN4012 in the
known concentration of rifampicin RS.
C43HssN4012.
2058
IP 2010
RIFAMPICIN AND ISONIAZID TABLETS

acetonitrile.

a quantity of the powder containing about 300.0 mg of
Rifa~picin, dissolve in 200.0 ml of acetonitrile,filter. Dilute
10.0 ml ofthe filtrate to 50.0 ml with acetonitrile. Dilute 5.0 ml
ofthis solution to 50.0 ml with the solvent mixture.
rifampicin RS in acetonitrile. Dilute 10.0 ml ofthis solution to
R~rerence
solution (b). A solution containing 0.01 per cent

acetonitrile. Dilute 5.0 ml of this solution to 50.0 ml with the
solvent mixture.
Use the chromatographic system and system suitability

parameters described under the test for Related substances.
than 2.0 per cent.
Calculate the content of C43HssN4012 in the tablets.

tablets containing 200 mg ofRifampicin in 100 ml of acetonitrile
and filter. Dilute 5 ml of the filtrate to 50 ml with the solvent
mixture.
Reference solution (a). Dissolve rifampicin RS in acetonitrile
to obtain a solution containing 0.2 mg per ml. Dilute 1 mlof
this solution to 100 ml with the solvent mixture.
solvent mixture.
- flowrate. 1.5 ml per minute,

Rifampin and Isonicotinylhydrazid Tablets
Rifampicin and Isoniazid Tablets contain not less than
amounts ofrifampicin, C43HssN4012 and isoniazid, C6H 7N 30.
Usual strength. Rifampicin 50 mg and Isoniazid 150 mg.
Identification
A. In the Assay, the chromatogram obtained with the test
solution shows peaks that correspond to the peaks due to
rifampicin RS and isoniazidRS in the chromatogram obtained
Tests
(2.4.14).

retention times are 1.0,0.55, 1.25, 1.51 and 2.61 for rifampicin,
rifampicin
quinone,
rifampicin
N-oxide,
3-formylrifamycin SV isonicotinyl hydrazone and
3-formylrifamycin SV respectively. Multiply the area ofeach
known impurity by its response factor. The response factors
are 1.00, 1.19, 1.03, 1.22 and 1.25 for rifampicin, rifampicin
quinone, rifampicin N-oxide, isonicotinyl hydrazone and
obtained with the reference solution (1.5 per cent), the area of
any peaks due to 3-formylrifamycin SV and isonicotinyl
hydrazone should not be more than 5 times the area of the
solution (5 per cent) and the area of any peak due to
3-formylrifamycin SV should not be more than the area ofthe
2059
RIFAMPICIN AND ISONIAZID TABLETS
IP 2010

solution (1.0 per cent). In the chromatogram obtained with the
test solution the area of any unknown peak should not be
cent).
Apparatus No.2,
Withdraw a suitable volume ofthe mediumand filter promptly
through a membrane filter discwith an average pore diameter
not greater than 0.8 Ilm. Reject the first few ml of the filtrate
and dilute a suitable volume of the filtrate with the medium.
Reference stock solution. A solution containing 0.0165 per

cent of rifampicin RS and 0.00825 per cent of isoniazid RS in
the dissolution medium.
For rifampicin
Measure the absorbance of the sample
solution and the reference stock solution suitably diluted with
the dissolution medium at 475 nm (2.4.7). Calculate the content
ofC43HssN40J2 in the medium from the absorbance obtained
from the reference stock solution.
For isoniazid (2.4.14).
Determine by liquid chromatography
Use the reference stock solution and sample solution suitably

diluted with a solution of 0.05 .M potassium dihydrogen
orthophosphate, adjust the pH to 6.2 with 0;1 M sodium
hydroxide to obtain the reference solution and the test
solution, respectively.
mobile phase: a mixture of99 volumes of 0.05 Mpotassium
dihydrogen phosphate and 1 volume of acetonitrile
with the pH adjusted to 4.0 0.05 with 2 per cent w/v
solution of phosphoric acid,
tailing factor is not more than 2.0, the column efficiency innot
Solvent mixture. Dissolve 1.4 g of disodium hydrogen

orthophosphate anhydrous in 1000 ml of water and adjust
the pH to 6.8 with dilute phosphoric acid.
a quantity ofthe powder containing about 40 mg ofIsoniazid,
dissolve in 100.0 ml of methanol and dilute to 500.0 m1 with the
solvent mixture.
rifampicin RS and 0.04 per cent w/v of isoniazid RS in
methanol. Dilute 10.0 ml of this solution to 50.0 ml with the
solvent mixture.
column. temperature 30,
solution pH 6.8 prepared by dissolving 1.4 g of disodium
hydrogen orthophosphate anhydrous in 1000 ml of
water and adjusting the pH to 6.8 0.05 with dilute
phosphoric acid, and 4 volumes of acetronitrile.
B. a mixture of 45 volumes ofthe buffer
. below,
Mobile phase B
(per cent v/v)
Mobile phase A
(per cent v/v)
100
100
15
16
20
0
100
100
100
100
Time
(in min.)
o
o
o
o
NOTE - Saturate the column with mobile phase B for about

1 hour before injection.
than 2.0 for rifampicin and isoniazid; the column efficiency for
the isoniazid peak is not less than 3000 and for rifampicin not
Calculate the content ofC6H1N30 in the dissolution medium;

The retention times are about 1.0 for rifampicin and about 0.3
for isoniazid.
D. Not less than 75 per cent of the stated amounts of

C43HssN40J2 and C6H7N 30.
Ca.lculaie the contents of C43HssN4012 and C 6H 7N 30 in the

tablets.
2060
IP 2010
RIFAMPICIN, ISONIAZIDN AND ETHAMBUTOL TABLETS

Rifampicin, Isoniazid and Ethambutol

Tablets
Rifampin, Isonicotinylhydrazid and Ethambutol
Rifampicin, Isoniazid and Ethambutol Tablets contain not less
stated amounts ofrifampicin, C43HssN4012' isoniazid, C6H7N30
and ethambutol hydrochloride CIQH24N 20 2.2HCl.
Usual strength. Rifampicin 300 mg, Isoniazid 200 mg and

Ethambutol 200 mg.
Identification
A. In the Assay for rifampicin and isoniazid, the principal
peaks in the chromatogram obtained with the test solution
correspond to the peaks in the chromatogram obtained with
B. In the Assay for ethambutol hydrochloride the principal

peak in the chromatogram obtained with the test solution
corresponds to the peak in the chromatogram obtained with
Tests
(2.4.14).

acetonitrile.
tablets containing 200 mg ofRifampicin, dissolve in 100 ml of
acetonitrile and filter. Dilute 5 ml ofthis solution to 50 ml with
acetonitrile. Dilute 5 ml of this solution to' 50 ml with the
solvent mixture.
octylsilane bonded to porous silica (5 flm),
retention times are 1.0,0.55, 1.25, 1.51 and 2.61 forrifampicin,
rifampicin quinone, rifampicin N-oxide, 3-formylrifamycin SV
Isonicotinyl hydrazone and 3-formyhifamycin SV respectively.
Multiply the areas of each known impurity by its response
factor. The response factors are 1.00, 1.19, 1.03, 1.22 and 1.25
for rifampicin, rifampicin quinone, rifampicin N-oxide,
Isonicotinyl hydrazone and 3-formylrifamycin SV respectively.
any peak due to 3-formylrifamycin SV Isonicotinyl hydrazone
should not be more than 5 times the area ofthe principal peak
in the chromatogram obtained with the reference solution
( 5 per cent) and the area ofany peak due to 3-formylrifamycin
SV should not be more than the area ofthe principal peak in
the chromatogram obtained with the reference solution (I per
cent ). In the chromatogram obtained with the test solution
the area of any unknown peak should not be more than
1.5 times the area ofthe principal peak in the chromatogram
Apparatus No.2,
not greater than 0.8 flm. Reject the first few ml ofthe filtrate.

cent w/v of rifampicin RS, 0.00825 per cent w/v of isoniazid
RS and 0.031 per cent w/v of ethambutol hydrochloride RS in
the dissolution medium. Keep this reference stock solution in
the dissolution bath during the test run.
For rifampicin - Dilute the filtrate, if necessary, with the
same solvent. Measure the absorbance (2.4.7) ofthe resulting
solution at the maximum at about 475 nm. Calculate the content
2061
RIFAMPICIN, ISONIAZID AND ETHAMBUTOL TABLETS
IP 2010
ofC43H58N40lz in the medium from the absorbance obtained

from suitably diluted reference stock solution.
For isoniazid- Determine by liquid chromatography (2.4.14).

Test solution. Suitably dilute the filtered medium with 0.05 M
potassium dihydrogen orthophosphate with the pH adjusted
to 6.2 with 0.1 M sodium hydroxide.
Reference solution. Suitably dilute the reference stock solution
with 0.05 M potassium dihydrogen orthophosphate with the
pH adjusted to 6.2 with 0.1 M sodium hydroxide.
- mobile phase: a mixture of99 volumes of0.05 M potassium
dihydrogen phosphate and 1 volume of acetonitrile
previously adjusted to pH 4.0 with a 2 per cent w/v

rifampicin RS and 0.04 per cent w/v of isoniazid RS in
methanol. Dilute 10.0 ml of this solution to 50.0 ml with the
diluent.
octadecy1si1ane bonded to porous silica (5 11m),
-column temperature. 30,
solution pH 6.8 (diluent) prepared by dissolving 1.4 g of
disodium hydrogen orthophosphate anhydrous in
1000 ml of water, and adjusting the pH to 6.8 0.05 with
dilute phosphoroic acid, and 4 volumes of acetonitrile,
B: a mixture of 45 volumes of the buffer
below,
Time
(in min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
100
100
o
o
100
Calculate the content ofC 6H 7N 30 in the dissolution medium.
15
100
For ethambutol hydrochloride chromatography (2.4.14).
16
100
22
100
o
o
Determine by liquid
Test solution. Suitably dilute the filtered medium with 0.05 M

potassium dihydrogen orthophosphate adjusted to pH 6.2
with 0.1 M sodium hydroxide.
NOTE - Saturate the column with mobile phase B for about

1 hour.
Calculate the content of CIOHz4NzOz.2HCl in the dissolution

medium.
tailing factor is not less than 2.0 for rifampicin and isoniazid,
the column efficiency determined from isoniazid peak is not
less than 3000 and that from rifampicin is not less than 25000
theoretical plates respectively, and the relative standard
The relative retention times are about 1.0 for rifampicin and
about 0.3 for isoniazid.

C43H58N401Z, C6H 7N 30 and CIOHz4NzOz.2HCl.
Calculate the contents of C43H58N40lZ and C 6H 7N30 in the

tablets.
For ethambutol hydrpchloride

Reference solution. Suitably dilute the reference stock solution

with 0.05 M potassium dihydrogen orthophosphate with the
pH adjusted to 6.2 with 0.1 M sodium hydroxide.
Use the chromatographic system described under the Assay
of ethambutol hydrochloride.
Assay. For rifampicin and isoniazid chromatography (2.4.14).
Determine by liquid

a quantity ofthe powdered tablets containing about 40 mg of
Isoniazid dissolve in 100.0 ml of methanol, dilute to 500.0 ml
with the diluent and mix.
Determine by liquid

tablets containing about 60mg of Ethambutol Hydrochloride
and dissolve in 100.0 ml ofthe diluent.
Reference solution. A 0.06 per cent w/v solution of ethambutol
hydrochloride RS in the diluent.
2062
IP 2010
RIFAMPICIN, ISONIAZID AND PYRAZINAMIDE TABLETS
- a stainless steel column 15 cm x 4.6 mm, packed with tablets containing about 200 mg of Rifampicin, dissolve in
nitrile groups chemically bonded to porous silica 100 ml of acetonitrile and filter. Dilute 5 ml ofthis solution to
particles 5 (/-lm) (such as Zorbax SB CN),
50 ml with the solvent mixture.
and 50 volumes ofa buffer solution pH 7.0 prepared by
w/v of rifampicin RS in acetonitrile. Dilute Iml ofthis solution
dissolving 1 ml of triethylamine in 1000 ml of water and
adjusting the pH to 7.0 with dilute phosphoric acid,
, Reference solution (b). A solution containing 0.01 per cent
w/v each of rifampicin RS and rifampicin qUinone RS in
solvent mixture.
relative standard deviation for replicate injections is not more Chromatographic system
than 2.0 per cent, the tailing factor is not more than 3.0 and the
column efficiency determined from Ethambutol hydrochloride
peak is not less than 1500 theoretical plates.
Calculate the content ofCIOH24N202.2HCl in the tablets.
dihydrogen phosphate, 35 volumes of acetonitrile,
Rifampicin, Isoniazid and
Rifampin, Isonicotinylhydrazid and Pyrazinamide Tablets
Rifampicin, .Isoniazid and Pyrazinamide Tablets contain not
less than 90.0 per cent and not more than 110.0 per cent ofthe
stated amounts ofrifampicin, C43HssN4012' isoniazid, C6 H7N30
and pyrazinamide CsHsN30.
Usual strength. Rifampicin 120 mg, Isoniazid 50 mg and
Pyrazinamide 300 mg.
Identification
In the Assay of rifampicin, isoniazid and pyrazinamide, the
principal peaks in the chromatogram obtained with the test
solution correspond to the peaks in the chromatogram
Tests
(2.4.14).

of 17.42 per cent w/v solution of dipotassium hydrogen
phosphate, 640 volumes of water and 250 volume,S of
acetonitrile.
column efficiency is not less than 2000 theoretical plates;
Multiply the area of each known impurity by its response
any peak due to 3-formylrifamycin SV Isonicotinyl hydrazone
(5 per cent) and the area ofany peak due to 3-formylrifamycin
SV should not be more than the area of the principal peak in
the chromatogram obtained with the reference solution ( 1 per
2063
RIFAMPICIN, ISONIAZID AND PYRAZINAMIDE TABLETS
IP 2010


with reference solution (b) the resolution between isonicotinic
acid and pyrazinamide is not more than 2.5 and between
pyrazinamide and isoniazid is not more than 4.0.
Apparatus No.2,
Mediuni. 900 nil ofa solution containing 2 g ofsodium chloride
and7.0 ml of hydf"Ochloric acid in 1000 illl of water, with a pH
of about 1.2,
than 0.8 flm. Reject the first few ml ofthe filtrate.
cent w/v of rifampicin RS, 0.00825 per cent w/v of isoniazid
RS and 0.04375 per cent w/v of pyrazinamide RS in the
dissolution medium and filtered. Keep this reference stock
solution in the dissolution bath during the test run.
Calculate the contents ofC6H7N30 and CsHsN 30 in the medium.

C43HssN4012, C6H7N30 and CsHsN30.
Assay. For rifampicin, isoniazid and pyrazinamide Detennine by liquid chromatography (2.4.14).
Test solution. Weigh and powder20 tablets. Weigh accurately
a quantity of the powdered tablets containing about 40 mg of
Isoniazid, dissolve in 100.0 ml of methanol, dilute to 500 ml
with the buffer solution pH 6.8 and mix.

rifampicin RS, 0.04 per cent w/v of isoniazid RS and 0.2 per
cent w/v of pyrazinamide RS in methanol. Dilute 10.0 ml of
For rifampicin ~ Dilute the filtrate, if necessary, with the .this solution to 50.0 ml with the buffer solution pH 6.8.
same solvent. Measure the absorbance (2.4.7) ofthe resulting
solution atthe maximum at about 475 urn. Calculate the content
ofC43HssN40,2 in the medium from the absorbance obtained
from suitably diluted reference stock solution.
column. temperature 30,
For isoniazid and pyrazinamide
Determine by liquid
solution pH 6.8 (diluent) prepared by dissolving 1.4 g of
dis odium hydrogen orthophosphate anhydrous in
NOTE - Use this solution within one hourfrom preparation.
1000 ml of water, adjusted to pH 6.8 0.05 with dilute
phosphoroic acid and 4 volumes of acetonitrile,
Testsolution. To 15.0 ml ofthe filtered medium, add 15 ml of
1 M dibasic potassium phosphate, dilute to 100.0 ml with the
mobile phase and mix.
Reference solution (a). To 15.0 ml of the reference stock
solution, add 15 ml of 1 M dibasic potassium phosphate,
below,
dilute to 100.0 ml with the mobile phase and mix.
Reference solution (b). To 10.0 ml of a 0.0125 per cent w/v
solution of isonicotinic acid in the dissolution medium, add
Time
Mobile phase A
Mobile phase B
(in min.)
(per cent v/v)
(per cent v/v)
4.0 ml ofthe reference stock solution and 15 ml of 1 M dibasic
potassium phosphate, dilute to 100.0 ml with the mobile phase
o
100
o
and mix.
5
100
o
100
6
0
15
0
100
16
100
o
mobile phase: a mixture of 860 volumes of water,
22
100
o
100 volumes of 1 M monobasic potassium phosphate NOTE - Saturate the column with mobile phase (B) for
about 1 hour.
Inject the test solution and the reference solution. The test is
not valid unless the tailing factor is not less than 2.0 for
rifampicin, isoniazid and pyrazinamide, the column efficiencies
Inject the test solution and reference solutions (a) and (b). determined from Isoniazid, pyrazinamide and that from
The relative retention times are about 0.7 for isonicotinic acid, rifampicin are not less than 3000, 5000 and 25000 theoretical
plates respectively, and the relative standard deviation for
1.0 forpyrazinainide and 1.8 for iSoniazid.
2064
IP 2010
RIFAMPICIN, ISONIAZID, PYRAZINAMIDE AND ETHAMBUTOL TABLETS
replicate injections is not more than 2.0 per cent. The relative
retention times are about 1.8 for rifampicin, about 0.7 for
isoniazid and about 1.0 for pyrazinamide.
Calculate the contents ofC43HssN4012, C6H7N30 and CsH sN 30
in the tablets.
Rifampicin, Isoniazid, Pyrazinamide

and Ethambutol Tablets
Rifampin, Isonicotinylhydrazid, Pyrazinamide and
Ethambutol Hydrochloride Tablets
Rifampicin, Isoniazid, Pyrazinamide and Ethambutol Tablets
contain not less than 90.0 per cent and not more than 110.0 per
cent ofthe stated amounts ofrifampicin, C43HssN4012' isoniazid,
C 6H 7N 30, pyrazinamide, C sH sN 30 and ethambutol
hydrochloride, CIOH24N202.2HCl.
Usual strength. Rifampicin 150 mg, Isoniazid 75 mg,
Pyrazinamide 400 mg and Ethambutol 275 mg.
Identification
A. In the Assay for rifampicin, isoniazid and pyrazinamide, the
principal peaks in the chromatogram obtained with the test
solution correspond to the peaks in the chromatogram
B. In the Assay for ethambutol hydrochloride, the principal
peak in the chromatogram obtained with the test solution
corresponds to. the peak in the chromatogram obtained with
Tests
. Related substances. Determine by liquid chromatography
(2.4.14).

acetonitrile.
Test solution. Weigh accurately a quantity ofpowdered tablets
containing 200 mg of Rifampicin, dissolve in 100 ml of
acetonitrile and filter. Dilute 5 ml ofthis solution to 50 ml with
Reference solution (a). Dissolve 20 mg of rifampicin RS in
100 ml of acetonitrile. Dilute 1 ml ofthis solution to a 100 ml

solvent mixture.
octylsilane bonded to porous silica (5 llm),
dihydrogen phosphate, 35 volumes of acetonitrile,
injection volume. 20 lll.
column efficiency is not less than.2000 theoretical plates.
Multiply the area of each known impurity by its response
any peak due to 3-fonnylrifamycin SV Isonicotinyl hydrazone
( 5 per cent) and the area ofany peak due to 3-formylrifamycin
SV should not be more than the area ofthe principal peak in
the chromatogram obtained with the reference solution (l per
Apparatus No.1,
Medium. 900 ml of sodium phosphate bufferpH 6.8 prepared
by dissolving 7 g of dibasic sodium phosphate anhydrous in
5000 ml of water and adjusting to pH 6.8 with phosphoric
acid,
2065
RIFAMPICIN, ISONIAZID, PYRAZINAMIDE AND ETHAMBUTOL TABLETS

than 0.8Ilm. Reject the first few ml ofthe filtrate. Determine the
amounts of rifampicin, C43Hs8N401Z, isoniazid, C 6H 7N 30,
pyrazinamide, CsHsNP and ethambutol hydrochloride,
CIQHz4NzOz.2HCl by using the methods described in the Assay
for rifampicin, isoniazid, pyrazinamide and ethambutol
hydrochloride.
C43Hs8N401Z, C6H7N 30, CsH sN 30 and CIQHZ4NzOz.2HCI.
Assay. For rifampicin, isoniazid and pyrazinamide -
IP 2010
efficiencies for isoniazid, pyrazinamide and rifampicin are not

less than 3000, 5000 and 25000 theoretical plates respectively.
retention time for rifampicin is 1.8, for isoniazid, 0.7 and for
pyrazinamide, 1.0.
Calculate the contents OfC43Hs8N401Z, C6H 7N 30 and CsH sN 30
in the tablets.
For ethambutol hydrochloride -
Determine by liquid

tablets containing about 60 mg of Ethambutol Hydrochloride
and dissolve in 100 ml ofthe solvent mixture.
Reference solution. A 0.06 per cent w/v solution of ethambutol
hydrochloride RS in the solvent mixture.

a quantity ofthe powdered tablets containing about 40 mg of
Isoniazid, dissolve in 100 ml of methanol, dilute to 500 ml with
the buffer solution pH 6.8, mix and filter.
rifampicin RS, 0.04 per cent w/v of isoniazid RS and 0.2 per
cent w/v of pyrazinamide RS in methanol. Dilute 10.0 ml of
this solution to 50.0 ml with the buffer solution pH 6.8.
a stainless steel column 25 cm X 4.6 mm, packed with
- column. temperature 30,
- mobile phase: A. a mixture of 96 volumes of buffer
solution pH 6.8 prepared by dissolving 1.4 g of disodium
hydrogen orthophosphate anhydrous in 1000 ml of
water, adjusting to pH 6.8 0.05 with dilute phosphoric
below,
Time
Solution A
SolutionB
(in min.)
(per cent v/v)
(per cent v/v)
o
100
o
5
100
o
6
0
100
100
15
0
16
100
o
22
100
o
nitrile groups chemically bonded to porous silica
particles (5Ilm),
and 50 volumes of buffer solution pH 7.0 (diluent)
prepared by dissolving 1 ml of triethylamine in 1000 ml
of water and adjusting the pH to 7.0 with dilute
-
phosphoric acid,
injection volume. 50 Ill..
than 2.0 per cent, the tailing fac:tor is not more than 3.0 and the
column efficiencydeterminedfrom ethambutol hydrochloride
peak is not less than 1500 theoretical plates.
Calculate the content ofCIQHz4NzOz.2HCl in the tablets.
Ritonavir
than 2.0 per cent, the tailing factor is not more than 2.0 for
rifampicin, isoniazid and pyrazinamide and the column
2066
Mol. Wt. 721.0
IP 2010
RlTONAVIR CAPSULES
Ritonavir is (5S,8S,lOS,IIS)-1 0-hydroxy-2-methyl5-(1-methylethyl)-I-[2-(1-methylethyl)-4-thiazolyl]-3,6-dioxo8,11-bis(phenylmethyl)-2,4,7, 12-tetraazatridecan-13-oic acid

5-thiazolylmethyl ester.
Ritonavir contains not less than 98.0 per cent and not more
than 102.0 per cent of C37H48N60SS2, calculated on the
anhydrous basis.
Dose. 100 mg twice daily.
dissolving 3.4 g ofsodium acetate and 0.94 g ofsodium

hexanesulphonate in 1000 ml of water and adjusting
the pH to 4.0 with hydrochloric acid,
column efficiency determined from the ritonavir peak is not
than 2.0, and the relative standard deviation for replicate
Identification
Calculate the content ofC37H48N60sS2.
A. Determine by infrared absorption spectrophotometrY (2.4.6).

Compare the spectrum with that obtained with ritonavir RS or
with the reference spectrum of ritonavir.

to ritonavir in the chromatogram obtained with the reference
solution.
Ritonavir Capsules
Ritonavir Capsules contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of ritonavir,
C37~8N60SS2'
C.Meltingpoint.119to 123(2.4.21).
Tests
Specific optical rotation (2.4.22). +7.0 to + 10.5, determined
(2.4.14), as described in the Assay and calculate the percentage
ofeach impurity peak in the chromatogram obtained with the
test solution. Not more than 0.5 per cent of any individual
impurity and not more than 1.0 per cent oftotal impurities is
found.
Sulphated ash (2.3.18). Not more than 0.1 per cent

examination in sufficient of a mixture of 45 volumes of
acetonitrile and 55 volumes of water to produce 100.0 ml.
Reference solution. A 0.025 per cent w/v solution ofritonavir
RS in a mixture of45 volumes ofacetonitrile and 55 volumes
ofwater.
and 55 volumes of a buffer solution prepared by
Identification
A. In the test for Assay, the principal spot in the chromatogram
B. When examined in the range 200 urn to 350 urn (2.4.7), a
maxima at the same wavelengths as the reference solution.
Tests
Apparatus No.1,
Medium. 900 ml of0.1 M hydrochloric acidwith 25 roM polyoxyethylene 10 lauryl ether prepared by dissolving
15.65 g ofpolyoxyethylene 10 lauryl ether in 950 ml ofwater.
Add 8.5 ml of hydrochloric acid and dilute to 1000 ml with
water,
Deterrriine by liquid chromatography (2.4.14).
Test solution. Use the filtrate and if necessary, dilute with the
dissolution medium.
Reference solution. A 0.1 per cent w/v solution of ritonavir
RS in methanol. Dilute 5 ml of the solution to 50 ml with the
dissolution medium.
2067
RITONAVIR CAPSULES
IP 2010

(5.0 per cent).

C37HtsN60sS2.
(2.4.14).
Solvent mixture. 40 volumes of 0.03M monobasic potassium

phosphate buffer and 60 volumes of acetonitrile.
contents of20 capsules containing about 25 mg of Ritonavir,
dissolve in 50 ml ofthe solvent mixture and filter.
ritonavir RS in the solvent mixture.
Reference solution (b). Dilute 1 ml of the solution to 100 ml
butyl silane chemically bonded to porous silica (3 /lm)
(Such as ACE C4),
- mobile phase: A. a mixture of69 volumes of0.03 M monobasic potassium phosphate buffer prepared by
in 1000 ml of water, 18 volumes ofacetonitrile, 8 volumes
of tetrahydrofuranand 5 volumes of n-butanol,
B. a mixture of40 volumes of0.03 M monobasic potassium phosphate buffer, 47 volumes of
acetonitrile, 8 volumes of tetrahydrojitran and 5 volumes
of n-butanol,
below,
Time
(in min.)
mobile phase A
(per cent v/v)
mobile phase B
(per cent v/v)
100
60
100
o
o
120
100
155
100


contents of20 capsules containing about 25 mg ofRitonavir,
disperse in 100.0 ml of methanol and filter.
Reference solution. A 0.025 per cent w/v solution ofritonavir
RS in methanol.
octadecylsilane bonded to porous silica (3.5 /lm),
prepared by dissolving 3.4 g of sodium acetate
trihydrate and 0.94 g of sodium l-hexanesulphonate in
1000 ml of water and adjusting the pH to 4.0 with
hydrochloric acid, and 45 volumes of acetonitrile,
Inject the reference solution. Run the chromatogram 1.5 times
the retention time (about 3 minutes)ofthe principal peak The
test is not valid unless the tailing factor is not more than 2.0,
the column efficiency in not less than 2000 theoretical plates
Calculate the content of C37H4sN60sS2 in the capsules.
Storage. Store protected from light in a refrigerator (2 to 8).
Ritonavir Tablets
Ritonavir Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of ritonavir,
C37HtsN60sS2.
the tailing factor is not more than 2.0 arid the column efficiency
in not less than 5000 theoretical plates.
. Inject the test solution and reference solution (b). In the
chromatogram obtained with the test solutiQn, the area ofany
Identification
Tests
Dissolution (2.5.2)
Apparatus No.1,
2068
IP 2010
RITONAVIR TABLETS
Medium. 900 ml ofa solution prepared by dissolving 15.7 g of

polyoxyethylene 1O-lauryl ether in 1000 ml of a 0.85 per cent
v/v solution of hydrochloric acid,
- injection volume. 100 f..11.

Time
(in min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
100
60
100
o
o
Determine by liquid chromatography (2.4.14)
120
100
Test solution. The filtrate obtained as given.above. Dilute the

filtrate if necessary, with the dissolution medium.
121
100
155
100
o
o

RS in methanol. Dilute 5 ml ofthe solution to 50 ml with the
dissolution medium.

(5.0 percent).
C37~8N60SSZ.
Assay. Determine by liquid chromatography(2.4.14)
Calculate the content of C37H48N60sSz.
(2.4.14).
Solvent mixture. A mixture of40 volumes of 0.03 Mmonobasic

potassium phosphate and 60 volumes of acetonitrile.

tablets containing 25 mg ofRitonavir and disperse in 100.0 ml
of methanol.
Test solution: Shake a quantity of the powdered tablets

containing 25 mg ofRitonavir with 50 m1 ofthe solvent mixture.

RS in methanol.
RefereYice solution (a). A 0.05 per cent w/v solution of

ritonavir RS in the solvent mixture.
silica gel consisting of porous spherical particles with
chemically bonded with nitrile group (3f..1m) (such as
YMCC4),
mobile phase: A. a mixture of69 volumes of 0.03 M monobasic potassium phosphate solution, 18 volumes of
acetonitrile, 8 volumes of tetrahydrofuran and
5 volumes of n- butanol,
B. a mixture of40 volumes of 0.03 M monobasic potassium phosphate solution, 47 volumes of
acetonitrile, 8 volumes of tetrahydrofuran and
5 volumes of n-butanol,
below,
phenyl stationary phase bonded to porous silica (3 f..1m),
prepared by dissolving 3.4 g of sodium acetate
trihydrate and 0.94 g of sodium l-hexanesulphonate
to 1000 ml with water and adjusting the pH to 4.0 with
dilute hydrochloric acid, and 45 volumes of
acetonitrile.
spectrophotometerset at 239 nm,
injection volume. 10 f..11.
than 2.0 per cent.
Calculate the content of C37H48N60sSz in the tablets.

exceeding 30.
2069
ROSIGLITAZONE MALEATE
IP 2010
(COOH
COOH
ClsHI9N303S,CJ1404
Mol. Wt. 473.5
Rosiglitazone Maleate is (RS)-5- {p-[2-(methyl-2-pyridylamino)

ethoxy]benzyl}-2,4-thiazolidinedione maleate (1: 1).
Rosiglitazone Maleate contains not less than 98.0 per cent
and not more than 102.0 per cent of ClsHI9N303S,C4H404,
Category. Antidibatic
Description. A white to off-white crystalline powder.
Identification
Compare the spectrum with that obtained with rosiglitazone
maleate RS.

examination in 100.0 ml ofmobile phase.
rosiglitazone maleate RS in mobile phase.
octadecylsilane bonded to porous silica (5 J.!m),
- mobile phase: a mixture of65 volumes of buffer solution
orthophosphate in 1000 ml of water and adjust the pH
to 3.0 with dilute phosphoric acid, 25 volumes of
- injection volume. 10 J.!l.
than 2.0 per cent.
Calculate the content ofClsHI9N303S,C4H404.
Tests

(2.4.14).

examination in 50 ml ofmobile phase.

rosiglitazone maleate RS in the mobile phase.
Rosiglitazone Maleate Tablets

100 ml with mobile phase.
Rosiglitazone Tablets contains not less than 90.0 per cent and
rosiglitazone, ClsH19N303S,

is not more than the area of the peak in the chromatogram
obtained with the reference solution (b) (1.0 per cent). Ignore
any peak (due to maleic acid) with a relative retention time of
about 0.4.
Heavy metals (2.3.13). Lgcomplies with the limit test for heavy
metals, Metboc1BJ20ppm).
Identification
Tests
Apparatus No.1,
not greater than 1.0 J.!m, rejecting the first 1 ml ofthe filtrate.
2070
IP 2010
ROSUVASTATIN CALCIUM

about 318 urn (2.4.7). Similarly measure the absorbance of a
standard solution of known concentration of rosiglitazone
maleate RS and calculate the content ofClsH,9N303S.

potassium hydrogen phosphate adjusted to pH 3.0 with
orthophosphoric acid, 25 volumes of acetonitrile and

ClsH19N303S.
(2.4.14).

containing 200 mg of Rosiglitazone, disperse in 100.0 ml of
mobile phase. Centrifuge and use clear supernatant liquid.
rosiglitazone maleate RS in the mobile phase.
Calculate the content ofClsH19N303S.
F

more than twice the area of the peak in the chromatogram
obtained with the reference solution (b) (2.0 per cent). Ignore
any peak (due to maleic acid) with a relative retention time of
about 0.4.
Tablets.
Disperse 1 tablet in 0.1 M hydrochloric acid to produce
0.004 per cent w/v solution. Measure the absorbance of the
Calculate the content of CIsHI9N303S from the absorbance
obtained from same concentration of rosiglitazone maleate
RS in the same medium.
OH OH 0
Ca++
N/'
H3C'N
ANI
0-
CH 3
S02CH 3
CH 3
(C22H27FN306S)2.Ca
2
Mol. Wt.l00l.l
Rosuvastatin Calcium is (E)-(3R,5S)-7- {4-(4-fluorophenyl)6-isopropyl-2-{methyl(methylsulphonylamino)]pyrimidin5-yl} -3,5-dihydroxyhepten-6-oic acid calcium.

Rosuvastatin Calcium contains not less than 98.0 per cent
and not more than 102.0 per cent of (C22H27FN306S)2.Ca.

a quantity of powdered tablets containing 20 mg of
RosiglitaZone, disperse in 100.0 ml ofmobile phase. Centrifuge
and use clear supernatant liquid.
Description. An off- white to creamish white crystalline

powder.
Identification

rosiglitaione maleate RS in mobile phase.
A. Determine by iDfrared absorption spectrophotometry(2.4.6).

Compare the spectrum with that obtained with fosuvastatin
calcium RS.
..
octadecysilane bonded to porous silica (5 11m),
B. Dissolve 20 mg in 25 ml of methanol, add 2 drops of methyl

red indicator neutralise with 6 M ammonium hydroxide. Add
3 M hydrochloric acid until the solution is acidic to the
2071
IF 2010
ROSUVASTATIN CALCIUM
indicator. Add ammonium oxalate solution, a white precipitate

is obtained.
Tests
than 2.0 per cent.

(2.4.14).

Calculate the content of(C22H27FN)06S)2.Ca.


rosuvastatin calcium RS in the mobile phase.

Rosuvastatin Calcium Tablets
- a stainless steel column 25 cm x 4.6 mrn packed with
- mobile phase: a mixture of 50 volumes of 0.2 per cent
w/v acetic acid in water, 25 volumes of acetonitrile
injection volume. 20 ~l.
Rosuvastatin Tablets contain not less than 90.0 per cent and
rosuvastatin, C44Hs4F2N6012S2.
Identification
Tests


examination in 100.0 ml ofmobile phase. Dilute 5.0 ml ofthe
solution to 25.0 ml with mobile phase, mix and filter.
Reference solution. A 0.05 per cent w/v of rosuvastatin
calcium RS in mobile phase. Dilute 5.0 ml of the solution to
25.0 ml with mobile phase.
acetic acid in water, 25 volumes of acetQn.itrile and
25 volumes of methanol, filter and degas.
Apparatus No.1,
Medium. 900 ml ofphosphate buffer pH 6.8,

rosuvastatin calcium RS in the mobile phase. Dilute 2 ml of
the solution to 100 ml with Dissolution medium.
Calculate the content ofC44Hs4F2N6012S2.
C44Hs4F2N6012S2.
Tablets.
under Assay.
Test solution. Disperse one tablet in 100 ml ofthe mobile phase.

Dilute 5 ml of the solution to 10 mlwith mobile phase and
filter.
(2.4.14).
2072
IP 2010
ROXITHROMYCIN

a quantity of powdered tablet containing 25 mg of
Rosuvastatin, disperse in 50 ml of mobile phase and filter.
Roxithromycin

rosuvastatin calcium RS in the mobile phase.
is not more than 3.0 times the area ofthe peak in the chromatogram obtained with the reference solution (b) (3.0 per cent).

a quantity of powdered tablet containing 25 mg of Rosuvastatin, disperse in 100.0 ml ofmobile phase. Dilute 5.0 ml of
the solution to 25.0 ml with mobile phase and filter.
rosuvastatin calcium RS in mobile phase. Dilute 5.0 ml ofthe
solution to 25.0 ml with mobile phase.
- mobile phase: a mixture of 585 volumes of a buffer
solution prepared by dissolving 1.54 g of ammonium
acetate in 900 ml water, adjust pH to 4.0 with glacial
acetic acid and dilute to 1000 ml with water, 360 volumes
of acetonitrile and 50 volumes of tetrahydrojitrane,
Mol. Wt. 837.0

Roxithromycin is (3R,4S,5S,6R,7R,9R,IIS,12R,13S,14R)4-[(2,6-dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-14-ethyl-7, 12,13-trihydroxy10-[(E)-[(2-methoxyethoxy)methoxy]imino]-3,5,7,9,11,
13-hexamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)f3-D-xylo-hexopyranosyl]oxy]oxacyclotetradecan-2-one;
erythromycin 9-(E)-[0-[(2-methoxyethoxy)methyl]oxime].
Roxithromycin contains not less than 96.0 per cent and not
more than 102.0 per cent of C41H76N201s, calculated on the
anhydrous basis.
Dose. 150 mg twice a day.
Identification
Compare the spectrum with that obtained with roxithromycin
RS.

Tests
Appearance ofsolution. A 1.0 per cent w/v solution in methanol
Specific optical rotation (2.4.22). - 93.0 to - 96.0, determined
in a 1.0 per cent w/v solution in acetone.
(2.4.14).

Calculate the content ofC44Hs4F2N6012S2.
equivalent amount of Rosuvastatin.
Solvent mixture. Mix 30 volumes of acetonitrile and

70 volumes of a 4.8 per cent w/v solution of ammonium
dihydrogen phosphate and adjust the pH to 5.3 with dilute
2073
IP 2010
ROXITHROMYCIN

roxithromycin RS in the solvent mixture.
Reference solution (c). Dissolve 2 mg of erythromycin 9-(E)[0-[[(2-methoxyethoxy)methoxy]methyl]oxime] (impurity
A) in 10 ml ofreference solution (a). Further dilute 1 mlofthis
solution to 10 ml with reference solution (a).
Reference solution (d). Dilute 1.0 ml of toluene to 100 ml with
acetonitrile. Dilute 0.2 ml of this solution to 200 ml with the
solvent mixture.
spherical end-capped octadecylsilane bonded to
porous silica (5 /lm), with a 10 nm pore size and a carbon
loading of about 19 per cent,
- mobile phase: A. a mixture of26 volumes of acetonitrile
ammonium dihydrogen phosphate, adjusted to pH 4.3
with dilute sodium hydroxide solution,
B. a mixture ono volumes of water and
- flow rate. 1. 1 ml per minute,
below,
Time
Mobile phase A
Mobile phase B
(in min.)
(per cent v/v)
(per cent vIv)
100
o
o
50
100
80
90
10
100
100
Inject reference solution (c). Relative retention time between

roxithromycin and erythromycln9-(E)-[O-[[(2-methoxyethoxy)
methoxy]methyl] oxime] (impurity A) is about 1.15. The test is
not valid unless the peak-to-valley ratio Hpl HI' is not more
than 2.0, where H p = height above the baseline ofthe peak due
to impurity A and HI' = height above the baseline ofthe lowest
point of the curve separating this peak from the peak due to
roxithromycin. .
Inject the test solution and reference solutions (b) and (d). In
the chromatogralll obtained with the test solution the area of
the impurity A peak obtained immediately after the peak
obtained with roxithromycin is not more than the area of the
solution (b) (1.0 per cent). The area of any other individual
impurity peak is not more than 0.5 times the area ofthe principal
(b) (0.5 per cent). The sum ofthe areas of all the peaks is not
chromatogram obtained with reference solution (b}(3.0 per
cent). Ignore any peak with an area 0.05 times the area ofthe
solution (b) (0.05 per cent). Ignore any peak due to toluene
identified using reference solution (d).
Heavy metals (2.3.13). Dissolve 2.0 g in a mixture of15 volumes

of water and 85 volumes of acetone and dilute to 20 ml with
the same solvent mixture. 12 ml ofthis solution complies with
the limit test for heavy metals, MethodA(10ppm). Use 1 mlof
lead standard solution (l0 ppm lead) prepared by diluting
lead standard solution (100 ppm lead) with a mixture of
15 volumes of water and 85 voiumes of acetone.
0.2g.

roxitliromycin RS in the solvent mixture.
Reference solution (b). Dissolve 2 mg of erythromycin
9-(E)-[0-[[(2-methoxyethoxy)methoxy]methyl]oxime]
(impurity A) in 10.0 ml ofreference solution (a). Further dilute
1.0 ml ofthis solution to 1O~O ml with reference solution (a).
- mobile phase: mix 307 volumes of acetonitrile and 693
volumes of a 4.91 per cent w/v solution of ammonium
dihydrogen phosphate adjusted to pH 5.3 with dilute
sodium lJydroxide solution,
flow rate.l.5 ml per minute,
- spectrophotometer set at 205 rirri,
- injection volume. 20 iiI.
peak-to-valley ratio Hpl HI' is not less than 1.5.
2074
IP20l0
ROXITHROMYCIN TABLETS

a quantity of the powder containing about 0.2 g of
Roxithromycin, add 80 ml ofthe solvent mixture and mix. Dilute
to 100.0 ml with the solvent mixtUre and filter.
Inject the test solution and reference solution (a)

Calculate the content ofC14Hz6Nz015'

roxithromycin RS in the solvent mixture.
Roxithromycin Tablets contain not less than 90.0 per cent and
roxithromycin, C 4 IH76N zO I 5.
Reference solution (b). Dissolve 2 mg of elythromycin 9-()[0-[[(2-methoxyethoxy)methoxy]methyl]oxime] (impurity

A) in 10 ml ofreference solution (a). Further dilute 1 ml ofthis
solution to 10 ml with reference solution (a).
Usual strengths. ISO mg; 300 mg.
-
Identification
Tests
Apprartus No.1,
Medium. 900 ml ofphosphate bujferpH 6.0,


roxithromycin RS in the dissolution medium.

mobile phase: mix 307 volumes of acetonitrile and
693 volumes of a 4.91 per cent w/v solution of
ammonium dihydrogen phosphate adjusted to pH 5.3
with dilute sodium hydroxide solution,
Inject reference solution (b). The relative retention time

between roxithromycin and erythromycin-(E)-[O-[[(2methoxyethoxy)methoxy]methyl]oxime] (impurity A) is about
1.15. The test is not valid unless the peak-to-valley ratio
Hpl ~, is not more than 1.5, where Hp = height above the
baseline of the peak due to impurity A and ~, = height above
the baseline of the lowest point of the curve separating this
peak from the peak due to roxithromycin.
Calculate the content OfC41H76NzOl5 in the medium.
than 2.0 per cent.
C41H76Nz015.
Calculate the content of C41H76NzOl5 in the tablets.


Labelling. If the tablets are dispersible, the label states that
the tablets should be dispersed in water immediately before
use.
2075
MONOGRAPHS
s
Saccharin
Saccharin Sodium
Salbutamol
Salbutamol Inhalation
Salbutamol Sulphate
SalbutamolInjection
Salbutamol Syrup
Salbutamol Tablets
SalicylicAcid
Salmeterol Xinafoate
Salmetrol and Fluticasone Propionate Inhalation
Salmetrol and Fluticasone Propionate Powder for Inhalation
Saquinavir
SaquinavirMesylate
SaquinavirMesylate Tablets
Secnidazole
Secnidazole Tablets
Serratiopeptidase
Serratiopeptidase Tablets
ColloidalSiliconDioxide
Sildenafil Citrate
Sildenafil Tablets
SilverNitrate
Simvastatin
SimvastatinTablets
SodiumAcetate
SodiumAlginate
SodiumAminosalicylate
SodiumAminosalicylate Tablets
SodiumAscorbate
Sodium Benzoate
2077
2081
2082
2083
2084
2085
2087
2088
2088
2089
2090
2091
2091
2092
2093
2094
2095
2096
2097
2098
2099
2100
2101
2102
2103
2104
2105
2106
2107
2108
2109
2110
MONOGRAPHS
2111
2111
Sodium Bicarbonate
Sodium Bicarbonate Injection
2112
2112
2113
Sodium Carbonate
Sodium CWoride
Sodium Chloride and Dextrose Injection
2114
2115
2116
2116
2117
2117
2118
2118
Sodium Chloride and Fructose Injection

Compound Sodium Chloride and Dextrose Injection
Sodium CWoride Hypertonic Injection
Sodium CWoride Injection
Compound Sodium Chloride Injection
Compound Sodium Chloride Solution
Sodium CWoride Irrigation Solution
Sodium Citrate
2119
2120
2121
2122
2122
2123
2124
2125
2125
Sodium Diatrizoate
Sodium Diatrizoate Injection
Sodium Dihydrogen Phosphate Dihydrate
Sodium Fluoride
Sodium Formaldehyde Sulphoxylate
Sodium Fusidate
Sodium Hydroxide
Sodium Lactate Injection
Compound Sodium Lactate and Dextrose Injection
Sodium Salicylate
2127
2128
2129
2130
2131
2132
2132
2133
2134
2134
Sodium Starch Glycollate
2137
HalfStrength Compound Sodium Lactate and Dextrose Injection

Modified Compound Sodium Lactate and Dextrose Injection
Compound Sodium Lactate Injection
Compound Sodium Lactate Solution for Irrigation
SodiumLauryl Sulphate
Sodium Metabisulphite
SodiumMethylparaben
Sodium Phosphate
SodiumPropylparaben
2078
MONOGRAPHS
Sodium Stibogluconate
2136
Sodium Stibogluconate Injection
2137
Sodium Thiosulphate
2138
Sodium Thiosulphate Injection
2138
Sodium Valproate
2139
Sodium Valproate Oral Solution
2140
Sodium Valproate Tablets
2140
SorbicAcid
2141
Sorbitan Oleate
2142
Sorbitol
2143
.Sorbitol Solution (70 Per cent) (Crystallising)
2144
Sorbitol Solution (70 Per cent) (Non-Crystallising)
2144
Spironolactone
2147
Spironolactone Tablets
2148
Stavudine
2149
Stavudine Capsules
2151
Stavudine Oral Solution
2152
Stavudine and Lamivudine Tablets
2153
Stearic Acid
2154
StearylAlcohol
2155
Stilboestrol
2156
Stilboestrol Tablets
2156
Streptokinase
2157
Streptokinase Injection
2159
Streptomycin Sulphate
2161
Streptomycin Injection
2162
Streptomycin Tablets
2163
Succinylcholine Chloride
2164
Succinylcholine Injection
2165
Sucralose
2165
Sucrose
2166
Sulphacetamide Sodium
2167
Sulphacetamide Eye Drops
2168
2079
MONOGRAPHS
2169
2170
2170
2171
2172
2173
2174
Sulphadiazine
Sulphadiazine Tablets
Sulphadoxine
Sulphamethizole
Sulphamethoxazole
Sumatriptan
Sumatriptan Injection
2080
IP 2010
SACCHARIN
Saccharin
o
S~O
-:?'
~
I \NH
o
C7HsN03S
Mol. Wt. 183.2
Saccharin is 1,2-benzisothiazol-3(2H)-one 1, I-dioxide.

Saccharin contains not less than 98.0 per cent and not more
than 101.0 per cent ofC 7HsN03S, calculated on the dried basis.
Category. Pharmaceutical aid (sweetening agent).
Description. White crystals or a white, crystalline powder;
odourless or with a faint, aromatic odour.
Identification

Compare the spectrum with that obtained with saccharin RS.
B. Mix 20 mg with 20 mg of resorcinol, add 05 ml ofsulphuric
acid and heat over a small flame until- a dark green colour is
produced; allow to cool and add 10 ml of water and an excess
of 2 M sodium hydroxide; a fluorescent green liquid is
produced.

sodium hydroxide, evaporate to dryness and gently fuse the
residue over a small flame until ammonia is no longer evolved.
Allow to cool, dissolve in 20 ml of water, make the solution
just acidic to litmus paper, filter and add 0.05 ml of ferric
chloride solution; a violet colour is produced.
D. A saturated solution is acidic to litmus paper.
Tests
Appearance ofsolution. Dissolve 5.0 g in 20 ml ofa 20 per cent
w/v solution of sodium acetate and dilute to 25 ml with the
same solution; the solution is clear (2.4.1), and colourless
(2.4.1).
(2.4.13).
Internal standard solution. Dissolve 25 mg of caffeine in
dichloromethane and dilute to 100 ml with the same solvent.
Test solution. Suspend 10.0 g ofthe substance under examination in 20 ml of water and dissolve using about 5 ml of strong
sodium hydroxide solution. Ifnecessary, adjust the pH of the
solution to 7.8 with 1 M sodium hydroxide or 1 M hydrochloric
acid and dilute to 50 ml with water. Shake the solution with 4

quantities, each of 50 ml, of dichloromethane. Combine the
lower layers, dry over anhydrous sodium sulphate and filter.
Wah the filter and the sodium sulphate with 10 ml of
dichloromethane. Combine the solution and the washings
and evaporate almost to dryness in a water-bath at a
temperature not exceeding 40. With a small quantity of
dichloromethane transfer quantitatively the residue into a
suitable 10-ml tube, evaporate to dryness in a current of
nitrogen and dissolve the residue in 1.0 ml of the internal
standard solution.
Blank solution. Evaporate 200 ml of dichloromethane to
dryness in a water-bath at a temperature not exceeding 40.
Dissolve the residue in I ml of dichloromethane.
Reference solution. Dissolve 20 mg of o-toluenesulphonamide
and 20 mg of p-toluene sulphonamide in dichloromethane
and dilute to 100 ml with the same solvent. Dilute 5 mlofthe
solution to 50 ml with dichloromethane. Evaporate 5 mlofthe
final solution to dryness in a current of nitrogen. Dissolve the
residue in 1 ml of the internal standard solution.
a fused silica column 10 m x 0.53 rom packed with
polymethylphenylsiloxane (film thickness 2 /lm),
- temperature:
column.! 80,
flow rate. 10 ml per minute ofnitrogen (carrier gas),
split ratio. 1:2.
Inject 1 /ll of each solution. The order of elution is
o-toluenesulphonamide, p-toluenesulphonamide, caffeine.
The test is not valid unless the resolution between the peaks
due to o-toluenesulphonamide and p-toluenesulphonamide
in the chromatogram obtained with the reference solution is
not less than 1.5 and the chromatogram obtained with the
blank solution does not show any peak with the same retention
times as the internal standard, o-toluenesulphonamide and
p-toluenesulphonamide.
In the chromatogram obtained with the test solution the ratio
of the area of the peak due to o-toluenesulphonamide to that
of the internal standard is not greater than the corresponding
ratio in the chromatogram obtained with the rference solution
(lOppm).
of the area of the peak due to p-toluenesulphonamide to that
ratio in the chromatogram obtained with the reference solution
(10 ppm).
Arsenic (2.3.10). Mix 5.0 g with 3.0 g of anhydrous sodium
2081
IP 2010
SACCHARIN SODIUM
Evaporate to dryness on a water-bath, gently ignite,and add

to the cooled residue a mixture of 16 ml of brominated
hydrochloric acid AsT and 5 ml of bromine solution. Add
40 ml of water and boil gently, adding sufficient bromine
solution from time to time to maintain a slight excess. Filter
and remove the excess ofbromine with a sufficient quantity of
stannous chloride solution AsT. The resulting solution
Identification
heavy metals, Method D (20 ppm). Use leadstandardsolution
(2 ppm Pb).
B. Dissolve 0.1 g in 5 ml of a 10 per cent w/v solution of

sodium hydroxide, evaporate to dryness and gently fuse the
residue over a small flame until ammonia is no longer evolved.
Allow to cool, dissolve in 20 ml of water, make the solution
just acidic to litmus paper, filter and add 0.05 ml of ferric
chloride solution; a violet colour is produced.
Readily carbonisable substances. Dissolve 0.2 g in 5 ml of

sulphuric acid (96 per cent w/w) and maintain at about 50
for 10 minutes. The solution is not more intensely coloured
Loss on drying (2.4.19). Not more than LOper cent, detennined
Assay. Weigh accurately about 0.5 g, dissolve in 75 ml ofhot
water, cool quickly and titrate with 0.1 M sodium hydroxide
using phenolphthalein solution as indicator.
C7HsN03S.

out.
A. Detennine by infrared absorption spectrophotomeuy (2.4.6).

Compare the spectrum with that obtained with saccharin
sodium RS.
C. Mix 20 mg with 20 mg of resorcinol, add 0.5 mlofsulphuric

acid and heat over a small flame until a dark green colour is
produced; allow to cool and add 10 ml of water and an excess
of 2 M sodium hydroxide; a fluorescent green liquid is
produced.
D. To 5 mlofa 10 per centw/v solution add 3 mlof2 Mhydrochloric acid; a white precipitate is produced. The precipitate,
after washing with water and drying at 105 melts at 226 to
230 (2.4.21).
E. 0.5 ml of a 10 per cent w/v solution gives reaction B of
sodium salts (2.3.1).
Tests
colourless (2.4.1).
Saccharin Sodium
Acidity or alkalinity. Dissolve 5.0 g in sufficient carbon

dioxide-free water to produce 50 ml (solution A). To 10 ml of
solution A add 5 ml of 0.005 M sulphuric acid, heat to boiling,
cool and add 0.1 ml of phenolphthalein solution. Not less
than 4.5 ml and not more than 5.5 ml of 0.01 M sodium
hydroxide is required tochange the colour of the solution to
pink.
Soluble Saccharin
s-:-o
~
-;:/'
N-Na
Related substances. Detennine by gas chromatography

(2.4.13).
C7~a03S
Mol. Wt. 205.2
Saccharin Sodium is the sodium salt of 1,2-benzisothiazol3(2H)-3-one 1, I-dioxide.

Saccharin Sodium contains not less than 99.0 per cent and not
more than 101.0 per cent ofC7H4NNa03S, calculated on the
anhydrous basis.
Category. Phannaceutical aid (sweetening agent).
Description. A white, crystalline powder or colourless crystals;
efflorescent in dry air.
Internal standard solution. Dissolve 25 mg of caffeine in

dichloromethane and dilute to 100 ml with the same solvent.
Testsolution. Suspend 10.Og of the substancel ul1<ier
examination in 50 ml of water. If necessary, adjust the pH of
the solution to 7.8 with 1 M sodium hydroxide or 1 M hydrochloric acid and dilute to 50 m1 with water. Shake the solution
with 4 quantities, each of50 ml, of dichloromethane. Combine
the lower layers, dry over anhydrous sodium sulphate and
filter. Wah the filter and the sodium sulphate with 10 ml of
dichloromethane. Combine the solution and the washings
and evaporate almost to dryness in a water-bath at a
2082
IP 2010
SALBUTAMOL
temperature not exceeding 40. With a small quantity of

dichloromethane transfer quantitatively the residue into a
suitable 10-ml tube, evaporate to dryness in a current of
nitrogen and dissolve the residue in 1,0 ml of the internal
standard solution.
Blank solution. Evaporate 200 ml of dichloromethane to
dryness in a water-bath at a temperature not exceeding 40.
Dissolve the residue in 1 ml of dichloromethane.
Reference solution. Dissolve 20 mg of o-toluenesulphonamide
and 20 mg of p-toluene sulphonamide in dichloromethane
and dilute to 100 ml with the same solvent. Dilute 5 ml of this
solution to 50 ml with dichloromethane. Evaporate 5 mlofthe
final solution to dryness in a current of nitrogen. Dissolve the
residue in 1 ml of the internal standard solution.
a fused silica column 10m x 0.53 mm packed with
polymethylphenylsiloxane (film thickness 2 !!m),
temperature:
column.180,
inlet port and detector. 250 0
flow rate. 10 ml per minute ofnitrogen (carrier gas),
split ratio. 1:2.
Inject 1 !!1 of each solution. The order of elution is
o-toluenesulphonamide, p-toluenesulphonamide, caffeine.
and'remove the excess ofbromine with a sufficient quantity of

stannous chloride solution AsT. The resulting solution
Readily carbonisable substances. Dissolve 0.2 g in 5 ml of
sulphuric acid (96 per cent w/w) and maintain at about 50
for 10 minutes. The' solution is not more intensely coloured
Heavy metals (2.3.13). 12 ml ofsolution A complies with the
standard solution (2 ppm Pb).
0.2g.
anhydrous glacial acetic acid, with slight heating ifnecessary.
C7H;NNa03S,
Salbutamol
Albuterol
due to .o-toluenesulphonamide and p-toluenesulphonamide
not less than 1.5 and the chromatogram obtained with the
blank solution does not show any peak with the same retention
times as the internal standard, o-toluenesulphonamide and
p-toluenesulphonamide.
of the area of the peak due to o-toluenesulphonamide to that
ratio in the chromatogram obtained with the reference solution
(10 ppm).
of the area of the peak due to p-t6luenesulphonamide to that
ratio in the chromatogram obtained with the rference solution
. (10 ppm).
Evaporate to dryness on a water-bath, gently ignite and add
to the cooled residue a mixture of 16 ml of brominated
hydrochloric acid AsT and 5 ml of bromine solution. Add
40 ml of water and boil gently, adding sufficient bromine
solution from time to time to maintain a slight excess. Filter
OH
HO
OH
Mol. Wt. 239.3
Salbutamol is (RS)-I-( 4-hydroxy-3-hydroxymethylphenyl)2-(tert-butylamino)ethanol.
Salbutamol contains not less than 98.0 per cent and not more
than 101.0 per cent ofC 13Hz1 N0 3, calculated on the dried basis.
Category. Beta-adrenoceptor agonist.
Dose. Orally, 6 to 16 mg daily, in divided doses; by inhalation,
for chronic bronchial asthma or as a prophylactic, 2 inhalations
of 100 !!g, 3 or 4 times daily; for the relief of acute
bronchospasm, 1 or 2 inhalations of 100 !!g as a single dose
when required, up to a maximumof8 inhalations in 24 hours;
by subcutaneous or intramuscular injection, 500 !!g, repeated
every 4 hours, if necessary; by slow intravenous injection,
250 !!g, repeated if necessary.
2083
SALBUTAMOL
IF 2010
Identification
ofany secondary peale is not more than the area ofthe principal
is not more than 3.3 times the area ofthe principal peak in the
cent). Ignore any peak with an area less than 0.05 per cent the

A. Detennine by infrared absorption spectrophotometry (2.4.6),
Compare the spectrum with that obtained with salbutamol RS
or with the reference spectrum of salbutamol.
B. When examined in the range 230 nm to 360 run (2.4.7), a

about 276 run, about 0.53 to 0.60.
chromatogram obtained with reference solution (a)
D. Dissolve 10 mg in 50 ml of a 2 per cent w/v solution of
borax, add 1ml ofa 3 per cent w/v solution of4-aminophenazone,
10 ml of a 2 per cent w/v solution of potassium ferricyanide
and 10 ml of chloroform, shake and allow to separate; an
orange-red colour is produced in the chlorofonn layer.
Tests
Appearance ofsolution. A2.0 per cent w/v solution in methanol
is clear (2.4.1), and not more intensely coloured than reference
(2.4.14).

each of salbutamol RS and (lRS)-2-[(l,1dimethylethyl)amino]-1-(4- hydroxyphenyl)ethanol RS
(salbutamol impurity A RS) in the mobile phase. Dilute 2.0 ml
ofthis solution to 100.0 ml with the mobile phase.
- a stainless steel column 15 cm x 3.9 mrn, packed with
endcapped octylsilane bonded to porous silica (5 /lm),
w/v of sodium heptanesulphonate and 0.25 per cent
w/v of potassium dihydrogen phosphate, adjusted to
pH 3.7 with orthophosphoric acid,
- flow rate; 1 ml per minute,
Inject the ref~rence solution. The test is not valid llnless the
r~s()lllti<:>.n _~e_t\\lee!1the peaks_~ue!()~ll('U()_s~llJllt~IIloLan~
Boron. To 50 mg add 5 ml ofa solution containing 1.3 per cent

w/v of anhydrous sodium carbonate and 1.7 per cent w/v of
potassium carbonate, evaporate to dryness on a water-bath
and dry at 120. Ignite the residue rapidly until the organic
matter has been destroyed, allow to cool, add 0.5 ml of water
and 3.0 ml ofa freshly prepared 0.125 per cent w/v solution of
curcumin in glacial acetic acid. Evaporate to dryness and
allow to cool. Add 3 ml ofa mixture prepared by adding 5 ml of
sulphuric acid, slowly and with stirring, to 5 ml of glacial
acetic acid. Mix and allow to stand for 30 minutes. Add
sufficient ethanol (95 per cent) to produce 100.0 ml, filter and
measure the absorbance ofthe filtrate at 555 run (2.4.7). Prepare
a reference solution in the following manner. Dissolve 0.572 g
of boric acid in 1000.0 ml of water. Dilute 1.0 ml to 100.0 ml
with water. To 2.5 ml of this solution add 5 ml of a solution
containing 1.3 per cent w/v of anhydrous sodium carbonate
and 1.7 per cent w/v of potassium carbonate and treat this
mixture in the same manner as described above beginning at
the words "Evaporate to dryness; ..". The absorbance of the
solution prepared from the substance under examination is
not more than that ofthe reference solution (50 ppm).
acid, detennining the end-point potentiometrically (2.4.25).
CI3HzIN03.
Salbutamol Inhalation Aerosol; Albuterol Inhalation
Aerosol
salbutamol impurity A is not less than 3.0. The relative retention

time with reference to salbutamo1 for salbutamol impurity A is . Salbutamol Inhalation is a suspension ofmicrofine Salbutamol
about 1.3.
or Salbutamol Sulphate in a suitable liquid in a suitable
2084
IP 2010
SALBUTAMOL SULPHATE
pressurised container. It may contain suitable pharmaceutical Assay. Carry out the test for Content of active ingredient
aids such as surfactants, stabilising agents etc.
delivered per actuation stated under Inhalation Preparations
(Pressurised metered-dose Preparations).
Salbutamol Inhalation delivers not less than 80.0 per cent and
not more than 120.0 per cent of the sfated amount of Use 30 ml of ethanol for preparations containing salbutamol
salbutamol, C 13H21 N0 3, per inhalation, by actuation ofthe valve. and 30 ml ofa mixture ofequal volumes of ethanol and water
for preparations containing salbutamol sulphate and dilute
Usual strength. 100 Jlg in each metered-dose.
the final solution and washings to 200.0 ml with ethanol. Dilute
a suitable volume of this solution with ethanol to produce a
Identification
solution containing 10 Jlgofsalbutamolperml. To 20ml ofthe
A. Discharge the container a sufficient number oftimes into a solution in a separating funnel add 180 ml of water and in the
mortar to obtain about 2 mg of salbutamol, grind the residue following order, 4 ml of N,N-dimethyl-4-phenylenediamine
thoroughly with 0.1 g of potassium bromide, add a further sulphate solution and 4 ml of a freshly prepared 8 per cent
w/v solution of potassium ferricyanide. Mix, allow to stand
0.2 g ofpotassium bromide and mix thoroughly.
for 15 minutes in subdued light and extract with two quantities,
On the resultant dispersion determine by infrared absorption each of 10 ml, of chloroform. Filter the extracts through a plug
spectrophotometry (2.4.6). Compare the spectrum with that of cotton wool, dilute to 25 ml with chloroform and measure
obtained with salbutamol RS or with the reference spectrum the absorbance of the resulting solution at 605 nm (2.4.7).
of salbutamol.
Calculate the content of C 13H 21 N0 3 in the solution from the
B. In the test for Related substances, the principal spot in the absorbance obtained by repeating the operation using a
chromatogram obtained with reference solution (a) suitable quantity ofa 0.001 per cent w/v solution ofsalbutamol
corresponds to that in the chromatogram obtained with RS in ethanol.
Calculate the amount OfCl3H21N03 delivered per actuation of

the valve.
Tests

30 volumes of 2-propanol, 16 volumes of water and 4 volumes
Test solution. Discharge the inhaler a sufficient number of
times into a small, dry beaker to obtain 10 mg of Salbutamol
and dissolve the residue in 0.5 ml of methanol.
Reference solution (aJ. Dilute 1.0 volume of test solution (a)
to 200 volumes with methanol.
Reference solution (bJ. A 0.010 per cent w/v solution of
salbutamol RS in methanol.
dry the plate in air until the odour of the solvent is no longer
detectable, place it for a few minutes in an atmosphere saturated
with diethylamine and spray with diazotised sulphanilic acid
any spot with an R r value higher than 0.85.
Other tests. Complies with the tests stated under Inhalation
Preparations (Pressurised metered-dose Preparations).
Follow the procedure described under Assay wherever the
amount of active substance is to be determined in any test.
Determine the content of active ingredient a second and third

time by repeating the procedure on the middle ten and on the
last ten successive combined actuations of the valve. For
each of the three determinations the average content of
Cl3H21N03 delivered per actuation of the valve meets the
requirements.
Labelling. The label states whether the preparation contains
Salbutamol or Salbutamol Sulphate.
When the active ingredient is Salbutamol Sulphate, the
quantity is stated in terms of the equivalent amount of
salbutamol.
Salbutamol Sulphate
Albuterol Sulphate
(CI3H21N03)2,H2S04
Mol. Wt. 576.7
Salbutamol Sulphate is (RS)-1-(4-hydroxy-3-hydroxymethylphenyl)-2-(tert-butylamino)ethanol sulphate.

Salbutamol Sulphate contains not less than 98.0 per cent and
not more than 101.0 per cent of(C 13H 21 N03)2, H 2S04, calculated
on the dried basis.
2085
SALBUTAMOLSULPHATE
IP 2010
Dose. Orally, the equivalent of6 to 16 mg ofsalbutamol daily,

in divided doses; by slow intravenous injection, the equivalent
of 250 Ilg of salbutamol or by intravenous infusion, the
equivalent of 3 to 20 Ilg of salbutamol per minute. (1 mg of
Salbutamol Sulphate is approximately equivalent to 830 Ilg of
salbutamol).
Identification
Test A may be omitted if tests B, C, D andE are carried out.
Tests Band C may be omitted if tests A, D and E are carried
out.
Compare the spectrum with that obtained with salbutamol
sulphate RS or with the reference spectrum of salbutamol
sulphate.
B. When examined in the range 230 Jim to 360 nm (2.4.7), a

an absorption maximum at about 276 run; absorbance at about
276 urn, 0.44 to 0.51.
D. Dissolve 10 mg in 50 ml of a 2 per cent w/v solution of
borax, add 1 ml of a 3 per cent w/v solution of 4-aminophenazone, lO ml of a 2 per cent w/v solution of potassium
ferricyanide and 10 ml of chloroform, shake and allow to
separate; an orange-red colour is produced in the chloroform
layer.
E. Gives reaction A ofsulphates (2.3.1).
Tests
Acidity or alkalinity. To 10 ml ofa 1.0 per cent w/v solution in
carbon dioxide-free water add 0.15 ml of methyl red solution

and 0.2 ml of 0.01 M sodium hydroxide. The solution is yellow
and not more than 0.4 ml of 0.01 M hydrochloric acid is
required to change the colour of the solution to red.
(2.4.14).

examination in 50.0 ml ofthe mobile phase..
R~ler~nce solution. Asolution contalnin.g()~02perceniw!v
each
of salbutamol RS and
(lRS)-2-[(l,Idimethylethyl)amino}-1-(4- hydroxyphenyl)ethanol RS
(salbutamol impurity A RS) in the mobile phase. Dilute 2.0 ml

of this solution to 100.0 ml with the mobile phase.
endcapped octylsilanebonded to porous silica (5 Ilm),
resolution between the peaks due to due to salbutamol and
salbutamol impurity Ais not less than 3.0. The relative retention
time with reference to salbutamol forsalbutamol impurity A is
about 1.3.
Inject the test solution and the reference. solution. Run the
is not more than 3.3 times the area ofthe principal peak in the
Boron. To 50 mg add 5 m1 ofa solution containing 1.3 per cent
w/v of anhydrous sodium carbonate and 1.7 per cent w/v of
potassium carbonate, evaporate to dryness on a water-bath
and dry at 120. Ignite the residue rapidly until the organic
matter has been destroyed, allow to cool, add 0.5 mlofwater
and 3.0 ml ofa freshly prepared 0.125 percentw/v solution of
curcumin in glacial acetic acid. Evaporate to dryness and
allow to cool. Add 3 m1 ofa mixture prepared by adding 5m1 of
sulphuric acid, slowly and with stirring, to 5 ml of glacial
acetic acid. Mix and allow to stand for 30 minutes. Add
sufficient ethanol (95 per cent) to produce 100.0 ml, filter and
measure the absorbance ofthe filtrate at 555 run (2.4.7). Prepare
a reference solution in the following manner. Dissolve 0.572 g
of boric acid in 1000.0 ml of water. Dilute 1.0 ml to 100.0 ml
with water. To 2.5 ml of this solution add 5 m1 of a solution
containing 1.3 per cent w/v of anhydrous sodium carbonate
and 1.7 per cent w/vofpotassium carbonate and treat this
mixture in the same manner as described above beginning at
the words" Evaponite to dryness::...".The absorbance onhe
solution prepared from the substance under examination is
not more than that of the reference solution (50 ppm).
Sulphated ash (2.3.18). Not more than O.lper cent.
2086
IP 2010
SALBUTAMOL INJECTION
anhydrousformic acid, add 35 ml of anhydrous glacial acetic
acid. Titrate with 0.1 M perchloric acid, determining the endpoint potentiometrically (2.4.25). Carry out a blank titration.
(CI3H2IN03)2, H2S04
detectable, place for a few minutes in an atmosphere saturated

with diethylamine and spray with diazotised nitroaniline
C. Dilute a volume containing 0.5 mg of salbutamol to 50 ml
with water, add 1 ml of dilute ammonia solution, 1 ml ofa 3 per
cent w/v solution of 4-aminophenazone, 10 ml of a 2 per cent
w/v solution of potassium ferricyanide and 10 ml of
chloroform. Shake and allow to separate; an orange-red colour
is produced in the chloroform layer.
Salbutamol Injection
D. A volume containing 1 mg ofsalbutamol gives the reactions

ofsulphates (2.3.1).
Albuterol Sulphate Injection; Salbutamol Sulphate

Injection
Tests
pH (2.4.24).3.4 to 5.0.
Salbutamol Injection is a sterile solution of Salbutamol

Sulphate in Water for Injections containing suitable stabilising
agents.
Salbutamol Injection contains not less than 90.0 per cent and
salbutamol, C 13 H2I N0 3.
Usual strengths. The equivalent of250 Ilg of salbutamol per
rnl; the equivalent of500 Ilg ofsalbutamol pennI; the equivalent

of5 mg ofsalbutamol in 5 ml (for intravenous infusion). (l mg
ofSalbutamol Sulphate is approximately equivalent to 830 Ilg
ofsalbutamol).
Identification
A. Dilute a volume with sufficient 0.1 M hydrochloric acid to
produce a solution containing 0.008 per cent w/v ofsalbutamol.
When examined in the range 230nm to 360 nm (2.4.7), the
resulting solution shows an absorption maximum only at about
276nm.

Test solution. Evaporatea suitable volume ofthe injection to
dryness using a rotary evaporator, wash the residue with four
quantities, each of5 ml, of ethanol, filter, evaporate the filtrate
to. dryness and dissolve the residue in sufficient water to
produce a solution containing the equivalent of 0.1 per cent
w/v of salbutamol.
Reference solution. A 0.12 per cent w/v solution of salbutamol
sulphate RS in water..

(2.4.14).
Test solution. Dilute a volume of injection containing about 5

mg ofSalbutamol in 100.0 ml ofthe mobile phase.
w/v of (lRS)-2-[(l,1-dimethylethyl)amino]-1-(4hydroxyphenyl)ethanol RS (salbutamol impurity A RS) and
0.0005 per cent w/v of salbutamol sulphate RSin the mobile
phase.
endcapped octylsilane bonded to porous silica (5 Ilm)
(such as Waters Symmetry C8),
3.0.
solution (a) (0.5 per cent) and the sum of all the secondary
peaks is not more than twice the area of the principal peak in
2087
SALBUTAMOL SYRUP
IP 2010
the chromatogram obtained with reference solution (a) (2.0

per cent).
Assay. Dilute a volume, accurately measured, containing about
0.15 mg ofsalbutamol with sufficient water to produce 80 ml,
add 4 ml of a 5 per cent w/v solution ofsodium bicarbonate,
4 ml of N,N-dimethyl-4-phenylenedial~inesulphate solution
and 4 ml of a freshly prepared 8 per cent w/v solution of
potassiumferricyanide. Mix, allow to stand for 15 minutes,
protected from light. Extract with two quantities, each of 10 ml,
of chloroform. Filter the extracts through a plug of cotton
wool and dilute to 25.0 ml with chloroform. Measure the
absorbance of the resulting solution at 605 run (2.4.7).
Calculate the content of C 13 H Z1 N03 from the absorbance
obtained by repeating the operation using 10.0 ml of a
0.0018 per cent w/v solution of salbutamol sulphate.

4 mg of salbutamol add 25 ml of 0.05 M sulphuric acid and
extract with two quantities, each of50 ml, of ether. Collect the
aqueous layers into a 250-ml volumetric flask and combine the
ether extracts. Wash the combined ether extracts with 50 ml of
water and add the aqueous layer to the solution in the 250 ml
volumetric flask. Discard the ether extracts and dilute the
aqueous solution with sufficient water to produce 250.0 ml.
To 10.0 ml of this solution add sufficient water to produce
80 ml and add 4 ml of a 5 per cent w/v solution of sodium
bicarbonate, 4 ml of N,N-dimethyl-4-phenylenediamine
sulphate solution and 4 ml of a freshly prepared 8 per cent
w/v solution of potassium ferricyanide. Mix, allow to stand
for 15 minutes, protected from light. Extract with two quantities,
each of 10 ml, of chloroform. Filter the extracts through a plug
ofcotton wool and dilute to 25.0 ml with chloroform. Measure
the absorbance ofthe resulting solution at 605 run (2.4.7).
Calculate the content of C 13 H Z1 N0 3 from the absorbance
obtained by repeating the operation using 10.0 ml of a
0.0018 per cent w/v solution of salbutamol sulphate.
Storage. Store protected from light, in single dose containers

in which the air has been displaced by nitrogen or other
suitable inert gas.

equivalent amount of salbutamol in a suitable dose-volume.

equivalent amount of salbutamol in a suitable dose-volume.
Salbutamol Syrup
Salbutamol Tablets
Albuterol Sulphate Syrup; Salbutamol Sulphate Syrup
Albuterol Sulphate Tablets; Salbutamol Sulphate Tablets
Salbutamol Syrup contains Salbutamol Sulphate equivalent

of the stated amount of salbutamol, C 13HZ1 N03
Usual strength. The equivalent of2 mg ofsalbutamol in 5 ml.
(1 mg of Salbutamol Sulphate is approximately equivalent to
830 f.!g ofsalbutamol).
Salbutamol Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent ofthe stated amount of salbutamol,
C13Hz IN03
Usual strengths. The equivalent of2 mg; 4 mg ofsalbutamol.
(1 mg of Salbutamol Sulphate is approximately equivalent to
830 f.!g ofsalbutamol).
Identification
Identification
A. To 5 ml add 50 ml ofa 2 per cent w/v solution of borax, 1 ml
of a 3 per cent w/v solution of 4-aminophenazone, 10 ml of a
2 per cent w/v solution ofpotassiumferricyanide and 10 ml of
chloroform. Shake and allow to separate; an orange-red colour
is produced in the chloroform layer.
B. To 5 ml add sufficient 1 M sodium hydroxide to make the

solution alkaline, add 1 ml of alkaline borate buffer pH 9.2
and 1 ml ofa 0.04 per cent w/v solution of2, 6-dichloroquinone
chlorimide in ethanol (95 per cent); a blue colour develops.
Tests
A. Carry out the method described under Related substances

applying separately to the plate 2 f.!l of each ofthe following
solutions. For the test solution shake a quantity of the
powdered tablets containing 10 mg of salbutamol with 10 ml
of methanol (80 per cent) and filter. The reference solution
containS 0.12 per cent 'ii/v ofSalbutamol sulphate RS The
ofsalbutalIlol witll50 1Il1 ofa 2per cent
solution oflJortix,
add 1 m1 of a 3 per cent w/v solution of 4-aminophenazone,
10 ml of a 2 per cent w/v solution of potassium ferricyanide
vv!v
pH (2.4.24).3.4 to 4.5.
2088
SALICYLIC ACID
IP 2010
and 10 ml of chloroform. Shake and allow to separate; an

orange-red colour is produced in the chloroform layer.
C. Shake a quantity of the powdered tablets containing 4 mg
of salbutamol with 10 ml of water and filter; the filtrate gives
the reactions of sulphates (2.3.1).
Tests

injection volume. 20 /-Ll.
The test is not valid unless resolution between two principal
peaks in the chromatogram obtained with reference solution
(b) is at least 1.5.
Calculate the content ofC,3H2IN03 in the tablet.


containing 10 mg of salbutamol with 1 ml of water for
15 minutes, centrifuge and use the supernatant liquid.
salbutamol sulphate RS in water.
Apply to the plate 20 /-Ll of each solution. After development,
detectable, place it for a few minutes in an atmosphere saturated
with diethylamine and spray with diazotised sulphanilic acid
chromatogram obtained with the reference solution. Ignore
any pink spot near the line of application.
Tablets.

Assay. Detelmine by liquid chromatography (2.4.14).
Test solution. Shake 10 tablets or a sufficient number oftablets

containing 5.0 mg ofsalbutamol with about 60 ml of water for
1 hour, add sufficient water to produce 250.0 ml, mix and
centrifuge 10 ml ofthe mixture and use the supernatant liquid.
Reference solution (a). A 0.0024 per cent wlv of salbutamol
sulphate RS in water..
wlv of 2-tert-butylamino-I-(4-hydroxy-3-methylphenyl)
ethanol sulphate RS and 0.0024 per cent wlv of salbutamol
sulphate RS in methanol (10 per cent).
Follow the chromatographic procedure described under
Uniformity ofcontent. The test is not valid unless the resolution
between the two principal peaks in the chromatogram obtained
with reference solution (b) is at least 1.5.
Calculate the content ofC 13 H 2I N0 3in the tablets.

equivalent amount of salbutamol.
Test solution. Add 50 ml of water to one tablet, shake for

1 hour, add sufficient water to produce 100.0 ml, mix and
centrifuge. Dilute further with water, ifnecessary, to produce
a solution containing 0.002 per cent wlv of salbutamol.
Salicylic Acid
Reference solution (a). A 0.0024 per cent. wlv of salbutamol

Reference solution (b). A 0.048 per cent wlv of 2-tertbutylamino-l-(4-hydroxy-3-methylphenyl) ethanol sulphate
RS and 0.048 per cent wlv of salbutamol sulphate RS in
methanol (10 per cent).
spherical particles ofsilica, 5 /-Lm in diameter, the surface
of which has been modified with chemically-bonded
nitrile groups (such as Spherisorb CN),
30 volumes of 0.05 M ammonium acetate and 5 volumes
of 2-propanol, the pH of the mixture being adjusted to
4.5 with glacial acetic acid,
COOH
~OH
V
C7H60 3
Mol. Wt. 138.1
Salicylic Acid is 2-hydroxybenzoic acid.

Salicylic Acid contains not less than 99.0 per cent and not
more than 100.5 per cent of C7H60 3, calculated on the dried
basis.
Category. Keratolytic.
Description. White or colourless, acicular crystals or a white,
crystalline powder.
2089
SALICYLIC ACID
IP 2010
Identification

OH
HO~N~o~
OH
~COOH
VV

Compare the spectmm with that obtained with salicylic acid
RS or with the reference spectIUm of salicylic acid.
C2sH37N04, C II H s0 3
B. Dissolve about 30 mg in 5 ml of 0.05 M sodium hydroxide,

neutralise if necessary and dilute to 20 ml with water. 1 ml of
the solution gives reaction A of salicylates (2.3.1).
Salmeterol Xinafoate is (RS)-4-hydroxy-d-[[[6-(4-phenylbutoxy)hexyl]amino]methyl]-1 ,3-benzenedimethanol

I-hydroxy-2-naphthoate.
C.Meltingpoint.158to 161(2.4.21).
Salmeterol Xinafoate contains not less than 97.0 per cent and
not more than 102.0 per cent ofC36H4sN07, calculated on the
anhydrous basis.
Tests
Appearance of solution, Dissolve 1.0 gin 10 ml of ethanol
(95 per cent). The resulting solution is clear, and colourless
(2.4.1).
Heavy metals (2.3.13). Dissolve 2.0 g in 15 ml of ethanol
(95 per cent) and add 5 ml of water. 12 ml of the resulting
D (20 ppm). Use lead standard solution (100 ppm Ph) diluted
with a mixture of 3 volumes of ethanol (95 per cent) and
1 volume of water to contain 2 Ilg ofPb per ml to prepare the
standard.
Iron (2.3.14). Boil 12.0 g with 14 rnl of dilute ammonia solution
and 35 ml of water. Cool and adjust the pH 5.0 to 6.0 by the
dropwise addition of dilute ammonia solution or dilute
sulphuric acid and dilute to 50 ml with water, if necessary.
Any pink colour produced is not more intense than that
obtained by boiling 2.0 g with 1 ml of iron standard solution
(20 ppm Fe), 2 ml of dilute ammonia solution and 45 ml of
water, adjusting the pH 5.0 to 6.0 and diluting to 50 ml with
water (2 ppm).
.
Chlorides (2.3.12). Dissolve 5.0 g in 50 ml ofboiling distilled
water, cool and filter (solution A). 20 ml ofsolution A complies
Sulphates (2.3.17). 7.5 ml ofsolution A complies with the limit
on 2.0 g.
on 1.0 g by drying over phosphorus pentoxide in a desiccator.
ethanol (95 per cent), add 20 ml of water and titrate with
0.1 M sodium hydroxide, using phenol red solution as
indicator, until a reddish violet colour is obtained.
HO)lJ
Mol. Wt. 603.7
Category. Bronchodilator.
Identification
Compare the spectIUm with that obtained with salmeterol
xinafoate RS or with the reference spectIUm of salmeterol
xinafoate.
Tests
(2.4.14).

Reference solution. A 0.002 per cent w/v solution ofsalmeterol
xinafoate RS in the mobile phase.
- fl stainless steel column 25 cm x 4.6 mm, packed with
phosphate in 1000 ml of water and adjusting the pH to
7.0 with triethylamine and 40 volumes of acetonitrile,
- spectrophotometer set at 220 TIm,
- injectionvolume. 10 Ill.
Inject the test solution. Any individual impurity is not more
than 0.5 per cent and the sum of all the impurities found is not
1 ml of 0.1 M sodium hydroxide is equivalenttoO:01381 gaf

C7H 60 3
Heavy metals (2.3.13). 1 g complies with limit test for heavy


2090
IP 2010
SALMETEROL AND FLUTICASONE PROPIONATE POWDER FOR INHALATION

0.5g.
Reference solution (b). A solution containing 0.5 mg of

fluticasone propionate per ml prepared by dissolving 10 mg of
jluticasone
propionate RS in 10 ml acetonitrile and adding
sufficient ofthe mobile phase to produce 20 ml.
determining the end-point potentiometrically (2.4.25). Cany Reference solution (c). Dilute suitable volumes of reference
solution (a) and reference solution (b) with the mobile phase
to obtain a solution containing 5 Ilg Salmeterol and 50 Ilg
1 m1 of 0.1 M perc/doric acid is equivalent to 0.06038 g of
Fluticasone propionate per ml or quantities as per the label
C36ILsN07
claim.
octylsilyl silica gel (5 Ilm),
Salmeterol and Fluticasone Propionate
Inhalation
prepared by dissolving 1.15 g ammonium dihydrogen
orthophosphate to 1000 ml of water and adjusting the
Salmeterol and F1uticasone Propionate Inhalation is a
pH to 3.5 with orthophosphoric acid, 25 volumes of
suspension ofmicrofine Salmeterol Xinafoate and Fluticasone
acetonitrile
Propionate in a suitable liquid filled in a suitable pressurised
flow
rate.
2
ml
per minute,
container. It may contain suitable pharmaceutical aids such as
spectrophotometer
set at 220 nm,
surfactants, stabilizing agents.
- inject 200 Ill.
Salmeterol and Fluticasone Propionate Inhalation delivers not
less than 80.0 per cent and not more than 120.0 per cent ofthe Inject reference solution (c). The test is not valid unless the
stated amounts of salmeterol, C 2s H 37N0 4 and fluticasone column efficiency for salmeterol and fluticasone propionate
propionate, C2sH31F30SS, per inhalation by actuation of the peak is not less than 1000 and 2500 theoretical plates
respectively and the tailing factor is not more than 2.0 for each
valve.
peak and the relative standard deviation for replicate injections
for each component is not more than 2.0 per cent.
Identification
with test solution correspond to the peaks in the
chromatogram obtained with reference solution (c).
Tests
Other tests. Comply with the tests stated under Inhalation
Preparations (Pressurised Metered-dose Preparations).
Follow the procedure described under Assay with suitable
dilution of the reference solution wherever the amount of
active substance is to be determined in any test.
Assay. CaD')' out the test for Content of active ingredient
(Pressurised Metered-dose Preparations).
Inject the test solution and reference solution (c).

Calculate the contents of C2s H37N0 4 and C2sH31F30SS in the
solution and the contents of C2sH37N04 and C2sH31FjOsS
delivered per actuation of the valve.
Determine the contents of the active ingredients a second
and third time by repeating the procedure on the middle ten
and on the last ten successive combined actuations of the
valve. For each of the three determinations the average
contents ofC 2s H37N04and C2sH31F30SS delivered per actuation
of the valve meet the requirements.
exceeding 30.
Labelling. The label states the amounts of active ingredients
delivered per inhalation.
Test solution. Prepare using the mobile phase as described

under the test for Content of active ingredient delivered per
actuation stated under Inhalation Preparations (Pressurised
Metered-dose Preparations).

Powder for Inhalation
Reference solution (a). A solution containing 0.5mg of

salmeterol per ml prepared by dissolving 10 mg of salmeterol
xinafoate RS in 10 ml acetonitrile and adding sufficient ofthe
mobile phase to produce 20 ml.
Salmeterol and Fluticasone Propionate Powder for Inhalation

consists of Fluticasone Propionate and Salmeterol Xinafoate
in microfine powder either alone oradmixed with Lactose in a
pre-metered unit for use in a suitable powder inhaler.
2091
IP 2010
SALMETEROL AND FLUTICASONE PROPIONATE POWDER FOR INHALATION
Salmeterol and Fluticasone Propionate Powder for Inhalation

125.0 per cent ofthe stated amounts ofsalmeterol C2sH37N04
and fluticasone propionate, C2sH31F30SS per unit dose.
Identification
Inject reference solution(c). The test is not valid unless the

column efficiency determined from the salmeterol and
fluticasone propionate peak is not less than 1000 and 2500
theoretical plates respectively, the tailing factor for each of
salmeterol and fluticasone propionate peaks is not more than

with the test solution correspond to the peaks in the
chromatogram obtained with reference solution (c).
Calculate the contents ofC2sH37N04 and C 2s H 31 F 30SS per unit.
Tests
Storage. Store protected from moisture, at temperature not

exceeding 30.
Other tests. Complies with the tests stated under the Inhalation
Preparations (Powders for Inhalation).
Inject the test solution and reference solution (c).
Labelling. The label states the quantities ofactive ingredients

per pre-metered unit.
Saquinavir
Test solution. Dissolve a quantity of the mixed contents of

20 capsules in sufficient ofthe mobile phase to get a solution
containing 5 Ilg per ml ofSalmeterol.
Reference solution (a). A solution containing 0.5 mg of
salmeterol per ml prepared by dissolving 10 mg of salmeterol
xinafoate RS in 10 ml acetonitrile and adding sufficient ofthe
mobile phase to produce 20 ml.
Reference solution (b). A solution containing 0.5 mg of
fluticasone propionate per ml prepared by dissolving 10 mg
ofjluticasone propionate RS in 10 ml acetonitrile and adding
sufficient ofthe mobile phase to produce 20 ml.
Reference solution (c). Dilute suitable volumes of reference
solution (a) and reference solution (b) with the mobile phase
to obtain a solution containing 5 Ilg Salmeterol and 50 Ilg
Fluticasone propionate per ml or quantities as per the label
claim..
Mol. Wt. 670.8

Saquinavir is (S)-N-[( fJS)-a-{(IR)-2-[(3S,4aS,8aS)3-(tert-butylcarbamoyl)octahydro-2(1H)-isoquinolyl]1-hydroxyethyl}phenethyl]-2-quinaldamidosuccinamide.
Saquinavir contains not less than 98.5 per cent and not more
than 101.0 per cent ofC38HsoN60s, calculated on the anhydrous
basis.
Dose. 1 g twice daily.
- a stainless steel column 15 cm x 3.9 mm, packed with.
Description. A white crystalline powder.
octylsilyl silica gel (5 Ilm),
prepared by dissolving 1.15 g ammonium dihydrogen
orthophosphate to 1000 ml of water and adjusting the
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6),
Compare the spectrum with that obtained with saquinavir RS
or with the reference spectrum of saquinavir.
to saquinavir in the chromatogram obtained with reference
solution (a).
2092
IP 2010
SAQUINAVIR MESYLATE
C. When examined in the range 220 nm to 350 nm (2.4.7), a

maxima at about 239 nm and 290 nm.
Tests
(2.4.14), as described in the Assay using the test solution and
reference solution (c).
Inject reference solution (c). Calculate the amount of related
substance by area normalisation method. In the chromatogram
than the principal peak is not greater than the area of the
principal peak obtained with reference solution (c) (0.1 per
cent) and the sum of the areas of all such peaks is not greater
obtained with reference solution (c) (0.5 per cent).

about 0.89 for saquinavir-related compound A ami about 1.0
for saquinavir. The test is not valid unless the resolution
between the peaks due to saquinavir-related compound A
and saquinavir is not less than 1.5, the column efficiency
determined from the saquinavir peak is not less than 500
theoretical plates and the relative standard deviation for
Separately inject the test solution and reference solution (a).
Record the chromatograms up to three times the retention
time of the principal peak and measure the responses for the
principal peak.
Calculate the content of C3sHsoN60s.
Saquinavir Mesylate
Heavy metals (2.3.13). 2.0 g complies with the limit tests for
0.5g.

saquinavir RS in the mobile phase.
Reference solution (b). Dissolve suitable quantities of
saquinavir-related compound A RS and saquinavir RS in the
mobile phase to obtain a solution containing 2 /lg per ml of
saquinavir-related compound A and 0.25 mg per ml of
saquinavir.
C3sHsoN60s,C~03S
Mol. Wt. 767.0
Saquinavir mesylate is (S)-N-[( as)-Cf..-{ (IR)-2-[(3S,4aS,8aS)3-(tert-butylcarbamoyl)octahydro-2(1H)-isoquinolyl]I-hydroxyethyl}phenethyl]-2-quinaldamidosuccinamide

methanesulphonate.
Saquinavir Mesylate contains not less than 98.5 per cent and
not more than 101.0 per cent ofC3sHsoN60s, CH40 3S, calculated

saquinavir RS in the mobile phase.
mobile phase: a mixture of 20 volumes of methanol,
50 volumes of acetonitrile and 30 volumes of a buffer
prepared by dissolving 4 g of sodium dihydrogen
phosphate in 1000.0 ml of water to which I ml of
diethylamine and 1 g of sodium octane sulphonate has
been added,
Dose. 1 g twice daily.
Identification
Compare the spectrum with that obtained with saquinavir
mesylate RS or with the reference spectrum of saquinavir
mesylate.
to saquinavir mesylate in the chromatogram obtained with
2093
SAQUINAVIR MESYLATE
IP 2010
Tests
triethylamine with water to make 1000 ml and adjusting

the pH of the solution to 25 with phosphoric acid,
injection volume. 20 ).Ll.

in a 0.5 per cent w/v solution in methanol at 436 run.
(2.4.14), as described in the Assay using the test solution and
Inject reference solution (c). Calculate the amount of related
substances by area normalisation method. In the
peak other than the principal peak is not greater than the area
of the principal peak in the' chromatogram obtained with
reference solution (c) (0.1 per cent) and the sum ofthe areas of
all such peaks is not greater than 5 times the area of the
solution (c) (0.5 per cent).
Methanesulphonic acid. 11.9 to 13.1 per cent w/w, calculated
on the anhydrous basis, determined by the following method.
Weigh accurately about 0.1 g of the substance under
examination, dissolve in 50 ml of methanol. Titrate with
0.1 M sodium hydroxide, determining the end-point potentiometrically (2.4.25). Carry out a blank titration.
CH3S03H.
Water (2.3.43). Not more than 1.0percent,determinedon 0.5 g.

about 0.89 for saquinavir-related compound A and about 1.0
for saquinavir mesylate. The test is not valid unless the
resolution between the peaks due to saquinavir related
compound A and saquinavir mesylate is not less than 1.5, the
column efficiency determined from the saquinavir mesylate
peak is not less than 500 theoretical plates and the relative
2.0 per cent.
Record the chromatograms upto three times the retention time
of the principal peak and measure the responses for the
principal peak.
Calculate the content ofC3sHsoN60s, CH40 3S.
Saquinavir Mesylate Tablets

Saquinavir Mesylate Tablets contain not less than 90.0 per
saquinavir C3sHsoN60s.
NOTE -Pelform the tests and assay using low-actinic

glassware.


saquinavirmesylate RS in the mobile phase.
Identification
Reference solution (b). Dissolve suitable quantities of

saquinavir-related compound A RS and saquinavir mesylate
RS in the mobile phase to obtain a solution containing 2 ).Lg
per ml ofsaquinavir- related compound A and 0.25 mg per ml
of saquinavir mesylate.
saquinavir mesylate RS in the mobile phase.
A. In the Assay, the principal spot in the chromatogram

obtained with the test solution corresponds -to that in the
B. When examined in the range 200 nm to 400 run (2.4.7),1 ml
of a 0.1 per cent w/v solution in methanoi diluted to 100 ml
with citrate buffer pH 3.0 (see under Dissolution), shows
absorption maxima at the same wavelengths as shown by the
reference solution.
NOTE - Store the btif.fer solution protectedfrom light. Make

adjustments ifnecessaly for system suitability.
Tests
octadecylsi1ane bonded to porous silica (5 ).Lm),
mobile phase: a mix.ture of 25 volumes of
tetrahydrofuran, 5 volumes of acetonitrile and
17 volumes of a buffer prepared by mixing 10. ml of
Apparatus. No 1
Medium. 900 ml of citrate bufferpH3.0 prepared by dissolving
5.82 mg of anhydrous dibasic sodium phosphate and 16.7 mg
of citiric acid monohydrate in 1000 ml of water and adjusting
the pH to 3.0 with orthophosphoric acid.
2094
IP 2010
SECNIDAZOLE
Calculate the content of C3sHsoN60S in the tablet.
Withdraw a suitable volume ofthe medium and filter promptly.

Dilute the filtrate, ifnecessary, with the medium. Measure the
absorbance (2.4.7) ofthe resulting solution at the maximum at
about 240 nm. Calculate the content of C38HsoN60S in the
lmown concentration of saquinavir mesylate RS.

C3sHsoN60S.
(2.4.14).
Test solution. Weigh and powder 20 tablets Weigh accurately

a quantity of the powder containing 100 mg of saquinavir in
100 ml ofthe mobile phase and filter.
saquinavir mesylate RS in the mobile phase.
- mobile phase: a mixture of 14 volumes of triethylamine
phosphate solution prepared by diluting 10 ml of
triethylamine to 1000 ml with water and adjusting the
tetrahydrofuran and 1 volume of acetonitrile,
- injection volume: 20 ~Ll.
Inject the test solqtion and reference solution (b). Run the
chromatogram for 5 times the retention time (about 12 minutes)
of the principal peale. In the chromatogram obtained with the
test solution, the area of any secondary peak is not more than
0.2 times the area of the peak in the chromatogram obtained
with the reference solution (b) (0.2 per cent) and the sum of
areas of all the secondary peaks is not more than the area of

0.5 g.

a quantity of the powder containing 100 mg of saquinavir,
dissolve in 100.0 ml ofmobile phase and filter. Dilute 5.0ml of
the filtrate to 20.0 ml with the mobile phase.
Reference solution. A 0.1 per cent w/v solution of saquinavir
mesylate RS in the mobile phase. Dilute 5.0 ml ofthe solution
prepared by diluting 10.0 ml of triethylamine to 1000 ml
with water, adjusting the pH to 2.5 with orthophosphoric
acid and filtering, 5 volumes of tetrahydrofitran and
1 volume of acetonitrile.
Calculate the content of C3sHsoN60S in the tablets.
equivalent amount of saquinavir.
Secnidazole

Tablets.
under Assay.
Test solution. Disperse one tablet in 500 ml ofthe mobile phase

and filter. Dilute 5 ml of the filtrate to 20 ml with the mobile
. phase.
Mol. Wt. 185.2

Secnidazole is (RS)-I-(2-methyl-5-nitroimidazol-l-yl)propan-
2-01.
2095
SECNIDAZOLE
IP 2010
Secnidazole contains not less than 98.0 per cent and not more
than 101.0 per cent of C7HIIN303, calculated on the anhydrous
basis.
Category. Antiamoebic.
Dose. Adults - 2g; Children - 30 mg per kg as single dose.

chromatogram obtained with the reference solution (a)
(0.5 per cent). Disregard any peak which is 0.1 times the area
Description. A white to yellowish white, crystalline powder.

Identification

0.001 per centw/v solution in 0.1 Mhydrochloric acid shows
an absorption maximum at about 277 nm, 0.325 to 0.355.
B. Heat about 10 mg in a water-bath with 10 mg ofzincpowder,

1 ml of water and 0.25 ml of 2 M hydrochloric acid for
5 minutes and cool. The solution gives the reaction ofprimary
Tests
Appearance of solution. A 5 per cent w/v solution in
1 M hydrochloric acid is not more opalescent than
opalescence standard OS2 (2.4.1) and not more intensely
(2.4.14).

100.0 ml with mobile phase. Dilute 1.0 ml of the solution to
w/v each of 2-methyl-5-nitroimidazole RS and secnidazole
RS in the mobile phase. Dilute 1.0 ml of the solution to
65 volumes of 0.14 per cent w/v solution of potassium
Inject reference solution (b). Tlie test is not valid unless the
resolution between 2-methyl-4-nitroimidazole and secnidazole
chmmaj:ogram obtained with the test solution, the area ofany
secondary peak is not more than 2 times the area ofthe peak in
the chromatogram obtained with reference solution (a)

examination in 25.0 ml ofthe mobile phase. Dilute 5.0 ml ofthe
secnidazole RS in mobile phase.
65 volumes of 0.14 per cent w/v solution of potassium
Calculate the content of C7HIIN303.
Secnidazole Tablets
Secnidazole Tablets contain not less than 95.0 per cent and
secnidazole, C7HIIN303'
Usual strengths. 1 g; 2 g.
Identification
Tests
Apparatus. No.1,
2096
IP 2010
SERRATIOPEPTIDASE

Medium. 900 ml of 0.1 M hydrochloric acid.

deviation of replicate injections is not more than 2.0 per cent.

not greater than 1.0 Ilm, rejecting the first I ml of the filtrate.
about 277 urn (2.4.7). Calculate the content ofC7HIIN303 in the
known concentration of secnidazole RS in the same medium.
D. Not less than 80 per cent ofthe stated amount ofC7HIIN303'
Labelling. The label states the strength of secnidazole.

Calculate the content ofC7HIIN303.

(2.4.14).
Serratiopeptidase

containing 50 mg ofSecnidazole, disperse in 100 ml ofmobile
phase and filter.
Serratiopeptidase is a proteolytic enzyme for oral use which is

produced by bacteria of genus Serratia.

secnidazole RS in the mobile phase.
Serratiopeptidase contains not less than 2000 units and not

more than 2600 units of serratiopeptidase activity per mg.

Category. Prolytic enzyme.
Description. A grayish white to pale brown pow4er with

characteristic odour.
Identification

more than 2.0 times the area ofthe peak in the chromatogram

a quantity ofpowdered tablet containing 50 mg ofSecnidazole,
disperse in 100.0 ml ofmobile phase and filter.. Dilute 5.0 ml of
the filtrate to 50.0 ml with mobile phase.
secnidazole RS in mobile phase.
(5Ilm),
mobile phase: a mixture of 85 volumes of
0.01 M potassium dihydrogen orthophosphate and
Dissolve 0.4 g of serratiopeptidase in 100 ml of acetic acid

sodium acetate buffer solution pH5.0, transfer exactly 1 mlof
this solution into 3 test tubes, refer them as A, Band C. Add 1
ml of water in tube A. In tube Band C add 1 ml of 0.04 ml of
disodium edetate solution. Mix gently and allow them to stand
at 410, for 1 hour. Add 2 ml of water in tube A and C. Add 2 ml
of 0.04 M zinc chloride solution in tube B. Mix gently and
allow them to stand at41 0, for 1 hour. Pipette 1 ml ofA and B
and make up the volume to 200 ml with borate bufferpH9.0.
Pipette 1 ml ofC and make up the volume to 50 ml with borate
bufferpH 9. O. Proceed with these solutions as sample solutions
as directed in the assay. The activities of solution A and Bare
almost same and the activity ofC is not more than 5 per cent of
that of activity of A.
Tests
Arsenic (2.3.10). Dissolve 0.4 g in 50 ml of water, and add 10
ml of stannated hydrochloric acid. The resulting solution
heavy metals, MethodB (50 ppm).
Sulphated Ash (2.3 .18). Not more than 1.5 per cent.
on 1 g by drying in an Qven at 105 for 4 hours.
2097
SERRATIOPEPTIDASE
IP 2010
Microbial contamination(2.2.9). Total viable aerobic count,

not more than 100 micro-organisms perml,determined by plate
count. 1 ml is free from Escherichia coli.
Assay. Unit defmition: one serratiopeptidase unit is defined
as the amount of enzyme required to liberate 1 I!m of free
tyrosine per minute under the specified assay conditions.
sodium borate hydrochloric acid buffer pH 9. O. Dissolve 19

gsodium borate in 900.ml of water. Adjust the pH 9.0 with 1
M hydrochloric acid and dilute with water to 1000 ml.
Substrate solution. Dissolve 1.2 g dried casein in 100 ml of
sodium borate hydrochloric acid buffer pH 9.0. Keep it on
boiling water-bath for 1-2 minutes to get clear solution. Cool
and filter through cotton and dilute with sodium borate
hydrochloric acid buffer pH 9. a to 200 ml.
Protein precipitating solution. To 18 g of trichloroacetic acid,
add 30 g of sodium acetate and 20 ml of glacial acetic acid,
and dilute with water to 100 ml.
Dilute folin s reagent. 1 ml of folin s reagent, add 2 ml of

water.
Reference tyrosine solution. Dissolve 10 mg of tyrosine in 1
ml of 1 M hydrochloric ac:id and dilute to 100 ml with sodium
borate hydrochloric acid buffer pH 9.0.
Reference tyrosine curve. To 1, 2, 3, 4 and 5 ml of reference
tyrosine solution, add 5, 4, 3, 2 and 1 ml of sodiul1~ borate
hydrochloric acid buffer pH 9.0 respectively. Add 5 ml of
protein precipitating reagent in each tube. To 2 ml of these
solutions, add 5 ml of sodium carbonate solution. Add 1 ml of
dilutedfolins reagent mix and allow to stand at 37 for 30
minutes, measure the absorbance at 660 nm (2.4.7). Plot a graph
of I!m of tyrosine per system against the absorbance.
Stock test solution. Weigh about 0.1 g ofthe substance under
examination, dissolve in 100 ml ofsodium borate hydrochloric
acid bufferpH 9. a(solution A). Mix and keep it for 5 minutes.
Take 1 ml ofsolution A and dilute to 200 ml with sodium borate
hydrochloric acid buffer pH 9.0 (solution B).
Test solution. Pipet 1.0 ml of stock test solution, allow to
stand in a water-bath at 37 for 5 minutes. Add 5 mlofsubstrate
solution shake immediately and allow to stand in water-bath
at 37 for 20 minutes. Add 5 ml of protein precipitating
solution. Mix and allow to stand in water-bath at 37 for 30
minutes and filter. Take 2 ml ofthe filtrate, add 5 ml of 0.6 per
cent w/v sodium carbonate solution. Add 1 ml of diluted
folin s reagent mix and allow to stand at 37 for 30 minutes.
Measure the absorbance at 660 nm (2.4.7).
Blank solution. Pipet 1.0 ml of stock test solution, allow to
stand in a 'Yater~bathat3rfor5 ll1inutes, add5 1l1l ofpr:ot~il1
precipitating solution, shake immediately and allow to stand
in water-bath at 37 for 20 minutes. Add 5 ml of substrate
solution. Mix and allow to stand in water-bath at 37 for 30
minutes and filter. Take 2 ml ofthe filtrate, add 50 ml of0.6 per

cent w/v sodium carbonate solution. Add 1 ml of diluted
folin s reagent mix and allow stand at 37 for 30 minutes.
Measure the absorbance at 660 mn (2.4.7).
Calculate the serratiopeptidase IV/mg by using concentration
of tyrosine from graph considering the reaction time and
dilution.
Storage. Store protected from light and moisture, at temperature
not exceeding 30.
Serratiopeptidase Tablets contain not less than 90.0 per cent
of the stated amount of Serratiopeptidase.
Tests
Assay. The tyrosine units are dissolved in sodium carbonate
solution, which is alkaline in nature. The Folin s ciocalteau
reagent helps in colour development where the tyrosine units
bind to the copper molecule in the reagent and causes the
reduction of phosphomolybdate which is present in the
reagent. There is formation of tyrosine-copper molybdate
coloured complex. The intensity of colour depends upon the
tyrosine units present which is read at 660 nm.
Disodium tetraborate buffer. Dissolve 19.0 gm of disodium

tetraborate in 900 ml of water, adjusted to pH 9.0 with 1 M
hydrochloric acid and dilute to 1000 ml with water.
Casein hammerstein substrate. Dissolve 1.2 gm of Casein
Hammerstein to 100 ml ofthe sodium tetraborate buffer. Allow
the casein to dissolve and form homogeneous solution. Boil
the solution in boiling water-bath for 2 minutes. After removing
from boiling water-bath, cool the casein substrate immediately
in ice cold water. Filter this solution through cotton plug to
get clear solution and dilute to 200 ml with water.
NOTE-Always add casein to the buffer while stirring. Do
not add buffer to casein as this will cause lumps and not go
into solution completely and giving incorrect values.
Trichloroacetic acid reagent. Dissolve 18 gm of
trichloroacetic Acid, 30 gm of anhydrous sodium acetate
and 20 ml of glacial acetic acid in 1000 ml of water.
NOTE-TCA should be in the crystal form and not in liquid
form as this will effect the quantity weighed and thereby
interfere with the complete precipitation of casein. Use
Sodium acetate anhydrous because if, sodium acetate
trihydrate should be used, the trihydrate forms gives lower
results.
2098
IP 2010
COLLOIDAL SILICON DIOXIDE
Diluted Folin 's Ciocalteau Reagent. Dilute 1 ml of Fo1in's

Ciocalteau reagent with 2 ml of water to get 3 ml ofthe reagent.
NOTE-Folin's Ciocalteau reagent should be ofthe protein
estimation grade and not the indicator grade. Folin 's reagent
. should be diluted to just before addition to the tubes since it
is sensitive to light. Do not prepare this solution at the
beginning ofthe assay.
Tyrosine reference solution. Dissolve 160 mg of tyrosine in
100 ml of 0.2 M hydrochloric acid.
1-
Serratiopeptidase Units/Tablets = AI - A 2 x 176 x

x
A 3 -A 4
20
100
SpI.Wt.(g)
L.A.
Where, 176
Conversion Co-efficient of Tyrosine to

Serratiopeptidase
20
Reaction time [minutes]
Absorbance of test solution
AI
Absorbance of blank solution
A2
Absorbance of tyrosine reference solution.
A3
A4
Absorbance ofO.2M hydrochloric acid
L.A. = 20000 Units/tablet.
Trisodium phosphate buffer pH 6.8. Weigh 19 g of trisodium

phosphate and 6.4 ml of hydrochloric acid in 1000 ml of water,
adjusted to pH 6.8.
For each sample, keep four test tubes. Mark two test tubes as
TEST and two as BLANK. Add 5 ml ofCasein Su,bstrate to the
tubes marked as TEST and 5 ml. ofTCA to the test tube marked
as BLANK. Prewarm these tubes for five minutes along with
the tubes containing the sample dilutions. Add 1 ml ofenzyme
solution to all these tubes and vertex the tubes for exactly 10
seconds. Keep the test tubes in the water-bath at 37 for 20
minutes for the reaction. After exactly 20 minutes, add 5 ml of
TCA to tubes marked as TEST and 5 ml ofCasein substrate to
the tube marked as BLANK. Vertex all the tubes exactly for 30
seconds. Keep the tubes in the water-bath at 37 for 30 minutes
for precipitation, filter.
25
% of LA = Serratiopeptidase Units/Tablet x 100
Dilute 1 ml of this solution to 100 ml with 0.2 Mhydrochloric

acid. Use 2 ml ofthis solution for colour development.

ofpowder containing about 120 mg ofSerratiopeptidase with
100 ml of trisodium phosphate btifferpH 6.8. Stir on magnetic
stirrer for 1 hour. Further dilute 5.0 ml to 25 ml with 5.0percent
ammonium sulphate solution, stir for 5 minutes and filter.
Dilute 2 ml of the filtrate to 100 ml with sodium tetraborate
btiffer. 1 ml of resulting solution is used for arialysis. Set the
water bath to 37.
100
- - - - x - x - x Avg.wt.mgm

exceeding 25.
Labeling. The label states the strength in terms of the
equivalent fo Enzyme activity units of Serratiopeptidase.
Colloidal Silicon Dioxide

ColloidalAnhydrous Silica
Si02
. Mol. Wt. 60.1
Colloidal Silicon Dioxide is a submicroscopic finned silica

prepared by the vapour-phase hydrolysis of a silicon
compound.
Colloidal Silicon Dioxide contains not less than 99.0 per cent
and not more than 100.5 per cent of Si02 , calculated on the
ignite basis.
Category. Pharmaceutical aid (tablet excipient).
NOTE- For tablets, the settlement may take a longer time

than 30 minutes. So filter the solution after the settlement is
clearly seen (Vertex in between for better precipitation).
Description. A light, fine, white, amorphous powder. It has a

particle size of about 15 nm.
Mark tubes as TEST, BLANK, TYROSINE and TYROSINE

BLANK.
Identification
About 20 mg gives the reaction of silicates (2.3.1).
Add the following respectively;

TEST - 2 m1 ofthe TEST filtrate.
Tests
BLANK- 2 ml ofBLANK filtrate.
pH (2.4.24).3.5 to 5.5, determined in a suspension of 1.0 g in

30 ml of carbon dioxide-free water.
TYROSINE - 2 ml ofTYROSINE standard solution.

TYROSINE BLANK- 0.2 M hydrochloric acid.
Add 5 ml of 6 per cent sodium carbonate solution to all tubes.
Add 1 ml of diluted Folin's reagent to all the tubes. Keep the
tubes at 37 for 30 minutes for colour development. Measure
the absorbance at about 660 nm (2.4.7).
Arsenic (2.3.10). To 2.5 g contained in a round-bottomed flask

add 50 ml of 3 M hydrochloric acid and heat under a reflux
condenser for 30 minutes. Cool, filter with the aid ofsuction
and transfer the filtrate to a 1OO-ml volumetric flask. Wash the
filter with several portions of hot water and add the washings
to the volumetric flask. Cool, dilute to volume with water and
2099
COLLOIDAL SILICON DIOXIDE
IF 2010
mix. To 50.0 ml ofthe solution add 3 ml of hydrochloric acid;

the resulting solution complies with the limit test for arsenic
(8 ppm).
Sildenafil Citrate is 5-[2-ethoxy-5-(4methylpiperazinylsulphonyl)phenyl]-I-methyl-3-n-propyl1,6-dihydro-7H-pyrazolo[4,3-dJpyrirnidin-7-one citrate.
Heavy metals (2.3.13). Suspend 2.5 g in sufficient water to

produce a semi-fluid slurry and dry at 140. When the dried
substance is white, break up the mass using a glass rod, add
25 ml of 1 M hydrochloric acid, boil gently for 5 minutes,
stirring frequently with the glass rod, centrifuge for 20 minutes
and filter the supernatant liquid through a membrane filter. To
the residue in the centrifuge tube add 3 ml of 2 M hydrochloric
acid and 9 ml of water, boil, centrifuge for 20 minutes and filter
the supernatant liquid through the same membrane filter. Wash
the residue with small quantities of watel; combine the filtrates
and washings and dilute to 50.0 ml with water. To 20.0 ml of
the solution add 50 mg of L-ascorbic acid and 1 ml of strong
ammonia solution, neutralise with 2 M ammonia and dilute to
25 ml with water. 12 ml ofthe solution complies with the limit
test for heavy metals, Method D (25 ppm). Use lead standard
solution (1 ppm Pb) to prepare the standard.
Sildenafil Citrate contains not less than 98.0 per cent and not
more than 102.0 per cent ofCzsH3sN601lS, calculated on the
dried basis.
Chlorides (2.3.12). To 1.0 g add a mixture of20 ml of 2 Mnitric

acid and 30 ml of water, heat on a water-bath for 15 minutes,
shaking frequently, dilute to 50 ml with water if necessary,
filter and cool. The filtrate complies with the limit test for
Loss on ignition (2.4.20). Not more than 5.0 per cent,
determined on 0.2 g by igniting at 900 in a platinum crucible
for 2 hours.
Assay. To the residue obtained in the test for Loss on ignition
add 0.2 ml of sulphuric acid and sufficient ethanol (95 per
cent) to moisten the residue completely, add 6 ml of
hydrofluoriC acid and evaporate to dryness on a hot plate at
95 to 105, avoiding loss from sputtering. Wash the sides of
the dish with 6 ml of hydrofluoric acid, evaporate to dryness
in a well-ventilated hood, ignite at 1000, allow to cool in a
desiccator and weigh. The difference between the weight of
the final residue and that ofthe residue obtained in the test for
Loss on ignition represents the amount of SiOz in the amount
of the substance taken for the test for Loss on ignition.
Sildenafil Citrate
o
(COOH
HO-C-COOH
t cooH
Mol. Wt. 666.7
Category. Used in erectyle dysfunction.

Description. A white to offwhite powder.
Dose. 50 mg per day.
Identification
Compare the spectrum that obtained with sildenafil citrate
RS or with the reference spectrum of sildenafil citrate.
B. Complies with the test for Citrate (2.3.1).
Tests
Citric acid. 28.0 per cent to 34.0 per cent.
Accurately weigh about 0.2 g of the substance under
examination and transfer into a 250-ml conical flask, add 50 ml
of methanol and sonicate to dissolve, then add 50 ml of water.
Titrate with 0.1 M sodium hydroxide solution using
phenolphthalein indicator until the colour changes from
colourless to pink.
C6Hs0 7

(2.4.14).
NOTE~Use
freshly prepared solution.

acetonitrile.
sildenafil citrate RS in the solvent mixture.
to 100.0 ml with the solvent mixture. Further dilute 3.0 ml of
this solution to 100.0 ml with the solvent mixture.
octadecylsilane bonded to porous silica (5 /-lm) (such as
Hypersil ODS C18),
- mobile phase: A. dissolve 3.85 g of ammonium acetate
in 1000 ml of water, adjusted to pH 7.5 with ammonia
solution,
B. acetonitrile,
below,
2100
IP 2010
SILDENAFIL TABLETS

prepared by dissolving 3.85 g of ammonium acetate in
1000 ml of water, adjusted to pH 7.5 with ammonia

Time
(in min.)
Mobile phase A
(per cen v/v)
Mobile phase B
(per cent v/v)
65
35
15
65
35
30
20
40
20
80
80
45
65
35
50
65
35
than 5.0 per cent. The relative retention time with reference to
sildenafil for N-oxide is about 0.29 and for chlorosulphonyl is
about 1.6.
chromatogram obtained with test solution the area ofthe peak
due to N-oxide impurity is not more than 1.67 times the area of
solution (b) (0.5 per cent), the area of peak due to
chlorosulphonyl is not more than 0.67 times the area of the
solution (b) (0.2 per cent). The area of any other secondary
cent). The sum of areas of all the secondary peaks is not more
than 3.3 times the area ofthe principal peale in the chromatogram
obtained with reference solution (b) (1.0 per cent). Ignore any
peak corresponding to citric acid.

Calculate the content ofC2sH3sN6011S.
SildenafIl Tablets
Sildenafil Citrate Tablets
Sildenafil Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of sildenafil
citrate, C2sH3SN6011 S.
Usual Strengths. 25 mg; 50 mg; 100 mg.
Identification
Tests
Apparatus No.1,
Medium. 900 ml of O. 01 M Hydrochloric acid,
NOTE-Use freshly prepared solution.

acetonitrile.
examination in 50.0 ml ofthe solvent mixture. Dilute 5.0 ml of
Reference solution. A 0.1 per cent w/v solution of sildenafil
citrate RS in the solvent mixture. Dilute 5.0 ml ofthis solution
octadecylsilane bonded to porous silica (5 /lm) (such as
Hypersil ODS CI8),

(2.4.7). Calculate the content of C2sH3SN60 IIS in the medium
concentration of sildenafil citrate RS in the same medium.
C2sH3SN6011 S.
(2.4.14)
Solvent mixture. 50 volumes ofmobile phase A and 50 volumes

of methanol.
ofpowder containing about 50 mg of sildenafil with 35 ml of
2101
SILDENAFIL TABLETS
IP 2010
the solvent mixture, sonicate for 20 minutes and dilute to 50 ml

with the solvent mixture, filter.

sildenafil citrate RS in the solvent mixture.
octadecylsilane chemically bonded to porous silica (5
Ilm) (Such as Zorbax Eclipse XDB C 18),
mobile phase: A. a solution containing 3.85 g of
ammonium acetate and 1.0 ml of triethylamine in 1000
of water, adjusted to pH 5.5 with acetic acid,
B. methanol,
a linear gradiept programme using the conditions given
below,
Time
(in min.)
Mobile Phase A
(per cent lv/v)
Mobile Phase B
(per cent lv/v)
47
53
20
47
53
35
10
90
45 .
10
90
50
47
53
ill
47
53
Test solution.N'Ieigh and powder 20 tablets. Disperse a quantity

ofpowder containing about 50 mg of sildenafil with 35 mlof
the solvent mixture, sonicate for 20 minutes and dilute to 50.0
ml with the solvent mixture, filter.
Reference solution. A 0.14 per cent w/v solution of sildenajil
citrate RS in the solvent mixture.
silica (5 Ilm) (Such as Zorbax Eclipse XDB C18),
- mobile phase: a mixture of 30 volumes of solution
containing 3.85 g of ammonium acetate and 1.0 ml of
triethylamine in 1000 ml of water, adjusted to pH 5.5
with acetic acid and 70 volumes of methanol,
Inject the reference solution the test solution.
Calculate the content of C2sH3sN60 I IS in the tablet.
equivalent amount of sildenafil.
than 5.0 per cent.
Silver Nitrate
Mol. Wt. 169.9
Silver Nitrate contains not less than 99.0 per cent and not
Inject reference solution (b) and the test solution. In the . more than 100.5 per cent ofAgN03.
chromatogram obtained with the test solution, the area of any Category. Local anti-infective.
principal peak in the chromatogram obtained with reference Description. Colourless crystals or a white, crystalline powder.
solution (b) (0.8 per cent), the area of peak cOlTesponding to
Identification
N-oxide impurity at relative retention time 0.54 is not more
than the area of the principal peak in the chromatogram A. Gives the reactions of silver salts (2.3.1).
obtained with reference solution (b) (1.0 per cent) and the sum
B. 10 mg gives reaction A ofnitrates (2.3.1).
of areas of all the secondary peaks is not more than 2.0 times
the area of the peak in the chromatogram obtained with
Tests
reference solution (b) (2.0 per cent). Ignore the peak due to
citric acid at retention time about 2.4 minutes.
Appearance of solution. A4.0 per cent w/v solution (solution
A) is clear (2.4.1), and colourless (2.4.1).
Acidity or alkalinity. To 2 ml of solution A add 0.1 mlof
bromocresol green solution; the solution is blue. To 2 ml of
Solvent mixture. 50 volumes ofmobile phase and 50 volumes this solution add 0.1 ml ofphenol red solution; the solution is
yellow.
of methanol.
2102
IP 2010
SIMVASTATIN
Aluminium, bismuth, copper and lead. Dissolve 1.0 g in a

mixture of4 ml of strong ammonia solution and 6 ml of water;
the resulting solution is clear (2.4.1), and colourless (2.4.1).
Foreign salts. Not more than 0.3 per cent, determined by the
following method. To 30 ml of solution A add 7.5 ml of
2 M hydrochloric acid, shake vigorously, heat for 5 minutes
on a water-bath, filter and evaporate 20 ml of the filtrate to
dryness on a water-bath. Dry the residue at 105 and weigh.
water, add 2 ml of2 M nitric acid and 2 ml ofjerric ammonium
sulphate solution and titrate with 0.1 M ammonium
thiocyanate until a reddish yellow colour is produced.
1 mlof 0.1 M ammonium thiocyanate is equivalent to
0.01699 gofAgN03.
Storage. Store protected from light and moisture, in nonmetallic containers.
Simvastatin
~SH380S
Mol.Wt.4l8.6
Simvastatin is (lS,3R, 7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6oxotetrahydro-2H-pyran-2-yl]ethyl]-3,7-dimethyl1,2,3,7,8,8a-hexahydronapthalen-l-y12,2-dimethylbutyrate.

Simvastatin contains not less than 97.0 per cent and not more
than 102.0 per cent ofC2sH380S, calculated on the dried basis.
A suitable antioxidant may be added.
Dose. 5 mg to 10 mg per day.
Identification
Compare the spectrum with that obtained with simvastatin RS
or with the reference spectrum of simvastatin.
Specific optical rotation (2.4.22). + 285 0 to + 3000 , determined

in a 0.5 per cent w/v solution in acetonitrile.
(2.4.14), as described under the Assay.
Inject test solution (a). Run the chromatogram 5 times the
retention time ofthe principal peak. The relative retention time
with reference to simvastatin for (3R,5R)-7-[(lS,2S,6R,8S,8aR)8-[(2,2-dimethylbutanoyl)oxy]-2,6-dimethyl-l ,2,6,7,8,8ahexahydronaphthalen-l-y1]-3 ,5-dihydroxyheptanoic acid
(hydroxy acid) (simvastatin impurity A) is about 0.45, for
lovastatin (simvastatin impurity E) and epilovastatin
(simvastatin impurity F) is about 0.6, for (lS,7S,8S,8aR)-8-[2[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl]ethyl]-7methyl-3-methylene-l ,2,3,"7,8,8a-hexahydronaphthalen-l-yl
2,2-dimethylbutanoate) (simvastatin impurity G) is about 0,8,
for (lS,3R, 7 S,8S,8aR)-8-[2-[(2R,4R)-4-(acetyloxy)-6oxotetrahydro-2H-pyran-2-yl]ethyl]-3,7-dimethyl-l ,2,3,7,8,8ahexahydronaphthalen-l-yl 2,2-dimethylbutanoate (acetate
ester) (simvastatin impurity B) is about 2.38, for
(lS,3R,7S,8S,8aR)-3,7-dimethyl-8-[2-[(2R)-6-oxo-3,6-dihydro2H-pyran-2-yl]ethyl]-1 ,2,3,7,8,8a-hexahydronaphthalen-l-yl
2,2-dimethylbutanoate (anhydrosimvastatin) (simvastatin
impurity C) is about 2.42 and for (2R,4R)-2-[[(lS,2S,6R,8S,8aR)8-[(2,2-dimethylbutanoyl)oxy]-2,6-dimethyl-l,2, 6,7,8,8ahexahydronaphthalen-l-yl]ethyl]-6-oxotetrahydro-2H-pyran4 - y 1(3 R, 5 R) -7 - [( 1 S, 2 S, 6 R, 8 S, 8 aR) - 8 - [( 2,2dimethylbutanoyl)oxy]-2,6-dimethyl-l ,2,6,7 ,8,8ahexahydronaphthalen-l-yl]-3,5-dihydroxyheptanoate (dimer)
(simvastatin impurity D) is about 3.8.
Inject test solution (a) and reference solution (b). In the
chromatogram obtained with test solution (a) the area of the
.secondary peaks corresponding to lovastatin and
epilovastatin is not more than twice the area of the principal
(b) (LOper cent), the area of any secondary peak other than
lovastatin and epilovastatin is not more than 0.8 times the
reference solution (b) (0.4 per cent). The sum ofthe areas ofall
the secondary peaks other than lovastatin and epilovastatin
Heavy metals (2.3.13).). 1.0 g complies with the limittest for
Tests

on 1.0 g by drying in an oven at 60 0 for 3 hours under high
vacuum.
2103
SIMVASTATIN
IP 2010
Simvastatin Tablets
Simvastatin Tablets contain not less than 90.0 per cent and
simvastatin, C2sH3s0s.
Solvent mixture. 40 volumes ofa 0.14 per centw/v solution of

potassium dihydrogen phosphate, adjusted to pH 4.0 with
orthophosphoric acid and 60 volumes of acetonitrile.
e~amination in 50.0 ml ofthe solvent mixture.

examination in 50.0 inl ofthe solvent mixture.
Reference solution (a). Dissolve 1 mg each of simvastatin RS
and lovastatin RS in 50.0 ml ofthe solvent mixture.
Reference solution (c). Dissolve 40 mg of simvastatin RS in
50.0 m1 ofthe solvent mixture.
Identification
Shake a quantity ofthe powdered tablets containing about 50
mg of Simvastatin with 20 ml of dichloromethane, filter and
evaporate the filtrate to dryness using a rotary evaporator
and a water-bath at 40. On the residue, determine by infrared
with that obtained with simvastatin RS or with the reference
spectrum of simvastatin.
Tests
J.UTI),
mobile phase: A. a mixture 50 volumes of acetonitrile
and 50 volumes of a 0.1 per cent v/v
solution of
B. 0.1 per cent v/v solution of
orthophosphoric acid in acetonitrile,
below,
injection volume. 5 J..tl.
(min)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
0-4.5
100
4.5-4.6
100 --795
o --75
4.6 - 8.0
95 --725
5 --775
8.0-11.5
25
75
11.5 -11.6
25 --7100
75 --70
11.6-13
100
Time
Apparatus No.1,
Medium. 900 ml of 0.01 M sodium dihydrogen orthophosphate
containing 0.5 per cent w/v of sodium dodecyl sulphate,
adjusted to pH 7.0 with 1 M sodium hydroxide,
a membrane filter and transfer 10 ml of the filtrate into a
centrifuge tube containing 0.1 g of pre-washed manganese
(IV) oxide. Shake the tube for 30 minutes, or until the
manganese (IV) oxide is completely dispersed, centrifuge and
measure the absorbance (2.4.7) ofthe clear supernatant liquid,
suitably diluted if necessary with dissolution medium at the
maximum at about 257 mn and the minimum at about 257 mn.
Calculate the content ofsimvastatin, C2sH3s0s.in the medium
concentration of simvastatin RS, using the differences in
absorbance at 247 nm and at 257 mn.
D. Not less than 70 per cent ofthe stated amount ofC2sH3s0s.
(2.4.14).
Inject reference solution (a). The test is not valid unless

resolution between the peaks due to lovastatin and
epilovastatin and simvastatin is not less than 5.0.
Calculate the content of simvastatin.
Storage. Store protected from light and moisture, under
nitrogen.
Buffer solution. A 0.51 per cent w/v solution of sodium

dihydrogen orthophosphate, adjusted to pH 4.5 with 25 per
cent v/v solution of orthophosphoric acid.
SolutionA. A mixture ofl volume of0.3 per cent v/v solution
of glacial acetic acid, adjusted to pH 4.0 with 1 M sodium
hydroxide and 4 volumes of acetonitrile.
Test solution. Shake a quantity Of the powdered tablets
containing about 25 mg ofSimvastatin with 20 ml ofsolution
A for 15 minutes, dilute to 100 m1 and filter.
2104
IP 2010
SODIUM ACETATE

ml with solution A.
Reference solution (b). A solution of 0.002 per cent w/v each
of simvastatin RS and lovastatin RS in solution A.
octadecylsilane bonded to porous silica (3 11m) (such as
HypersilODS),
- mobile phase: A. a mixture of40 volumes of acetonitrile
and 60 volumes of buffer solution,
B. a mixture of 20 volumes of buffer
below,
Time
Mobile phase A
Mobile phase B
(in min.)
(per cent v/v)
(per cent v/v)
0-5
85
15
5 -30
85 -70
15 -7100
30-45
0
100
45~46
0 -785
100 -715
46-50
85
15
SolutionA. Add 3 ml ofglacial acetic acid to 900 ml of water;

adjust the pH to 4.0 with 1 M sodium hydroxide and add
sufficient water to produce 1000 ml. Mix 1 volume of this
solution with 4 volumes of acetonitrile.
Test solution. Disperse a quantity ofwhole tablets containing
about 0.16 g ofSimvastatin in water and mix with the aid elf
ultrasound and shaking. Add sufficient of solution A to
produce about 75 ml, mix with the aid ofultrasound and shaking
for 15 minutes, dilute to 100 ml with solution A and centrifuge.
Dilute 1 ml ofthe clear supernatant solution with sufficient of
solution A to produce a solution containing 0.01 per cent
w/v ofSimvastatin.
Reference solution. A 0.01 per cent w/v solution ofsimvastatin
RS in solution A.
octadecylsilane bonded to porous silica (5 11m) (such as
HypersilODS),
prepared by dissolving 5.1 g of sodium dihydrogen
orthophosphate in 900 ml of water, adjusted to pH 4.5
with orthophosphoric acid or 1 M sodium hydroxide
and add sufficient water to produce 1000 ml and 13
flow rate. 1.5 mlperminute,
resolution between the peaks due to simvastatin and lovastatin
is not less than 3.0. The relative retention time with reference
to simvastatin for simvastatin hydroxy acid (simvastatin
impurity A) is about 0.52, for lovastatin (simvastatin impurity . Inject the reference solution. The test.is not valid unless the
E) is about 0.71, for epilovastatin (simvastatin impurity F) is tailing factor ofthe principal peak is not more than 2.0.
about 0.74, for anhydrosimvastatin (simvastatin impurity C) is Inject the test solution and the reference solution.
about 1.74 and for simvastatin dimer (simvastatin impurity D)
Calculate the content of CZSH380S in the tablets.
is about 2.42.
chromatogram obtained with the test solution the area ofpeaks
corresponding to lovastatin and epilovastatin is not more than
five times the area of the principal peak in the chromatogram
obtained with reference solution (a) (1.0 per cerit), the area of
any secondary peak other than lovastatin and epilovastatin is
cent). The sum of the areas of all the secondary peaks other
than lovastatin and epilovastatin is not more than five times
with reference solution (a) (1.0 per cent). Ignore any peak with
an area less than 0.25 times ofthe area ofthe principal peak in
the chromatogram obtained with reference solution (a) (0.05 .
per cent).

Sodium Acetate
CHaCOONa,3H20
Mol. Wt. 136.1
Sodium Acetate contains not less than 99.0 per cent and not
more than 101.0 percent ofC zH 3NaOz, calculated on the
dried basis.
Category. Pharmaceuticalaid (for peritoneal dialysis fluids).
Description. Colourless crystals or a white, crystalline powder.
Identification
Dissolve 10.0 g in sufficient carbon dioxide-free water to
produce 100.0 ml (solution A). 1 ml ofsolution A gives reaction
B of acetates and reaction A of sodium salts (2.3.1).
2105
IP 2010
SODIUM ACETATE
Tests
colourless (2.4.1).
pH (2.4.24).7.5 to 9.0, determined in a 5.0per cent w/v solution.
Arsenic (2.3.1 0). Dissolve 5.0 g in 50ml of water and add 15 ml
Calcium and magnesium. Not more than 50 ppm, calculated
as Ca, determined by the following method. Mix 200 ml of
water with 10mlofammonia bufferpH10.0,0.1 gofmordant
black 11 mixture and 2 ml of 0.05 M zinc chloride. Add
dropwise 0.02 M disodium edetate until the colour changes
from violet to blue. To this solution add 109 ofthe substance
under examination, shake to dissolve and titrate with
0.02 M disodium edetate until the blue colour is restored. Not
more than 0.65 ml of 0.02 M disodium edetate is required.
Heavy metals (2.3.13).12 ml of solution A complies with the
limit test for heavy metals, Method D (10 ppm).
Iron (2.3.14). 20 ml ofsolution A complies with the limittestfor
iron (20 ppm).
Chlorides (2.3.12). 10 ml ofsolutionA complies with the limit
Reducing substances. Dissolve 1.0 g in 100 ml ofboiling water,
add 5 ml of1 M sulphuric acid and 0.5 ml of 0.002 Mpotassium
permanganate, mix and boil gently for 5 minutes; the pink
colour is not completely discharged.
Loss on drying (2.4.19).39.0 to 40.5 percent, determined on
0.2 g by drying in an oven at 130. Place the substance under
examination in the oven while the oven is still cold.
anhydrous glacial acetic acid, add 5 ml of acetic anhydride,
mix and allow to stand for 30 minutes. Titrate with
0.1 M perchloric acid, using 0.3 ml of 1-naphtholbenzein
solution as indicator, until a green colour is produced. Carry
CzH3Na02.
Sodium Acetate intended for use in the preparation ofdialysis
solutions complies with the following additional requirement
Aluminium. Dissolve 20 g in 100 ml of water and adjust to
pH 6.0 by the addition of about 10 ml of lA{hydrqchloric
acid. Extract with successive quantities of20, 20 and 10 ml of
a 0.5 per cent w/v solution of 8-hydroxyquinoline in
chloroform and dilute the combined extracts to 50 ml with
chloroform. Use as the standard solution a mixture of0.4 ml of
aluminium standard solution (2 ppm Al), 10 ml of acetate

buffer pH 6.0 and 98 ml of water treated in the same manner
and as the blank solution a mixture of 10 ml of acetate buffer
pH6.0 and 100 ml of water treated in the same manner. Measure
the fluorescence (2.4.5) of the test solution and the standard
solution, using an excitation wavelength of about 392 nm and
emission wavelength of about 518 nm, and setting the
instrument to zero with the blank solution in each case. The
fluorescence ofthe test solution is not greater than that ofthe
standard solution (0.2 ppm).
intended for use in the manufacture of dialysis solutions.
Sodium Alginate
Sodium Polymannuronate
Sodium Alginate consists mainly ofthe sodium salt of alginic
acid which is a mixture of polyuronic acids [(C6H s0 6)n]
composed of residues ofD-mannuronic acid and L-guluronic
acids and is obtained mainly from algae belonging to the order
Phaeophyceae.
Category. Pharmaceutical aid (viscosity-increasing agent).
Description. A cream-coloured to pale yellowish brown
powder; almost odourless.
Identification
A. Dissolve 0.2 gwith shaking in 20ml of water and to 5 ml of
the resulting solution add 1 ml of calcium chloride solution;
a voluminous gelatinous precipitate is produced.
B. To 10 ml of the solution obtained in test A add 1 ml of
1 M sulphuric acid; a gelatinous mass is produced.
C. To 5 mg add 5 ml of water, 1 ml ofa freshly prepared 1 per

cent w/v solution of naphthalene-1,3-diol in ethanol (95 per
cent) and 5 ml of hydrochloric acid. Boil for 3 minutes, cool,
add 5 ml of water and shake with 15 ml of di-isopropyl ether.
The upper layer exhibits a deeper bluish red colour than the
upper layer obtained by repeating the procedure without the
D. The residue obtained in the test for Sulphated ash gives
reaction A ofsodium salts (2.3.1).
Tests
Heavy metais (2.3.13). 0.5 g complies with the limiftestfor
heavy metals, MethodB (40 ppm), using nitric acid instead
of sulphuric acid for wetting the sample.
2106
IP 2010
SODIUM AMINOSALICYLATE
Chloride. Not more than 1.0 per cent, determined by the

following method. Shake 2.5 g with 50 ml of2 M nitric acid for
1 hour, dilute to 100 ml with 2M nitric acid and filter. To 50 ml of
the filtrate add 10.0 ml of 0.1 M silver nitrate and 5 ml of
toluene. Titrate with 0.1 M ammonium thiocyanate using 2 ml
offerric ammonium sulphate solution as indicator and shaking
vigorously towards the end-point.
1 ml of 0.1 M silver nitrate is equivalent to 0.00355 g ofCI.
Microbial contamination (2.2.9). Not more than 103 microorganisms per gram. It complies with the test for Escherichia
coli and Salmonella.
Sulphated ash (2.3.18). 30.0 to 36.0 per cent, determined on
0.1 g and calculated on the dried basis.
Loss on drying (2.4.19). Not more than 15.0 per cent,
Sodium Aminosalicylate
Sodium PAS
COON a
~OH
Y
,2H zO
Tests
pH (2.4.24).6.5 to 8.5, determined in a2.0 percentw/v solution.
3-aminophenol. Determine by liquid chromatography (2.4.14).

Reference solution (a). Dissolve 25.0 mg of 3-aminophenol
RSin 100.0 ml ofthe mobile phase (solution A). Dilute5.0mlof
the solution to 100.0 ml with the mobile phase. Dilute 5.0 ml of
the resulting solution to 50.0 ml with the mobile phase.
Reference solution (b). Dissolve 25.0 mg of sodium
aminosalicylate RS in 100.0 ml of the mobile phase. Dilute
5.0 ml ofthis solution and 5 ml of solution A to 100.0 ml with
the mobile phase.
octadecylsilane bonded to porous base deactivated
silica (5 11m) (such as Hypersil BDS),
mobile phase: dissolve 6.0 g of disodium hydrogen
orthophosphate and 6.6 g of sodium dihydrogen
orthophosphate dihydrate in 1600 ml with water, add
19 ml of tetra n-butyl ammonium hydrOXide (20 per
cent solution) and make the volume to 1700 ml with
water and add 300 ml of methanol,
for replicate injections is not more than 5.0 per cent
NH z
Mol. Wt. 211.2
Sodium Aminosalicylate is sodium 4-amino-2-hydroxybenzoate dihydrate.
Sodium Aminosalicylate contains not less than 99.0 per cent
and not more than 101.0 per cent ofC7H6NNa03' calculated on
Dose. 10 to 15 g daily, in divided doses.
Description. A white to cream coloured crystalline powder.
Identification
Compare the spectrum with that obtained with sodium
aminosalicylate RS or with the reference spectrum ofsodium
aminosalicylate.
B. A 5 per cent w/v solution complies with the tests for sodium
salts (2.3.1).
resolution between m-aminophenol and sodium
aminosalicylate is at least 5.0.
chromatogram obtained with the test solution, the area of the
peak corresponding to 3-aminophenol is not more than the
area ofthe corresponding peak in the chromatogram obtained
with reference solution (a) (0.25 per cent).
Hydrogen Sulphide and Sulphur dioxide. Dissolve 0.5 gin
5 ml of 1 M sodium hydroxide, add 6 ml of 3 M hydrochloric
acid and stir vigorously. No odour of hydrogen sulphide or
sulpher dioxide is perceptible, and not more than a faint odour
of Amyl Alcohol is perceptible. A piece of moistened lead
acetate paper held over the mixture does not become
discoloured.
Arsenic (2.3.1 0). Mix 5.0 g with 10 ml of bromine solution and
evaporate to dryness on a water-bath, i~ite gently, dissolve
the cooled residue, ignoring any carbon, in 50 ml of water and
14 ml of brominated hydrochloric acid AsT and remove the
2107
SODIUM AMINOSALICYLATE
IP 2010
excess bromine with a few drops of stannous chloride solution

AsT. The resulting solution complies with the limit test for
Sodium Aminosalicylate Tablets
arsenic (2 ppm).
Sodium PAS Tablets
Sodium aminosalicylate tablet contains not less than 95.0 per

cent not more than 105.0 per centofC7H6NNa03.2HzO..
Chlorides (2.3.12). Dissolve 1.0 g in 10 rn1 ofwater, add 3 rn1 of

acetic acid and filter, dilute the filtrate to 50 ml with water.
10 ml ofthe solution complies with the limit test for chlorides
(0.25 per cent).
Usual strengths. 500 mg; 1 g.
Sulphates (2.3.17).0.1 g complies with the limit test for

sulphates (0.15 per cent).
Loss on drying (2.4.19). 16.0 to 17.5 per cent, determined on

Reference solution (a). Dissolve 50.0 mg of sodium
aminosalicylate RS in 100.0 ml of the mobile phase (solution
A). Dilute 5.0 ml ofthis solution to 100.0 ml with the mobile
phase.
Reference solution (b). Dissolve 25.0 mg of 3-aminophenol

RS in 100.0 ml ofthe mobile phase. Dilute 5.0 rn1 ofthis solution
and 5 ml ofsolution A to 100.0 ml with the mobile phase.
- a stainless steel column 25 cm X 4.6 rom, packed with
octadecylsilane bonded to porous silica (5 ).tm),
- mobile phase: dissolve 6.0 g of disodium hydrogen
19 ml of tetra n-butyl ammonium hydroxide (20 per
cent solution) and make the volume to 1700 ml with
water and add 300 ml of methanol,
- injectionvolume. 20 ).tl.
than 2.0. The column efficiency in not less than 3000 theoretical
plates. The relative standard deviation for replicate injections
resolution between 3-aminophenol and sodium
aminosalicylate is at least 5.0.
Calculate the content of C7H6NNa03'
Identification
Digest a quantity of powdered tablets containing about 3.0 g
ofsodium aminosalicylate, with 40 ml of water, and filter. Add
to the filtrate 15 ml of 1M acetic acid, and allow it to stand
until precipitation has occurred. Collect the precipitate on a
filter, wash well with water and dry at 105C for 30 min. The
residue complies with the following tests.
A. Place about 1 g of the residue in a small, round-bottom
flask, and add 10 ml of acetic anhydride. Heat the flask on a
steam bath for 30 minutes, and add 40ml of water, mix, filter,
cool, and allow to stand until diacetyl derivative crystallizes,
wash well with water, and dry at 105 for 1 hour. The diacetyl
derivative so obtained melts at 191 to 197.
B. Shake 0.1 g ofthe residue with 10 ml ofwatel; and filter. To

5 ml of the filtrate add 1 drop offerric chloride solution. A
violet colour is produced.
C. In the Assay, the principal peak in the chromatogram
Tests
3-aminophenol. Determine by liquid chromatography (2.4.14).
Test solution. Weigh and powder 20 tablets. Weigh a quantity

of the powder containing about 50 mg of Sodium
Aminosalicylate and dissolve in 100 ml ofthe mobile phase.
m-aminophenol RS in the mobile phase. Dilute 5 ml to 100 ml
with the mobile phase. Dilute 5 ml ofthe resulting solution to

sodium aminosalicylate RS in the mobile phase.
Reference solution (c). Mix 5 ml each ofreference solution (a)
and reference solution (b) and dilute to 100 ml with the mobile
phase.
octadecylsilane bonged tQ PQrous .pase .de().ctivated
silica (5 ).tm) (such as HypersilBDS),
- mobile phase: dissolve 6.0 g of disodium hydrogen
2108
IP 2010
SODIUM ASCORBATE

cent aqueous solution) and make the volume to 1700 ml
with water, add 300 ml of methanol, mix well and degas,
Inject reference solution (c). The resolution between
3-aminophenol and sodium aminosalicylate is not less
than 5.0.
chromatogram obtained.with the test solution, the area of the
peak corresponding to 3-aminophenol is not more than the
solution (a)( 1.0 per cent):
Apparatus. No.2,
Medium. 900 m1 water,

a quantity of the powder containing about 0.5 g of sodium
aminosalicylate, add sufficient mobile phase to produce
100.0 ml and filter. Dilute 5.0 ml ofthis solution to 50.0 ml with
the mobile phase.
Reference solution. A 0.05 per cent w/v solution of sodium
aminosalicylate RS in the mobile phase.
octadecylsilanebonded to porous silica (5 Ilm),
19 ml of tetra n~butyl ammonium hydroxide (20 per
injections is not more than 2.0 per cent

Test solution. The filtrate diluted with the mobile phase to

produce a 0.055 per cent w/v solution.
Reference solution. A 0.055 per cent w/v solution of sodium
aminosalicylate RS in the mobile phase.
injections is not more than 2.0 per cent
Calculate the content ofC7H6NNa03.2H20 in the medium.
Calculate the content OfC7H6NNa03. 2H20 in the tablets.

Sodium Ascorbate
CHzOH
I
HCOH
RO
Nad
OH
Mol. Wt. 198.1
Sodium Ascorbate is L-ascorbic acid monosodium salt.

Sodium Ascorbate contains not less than 99.0 per cent and
not more than 101.0 per cent ofC6H7Na06, calculated on the
dried basis.
Category. Vitamin C.
Dose. The equivalent of up to 1 g of ascorbic acid daily.

C7H6NNa03.2H20.
Description. White or faintly yellow crystals or a crystalline

powder; odourless or almost odourless. It darkens gradually
on exposure to light.
2109
SODIUM ASCORBATE
IP 2010
Identification
Category. Phannaceutical aid(preservative).
Test A may be omitted iftests B, C and D are carried out. Test

Description. A white, crystalline or granular powder or flakes;

odourless or with a faint odour; hygroscopic.

ascorbate RS.
B. T04mlofa2percentw/vsolutionadd 1 mlofO.1 Mhydrochloric acid, add a few ml of 2,6-dichlorophenol-indophenol
solution; the solution is decolorised.
C. To 4mlofa2percentw/v solution add 1 mlofO.1 Mhydrochloric acid, add 1 drop of a freshly prepared 5 per cent w/v
solution of sodium nitroprusside and 2 ml of dilute sodium
hydroxide solution. Add 0.6 ml of hydrochloric acid dropwise
and stir; the yellow colour turns blue.
D. A 2 per cent w/v solution gives the reactions of sodium
salts (2.3.1).
Identification
A. To a 10 per cent w/v solution add ferric chloride test
solution; a buff coloured precipitate is fonned. Add dilute
hydrochloric acid; a white crystalline precipitate is produced.
B. Gives the reactions of sodium salts and reactions Band C
ofbenzoates (2.3.1).
Tests
Appearance of solution. A5.0 per cent w/v solution in carbon

dioxide-free water is clear (2.4.1) and not more intensely
Acidity or alkalinity. To 20 ml ofa 5.0 per cent w/v solution in

carbon dioxide-free water add 0.2 ml of phenolphthalein
solution. Not more than 0.2 ml of 0.1 M hydrochloric acid or
0.2 ml of 0.1 M sodium hydroxide is required to change the
colour of the solution.
pH (2.4.24). 7.0 to 8.0, detennined in a 10.0 per centw/v solution.
~rsenic (2.3.10).
Tests
Specific optical rotation (2.4.22). +103 to +108, detennined

detennined on 1.0 g by drying over phosphorus pentoxide at
a pressure not exceeding 0.7 kPa for 24 hours.
100 ml of carbon dioxide-free water and 25 rnl of1 M sulphuric
acid. Titrate immediately with 0.05 M iodine, using 1 ml of
starch solution as indicator, until a persistent violet-blue
colour is obtained.
1 ml of 0.05 M iodine is equivalentto 0.009905 g ofC6H7Na06.
Heavy metals (2.3.13). Dissolve 2.0 g in 45 rnl of water and 5 ml

of hydrochloric acid. 25 ml of the solution complies with the
limit test for heavy metals, Method B (20 ppm).
Chlorinated compounds. Dissolve 0.33 g in 5 ml of
0.5 M sodium carbonate, evaporate to dryness and heat the
residue until completely charred, keeping the temperature
below 400. Extract the residue with a mixture of 10 ml of water
and 12 ml of dilute nitric acid and filter; the filtrate complies
with the limit test for chlorides (2.3.12).
Sodium Benzoate

anhydrous glacial acetic acid, wanning to 50 if necessary,
cool. Titrate with 0.1 M perchloric acid, using 0.05 ml of
titration.
COONa
6
C7HsNaOz
Mix 5.0 gwith 10ml of bromine solution and

evaporate to dryness on a water-bath. Ignite gently, dissolve
the cooled residue, ignoring any carbon, in 50 ml of water and
14 ml of brominated hydrochloric acid AsT, and remove the
excess of bromine with 2 ml of stannous Chloride solution
arsenic (2 ppm).
Mol. Wt. 144.1
Sodium Benzoate contains not less than 99.0 per cent and not
more than 100.5 per cent ofC7HsNaOz, calculated on the dried
basis.
1 mlofO.1 M perchloric acid is equivalent to 0.01441-g of

C7HsNaOz.
2110
IP 2010
SODIUM BICARBONATE INJECTION
neutralise with hydrochloric acid and dilute to 15 ml with

distilled water. The resulting solution complies with the limit
Sodium Bicarbonate
Sodium Hydrogen Carbonate
NaHC0 3
Mol. Wt. 84.0
Sodium Bicarbonate contains not less than 99.0 per cent and
not more than 101.0 per cent ofNaHC03

carbon dioxide-free water and titrate with 1 M hydrochloric
acid using 0.2 ml of methyl orange solution as indicator.
Category. Electrolyte replenisher; systemic alkaliser.
1 ml of 1 M hydrochloric acid is equivalent to 0.08401 g of

NaHC03
Dose. 1 to 4 g.
Description. A white, crystalline powder or small, opaque,

monoclinic crystals. It gradually forms sodium carbonate on
heating in the dry state or in solution.
Sodium Bicarbonate Injection
Identification
Sodium Bicarbonate Intravenous Infusion
A. To 5 ml of a 5.0 per cent w/v solution in carbon dioxidefree water (solution A) add 0.1 m1 ofphenolphthalein solution;
a pale pink colour is produced. On heating, a gas is evolved
and the solution becomes red.
Sodium Bicarbonate Injection is a sterile solution of Sodium

Bicarbonate in Water for Injections.
B. Gives reaction A ofbicarbonates (2.3.1).
Sodium Bicarbonate Injection contains not less than 94.0 per

sodium bicarbonate, NaHC0 3
C. Solution A gives the reactions of sodium salts (2.3.1).
Category. Electrolyte replenisher; systemic allcaliser.
Tests
Usualstrengths. 1.4,4.2,5.0,7.5 and 8.4 percentw/v.

colourless (2.4.1).
Arsenic (2.3.10). Dissolve 5.0 gin 50 rnl ofwater, add 15 ml of
brominated hydrochloric acid AsT and remove the excess of
bromine with a few drops ofstannous chloride solution AsT.
The resulting solution complies with the limit test for arsenic
(2 ppm).
Calcium. A2.0 per centw/v solution, when boiled for 5 minutes,
is clear.
Heavy metals (2.3.13). Mix 4.0 g wi1h 5 rnl ofwater and 18 rnl of
dilute hydrochloric acid, heat to boiling and maintain that
temperature for 1 minute. Add 0.05 ml of phenolphthalein
solution and sufficient 5 M ammonia dropwise to give the
solution a faint pink colour, cool and dilute to 25 ml with water.
The solution complies with the limit test for heavy metals,
MethodA(5 ppm).
Iron (2.3.14). Dissolve 2.0 g in 20 ml of water and 4 ml of
hydrochloric acid and dilute to 40 ml with water; the resulting
solution complies with the limit test for iron (20 ppm).
Carbonate. pH ofsolution A, when freshly prepared, not more
than 8.6.
Chlorides (2.3.12).1.25 g dissolved in 15 ml ofwater and 2 ml
of nitric acid complies with the limit test for chlorides
(200 ppm).
Sulphates (2.3.17). Suspend 1.0 g in 10 ml ofdistilled water,
Identification
A. The residue on evaporation, when moistened with
hydrochloric acid and introduced on a platinum wire into a
flame, imparts a yellow colourto the flame.
B. Gives reaction A of sodium salts and the reactions of
bicarbonates (2.3.1).
Tests
pH (2.4.24). 7.0 to 8.5.
perrnl.
Assay. Titrate a volume containing 1.5 g ofSodium Bicarbonate
with 1 M hydrochloric acid using methyl orange solution as
indicator.
NaHC03
Labelling. The label states (1) the strength as the percentage
w/v of Sodium Bicarbonate; (2) the approximate
concentrations, in millimoles per litre, ofthe sodium ions and
the bicarbonate ions; (3) that an injection containing visible
particles in the solution should not be used.
2111
SODIUM CARBONATE
IP 2010
Sodium Carbonate
Na2C03
Na2C03,H20

Mol.Wt.124.0 (monohydrate)
Sodium Carbonate is anhydrous or contains one molecule

ofwater of hydration.
Assay. Weigh accurately about l.0 g,dissolve in 25 ml of

water and titrate with 1 M hydrochloric acid using methyl
orange solution as indicator.
Na2C03'
Storage. Store in tightly-closed, non-metallic containers.
Sodium Carbonate contains not less than 99.5 per cent and
not more than 100.5 per cent of Na2C03, calculated on the
dried basis.
Labelling. The label states whether the material is anhydrous

or monohydrate.
Category. Pharmaceutical aid (alkalising agent)

Description. Anhydrous - A white or almost white, slightly
granular powder, hygroscopic.
Sodium Chloride
NaCI
Monohydrate crystals.
A white, crystalline powder or colourless
Mol. Wi. 58.4
Sodium Chloride contains not less than 99.0 per cent and not
more than 100.5 per cent ofNaCl, calculated on the dried basis.
Identification
A. Dissolve 1 g in water and dilute to 10 ml with water; the
solution is strongly alkaline.
B. The solution prepared for test A gives reaction A of
carbonates and reactions of sodium salts (2.3.1)
Category. Pharmaceutical aid (tonicity agent); fluid and

electrolyte replenisher.
Description. White or colourless crystals or a white crystalline
powder.
Identification
Tests
A. Gives the reactions of chlorides (2.3.1).

Appearance ofsolution. Dissolve 2.0 g in 10 ml of water. The
resulting solution is clear and not more intensely coloured
thanreference solution YS6, (2.4.1).
Alkali hydroxides and bicarbonates. Dissolve 0.4 g in 20 ml of
water, add 20 ml of barium chloride solution and filter. To
10 ml of the filtrate add 0.1 ml of phenolphthalein solution.
The solution does not become red. Heat the remainder ofthe
filtrate to boiling for 2 minutes. The solution remains clear.
Heavy metals (2.3.13). Dissolve 2.0 g in portions in a mixture of
5 ml ofhydrochloric acid and 25 ml ofwater. Heat the solution
to boiling and cool. Add dilute sodium hydroxide solution
until the solution is neutral. Dilute to 50 ml with water (solution
A). 12 ml ofthe resulting solution complies with the limit test
for heavy metals, Method A (50 ppm). Use lead standard
solution (2 ppm Pb) for the standard.
Iron (2.3 .14). Dilute 5 ml ofsolution A to 10 ml with water. The
Chlorides (2.3.12). Dissolve 0.4 g in water, add 4 ml ofdilute
nitric acidand dilute to 15 ml with water. The solution complies
with the limit test fOfchlorides (125 ppm).
test for sulphates, (250 ppm).
Loss on drying (2.4.19). Not more thanl.O per cent (for
anhydrous form) or between 12.0 per cent and 15.0 per cent
(for monohydrate form), determined on 2.0 g by drying in an
oven at 300.
B. A 20 per cent w/v solution in carbon dioxide-free water

prepared from distilled water (solution A) gives the reactions
ofsodium salts (2.3.1).
Tests
colourless (2.4.1).
bromothymol blue solution; not more than 0.5 ml of
0.01 M hydrochloric acid or of 0.01 M sodium hydroxide is
required to change the colour of the solution.
Arsenic (2.3.10). Dissolve 10.0 gin 50 ml of water and 12 mlof
stannated hydrochloric acid AsT. The resulting solution
Barium. Dissolve 2 g in 10 ml of water, and add 2 ml of dilute
sulphuric acid; no turbidity is produced within 2 hours.
Bromide. To 0.5 ml ofsolution A add 4.0 ml of water, 2.0 ml of
phenol red reagent and l.Oml of 0.01 per cent w/v solution of
chloramine Tand mix immediately. After exactly 2 minutes,
add 0.15 ml of 0.1 M sodium thiosulphate, mix and dilute to
10.0 ml with water. The absorbance ofthe solution measured
a:t!l1J()ut~9QI!Ill C2,4})'lIsiilg ~ateras theblailk, is notIllore
than that of the standard solution prepared at the same time
and in the same manner, using 5.0ml ofa 0.0003 percentw/v
solution of potassium bromide (100 ppm).
2112
IP 2010
SODIUM CHLORIDE AND DEXTROSE INJECTION
Calcium and magnesium. Not more than 50 ppm, calculated

as Ca, determined by the following method. Dissolve 20.0 gin
200 ml of water, and add 0.1 ml of hydrochloric acid, 5 ml of
strong ammonia-ammonium chloride solution, 5 drops of
eriochrome black T solution and titrate with 0.005 M disodium
edetate to a blue end-point.
1 ml of 0.005 M disodium edetate is equivalent to 0.0002004 g
ofCa.
Ferrocyanide. Dissolve 2.0 g in 6 ml ofwater and add 0.5 ml of
a mixture of5 ml ofa 1per cent wIv solution ofjerric ammonium
sulphate in a 0.25 per cent w/v solution of sulphuric acid, and
95 ml ofa 1 per cent w/v solution offerrous sulphate; no blue
colour is produced within 10 minutes.
Heavy metals (2.3.13). 4.0 g in 2 ml of dilute acetic acid and
Iodide. Moisten 5 g by adding dropwise, a solution freshly
prepared by mixing 25 ml of iodide-free starch solution 2 ml of
0.5 M sulphuric acid, 0.15 ml of sodium nitrite solution and
25 ml of water and examine the mixture in daylight; the
substance shows no blue colour after 5 minutes.
Iron (2.3.14). 2.0 g dissolved in 20 ml of water complies with
the limit test for iron (20 ppm).
the combined extracts to 50 ml with chloroform. Use as the

blank solution a mixture of 10 ml of acetate bufferpH 6.0 and
100 ml of water treated in the same manner and as the standard
solution a mixture of 2 ml of aluminium standard solution
(2 ppm AI), 10 ml of acetate bufferpH 6.0 and 90 ml of water
treated in the same manner. Measure the fluorescence of the
test solution and of the standard solution (2.4.5), using an
excitation wavelength of392 nm and a secondary filter with a
transmission band centered at 518 nm, or a monochromator
set to transmit at this wavelength, and setting the instrument
to zero with the blank solution in each case. The fluorescence
of the test solution is not greater than that of the standard
solution.
suitable for use in the manufacture of parenteral preparations
or for the preparation of dialysis solutions.
Sodium Chloride and Uextrose

Injection
Sulphates (2.3.17). 2.5 ml of solution A diluted to 15 ml with

water complies with the limit test for sulphates (300 ppm).
Sodium Chloride and Dextrose Intravenous Infusion;

Sodium Chloride and Glucose Injection; Sodium Chloride
and Glucose Intravenous Infusion
Sodium Chloride and Dextrose Injectionis a sterile solution of

Sodium Chloride and pextrose in Water for Injections.

water in a glass-stoppered flask. Add 50.0 ml of 0.1 M silver
nitrate, 5 ml of 2 M nitric acid and 2 ml of dibutyl phthalate,
shake well and titrate with 0.1 M ammonium thiocyanate using
2 ml offerric ammonium sulphate solution as indicator, until
the colour becomes reddish yellow.
Sodium Chloride and Dextrose Injection contains not less than

amounts ofsodium chloride, NaCl, and dextrose, C6H 120 6
Usual strengths. Injections containing the following amounts
ofSodium Chloride, NaCI and Dextrose, C6H 1Z0 6
Per centage
w/vof
sodium
chloride
(NaCl)
Percentage
w/vof
dextrose
(C6H 1Z0 6)
0.11
0.18
0.45
0.2
0.45
10
Sodium Chloride intended for use in the preparation ofdialysis

solutions complies with the fpllowing additionai requirement.
0.225
0.9
2.5
0.3
0.9
Aluminium. Not more than 0.2 ppm, determined by the

following method. Dissolve 20 g in 100 ml of water and add
10 ml of acetate buffer pH 6.0. Extract the resulting solution
with successive quantities of20, 20 and 10 ml ofa 0.5 per cent
w/v solution of 8-hydroxyquinoline in chloroform and dilute
0.33
0.9
0.45
2.5
0.9
10
25
1 ml of 0.1 M silver nitrate is equivalentto 0.005844 gofNaCl.

Sodium Chloride intended for use in the manufacture of
parenteral preparations or in the manufacture of dialysis
solutions complies with the following additional requirement.
Potassium. Not more than 0.1 per cent, determined by flame
photometry (2.4.4), using a 1.0 per cent w/v solution and
measuring at 768 nm. Use suitable dilutions in water of
potassium solution FP for the standard solution.
Percentage
w/vof
sodium
chloride
(NaCl)
Percentage
w/vof
dextrose
(C6H 1Z0 6)

solution.
2113
IP 2010
SODIUM CHLORIDE AND DEXTROSE INJECTION
Identification
A. To 1 ml add 0.05 mlofpotassium cupri-tartrate solution;
B. Gives reaction B ofsodium salts and reaction A ofchlorides

(2.3.1).
Sodium Chloride and Fructose Injection contains not less than

amounts of sodium chloride, NaCI, and fructose, C6H 1Z0 6 It
contains .no antimicrobial agent.
Usual strengths. Injections containing the following amounts
ofSodium Chloride, NaCl, and Fructose, C6H 1z06
Per cent
w/v of
Sodium
Chloride
(NaCl)
Tests
pH (2.4.24).3.5 to 6.5.
5-Hydroxymethylfurfural and related substances. Dilute a
volume containing 1.0 g ofdextrose, C6H 1Z0 6, to 500 ml with
water and measure the absorbance of the resulting solution
at the maximum at about 284 nm (2.4.7); absorbance at about
284 nm, not more than 0.25.
Bacterial endotoxins (2.2.3). Not more than 10 Endotoxin Units
per g of dextrose.
Preparations (Intravenous Infusions).
Assay. For sodium chloride- Titrate an accurately measured
volume containing 0.1 g ofSodium Chloride with 0.1 M silver
nitrate using potassium chromate solution as indicator.
I ml of0.1 M silver nitrate is equivalentto 0.005844 g ofNaCl.
2 to 5 g of anhydrous dextrose, C6H 1Z 0 6, add 0.2 ml of
5 M ammonia and sufficient water to produce 100.0 ml. Mix
well, allow to stand for 30 minutes and measure the optical
rotation in a 2-dm tube (2.4.22). The observed rotation in
degrees multiplied by 0.9477 represents the weight, in g, of
dextrose, C6H J2 0 6, in the volume ofthe injection taken for assay.
Storage. Store in single dose containers. On keeping, small
solid particles may separate from a glass container.
Labelling. The label states (I) the strength as the percentages
w/v of Sodium Chloride and Dextrose; (2) that a solution
containing visible particles must not be used.
When the preparation is intended for intravenous infusion,
the label states the approximate concentrations, in millimoles
per litre, of the sodium and chloride ions and the number of
grams per litre ofdextrose, C 6H 1Z0 6
Per cent
w/vof
Fructose
(C6H 1Z0 6)
Per cent
w/v of
Sodium
Chloride
(NaCI)
0.11
0.45
0.18
0.45
0.225
0.9
0.33
0.9
0.45
0.9
Per cent
w/vof
Fructose
2.5
10
2.5
5
10

solution.
Identification
B. The solution prepared in the Assay is laevo-rotatory.
C. Gives reaction B ofsodium salts and reaction A ofchlorides
(2.3.1).
Tests
pH (2.4.24).3.0 to 6.0.
volume containing 1.0 g of Fructose to 500.0 ml with water
maximum at about 284 nm (2.4.7); absorbance at about 284 nm,
not more than 0.50.
Heavy metals (2.3.13). Evaporate a volume containing 4.0 g of
Fructose to 10 ml and add 2 ml of dilute acetic acid and
Sodium Chloride and Fructose

Injection
Sodium Chloride and Fructose Intravenous Infusion;
Sodium Chloride and Fructose Infusion; Fructose and
Sodium CWoride Injection.
Sodium Chloride and Fructose Injection is a sterile solution of
Sodium Chloride and Fructose in Water for Injections.

perrol.
..
Assay. For sodium chloride "- Titrate an accurately measured
volume containing about 0.1 g ofSodium Chloride with 0.1 M
silver nitrate using potassium chromate solution as indicator.
1 ml of0.1 M silver nitrate is equivalentto 0.005844 g ofNaCl.
2114
IP 2010
COMPOUND SODIUM CHLORIDE AND DEXTROSE INJECTION
Forfructose - To an accurately measured volume containing

about 5.0 g of Fructose, add 0.2 ml of 6 M ammonia and
sufficient water to produce 100.0 ml. Mix well, allow to stand
for 30 minutes and measure the optical rotation in a 2-dm tube
(2.4.22). The observed rotation in degrees multiplied by 0.5427
represents the weight, in g, offructose, C6H 120 6, in the volume
taken for assay.
Storage. Store in single dose containers. On keeping, small
solid particles may separate from glass containers.
w/v ofSodium Chloride and Fructose; (2) when the preparation
is intended for intravenous infusion, the approximate
concentrations, in millimoles per litre, of the sodium and
chloride ions and the number ofgrams per litre offructose; (3)
that a solution containing visible particles should not be used.
C. After evaporation to one half of its original volume, the

solution gives reaction A ofpotassium salts and reaction B of
calcium salts (2.3.1).
Tests
pH (2.4.24). 3.5 to 6.5.
maximum at about 284 nm; absorbance at about 284 nm, not
more than 0.25 (2.4.7).
perml.
Assay. For sodium chloride - Dilute suitably with water and
measuring at 589 nm and using sodium solution FP or sodium
standard solutions.
Compound Sodium Chloride and

Dextrose Injection
Compound Sodium Chloride and Dextrose Intravenous
Infusion .
1 g ofNa is equivalent to 2.542 g ofNaCl.
Compound Sodium Chloride and Dextrose Injection is a sterile

solution containing 0.86 per cent w/v of Sodium Chloride,
0.03 per cent w/v ofPotassium Chloride, 0.033 per cent w/v of
Calcium Chloride and 5. per cent w/v ofDextrose in Water for
Injections.
For potassium chloride - Dilute suitably with water and

measuring at 767 nm and using potassium solution FP or
potassium solution AAS respectively, suitably diluted with
water for the standard solutions.
Compound Sodium Chloride and Dextrose Injection contains

not less than 0.82 per cent and not more than 0.90 per cent
w/v of sodium chloride, NaCI, not less than 0.0285 per cent
and not more than 0.0315 percent w/v ofpotassium chloride,
KCI, not less than 0.030 per cent and not more than 0.036 per
cent w/v of calcium chloride, CaCh,2H 20, and not less than
0.523 per cent and not more than 0.580per cent w/v ofchloride,
CI, and not less than 4.75 per cent and not more than 5.25 per
cent w/v of dextrose, C6H I2 0 6 It contains no antimicrobial
agent.
Category. Fluid and electrolyte replenisher.
solution.
Identification
B. Gives reaction B ofsodium salts and reaction A ofchlorides

(2.3.1).
I g ofK is equivalent to 1.907 g ofKCl.
For calcium chloride - To 50.0 ml add 5.0 ml of

0.01 M magnesium sulphate and 5 ml of ammonia buffer
pH 10.9 and titrate with 0.01 M disodium edetate using
eriochrome black T mixture as indicator. From the volume of
0.01 M disodium edetate required subtract the volume of
0.01 M magnesium sulphate added.
1 ml ofthe remainder of O. 01 M disodium edetate is equivalent
to 0.00147 gofCaCh,2H20.
For total chloride- To 20.0 ml add 30 ml of water, 50.0 ml of

a blank titration.
1 ml of 0.1 M silver nitrate is equivalentto 0.003545 goftotal
Chloride, calculated as Cl.
For dextrose - To an accurately measured volume containing

2115
IP 2010
SODIUM CHLORIDE HYPERTONIC INJECTION
water to produce 100.0 ml.Mix well, allow to stand for
sulphate solution as indicator, until the colour becomes reddish

represents the weight, in g, ofdextrose, C6H 1Z0 6, in the volume
taken for assay.
yellow.
Storage. Store in single dose containers of glass or plastic at

a temperature not exceeding 30. On keeping, small solid
particles may separate from the solution i~ glass containers.
Labelling. The label states (1) the.strength as the percentages
w/v ofSodium Chloride, Potassium Chloride, Calcium Chloride
and Dextrose; (2) that the injection contains, in millimoles per
litre, the following approximate amounts ofthe ions. sodium,
147.5, potassium, 4, calcium, 4.5, and Chloride, 156; (3)the
total osmolar concentration in mOsmol per litre; (4) that the
injection should not be used if it contains visible partiCles.
1ml of 0.1 M silver nitrate is equivalentto 0.005844 g ofNaCl.

Storage. Store in single dose containers of glass or plastic.
On keeping, small solid particles may separate from a glass
container.
Labelling. The label states{l) the strength as the percentage
w/v ofSodiumChloride; (2) that a solution containing visible
solid particles must not be used.
When the preparation is .intended for intravenous. infusion,
the label states that the injection contains approximately
270 millimoles each ofsodium and chloride ions per litre.
Sodium Chloride Injection

Sodium Chloride Intravenous Infusion
Sodium Chloride Hypertonic Injection
Sodium Chloride Injection is a sterile 0.9 per cent w/v solution

of Sodium Chloride in Water for Injections. It contains no
antimicrobial agent.
Hypertonic Saline
Sodium Chloride Hypertonic Injection is a sterile 1.6 per cent
, w/v solution of Sodium Chloride in Water for Injections. It
contains no antimicrobial agent.
Sodium Chloride Hypertonic Injection contains not less than
1.52 per cent w/v and not more than 1.68 per cent w/v of
sodium chloride, NaCI.
Sodium Chloride Injection contains not less than 0.85 per cent
w/v and not more than 0.95 per cent w/v of sodium Chloride,
NaCl.
Identification
Gives the reactions ofsodium salts and reaction A ofchlorides
(2.3.1).
Identification
(2.3.1).
Tests
pH (2.4.24). 5.0to 7.5.
Heavy metals (2.3.13). Evaporate 40 ml to about 20 mI, add 2 ml
of dilute acetic acid and dilute to 25 ml with water. The solution
(0.5 ppm).
perml.
6.1615 ofSodium Chloride ill a glass-stoppered flask add 50 mI
6fwater arid 50.0 rril ofO.l MsilvernTtrate,Srrilof 2.MtiiiiTc
acid and 2 ml of dibutylphthalate. Shake well and titrate with
0.1 M ammonium thiocyanate using 2 ml ofjerric ammonium
Tests
pH (2.4.24).4.5 to 7.0.
Heavy metals (2.3.13). Evaporate 67 mI to about 20 mI, add 2 mI
(0.3 ppm).
perml.
0.16 g ofSodium Chloride in a glass-stoppered flask add 50 ml
of water and 50.0 ml of 0.1 M silver nitrate, 5 ml of2 M nitric
acid and 2 ml of dibutylphthalate. Shake well and titrate with
O;lMammonium thiocyanate using2 ml ofjerric ammonium
sulphate solution as indicator, until the colour becomes reddish
yellow.
1ml of 0.1 M silver nitrate is equivalentto 0.005844 g ofNaCI.
2116
IP 2010
COMPOUND SODIUM CHLORIDE SOLUTION

container.
w/v of Sodium Chloride; (2) that a solution containing visible
solid particles must not be used.
the label states that the injection contains approximately
150 millimoles each ofsodium and chloride ions per litre.

Ringer's Injection
Compound Sodium Chloride Injection isa sterile solution
containing 0.86 per cent w/v ofSodium Chloride, 0.03 per cent
w/v ofPotassium Chloride and 0.033 per cent w/v ofCalcium
Chloride in Water for Injections. It contains no antimicrobial
agent.
Compound Sodium Chloride Injection contains not less than
0.82 per cent wIV and not more than 0.90 per cent w/v of
sodium chloride, NaCl, not less than 0.0285 percent w/v and
not more than 0.0315 per cent w/v ofpotassium chloride, KCl,
and not less than 0.030 per cent w/v and not more than
0.036 per centw/v ofcalcium chloride, CaClz, 2H20.
Description. A clear, coloUrless solution.
1 g ofNa is equivalent to 2.54 g ofNaCl.
For potassium Chloride - Dilute appropriately with water

and determine by Method A for flame photometry (2.4.4),
measuring at 767 nm or by Method A for atomic absorption
spectrophotometry (2.4.2), using potassium solution Fp,
suitably diluted with water for the standard solutions.
1 g ofK is equivalent to 1.007 g ofKCl.
For Calcim Chloride - To 50.0 mladd 5.0 ml of 0.01 M

magnesium sulphate and 5 ml of ammonia bufferpH 10.9 and
titrate with O.OlM disodium edetate using mordant black II
mixture as indicator. From the volume of 0.01 M disodium
edetate required subtract the volume of O.OlM magnesium
sulphate added.
1 ml ofthe remainder of 0.01 M disodium edetate is equivalent
to 0.00147 g ofCaClz, 2H20.
container.
w/v of Sodium Chloride, Potassium Chloride and Calcium
Chloride; (2) that a solution containing visible solid particles
must not be used.
the label states that the Injection contains in millimolesper
litre, the following approximate amounts ofions; sodium, 147.5;
potassium, 4; calcium, 2.25 and chloride, 156.
Identification
Gives reaction B of sodium salts and reaction A of chlorides
(2.3.1). When concentrated to one halfof its original volume,
it gives reaction A ofpotassium salts and reaction B ofcalcium
salts (2.3.1).
Tests
pH (2.4.24). 5.0to 7.5.
Heavy metals (2.3 .13). Evaporate 67 ml to about 20 ml, add 2 ml
(O.3'ppm).
Unitperml.
Assay. For sodium chloride -Dilute appropriately with water
measuring at 589 urn or by Method A for atomic absorption
spectrophotometry (2.4.2), using sodium solution FP, suitably
diluted with water for the standard solutions.

Ringer's Solution
Compound Sodium Chloride Solution is a solution containing
0.86 per cent w/v of Sodium Chloride, 0.03 per cent w/v of
Potassium Chloride and 0.033 per cent w/v ofCalcium Chloride
in Purified Water. The solution may be clarified by filtration.
Compound Sodium Chloride Solution contains not less than
0.82 per cent w/v and not more than 0.90 per cent of sodium
chloride, NaC!, not less than 0.025 per cent and not more than
0.035 per cent w/v of potassium chloride, KCl, and not less
than 0.030 per cent and not more than 0.036 per cent wlv of
calcium chloride, CaC12 , 2H20.
Category. Irrigation solution (forextemal use).
Identification
Gives reaction B of sodium salts and reaction A of chlorides
(2.3.1). When concentrated to one half of its original volume,
2117
COMPOUND SODIUM CHLORIDE SOLUTION
IP 2010
it gives reaction A ofpotassium salts and reaction B ofcalcium

salts (2.3.1).
Identification
Tests

(2.3.1).
pH (2.4.24). 5.0to 7.5.
Tests
Heavy metals (2.3.13). Evaporate 67 ml to about 20 rnI, add 2 rnl

ofdilute acetic acid and dilute to 25 ml with water. The solution
(0.3 ppm).
pH (2.4.24). 4.5 to 7.0.

Heavy metals (2.3.13). Evaporate 67 ml to about20 ml, add 2 rnl
ofdilute acetic acidand dilute to 25 rnl with water. The solution
complies with limit test for heavy metals, MethodA( 0.3 ppm).
Assay. For sodium chloride - Dilute appropriately with water

and determine by Method A for flame photometry (2.4.4), Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit
measuring at 589 nm or by Method A for atomic absorption . perml.
spectrophotometry (2.4.2), using sodium solution Fp, suitably Other tests. Complies with the tests stated under Parenteral
diluted with water for the standard solutions.
1 g ofNa is equivalent to 2.54 g ofNaCI.
Assay. Titrate an accurately measured volume containing
For potassium chloride - Dilute appropriately with water
measuring at 767 nm or by Method A for atomic absorption
spectrophotometry (2.4.2), using potassium solution Fp,
suitably diluted with water for the standard solutions.
1 g ofK is equivalent to 1.007 g ofKCl.
For calcium Chloride - To 50.0 ml add 5.0 ml of 0.01 M
magnesium sulphate and 5 ml ofammonia bufferpH 10.9 and
titrate with 0.01 M disodium edetate using mordant black II
mixture as indicator. From the volume of 0.01 M disodium
edetate required subtract the volume of O.OIM magnesium
sulphate added.
about 0.135 g of Sodium Chloride with 0.1 M silver nitrate

using potassium chromate solution as indicator.
1 ml of0.1 M silver nitrate is equivalentto 0.005844 g ofNaCI.
Storage. Store in single dose containers. The container may
be designed to empty rapidly and may contain a volume of
more than 1 litre.
w/v of Sodium Chloride; (2) that the solution should not be
used if it contains visible particles; (3) 'For Irrigation only'
and 'Not for Injection'; (4) that once the container is opened,
the unused portion should be discarded.

to 0.00147 g ofCaClz, 2HzO.
Sodium Citrate
Trisodium Citrate
Labelling. The label states the strength as the percentages

w/v of Sodium Chloride, Potassium Chloride and Calcium
Chloride. If the contents ofthe container are sterile, the label
states (1) Sterile Compound Sodium Chloride Solution; (2)
that the solution is not intended for injection.
HJC-0
GOONa
2H 20
,
NaOOG
GOONa
.
C6HsNa307,2HzO
Sodium Citrate is trisodium 2-hydroxypropane1,2,3-tricarboxylate dihydrate.
Sodium Chloride Irrigation Solution

Sodium Chloride Irrigation Solution is a sterile solution
containing 0.9 per cent w/v of Sodium Chloride in Water for
Injections.
Sodium Chloride Irrigation Solution contains not less than
sodium Chloride, NaCl. It contains no antimicrobial agent.
Mol. Wt. 294.1
Sodium Citrate contains not less than 99.0 per cent and not
more than 101.0 per cent of C6HsNa307, calculated on the
anhydrous basis.
Category. Systemic alkalinising agent.
Dose. 1 to 10 g.
Description. White, granular crystals or a White, crystalline
powder; odourless; slightly deliquescent in moist air.
2118
IP 2010
SODIUM DIATRIZOATE
Identification
A. 10.0 per cent w/v solution in carbon dioxide-free water
(solution A) gives the reactions of sodium salts and reaction
A ofcitrates (2.3.1).

anhydrous glacial acetic acid, warming ~o about 50. Allow
to cool. Titrate with 0.1 M perchloric acid, using 0.25 ml of
l-naphtholbenzein solution as indicator. Carry out a blank
titration.
Tests

C6HsNa307.

colourless (2.4.1).
Acidity or alkalinity. Titrate 20 ml of solution A with

0.05 M sulphuric acid or 0.1 M sodium hydroxide using
thymol blue solution as indicator; not more than 0.5 ml of
0.05 M sulphuric acid or 0.1 M sodium hydroxide is required.
Sodium Diatrizoate
Diatrizoate Sodium
Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and add 15 ml

Heavy metals (2.3.13). Dissolve 2.0 g in lOrnlofwater, 5 mlof
dilute hydrochloric acid and sufficient water to produce
Method A ( 10 ppm).
Oxalate. Dissolve 0.5 g in 4 rnl ofwater, add 3 rnl ofhydrochloric
acid and 1 g of zinc, in granules, and heat on a water-bath for
1 minute. Allow to stand for 2 minutes, decant the liquid into a
test-tube containing 0.25 ml of a 1 per cent w/v solution of
phenylhydrazine hydrochloride and heat to boiling. Cool
rapidly, transfer to a graduated cylinder a,nd add an equal
volume of hydrochloric acid and 0.25 ml of potassium
ferricyanide solution. Shake and allow to stand for 30 minutes.
Any pink colour produced is not more intense than that
obtained by treating at the same time and in the same manner
4 ml of a 0.005 per cent w/v solution of oxalic acid (300 ppm,
calculated as anhydrous oxalic acid).
Sulphates (2.3.17). To 10 ml of solution A add 2 ml of
7 M hydrochloric acid and dilute to 15 ml with distilled water;
the resulting solution complies with the limit test for sulphates
(150 ppm).
Tartrates. To a solution of 1 g in 2 ml of water in a test-tube,
add 1 ml of a 10 per cent w/v solution of potassium acetate
and 1 ml of 6 M acetic acid. Scratch the walls ofthe test-tube
with a glass rod; no crystalline precipitate is formed.
Readily carbonisable substances. Heat 0.2 g, in powder, with
10 ml of sulphuric acid (96 per cent w/w) in a water-bath at
90 1 for J hour and cool rapidly. The solution is not more
intensely coloured than reference solution YS2 or GYS2 (2.4.1).
Water (2.3.43). 11.0 to 13.0 per cent, determined on 0.3 g and
after stirring for 15 minutes before titrating.
CIIHsI3N2Na04
Mol. Wt. 635.9
Sodium Diatrizoate is sodium 3,5-diacetamido-2,4,6-triiodobenzoate.

Sodium Diatrizoate contains not less than 98.0 per cent and
not more than 101.0 per cent ofC II H sI3N 2N aO4, calculated on
the anhydrous basis. .
.
Category. Urographic radio-opaque substance.
Dose. To be decided by the physician in accordance with the
needs of the patient.
Description. A white powder; odourless or almost odourless.
Identification
Tests A andD may be omitted iftests B, C, E andFare carried
out. Tests B, C and F may be omitted if tests A, D and E are
carried out.
diatrizoate RS or with the reference spectrum of sodium
diatrizoate.

solution.
examination in 100 ml of0.08 per cent w/v solution ofsodium
hydroxide in methanol.
2119
SODIUM DIATRIZOATE
IP20IO
Reference solution. A 0.1 per cent w/v of sodium diatrizoate

RS in 0.08 per, cent w/v solution of sodium hydroxide in
methanol~
Apply to the plate 10 'Ill of each solution. After development,

C. Heat 0.5 g in a crucible; violet vapours ofiodine are evolved.
D. To 20 mg add 5 ml of1 M sodium hydroxide and boil gently
under a reflux condenser for 10 minutes. Cool, add 5 ml of
2 M hydrochloric acid and cool in ice for 5 minutes. Add 4 ml
of a 1 per cent w/v solution of sodium nitrite, cool in ice for
5 minutes, add 0.3 g of sulphamic acid, shake gently until
effervescence ceases and add 2 ml ofa 0.4 per cent w/v solution
of N-(l-naphthyl) ethylenediamine dihydrochloride; an
orange-red colour is produced.
E. Heat 0.5 g with 1 ml of sulphuric acid on a water-bath until
a pale violet solution is produced, add 2 ml of ethanol (95 per
cent) and heat again; odour of ethyl acetate is produced.
F. Gives the reactions of sodium salts (2.3.1).
Tests
pH (2.4.24).7.5 to 9.5, determined in a 50 per centw/v solution.
Free amine. Place 1.0 g in a 50-ml glass-stoppered volumetric
flask, add 5 ml of water, 10 ml of 0.1 M sodium hydroxide and
25 ml of dimethyl sulphoxide. stopper the flask, mix the
contents gently and cool in ice, protected from light. After
5 minutes, slowly add 2 ml of hydrochloric acid, mix and allow
to stand for 5 minutes. Add 2 ml ofa 2 per cent w/v solution of
sodium nitrite, mix and allow to stand for 5 minutes. Add 1 ml
ofan 8 per cent w/v solution of sulphamic acid, mix and allow
to stand for 5 minutes. Add 2 ml of a 0.1 per cent w/v solution
of N-(l-naphthyl)ethylenediamine dihydrochloride in a
70 per cent w/v solution of 1,2-propanediol and mix. Rem~ve
the flask from the ice and allow to stand in water at 25 2 for
10 minutes, with occasional shaking. Add sufficient dimethyl
sulphoxide to produce 50 ml and mix. Within 5 minutes, measure
about 470 nm (2.4.7), using as the blank a solution prepared
by treating 5 ml of water in the same manner; absorbance, not
more than 0.40, calculated on the anhydrous basis.
Free iodine and iodide. Dissolve 2.0 g in 24 ml of water taken
in a 50-ml glass-stoppered centrifuge tube. Add 5 ml of toluene
and 5 ml of 1 M sulphuric acid, shake well and centrifuge; no
red colour appears in the toluene layer. To the mixture add 1 ml
of a. 2 per cent w/v solution of sodium nitrite, shake arid
centrifuge; any red colour in the toluene layer is not more
intense than that produced in a solution prepared in the same
manner using 2.0 ml of a 0.025 per cent w/v solution of

potassium iodide and 22 ml of water (220 ppm).
Heavy metals. Dissolve 1.0 g in 20.0 ml of water and 5 ml of
1 M sodium hydroxide, transfer the solution to a 50-ml Nessler
cylinder, dilute with water to 40 ml and mix. Add 10 ml of
sodium sulphide solution, shake and allow to stand for
5 minutes (20 ppm), the colour of the solution when viewed
downward over a white surface is not,more intense than that
produced by treating 2.0 ml of lead standard solution
(10 ppm Ph) in the same manner in place of the substance
under examination..
Water (2.3.43). 4.0 to 7.0 percent,determined on 0.4 g.
Assay. Weigh accurately about 0.4 g in a glass-stoppered

conical flask, add 12 ml of 5 M sodium hydroxide and 1 g of
zinc powder and boil under a reflux condenser for 30 minutes.
Cool, rinse the condenser with 30 ml of water, filter through
cotton and wash the flask and filter with two quantities, each
of20 ml, of water. To the combined filtrate and washings add
80 ml of hydrochloric acid, cool and titrate with
0.05 Mpotassium iodate until the dark brown colour becomes
pale brown. Add 5 ml of chloroform and continue the titration,
shaking well after each addition, until the chloroform becomes
colourless.
1 ml of 0.05 Mpotassium iodate is equivalent to 0.02120 g of
CIIHsI3N2Na04.

Diatrizoate Sodium Injection
Sodium Diatrizoate Injection is a sterile solution of Sodium
Diatrizoate in Water for Injections. It may contain small
amounts ofsuitable buffers, stabilisers and antimicrobial agents
but the preparation intended for intravenous administration
contains no antimicrobial preservative.
Sodium Diatrizoate Injection contains not less than 95.0 per
sodium diatrizoate, CIIHsI3N2Na04'
Usual strengths. 25 per cent w/v; 40 per cent w/v.
Identification
Evaporate a volume ofthe injection containing 1 g ofSodium
Diatrizoate to dryness. The residue complies with following
tests.
A; Heat 0.5 g ofresidue in a crucible; violet vapours ofiodine
are evolved.
B. To 20 mg ofthe residue add 5 ml of 1 M sodium hydroxide

and boil gently under a reflux condenser for 10 minutes. Cool,
2120
IP 2010
SODIUM DIHYDROGEN PHOSPHATE DIHYDRATE
add 5 ml of 2 M hydrochloric acid and cod I in ice for 5 minutes.

Add 4 ml ofa I per cent w/v solution of sodium nitrite, cool in
ice for 5 minutes, add 0.3 g of sulphamic acid, shake gently
until effervescence ceases and add 2 ml of a 0.4 per cent w/v
solution of N-(l-naphthyl) ethylenediamine dihydrochloride;
an orange-red colour is produced.
C. Heat 0.5 g ofresidue with I ml of sulphuric acid on a waterbath until a pale violet solution is produced, add 2 ml of ethanol
(95 per cent) and heat again; odour of ethyl acetate is
produced.
Cool, rinse the condenser with 30 ml of water, filter through

cotton and wash the flask and filter with two quantities, each
of20 ml, of water. To the combined filtrate and washingsadd
80 ml of hydrochloric acid, cool and titrate with
0.05 M potassium iodate until the dark brown colour becomes
pale brown. Add 5 ml of chloroform and continue the titration,
shaking well after each addition, until the chlorofonn becomes
colourless.
1 ml of 0.05 Mpotassium iodate is equivalent to 0.02120 g of
CllHgI3N2Na04.
Tests
pH (2.4.24). 6.6 to 7.6.
Free amine. To a volume containing 1.0 g of Sodium Diatrizoate
in a 50-ml glass-stoppered volumetric flask, add 5 ml of water,
10 ml of 0.1 M sodium hydroxide and 25 ml of dimethyl
sulphoxide. Stopper the flask, mix the contents gently and
cool in ice, protected from light. After 5 minutes, slowly add
2 ml of hydrochloric acid, mix and allow to stand for 5 minutes.
Add 2 ml of a 2 per cent w/v solution of sodium nitrite, mix
and allow to stand for 5 minutes. Add 1 ml of an 8 per cent
w/v solution of sulphamic acid,mix and allow to stand
for 5 minutes. Add 2 ml of a 0.1 per cent w/v solution of
N-(l-naphthyl)ethylenediamine dihydrochloride in a 70 per
cent w/v solution of 1,2-propanediol and mix. Remove the
flask from the ice and allow to stand in water at 25 2 for
10 minutes, with occasional shaking. Add sufficient dimethyl
sulphoxide to produce 50 ml and mix. Within 5 minutes, measure
about 470 nm (2.4.7), using as the blank a solution prepared
by treating 5 ml of water in the same manner; absorbance, not
more than 0.30, calculated on the anhydrous basis.
Free iodine and iodide. To a volume containing 2.0 g ofSodium
Diatrizoate in 24 ml of water taken in a 50-ml glass~stoppered
centrifuge tube add 5 ml of toluene and 5 ml of 1 M sulphuric
acid, shake well and centrifuge; no red colour appears in the
toluene layer. To the mixture add I ml of a 2 per cent w/v
solution of sodium nitrite, shake and centrifuge; any red colour
in the toluene layer is not more iritense than that produced in
a solution prepared in the same manner using 2.0 ml of a
0.025 per cent w/v solution ofpotas~ium iodide and 22 ml of
water (220 ppm).
Pyrogens (2.2.8). Complies with test for pyrogens, using per
kg ofthe rabbit's weight a volume containing 2.5 g ofSodium
Diatrizoate.
Labelling. The label states (1) the concentration ofthe active

ingredient; (2) whether the contents are intended for
intravenous injection and, if so, that the unused portion
remaining in the container after use must be discarded.
Sodium Dihydrogen Phosphate

Dihydrate
Sodium Acid Phosphate
NaH2P04,2H20
Mol. Wt. 156.0
Sodium Dihydrogen Phosphate Dihydrate contains not less

than 98.0 per cent and not more than 100.5 per cent of
NaH2P0 4, calculated on the dried basis.
Category. Urinary acidifier.
Dose. 2 to 4 g.
Description. Colourless crystals or a white powder; odourless.
Identification
A. Dissolve 10.0 g in sufficient carbon dioxide-free water to
produce 100 ml (solution A). Solution A is faintly acid.
B. Solution A neutralised with a 10 per cent w/v solution of

potassium hydroxide gives reaction A of sodium salts (2.3.1).
C. Solution A gives the reactions of phosphates (2.3.1).
Tests
colourless (2.4.1).
pH (2.4.24). 4.2 to 4.5, determined in a mixture of5 ml ofsolution
A and 5 ml of carbon dioxide-free water.

Arsenic (2.3.10). Dissolve 0.5 gin 50 ml of water and add 10 ml

Assay. Weigh accurately about 0.5 g in a glass-stoppered

conical flask, add 12 ml of 5 Msodium hydroxide and 1 gof
zinc powder and boil under a reflux condenser for 30 minutes.
Heavy metals (2.3.13).12 ml of solution A complies with the

standard solution (l ppm Pb) to prepare the standard.
2121
SODIUM FLUORIDE
IP 2010
Iron (2.3.14). 20 ml ofsolution A complies with the limit test for

iron (20 ppm).
Chlorides (2.3.12).10 ml of solution A diluted to 20 ml with
water complies with the limit test for chlorides (250 ppm)
Sulphates (2.3.17). To 5 ml of solution A add 0.5 ml of
hydrochloric acid and dilute to 15 ml with distilled water; the
solution complies with the limit test for sulphates (300 ppm).
Reducing substances. To 5 ml of solution A add 0.25 ml of
0.02 M potassium permanganate and 5 ml of 1 M sulphuric
acid and heat in a water-bath for 5 minutes; the pink colour is
not completely discharged.
Disodium phosphate. Dilute 10 ml ofsolution A to 50 ml with
water and titrate with 0.05 M sulphuric acid using
bromocresol green solution as indicator; not more than 1 ml
of 0.05 M sulphuric acid is required.
Loss on drying (2.4.19). 21.5 to 24.0 per cent, determined on
water and titrate with carbonate-free 1 M sodium hydroxide,
NaHZP04
Sodium Fluoride
NaF
Mol. Wt. 42.0
Sodium Fluoride contains not less than 98.5 per cent and not
more than 100.5 per cent ofNaF, calculated on the dried basis.
Acidity or alkalinity. Dissolve 2.5 g of potassium nitrate in

40 ml of solution A, dilute to 50 ml with carbon dioxide-free
water, cool to 0 and add 0.2 ml of dilute phenolphthalein
solution. If the solution is colourless, not more than 1.0 ml of
0.1 M sodiul/1 hydroxide is required to produce a red colour
that persists for not less than 15 seconds. If the solution is
red, not more than 0.25 ml of 0.1 M hydrochloric acid is
required to change the colour of the solution. Reserve the
neutralised solution for the test for Fluorosilicate.
Fluorosilicate. Heat to boiling the solution reserved in the
test for Acidity or alkalinity and titrate while hot with
0.1 M sodium hydroxide until a red colour is produced. Not
more than 1.5 ml of 0.1 M sodium hydroxide is required.
Sulphates (2.3.17). Dissolve 0.25 gin 10 ml of a saturated
solution of boric acid in distilled water and add 5 ml of
distilled water and 0.6 ml of 7 M hydrochloric acid. The
solution complies with the limit test for sulphates (200 ppm).
Prepare the standard by mixing together 0.6 ml of 7 M hydrochloric acid, 5 ml of sulphate standard solution
(10 ppm SO4) and 10 ml of a saturated solution of boric acid
in distilled water.
Assay. Weigh accurately about 80 mg, add a mixture of5 ml of
acetic anhydride and 20 ml of anhydrous glacial acetic acid
and heat to dissolve. Cool, add 20 ml of dioxan. Titrate with
indicator, until a green colour is produced. Carry out a blank
titration.
Category. Preventive for dental caries.
NaF.
Description. A white powder or colourless crystals.
Identification
A. Dissolve 2.5 g in sufficient carbon dioxide-ji-ee water
without heating to produce 100 ml (solution A). To 2 ml of
solution A add 0.5 ml of calcium chloride solution; a gelatinous
white precipitate is produced which dissolves on adding 5 ml
of ferric chloride solution.
II
N~ -O/S~OH ,2H 2 0
B. Add about 4 mg to a mixture of 0.1 ml of alizarin red S

solution and 0.1 ml of zirconyl nitrate solution and mix; the
colour changes to yellow.
. Mol Wt. 154.1
Tests
Sodium Formaldehyde Sulphoxylate is monosodium

hydroxymethane sulphinate dihydrate. It may contain a
suitable stabilising agent such as sodium carbonate.

colourless (2.4.1).
Sodium Formaldehyde Sulphoxylate contains an amount of

CH3Na03S equivalent to not less than 45.0 per cent and
C. Gives reaction A ofsodium salts (2.3.1).
2122
IP 2010
SODIUM FUSIDATE
not more than 55.0 per cent of S02, calculated on the dried
basis.


water, add sufficient water to produce 50.0 ml and mix. To 4.0
ml of this solution add 100 ml of water and titrate with 0.1 M
iodine using 3 ml of starch solution, added towards the end
of the titration, as indicator.
Description. White crystals or hard white masses; odour,

characteristic and garlic-like.
Identification
A. Dissolve about 4 g in 10 ml ofwater in a test-tube and add
1 ml of ammoniacal silver nitrate solution; metallic silver is
produced either as a finely divided grey precipitate or as a
bright metallic mirror on the inner surface of the tube.
B. Add about 50 mg to a solution of40 mg ofsalicylic acid in
5 ml of sulphuric acid and warm very gently; a permanent
deep red colour develops.
1 ml of 0.1 M iodine is equivalent to 0.001602 g ofS0 2.

Sodium Fusidate
COONa
Tests
Alkalinity. Dissolve 1.0 g in 50 ml ofwater and add 0.15 mlof
dilute phenolphthalein solution; not more than 3.5 ml of
0.05 M sulphuric acid is required to change the colour ofthe
solution.
pH (2.4.24). 9.5 to 10.5, determined in a2.0 per centw/v solution
Iron (2.3.14). Ignite 1.0 g, initially at a low temperature until
thoroughly charred and finally at about 600, preferably in a
muffle furnace, until all the carbon has been burnt off. Cool,
dissolve the residue in 2 ml ofhydrochloric acid and dilute to
50 ml with water. Add about 50 mg ofammonium persulphate
and 5 ml ofammonium thiocyanate solution, mix and transfer
to a Nessler cylinder. The red colour of the solution is not
more intense than that of 1.0 ml of iron standard solution
(10 ppm) treated in the same manner.
Mol. Wt. 538.7

Sodium Fusidate is sodium(17Z)-16~-acetoxy-3a,11a-di
hydroxyfusida-17(20),24-diene-2l-oate, produced by the
growth of certain strains of Fusidium coccineum or by any
other means.
Sodium Fusidate contains not less than 97.5 per cent and not
more than 101.0 per cent of C31 H 47Na06, calculated on the
anhydrous basis.
Dose. 1.5 g daily, in divided doses.
Sulphides. Dissolve 6 g in 14 ml of water in a test-tube and

wet a strip of lead acetate paper in the clear solution; no
discolouration is evident within 5 minutes.

slightly hygroscopic.
Sodium sulphite. Not more than 5.0 per cent, calculated as

Na2S03, determined by the following method. Transfer 4.0 ml
of the solution obtained in the Assay to a flask, add 2 ml of
formaldehyde solution and titrate with 0.1 M iodine that is
used for the Assay, adding starch solution towards the end
of the titration as indicator.
A.Dissolve 0.1 gin 5 ml ofwater, add 5 ml ofchloroform and

0.1 ml ofa 10 per cent w/w solution ofphosphoric acid, shake
vigorously for 1 minute, allow to separate and filter the lower
layer through absorbent cotton covered with anhydrous
sodium sulphate. Repeat the extraction with two quantities,
each of 5 ml, of chloroform, evaporate the combined extracts
at a pressure of 2 kPa, dry the residue over phosphorus
pentoxide at a pressure not exceeding 0.7 kPa for 2 hours and
dissolve in 1 ml of chloroform IR.
Calculate the percentage of Na2S03 from the expression

78.775(V2- vyw,
where VI and V 2are the volumes, in mI, of0.1 M iodine consumed
in this test and in the Assay respectively and W is the weight,
in g, of the substance under examination taken for the Assay.
Identification

2123
SODIUM FUSIDATE
IP 2010
obtained withfitsidic acid RS or with the reference spectrum

offusidic acid.

cent).

chromatogram obtained with the test solution (b) corresponds
to that in the chromatogram obtained with reference solution
(a).

0.5g.
C. Ignite 1 g. The residue gives the reactions of sodium salts

(2.3.1).

of 15 ml of water and 20 ml of ethanol (95 per cent). Titrate
with 0.1 M hydrochloric acid to pH 4.1, stirring continuously,
Tests
1 ml of 0.1 M hydrochloric acid is equivalent to 0.05387 g of

C31H47Na06.

dioxide-free water is not more intensely coloured than
reference solution BS6 (2.4.1).
Sodium Hydroxide
Specific optical rotation (2.4.22). +5.0 to +8.0, determined at

20 by dissolving 1.5 g in 25 ml of water, adding 0.1 ml of
5 M ammonia and diluting to 50 ml with water.
Caustic Soda
NaOH

(2.4.14).
Sodium Hydroxide contains not less than 97.0 per cent and
not more than 100.5 per cent oftotal alkali, calculated as NaOH.

Category. Pharmaceutical aid (allcalising agent).
Reference solution (a). Dissolve 5 mg of 3-ketofilsidic acid

RS in 5 ml ofthe mobile phase. To 1.0 ml of this solution add
0.2 ml ofthe test solution and dilute to 20.0 ml with the mobile
phase.
Reference solution (b). Dilute 20 III of the test solution to
- mobile phase: a mixture of 10 volumes of methanol, 20
volumes of 1.0 per cent w/v solution of orthophosphoric
acid, 20 volumes of water and 50 volumes of
acetonitrile,
resolution between the peaks due to 3-ketofusidic acid and
sodium fusidate is not less than 2.5.
Mol. Wt. 40.0
Description. White, crystalline masses supplied as sticks,

pellets or slabs; deliquescent. Readily absorbs carbon dioxide.
CAUTION - Great care should be exercised in handling

Sodium Hydroxide as it rapidly destroys tissues.
Identification
A. Carefully dissolve 5.0 g in 12 ml of distilled water, add
17 ml of 7 M hydrochloric acid, adjust the pH to 7.0 with
1 M hydrochloric acid and add sufficient distilled water to
produce 50 ml (solution A). 2 ml ofsolution A gives reaction A
of sodium salts (2.3.1).
B. pH ofa 0.01 percentw/v solution, not less than 11.0 (2.4.24).
Tests
Arsenic (2.3.10). Dissolve 2.5 gin 50 ml of water, add 16ml of
brominated hydrochloric acid AsT, and remove the excess of
bromine with a few drops of stannous chloride solution AsT.
The resulting solution complies with the limit test for arsenic
(4 ppm).
Inject the test solution, reference solution (a) and (b). The
signal-to-noise ratio of the principal peak is not less than 3. Heavy metals (2.3.13). Dissolve 1.0 ginS ml of water and 10 ml
Run the chromat()gram 3.5 times the retention time ofprincipal of 3 M hydrochloric acid, heat to boiling, cool and dilute to
peak. The sum of all the secondary peaks is not more than 4 25 ml with water. The solution complies with the lirriit test f6r
times the area of the prinCIpal peak in the c1ITolTlatogtam , heavy metals, Method A ( 20 ppm).
obtained with reference solution (a) (4.0per cent). Ignore any Iron (2.3.14). 20 ml ofsolution A complies with the limittest for
peak with an area less than the area ofthe principal peak in the iron (20 ppm).
2124
IP 2010
COMPOUND SODIUM LACTATE AND DEXTROSE INJECTION
Carbonates. Not more than 2.0 per cent, calculated as Na2C03,

C. Carry out reaction C of calcium salts (2.3.1); no white

Chlorides (2.3.12). Dissolve 2.0 g in 5 ml of water, acidify with

about 8 ml of nitric acid and dilute to 20 ml with water. The
solution, without the addition of dilute nitric acid, complies
Tests
Sulphates (2.3.17). Dissolve 2.0 gin 6 ml of distilled water,

adjust the pH to 7 with hydrochloric acid and dilute to 20 ml
with distilled water. The resulting solution complies with the
limit test for sulphates (75 ppm).
pH (2.4.24).5.0 to 7.0.
per millimole.
Assay. Measure accurately 10 ml, evaporate to dryness in a

platinum dish and ignite very gently until completely
carbonised. Boil the residue with 25.0 ml of 0.05 M sulphuric
acid, filter and wash thoroughly with hot water. Titrate the
Assay. Weigh accurately about 2.0 g, dissolve in about 80 ml .excess of acid in the combined filtrate and washings with
of carbon dioxide-free water, add '0.3 ml of phenolphthalein 0.1 M sodium hydroxide using methyl orange solution as
solution and titrate with 1 M hydrochloric acid. Add 0.3 ml indicator.
of methyl orange solution and continue the titration with
1 ml of 0.05 M sulphuric acid is equivalent to 0.Q1l21 g of .
1 M hydrochloric acid.
Potassium. Acidify 2.5 ml of solution A with acetic acid and
add 0.15 ml of sodium cobaltinitrite solution; no precipitate
is formed.
C3HsNa03.
1 ml of 1 M hydrochloric acid used in the second part of the

titration is equivalent to 0.1060 g ofNa2C03'
1 ml of 1 M hydrochloric acid used in the combined titrations
is equivalent to 0.0400 g oftotal alkali, calculated as NaOH.
. Storage. Store protected from moisture, in non-metallic
containers.

On keeping, small solid particles may separate from the solution
in glass containers.
Labelling. The label states (1) that the Injection is one-sixth
molar and contains, in one litre, approximately 167 millimoles
each of sodium ions and of bicarbonate ions (as lactate); (2)
that the injection should not be used if the solution contains
visible solid particles.
Sodium Lactate Intravenous Infusion

Sodium Lactate Injection is a sterile solution containing
1.85 per cent w/v ofsodium lactate in Water for Injections. It is
prepared from Lactic Acid with the aid ofSodium Hydroxide
and sufficient Dilute Hydrochloric Acid to adjust the pH of
the solution.
Sodium Lactate Injection contains not less than 1.75 per cent
and not more than 1.95 per cent w/v of sodium lactate,
C3HsNa03.
Dose. By intravenous infusion at the rate of 5 ml or less per
minute.
Identification
A. When warmed with potassium permanganate, gives
acetaldehyde, recognisable by its odour.
B. The residue on evaporation, when moistened with
flame, imparts a yellow colour to the flame.
Compound Sodium Lactate and

Dextrose Injection
Compound Sodium Lactate with Dextrose Intravenous
Infusion; Ringer-Lactate Solution with Dextrose for
Injection; Hartmann's Solution with Dextrose for
Injection.
Compound Sodium Lactate and Dextrose Injection is a sterile
solution containing 0.24 per cent v/v ofLactic Acid (equivalent
to 0.32 per cent wIv ofsodium lactate) with 0.6 per cent w/v of
Sodium Chloride, 0.04 per cent w/v of Potassium Chloride,
0.027 per cent w/v of Calcium Chloride and 5 per cent w/v of
Dextrose in Water for Injections.
Compound Sodium Lactate and Dextrose Injection contains
w/v of sodium, Na, not less than 0.019 per cent and not more
than 0.022 percentw/v ofpotassium, K, not less than 0.37 per
cent and not more than 0.42 per cent w/v oftotal chloride, Cl,
w/v of calcium chloride, CaCh,2H 20, and not less than
2125
COMPOUND SODIUM LACTATE AND DEXTROSE INJECTION
0.23 per cent and not more than 0.28 per cent w/v oflactate,
calculated as C3H60 3 , and not less than 4.50 per cent and not
more than 5.25 per cent w/v of dextrose, C6H 120 6 It contains
no antimicrobial agent.
solution.
Identification
A. To 1 ml add 0.05 ml of potassium cupri-tartrate solution;

B. When warmed with potassium permanganate gives
C. The residue on evaporation, when moistened with
flame imparts a yellow colour to the flame. When viewed
through a suitable blue glass, the flame is tinged reddish
purple.
D. Gives reaction C ofcalcium saltsand reaction A ofchlorides
(2.3.1).
IPlDID

standard solutions.
For total chlorides- To 20.0 ml add 30 ml of water, 50.0 ml of
a blank titration.
I ml of 0.1 M silver nitrate is equivalent to 0.003545 g oftotal
chloride, calculated as CL

0.01 M magnesium sulphate and 5 ml of ammonia buffer
to 0.00147 gofCaClz,2H20.
For lactate - Determine by liquid chromatography (2.4.14).

Test solution. The preparation under examination.
Reference solution. A 0.28 per cent w/v solution of lithium
lactate RS in the mobile phase.
Tests
pH (2.4.24). 4.0 to 6.5.
morethan0.25 (2.4.7).
Heavy metals (2.3.13). Evaporate a volume containing 4 g of
dextrose to 10 ml and add 2 ml of dilute acetic acid and
sufficient water to produce 25 mL The solution complies with
the limit test for heavy metals, Method A ( 5 ppm).
permL
(Injections).
Prepar~tions

589 nm and using sodium solution FP or sodium solution
solutions.

767 nm and using potassium solution FP or potassium
10 volumes of a 2 per cent v/v solution of ocfylamine in
acetonitrile, the pH of which is adjusted to 7.0 with a
10 per cent v/v solution of phosphoric acid,
and measure the responses for the major peak.
Calculate the content of C3H60 3, in the injection.

(2.4.22). The observed rotation in degrees multiplied by
0.9477 represents the weight, in g, ofdextrose, C6H 120 6, in the
volume taken for assay.
a temperature not exceeding 30. On keeping, small particles
may separate from the solution in glass containers.
Labelling. The label states (1) that the injection contain's, in
millimoles per litre, the following approximate amounts ofthe
2126
IP 2010
HALF STRENGTH COMPOUND SODIUM LACTATE AND DEXTROSE INJECTION
ions. sodium, 131, potassium, 5, calcium, 2; bicarbonate

(as lactate), 29 and Chloride, 111; (2) the total osmolar
concentration in mOsmol per litre; (3) that the injection should
not be used if it contains visible particles.

morethan0.25 (2.4.7).
perml.
Half Strength Compound Sodium

Lactate and Dextrose Injection
Half Strength Compound Sodium Lactate with Dextrose
Intravenous Infusion; Half Strength Ringer-Lactate
Solution with Dextrose Injection
Half Strength Compound Sodium Lactate and Dextrose
Injection is a sterile solution containing 0.12 per cent v/v of
Lactic Acid (equivalent to 0.16 per cent wIv ofsodium lactate)
with 0.3 per cent w/v ofSodium Chloride, 0.02 per cent w/v of
Potassium Chloride, 0.0135 per cent w/v ofCalcium Chloride
and 5 per cent w/v of Dextrose in Water for Injections.
Compound Sodium Lactate and Dextrose Injection contains
w/v ofsodium, Na, not less than 0.0095 per cent and not more
than 0.011 per cent w/v of potassium, K, not less than
0.185 per cent and not more than 0.210 per cent w/v of total
chloride, Cl, not less than 0.0125 per cent and not more than
0.0145 per cent w/v ofcalcium chloride, CaClz,2HzO, and not
less than 0.115 per cent and not more than 0.140 per cent wIv
oflactate, calculated as C3H 60 3, and not less than 4.5 per cent
and not more than 5.25 per cent w/v of dextrose, C6H 1Z0 6 It
contains no antimicrobial agent.
solution.

Assay. For sodium - Dilute suitably with water and detennine
solutions.
For potassium - Dilute suitably with water and detennine

standard solutions.
a blank titration.
1 ml of 0.1 Msilvernitrate is equivalent to 0.003545 g oftotal
A. To 1 ml add 0.05 ml of potassium cupri-tartrate solution;

red precipitate is fonned.
For calcium chloride

To 50.0 ml add 5.0 ml of
0.01 M magnesium sulphate and 5 ml of ammonia buffer pH
10.9 and titrate with 0.01 M disodium edetate using eriochrome
black T mixture as indicator. From the volume of


to 0.00147 gofCaClz,2HzO.

purple.
Identification
D. Gives reaction C ofcalcium salts and reaction A ofchlorides

(2.3.1).
Tests
pH (2.4.24). 4.0 to 6.5.

10 volumes of a 2 per cent v/v solution of ocfylamine in
2127
MODIFIED COMPOUND SODIUM LACTATE AND DEXTROSE INJECTION

flow rate. 2 m1 per minute,
Calculate the content of C3H60 3 in the injection.

represents the weight, in g, ofdextrose, C6H 120 6, in the volume
taken for assay.
Labelling. The label states (1) that the injection contains, in
ions. sodium, 65.5, potassium, 2.5, calcium, 1, bicarbonate
(as lactate), 14.5, and chloride, 55.5; (2) the total osmolar
Modified Compound Sodium Lactate

and Dextrose Injection
IP 2010

Description. A clear, colourless or faintly. straw-coloured
solution.
Identification
A. To 1 ml add 0.05 ml ofpotassium cupri-tartrate solutiori;
purple.
D. Gives reaction C ofcalcium salts and reaction A ofchlorides
(2.3.1).
Tests
pH (2.4.24). 4.0 to 6.5.
maximum at about 284 nm; absorbance at about 284 nm (2.4.7),
not more than 0.25.
perml.
Modified Compound Sodium Lactate with Dextrose

Intravenous Infusion; Modified Lactated Ringer's and
Dextrose Injection.
is a sterile solution containing 0.048 per cent v/v of Lactic
Acid (equivalent to 0.064 per cent w/v ofsodium lactate) with
0.12 per cent w/v of Sodium Chloride, 0.008 per cent w/v of
Potassium Chloride, 0.0054 per cent w/v ofCalcium Chloride
and 5 per cent w/v of Dextrose in Water for Injections.
contains not less than 0.054 percent and not more than
0.064 per cent w/v ofsodium, Na, not less than 0.0038 per cent
and not more than 0.0044 per cent w/v of potassium, K, not
less than 0.074 per cent and not more than 0.084 per cent w/v
oftotal chloride, Cl, not less than 0.005 per cent and not more
than 0.0058 per cent w/v ofcalcium chloride, CaCIz,2H20, and
w/v oflactate, calculated as C3H60 3, and not less than 4.5 per
cent and not more than 5.25 per cent wIv ofdextrose, C6H 1206.
It contains no antimicrobial agent.

solutions.
For potassium
Dilute suitably with water and determine
standard solutions.
For total chlorides - To 20.0 ml add 30 m1 ofwater, 50.0 mlof
thiocyanate using ferriC ammonium sulphate solution as
2128
IP 2010
COMPOUND SODIUM LACTATE INJECTION

a blank titration.

Compound Sodium Lactate IntrFlvenous Infusion; RingerLactate Solution for Injection; Hartrnann's Solution for
Injection
For calcium chloride - Evaporate 100.0 ml on a water-bath

to 50 ml, add to this solution 5.0 ml of 0.01 M magnesium
sulphate and 5 ml of ammonia bufferpH 10.9 and titrate with
0.01 M disodium edetate using eriochrome black T mixture
as indicator. From the volume of 0.01 M disodium edetate
required subtract the volume of 0.01 M magnesium sulphate
added.
1 ml ofthe remainder of O. DIMdisodium edetate is equivalent
to 0.00147 gofCaCI2,2H20.

.
octadecylsilane bonded to porous silica (5 f.Lm),
10 volumes of a 2 per cent v/v solution of octylamine in
injection volume. 10 f.Ll.
Calculate the content of C3H 60 3 in the injection.
Compound Sodium Lactate Injection is a sterile solution

containing 0.24 per cent v/v of Lactic Acid (equivalent to
0.32 per cent w/v of sodium lactate) with 0.6 per cent w/v of
Sodium Chloride, 0.04 per cent w/v ofPotassium Chloride and
0.027 per cent w/v ofCalcium Chloride in Water for Injections.
Compound Sodium LaCtate Injection contains not less than
0.27 per cent and not more than 0.32 per cent w/v of sodium,
Na, not less than 0.019 per cent and not more than 0.022 per
cent w/v of potassium, K, not less than 0.37 per cent and not
more than 0.42 per cent w/v oftotal chloride, CI, not less than
0.025 per cent and not more than 0.029 per cent wIv ofcalcium
chloride, CaCh,2H20, and not less than 0.23 per cent and not
more than 0.28 per cent w/v oflactate, calculated as C 3H 60 3
Identification
A. When warmed with potassium permanganate gives

flame, imparts a yellow colour to the flame. When viewed
through a suitable blue glass, the flame is tinged with reddish
purple.
C. Gives reaction C ofcalcium salts (2.3.1).
Tests

30 minutes and determine the optical rotation in a 2-dm tube
(2.4.22). The observed rotation in degrees multiplied by
0.9477 represents the weight, in g, ofdextrose, C6H 120 6 , in the
volume taken for assay.
ions. sodium, 26.2, potassium, 1, calcium, 0.4, bicarbonate (as
lactate), 5.8, and chloride, 22.2; (2) the total osmolar
pH (2.4.24). 5.0to 7.0.

Unit per ml.
by Method A for flame photometry (2.4.4), or by atomic
and using sodium solution FP or sodium solution AAS,
suitably diluted for the standard solutions.

by Method A for flame photometry (2.4.4), or by atomic
and using potassium solution FP or potassium solution AAS,
suitably diluted for the standard solutions.
2129
COMPOUND SODIUM LACTATE INJECTION
IP 2010
For total chlorides- To 20 ml add 30 mlofwater, 50.0 ml of

0.1 M silver nitrate and 2 ml of nitric acid, filter, wash the
titrate the excess of silver nitrate with 0.1 Mammonium
than 0.022 percentw/v ofpotassium, K, not less than 0.37 per

cent and not more than 0.42 per cent w/v oftotal chloride, Cl,
w/v of calcium chloride, CaClz,2HzO, and not less than
0.23 per cent and not more than 0.28 per cent w/v oflactate,
calculated as C3H60 3 It contains no antimicrobial agent.

Category. Irrigation fluid.
For calcium chloride- To 50 ml add 5.0 ml ofO. 01 M magnesium

sulphate and 5 ml ofammonia bufferpH 10.9 and titrate with
0.01 M disodium edetate using mordant black 11 mixture as
indicator. From the volume of 0.01 M disodium edetate
required subtract the volume of 0.01 M magnesium sulphate
added.
to 0.00147 g ofCaCh, 2HzO.
For lactate, calculated as C3H 60 3 - Evaporate 50 ml to

dryness in a platinum dish and ignite very gently until
completely carbonised. Boil the residue with 25.0 ml of
0.05 M sulphuric acid, filter, and wash thoroughly with hot
water.Titrate the excess of acid in the combined filtrate and
washings with 0.1 M sodium hydroxide using methyl orange
1 ml of O. 05 M sulphuric acid is equivalent to 0.009008 g of
C3H60 3

in glass containers.
ions. sodium, 131, potassium, 5, calcium, 2, bicarbonate
(as lactate), 29 and chloride, Ill; (2) that the injection should
not be used if the solution contains visible solid particles.

Identification
A. When warmed with potassium permanganate gives
purple.
C. Gives reaction C ofcalcium salts and reaction A ofchlorides
(2.3.1).
Tests
pH (2.4.24). 5.0to 7.0.
perml.
solutions.
standard solutions.
Compound Sodium Lactate Solution

for Irrigation
Ringer-Lactate Solution for Irrigation; Hartmann's
Solution for Irrigation
Compound Sodium Lactate Solution for Irrigation is a sterile
solution containing 0.24 per cent v/v ofLactic Acid (equivalent
to 0.32 per cent wIv ofsodium lactate) with 0.6 per cent wIv of
Sodium Chloride, 0.04 per cent w/v ofPotassium Chloride and
0.027 per cent w/v ofCalcium Chloride in Water for Injections.
Compound Sodium Lactate Solution for Irrigation contains
w/v of sodium, Na, not less than 0.019 per cent and not more

O.O} M magnesium sulphate and 5 ml of ammonia buffer

to 0.00147 g ofCaCh, 2HzO.
2130
IP 2010
SODIUM LAURYL SULPHATE

a blank titration.
1 ml of0.1 M silver nitrate is equivalent to 0.003545 g oftotal
10 volumes ofa 2 per cent v/v solution ofoctylamine in
10 per cent v/v solution ofphosphoric acid,
Calculate the content of C3H60 3, in the injection.
in glass containers. The container may be designed to empty
rapidly and may contain a volume ofmore than one litre.
Labelling. The label states (1) that the irrigation solution
contains, in millimoles per litre, the following approximate
amounts of the ions. sodium, 131, potassium, 5, calcium,
2, bicarbonate (as lactate), 29, and chloride, 111; (2) that the
solution should not be used ifit contains visible particles; (3)
'For irrigation only' and 'Not for injection';-(4) that once the
container is opened, the unused portion should be discarded.
Identification
A. A 1 per cent w/v solution, when shaken, produces plenty
offoam.
B. Mix 0.1 mlofa 1percentw/v solution with 0.1 mlofaO.1 per
cent w/v solution ofmethylene blue and 2 ml of 1 M sulphuric
acid, add 2 ml of dichloromethane and shake; the
dichloromethane layer is intensely blue.
C. Mix about 10 mg with 10 ml of ethanol and heat to boiling

on a water-bath, shaking frequently. Filter immediately and
evaporate the ethanol. Dissolve the residue in 8 ml of water,
add 3 ml of 2 M hydrochloric acid, evaporate the solution to
half its volume and cool. Filter and to the filtrate add 1 ml of
barium chloride solution; a white, crystalline precipitate is
produced.
D. Gives reaction B ofsodium salts (2.3.1).
Tests
Alkalinity. Dissolve 1.0 g in 100 ml of carbon dioxide-free
water and add 0.1 ml ofphenol red solution. Not more than
0.5 ml of 0.1 M hydrochloric acid is required to change the
colour of the solution.
Non-esterified alcohols. Not more than 4 per cent, detelmined
by the following method. Dissolve 10.0 g in 100 ml bfwater,
add 100 ml of ethanol (95 per cent) and extract the solution
with three quantities, each of 50 ml, of n-pentane, adding
sodium chloride, if necessary, to promote separation of the
two layers. Wash the combined organic layers with three
quantities, each of 50 ml, of water. Dry the organic solution
over anhydrous sodium sulphate, filter and evaporate on a
water-bath until the odour ofpentane is no longer detectable.
Heat the residue at 105 for 15 minutes, cool and weigh.
Sodium Chloride and Sodium Sulphate. Not more than a total
of8.0 per cent, determined by the following methods.
For sodium chloride-Dissolve 5.0 gin 50 ml of water, add
2 M nitric acid dropwise until the solution is neutral to litmus
paper, add 2 ml of potassium chromate solution and titrate
with 0.1 M silver nitrate.
1 ml of0.1 M silver nitrate is equivalent to 0.005844 g ofNaCl.
Sodium Lauryl Sulphate

Sodium Lauryl Sulphate is a mixture ofsodium alkyl sulphates
consisting mainly of sodium dodecyl sulphate,
CH3 [CH z]'oCHzOS03Na.
Sodium Lauryl Sulphate contains not less than 85.0 per cent
ofsodium alkyl sulphates, calculated as ClzHzsNa04S,
Category. Pharmaceutical aid (anionic emulsifying agent).
Description. A white or pale yellow powder or crystals.
For sodium sulphate - Dissolve 0.5 g in 20 ml of water,

warming gently if necessary, and add 1 ml of a 0.05 per cent
w/v solution of dithizone in acetone. If the solution is red,
add 1 M nitric acid, dropwise, until it becomes bluish green.
Add 2 ml ofdichloroacetic acidsolution and 80 ml ofacetone
and titrate with 0.01 M lead nitrate until a permanent orangered colour is obtained.
1 ml of 0.01 M lead nitrate is equivalent to 0.001420 g of
NazS04'
2131
SODIUM METABISULPHITE
IP 2010

water to produce 1000.0 ml, warming ifnecessary. To 20.0 ml
add 15 ml of chloroform, 10 ml of dilute sulphuric acid and
1 ml of dimethyl yellow-oracet blue B solution and titrate
with 0.004 M benzethonium chloride, shaking vigorously
and allowing the layers to separate after each addition, until
the chloroform layer acquires a permanent clear green colour.
1 ml of 0.004 M benzethonium chloride is equivalent to
0.001154 g of sodium alkyl sulphates, calculated as
ClzHzsNa04S,
hydrochloric acid and 20 ml ofwater and add a few drops of

bromine solution, cool, dilute with water to 25 mI, then add
50 mg of ammonium persulphate and 5 ml of ammonium
thiocyanate solution. Any colour produced is not more intense
than that obtained by adding 5 ml of ammonium thiocyanate
solution to a mixture of iron standard solution (20 ppm Fe),
2 ml of hydrochloric acid, 50 mg of ammonium persulphate
and sufficient water to produce 25 ml (40 ppm).
Thiosulphate. Dissolve 1.0 g in 10 ml of 2 M hydrochloric

acid and heat on a water-bath for 10 minutes; not more than a
faint opalescence is produced.
Assay. Weigh accurately about 0.1 g and dissolve in 50.0 ml

0.05 M iodine, add 1 ml of hydrochloric acid and titrate the
excess of iodine with 0.1 M sodium thiosulphate using starch
solution, added towards the end of the titration, as indicator.
Sodium Pyrosulphite; Sodium Disulphite
1 ml of 0.05 Miodine is equivalent to 0.004753 g ofNazSzOs.
NazSzOs
Mol. Wt. 190.1
Sodium Metabisulphite may be prepared by saturating a

solution of Sodium Hydroxide with sulphur dioxide and
allowing crystallisation to occur.
Sodium Metabisulphite contains not less than 95.0 per cent
and not more than 100.5 per cent ofNazSzOs.
Storage. Store protected from light and moisture. On exposure

to air and moisture it is slowly oxidised to sulphate with
disintegration of the crystals.
Sodium Methylparaben
Sodium Methyl Hydroxybenzoate
Description. Colourless, prismatic crystals or a white or creamy

white powder; odour, sulphurous.
Identification
A. Gives the reactions of sodium salts (2.3.1).
B. A solution decolorises iodinatedpotassium iodide solution
and the resulting solution gives the reactions of sulphates
(2.3.1).
Tests
Acidity. A solution is acidic to phenol red solution.
Arsenic (2.3.10). Mix 2.5 g in a porcelain dish with 10 ml of
water, 1.25 g ofpotassium chlorate and 16 ml ofhydrochloric
acid and heat to expel chlorine, remove the last traces of
chlorine with a few drops of stannous chloride solution AsT
and add 35 ml of water. The resulting solution complies with
Heavy metals (2.3.13). Dissolve 1.0 g in 10 mi ofwater, add 5 ml
of hydrochloric acid and evaporate to dryness on a waterbath. Dissolve the residue in 25 ml ofwater containing 2 ml of
dilute acetic acid. The solution complies with the limit test
for heavy metals, Method A ( 20 ppm).
Iron. To 0.5 g add 2 ml ofhydrochloric acid and evaporate on
a water-bath to dryness. Dissolve the residue in 2 ml of
CsH7Na03
Mol. Wt. 174.1
Sodium Methylparaben is the sodium salt of methyl

4-hydroxybenzoate.
Sodium Methylparaben contains not less than 99.0 per cent
and not more than 102.0 per cent ofCsH7Na03' calculated on
Identification
A. Dissolve 0.5 g in 5 ml ofwater and acidify to litmus paper
with hydrochloric acid; a white precipitate is produced. Wash
the precipitate with water and dry.
Compare the spectrum with that obtained with methylparaben
RS.
2132
IP 2010
SODIUM PHOSPHATE
B. The residue on ignition gives the reactions of sodium salts

(2.3.1).
Sodium Phosphate
Tests
Disodium Hydrogen Phosphate; Disodium Hydrogen

Phosphate Dodecahydrate
Appearance of solution. Al 0.0 per cent w/v solution in water

is clear (2.4.1).
NalIP04, 12HzO
pH (2.4.24). 9.5 to 1O.5,determined in a 0.1 per cent w/v solution.
Mol. Wt. 358.1
Sodium Phosphate contains not less than 98.0 per cent and
not more than 101.0 per cent ofNazHP0 4, calculated on the
anhydrous basis.

Category. Cathartic; pharmaceutical aid (buffering agent).

Description. Colourless, transparent crystals; very

efflorescent.

examination in 10 ml of water. Immediately add 2 ml of
hydrochloric acid and shake with 50 illl of ether. Evaporate
the upper layer to dryness and take up the residue with 10 ml
of acetone.
Reference solution (a). Dissolve 34.3 mg of 4-hydroxybenzoic
acid (sodium propylparaben impurity A) in 100.0 of acetone.
100.0 ml with acetone.
Reference solution (c). Dissolve 10 mg of ethyl
parahydroxybenzoate RS in 1 ml oftest solution (a) and dilute
to 10.0 ml with acetone.
Apply 5 IJ.I ofeach solution. Allow the mobile phase to rise 15
spot due to 4- hydroxybenzoic acid is not more intense than
the corresponding spot in the chromatogram obtained with
reference solution(a) (4.0 per cent) and any other secondary
spot is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.5 per cent). The test is
solution (c) shows two clearly separated principal spots.
Identification
A 10.0 per cent w/v solution in distilled water (solution A)
gives the reactions of sodium salts and ofphosphates (2.3.1).
Tests
colourless (2.4.1).
Arsenic (2.3.10). Dissolve 5.0 gin50mlofwaterandadd IOrnl
of stannated hydrochloric acid AsT. The resulting solution '
Heavy metals (2.3.13). Dissolve 2.0 g in 10 rnl of water, adding
4 ml of 1 M acetic acid and diluting to 25 ml with water. The
A(lOppm).
Iron (2.3.14). 20 ml ofsolutionA complies with the limittest for,
iron (20 ppm).
Chlorides (2.3.12). To 10 ml of solution A add 10 ml of
2 M nitric acid and dilute to 20 ml with water. The resulting
solution complies with the limit test for chlorides (250 ppm).
Chlorides (2.3.12). Dissolve 1.0 g in 20 ml of water, add 0.2 ml

of nitric acid and filter. 15 ml ofthe filtrate complies with the
limit test for chlorides (330 ppm).
Sulphates (2.3.17). To 2.5 ml of solution A add 2 ml of

2 M hydrochloric acid and dilute to 15 ml with distilled water.
The resulting solution complies with the limit test for sulphates
(600 ppm).
Sulphates (2.3.17). Dissolve 0.5 gin 40 ml of water, add 3.5 ml

of 2 M hydrochloric acid, dilute to 60 ml with water and filter.
15 ml of the filtrate complies with the limit test for sulphates
(0.12 per cent).
Reducing substances. To 5 ml of solution A add 5 ml of

1 M sulphuric acid and 0.25 ml of 0.02 M potassium
permanganate and heat on a water-bath for 5 minutes; the red
colour is not completely discharged.
'
Water (2.3.43). Not more tban 5.0 percent, determined on 1.0 g.
Sodium dihydrogen phosphate. The value ofthe expression

(nr 25)/(25 - nl),
Assay. Dissolve 0.15 gin 50 ml of anhydrous acetic acid.

CgH7Na03'
where nl and n2 are the titres of 1 M sodium hydrOXide obtained

in the Assay, does not exceed 0.025.
Water (2.3.43). 57.0 to 61.0 per cent, determined on 0.1 g
dissolved in a mixture of 10 volumes of methanol and 40
volumes of dimethylformamide.
2133
SODIUM PROPYLPARABEN
IP 2010
Assay. Weigh accurately about 4.0 g (w), dissolve in 25 ml of

water, add 25.0 ml of i M hydrochloric acid and titrate
potentiometrically with 1 M sodium hydroxide to the first
inflection ofthe pH curve (nl mI). Continue the titration until
the second inflection ofthe curve is reached; the total volume
of sodium hydroxide required is n2 ml.
Calculate the content of NazHP0 4 from the expression
1420(25 - n l)/W (100 - d),
where d is the percentage water content.

examination in 10 ml of water. Immediately add 2 ml of
hydrochloric acid and shake with 50 m1 of ether. Evaporate
the upper layer to dryness and take up the residue with 10 ml
of acetone.
Reference solution (a). Dissolve 34.3 mg of4-hydroxybenzoic
acid (sodium propylparaben impurity A) in 100.0 of acetone.
Reference solution (b). Dilute 0.5 ml oftest solution (a) to 100
ml with acetone.
Reference solution (c). Dissolve 10 mg of ethyl

parahydroxybenzoate RS in 1 m1 oftest solution (a) and dilute
to 10.0 m1 with acetone.
Sodium Propylparaben
Sodium Propyl Hydroxybenzoate
Mol. Wt. 202.2

Sodium Propylparaben is the sodium salt of propyl
4- hydroxybenzoate.
Sodium Propylparaben contains not less than 99.0 per cent
and not more than 102.0 per cent ofCIOHIINa03, calculated on
Apply 5 f-LI ofeach solution. Allow the mobile phase to rise 15

spot due to 4- hydroxybenzoic acid is not more intense than
the corresponding spot in the chromatogram obtained with
reference solution (a) (4.0 per cent) and any other secondary
obtained with reference solution (b) (0.5 per cent). The test is
solution (c) shows two clearly separated principal spots.
Chlorides (2.3.12). Dissolve 1.0 g in 20 ml ofwater, add 0.2 ml
of nitric acid and filter. 15 ml ofthe filtrate complies with the
Sulphates (2.3.17). Dissolve 0.5 g in 40 ml ofwater; add 3.5 ml
of 2 M hydrochloric acid, dilute to 60 ml with water and filter.
15 ml ofthe filtrate complies with the limit test for sulphates
(0.12 percent).
Water (2.3.43). Not more than 5.0 per cen, determined on 1.0 g.
Identification
A. Dissolve 0.5 gin 5 ml of water and acidify to litmus paper
with hydrochloric acid; a white precipitate is produced. Wash
the precipitate with water and dry.
Compare the spectrum with that obtained withpropylparaben
RS.
B. The residue on ignition gives the reactions ofsodium salts
(2.3.1).
Assay. Dissolve 0.15 g in 50 ml of anhydrous acetic acid.

potentiometrically (2.4.25). Carry out the blank titration.
CIOHIINa03'
Sodium Salicylate
Tests
COONa
~OH
Appearance ofsolution. A 10.0 per cent w/v solution in water

is clear (2.4.1).
pH (2A24); 9;5 to 105, determined in a 0.1 per cent w/v solution.

C7HsNa03
Mol. Wt. 160.1
Sodium Salicylate is sodium 2-hydroxybenzoate.
2134
IP 2010
SODIUM STARCH GLYCOLLATE
Sodium Salicylate contains not less than 99.0 per cent and not
more than 101.0 per cent ofC7HsNa03' calculated on the dried
basis.
Category. Anti-inflammatory; analgesic.
Dose. 500 mg to 2 g, with food; in the treatment of acute
rheumatism, 5 to 109 daily, in divided doses.
Description. Colourless, small crystals or shiny flakes or a
white, crystalline powder.

Cany out a blank titration.
C7HsNa03.
Identification
salicylate RS or with the reference spectrum of sodium
salicylate.

Sodium Carboxymethyl Starch
Sodium Starch Glycollate is the sodium salt of a poly-aglucopyranose in which some of the hydroxyl groups are in
the form ofcarboxymethyl ether.
B. A 10.0 per cent w/v solution in carbon dioxide-free water

prepared from distilled water (solution A) gives the reactions
ofsalicylates (2.3.1).
Sodium Starch Glycollate contains not less than 2.8 per cent
and not more than 4.5 per cent of sodium', Na, calculated on
the material washed with Ethanol (95 per cent) and dried as
described under Assay.
C. Gives reaction B ofsodium salts (2.3.1).
Category. Phannaceutical aid (tablet disintegrant).
Tests
Description. A very fine, white or off-white, free-flowing

powder; odourless or almost odourless.

more intensely coloured than reference solution BYS6 (2.4.1).
Identification
Acidity. To 20 ml ofsolutionA add 0.1 ml ofphenol redsolution;

the solution is yellow. Titrate with 0.01 M sodium hydroxide
to a reddish violet colour; not more than 2.0 ml of
0.01 M sodium hydroxide is required.

Compare the spectrum with that obtained with sodium starch
glycol/ate RS or with the reference spectrum ofsodium starch
glycollate.
Arsenic (2.3 .10). Mix 5.0 g with 10 ml of bromine solution and

evaporate to dryness on a water-bath. Ignite gently, cool,
dissolve the residue, ignoring any carbon, in 50 ml of water
and 14 ml of brominated hydrochloric acid AsT and remove
the excess ofbromine with 2 ml of stannous chloride solution
arsenic (2 ppm).
B. to 5 ml ofa 2 per cent w/v dispersion in water add 0.05 ml

of 0.005 M iodine; a dark blue colour is produced.
Heavy metals (2.3.13). Dissolve 2.0 g in 46 ml of water, add

with constant stirring 4 ml of dilute hydrochloric acid, filtering
and using 25 ml ofthe filtrate. The solution complies with the
limit test for heavy metals, Method A ( 20ppm).
pH (2.4.24).5.5 to 7.5, detennined in a2.0 percentw/v dispersion

Chlorides (2.3 .12). To 25 ml of solution A add 15 ml of water

and 10 ml of 2 M nitric acid and filter. 25 ml of the filtrate
complies with ~he limit test for chlorides (200 ppm).
Sulphates (2.3 .17). 2.5 ml of solution A diluted to 15 ml with
distilled water complies with the limit test for sulphates
(600 ppm).
C. The solution obtained in the test for Heavy metals gives

the reactions of sodium salts (2.3.1).
Tests
Heavy metals (2.3.13). To 4.0 g in a silica or platinum dish add

2 ml of a 50 per cent w/v solution of sulphuric acid, heat in a
water-bath and then cautiously over a flame at about 600.
Continue heating until all black particles have disappeared,
allow to cool, add 0.1 rol of 1 M sulphuric acid, heat to ignition
once again and allow to cool. Add 0.1 ml of 2 M ammonium
carbonate, evaporate to dryness and cautiously ignite. To
the residue add 5 ml of hydrochloric acid, evaporate to
dryness on a water-bath and dissolve the residue in 100 ml of
water. 25 ml ofa solution complies with the limit test for heavy
2135
SODIUM STARCH GLYCOLLATE
IP 2010
Iron (2.3.14). 50 ml of the solution obtained in the test for

Heavy metals complies with the limit test for iron (20 ppm).
Sodium Chloride. Not more than 10.0 per cent, determined by
the following method. To 1.0 g add 20.0 ml of 0.1 M silver
nitrate and 30 ml ofnitric acidand boil carefully for 30 minutes.
Cool and add a sufficient volume of a saturated solution of
potassium permanganate to change the colour ofthe solution
to red. Discharge the colour by the dropwise addition of
hydrogen peroxide solution (10 vol), add 3 m1 of dibutyl
phthalate and titrate with 0.1 M ammonium thiocyanate using
ferric ammonium sulphate solution as indicator, shaking
vigorously after each addition of titrant. Carry out a blank
titration.
SodiumAntimony Gluconate
Microbial Contamination (2.2.9). 1.0 g is free from Escherichia

coli and Salmonellae.
determined on 0.5 g by drying in an oven at 105.
Assay. Weigh accurately about 4.0 g, add 350 ml ofa mixture
of4 volumes of ethanol (95 per cent) and 1 volume of water,
add 0.25 ml of phenolphthalein solution and mix. Add
1 M sodium hydroxide dropwise until the colour of the
suspension becomes faintly pink, shake for 30 minutes and
decant through a sintered glass crucible. Repeat the extraction
three times, or until a test for chloride ions is negative. Transfer
the bulk of the residue to the crucible, wash the residue with
ethanol (95 per cent) and dry at 110 to constant weight.
Weigh accurately 0.5 g ofthe residue, add 80 ml ofanhydrous
glacial acetic acid, heat under a reflux condenser for 2 hours,
cool. Titrate with 0.1 Mperchloric acid, determining the endpoint potentiometrically (2.4.25).
1 ml of 0.1 M perchloric acid is equivalent to 0.0023 g ofNa.
OH
HO\/0
0\ O-Sb-O
o-stf
\0
/\
o OH
HO
1 ml of0.1 M silver nitrate is equivalent to 0.005844 g ofNaCI.

Sodium glycollate. To 0.2 g add 5 m1 of glacial acetic acid,
mix well and add 5 ml of water, stirring occasionally until
solution is complete. Slowly add 50 ml ofacetone with stirring
and then add 1 g of sodium chloride. Filter, wash the residue
with acetone and dilute the filtrate to 100 m1 with acetone.
Transfer 2 ml ofthis solution to an open flask, heat on a waterbath for exactly 20 minutes, cool, add 5 ml ofnaphthalenediol
reagent and mix thoroughly. Add a further 15 ml ofthe same
reagent, mix, cover the flask with aluminium foil and heat on a
water-bath for 20 minutes. Cool and dilute to 25 ml with
sulphuric acid. The absorbance (2.4.7) ofthe resulting solution
at the maximum at about 540 om using water as the blank, is
not more than that of a solution prepared in the following
manner. To 5 ml of a 0.062 per cent w/v solution ofglycollic
acid, previously dried at a pressure not exceeding 2 kPa for
16 hours, add 5 ml ofglacial.acetic acid, dilute to 100 ml with
acetone and complete the procedure described above
beginning at the words "Transfer 2 ml...." (2.0 per cent).
OH
HO
NaO
ONa
Sodium Stibogluconate is mainly the disodium salt of

~-oxy-bis[gluconato(3)_02,()3 ,()4-hydroxoantimony].
The method of manufacture is such as to ensure consistently
controlled reaction stoichiometry in order to yield sodium
stibogluconate that is satisfactory with regard to intrinsic
toxicity.
Sodium Stibogluconate contains not less than 30.0 per cent
and not more than 34.0 per cent of pentavalent antimony,
calculated on the dried and methanol-free basis.
Dose. By intramuscular or intravenous injection, 600 mg to 2 g
daily, for 10 to 30 days.
Description. A colourless, mostly amorphous powder;
Identification
A. An aqueous solution is dextro-rotatDly.
B. Pass hydrogen sulphide into a 5 percent w/v solution for
several minutes; an orange precipitate is produced.
C. When heated, it chars without melting, emitting an odour

ofburnt sugar and leaving a residue which gives the reactions
of antimony compounds and the reactions of sodium salts
(2.3.1).
.
Tests
pH (2.4.24),5.0 to 5.6, determined in the solution obtained in
the test for Stability of solution.
StabiliiYor solution. Heat a solution contaming 10.0 per cent

wlv ofpentavalent antimony in an autoclave at 115.5 arid at a
pressure of 70 kPa for 30 minutes. The resulting solution is
colourless or almost colourless.
2136
IP 2010
SODIUM STIBOGLUCONATE INJECTION
Trivalent antimony. Dissolve 2.0 g in 30 ml ofwater, add 15 ml

of hydi-ochloric acid and titrate with 0.00833 M potassium
bromate using methyl orange solution as indicator. Not more
than 1.3 ml of 0.00833 M potassium bromate is required.
Chlorides. Dissolve 2.5 g in 50 ml of water and add 2 ml of
2 M nitric acid and 75 ml ofacetate bufferpH 5.0. Titrate with
0.1 M silver nitrate, detennining the end-point potentiometrically (2.4.25). Not more than 3.0 ml of0.1 M silver nitrate
is required.
Methanol. Not more than 2.0 per cent w/w, determined by gas
Test solution. Add 5 ml of water to 0.5 g of the substance
under examination and mix with the aid ofultrasound until the
solution is complete.
Reference solution (a). Add 5 ml ofa 0.2 per centv/v solution
of ethanol (internal standard) to 0.5 g of the substance under
examination and mix with the aid ofultrasound Until the solution
is complete.
Reference solution (b). Add 1 ml ofa 1.0 per centv/v solution
ofmethanol to 5 ml ofa 0.2 per cent v/v solution ofthe internal
standard.
- a glass column 1.5 m x 4 mm, packed with porous polymer
beads (80 to 100 mesh),
- temperature: column. 130,
Calculate the percentage w/w ofmethanol taking 0.792 g as its
weight per ml at 20.

Sodium Antimony Gluconate Injection
Sodium Stibogluconate Injection is a sterile solution ofSodium
Stibog1uconate in Water for Injections. Injection in multiple
dose containers must not contain phenol as preservative.
Sodium Stibogluconate Injection contains not less than
pentavalent antimony, Sb.
Usual strength. The equivalent of 100 mg of pentavalent
antimony per m!.
solution.
Identification
A. It is dextro-rotatOlY.
B. Dilute 2 ml to 10 ml with water and pass hydrogen sulphide
through the solution; no immediate precipitate is produced.
Continue to pass hydrogen sulphide through the solution for
several minutes; an orange precipitate is produced.
C. The residue obtained by evaporation to dryness, after
incineration, gives the reactions of antimony compounds and
the reactions of sodium salts (2.3.1).
Tests
pH (2.4.24).5.0 to 5.6.
Undue toxicity. Dissolve a suitable quantity ofthe substance

under examination in water for injections to give a solution
containing 28 mg of pentavalent antimony per ml. Inject
intravenously 0.3 ml ofthe solution into each of 10 mice that
have been deprived of food for not less than 17 hours. After
injections allow the mice access to food and water. None of
the mice dies within 24 hours. If one of the mice dies within
24 hours, repeat the test. None of the second group of mice
dies within 24 hours.
Undue toxicity. Dilute a suitable volume ofthe injection under

examination with sufficient water for injections to give a
solution containing the equivalent of 28 mg of pentavalent
antimony per m!. Inject intravenously 0.3 ml of the solution
into each of 10 mice that have been deprived of food for not
less than 17 hours. After injections allow the mice access to
food and water. None of the mice dies within 24 hours. If one
of the mice dies within 24 hours, repeat the test. None of the
second group of mice dies within 24 hours.
Loss on drying (2.4.19). Not more than 15.0 percent,

determined on 0.25 g by drying in an oven at 130 at a pressure
not exceeding 0.7 kPa.


hydrochloric acid, add 70 ml of phosphoric acid and stir
carefully until completely mixed. Titrate with 0.05 Mferrous
ammonium sulphate prepared using sulphuric acid (1 per
cent), determining the end-point potentiometrically (2.4.25),
using a platinum electrode and a silver-silver chloride reference
electrode.
1 ml of 0.05 Mferric ammonium sulphateis equivalent to
0.003044 g ofpentavalent antimony.
Assay. To 1.0 ml, accurately measured, add 30 ml of

hydrochloric acid and 70 ml of phosphoric acid and mix.
Titrate with 0.05 Mferric ammonium sulphate prepared using
sulphuric acid (l per cent), determining the end-point
potentiometrically (2.4.25), using a platinum electrode and a
silver-silver chloride reference electrode.
1 ml of 0.05 M ferric ammonium sulphate is equivalent to
0.003044 g ofpentavalent antimony.
equivalent amount of pentavalent antimony per ml.
2137
SODIUM THIOSULPHATE
IP 2010
Sodium Thiosulphate
Sulphides. To 10 ml of solution A add 0.05 ml of a "!'reshly

prepared 5 per cent w/v solution of sodium nitroprusside; the
solution does not become violet.
Sodium Hyposulphite
Na2S203,5H20
Mol. Wt. 248.2
Sodium Thiosulphate contains not less than 99.0 per cent

and not more than 101.0 per cent ofNa2S203,5H20.
Category. Antidote to cyanide poisoning
Dose. By intravenous or intramuscular injection, 300 mg to 1 g.
Sulphates and sulphites (2.3.17). Dilute 2.5 ml ofsolutionA to

10 ml with distilled water. To 3 ml ofthis solution add 2 ml of
iodine solution and gradually add more iodine solution and
dilute to 15 ml with distilled water. The resulting solution
complies with the limittest for sulphates (0.2 per cent).
water and titrate with 0.05 M iodine using starch solution,
added towards the end of the titration, as indicator.
Description. Colourless large crystals or a coarse, crystalline

powder; odourless; deliquescent in moist air and effloresces
in dry air at temperature above 33. It dissolves in its water of
crystallisation at about 49.
1 ml of 0.05 M iodine is equivalent to 0.02482 g of

Na2S203,5H20.
Identification
A. To 0.5 ml ofa 10.0 per cent w/v solution in carbon dioxidefree water (solution A) add 0.5 ml of water and 2 ml of
0.1 M silver nitrate; a white precipitate is produced which
quicldy becomes yellowish and [mally black.
Sodium Thiosulphate Injection
B. To a portion of solution A add a few drops of iodine

solution; the colour is discharged.
C. Dilute 2.5 mlofsolutionAto 5 ml with water and add 1 ml of
hydrochloric acid; a gas is evolved which turns starch-iodate
paper blue and a precipitate of sulphur is produced.
D. 1 ml of solution A gives reaction Aof sodium salts (2.3.1).
Sodium Hyposulphite Injection

Sodium Thiosulphate Injection is a sterile solution of Sodium
Thiosulphate in Water for Injections.
Sodium Thiosulphate Injection contains not less than
amount of sodium thiosulphate, Na2S203,5H20.
Tests

colourless (2.4.1).
Identification
pH (2.4.24).6.0 to 8.4, determined in solution A.

Arsenic (2.3.1 0). Dissolve 5.0 g in 50 ml of water and add 10 ml
Heavy metals (2.3.13). Dissolve 1.0 g in 10 ml ofwater. Slowly
add 5 ml of dilute hydrochloric acid and evaporate the mixture
to dryness on a water-bath. Gently boil the residue with 15 ml
of water for 2 minutes and filter. Heat the filtrate to boiling,
add sufficient bromine solution to the hot filtrate to produce
a clear solution and add a slight excess of bromine solution.
Boil the solution to expel the bromine completely, cool to room
temperature and add a drop of phenolphthalein solution and
sodium hydroxide solution until a slight pink colour is
produced. Add 2 ml of dilute acetic acid and dilute with water
to 25 ml. The solution complies with the limit test for heavy
metals;MethodA (20 ppm);
Chlorides (2.3.12). To 12.Sri:il orsoliitioiiA add IS fir of
2 M nitric acid, boil gently for 3 to 4 minutes, cool and filter.
The filtrate complies with the limit test for chlorides (200 ppm).
A. To 0.5 ml ofa 10.0 per cent w/v solution in carbon dioxidefi-ee water (solution A) add 0.5 ml of water and 2 ml of
0.1 M silver nitrate; a white precipitate is produced which
quickly becomes yellowish and finally black.
B. To a portion of solution A add a few drops of iodine
solution; the colour is discharged.
C. Dilute 2.5 ml ofsolution A to 5ml with water and add 1 mlof
hydrochloric acid; a gas is evolved which turns starch-iodate
paper blue and a precipitate of sulphur is produced.
D. 1 ml of solution A gives reaction A of sodium salts (2.3.1).
Tests
pH (2.4.24). 7.0t09.0.
0.5 g of Sodium Thiosulphate add about 20 ml of water and
titrate with 0.05 M iodine, using 3 ml of starch solution, added
towards the end of the titration, as indicator.
2138
IP 2010
SODIUM VALPROATE

NaZSZ03,5RzO.
Sodium Valproate
Divalproex Sodium
Mol. Wt. 166.2
Test solution (a). Add 5 ml of 1 Msulphuric acid to 10 ml ofa

0.04 per cent w/v solution ofthe substance under examination
and shake with three quantities, each of20 ml, of ether. Add
10 ml of a 0.02 per cent w/v solution of butyric acid (internal
standard) in ether to the combined ether extracts, shake with
anhydrous sodium sulphate, filter and evaporate the filtrate
to a volume of about 10 ml at a temperaturt;l not exceeding 30
using a rotary evaporator.
Test solution (b). Add 0.5 ml of 1 M sulphuric acidto 10 ml of
a 0.04 per centw/v solution ofthe substance under examination
and shake with three quantities, each of 5 ml, of ether. Shake
the combined ether extracts with anhydrous sodium sulphate,
filter and evaporate the filtrate to a volume ofabout 10 ml at a
temperature not exceeding 30 using a rotary evaporator.
Sodium Valproate is sodium 2-propylpentanoate.
Test solution (c). Dissolve 0.5 g of the substance under

Sodium Valproate contains not less than 98.5 per cent and not . examination in 10 ml ofwater, add 5 ml of 1 M sulphuric acid
more than 101.0 per cent ofCaR,sNaOz, calculated on the dried and treat as described for test solution (a) beginning at the
words "shake with three...".
basis.
Dose. 600 mg to 1.6 g daily, in divided doses.
hygroscopic.
Identification

valproate RS or with the reference spectrum of sodium
valproate.
B. In the test for Related substances, the principal peak in the
solution.
C. Dissolve 1.25 g in 20 ml of distilled water in a separating
funnel, add 5 ml of2 M nitric acid, shake and allow the mixture
t6 stand for 12 hours; the lower layer (solution A) gives
Tests
Appearance of solution. A 20.0 per cent w/v solution is not
more opalescent than opalescence standard OS2 (2.4.1), and
is not more intensely coloured than reference solution
, YS6(2.4.l).
Acidity or alkalinity. To 10 ml ofa 10.0 per cent w/v solution
in carbon dioxide-free water add 0.1 ml of dilute
phenolphthalein solution; not more than 0.75 ml of either
0.1 M sodium hydroxide or 0.1 M hydrochloric acid is
(2.3.13).
Reference solution. Prepare in the same manner as test solution

(b) but using sodium valproate RS in place of the substance
under examination.
- a glass column 2.6 m x 2 rom, paclced with silariised
diatomaceous support (125 to 180 mesh) impregnated
with 5 per cent w/w of polyethylene glycol 20,000
2-nitroterephthalate and 1 per cent w/w ofphosphoric
acid,
- temperature. colurnn.150 to 170 to obtain a retention
time ofabout 12 minutes for valproic acid [the principal
peak in test solution (b)],
inlet port at 200 and detector at 300,
Allow the chromatography to proceed for 2.5 times the
retention time of valproic acid. Adjust the sensitivity so that
the height of the peak due to the internal standard in the
chromatogram obtained with test solution (a) is not less than
70 per cent of the full-scale deflection on the recorder. In the
chromatogram obtained with test solution (c), the sum of the
areas of any secondary peaks is not greater than the area of
the peak due to the internal standard. Ignore any peaks the
area ofwhich is less than I per cent ofthe area ofthe peak due
to the internal standard. The test is not valid unless the
resolution between the peak due to the internal standard and
the principal peak in the chromatogram obtained with test
solution (a) is at least 12.
Chlorides (2.3.12). Dissolve 1.25 gin 10 ml of water. The
resulting solution complies with the limit test for chlorides
(200 ppm).
Sulphates (2.3.17). Solution A complies with the limit test for
2139
SODIUM VALPROATE ORAL SOLUTION
IP 2010
heavy metals, Method B. Use 2 ml of lead standard solution
(10 ppm) to prepare the standard (20 ppm).
anhydrous glacial acetic acid Titrate with 0.1 M perchloric

CSH1SNaOz.

Sodium Valproate Elixir
Sodium Valproate Oral Solution is a solution of Sodium
Valproate in a suitable flavoured vehicle.
Sodium Valproate Oral Solution contains not less than
amount ofsodium valproate, CSH1SNaOz.
Identification
A. Shake a quantity containing about 0.25 g of Sodium
Valproate with two quantities, each of 25 ml, of chloroform
and discard the chloroform extracts. Add 10 ml of a saturated
solution of sodium chloride and 10 ml of 2 M hydrochloric
acid, mix and shake with 25 ml of chloroform. Wash the
chloroform layer with 5 ml of water, shake with anhydrous
sodium sulphate, filter and evaporate to dryness.
obtained with valproic acidRS or with the reference spectrum
ofvalproic acid.
B. Shake a quantity containing about 0.25 g of Sodium
Valproate with a mixture of 10 ml of a saturated solution of
sodium chloride, 10 ml of2 M hydrochloric acid and 25 ml of
chloroform. Evaporate the chloroform layer to dryness,
dissolve the residue in 2 ml of water, make just alkaline with
2 M sodium hydroXide and add 0.5 ml of a 10 per cent w/v
solution of cobalt nitrate; a purple precipitate is produced
which is soluble in dichloromethane.
Test solution (a). Mix a quantity ofthe oral solution containing

0.50 g of Sodium Valproate with 10 ml of water, acidify with
2 M sulphuric acid and shake with three quantities, each of
20 ml, of dichloromethane. Wash the combined
dichloromethane extracts with 10 ml of water, shake with
to a volume ofabout 10 ml at a temperature not exceeding 30
Test solution (b). Mix a quantity ofthe oral solution containing
0.5 g of Sodium Valproate with 10 ml of a 0.02 per cent w/v
solution of octanoic acid (internal standard) in 0.1 M sodium
hydroxide and continue as described for test solution (a)
beginning at the words "acidify with 2 M sulphuric acid...".
Reference solution. A 0.02 per cent w/v solution ofthe internal
standard in dichloromethane.
- a glass column 1.5 m x 4 mm. packed with acid-washed,
silanised diatomaceous support (80 to 180' mesh)
coated with 15 per cent w/w of free fatty acid phase
(such as Supelco FFAP 2-1063) and 1 per cent w/w of
phosphoric acid,
- temperature. column. 170.
In the chromatogram obtained with test solution (b) the sum
of the areas of any secondary peaks is not greater than the
area of the peak due to the internal standard.
Assay. Weigh accurately a quantity containing about 0.15 g
ofSodium Valproate, add 50 ml ofwater, mix thoroughly and
add 10 ml of saturated solution of sodium chloride, 10 ml of
2 M hydrochloric acid and 40 ml ofa mixture of2 volumes of
ether and 1 volume of light petroleum (40 to 60), shake,
allow to separate and reserve the ether layer. Shake the
aqueous layer with a further 40 ml ofthe ether-lightpetroleum
mixture and discard the aqueous layer. Shake each of the
ether extracts with the same three quantities, each of 10 ml, of
a saturated solution ofsodium chloride, combine the extracts
and evaporate to a volume of about 1 ml at a temperature not
exceeding 30. Add 50 ml ofethanol (95 per cent), previously
neutralised with 0.01 M sodium hydroxide using dilute
phenolphthalein solution as indicator, and titrate with
0.1 M sodium hydroxide.
CSH1SNaOz.
Determine the weight per ml of the preparation (2.4.29) and
calculate the content ofCsHlsNaOz weight in volume.
Sodium Valproate Tablets
Tests
(2.4.13).
Sodium Valproate Tablets contain not less than 95.0 per cent
sodium valproate, CSHlSNaOz.
2140
IP 2010
SORBIC ACID
Identification
A. Shake a quantity of the powdered tablets containing about
0.5 g ofSodium Valproate with 10 ml of water and centrifuge.
AcidifY 5 ml of the supernatant liquid with 2 M sulphuric
acid, shake with 25 ml of chloroform and wash the chloroform
layer with 5 ml of water. Dry by shaking with anhydrous
sodium sulphate, filter and evaporate the chloroform.
obtained with valproic acidRS or with the reference spectrum
ofvalproic acid.
B. Shake a quantity of the powdered tablets containing about
0.25 g ofSodium Valproate with 3 ml of water and centrifuge.
To 2 ml of the supernatant liquid add 0.5 ml of a 10 per cent
w/v solution of cobalt nitrate; a purple precipitate is produced
which is soluble in dichloromethane.

quantity of the powder containing about 0.3 g of Sodium
Valproate, add 70 ml of water, shake for 10 minutes, dilute to
100.0 ml with water and filter. To 50.0 ml ofthe filtrate add 10 ml
of a 30 per cent w/v solution of sodium chloride and 10 ml of
2 M hydrochloric acid. Extract with 40 ml of a mixture of
2 volumes of ether and 1 volume of light petroleum (boiling
range 40 to 60) and allow to separate. Shake the aqueous
layer with a further 40 ml ofthe ether-light petroleum mixture.
Shake each ofthe ether extracts with the same three quantities,
each of 10 ml, of a saturated solution of sodium chloride,
combine the ether extracts and evaporate to a volume ofabout
1 m1 at a temperature not exceeding 30. Add 50 ml of ethanol
(95 per cent), previously neutralised with 0.01 M sodium
hydroxide using dilute phenolphthalein solution as indicator,
and titrate with 0.1 M sodium hydroxide.
CSHISNa02.
Tests
(2.4.13).
Test .solution (a). Shake a quantity of the powdered tablets

containing 0.50 g of Sodium Valproate with 10 ml of water,
acidifY with 2 M sulphuric acid and shake with three quantities,
each of 20 ml, of dichloromethane. Wash the combined
dichloromethane extracts with 10 ml of water, shake with
to a volume ofabout 10 ml at a temperature not exceeding 30
Test solution (b). Shake a quantity of the powdered tablets
containing 0.50 g ofSodiurn Valproate with 10 ml ofa 0.020 per
cent w/v solution of octanoic acid (internal standard) in
0.1 M sodium hydroxide and continue as described for test
solution (a) beginning at the words "acidifY with 2 M sulphuric
acid...".
Reference solution. A 0.02 per cent w/v ofthe internal standard
in dichloromethane.
silanised diatomaceous support (80 to 180 mesh) coated
with 15 per cent w/w of free fatty acid phase (such as
Supelco FFAP 2-1063) and 1 per cent w/w ofphosphoric
acid,
- temperature. column. 170,
In the chromatogram obtained with test solution (b) the sum
area of the peak due to the internal standard.
Sorbic Acid
MoL Wt. 112.1

Sorbic Acid is (2E,4E)-hexa-2,4-dienoic acid.
Sorbic Acid contains not less than 99.0 per cent and not more.
than 101.0 per cent of C 6Hs0 2 , calculated on the anhydrous
basis.
Identification

Compare the spectrum with that obtained with sorbic acid RS
or with the reference spectrum of sorbic acid.
B. Dissolve 50 mg in sufficient water to produce 250 ml and
dilute 2 ml ofthis solution to 200 ml with 0.1 M hydrochloric
acid.
When examined in the range 230 urn to 360 urn (2.4.7), the
264 urn; absorbance at about 264 urn, 0.43 to 0.51.
C. Dissolve 0.2 g in 2 ml of ethanol (95 per cent) and add
0.2 ml of bromine water; the solution is decolorised.
2141
SORBIC ACID
IP 2010
Tests
B. Complies with the test for iodine value (2.3.28).
Appearance ofsolution. A 5.0 per cent w/v'solution in ethanol

(95 per cent) is clear (2.4.1), and colourless (2.4.1).
C. Complies with the test for composition offatty acids.
D. Margaric acid. Not more than 0.2 per cent for oleic acid of
vegetable origin and not more than 4.0 per cent for oleic acid
ofanimal origin.
Aldehydes. Not more than 0.15 per cent, determined by the

following method. Dissolve 1.0 g in a mixture of 50 ml of
2-propanol and 30 ml of water, adjust the pH of the solution
to 4.0 with 0.1 M hydrochloric acid or 0.1 M sodium
hydroxide and dilute to 100 ml with water. To 10 ml of the
resulting solution add 1 ml of decolorised filchsin solution
and allow to stand for 30 minutes. Any colour produced is not
more intense than that obtained in a solution prepared
simultaneously by adding 1 ml of decolorisedfilchsin solution
to a mixture of 1.5 ml of acetaldehyde standard solution
(100 ppm C2H 40), 4 ml of 2-propanol and 4.5 mlofwater.
Tests
Composition offatty acids. Determine by gas chromatography

(2.4.13).

2.0g.
ethanol (95 per cent) and titrate with 0.1 M sodium hydroxide,
using 0.2 ml of phenolphthalein solution as indicator, until a
pink colour is produced.
C6HsOz.
Relative density (2.4.29). About 0.99.

Saponification value (2.3.37). 145 to 160. Carry out the
saponification for 1 hour.
Composition of the fatty acid fraction of the substance:
myristic acid: Not more than 5.0 per cent;
- palmitic acid: Not more than 16.0 per cent;

-palmitoleic acid: Not more than 8.0 per cent;
stearic acid: Not more than 6.0 per cent;
-
oleic acid: 65.0 per cent to 88.0 per cent;
-linoleic acid: Not more than 18.0 per cent;

-linolenic acid: Not more than 4.0 per cent;
- fatty acids with chain length greater than C 18: Not more
than 4.0 per cent.
Sorbitan Oleate
Heavy metals (2.3.13). In a silica crucible, mix thoroughly 2.0 g

of the substance under examination with 0.5 g of magnesium
oxide. Ignite to dull redness until a homogeneous white or
greyish-white mass is obtained.
Mol. Wt. 428.6

Sorbitan Oleate is mixture usually obtained by esterification
of 1 mole ofsorbitol and its mono-and di- anhydrides per mole
of oleic (cis-9-octadecenoic) acid. A suitable antioxidant may
be added.
NOTE-When sorbitan mono-oleate is demanded, Sorbitan

Oleate shall be supplied.
Description;Abrownish;yellow, viscous liquid.
Identification
A. Complies with the test for hydroxyl value (2.3 .27).
Ifafter 30 min ofignition the mixture remains coloured, allow

to cool, mix using a fine glass rod and repeat the ignition. If
necessary repeat the operation. Heat at 8000 for about 1 hour.
Take up the residue in 2 quantities, each of5 mI, of a mixture of
equal volumes of hydrochloric acid and water. Add 0.1 mlof
phenolphthalein solution and then concentrated ammonia
until a pink colour is obtained. Cool, add glacial acetic acid
until the solution is decolourised and add 0.5 ml in excess.
Filter if necessary and wash the filter. Dilute to 20 ml with
wate/: 12 ml of resultant solution complies with the limit test
for heavy metals, Method D (10 ppm).
Total ash (2.3.19). Not mOre than OSper cent, detefifiified ofi
1.5 g.
LOg.
2142
SORBITOL
IP 2010

Labelling. The label states the origin of the oleic acid used
(animal or vegetable).
Sorbitol
D-Glucitol
50 ml (solution A). To 10 ml ofsolution A add 10 ml of carbon

dioxide-fi-ee water. To 10 ml of the resulting solution add
0.05 ml of phenolphthalein solution; not more than 0.2 ml of
0.01 M sodium hydroxide is required to change the colour of
the solution to pink. To a further 10 ml of the solution add
0.05 ml of methyl redsolution; Not more than 0.3 ml of 0.01 M
solution to red.
Specific optical rotation (2.4.22). +4.0 to +7.0, determined in

a solution prepared in the following manner. Dissolve a mixture
of5.0 g ofthe substance under examination and 6.4 g of borax
in 40 ml of water, allow to stand for 1 hour, shaking occasionally,
dilute to 50 ml with water and filter, if necessary.
CHzOH
I
H-C-OH
I
HO-C-H
I
H-C-OH
I
H-C-OH
I
CHzOH

(2.4.14).
examination in 100.0 ml of water.
Mol. Wt. 182.2
Sorbitol is o-glucitol, a hexahydric alcohol related to

glucose.
Sorbitol contains not less than 98.0 per cent and not more
than 101.0 per cent ofC 6H 140 6, calculated on the anhydrous
basis.
Reference solution (a). Dissolve 0.5 g of sorbitol RS in 10.0

mlofwatel:
100.0 ml with water.
Category. Nutrient (for parenteral administration);

pharmaceutical aid (sweetening agent; excipient).
Reference solution (d). Dissolve 0.5 g each of sorbitol and

mannitol (sorbitol impurity A) in 10.0 ml of water.
strong cation exchange resin (calcium form) (9 Jlm),
mobile phase: water,
refractometer at a constant temperature,
Identification
(a).
B. Dissolve 50 mg in 3 ml of water, add 3 ml of catecholsolution

and pour the mixture into 6 ml ofsulphuric acid; a pink colour
is produced.
C. Dissolve 5 g in 3 ml of water with the aid of gentle heat,
cool, add 7 ml of methanol, 1 ml of benzaldehyde and 1 ml of
hydrochloric acid, mix and shake continuously for 2 hours.
Filter, dissolve the crystals in 20 ml of boiling sodium
bicarbonate solution and allow to crystallise. The residue,
after washing rapidly with 5 ml ofa mixture ofequal volumes
of methanol and water and drying in a current of air, melts at
about 175(2.4.21).
Tests
colourless (2.4.1).
Acidity or alkalinity. Dissolve 5.0 g in sufficient carbon
dioxide-free water prepared from distilled water to produce
resolution between the peaks due to sorbitol and sorbitol
impurity A is not less than 2.0. The relative retention time with
reference to sorbitol for maltitol (sorbitol impurity C) is about
0.6, for mannitol (sorbitol impurity A) is about 0,8 and for iditol
(sorbitol impurity B) is about 1.1.
Inject the test solution, reference solution (b), (c) and (d). Run
the chromatogram 3 times the retention time of the principal
peale. In the chromatogram obtained with the test solution the
area of any secondary peak is not more than the area of the
solution (b) (2.0 per cent) and the sum of aJI the secondary
peaks is not more than 1.5 times the area ofthe principal peak
per cent). Ignore any peak with an area less than the area of
2143
SORBITOL
IP 2010
Arsenic (2.3.1 0). Dissolve 5.0 g in 50 ml ofwater and add 10 ml

Chlorides (2.3.12). 5.0 g complies with the limit test for chlorides
(50 ppm).
Nickel. Dissolve 10.0 g in sufficient water to produce 20 ml,
add 3 ml of bromine water and 2 ml of a 20 per cent w/v
solution of citric acid, mix and add 10 ml of 6 M ammonia and
1 rn1 ofa 1per cent w/v solution of dimethylglyoxime in ethanol
(95 per cent). Mix, dilute to 50 ml with water and allow to
stand for 5 minutes; any colour produced is not more intense
than that produced by treating in the same manner and at the
same time 1.0 ml of nickel standard solution (10 ppm Ni)
diluted to 20 ml with water (1 ppm).
Reducing sugars. Dissolve 5.0 g in 3 ml of water with the aid
of gentle heat, cool, add 20 ml of cupri-citric solution and a
few glass beads, heat in such a manner that the solution boils
in 4 minutes and continue boiling for a further 3 minutes. Cool
rapidly and add 100 ml ofa 2.4 per cent v/v solution ofglacial
acetic acid followed by 20.0 ml of 0.025 M iodine. Add,
shaking continuously, 25 ml of a 6 per cent v/v solution of
hydrochloric acid and, when any precipitate has redissolved,
titrate the excess iodine with 0.05 M sodium thiosulphate
titration, as indicator. Not less than 12.8 ml of 0.05 M sodium
LOg.
described in the test for Related substances with the following
modification.
Calculate the content ofC6H 140 6
Sorbitol intended for use in the manufacture ofparenteral

preparations without a further appropriate procedure for
per g for parenteral preparations having a concentration of
less than 10 per cent WIY 6fS6rbit61 and not more than 2.5
Endotoxin Units per g for parenteral preparations containing
10 per cent w/v or more of sorbitol.

Sorbitol Solution (70 Per cent)

(Crystallising)
Sorbitol (70 Per cent) (Crystallising)
Sorbitol Solution (70 per cent) (Crystallising) is an aqueous
solution of hexitoIs.
Sorbitol Solution ( 70 per cent) (Crystallising) contains not
less than 68.0 per cent w/w and not more than 72.0 per cent
w/w ofhexit0 Is, calculated as D-glucitol (C 6H I40 6).
Category. Pharmaceutical aid (sweetening vehicle); humectant.
Description. A clear, colourless, syrupy liquid.
Identification
A. Dilute 7.0 gwith40 ml of water, add 6.4 gofborax, allow to
stand for 1 hour, shaking occasionally, and dilute to 50.0 ml
with water. Filter if necessary. The optical rotation of the
resulting solution is 0 to +1,50 (2.4.22).
B. Dry 1 g over phosphorus pentoxide at a pressure of 1.5 to
2.5 kPa. Heat 0.5 g of the residue with a mixture of 5 ml of
acetic anhydride and 0.5 ml pyridine with the aid ofheat and
allow to stand for 10 minutes. Pour the mixture into 25 ml of
water, allow to stand in ice for 2 hours and filter. The precipitate,
after recrystallisation from a small volume of ethanol (95 per
cent) and drying over phosphorus pentoxide at a pressure of
1.5 to 2.5 kPa, melts at about 100 (2.4.21).

the plate with a uniform 0.75-mm thick layer ofthe following
mixture. Mix 0.1 g of cm'bomer with 110 ml of water and allow
to stand, with gentle stirring, for 1 hour. Adjust to pH 7.0 by
the gradual addition, with continuous shaking, of 2 M sodium
hydroxide and add 30 g of silica gel H. Heat the plate at 110
for 1 hour, allow to cool and use immediately.
Mobile phase. A mixture of 85 volumes of 2-propanol and

15 volumes of a 0.2 per cent w/v solution of boric acid.
Reference solution. A 0.25 per cent w/v solution of sorbitol
RS in ethanol (95 per cent).
dry the plate at 100 to 105 for 15 minutes, allow to cool, spray
with a 0.5 per cent w/v solution of potassium permanganafe
in 1 M sodium hydrOXide and heat at 100 for 2 minutes. The
2144
IP 2010
SORBITOL SOLUTION ( 70 PER CENT) ( NON-CRYSTALLISING)


examination in 50.0 ml of wate!:
Tests
Reference solution (a). Dissolve 65 mg of sorbitol RS in 5.0

mlof wate!:

dioxide-free water (solution A) is clear (2.4.1), and colourless
(2.4.1).
phenolphthalein solution; not more than 0.2 ml of 0.01 M
solution to pink. To a further 10 ml ofthe solution add 0.05 ml
of methyl red solution. Not more than 0.3 ml of 0.01 M
solution to red.
Relative density (2.4.29). Not less than 1.290.
Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and add 10 ml
CWorides (2.3.12). 5.0 g complies with the limit test for chlorides
(50 ppm).
add 3 rn1 of bromine water and 2 ml of a 20 per cent w/v
solution of citric acid, mix and add 10 ml of 6 M ammonia and
1 mlofa 1 percentw/v solution ofdimethylglyoxime in ethanol
same time 1.0 m1 of nickel standard solution (10 ppm Ni)
Sulphates (2.3 .17). 12 ml ofsolution A complies with the limit
Reducing sugars. Dissolve 5.0 g in 3 ml of water with the aid
of gentle heat, cool, add 20 ml of cupri~citric solution and a
few glass beads, heat in such a manner that the solution boils
titration, as indicator. Not less than 12.8 ml of 0.05 M sodium
Reference solution (b). Dissolve 65 mg each of mannitol and

sorbitol in 5.0 ml of wate!:
strong cation exchange resin (calcium form) (9 /lm),
- mobile phase: water,
- refractometer at a constant temperature,
resolution between the peaks due to sorbitol and mannitol
(sorbitol impurity A) is not less than 2.0. The relative retention
time with reference to sorbitol for mannitol (sorbitol impurity
C) is about 0.8.
Calculate the content of D-sorbitol, C 6H 140 6 .
Sorbitol Solution (70 Per cent)

(N on-Crystallising)
Sorbitol (70 per cent) (Non-crystallising)
Sorbitol Solution (70 per cent) (Non-crystallising) is an aqueous
solution of hydrogenated, partly hydrolysed starch.
Sorbitol Solution (70 per cent) (Non-crystallising) contains
not less than 68.0 per cent w/w and not more than 72.0 per
cent w/w ofsolid matter and not less than 62.0 per cent w/w of
polyols expressed as D-glucitol (C 6H I4 0 6).
Category. Pharmaceutical aid (sweetening vehicle); humectant.
Description. A clear, colourless or faintly yellow, syrupy liquid;
odourless.
Identification
A. Dilute 7.0 g with sufficient carbon dioxide-free water to

produce 50 ml (solution A). To 3 ml ofa freshly prepared 10 per
cent w/v solution of catechol add 6 ml of sulphuric acid while
cooling in ice. To 3 ml ofthe mixture add 0.3 ml of solution A
and heat gently over a naked flame for 30 seconds; a pink
colour is produced which becomes deep brownish red.
R Dry I g over phosphorus pentoxide at 80 at a pressure of
1.5 to 2.5 kPa. Dissolve 0.5 g ofthe residue in a mixture of5 mi
2145
SORBITOL SOLUTION ( 70 PER CENT) ( NON-CRYSTALLISING)
ofacetic anhydride and 0.5 ml ofpyridine with the aid ofheat

and allow to stand for 10 minutes: Pour the mixtureinto 25 ml
ofwater, allow to stand in ice for 2 hours and filter. The residue,
after recrystallisation from a small volume of ethanol (95 per
cent) and drying over phosphorus pentoxide at a pressure of
1.5 to 2.5 kPa, melts at about 100 (2.4.21).
the plate with a uniform 0.75-mm thick layer ofthe following
mixture. Mix 0.1 g ofcarbomer with 110 ml ofwater and allow
to stand, with gentle stirring, for 1 hour. Adjust to pH 7.0 by
the gradual addition, with continuous shaking, of 2 M sodium
hydroxide and add 30 g ofsilica gel H. Heat the plate at 110
for 1 hour, allow to cool and use immediately.
Mobile phase. A mixture of 85 volumes of 2-propanol and
15 volumes of a 0.2 per cent w/v solution of boric acid.
Reference solution. A 0.25 per cent w/v solution of sorbitol
Apply to the plate 2 f!l of each solution. After development,
dry the plate at 100 to 105 for 15 minutes, allow to cool, spray
with a 0.5 per cent w/v solution ofpotassium permanganate
in 1 M sodium hydroxide and heat at 100 for 2 minutes. The
IP 2010
Chlorides (2.3 .12). 5.0 g cotDplies with the limit test for cWorides
(50 ppm).
add 3 ml of bromine water and 2 ml of a 20 per cent w/v
solution ofcitric acid, mix and add 10 ml of 6 M ammonia and
1 ml ofa 1per cent w/v solution ofdimethylglyoxime in ethanol
same time 1.0 ml of nickel standard solution (10 ppm Ni)
Reducing sugars. Dissolve 5.0 g in 3 ml ofwater with the aid
of gentle heat, cool, add 20 ml of cupri-citric solution and a
few glass beads; heat in such a manner that the solution boils
titration, as indicator. Not less than 12.8 ml of0.05 M sodium
Tests
colourless (2.4.1).
Acidity or alkalinity. To 10 ml of solution A add 10 ml of
carbon dioxide-free water. To 10 ml of the resulting solution
add 0.05 ml ofphenolphthalein solution; not more than 0.2 ml
of 0.01 M sodium hydroxide is required to change the colour
ofthe solution to pink. To a further 10 ml of the solution add
0.05 ml of methyl red solution; Not more than 0.3 ml of
0.01 M hydrochloric acid is required to change the colour of
the solution to red.
Optical rotation (2.4.22). +1.5 to + 3.so, determined in a solution
prepared in the following manner. Dilute 7.0 g with 40 ml of
water, add 6.4 g of borax, allow to stand for 1 hour, shaking
occasionally, dilute to 50 ml with water and filter, ifnecessary.

examination in 50.0 ml ofwater.
Reference solution (a). Dissolve 55 mg of sorbitol RS in 5.0
mlof water.
Reference solution (b). Dissolve 65 mg of mannitol and 55
mg of sorbitol in 5.0 ml of water.
strong cation exchange resin (calcium form) (9 f!m),
- mobile phase: water,
- flow rate. 0.5 rnl per minute,
- refractometer at a constant temperature,
- injection volume. 20 f!l.
resolution between the peaks due to sorbitol and mannitol
(sorbitol impurity A) is not less than 2.0. The relative retention
Relative density (2.4.29). Not less than 1.285.
time with reference to sorbitol for mannitol (sorbitol impurity
Arsenic (2.3.1 0). Dissolve 5.0 g in 50 rnl ofwater and add 10 rnl .
C) is about 0.8.
oCsfarznated hydrochloric aCid AsT. The resulting solution
Heavy metals (2.3.13). 2.0 g complies with the limit test for Calculate the content of D-sorbitol, C6H 140 6. .
heavy metals, MethodA(lO ppm).

2146
IP 2010
SPIRONALACTONE
was done.
Spironolactone
Mol. Wt. 416.6

Spironolactone is 7a-acetylthio-3-oxo-17a-pregn-4-ene21,17~-carbolactone.
Spironolactone contains not less than 97.0 per cent and not
more than 102.0 per cent ofC 24 H320 4S, calculated on the dried
basis.
Category. Diuretic.

in d'aylight and in ultraviolet light at 365 nm. The principal
compact spot.
C. Shake about 10 mg with 2 ml of sulphuric acid
(50 per cent); an orange solution with an intense yellowish
fluorescence is produced. Heat the solution gently; the colour
becomes deep red and hydrogen sulphide is evolved which
turns lead acetate paper black. Add the solution to 10 ml of
water; a greenish yellow solution is produced which shows
opalescence or a precipitate.

Description. A yellowish white to buff coloured powder;
odourless or with a slight odour ofthioacetic acid. It exhibits
polymorphism.
Tests
Identification

(2.4.14).

Compare the spectrum with that obtained with spironolactone
RS or with the reference spectrum ofspironolactone. Examine
the substances as 5 per cent w/v solutions in chloroform.

Mobile phase. A mixture of 40 volumes of cyclohexane and
10 volumes of toluene.
examination in 10 m1 ofthe solvent mixture.
Reference solution (a). Dissolve 25 mg of spironolactone RS

in a 1.0 per cent w/v solution in chloroform.

examination in 2.5 ml oftetrahydrojUran and dilute to 25.0 rnl
Reference solution (b). Dissolve 25 mg of canrenone RS in
1.0 ml of tetrahydrojUran and dilute to 10.0 ml with the mobile
phase.
Reference solution (c). Dilute 1.0 rnl ofreference solution (b)
Reference solution (d). Dilute 1.0 ml ofthe test solution and 1
ml ofreference solution (b) to 100.0 ml with the mobile phase.
Reference solution (e). Dilute 0.5 ml ofreference solution (a)
- mobile phase: a mixture of8 volumes of acetonitrile, 18
volumes of tetrahydrofitran and 74 volumes of water,
2147
SPIRONALACTONE
IP 2010
- spectrophotometer set at 254 urn and283 nm,

- injection volume. 20 l.tI.
resolution between the peaks due to canrenone and
spironolactone is not less than 1.4 and the signal-to-noise
ratio of the principal peak is not less than 6.
Spironolactone Tablets contain not less than 95.0 per cent

spironolactone, Cz4H32 0 4S. The tablets may contain added
flavouring agents.
Inject the test solution, reference solution (a), (c), (d) and (e).
At 254 urn, run the chromatogram twice the retention time of
the principal peale. In the chromatogram obtained with the test
solution, the sum ofthe areas ofall the secondary peaks other
than canrenone, is not more than the area ofthe principal peak
per cent). Ignore any peak with an area less than the area of
solution (e). At 283 rim, the area ofthe peak due to canrenone
is not more than the area of the corresponding peak in the
cent). Calculate the content of canrenone at 283 urn and the
content ofthe other impurities at 254 nm. The sum ofareas of
all the peaks is not more than 1.0 per cent.
Chromium. Mix 0.2 g with 1 g of potassium carbonate and
0.3 g of potassium nitrate in a platinum crucible, heat gently
until fused and ignite at 6000 to 6500 until the carbon is removed.
Cool, dissolve the residue as completely as possible in 10 ml
of water with the aid of gentle heat, filter and dilute to 20 ml
with water. To 10 ml ofthe solution add 0.5 g of urea and just
acidify with sulphuric acid (14 per cent). When effervescence
ceases add a further 1 ml of sulphuric acid (14 per cent),
dilute to 20 ml with water and add 0.5 ml of diphenylcarbazide
solution. Any colour produced is not more intense than that
obtained by adding 1 ml of sulphuric acid (14 per cent) to
potassium dichromate, diluting to 20 ml with water and adding
0.5 ml of diphenylcarbazide solution (50 ppm).
Free mercapto compounds. Shake 2.0 g with 20 ml ofwater for
1 minute and filter. To 10 ml of the filtrate add 0.05 ml of
0.01 M iodine and 0.1 ml of starch solution and mix; a blue
colour develops.
on 1.0 g by drying in an oven at 105 0 for 3 hours,
methanol to produce 250.0 ml, dilute 5.0 ml to 100.0 ml with
methanol and measure the absorbance of the resulting
solution at the maximum at about 238 urn (2.4.7).
Calculate the content ofC z4H 32 0 4S talcing 470 as the specific
Identification
0.125 g of Spironolactone with two quantities, each oflO ml,
of chloroform, filter, evaporate the combined filtrates to
dryness and dissolve the residue in 2.5 ml of chloroform.
On the resulting solution determine by infrared absorption
obtained with spironolactone RS or with the reference
spectrum of spironolactone.

10 volumes of toluene.
containing 50 mg ofSpironolactone with 10 ml of chloroform,
filter and evaporate the filtrate to dryness. Dissolve 25 mg the
residue in 10 ml ofthe solvent mixture.
Reference solution (a). Dissolve 25 mg of spironolactone RS
Place the dry plate in a tank containing a shallow layer ofthe
was done.
phase to rise 12 em. Dry the plate in a current ofwarm air, allow
spot in the chrOlnatogram obtained \Vith the test solution
cOl:responds to that in -the chroIIl-atogram obtained with
reference solution (a). The pnnCipalspofintlie Clrromatogram
compact spot.
2148
IP 2010
STAVUDINE
C. Shake about 10 mg of the residue obtained in test B with

2 ml of sulphuric acid (50 per cent); an orange solution with
an intense yellowish fluorescence is produced. Heat the
solution gently; the colour becomes deep red and hydrogen
sulphide is evolved which turns lead acetate paper black.
Add the solution to 10 ml ofwater;'a greenish yellow solution
is produced which shows opalescence or a precipitate.
Tests
Apparatus. No.1,
Medium. 1000 ml of 0.1 M hydrochloric acid containing
0.1 per cent w/v of sodium dodecyl sulphate,
Withdraw a suitable volume of the medium, filter through a
than 1.0 f..lm. Measure the absorbance of the filtrate, suitably
Calculate the content of Cz4H 32 0 4S in the medium taking 445
D. Not less than 70.0 per cent ofthe stated amount ofCz4 H32 0 4S.,

- spectrophotometer set at 254 nm and 283 nm,
- injection volume. 20 f..ll.
resolution between the peaks due to canrenone and
spironolactone is not less than 1.4 and the signal-to-noise
ratio of the principal peak is not less than 6.
Inject the test solution, reference solution (a), (c), (d) and (e).
At 254 nm, run the chromatogram twice the retention time of
solution, the sum ofthe areas ofall the secondary peaks other
than the canrenone, is not more than the area of the principal
(a) (1.0 per cent). Ignore any peak with an area less than the
reference solution (e). At 283 nm, the area of the peak due to
canrenone is not more than the area of the corresponding
(c) (1.0 per cent). Calculate the content ofcanrenone at 283 nm
and the content ofthe other impurities at 254 nm. The sum of
areas of all the peaks is not more than 1.0 per cent.
(2.4.14).

containing about 62.5 mg of Spironolactone with 25 ml of
chloroform, sonicate for 15 minutes, centrifuge and filter the
supernatant. Repeat the procedure on the residue with a
further 25 ml of chloroform. Combine the chloroform extracts
and evaporate to dryness using a rotary evaporator. Add 2.5
ml of tetrahydrojUran and 22.5 ml of the mobile phase to the
residue.
quantity of the powder containing about 25 mg of Spironolactone, add 100 ml of methanol and heat just to boiling, with
swirling. Cool, add sufficient methanol to produce 250.0 ml,
dilute 10.0 ml to 100.0 ml with methanol and measure the
238 nm (2.4.7).

Reference solution (b). Dissolve 25 mg of canrenone RS in
1.0 ml oftetrahydrojUran and dilute to 10.0 ml with the mobile
phase.
Calculate the content ofC z4 H32 0 4S taking 470 as the specific

Stavudine

Reference solution (d). Dilute 1.0 ml of the test solution and
1.0 ml of reference solution (b) to 100.0 ml with the mobile
phase.
Reference solution (e). Dilute 0.5 ml ofreference solution (a)
endcapped octylsilane bonded to porous silica (5 f..lm)
(such as Spherisorb C8),
- mobile phase: a mixture of8 volumes of acetonitrile, 18
volumes of tetrahydrojUran and 74 volumes of water,
Mol. Wt. 224.2

Stavudine is 2',3'-didehydro-3'-deoxythymidine.
Stavudine contains not less than 98.0 per cent and not more
than 102.0 per cent of C IOH 12N z0 4, calculated on the dried
basis.
2149
STAVUDINE
IP 2010
Dose. 30 to 40 mg twice daily depending on body weight of
the patient.
Identification
Compare the spectrum with that obtained with stavudine RS
or with the reference spectrum of stavudine.
to stavudine in the chromatogram obtained with reference
solution (c).
C. Melts at about 164 (2.4.21).
Tests
(2.4.14), as described in the Assay.
Separately inject the test solution, reference solutions (a) and
(b) and record the chromatograms for at least three times the
obtained with the test solution, the area of any peak
corresponding to thymine is not greater than the area of the
(b) (1 per cent); the area of any peak corresponding to
~-thymidine is not greater than the area of the corresponding
(a) (1 per cent); the area ofany individual impurity peak other
than thymine and ~-thymidine is not more than 0.5 per cent
and the sum of the areas of all the impurity peaks other than
thymine and ~-thymidine is not more than 1.0 per cent.
on 1.0 g by drying at 105 for 3 hours.

w/v of stavudineRS;--O;Ol 'percent-w/v of thymine and
0.0005 per cent w/v of a-thymidine in the mobile phase;
thymine in the mobile phase.

stavudine RS in the mobile phase.
octadecylsilane bonded to porous silica (5 !-Lm),
80 volumes of water,
- spectrophotometer set at 265 rrm,
- injection volume. 20 !-Ll.
Inject separately reference solutions (a) and (c). The order of
elution in the chromatogram obtained with reference solution
(a) is thymine, a-thymidine and stavudine with relative
retention times of about 0.5, 0.7 and 1.0 respectively. The test
is not valid unless the column efficiency determined from the
stavudine peak in the chromatogram obtained with reference
solution (a) is not less than 3000 theoretical plates, the tailing
factor is not more than 1.5 and the relative standard deviation
for replicate injections of reference solution (c) is not more
than 2.0 per cent.
Inject separately the test solution and reference solution (c)
and measure the responses of the principal peak.
Calculate the content OfCIOHlzNz04.
Stavudine Capsules
Stavudine Capsules contain not less than 90.0 per cent and
not more than 110.0 per cent ofthe stated amount ofstavudine,
CIOHIZNz04.
Identification
A.Shake a quantity of the mixed contents of the capsules
containing about 50 mg of Stavudine with 80 ml of water for
10 minutes. Add sufficient water to produce 100 ml, mix and
filter. Dilute 10 ml ofthe filtrate to 100 ml with water.
265rrm.
to stavudine in the chromatogram obtained with the reference
solution.
Tests
(2.4.14).
2150
IP 2010
STAVUDINE CAPSULES
Test solution. Mix well the contents of20 capsules and transfer
an accurately weighed quantity containing about 50 mg of
Stavudine to a 100-ml volumetric flask. Add about 60 ml of
water, mix with the aid ofultrasound for 10 minutes, dilute to
volume with water, mix and filter.
Reference solution. Weigh 5 mg each of stavudine RS and
thymine RS and transfer to a 25-ml volumetric flask. Add 5 ml
of methanol, mix with the aid of ultrasound to dissolve and
dilute to volume with water.
mobile phase: gradient mixtures of acetonitrile and
0.1 M ammonium acetate,
below,
Time
(in min.)
0.1 M Ammonium acetate Acetonitrile

(per cent v/v)
(per cent v/v)
95
95
20
80
30
20
80
31
95
35
95
Inject separately water and the test solution. Examine the

water chromatogram for any extraneous peaks and ignore the
corresponding peaks observed in the chromatogram obtained
with the test solution.
with the test solution corresponding to thymine is not more
than 3.0 per cent. Any other secondary. peak observed in the
than 0.5 per cent and the sum ofthe areas of all the secondary
peaks is not more than 4.0 per cent when calculated by.
percentage area normalisation.
Withdraw asuitable volume ofthe medium and filter.


and measure the responses for the major peale
column efficiency determined from the peaks of stavudine
and thymine is not less than 7000 theoretical plates and the
tailing factor is not more than 2.0.
Apparatus No.1,
Reference solution. In the case of capsules containing 30 mg

of Stavudine, weigh accurately about 30 mg of stavudine RS
and transfer to a 1OO-ml volumetric flask. Dissolve and dilute
to volume with 0.01 M hydrochloric acid. Dilute 5.0 ml ofthis
solution to 50.0 ml with 0.01 M hydrochloric acid; in the case
of capsules containing 40 mg of stavudine, weigh accurately
about 40 mg ofstavudine RS and transfer to a 1OO-ml volumetric
flask. Dissolve and dilute to volume with 0.01 Mhydrochloric
acid. Dilute 5.0 ml of this solution to 50.0 ml with
Inject the reference solution and record the chromatograms

for twice the retention time of stavudine. The test is not valid
unless the column efficiency detelmined from the stavudine
peak is not less than 3000 theoretical plates, the tailing factor
is not more than 1.5 and the relative standard deviation for
replicate injections is not more than 1.5 per cent
25
Calculate the content ofCIOHI2N204 in the medium.

CIOH12N204.


contents of20 capsules containing about 50 mg ofStavudine
and transfer to a 1OO-ml volumetric flask. Add about 60 ml of
water, mix with the aid ofultrasound for 10 minutes, dilute to
volume with water, mix and filter. Further dilute 10.0 ml ofthe
filtrate to 25.0 ml with water.
Reference solution. Weigh accmately about 50 mg ofstavudine
RS and transfer to a 1OO-ml volumetric flask. Add about 60 ml
of water, mix with the aid ofultrasound to dissolve and dilute
to volume with water. Dilute 10.0 ml ofthis solution to 25.0 ml
with water.
and 95 volumes of 0.1 M ammonium acetate,
2151
STAVUDINE ORAL SOLUTION
IP 2010

- injection volume. 20 III
Reference.solution (a). A 0.05 per cent w/v solution of

stavudine RS in water.
column efficiency determined from the stavudine peak is not
Calculate the content of ClOHIZNz04 in the capsules.
Reference solution (b). Dilute 1ml ofreference solution (a) to

100 ml with water.
w/v each ofthymine and thymidine in water. Dilute 2 ml ofthe
solution to 100 ml of water.
Iriject reference solution (a). The test is not valid unless the
resolution between thymine and thymidine peak is not less
than 8.4.

Stavudine Oral Solution is a mixture consisting of Stavudine
with buffering agents and other excipients. It contains a
suitable flavouring agent. It is filled in a sealed container.
The oral solution is constituted by dispersing the contents of
the sealed container in the specified volume of water just
before issue.
Stavudine Oral Solution contains not less than 90.0 per cent
stavudine CIOHIZNz04.
Storage. Store the constituted solution in a refrigerator

(2 to 8). Discard any unused portion after 30 days of
reconstitution..
chromatogram for 4 times the retention time (about 9 minutes)
of the principal peak. In the chromatogram obtained with the
test solution, the area of any secondary peak is not more than
with the reference solution (b) (1.5 per cent) and the sum of
areas of all the secondary peaks is not more than 3 times the
area of the peak in the chromatogram obtained with the
0.5g.
The contents of the sealed container comply with the . Test solution. Weigh accurately a quantity ofthe reconstituted
requirements stated under Oral Liquids and with the solution containing 10 mg of Stavudine, disperse in sufficient
water and diluted to 100.0 ml with water.
following requirements.
Identification

stavudine RS in water.


w/v each ofthymine and thymidine in water. Dilute 2 mlofthe
solution to 100 ml with water.
Tests
pH (2.4.24).5.0 to 7.0, determined in the reconstituted solution.

(2.4.14).
Test solution. Weigh accurately a quantity ofthe reconstituted
solution containing 10 mg of stavudine and dissolve in 20 ml
of water.
mobile phase: A. 95 volumes of 25 mM ammonium
acetate and 5 volumes of methanol,
B. 50 volumes of 25 mM ammonium
acetate and 50 volumes of methanol.
flow rate. ml per minute,
below,
2152
IP 2010
STAVUDlNE AND LAMIVUDINE TABLETS
25-ml volumetric flask. Add 5 ml of methanol, sonicate to

dissolve, dilute to volume with water and mix.

Time
(in min.)
mobile phase A
(per cent v/v)
mobile'phase B
(per cent v/v)
100
100
0
100
o
o
60
120
155
Reference solution (b). Weigh accurately about 100 mg of

lamivudine RS, transfer to a 200 ml volumetric flask, add about
100 ml ofwater and mix with the aid ofultrasound to dissolve.
Add 10 ml ofreference solution (a) to this solution and dilute
to volume with water and mix.
100
resolution between thymine and thymidine is not less than
8.4.
Calculate the content ofC IOH 12N 20 4 in the oral solution.
exceeding 30.
,Stavudine and Lamivudine Tablets

Stavudine and Lamivudine Tablets contain not less than
amounts of stavudine, CIOHI2N204 and lamivudine,
CSHIIN303S,
Usual strengths. Stavudine, 30 mg and Lamivudine, 150 mg;
Stavudine, 40 mg and Lamivudine, 150 mg.
Identification
In the Assay, the two principal peaks in the chromatogram
obtained with the test solution correspond to the peaks due
to stavudine and lamivudine in the chromatogram obtained
Tests
(2.4.14).
tablets containing about 100 mg oflamivudine and transfer to
a 200-ml volumetric flask. Add about 100 ml ofwater, mix with
the aid of ultrasound for 10 minutes with occasional shaldng
to obtain a uniform dispersion, cool to room temperature, dilute
to volume with water and mix. Filter through a membrane filter
disc with an average pore diameter not greater than
1.0 /lm, rejecting the first few ml ofthe filtrate.
stavudine RS and 6.5 mg of thymine RS, and transfer to a
mobile phase: gradient mixtures of acetonitrile and
0.1 M ammonium acetate,
below,
Time
0.1 M Ammonium acetate Acetonitrile
(per cent v/v)
(in min.)
(per cent v/v)
5
o
95
5
5
95
80
25
20
80
30
20
5
31
95
5
35
95
Inject separately reference solutions (b) and (c). The test is
not valid unless the column efficiency determined from the
thymine, stavudine and lamivudine peaks is not less than
3000 theoretical plates and the tailing factor for the same peaks
is not more than 2.0.
chromatogram obtained with water for any extraneous peaks
and ignore the corresponding peaks observed in the
with the test solution corresponding to thymine is not more
than 3.0 per cent. Any other secondary peak observed in the
than 0.5 per cent and the sum ofthe areas ofall the secondary
peaks is not more than 4.0 per cent when calculated by
percentage area normalisation.
Apparatus No.1,
2153
STAVUDINE AND LAMIVUDINE TABLETS
IP 2010
Reference solution. In the case of tablets containing 30 mg of

stavudine, weigh accurately about 33 mg of stavudine RS and
167 mg of lamivudine RS and transfer to a 100 ml volumetric
flask. Add about 20 ml of methanol, sonicate to dissolve and
dilute to volume with a solvent mixture of equal volumes of
methanol and water (diluent). Dilute 5.0 ml of this solution to
50.0 ml with 0.01 M hydrochloric acid; in the case oftablets
containing 40 mg ofstavudine, weigh accurately about 44 mg
of stavudine RS and 167 mg of lamivudine RS and transfer to
a 100-ml volumetric flask. Add about 20 ml of methanol, mix
with the aid of ultrasound to dissolve and dilute to volume
with the diluent. Dilute 5.0 ml ofthis solution to 50.0 ml with
65 volumes of a buffer prepared by dissolving 0.68 g of
potassium dihydrogen phosphate and 1.0 g of sodium
octanesulphonate in 1000.0 ml of water to which 1 mlof
triethylamine is added and the pH of which is adjusted
to 2.5 with phosphoric acid.
column efficiency determined from the lamivudine peak is not
less than 2000 theoretical plates, the tailing factor for the
individual stavudine and lamivudine peaks is not more than
for each of the peaks corresponding to stavudine and
lamivudine is not more than 1.0 per cent.
and measure the responses for the major peaks due to
stavudine and lamivudine.
Calculate the contents ofCIOHlzNz04and CgHIIN303S in the
medium.
CIOH 1zNZ0 4and CgHIIN303S.
than 1.0 11m, rejecting the first few ml of the filtrate. Dilute
suitably with the same solvent mixture to obtain a solution
with a concentration of0.15 mg per ml oflamivudiIie.
Reference solution. A similarly prepared solution containing

0.015 per cent w/v of lamivudine RS and a concentration of
stavudine RS similar to that ofthe concentration ofstavudine
in test solution.
Use the chromatographic system described under Dissolution.
column efficiency determined from the lamivudine peak is not
less than 2000 theoretical plates, the tailing factor for the
individual peaks due to lamivudine and stavudine is not more
injections for each of the peaks corresponding to stavudine
and lamivudine is not more than 2.0 per cent.
and measure the responses for the major peaks.
Calculate the contents of CIOHIZNz04 and CgHIIN303S in the
tablets.
Stearic Acid
COOH
Stearic Acid consists ofa mixture offatty acids, chiefly stearic

acid (ClgH360Z) and palmitic acid (C I6H 32 0 Z). It may contain a
suitable antioxidant.
Stearic Acid contains not less than 40.0 per cent of CIgH360Z
and the sum of the contents of CIgH360Z and C I6H 320 Zis not
less than 90.0 per cent.
Category. Pharmaceutical aid (tablet lubricant; emulsion
adjunct).
Description. A white powder or white, greasy, flaky crystals
or hard masses showing signs of crystallisation.

Identification
Test solution. Weigh and powder 20 tablets. Transfer an

accurately weighed quantity of the powder containing about
150 mg ofLamivudine to a 100-ml volumetric flask, add about
20 ml of methanol and 50 ml ofa mixture ofequal volumes of
water and methanol,!!li;x,,,,,i!h _!h~lliQor1l1tra.~()_1l1!.c! J()!
5 minutes with occasional shaking to obtain a uniform
dispersion:CooltoroomtemperaiiifeanddHiite to
with the same solvent mixture. Filter the solution through a
In the Assay, the chromatogram obtained with the test solution

shows two principal peaks which correspond to the two
principal peaks in the chromatogram obtained with reference
solution (c).
volume
Tests
Congealing temperature (2.4.1 0). Not lower than 54.
Acid value (2.3.23). 200 to 212, determined on 1.0 g.
2154
IP 2010
STEARYL ALCOHOL
Iodine value (2.3.28). Not more than 4.0 (iodine monochloride

method).
Mineral acid. Shake 5 g ofthe melted substancewith an equal
volume of hot water for 2 minutes, cool and filter. To the
filtrate add 0.05 ml of methyl orange solution; no red colour is
produced.
on2.0 g.
Description. A white, unctuous mass or almost white flakes or

granules; odour, faint and characteristic.
Tests
Appearance of solution. Dissolve 0.5 g in 20 ml of ethanol
(95 per cent) by heating to boiling and allow to cool. The
solution is clear (2.4.1), and not more intensely coloured than
reference solution BS6 (2.4.1).
Melting range (2.4.21). 55to 60.
Test solution. Add 5 ml of boron trifluoride solution to 0.1 g

of the substance under examination and heat under a reflux
condenser for 15 minutes, cool and transfer to a separating
funnel with the aid of 10 ml of hexane. Add 10 ml ofa saturated
solution of sodium chloride and 10 ml of water, shake, discard
the lower aqueous layer and dry the upper layer over
anhydrous sodium sulphate.
Reference solution (a). Add 5 ml of a 1 per cent w/v solution
of nonadecanoic acid (internal standard) in toluene to a
mixture of50 mg ofstearic acidRS and 50 mg ofpalmitic acid
RS and evaporate to dryness. Add 5 m1 of boron trifluoride
solution and complete the procedure described for the test
solution beginning at the words "heat under a reflux
condenser...." ..
Reference solution (b). Add 5 ml of the internal standard
solution to 0.1 g ofthe substance under examination, evaporate
to dryness, add 5 ml of boron trifluoride solution and complete
the procedure described for the test solution beginning at the
words "heat under a reflux condenser......".
Reference solution (c). Prepare in the same manner as reference
solution (a) but omitting the internal standard.
a glass column 1.5 m x 4 rom, packed with acid-washed,
with 15 per cent w/w of diethylene glycol succinate
polyester,
temperature: column 170,

Iodine value (2.3.28). Not more than 2.0 (iodine bromide
method), determined on 2.0 g dissolved in 25 ml of chloroform,
warming if necessary to effect solution.
Saponification value (2.3.37). Not more than 2.0, determined
on 10.0 g.
Test solution. Dissolve 0.1 g 'of the substance under

examination in 10.0 ml.of ethanol (95 per cent).
Reference solution (a). Dissolve 50 mg of cetyl alcohol in
10.0 ml of ethanol (95 per cent).
Reference solution (b). Dissolve 50 mg of stealyl alcohol RS
in 5.0 ml of ethanol (95 per cent).
Reference solution (c). To 1.0 ml ofreference solution (a), add
1.0 ml of reference solution (b) and dilute to 10.0 ml with .
a stainless steel column 30 m x 0.32 rom, packed with
poly(dimethyl)siloxane (lllm),
temperature:
Temperature
Time
(min)
(0)
Column
0-20
150-250
20-40
250
injector port and detector at 250 0 ,
flow rate. 1 ml per minute using nitrogen as the carrier
gas.
Calculate the content OfClSH360z, and C 16H 32 0 Z

resolution between the peaks due to cetyl alcohol and stearyl
alcohol is not less than 5.0.
Stearyl Alcohol
Inject 1 III of the test solution, reference solution (b) and (c).
StearylAlcohol is a mixture ofsolid alcohols consisting chiefly
of l-octadecanol, (C 1sH380).
Calculate the content ofC 1s H38 0.
Category. Pharmaceutical aid (stiffening agent).

2155
STILBOESTROL
IP 2010
Stilboestrol

Diethylstilboestrol

OH

stilboestrol RS in ethanol (95 per cent).
stilboestrol monomethyl ether RS in ethanol (95 per cent).
HO
Mol. Wt. 268.4
Stilboestrol is (E)-a,~-diethylstilbene-4,4'-diol.
Stilboestrol contains not less than 97.0 per cent and not more
than 101.0 per cent ofClsHzoOz, calculated on the dried basis.
Category. Oestrogen.
Dose. In the treatment of menopausal symptoms, 100 Ilg to
I mg daily; for the suppression oflactation, 5 mg thrice daily
for 3 days, followed by 5 mg daily for 6 days; in the treatment
ofcarcinoma ofthe prostate and mammary carcinoma, 10 to 20
mgdaily.
odourless.
Identification
Compare the spectrum with that obtained with stilboestrol RS
or with the reference spectrum of stilboestrol.

irradiated solution prepared as directed in the Assay shows
C. In the test for Mono- and di-methyl ethers, the principal
D. Dissolve 0.5 mg in 0.2 ml ofglacial acetic acid, add I mlof
phosphoric acid and heat in a water-bath for 3 minutes; a
deep yellow colour is produced which almost disappears on
dilution with 3 ml of glacial acetic acid (distinction from
dienoestrol).
Tests
4,4'-Dihydroxystilben and related ethers. Absorbance of a
1.0 per cent w/v solution in ethanol at about 325 run, not more
than 0.50 (2.4.7).
Mono- and di-methyl ethers. Determine by thin-layer
chromatography (2.4.1 7), coating the plate with silica gel.

stilboestrol dimethyl ether RS in ethanol (95 per cent).
w/v each of dienoestrol RS and stilboestrol RS
(20 per cent) and heat at 120 for 10 minutes. Any secondary
spots in the chromatogram obtained with the test solution
corresponding to the mono-and di-methyl ethers of
stilboestrol are not more intense than the spots in the
chromatograms obtained with reference solutions (b) and (c)
respectively. Stilboestrol sometimes produces two spots. The
test is not valid unless the chromatogram obtained with
reference solution (d) shows two clearly separated spots of
approximately the same intensity.
ethanol to produce 100.0 ml and dilute 10.0 ml ofthis solution
to 100.0 ml with ethanol. To 25.0 ml of the resulting solution
add 25.0 ml of a solution prepared by dissolving 1 g of
dipotassium hydrogen phosphate in 55 ml of water, transfer a
portion of the mixture to a l-cm closed quartz cell, place the
cell 10 cm from a 15-watt short-wave, ultraviolet light ofmercury
lamp and irradiate for 10 minutes. Measure the absorbance of
the irradiated solution at the maximum at about 418 nm (2.4.7).
Calculate the content of ClsHzoOz from the absorbance
obtained by repeating the operation using stilboestrol RS in
Diethylstilboestrol Tablets
Stilboestrol Tablets contain not less than 90.0 per cent and
stilboestrol, ClsHzoOz.
2156
STREPTOKINASE
IP 2010
Usual strengths. 500 Ilg; 1 mg; 5 mg; 25 mg.
Streptokinase
Identification
Streptokinase is a preparation ofa protein obtained from culture

filtrat",s ofcertain strains of Streptococcus haemolyticus group
C. It has the property of combining with human plasminogen
to form plasminogen activator and is purified to contain not
less than 600 Units ofStreptokinase activity per Ilg ofnitrogen
before addition of any stabiliser or carrier. It usually contains
a buffer and may be stabilised by the addition of suitable
substances such as Human Albumin.

irradiated solution prepared as directed in the Assay shows
of Stilboestrol with ether, filter and evaporate the filtrate to
dryness. Dissolve 0.5 mg of the residue in 0.2 ml of glacial
acetic acid, add 1 ml of phosphoric acid and heat in a waterbath for 3 minutes; a deep yellow colour is produced which
almost disappears on dilution with 3 ml of glacial acetic acid
(distinction from dienoestrol).
Category. Fibrinolytic enzyme.

Dose. By intravenous infusion, 250,000 to 600,000 Units over
30 minutes followed by 100,000 Units every hour for upto 1
week.
Description. A white powder or a. white, friable solid;
hygroscopic.
Tests
Uniformity of content. For tablets containing 10 mg or less
Finely crush one tablet, add 10 ml of ethanol, shake for
30 minutes, add sufficient ethanol to produce 25.0 ml and
centrifuge. Pipette an aliquot of the supernatant liquid
containing 0.5 mg of Stilboestrol add 25.0 ml of a solution
prepared by dissolving 1 g of dipotassium hydrogen
phosphate in 55 ml of water, transfer a portion ofthe mixture
to a l-cm closed quartz cell, place the cellI cm from a 15-watt
short-wave, ultraviolet light ofmercury lamp and irradiate for
10 minutes. Measure the absorbance ofthe iITadiated solution
at the maximum at about 418 nm (2.4.7).
Calculate the content of ClsHzoOz in the tablet from the

stilboestrol RS in place of the substance under examination.
quantity ofthe powder containing about 5 mg of Stilboestrol,
add 5 ml of ethanol, shake for 15 minutes, add sufficient
ethanol to produce 100.0 ml and centrifuge. Dilute 20.0 ml of
the clear, supernatant liquid to 50.0 ml with ethanol and mix.
To 25.0 ml ofthe resulting solution, add 25.0 ml of a solution
prepared by dissolving 1 g of dipotassium hydrogen
phosphate in 55 ml of water, transfer a portion ofthe mixture
to a l-cm closed quartz cell, place the cellI 0 cm from a 15-watt
short-wave, ultraviolet light ofmercury lamp and iITadiate for
10 minutes. Measure the absorbance ofthe irradiated solution
at418nm(2.4.7).
Calculate the content of ClsHzoOz in the tablet from the
stilboestrol RS in place of the substance under examination.
Identification
A. Place 0.5 ml ofcitrated human, canine or rabbit plasma in a
haemolysis tube maintained in a water-bath at 37. Add 0.1 ml
of a solution of the substance under examination containing
10,000 Units per ml in citro-phosphate buffer pH 7.2 and
0.1 ml ofa solution of thrombin containing 20 Units per ml in
citro-phosphate buffer pH 7.2 and shake immediately; a clot
forms and lyses within 30 minutes. Repeat the procedure using
citrated bovine plasma; lysis does not occur within 1 hour.
B. Dissolve 0.6 g of agar in 50.0 ml of mixed barbitone buffer
pH 8.6, heating until a clear solution is obtained. Place glass
plates (50 mm x 50 mm) that are free from traces ofgrease on a
level surface. Apply to each plate 4 ml ofthe agar solution and
allow to cool until set. Bore a hole 6 mm in diameter in the
centre of the agar and an appropriate number of holes (not
exceeding six) at distances of 11 mm from the central hole
removing the residual agar by means of a cannula connected
to a vacuum pump. Place a quantity of 80 III of goat or rabbit
anti streptokinase serum containing 10,000 Units of
antistreptokinase activity per ml in the central hole and 80 III
of a solution of the substance under examination containing
125,000 Units of streptokinase activity per ml of each of the
sUITounding holes. Place the plates in a humidified tame for
24 hours.
Only one precipitation arc is produced which is well-defined
and localised between the application point of the serum and
each hole containing the solution of the substance under
examination.
Tests
pH (2.4.24). 6.8 t07.5, determined on a solution prepared in

freshly boiled and cooled water containing 5000 Units per m!.
2157
STREPTOKINASE
IP 2010
Streptodornase. Introduce 0.5 ml ofa 0.1 per cent wIv solution

of sodium deoxyribonucleate in imidazole buffer pH 6.5 into
each of eight centrifuge tubes. To each of the first two tubes
add 0.25 ml of imidazole bufferpH 6.5 and 0.25 ml ofsolution
of the substance under examination in imidazole bz!ffer
pH 6.5 containing 150,000 Units per ml (solution A) followed
immediately by 3.0 ml of 0.25 M perchloric acid. Mix the
contents of each tube, centrifuge for 5 minutes at 3000 rpm
and measure the absorbance ofeach ofthe supernatant liquids
at about 260 nm (2.4.7), using as the blank a mixture of 1.0 ml of
imidazole bufferpH 6.5 and 3.0 ml of 0.25 Mperchloric acid.
Calculate the sum ofthe two absorbances (AI)' To each of the
remaining six tubes add, respectively, 0.25, 0.25, 0.125, 0.125, 0
and 0 ml of imidazole buffer pH 6.5 followed by 0.25 ml of
solution A and finally 0, 0, 0.125, 0.125, 0.25 and 0.25 ml
respectively of a solution of the Standard Preparation
containing 20 Units of streptodornase activity per ml in
imidazole buffer pH 6.5. [The Standard Preparation is the
1st International Standard Preparation for Streptodornase,
established in 1964, consisting of a freeze-dried mixture of
streptodornase and streptokinase with lactose (supplied in
ampoules containing 2400 Units of streptodornase activity),
or another suitable preparation the activity of which has been
determined in relation to the International Standard]. Mix the
contents of each tube, incubate at 37 for 15 minutes and add
to each tube 3.0 ml of 0.25 M perchloric acid. Mix the contents
of each tube, centrifuge and measure the absorbance of each
ofthe supernatant liquids at about 260 nm (2.4.7), using as the'
blank the mixture specified above. Ifthe sum ofthe absorbances
of the liquids in the third and fourth tubes is A], that of the
liquids in the fifth and six tubes is A 3 , and that ofthe liquids in
the seventh and eighth tubes is (A 4 ), (AI-A]) is less than
0.5(A 3 +A 4)-A].
Streptolysin. Dissolve a quantity of the substance under

examination containing 500,000 Units in 0.5 ml ofa mixture of
90 volumes of saline solution and 10 volumes of citrophosphate bufferpH 7.2 in a haemolysis tube. Add 0.4 ml ofa
2.3 per cent w/v solution of sodium thioglycollate and
incubate in a water-bath at 37 for 10 minutes. Add 0.1 mlofa
solution ofa reference preparation of human antistreptolysin
o containing 5 Units per ml and incubate at 37 for 5 minutes.
Add 1 ml of rabbit elythrocyte suspension, continue the
incubation for 30 minutes and centrifuge at about 1000 rpm.
The absorbance of the supernatant liquid at about 550 nm
(2.4.7), is not more than 1.5 times the absorbance obtained
by repeating the above procedure using 0.5 ml of the mixture
of saline solution and citro-phosphate buffer pH 7.2 in
place of the solution containing the substance under
examination,
Loss Oil dryillg (2.4.19). Not more thal14.0 per cent, deterrriiIled
Assay. The potency of streptokinase is determined by

comparing its ability to activate human plasminogen to form
plasmin with that of the Standard Preparation. The plasmin
generated is detefll1ined by measurement of the time taken to
lyse a fibrin clot under the conditions of a suitable method of
assay.
Standard Preparation
The Standard Preparation is the 2nd International Standard
for Streptokinase, established in 1989, consisting of freezedried streptokinase (supplied in ampoules containing
700 Units of streptokinase activity), or another suitable
preparation the activity of which has been detefll1ined in
relation to the International reference preparation.
Method
Use citro-phosphate buffer pH 7.2 containing 3 per cent w/v
of bovine serum albumin for the preparation of solutions and
dilutions.
Prepare a solution of the Standard Preparation to contain
1000 Units of streptokinase activity per ml and prepare a
solution of the preparation under examination expected to
have the same concentration; keep the solutions in ice and
use within 6 hours. Prepare three 1.5-fold serial dilutions of
the solution of the Standard Preparation so that the longest
clot-lysis time is less than 20 minutes and prepare three similar
dilutions ofthe solution ofthe preparation under examination.
Keep the solutions in ice and use within 1 hour. Using
24 tubes, 8 mm in diameter, label the tubes S" S2, S3 for the
dilutions of the Standard Preparation and T 1, Ti, T3 for the
dilutions ofthe preparation under examination, allocating four
tubes to each dilution. Place the tubes in ice. Into each tube
introduce 0.2 ml of the appropriate dilution, 0.2 ml of citrophosphate buffer pH 7.2 containing 3 per cent w/v of bovine
serum albumin and 0.1 ml ofa solution containing 20 Units of
thrombin per ml. Place the tubes in a water-bath at 37 and
allow to stand for 2 minutes to attain temperature equilibrium.
Using an automatic pipette, introduce into the bottom of the
first tube 0.5 ml of a 1 per cent w/v solution to human
euglobulins ensuring mixing. At 5-second intervals introduce
successively into the remaining tubes 0.5 ml of a 1 per cent
w/v solution of human euglobulins. Using a stop-watch,
measure for each tube the time in seconds that elapses between
the addition of the euglobulin and the lysis of the clot.
Using the logarithms ofthe lysis times, calculate the result of
the assay by standard statistical methods.
The estimated potency is not less than 90 per cent and not
iiiore tha.n III per cent of the sta.ted potency. The fiducial
limits of error are not less than 80 per cent and not more than
125 per cent of the stated potency.
2158
IP 2010
STREPTOKINASE INJECTION
Streptokinase intended for use in the manufacture ofparenteral

preparations complies with the following additional
requirements.
Abnormal toxicity (2.2.1). Determine by Method A, using a
solution containing 50,000 Units in 0.5 ml of water for
injections administered in 15 to 20 seconds.
Bacterial endotoxins (2.2.3). Dissolve the contents of the
sealed container in water BET to give a solution containing
10,000 Units ofStreptokinase per ml. Carry out the test on the
resulting solution; the maximum allowable endotoxin
concentration of the solution is 23.33 Units of endotoxin per
ml. Carry out the test using the maximum valid dilution ofthe
prepared solution calculated from the declared sensitively of
the lysate used in the test.
Storage. Store in sealed containers, protected from light. The
containers should be sterile; tamper-evident and sealed so as
to exclude micro-organisms. Under these conditions the
contents may be expected to retain their potency for 2 years.
Labelling. The label states (1) the number of Units of
streptokinase activity in the container; (2) the number ofUnits
of streptokinase activity per mg, calculated with reference to
the dried preparation; (3) the name and quantity of any
added substances; (4) the storage conditions; (5) whether or
not it is intended for use in the manufacture of parenteral
preparations.
Identification
A. Place 0.5 ml ofcitrated human, canine or rabbit plasma in a
haemolysis tube maintained in a water-bath at 37. Add 0.1 ml
of a solution of the contents of the container containing
10,000 Units per ml in citro-phosphate buffer pH 7.2 and
0.1 ml ofa solution of thrombin containing 20 Units per ml in
citro-phosphate buffer pH 7.2 and shake immediately; a clot
citrated bovine plasma; lysis does not occur within 1 hour.
B. Dissolve 0.6 g of agar in 50.0 ml of mixed barbitone buffer

pH 8.6, heating until a clear solution is obtained. Place glass
plates (50 mm x 50 mm) that are free from traces ofgrease ona
level surface. Apply to each plate 4 ml ofthe agar solution and
allow to cool until set. Bore a hole 6 mm in diameter in the
centre of the agar and an appropriate number of holes
(not exceeding six) at distances of 11 mm from the central hole
removing the residual agar by means of a cannula connected
to a vacuum pump. Place a quantity of 80 III of goat or rabbit
anti streptokinase serum containing 10,000 Units of
antistreptokinase activity per ml in the central hole and 80 III
of a solution of the contents of the container containing
125,000 Units of streptokinase activity per ml of each ofthe
surrounding holes. Place the-plates in a humidified tanle for
24 hours.
Only one precipitation arc is produced which is well-defined
and localised between the application point of the serum and
each hole containing the solution of the substance under
examination.
Tests
Streptokinase Injection is a sterile material consisting of
Streptokinase with or without auxiliary agents. It is filled in a
sealed container.
'

requirements stated under Parenteral Preparations (Powders
for Injection) and with the following requirements.
Usual strengths. 50,000 Units; 100,000 Units; 750,000 Units;
1,500,000 Units.
pH (2.4.24). 6.8 to 7.5, detennined on a freshly constituted

injection containing 5000 Units per ml.
Streptodornase. Introduce 0.5 ml ofa 0.1 per centw/v solution
of sodium deoxyribonucleate in imidazole buffer pH 6.5 into
each of eight centrifuge tubes. To each of the first two tubes
add 0.25 ml of imidazole bufferpH 6.5 and 0.25 ml ofsolution
of the contents of the container with the substance under
examination in imidazole buffer pH 6.5 containing
150,000 Units per ml (solution A) followed immediately by
3.0 ml of 0.25 M perchloric acid. Mix the contents of each
tube, centrifuge for 5 minutes at 3000 rpm and measure the
absorbance of each of the supernatant liquids at about
260 mn (2.4.7), using as the blank a mixture of 1.0 ml ofimidazole
bufferpH 6.5 and 3.0 ml of 0.25 M perchloric acid. Calculate
the sum ofthe two absorbances (AI)' To each ofthe remaining
six tubes add, respectively, 0.25, 0.25, 0.125, 0.125, 0 and 0 ml
of imidazole btiffer pH 6.5 followed by 0.25 ml of solution A
and finally 0, 0, 0.125, 0.125, 0.25 and 0.25 ml respectively ofa
solution of the Standard Preparation containing 20 Units of
streptodornase activity per ml in imidazole btiffer pH 6.5.
2159
STREPTOKINASE INJECTION
IP 2010
[The Standard Preparation is the 1st International Standard

Preparation for Streptodornase, established in 1964, consisting
of a freeze-dried mixture of streptodornase and streptokinase
with lactose (supplied in ampoules containing 2400 Units of
streptodornase activity), or another suitable preparation the
activity of which has been determined in relation to the
International Standard]. Mix the contents of each tube,
incubate at 37 for 15 minutes and add to each tube 3.0 ml of
0.25 M perchloric acid. Mix the contents of each tube,
centrifuge and measure the absorbance of each of the
supernatant liquids at about 260 nm (2.4.7), using as the blank
the mixture specified above. Ifthe sum of the absorbances of
the liquids in the third and fourth tubes is A 2 , that of the
liquids in the fifth and six tubes is A 3 , and that ofthe liquids in
the seventh and eighth tubes is (A 4), (A I-A 2) is less than
0.5(A 3 +A,J-A 2
Streptolysin. Dissolve a quantity of the contents of the
container containing 500,000 Units in 0.5 ml of a mixture of
90 volumes of sali;1e solution and 10 volumes of citrophosphate bufferpH 7.2 in a haemolysis tube. Add 0.4 ml ofa
2.3 per cent w/v solution of sodium thioglycollate and
incubate in a water-bath at 37 for 10 minutes. Add 0.1 mlofa
solution of a reference preparation of human antistreptolysin
o containing 5 Units per ml and incubate at 37 for 5 minutes.
Add 1 ml of rabbit elythrocyte suspension, continue the
incubation for 30 minutes and centrifuge at about 1000 rpm.
The absorbance of the supernatant liquid at about 550 nm
(2.4.7), is not more than 1.5 times the absorbance obtained
by repeating the above procedure using 0.5 ml ofthe mixture
of saline solution and citro-phosphate buffer pH 7.2 in
place of the solution containing the substance under
examination.
sealed container in water BET to give a solution containing
10,000 Units ofStreptokinase per ml. Can)' out the test on the
resulting solution; the maximum allowable endotoxin
concentration of the solution is 23.33 Units of endotoxin per
ml. Carry out the test using the maximum valid dilution ofthe
prepared solution calculated from the declared sensitively of
the lysate used in the test.
Assay. Determine on the mixed .contents often containers.
The potency of streptokinase is determined by comparing
its ability. to activate human plasiUinogen to fOrm plaslnin
with that of the Standard Preparation. The plasmin
generated is determined by measurement ()fthe time taken to
lyse a fibrin clot under the conditions of a suitable method of
assay.
The Standard Preparation is the 2nd' International. Standard
for Streptokinase, established in 1989, consisting of freezedried streptokinase (supplied in ampoules containing
700 Units of streptokinase activity), or another suitable
preparation the activity of which has been determined in
relation to the International reference preparation.
Method
Use citro-phosphate buffer pH 7.2 containing 3 per cent w/v
of bovine serum albumin for the preparation of solutions and
dilutions.
Prepare a solution of the Standard Preparation to contain
1000 Units of streptokinase activity per ml and prepare a
solution ofthe contents ofthe container expected to have the
same concentration; keep the solutions in ice and use within
6 hours. Prepare three 1.5-fold serial dilutions ofthe solution
of the Standard Preparation so that the longest clot-lysis time
is less than 20 minutes and prepare three similar dilutions of
the solution of the preparation under examination. Keep the
solutions in ice and use within 1 hour. Using 24 tubes, 8 mm in
diameter, label the tubes S], S2, S3 for the dilutions of the
Standard Preparation and T], T2 , T3 for the dilutions of the
preparation under examination, allocating four tubes to each
dilution. Place the tubes inice. Into each tube introduce 0.2 ml
of the appropriate dilution, 0.2 ml of citro-phosphate buffer
pH 7.2 containing 3 per cent w/v of bovine serum albumin
and 0.1 ml of a solution containing 20 Units of thrombin per
ml. Place the tubes in a water-bath at 37 and allow to stand for
2 minutes to attain temperature equilibrium. Using an automatic
pipette, introduce into the bottom of the first tube 0.5 ml of a
1 per cent w/v solution to human euglobulins ensuring mixing.
At 5-second intervals introduce successively into the
remaining tubes 0.5 ml of a 1 per cent w/v solution of human
euglobulins. Using a stop-watch, measure for each tube the
time in seconds that elapses between the addition of the
euglobulin and the lysis of the clot.
Using the logarithms of the lysis times, calculate the result of
more than 111 per cent of the stated potency. The fiducial
limits of error are not less than 80 per cent and not more than
125 per cent of the stated potency.
Storage. Store in sealed containers, protected from light in a
refrigerator (2 to 8). The containers should be sterile and
sealed so as to exclude micro-organisms. Under these
co~ditions -the contents may -be expected to retain their
potency for 2 years.
Labelling. The label states the total number of Units of
streptokinase activity contained in it.
2160
IP 2010
STREPTOMYCIN SULPHATE

Reference solution (a). A 0.1 per cent w/v of streptomycin
w/v of streptomycin sulphate RS, 0.1 per cent w/v of neomycin
sulphate RS and 0.1 per cent w/v of kanamycin monosulphate
RSin water.
phase to rise 12 cm. Dry the plate in warm air, spray with a
mixture of equal volumes of a 0.2 per cent w/v solution of
naphthalene-l,3-diol in ethanol (95 per cent) and sulphuric
acid (45 per cent) and heat at 150 for 5 to 10 minutes. The
2
(CzIH39N7012)z,3HzS04
Mol. Wt. 1457.4
Streptomycin Sulphate is the sulphate of 0-2-deoxy2-methylamino-a-L-glucopyranosyl-( 1-72)-O-5-deoxy3-C-formyl-a-L-lyxofuranosyl-(I-4)-NJ,N3-diamidinoD-streptamine, a substance produced by the growth of
certain strains of Streptomyces griseus or obtained by any
other means.
B. Dissolve 5 to 10 mg in 4 ml of water and add 1 ml of

1 M sodium hydroxide. Heat in a water-bath for 4 minutes. Add
a slight excess of2 Mhydrochloric acid and 0.1 ml ofa 10 per
cent w/v solution ofjerric chloride; a violet colour is produced.
C. Dissolve 0.1 g in 2 ml of water and add 1 ml of dilute
I-naphthol solution and 2 ml ofa mixture ofequal volumes of
dilute sodium hypochlorite solution and water; a red colour
is produced.
Streptomycin Sulphate has a potency equivalent to not less

than 700 flg and not more than 850 flg ofstreptomycin per mg.
It contains not less than 90.0 per cent of the stated amount of
streptomycin, C21H39N7012, calculated on the dried basis.
D. Dissolve 10 mg in 5 ml ofwater and add 1 ml of1 Mhydrochloric acid. Heat in a water-bath for 2 minutes. Add
2 ml ofa 0.5 percentw/v solution of I-naphthol in 1 Msodium
hydroxide and heat in a water-bath for 1 minute; a faint yellow
colour is produced.
E. Gives the reactions of sulphates (2.3.1).
Dose. By intramuscular injection, the equivalent of 500 mg to
1 g of streptomycin daily, or at longer intervals.
with slight odour; hygroscopic.
Identification
A. Determine by thin-layer chromatography (2.4.17). Prepare
the plate by mixing 0.3 g of carbomer with 240 ml of water,
allowing to stand with moderate stirring for 1 hour, adjusting
the pH to 7.0 by the gradual addition with constant shaking of
2 M sodium hydroxide and adding 30 g of silica gel H. Spread
a uniform layer of the resulting suspension 0.75 mm thick.
Heat the plate at 110 for 1 hour, allow to cool and use
immediately.
Mobile phase. A 7 percent w/v solution of potassium

dihydrogen phosphate.
Tests
Appearance ofsolution. A25.0 per cent w/v solution in carbon
dioxide-ji'ee water is not more intensely coloured than degree
3 of the appropriate range of reference solutions (2.4.1). The
solution, after standing protected from light at a temperature
of about 20 for 24 hours, is not more opalescent than
opalescence standard OS2 (2.4.1).
pH (2.4.24). 4.5 to 7.0, determined in a25.0percentw/v solution.
Sulphates. 18.0 to 21.5 per cent, calculated on the dried basis,
when determined by the following method. Dissolve 0.25 g in
100 ml of water, adjust the pH to 11 with strong ammonia
solution and add 10.0 ml of 0.1 Mbarium chloride and 0.5 mg
of metalphthalein. Titrate the excess of barium chloride with
0.1 M disodium edetate, adding 50 ml of ethanol (95 per
cent) when the colour of the solution begins to change and
continuing the titration until the violet-blue colour disappears.
2161
STREPTOMYCIN SULPHATE
IP 2010
1 ml of 0.1 M barium chloride is equivalent to 0.009606 g of

sulphate, S04.
Colorimetric test. Dissolve 0.1 g in sufficient water to produce
100 ml. To 5 ml, add 5 ml of 0.2 M sodium hydroxide and heat
in a water-bath for exactly 10 minutes. Cool in ice for exactly
5 minutes, add 3 ml of a 1.5 per cent w/v solution of ferric
ammonium sulphate in 0.25 M sulphuric acid and sufficient
water to produce 25 ml and mix. Exactly 20 minutes after the
addition of the ferric ammonium sulphate solution, measure
the absorbance ofa 2-cm layer ofthe solution atthe maximum
at about 525 nm (2.4.7), using as the blank a solution prepared
in the same manner but omitting the substance under
examination. The absorbance is not less than 90.0 per cent of
that obtained by carrying out the procedure at the same time
and in the same manner using streptomycin sulphate RS in
place of the substance under examination, each absorbance
being calculated on the dried basis.
Streptomycin B. Determine by thin-layer chromatography

of glacial acetic acid and 25 volumes of methanol.
examination in 5 ml ofa freshly prepared mixture of97 volumes
of methanol and 3 volumes of sulphuric acid, heat under a
reflux condenser for I hour, cool, wash down the condenser
with methanol and add sufficient methanol to produce 20 m!.
Reference solution. Dissolve 36 mg of D-mannose in 5 ml ofa
freshly prepared mixture of 97 volumes of methanol and
3 volumes of sulphuric acid, heat under a reflux condenser
for 1 hour, cool, wash down the condenser with methanol and
add sufficient methanol to produce 50 m!. Dilute 5 rnl of the
resulting solution to 50 ml with methanol; this solution
contains the equivalent of0.03 per cent w/v ofstreptomycin B
(1 mg of D-mannose is equivalent to 4.13 mg of streptomycinB).
- a glass column 1.5 to 2.0 m x 2 to 4 mm, packed with
ethylvinylbenzene-divinylbenzene copolymer (150 to
180 Ilm) porous polymer beads (such as Porapak Q),
- temperature: column 120 to 140,
inlet port and detector at least 50 higher than that of
the column,
flow rate. 30 to 40 ml per minute ofthe carrier gas.
The area of any peak corresponding to methanol in the
chromatogram obtained with the test solution is not greater
on 1.0 g by drying over phosphorus pentoxide at 60 at a
pressure not exceeding 0.1 kPa for 24 hours.
Method A or B (2.2.10), and express the results in Ilg of
streptomycin per mg.
Streptomycin Sulphate intended for use in the manufacture

ofparenteral preparations without a further appropriate
procedurefor removal ofbacterial endotoxins complies with
the following additional requirement.
Unit per mg ofstreptomycin.
Streptomycin Sulphate intended for use in the manufacture

of parenteral preparations without a further appropriate
intended for use in the manufacture ofparenteral preparations
micro-organisms.

phase to rise 13 to 15 cm. Dry the plate in air, and spray with a
freshly prepared mixture of equal volumes of a 0.2 per cent
w/v solution ofnaphthalene-1,3-diol in ethanol (95 per cent)
and a 20 per cent v/v solution of sulphuric acid and heat at
110 for 5 minutes. Any spot in the chromatogram obtained
with the test solution is not more intense than the
corresponding spot in the chromatogram with the reference
solution.
Labelling. The label states (1) the equivalent weight of

streptomycin contained in it; (2) whether or not the contents
are intended for use in the manufacture of parenteral
preparations; (3) the name and quantity ofany added stabiliser.
Methanol. Determine by gas chromatography (2.4.13).
Streptomycin Injection is a sterile material consisting of

Streptomycin Sulphate with or without auxiliary agents. It is

Reference solution. A 0.012 per cent wlV solution of methanol
in water.
Streptomycin Sulphate Injection
filredin-a-seah:~a-container~----'--
--- '-'"--'-"-'--'"--'-'._.__._._.,-

2162
IP 2010
STREPTOMYCIN TABLETS

Storage. The constituted solution'should be used immediately
Streptomycin Injection contains not less than 90.0 per cent
streptomycin, CZIH39N70Iz.

(Powders for Injection) and with the following requirements.
B. Dissolve 5 to 10 mg in 4 ml ofwater and add 1 ml of1Msodium

hydroxide, Heat in a water-bath for 4 minutes. Add a slight
excess of 2 M hydrochloric acid and 0.1 ml of a 10 per cent
w/v solution offerric chloride; a violet colour is produced.
C. Dissolve 0.1 g in 2 ml of water and add 1 ml of dilute
dilute sodium hypochlorite solution and water; a red colour
is produced.
D. Dissolve 10mgin.5 mlofwaterandadd 1 mlof I Mhydrochloric acid. Heat in a water-bath for 2 minutes. Add
2 ml ofa 0.5 per cent w/v solution of I-naphthol in I M sodium
hydroxide and heat in a water-bath for 1 minute; a faint yellow
colour is produced.
Usual strengths. The equivalent of 750 mg and 1 g of

streptomycin.
Tests
Description. A white or almost white powder which yields a

clear, colourless or faintly yellow coloured solution when
dissolved in water.
pH (2.4.24). 4.5 to 7.0, determined in a25.0 per cent w/v solution.
Identification
A, Determine by thin-layer chromatography (2.4.17). Prepare
the plate by mixing 0.3 g of carbomer with 240 ml of water,
allowing to stand with moderate stirring for 1 hour, adjusting
the pH to 7.0 by the gradual addition with constant shaking of
2 M sodium hydroxide and adding 30 g of silica gel H. Spread
a uniform layer of the resulting suspension 0.75 rom thiclc.
Heat the plate at 110 for 1 hour, allow to cool and use
immediately.
Mobile phase. A 7 per cent w/v solution of potassium

Reference solution (a). A 0.1 per cent w/v of streptomycin
Assay. Determine on the mixed contents often containers by
the microbiological assay ofantibiotics, MethodA or B (2.2.10).
Bacterial endotoxins (2.2.3): Not more than 0.25 Endotoxin
Unit per mg ofstreptomycin.
equivalent amount of streptomycin.
Streptomycin Sulphate Tablets

w/v of streptomycin sulphate RS, 0.1 per cent w/v of neomycin
sulphate RS and 0.1 per cent w/v of kanamycin monosulphate
RSin water.
Streptomycin Tablets contain not less than 90.0 per cent and
streptomycin, CZIH39N7012.

phase to rise 12 cm. Dry the plate in warm air, spray with a
mixture of equal volumes of a 0.2 per cent w/v solution of
naphthalene-I,3-diol in ethanol (95 per cent) and sulphuric
acid (45 per cent) and heat at 150 for 5 to 10 minutes. The
Identification
Usual strength. The equivalent of500 mg ofstreptomycin.
A. Triturate a quantity of the powdered tablets containing

0.2 g of streptomycin in a mixture of 2 ml of methanol and
0.1 ml of sulphuric acid, filter, ifnecessary, and allow to stand
at about 25; crystals of streptidine sulphate separate in the
course of 2 to 3 days. Dissolve the crystals in a solution of
0.1 g of picric acid in 10 m1 of hot water and cool; the
precipitate, after recrystallisation from hot water, melts at about
283 (2.4.21).
2163
SUCCINYLCHOLINE CHLORIDE
IP 2010
B. Boil a small quantity of the powdered tablets containing

0.1 g ofstreptomycin with 5 ml of1 M sodium hydroxide for a
few minutes, add a slight excess of 2 M hydrochloric acid
and 0.15 ml of a 10 per cent w/v solution offerric chloride; a
brilliant violet colour is produced.
Tests
quantity ofthe powder containing about 0.3 g ofstreptomycin
and, triturate with 20 ml of buffer solution pH 8.0. Dilute to
A or B (2.2.10).
equivalent amount of streptomycin.
B. Dissolve about 25 mg in 1 ml of water, add 0.1 ml ofal per

cent w/v solution of cobalt chloride and 0.1 ml of potassium
ferrocyanide solution; a green colour is produced.
C. Dissolve 1 g in sufficient carbon dioxide-.free water to
produce 20 ml. To 1 ml ofthis solution add 9 ml of water, 10 ml
of 1 M sulphuric acid and 30 ml of ammonium reineckate
solution; a pink precipitate is produced. Allow to stand for
30 minutes, filter and wash with water, then with ethanol
(95 per cent) and finally with ether. The residue, after drying
at 80 melts at l800 to 185(2.4.21).
D. Gives the reactions of chlorides (2.3.1).
Tests
Appearance of-solution. A 5.0 per cent w/v solution in carbon
dioxide-free water (solution A) is clear (2.4.1).4 ml ofsolution
A diluted to 10 ml with water is colourless (2.4.1).
pH (2,4,24). 4.0 to 5.0, determined in a 0.5 per cent w/v solution.
Choline chloride. Determine by thin-layer chromatography
(2.4.17), coating the plate with microclystalline cellulose.
Mobile phase. A mixture of 50 volumes of I-butanol,

40 volumes of water and 10 volumes of anhydrous formic
acid, shake for 10 minutes, allow to separate. Use the upper
layer as the mobile phase.
Suxamethonium Chloride

Mol. Wt. 397.3

Succinylcholine Chloride is 2,2'-succinyldioxybis(ethyltrimethylammonium) dichloride dihydrate.
Succinylcholine Chloride contains not less than 98.0 per cent
and not more than 101.0 per cent ofC14H30ChN204, calculated
Category. Depolarising skeletal muscle relaxant.
Dose. By intravenous injection, 20 to 100 mg, according to the
needs of the patient.
almost odourless; hygroscopic.
Identification
Band C may be omittediftests A and D are carried out.
Compare the spectrum with that obtained with succinylcholine
chloride RS or with the reference spectrum ofsuccinylcholine
chloride.
Reference solution. A solution containing 4 per cent w/v of

succinylcholine chloride RS and 0.02 per cent w/v of choline
chloride in methanol.
dry the plate in air, spray with potassium iodobismuthate
with the test solution is not more intense than the spot due to
choline chloride in the chromatogram obtained with the
reference solution. The test is not valid unless the
chromatogram obtained with the reference solution shows
anhydrous glacial acetic acid, add 30 ml of acetic anhydride
and 10 ml of mercuric acetate solution. Titrate with
indicator, until a bluish green colour is produced. Carry out a
blank titration.
C14H30ChN204.
2164
IP 2010
SUCRALOSE
Sucralose
Suxamethonium Chloride Injection
~
O:
Succinylcholine Injection is a sterile solution of

Succinylcholine Chloride in Water for Injections.
HO
Succinylcholine Injection contains not less than 90.0 per cent

succinylcholine chloride, C,4H30CIzN204,2H20.
CI
0
OHo~CI
OH
Usual strength. 50 mg per m!.
Identification
Mol. Wt. 397.6

Sucralose is 1,6-dichloro-l ,6-dideoxy-~-o-fructofuranosyl-4
chloro-4-deoxy-0'.-o-galactopyranoside.
Dilute a volume containing 20 mg ofSuccinylcholine Chloride

to 50 ml with water. To 0.5 ml add 2 ml of chloroform, 2 ml ofa
solution containing 0.16 per cent w/v of citric acid and 6.6 per
cent w/v of disodium hydrogen phosphate and 0.1 ml of a
solution containing 0.15 per cent w/v ofeach of bromothymol
blue and anhydrous sodium carbonate. Shake for 2 minutes
and allow to separate. The chloroform layer is yellow.
Sucralose contains not less than 98.0 per cent and not more
than 102.0 per cent ofC12H19ChOs, calculated on the anhydrous
basis.
Tests
Identification
pH (2.4.28).3.0 to 5.0.
Hydrolysis products. The volume of 0.1 M sodium hydroxide

required for the preliminary neutralisation in the Assay is not
more than one tenth of the total volume of 0.1 M sodium
hydroxide required for the preliminary neutralisation and the
hydrolysis.
0.25 g ofSuccinylcholine Chloride add 30 ml of carbon dioxidefi-ee water and shake with five quantities, each of 25 ml, of
ether. Wash the combined ether solutions with two quantities,
each of 10 ml, of water and discard the ether. Shake the
combined washings with two quantities, each of 10 ml, of
ether, add the washings to the original aqueous solution and
neutralise with 0.1 M sodium hydroxide using bromothymol
blue solution as indicator. Add 25.0 ml of 0.1 M sodium
hydroxide, heat under a reflux condenser for 40 minutes, allow
to cool and titrate the excess ofallcali with 0.1 M hydrochloric
acid using bromothymol blue solution as indicator. Repeat
the operation using 40 rol of carbon dioxide-free water
beginning at the words "Add 25.0 ml of 0.1 M sodium
hydroxide .....". The difference between the titrations
represents the amount of sodium hydroxide required.
CI~30CIzN204,2H20.
Storage. Store protected from light, at a temperature between

2 to 8. The injection should not be allowed to freeze.
Category. Pharmaceutical aid (sweetening vehicle).
Compare the spectrum with that obtained with sucralose RS

or with the reference spectrum of sucralose.
Tests
Specific optical rotation (2.4.22). +84.0 to +87.5, determined'
at 20 in a 1.0 per cent w/v solution in wate!:
(2.4.17), coating the plate with silica gel.
Mobile phase. A mixture of 70 volumes of 5 per cent w/v
solution of sodium chloride and 30 volumes of acetonitrile.
Reference solution (a). A 1.0 per cent w/v solution ofsucralose
RS in methanol.

dry the plate in air and spray the plate with 15 per cent w/v
solution of sulphuric acid in methanol. Heat the plate at 125
for 10 minutes. The Rf value of the principal spot obtained
with the test solution corresponds to the spot in the
chromatogram obtained with reference solution (a). Any
2165
IP 2010
SUCRALOSE

Hydrolysis products. Determine by thin-layer chromatography
(2.4.17), coating the plate with silica gel.
Sucrose
Refilled Sugar
HO
NOTE-This test does not require a developing solvent.
.)---0

examination in 10 mtofmethanol.
OH
OH

mannitol in water.
Hl.----O~
~l
OH
OH

w/v offructose and 10 per cent w/v of mannitol in water.
Apply to the plate 5 J.ll of each solution. Spray with a solution
containing 1.23 g ofp-anisidine and 1.66 g ofphthalic acid in
100 rn1 ofmethanol, heatthe plate at 105 for 15 minutes. Ifthe
spot from reference solution (a) has darkened, repeat the test,
heating for a shorter period oftime. Immediately after heating,
view the plate against a dark background: the color of the
spot obtained from the test solution is not more intense than
that obtained from reference solution (b) (0.1 per cent).
Description. An almost white or colourless crystals, dry

crystalline powder; odourless; taste, sweet.
Identification

LOg.
Reference solution. A 0.1 per cent w/v solution of sucralose
octadecylsilane bonded to porous silica (5 J.lm),
refractive index detector
injection volume. 20 J.ll.
than 2.0 percent.
ClzHn011
Mol. Wt. 342.3
Sucrose is ~-D-fructofuranosyl a-D-glucopyranoside.

Category. Pharmaceutical aid (sweetening agent; tablet
excipient).
Dissolve 150.0 g in sufficient carbon dioxide-free water

prepared from distilled water to produce 300 ml (solution A).
Dilute 1 ml of solution A to 100 ml with water. To 5 ml of the
solution add 2 ml of freshly prepared 2 M sodium hydroxide
and 0.15 ml of a freshly prepared copper sulphate solution;
the solution is clear and blue and remains so on boiling. To
the hot solution add 4 ml of 2 M hydrochloric acid, heat to
boiling and add 4 ml of 2 M sodium hydroxide; an orange
precipitate is produced immediately.
Tests
phenolphthalein solution. The solution is colourless and not
more than 0.6 m1 of 0.01 M sodium hydroxide is required to
change the colour of the solution to pink.
in a 10 per cent w/v solution.
Barium. To 10 ml ofsolution A add I ml of 1 M sulphuric acid.
When examined immediately and after 1 hour any opalescence
is not more intense than that of a mixture of 1 nil of distilled
water and 10 ml of solution A.
Calculate the cOntent ofClzH19ChOso
Calcium. To 1 ml of solution A add 9 ml ofwater and 1 mlof

Cfrffrff6f11Uftfoxalate-S6Itttiol1;-tlre-so1utionremains clear-for at
least 1 minute.

exceeding 21.
Heavy metals (2.3.13). Add 0.1 ml ofdilute hydrochloric acid

to 4 ml ofsolution A and dilute with sufficient water to produce
test solution and the reference solution.
2166
IP 2010
SULPHACETAMIDE SODIUM
Method A (10 ppm).
Sulphacetamide Sodium is the monohydrate ofthe sodium

salt ofNI-acetylsulphanilamide.
Sulphites. To 4 ml ofsolution A add sufficient water to produce

20 ml, add 0.05 ml of 0.1 M iodine and 0.05 ml ofstarch solution;
a blue colour develops.
Sulphacetamide Sodium contains not less than 99.0 per cent

and not more than 101.0 per cent ofCsH9N2Na03S, calculated
Dextrins. To 2 ml of solution A add 8 ml of water, 0.05 ml of

2 M hydrochloric acid and 0.05 ml of 0.05 M iodine; the
solution remains yellow or becomes faint bluish green.
Glucose and invert sugar. Dissolve 20 g in sufficient water to

make 100 ml and filter if necessary. Place 50 ml of the clear
solution in a 250-ml beaker, add 50 ml of alkaline cupric
tartarate solution, cover the beaker with a watch glass, heat
the mixture at such a rate that it comes to a boil in approximately
4 minutes and continue boiling for exactly 2 minutes. Add
immediately 100 ml of recently boiled and cooled water and
collect the precipitated cuprous oxide on a tared sintered.glass crucible. Wash the residue with the hot water, then with
10 ml of ethanol (95 per cent) and fmally with 10 ml of ether.
Dry at 105 for 1 hour; the weight of the cuprous oxide is not
more than 112 mg.
Colouring matter. A. To 100 mLof solution A in a groundglass~stoppered tube add 1 ml of dilute hypophosphorous
acid and allow to stand for 1 hour; no unpleasant odour is
detectable.
B. Examine solution A in ultraviolet light at 365 nm. Any
fluorescence is not more intense than that of a solution
containing 0.4 Ilg per ml of quinine sulphate in 0.005 M
sulphuric acid.
.
Sulphated ash (2.3.18). Not more than 0.1 per cent detennined
by dissolving 5.0 g in 5 ml of water, adding 2 ml of sulphuric
acid, evaporating to dryness and igniting to constant weight.
Sucrose intended for use in the manufacture ofparenteral

preparations without a fitrther appropriate procedure for
unit per mg of Sucrose.
Description. A white or yellowish white, crystalline powder;

odourless.
Identification
Test A may be omitted iftests B, C, D, E and F are carried out.
Tests B, C, D and E may be omitted iftests A andF are carried
out.
Compare the spectrum with that obtained with sulphacetamide
sodium RS or with the reference spectrum of sulphacetamide
sodium.
reference solution.
C. Dissolve 1 g in 10 ml of water, add 6 ml of2 M acetic acid
and filter. Wash the precipitate with a small volume of water
and dry at 105 for 4 hours. The melting range ofthe precipitate
is 181to 185 (2.4.21).
D. Dissolve 0.1 g ofthe precipitate obtained in test C in 5 mlof
ethanol (95 per cent), add 0.2 ml of sulphuric acid and heat;
ethyl acetate, recognizable by its odour, is produced.
E. Dissolve 1 mg ofthe precipitate obtained in test C in 5 mlof
water with the aid of heat. The solution gives the reaction of
primary aromatic amines (2.3.1), producing an orange-red
precipitate.
F. A 5 per cent w/v solution gives the reactions of sodium

salts (2.3.1).
Tests

SuIphacetamide Sodium
pH (2.4.28).8.0 to 9.5, detennined in a 5.0 per centw/v solution.

(2.4.14).
Note-Prepare the solutions immediately before use and

Mol. Wt. 254.2

2167
IP 2010
SULPHACETAMIDE SODIUM
Reference solution (a). Dissolve 5 mg each of sulphacetamide

sodium RS and sulphanilamide (sulphacetamide sodium
impurity A) in 1.0 m1 ofthe mobile phase.
Sulphacetamide Sodium Eye Drops
Reference solution (b). Dilute 1.0 m1 of the test solution to

100.0 ml with the mobile phase. Further dilute 1.0 ml ofthis
Sulphacetamide Eye Drops are a sterile solution of

Su1phacetamide Sodium in Purified Water. It may contain a
suitable antimicrobial agent.
Sulphacetamide Eye Drops contain not less than 95.0 per cent
sulphacetamide sodium, CSH9NzNa03S,HzO.
Jill1)
-
mobile phase: a mixture of I volumes of glacial acetic

acid, 10 volumes of methanol and 89 volumes of water,
flow rate. 0.8m1perminute,
resolution between the peaks due to sulphacetamide impurity
A and sulphacetamide is not less than 5.0. The relative retention
time with reference to sulphacetamide for sulphacetamide
impurity A is about 0.5.
ofthe peak due to sulphacetamide impurity A is not more than
any secondary peak is not more than the area of the principal
(b) (0.1 per cent). The sum of all the secondary peaks is not
Sulphates (2.3 .17). Dissolve 1.5 g in sufficient distilled water
to produce 25ml, add 25 ml of 2 M acetic acid, shake for
30 minutes and filter. 25 m1 ofthe filtrate complies with the limit
50 ml of water and 20 ml of2 M hydrochloric acid, add 3 g of
potassium bromide, cool in ice and carry out the nitrite.titration
(4,JJl).
1 ml of 0.1 M sodium nitrite is equivalent to 0:02362 g of
CSH9NzNa03S,
Usual strengths. 10 per cent w/v; 20 per cent w/v; 30 per cent
w/v.
Identification
To a volume containing 0.5 g of Suiphacetamide Sodium add
6 m1 of 5 M acetic acid, stirring constantly. Filter the precipitate,
wash with water and dry at 105 for 4 hours. The residue
Compare the spectrum with that obtained with sulphacetamide
RS or with the reference spectrum of sulphacetamide.

reference solution.
C. Dissolve 10 mg in 2 ml of 2 M hydrochloric acid. The
solution gives the reaction ofprimary aromatic amines (2.3.1).
Tests
Appearance of solution. Dilute the eye drops, ifnecessary, to
contain 10.0 per cent w/v of Sulphacetamide Sodium. The
solution is not more intensely coloured than reference solution
BYS4(2.4.1).
pH (2.4.24).6.6 to 8.6.
(2.4.14).
Note-Prepare the solutions immediately before use and

Test solution. Dissolve a volume of solution containing about
0.2 g ofSuiphacetamide Sodium in 10.0 ml ofthe mobile phase.
Reference solution (a). Dissolve 5 mg each of sulphacetamide
sodium RS and sulphanilamide (sulphacetamide sodium
impurity A) in 1.0 ml ofthe mobile phase.
Reference solution (b). Dilute 1.0 ml of the test solution to .

100.0 ml with the mobile phase. Further dilute 1.0 m1 of this
- a stainless steel column 12.5 cm x 4 rom, packed with
(5 flm)
2168
IP 2010
SULPHADIAZINE
- mobile phase: a mixture of I volumes of glacial acetic

acid, 10 volumes ofmethanol and 89 volumes ofwater,
resolution between the peaks due to sulphacetamide impurity
A and sulphacetamide is not less than 5.0. The relative retention
time with reference to sulphacetamide for sulphacetamide
impurity Ais about 0.5.
ofthe peak due to sulphacetamide impurity A is not more than
any secondary peak is not more than the area ofthe principal
(b) (0.1 per cent). The sum of all the secondary peaks is not
0.5 g ofSuiphacetamide Sodium add 75 ml ofwater and 10 ml
of hydrochloric acid. Add 3 g ofpotassium bromide, cool in
ice and carry out the nitrite titration (2.3.31).
CgH9N2Na03S,H20.
Storage. Store protected from light and moisture. The Eye
Drops should not be allowed to freeze.
Labelling. The label states (1) the name and concentration of
any antimicrobial agent used; (2) that it is not meant for
injection; (3) that the solution should be used within one
month of opening the container; (4) that the solution should
not be used ifit is dark brown in colour; (5) that it should not
be allowed to freeze.
Sulphadiazine
q~ IIo
C IOH ION40 2S
Dose. Initial dose, 3 g; subsequent doses, upto 4 g daily, in
divided doses..
Description. White, yellowish white or pinkish white crystals
or crystalline powder; almost odourless.
Identification
C and D may be omitted if tests A and B are carried out.
Compare the spectrum with that obtained with sulphadiazine
RS or with the reference spectrum of sulphadiazine.
C. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid and
dilute 1 ml of this solution to 10 ml with water. The solution,
without further acidification, gives the reaction of primary
D. Heat 3 g in a test-tube inclined at an angle of 45 with the
lower part immersed in a silicone oil-bath at about 270. It
decomposes and a white or yellowish white sublimate is
produced. The sublimate, after recrystallisation fromtoluene
and drying at 100 melts at 123 to 127(2.4.21).
Tests
Appearance of solution. Dissolve 0.8 gin 10 ml of1 M sodium
hydroxide. The solution is not more intensely coloured than
reference solution YS5, BYS5 or GYS5 (2.4.1).
Acidity. Heat 1.25 g ofthe finely powdered substance at about
70 with 25 ml of carbon dioxide-free water for 5 minutes.
Cool for about 15 minutes in ice and filter. To 20 ml of the
filtrate add 0.1 ml of bromothymol blue solution. Not more
than 0.2 ml of 0.1 M sodium hydroxide is required to change
the colour of the solution.
Related substances (2.3.7). Complies with test C.
N:)
I
I
r(YS'N~N
H2 N
Sulphadiazine contains not less than 99.0 per cent and not
more than 101.0 per cent of C IO H ION 40 2S, calculated on the
dried basis.
Mol. Wt. 250.3
Sulphadiazine is N -(pyrimidin-2-yl)sulphanilamide.

20 ml of2 M hydrochloric acid and 50 ml ofwater. Add 3 gof
2169
SULPHADIAZINE TABLETS
IP 2010
potassium bromide, cool in ice and carry out the nitrite titration
(2.3.31).


C IOH ION 40 ZS.
Withdraw a suitable volume ofthe sample and filter promptly

not greater than 1.0 mIll. Rej ect the first few ml ofthe filtrate
and dilute a suitable volume of the filtrate with the same
solvent. Dilute suitably with 0,01 M sodium hydroxide.
Measure the absorbances of the resulting solution and of a
standard solution of sulphadiazine RS ofsimilar concentration
in the same medium atthe maximum at about 254 urn (2.4.7).
Sulphadiazine Tablets contain not less than 95.0 per cent and
sulphadiazine, C IOH ION 40 ZS.
Calculate the content ofC IOH lON 40 ZS in the medium.

D. Not less than 70.0 per cent of the stated amount of
C IOH ION 40 ZS.
Identification
0.5 g Sulphadiazine with two successive quantities, each of
5 ml, of chloroform and reject the chloroform. Triturate the
residue with 10 ml of dilute ammonia solution for 5 minutes,
add 10 ml of water and filter. Warm the filtrate until most ofthe
ammonia has been expelled, cool and acidifY with acetic acid.
Collect the precipitate, wash with water and dry at about 100;
the residue melts at about 256, with decomposition (2.4.21).

quantity ofthe powder containing about 0.5 g ofSulphadiazine
and dissolve as completely as possible in amixture of50 ml of
water and 10 ml of hydrochloric acid. Carry out the nitrite
titration (2.3.31).

C IOH ION 40 ZS.
B. On the residue obtained in test A determine by infrared

with that obtained with sulphadiazine RS or with the reference
spectrum of sulphadiazine.
Sulphadoxine
Sulphonnethoxine; Sulphoethomidine

Tests
Related substances (2.3.7). Complies with test C, but using
the following solutions.
Test solution (a). Extract a quantity of the powdered tablets

containing 0.5 g of Sulphadiazine with 25 ml ofa mixture of
solution by shaking for 10 minutes, filter and use the filtrate.
Test solution (b). Dilute I volume of test solution (a) to
5 volumes with a mixture of 24 volumes of methanol and
I volume of strong ammonia solution.
Test solution (c). Dilute I volume of test solution (a) to
Mol. Wt. 310.3

Sulphadoxine is NI-( 5,6-dimethoxypyrirnidin4-yl)sulphanilamide.
Sulphadoxine contains not less than 99.0 per cent and not
more than 101.0 per cent ofClzH14N404S, calculated on the
dried basis.
Category. Antibacterial; antimalarial in combination with
pyrimethamine.
Dose. Orally, initially, 2 g; subsequent doses, I to 1.5 g weekly.

sulphadiazine RS in the same solvent mixture.
By deep intramuscUlar of slow intravenous injeCtion, 2.5 g
Description. White or yellowish white crystals or crystalline

powder.
Apparatus. No. I,
initially, followed by 1.5 g after four days.
2170
IP 2010
SULPHAMETHIZOLE
Identification

CI2HI4N404S.


Compare the spectrum with that obtained with sulphadoxine
RS or with the reference spectrum of sulphadoxine.
Sulphamethizole

C. Dissolve 0.5 g in 1 ml of sulphuric .acid (40 per cent),
heating gently to effect solution, and continue heating until a
crystalline precipitate is produced. Allow to cool, add 10 ml of
2 M sodium hydroxide, cool again, add 25 ml of ether and
shake for 5 minutes. Dry the upper layer over anhydrous
sodium sulphate, filter and evaporate the solvent by heating
on a water-bath. The residue melts either at 80 to 82 or at 90
to 92(2.4.21).
D. Dissolve about 5 mg in 10 ml of1 M hydrochloric acid and
dilute 1 ml to 10 ml with water. The resulting solution, without
further acidification, gives the reaction of primary aromatic
amines (2.3.1 ).
Tests
Appearance ofsolution. Dissolve 0.8 gin 10 ml of1 Msodium
70 with 25 ml of carbon dioxide-free water for 5 minutes.
substances in sulphonamides (2.3.7), Method C, but using
the following solution.

under examination in a mixture of24 volumes of methanol and
1 volume of strong ammonia solution.
Mol. Wt. 270.3

Sulphamethizole is Nl-(5-methyl-l ,3,4-thiadiazol2-yl)sulphanilamide.
Sulphamethizole contains not less than 99.0 per cent and not
more than 101.0 per cent of C9HION402S2, calculated on the
dried basis.
Dose. 100 to 200 mg every four to six hours.
Description. White or yellowish white crystals or crystalline
powder; odourless.
Identification
C and D may be omitted if tests A and B are carried out.
Compare the spectrum with that obtained with sulphamethizole
RS or with the reference spectrum of sulphamethizole.
(b).
C. Dissolve 50 mg in 4 ml of methanol and add 0.2 ml of a
4.0 per cent w/v solution of cupric acetate; a flocculent,
yellowish green precipitate is produced which becomes dark
green.
D. Dissolve about 5 mg in 10 ml of1 M hydrochloric acid and

dilute 1 ml ofthis solution to 10 ml with water. The solution,
without further acidification, gives the reaction of primary
Tests

2 M hydrochloric acid, add 3 g ofpotassium bromide, cool in
Appearance ofsolution. Dissolve 0.8 gin 10 ml of1 M sodium


2171
SULPHAMETHIZOLE
IP 2010

70 with 25 ml of carbon dioxide:free water for 5 minutes.

sulphamethizole RS in acetone.
Apply to the plate 2 f..Ll of each solution. After development,
dry the plate at 105 and examine in ultraviolet light at 254 nm.
Sulphamethoxazole contains not less than 99.0 per cent and

not more than 101.0 per cent ofC IOH"N30 3S, calculated on the
dried basis.
Dose. Initial dose, 2 g; subsequent doses, 1 g two or three
times daily.
almost odourless.
Identification
Compare the spectrum with that obtained with sulphamethoxazole RS or with the reference spectrum of sulphamethoxazole.
C. Dissolve about 5 mg in 10 ml of1 M hydrochloric acid and
dilute 1 ml to 10 ml with water. The resulting solution, without
further acidification, gives the reaction of primary aromatic
amines (2.3.1).

Assay. Weigh accurately about 0.25 g, dissolve in 50 ml of2 M
hydrochloric acid, add 3 g of potassium bromide, cool in ice
and carry out the nitrite titration (2.3.31).
C9HION40zSz.
Sulphamethoxazole
Tests
Appearance ofsolution. Dissolve 0.8 gin 10 ml of1 M sodium
Acidity. Heat 1.25 g of the finely powdered substance with
25 ml of carbon dioxide:free water at 70 for 5 minutes. Cool
for about 15 minutes in ice and filter. To 20 ml ofthe filtrate add
0.1 ml of bromothymol blue solution. Notmore than 0.3 ml of
the solution.
Substances in Sulphonamides (2.3.7), Method C, but using
the following solution.

examination in sufficient of a mixture of 1 volume of strong
ammonia solution and 24 volumes of methanol to produce
5ml.
C IOH lI N 30 3S
Sulphamethoxazole is Nl-(5-methylisoxazol3-yl)sulphanilarnide.
Mol. Wt. 253.3
2172
IP 2010
SUMATRIPTAN
2 M hydrochloric acid, add 3 g ofpotassium bromide, cool in
I ml of 0.1 M sodium nitrite is equivalent to 0.02533 g of
CIQH 11 N30 3S.
Inject the reference solution. Run the chromatogram 5 times

the retention time of the principal peak. The test is not valid
unless the resolution between [3-[2-(dimethylamino)ethyl]2-[[3-[2-(dimethylamino)ethyl]-IH-indol-5-yl]methyl]-IHindol-5-yl]-N- methylmethanesulphonamide (sumatriptan
impurity A) and sumatriptan is not less than 1.5.
Sumatriptan
CIJ1ZIN30ZS
silica gel (5 j.Lm) (such as Spherisorb silica S5W),
- mobile phase: a mixture of 10 volumes of 10 M
ammonium acetate and 90 volumes of methanol,
- injection volume. 20 j.Ll.
Mol. Wt. 295.4
Sumatriptan is 3-(2-dimethylaminoethyl)indol-5-yl-Nmethylmethanesulphonamide.
Sumatriptan contains not less than 97.5 per cent and not more
than 102.0 per cent of CI4HzIN30ZS, calculated on the
anhydrous basis.
Inject test solution (a) and (b). In the chromatogram obtained

with test solution (a) the area of any peak corresponding to
sumatriptan impurity A is not more than the area ofthe principal
peak in the chromatogram obtained with test solution (b) (0.6
per cent, taking into account the correction factor of 0.6 for
impurity A) and the area of any peak due to [3-[2(dimethylamino)ethyl]-I-[[3-[2-(dimethylamino)ethyl]-IHindol-5-yl]methyl]-IH-indol-5-yl]-N- methylmethanesulphonamide (sumatriptan impurity H) is not more than 0.3 times the
test solution (b) (0.3 per cent).
(2.4.14).

of 0.025 M sodium dihydrogen orthophosphate, adjusted to
pH6.5.
Category. Antimigraine.
Description. A white to pale yellow powder.
Identification
Compare the spectrum with that obtained with sumatriptan
RS.
Tests
Impurities A and H. Determine by liquid chromatography
(2.4.14).
Solvent mixture. I volume of acetonitrile and 3 volumes of

0.025 M sodium dihydrogen orthophosphate, adjusted to
pH 6.5.
Test solution (a). A 0.2 per cent w/v solution of the substance
Test solution (b). Dilute I ml oftest solution (a) to 100 inl with
Reference solution. Dissolve the contents of a vial of
sumatriptan for system suitability RS to I ml with 1 M
hydrochloric acid.
Test solution (a). A 0.2 per cent w/v solution of the substance
Test solution (b). Dilute I ml oftest solution (a) to 100 ml with
the solvent mixture and further dilute I ml of the resulting
solution to 10 ml with the solvent mixture.
Reference solution. Dilute the contents ofa vial of sumatriptan
impurity mixture RSto I ml with 0.1 M hydrochloric acid.
octadecylsilane bonded to porous silica (5 j.Lm) ( such
as Spherisorb ODS I),
containing 0.97 g of dibutylamine, 0.735 g of sodium
orthophosphoric acid and 2.93 g of sodium dihydrogen
orthophosphate in 750 ml water; adjusted to pH 6.5
with 10M sodium hydroxide and diluted to 1000 ml
with water;
2173
SUMATRIPTAN
IP 2010
- injection volume. 20 ,.ti.
resolution between the peaks due to [3-[2-(dimethylamino)
ethyl]-I-(hydroxymethyl)-IH-indol-5-yl]-N- methylniethanesulphonamide (sumatriptan impurity C) and sumatriptan is
not less than 1.5.
Sumatriptan Injection is a sterile isotonic solution of

Sumatriptan Succinate in Water for Injections.
Sumatriptan Injection contains not less than 95.0 per cent and
sumatriptan succinate, CI4H2IN302S,C4H604.
Inject test solution (a) and (b). In the chromatogram obtained

with test solution (a) the areas of any peaks corresponding to
N-methyl[3- [2-(methylamino )ethyl] -lH-indol-5yl]methanesulphonamide (sumatriptan impurity B), [3-[2(dimethylamino)ethyl]-I-(hydroxymethyl)-lH-indol-5-yl]-Nmethylmethanesulphonamide (sumatriptan impurity C) and
N ,N-dimethyl-2-[5-[(methyIsulphamoyl)methyl]-IH-indol-3yl]ethanamine N-oxide (sumatriptan impurity D) is not more
obtained with test solution (b) (0.5 per cent); the area of any
peak corresponding to [3-(2-aminoethyl)-lH-indol-5-yl]-Nmethylmethanesulphonamide (sumatriptan impurity E) is not
more than the principal peak in the chromatogram obtained
with test solution (b) (0.1 per cent); the area of any other
secondary peak is not more than the principal peak in the
chromatogram obtained with test solution (b) (0.1 per cent).
The total impurity content in the test for Impurities A and H
and in the test for Related substances is not more than 1.5 per
cent. Ignore any peak with an area less than 0.5 times the area
of the peak in the chromatogram obtained with test solution
(b) (0.05 per cent).
For impurities A and H. Determine by liquid chromatography

(2.4.14).

LOg.
Solvent mixture. 75 volumes of 0.025 M sodium dihydrogen

orthophosphate, adjusted to pH 6.5 and 25 volumes of
acetonitrile.

described under Related substances, using the following
solutions.
examination in 100.0 ml ofthe
solvent mixture.
I
sumatriptan succinate RS in the solvent mixture.
Reference solution (b). Dilute the contents of a vial of
sumatriptan impurity mixture RS to 1 ml with 0.1 M
hydrochloric acid.
resolution between the peaks due to sumatriptan and
sumatriptan impurity C is not less than 1.5.
Inject reference solution (a) and the test solutioR

Description. A clear, colourless to pale yellow solution.
Identification
To a volume ofthe injection containing 40 mg of sumatriptan
succinate add 1 ml of saturated sodium chloride solution and
1ml of saturated sodium carbonate solution. Shake
vigorously for 30 seconds, add two quantities of2 rn1 ofpropan2-01, shake, allow to separate (this may take up to 24 hours)
and discard the aqueous layer. Evaporate under a stream of
nitrogen and dry at 100. On the residue, determine by infrared
with that obtained with sumatriptan succinate RS.
Tests
pH (2.4.24). 4.2 to 5.3.
Test solution. Dilute a volume ofinjection containing 30 mg of

Sumatriptan Succinate to 10.0 ml with the solvent mixture.
sumatriptan for system suitability RS (containing impurities
A ([3-[2-(dimethylamino)ethyl]-2-[[3-[2-(dimethylamino)ethyl]1H- indol-5-yl]methyl]-1 H-indol-5- yl]-N-methy Imethane
sulphonamide) and H ([3-[2-(dimethylamino)ethyl]-I-[[3-[2(dimethylamino)ethyl]-lH-indol-5-yl]methyl]-lH-indol-5-yl]N-methylmethanesulphonamide) to 1 ml with 1 Mhydrochloric
acid.
silica (such as Spherisorb silica S5W),
mobile phase: a mixture of 10 ~()lu_IIl~~~iJO)\i
ammonium acetate and 90 volumes of methanol,
2174
SUMATRIPTAN INJECTION
IP 2010
resolution between the peaks due to sumatriptan and
sumatriptan impurity A is not less than 1.5.
of any peak corresponding to sumatriptan impurity A is not
cent) and the area of secondary peak corresponding to
sumatriptan impurity H is not more than the area ofthe principal
(a) (1.0 per cent).
(2.4.14).
Solvent mixture. 75 volumes of 0.025 M sodium dihydrogen

orthophosphate, the pH of which has been adjusted to 6.5
and 25 volumes acetonitrile.
Test solution (a). Dilute a volume of injection containing 30
mg ofSumatriptan Succinate in 10 ml ofthe solvent mixture.
Test solution (b). Dilute 1.0 ml oftest solution (a) to 100.0 ml
Reference solution (a). Dilute the contents of a vial of
sumatriptan impurity mixture RS (contains sumatriptan
impurity B (N-methyl[3-[2-(methylamino)ethyl]-IH-indol-5yl]methanesulphonamide), C ([3-[2-(dimethylamino)ethyl]-l(hydroxymethyl)- iH- indol-5-yl]-N-methylmethanesulphonamide), D (.N,N-dimethyl-2-[5-[(methylsulphamoyl)methyl]-IHindol-3-yl]ethanamine N- oxide), and E ([3-(2-aminoethyl)-IHindol-5-yl]-N-methylmethanesulphonamide) to 1 ml with 0.1
M hydrochloric acid.
Reference Solution (b). A 0.3 per cent w/v solution of
sumatriptan impurity standard RS in the solvent mixture.
octadecylsilane bonded to porous silica (5 flm) (such as
SpherisorbODS 1),
and 75 volumes of a solution containing 0.97 g of
dibutylamine, 0.735 g of orthophosphoric acid and 2.93
g of sodium dihydrogen orthophosphate, adjust the
pH to 7.5 with 10M sodium hydroxide and diluted to
1000 ml with water,

resolution between the peaks due to sumatriptan impurity C
and sumatriptan is not less than 1.5.
Inject test solution (a), (b) and reference solution (b). In the
chromatogram obtained with test solution (a) the area of any
peak corresponding to (3a-hydroxy,l, I-dimethyl-5[(methylamino )sulfonyl]methyl-l ,2,3,3 a,8,8 ahexahydropyrrolo[2,3-b ]indol-l-ium trifuoroacetate)
(sumatriptan impurity A) is not more than 1.5 times the area of
the principal peak in the chromatogram obtained with test
solution (b) (1.5 per cent), the area ofany peak corresponding
to 2 (1-3-[2-(dimethylamino)ethyl]-3-hydroxy-2-oxo-2,3dihydro-l H-indol-5-y I-N-methy Imethanesulfonamide)
(sumatriptan impurity H) is not more than the area of the
principal peak in the chromatogram obtained with test solution
(b) (1.0 per cent). The area of any other secondary peak is not
more than the area of the principal peak in the chromatogram
obtained with test solution (b) (1.0 per cent) and sum of areas
ofthe peaks due to sumatriptan impurity A and H is not more
obtained with test solution (b) (4.0 per cent).
Bact,erial endotoxins (2.2.3). Notmore than 350.0 Endotoxin
Units per ml of sumatriptan succinate.
Test solution. Dilute the volume of injection containing 3 mg

ofSumatriptan succi~ate in 100 ml ofthe solvent mixture.
sumatriptan succinate RS in the solvent mixture.
sumatriptan impurity mixture RS (contains impurities B, C, D
and E) to 1 ml with 0.1 M hydrochloric acid.
substances.
Inject reference solution (b). The test is not valid unless
resolution between the peaks due to sumatriptan succinate
and sumatriptan succinate impurityC is not less than 1.5.
Calculate the content ofC14Hz1N30zS,C4H604in the injection.
2175
MONOGRAPHS
T
2181
2181
2183
2184
2185
2186
Talc
Tamoxifen Citrate
TamoxifenTablets
Tamsulosin Hydrochloride
Tartaric Acid
Telmisartan
2187
2188
TelmisartanTablets
Tenofovir Disoproxil Fumarate
2190
2191
2193
2193
Tenofovir Disoproxil Fumarate Tablets

Tenofovir and Emtricitabine Tablets
Temozolomide
Temozolomide Capsules
2194
2195
Terazosin Hydfochloride Dihydrate

Terbutaline Sulphate
2197
2197
2198
2199
2200
2201
Terbutaline Inhalation
Terbutaline Injection
Terbutaline Tablets
Testosterone Propionate
Testosterone Propionate Injection
Tetracycline
2202
2203
2204
2205
2206
2206
2207
2208
2208
2209
Tetracycline Hydrochloride
Tetracycline Capsules
Tetracycline Ointment
Theophylline
Theophylline Injection
Thiabendazole
Thiabendazole Tablets
Thiacetazone
Thiacetazone and Isoniazid Tablets
Thiamine Hydrochloride
2177
MONOGRAPHS
2210
2211
2212
2213
2214
2215
2216
2217
2218
2219
Thiamine Injection
Thiamine Tablets
Thiamine Mononitrate
Thiocolchicoside
Thiocolchicoside Capsules
Thiomersal
Thiopentone Sodium
Thiopentone Injection
Thiotepa
ThiotepaInjection
2220
2221
2222
2223
2224
2224
2225
2226
2227
2227
2228
2229
2230
2231
2232
Thymol
Thyroxine Sodium
ThyroxineTablets
Ticarcillin and ClavulanicAcidInjection
Timolol Maleate
Timolol Eye Drops
Timolol Tablets
Tinidazole
Tinidazole Tablets
Tiotropium Bromide Monohydrate
Tiotropium Powder for Inhalation
TitaniumDioxide
Tizanidine Hydrochloride
TizanidineTablets
Tobramycin
2233
2234
2235
2235
2236
2237
2238
2239
Tobramycin Injection
Tocopheryl Acetate
Tolazarnide
Tolazarnide Tablets
Tolbutamide
Tolbutamide Tablets
Tolnaftate
Tolnaftate Cream
2178
MONOGRAPHS
2239
2239
2240
2240
2242
2243
2243
2245
2245
Tolnaftate Gel
Tolnaftate Topical Powder
Tolnaftate Topical Solution
Tolterodine Tartrate
Topiramate
Topiramate Tablets
Topotecan Hydrochloride
Topotecan Injection
Tramadol Hyrochloride
2246
2247
2248
2250
2251
2252
2252
2253
2255
2256
2256
2257
2257
2258
2259
2260
2260
2261
Tramadol Capsules
Trandolapril
Trandolapril Tablets
Travoprost
Travoprost Eye Drops
Triamcinolone
Triamcinolone Tablets
TriamcinoloneAcetonide
TriamcinoloneAcetonide Injection
Triamterene
.Triamterene Capsules
Tributyl Citrate
Trichloromonofluoromethane
Triethyl Citrate
Trifluoperazine Hydrochloride
Trifluoperazine Injection
Trifluoperazine Tablets
TriflupromazineHydrochloride
Triflupromazine Injection
Triflupromazine Tablets
Trimetazidine Hydrochloride
Trimethoprim
Trimethoprim and Sulphamethoxazole Oral Suspension
Trimethoprim and Sulphamethoxazole Tablets
2179
2262
2262
2263
2264
2265
2266
MONOGRAPHS
2267
2268
2268
2269
2270
2270
TrimethoprimTablets
Triprolidine Hydrochloride
Triprolidine Tablets
Trisodium Edetate Concentrate for Injection
Tropicamide
Tropicamide Eye Drops
Tubocurarine Chloride
2271
2272
2272
Tubocurarine Injection
2273
Troxidone
Troxidone Capsules
2180
IP 2010
TAMOXIFEN CITRATE
Talc
C. The residue obtained in identification test B gives the

reaction ofsilicates (2.3.1).
Purified Talc; Talcum

Talc is a powdered, selected natural hydrated magnesium
silicate. It may contain varying amounts of aluminium and
iron in forms insoluble in 1 M sulphuric acid.
Category. Pharmaceutical aid (dusting powder).
Description. A white or almost white powder, free from
grittiness; readily adheres to the skin; unctuous to the touch;
odourless.
Production
Talc derived from deposits that are known to contain associated
asbestos is not suitable for pharmaceutical use. The
manufacturer is responsible for demonstrating by the test for
amphiboles and serpentines that the product is free from
asbestos. The presence of amphiboles and of serpentines is
revealed by infrared spectrophotometry. If detected, the
specific morphological criteria of asbestos are investigated
by a suitable method of optical microscopy to determine
whether tremolite asbestos or chrysotile is present, as
described below.
Tests
Acidity or allmUnity. Shake 5.0 g with 25 ml of carbon dioxidefree water for 1 minute, filter and add to the filtrate 0.5 ml of
bromothymol blue solution; the solution is not acidic and
requires not more than 0.3 ml of 0.1 M hydrochloric acid to
make it acidic.
Iron (2.3.14). Boil 4.0 g with 25 ml of water for 30 minutes,
replacing the water lost by evaporation, and filter. The filtrate,
after the addition of5 ml of nitric acid, complies with the limit
test for iron (10 ppm).
Acid-soluble substances. Not more than 2.0 per cent,
determined by the following method. Digest 2.0 g with 40 ml of
dilute hydrochloric acid for 15 minutes, filter, evaporate the
filtrate; to the residue add 0.1 ml of sulphuric acid and ignite
to constant weight.
Water-soluble substances. Shake 5.0 g with 25 ml of water for
1 minute, filter, evaporate the filtrate and dry to constant weight;
the residue weighs not more than 10 mg.
Carbonates. To 1 g add 20 ml of dilute hydrochloric acid; no
produced.
effervesc~nce is
In the range 740 em-I to 760 em-I using scale expansion, any
absorption band at 758 cm- I may indicate the presence of
tremolite or of chlorite. If the absorption band remains after
ignition ofthe substance under examination at 850 500 for at
least 30 minutes, it indicates the presence ofthe tremolite. In
the range 600 em-I to 650 cm- 1 using scale expansion, any
absorption band or shoulder may indicate the presence of
serpentines.
Chlorides (2.3.12). Suspend 2.0 gin 10 mlofwater, add lOml

of2 M nitric acid, shake for 15 minutes and filter. 10 ml ofthe
filtrate complies with the limit test for chlorides (250 ppm).
Organic compounds. The residue obtained in the test for Loss
on drying is not more than slightly yellow or grey.
Microbial contamination (2.2.9).
Identification
TestA may be omitted if tests B, Care carried out. Tests Band
C may be omitted if test A is carried out.
shows absorption bands at 3677 em-I, 1018 em-I and 669 em-I.
B. In a platinum crucible, melt a mixture of0.2 g of anhydrous

sodium carbonate and 2.0 g of potassium carbonate. To the
melted mass add 0.1 g of the substance under examination
and heat until the mixture is completely melted. Allow to cool
and transfer the melted mass into an evaporating dish with 50
mlofhot water. Add hydrochloric acid until effervescence
ceases. Add 10 ml of hydrochloric acid and evaporate to
dryness on a water-bath. Allow to cool. Add 20 ml of water,
heat to boiling and filter (the residue is used for identification
test C). To 5 ml ofthe filtrate add 1 ml of ammonia and 1 mlof
ammonium chloride solution and filter. To the filtrate add 1
ml of disodium hydrogen phosphate solution. A white,
crystalline precipitate is formed.
If intended for topical administration, the total viable aerobic

count is not more than a total of 102 bacteria and fungi per gram.
If intended for oral administration, the total viable aerobic
count is not more than 103 bacteria and not more than 102 fungi
per gram.
Tamoxifen Citrate
2181
HO
COOH
HOOC~COOH
Mol. Wt. 563.7
TAMOXIFEN CITRATE
IP 2010
Tamoxifen Citrate is (Z)-2-[4-(1,2-diphenylbut-l-enyl)1- phenoxy]ethyldimethylamine citrate.

Tamoxifen Citrate contains not less than 99.0 per cent and not
more than 101.0 percent ofC26H 29NO, C 6H s0 7, calculated on
the dried basis.
.Category. Antioestrogen.
Dose: The equivalent of 20 to 40 mg of tamoxifen daily as a
single dose or in 2 divided doses.
Identification

C and D may be omitted iftests A and B are carried out.
Compare the spectrum with that obtained with tamoxifen
citrate RS or with the reference spectrum oftamoxifen citrate.
B. When examined in the rang~ 220 nm to 360 nm (2.4.7), a

maxima at about 237 nm and 275 nin.

10 volumes of triethylamine.
Reference solution. A 1.0 per cent w/v solution of tamoxifen
citrate RS in methanol.
Apply to the plate 5 I.d of each solution. After development,
D. To 10 mg add 4 ml ofpyridine and 2 ml of acetic anhydride
and shake; a yellow colour is produced. Heat in a water-bath
for 2 minutes; a pink to red colour is produced.
Tests
E-Isomer and related substances. Determine by liquid

examination in 100 ml ofa mixture of60 volumes ofacetonitrile,
25 volumes of water and 15 volumes of tetrahydrofilran.
acetonitrile, 25 volumes of water and 15 volumes of
tetrahydrofuran.

tamoxifen citrate impurity standard RS in the same solvent
mixture.
a stainless steel column 20 cm x 5 rom, packed with
25 volumes of water, 15 volumes of tetrahydrojitran
and 0.4 volume of strong ammonia solution,
In the chromatogram obtained with reference solution (b) a
peak due to E-isomer appears immediately following the peak
due to Z-tamoxifen. Adjust the sensitivity of the instrument
so that the height ofthe peak due to E-isomer is about 15 per
cent ofthe full-scale deflection on the recorder. Measure the
height ofthe peak due to E-isomer by dropping a perpendicular
from the top of the peak to a line drawn tangentially between
the troughs on each side of the E-isomer peak or the trough
between the E- and Z-isomer peaks and the baseline, whichever
is appropriate.
The test is not valid unless the height ofthe trough separating
the peaks due to E- and Z-tamoxifen in the chromatogram
obtained with reference solution (b) is less than 7 per cent of
full-scale deflection on the recorder and the retention time of
the principal peak is less than 30 minutes. (The retention time
decreases with increasing concentration of ammonia in the
mobile phase).
The content of E-isomer in the substance under examination
is not more than 1.0 per cent calculated from the declared
content of E-isomer in tamoxifen citrate impurity standard
RS. The area of any secondary peak in the chromatogram
obtained with the test solution, other than a peak due to Eisomer, is not greater than halfthat ofthe peak due to tamoxifen
citrate in the chromatogram obtained with reference solution
(a) and the sum of the areas of all such peaks is not greater
than the area ofthe peak due to tamoxifen in the chromatogram
obtained with reference solution (a). Ignore any peak with a
retention time less than 2.5 minutes and any peak with an area
less than 0.05 times the area of the principal peak in the
Loss on drying(2.4; 19);Notmore than05percent;determined
2182
IP 2010
TAMOXIFEN TABLETS
acid, using 1-naphtholbenzein solution as indicator. Carry



C26H29NO, C6Hs0 7
D. Dissolve 10 mg of the residue obtained in the preparation

of the test solution in test C in 4 ml of pyridine, add 2 mlof
acetic anhydride and heat on a water-bath for 2 minutes; a
pink to red colour is produced.
Tests
Tamoxifen Tablets
E-Isomer and Related substances. Determine by liquid

Tamoxifen Citrate Tablets

Tamoxifen Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount oftamoxifen,
C26H29NO.
Usual strengths. The equivalent of 10 mg, 20 mg and 40 mg of

tamoxifen.
Test solution. Mix a quantity of the powdered tablets

containing 60 mg of tamoxifen with 60 ml of a mixture of
60 volumes of acetonitrile, 25 volumes of water and
15 volumes oftetrahydroJuran with the aid of ultrasound for
5 minutes, dilute to 100 ml with the same solvent mixture and
filter through Whatrnan No.1 filter paper.
100 ml with the same solvent mixture.
Identification
A. To a quantity of the powdered tablets containing 0.1 g of
tamoxifen add 20 ml of water, warm, add 2 ml of 5 M sodium
hydroxide and cool. Extract with two quantities, each of 10 ml,
of ether, filtering after each extraction. Combine the ether
extracts and evaporate to dryness in a stream of nitrogen at
room temperature. Dry the residue at a pressure not exceeding
0.7 kPa for 30 minutes.
obtained with tamoxifen citrate RS or with the reference
spectrum oftamoxifen citrate.
B. When examined in the range 220 om to 360 nm (2.4.7), a
0.002 per cent w/v solution in methanol ofthe residue obtained
in test A shows absorption maxima at about 237 nm and
275nm.

solution.
Test solution. Shake a quantity or the powdered tablets
containing 0.1 g tamoxifen with 10 ml of methanol, filter,
evaporate the filtrate to dryness on a water-bath, dry the
residue at 100 for 30 minutes and dissolve 10 mg ofthe residue
Reference solution. A 0.1 per cent w/v solution of tamoxifen
citrate RS in methdnol.
Apply to the plate 10 ILl of each solution. After development,

tamoxifen citrate impurity standard RS in the same solvent
mixture.
a stainless steel column 20 em x 5 rom, packed with
octadecylsilane bonded to porous silica (5/Lm),
25 volumes of water, 15 volumes of tetrahydrofilran
and 0.4 volume of strong ammonia solution,
injection volume. 20 ILL
In the chromatogram obtained with reference solution (b) a
peak due to E-isomer appears immediately following the peak
due to Z-tamoxifen. Adjust the sensitivity of the instrument
so that the height of the peak due to E-isomer is about 15 per
cent offull-scale deflection on the recorder. Measure the height
ofthe peak due to E-isomer by dropping a perpendicular from
the top of the peak to'a line drawn tangentially between the
troughs on each side of the E-isomer peak or the trough
between the E- and Z-isomer peaks and the baseline, whichever
is appropriate.
The test is not valid unless the height ofthe trough separating
the peaks due to E- and Z-tamoxifen in the chromatogram
obtained with reference solution (b) is less than 7 per cent of
the full-scale deflection on the recorder and the retention time
of the principal peak is less than 30 minutes. (The retention
time decreases with increasing concentration of ammonia in
the mobile phase).
The content of E-isomer in the substance under examination
is not more than 1 per cent calculated from the declared content
2183
TAMOXIFEN TABLETS
IP 2010
of E-isomer in tamoxifen citrate impurity standard RS. The

area of any secondary peak in the chromatogram obtained
with the test solution, other than a peak due to E-isomer, is
not greater than half that of the peak due to tamoxifen citrate
in the chromatogram obtained with reference solution (a) and
the sum of the areas of all such peaks is not greater than the
area ofthe peak due to tamoxifen in the chromatogram obtained
with reference solution (a). Ignore any peak with a retention
time less than 2.5 minutes and any peak with an area less than
obtained with reference solution (a).
Crush one tablet and transfer to a 1OO-ml volumetric flask with
the aid of 75 ml of methanol. Shake well for 5 minutes, add
sufficient methanol to produce 100.0 ml and filter. Dilute
10.0 ml ofthe filtrate to 100.0 ml with methanol. Measure the
275 urn (2.4.7). Calculate the content ofC z6H z9NO in the tablet
Tamsulosin Hydrochloride contains not less than 98.0 per

cent and not more than 102.0 per cent of CzoHz8NzOsS,HCI,
Description. A white to offwhite powder.
Dose. 400 to 800 Jlg daily.
Identification
Compare the spectrum with that obtained with tamsulosin
tamsulosin hydrochloride.
maxima at about 279 and 225 urn.
C. Dissolve with heating 0.75 g in water and dilute to 100 ml

with water. To 5 ml of the solution, cooled in an ice-bath add
3 ml of dilute nitric acid and shake. Allow to stand at room
temperature for 30 minutes and filter. The filtrate gives reaction
A ofchlorides (2.3.1).
Tests

quantity of the powder containing about 25 mg oftamoxifen,
shake with 100 ml of methanol for 15 minutes, add sufficient
methanol to produce 250.0 ml and filter. Dilute 10.0 ml ofthe
filtrate to 100.0 ml with methanol and measure the absorbance
ofthe resulting solution at the maximum at about 275 run (2.4.7).

Calculate the content of Cz6H z9NO taking 325 as the specific

equivalent amount oftamoxifen.
_ .. _._
A. Test solution. Dissolve 50.0 mg of the substance under

Reference solution(a). Dilute 1.0 ml of the test solution to

Reference solution (b). Dissolve 4 mg of 2-methoxy-5-[(2R)2-[(2-(2-methoxyphenoxy)ethyljaminoJpropyljbenzenesulphonamide RS (tamsulosin impurity A RS) and 4 mg of the
substance under examination in the mobile phase and dilute
to 20.0 ml with the mobile phase. Dilute 2.0 ml ofthis solution
Reference solution (c). Dissolve 4 mg of (2R)-N- [2-(2ethoxyphenoxy)ethyIJ-l-(4-methoxyphenyl)propan-2-amine
RS (tamsulosin impurity B RS) and 4 mg of the substance
under examination in the mobile phase and dilute to 20.0 ml
with the mobile phase. Dilute 2.0 ml ofthis solution to 20.0 ml
with the mobile phase
, Hel
CzOHZ8NzOsS,HCL_..

(2.4.14).
_MoLWt.A45.0.
- a stainless steel column 15 cm x 4.6 rum, packed with
octadecylsilane bonded to porous silica (5 !!m),
Tamsulosin Hydrochloride is (R)..5..(2 ([2"(0"ethoxyphenoxy)

ethyl]aminopropyl)-2-methoxybenzenesulfonamide
hydrochloride.
2184
(il'liiinn"temp'eiilfiiTe'.40d~
-.-.-.--.-.,----"--,-",','-,-',',-~,,',',"-,,---,-'_
. _._-
mobile phase: a mixture of 600 ml of acetiiizifrile arid

1400 ml of a solution prepared by dissolving 3.0 g of
sodium hydroxide in a mixture of 8.7 ml of perchloric
TARTARIC ACID
IP 2010
acid and 1900 ml of water, adjusting the pH to 2.0 with

0.5 M sodium hydroxide and diluting to 2000 ml with
water,
- spectrophotometer set at 225 nm, .
Inject the test solution and reference solutions (a) and (b) and
record the chromatograms for 1.5 times the retention time of
tamsulosin (retention time = about 6 minutes). In the
chromatogram obtained with reference solution (b), the
resolution between the peaks due to tamsu10sin impurity A
and tamsulosin is not less than 6.
In the chromatogram obtained with the test solution, for each
unspecified impurity eluting before tamsuIosin, the area of
the peak is not more than the area of the principal peak in the
Reference solution (b). Dissolve 5.0 mg of tamsulosin

racemate RS in methanol and dilute to 25.0 ml with methanol.
Dilute 2.0 ml ofthis solution to 10.0 ml with methanol.
astainless steel column 25 cm x.4.6 mm packed with
silica gel OD for chiral separations (Such as Chiralpak
AD-H) (5 Ilm),
- mobile phase: a mixture of 1 volume of diethylamine,
150 volumes of methanol, 200 volumes of anhydrous
ethanol and 650 volumes of hexane,
- flow rate. 0.5 mlperminute,
Inject reference solution (b). Record the chromatogram for
about 20 minutes. The resolution between tamsulosin and
impurity C (5-[(2S)-2-[[2-(2-ethoxyphenoxy) ethyl]amino]
propyl]-2-methoxybenzenesulphonamide) is not less than 2.
The relative retention with reference to tamsulosin (retention
time = about 14 minutes) oftamsulosin impurity C is about 0.8.
B. Repeat the chromatographic procedure with the following

modifications.
- mobile phase: dissolve 3.0 g of sodium hydroxide in a
mixture of8.7 ml ofperch10ric acidand 900 ml ofwater,
adjust the pH to 2.0 with 0.5 M sodium hydroxide and
dilute to 2000 ml with water; add 2000 ml ofacetonitrile,

chromatogram obtained, the area ofthe peak obtained due to
tamsulosin impurity C is not more than the area ofthe principal
(a) (0.1 percent).
Inject the test solution and reference solutions (a) and (c).
Run the chromatograms for 5 times the retention time of
tamsulosin (retention time = 2.5 minutes). In the chromatogram
obtained with reference solution (c), the resolution between
the peaks due to tamsulosin and tamsuIosin impurity B is not
less than 2. .
In the chromatogram obtained with the test solution, for each

unspecified impurity eluting after tamsulosin, the area of any
cent). The sum of the areas of the impurities eluting before
tamsulosin in test A and after tamsulosin in test B is not more
than twice the area ofthe principal peak in the chromatogram
S-isomer. Determine by liquid chromatography (2.4.14).
Assay. Weigh accurately about 0.35 g and dissolve in 5.0 ml of

anhydrousformic acid, add 75 ml ofa mixture of2vo1umes of
acetic anhydride and 3 volumes ofglacial acetic acid Titrate
immediately with 0.1 M perchloric acid, determining the endpoint potentiometrically (2.4.25). Carry out a blank titration.
CzoHzsNzOsS,HCl.
Tartaric Acid
L- Tartaric
,,'

examination in methanol and add sufficient methanol to
produce 25.0 ml.
Reference solution (aj. Dilute 1.0 ml of the test solution to
100.0 ml with methanol. Dilute 1.0 m1 ofthis solution to 10.0 ml
with methanol.
Acid
OH
HOOC/\ /COOH.
H;<'OH
C4H60 6
Mol. Wt. 150.1
Tartaric Acid is (2R,3R)-2,3-dihydroxybutanedioic acid.
2185
TARTARIC ACID
IP 2010
Tartaric Acid contains not less than 99.5 per cent and not
more than 101.0 per cent ofC"H60 6, calculated on the dried
basis.
Telmisartan
Category. Pharmaceutical aid (buffering agent).

Description. Colourless crystals or a white or almost white
crystalline powder.
Identification
A. Ignite a few mg; it gradually decomposes giving off an
odour resembling that of burnt sugar (distinction from citric
.
acid).
B. A 10 per centw/v solution in distilled water (solution A) is
strongly acidic.
C. Gives reactions A and B oftartrates (2.3.1).
Tests
more intensely coloured than reference solution YS6 (2.4.1).
Arsenic (2.3.1 0). Dissolve 5.0 g in 50 ml ofwater and add 10 ml
Oxalate. Neutralise 10 ml of solution A with dilute ammonia
solution, add 0.1 ml of dilute acetic acid and 10 ml of calcium
sulphate solution; no opalescence is produced within
20 minutes.
water and titrate with 1 M sodium hydroxide using 0.5 ml of
phenolphthalein solution. as indicator.
CJI60 6.
Mol. Wt. 514.6

Telmisartan is 4' - ([4-methyl-6-(1-methyl-lH-benzimidazol-2yl)-2-propyl-lH-benzimidazol-l-yl]methyl}-2biphenylcarboxylic acid.
Telmisartan contains not less than 98.0 per cent and not more
than 102.0 per cent of C33H30N40z, calculated on the dried
basis.
Dose. 40 mg once a day.
Identification
Compare the spectrum with that obtained with telmisartan RS
or with the reference spectrum oftelmisartan.
Tests
Appearance of solution. A 5 p~r cent w/v solution in 1 M
sodium hydroxide is not more intensely coloured than
reference solution YS4(2.4.1).
(2.4.14).

examination in 5 ml of methanol, add 100 III ofa 4 per cent w/v
solution of sodium hydroxide and dilute to 50.0 ml with
methanol.
Reference solution. Dilute 1.0 ml ofthe test solution to 10.0 ml
with methanol. Dilute 1.0 ml ofthis solution to 100.0 ml with
methanol.
octadecylsilane bonded to porous silica (5 11m), (Such
as Thennoquest),
colurnntemperature. 40,
mobile phase: A. dissolve 2.0 g ofpotassium dihydrogen
phosphate and 3.8 g of sodium pentanesulphonate
monohydrate in water, adjusted to pH 3.0 with
orthophosphoric acid and dilute to 1000 ml with water,
2186
IP 2010
TELMISARTAN TABLETS
B. a mixture of 20 volumes of methanol

below,
- spectrophotometer set at 230 nID,
Time
(min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
0-3
70
30
3-28
70-+20
30-+80
30
70
30

theoretical plates is not less than 3000, the,tailing factor is not
Calculate the content ofC33H30N40z.
theoretical plates is not less than 2000, tailing factor is not
secondary peak is not more than 2 times the area ofthe principal
(0.2 per cent). The sum of areas of all the secondary peaks is
not more than 10 times the area of the principal peak in the
cent). Ignore any peak with an area less than 0.5 times thearea
Telmisartan Tablets
Telmisartan Tablets contain not less than 90.0 per cent and
not more than 11 0.0 per cent of the stated amount of
telmisartan, C33H30N40z.
Identification
In the Assay, the retention time of the principal peak in the
solution.
Tests

on 1 g by drying in oven at 105.
Solvent mixture. 80 volumes of buffer solution prepared by

diluting 5.0 ml of triethylamine to 2000 ml with water and 20
in 100.0 ml ofthe solvent mixture. Dilute 5.0 ml ofthe solution
tebnisartan RS in the solvent mixture.
octadecylsilane bonded to porous silica (5 /-lm) (such
as Inertsil ODS-3),
phosphate in 1000 ml ofwater; add 2 ml of triethylamine
Apparatus No.1,
under Assay using the following solutions;

Test solution. Use the filtrate, dilute if necessary with the
dissolution medium.
telmisartan RS in the solvent mixture. Dilute 5,0 ml of this
solution to 100.0 ml with the dissolution medium.
C33H30N40Z.
(2.4.14).
2187
IP 2010
TELMISARTAN TABLETS

diluting 5.0 ml of triethylamine to 2000ml with water and 20


ofpowder containing about 100 mg ofTelmisartan in 100.0 ml
ofsolvent mixture, sonicate for 45 minutes and filter.
Test solution. Shake 5 intact tablets with 100 ml ofthe solvent

mixture and sonicate for 45 minutes. Dilute to 250 ml with the
solvent mixture and filter. Further dilute to obtain 0.004 per
cent w/v solution with the solvent mixture.

telmisartan RS in the solvent mixture.
a stainless steel column l5cm x 4.6 mm, packed with
octadecylsilane bonded to porous silica (5 /lm) (suc4 as
InertsilODS-3),
mobile phase: A. dissolve 0.5 g of potassium
dihydrogen phosphate in 1000 ml of water; add 2 ml of
triethylamine, adjusted to pH 3.2 with orthophosphoric
acid,
B. acetonitrile,
below,
spectrophotometer set at 298 nIn,
lleference solution. A 0.004 per cent w/v solution of

telmisartan RS in the solvent mixture.
octadecylsilane bonded to porous silica (5 /lm) (such
as Inertsil ODS-3),
phosphate in 1000 ml of water; add 2 ml of triethylamine
- spectrophotometer set at 298 nIn,
Time
(min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
78
22
injections is not more than 2.0 percent.
80
20
70
30
Calculate the content of C33H30N40z in the tablets.
15
60
40
25
60
40
26
40
60
35
20
80
40
78
22
theoretical plates is not less than 3000, tailing factor is not
more than 2.0 and relative standard deviation for replicate
per cent), the sum ofthe area of all the secondary peaks is not
Ignore any peak with an area less than 0.1 times the area ofthe
-principal peakinthe chromatogram obtained with the reference
solution (0.05 per cent).
HXGaaH
HaaG
Mol. Wt. 635.5

Tenofovir Disoproxil Fumarate salt ofbis(isopropyloxycarbonyloxymethyl ester of (R)-9-(2-phosphonomethoxypropyl)adenine with fumaric acid.
Tenofovir Disoproxil Fumarate contain not less than 97.0 per
cent and not more than 102. 0 per cent ofCI9H30N501OP,C4~04,
c!ll~lll_at~~ on tl1eanb:ydr()us basis.
Dose. 300 mg daily.
Description. A white to off- white crystalline powder.

2188
IP 2010
TENOFOVIR DISOPROXIL FUMARATE
Identification
Compare the spectrum with that obtained with tenofovir
disoproxil jitmarate. RS or with the reference spectrum of
tenofovir disoproxil fumarate.
Tests
Inject the test solution, reference solution (b) and reference

solution (c). In the chromatogram obtained with the test
solution, the area of any secondary peak is not more than
the area of the peak in the chromatogram obtained with the
reference solution (b) (1.0 per cent) and the sum of all the
secondary peaks is not more than 2.5 times the' area of the
(b) (2.5 per cent). Ignore any peak corresponding to the peak
obtained in the chromatogram in the reference solution (c)
and any peak having area less than 0.02 times the area of the

(2.4.14).
Fumaric Acid. 17.5 per cent to 19.0 percent.


Reference solution (a). A 0.2 per cent w/v solution of tenofovir

disoproxil jitmarate RS in methanol.
Reference solution. Dissolve 25 mg of the jitmaric acid in

50 ml of the mobile phase. Dilute 10 ml of the solution to

Reference solution (c). Dissolve 10 mg ofthejUmaric acid in
50 ml of methanol.
asODS3V),
mobile phase: A. D.1 M ammonium acetate solution
with the pH adjusted to 3.8 with glacial acetic acid,
B. a mixture of 70 volumes of methanol
below,
Time
(in min.)
mobile phase A
(per cent v/v)
mobile phase B
(per cent v/v)
95
10
50
50
25
50
50
50
20
80
60
95
65
95
and 60 volumes ofa buffer solution prepared by adding
0.1 per cent v/v of triethylamine in D.D5M sodium
dihydrogen phosphate with the pH adjusted to 2.3 with
orthophosphoric acid imd filtered,
Calculate the content of fumaric acid.
1.0g.
NOTE - Prepare the solutions immediately before use and

carry out the test protectedfrom light.
Reference solution. A 0.1 per cent w/v solution of tenofovir

disoproxiljillnarate RS in the mobile phase. Dilute 5.0 ml of
2189
TENOFOVIR DISOPROXIL FUMARATE TABLETS
IF 2010
and 60 volumes ofa buffer solution prepared by adding
1 ml trieti1ylamine to 0.05M sodium dihydrogen
phosphate with the pH adjusted to 2.3 with orthophosphoric acid and :filtered,
Calculate the content ofCI9H30NsOIOP,C4H404.

(2.4.14).

a quantity of the powder containing 100 mg of Tenofovir
Disoproxil Fumarate and disperse in 50 ml.of methanol and
filter.
Reference solution (a). A solution of tenofovir disoproxil
fumarate RS containing 0.2 per cent of tenofovir disoproxil in
methanol.
Reference solution (c). Dissolve 10 mg of fumaric acid in
50 ml of methanol.
mobile phase: A. dissolve 1.9 g of ammonium acetate
in 1000 ml of water and adjust the pH to 3.8 with glacial
acetic acid,
B. methanol,
below,
Tenofovir Disoproxil Fumarate Tablets contain not less than

amount oftenofovir disoproxil fumarate, CI9H30NsOIOP,C4H404.
Usualstrength. 300 mg.
Identification
maximum at the same wavelength as the reference solution.
Time
(in min.)
mobile phase A
(per cent v/v)
mobile phase B
(per cent v/v)
95
10
50
50
25
50
50
50
20
80
60
95
Tests
Apparatus No.1,
Speed and time. 50 rpm and 45 minutes
Withdraw a suitable volume ofthe medium and :filter promptly.
at about 260.nm. Calculate the content ofC19H30NsOIOP,C4:I-404
in the mediiim from the absorbance obtained fromasoliition
oflmown concentration of tefzofovir dis6pt6xilfutJ1atate RS.
CI9H30NsOIOP,C4:I-404'
Inject the test solution, reference solutions (b) and (c). In the
(6.0 per cent). Ignore the peak corresponding to the peak in
the chromatogram obtained in the reference solution (c).
0.5g.
2190
IP 2010
TENOFOVIR AND EMTRICITABINE TABLETS
Tests
Solvent mixture. Equal volumes of water and methanol.
Apparatus No.1

Disoproxil Fumarate, dissolve in 100 ml ofsolvent mixture and
filter. Dilute 5.0 ml of the filtrate to 50.0 ml with the mobile
phase.
Reference solution. A 0.120 per cent w/v solution of tenofovir
disoproxilfitmarate RS in the solvent mixture. Dilute 5.0 ml of

as Inertsil ODS 3V),
prepared by dissolving 7.8 g of sodium dihydrogen
orthophosphate in 1000 ml of water, adding 1 ml of
triethylamine and adjusting the pH to 2.3 with
orthophosphoric acid (1 0 per cent v/v), and 40 volumes
of acetonitrile,
flow rate. 1 m1 per minute,
spectrophotometer set at 260 llill,
Calculate the content OfC19H30NsOIOP,C4H404in the tablets.
exceeding 30.

Determine by liquid chromatography (2.4.14)
Test solution. The filtrate obtained as given above. Dilute the
filtrate if necessary, with the dissolution medium.
Reference solution. Asolution containing 0.32 per cent w/v of
tenofovir disoproxil fillnarate RS and 0.24 per cent w/v of
emtricitabine RS in methanol. Dilute 5 ml of the solution to
50 ml with the dissolution medium.

Use the chromatographic system given under Assay.
Calculate the contents of CI9H30NsOIOP,C4H404 and
CgH IOFN30 3S.
C19H30NsOIOP.C~04and CgH IOFN30 3S.
(2.4.14).
For Emtricitabine
a quantity of the powder containing 50 mg of Emtricitabine,
disperse in 5 ml of methanol, dilute to 50 ml with mobile phase
A and filter.
emtricitabine RS in mobile phase A.
100 ml with mobile phase A.

Tenofovir Disoproxil Fumarate and Emtricitabine Tablets
Tenofovir and Emtricitabine Tablets contain not less than
amounts oftenofovir disoproxil fumarate, CI9H30NsOIOP,C4~04
and emtricitabine, CgHIO FN30 3S.
Usual strength. Tenofovir 300 mg and Emtricitabine 200 mg.
Identification
Chromatographic system,
octadecylsilane bonded to porous silica (5Ilm),
mobile phase: A. a mixture of 95 volumes of a buffer
solution prepared by dissolving 1.9 g of ammonium
acetate in 1000 ml of water, adjusted the pH to 3.8 with
glacial acetic acid, and 5 volumes of methanol,
B. a filtered mixture oBO volumes ofthe
buffer solution and 70 volumes of methanol,
below,
2191
TENOFOVIR AND EMTRICITABINE TABLETS

Time
(in min.)
o
30
35
55
60
75
IP 2010
Mobile phase A
(per cent v/v)
100
100
Mobile phase B
(per cent v/v)
o
o
100
100
100
100
o
o
o
o
(3.0 per cent).
For Tenofovir Disoproxil Fumarate

Disoproxil Fumarate and disperse in 50 ml of methanol.
Reference solution (a). A 0.1 per cent w/v solution of tenofovir
disoproxil fumarate RS in methanol.
Reference solution (c). Dissolve 10 mg of ji/maric acid in 50
ml of methanol.
mobile phase: A. a buffer solution prepared by
dissolving 1.9 g of ammonium acetate in 1000 ml of
water and adjusting the pH to 3.8 with glacial acetic
acid,
B. methanol,
- a linesr gradient programme using the conditions given
below,
Time
Mobile phase A
Mobile phase B
(in mins)
(per cent)
(per cent)
o
95
5
25
50
60
75
50.
20
95
95
Inject the test solution, reference solutions (b) and (c). In the
(6.0 per cent). Ignore the peak corresponding to the peak in
the chromatogram obtained in the reference solution (c).
Other tests. Comply with the tests stated under the Tablets.

methanol.
Disoproxil Fumarate, disperse in 10 ml of water and dilute to
250.0 ml with methanol. Dilute 5.0 ml ofthe solution to 25.0 ml
of emtricitabine RS and 0.04 per cent w/v of tenofovir
disoproxil ji/marate RS in methanol. Dilute 5.0 ml of the
solution to 25.0 rill with the solvent mixture.
- a stainless steel column 4.6 mm ~ 5 cm packed with
mobile phase: A. a buffer solution prepared by
in 1000 ml of water and adjusting the pH to 3.0 with
B. a mixture of20 volumes ofthe buffer
below,
50
80
5
5
2192
Time
(in mins)
Mobile phase A
(per cent)
Mobile phase B
(per cent)
94
94
50
6
-- -- -50--~------
35
65
94
12
94
6
6
IP 2010
TEMOZOLOMIDE CAPSULES
less than 2000 theoretical plates for the peak due to tenofovir
disoproxil, 500 theoretical plates for the peak due to
emtricitabine and the relative standard deviation for replicate
injections is not more than 2.0 per cent for each component.
Calculate the contents of C19H30NsOIOP,C4H404 and
CgHIQFN30 3S in the tablets.
octadecylsilane bonded to porous silica (5 Ilm), (Such
as Hypersil BDS),
- mobile phase: a mixhJre of90 volumes of 0.5 per cent
w/v solution of acetic acid and 10 volumes of
acetronitrile,
Storage. Store protected from moisture, at a temperahlre not

exceeding 30.
theoritical plates is not less than 2000 and the tailing factor is
not more than 2.0.
Temozolomide

chromatogram obtained with the test solution, area of any
solution (0.5 per cent). The sum of areas of all the secondary
peaks is not more than the area of the principal peak in the
Mol. Wt. 194.2
Heavy metals (2.3.13). 1.0 gm complies with the limit test for
Temozolornide is 3,4-dihydro-3-methyl-4-oxoimidazo[5, I-d]1,2,3,5-tetrazine-8-carboxarnide.
Temozolomide contains not less than 98.0 per cent and not
more than 102.0 per cent ofC 6H 6N 60 2, calculated on the dried
basis.
on 1.0 gm by drying in an oven at 105 for 3 hours.
Dose. 75 mg daily.

CA UTION- Temozolomide is cytotoxic; extra care required

to prevent inhaling particles and exposing the skin to it.

temozolomide RS in the mobile phase.
Identification
Use the chromatographic system as described under Related

substances.

Compare the spectrum with that obtained with temozolomide
RS or with the reference spectrum oftemozolomide.
than 2.0 per cent.

obtained with test solution (b) corresponds to the peak in the

Calculate the content ofC6H 6N 60 2
Tests
(2.4.14).

Reference solution. Dilute 1.0 ml of the test solution to
Temozolomide Capsules contain not less than 90.0 per cent
temozolornide, C6H6N 60 Z .
2193
TEMOZOLOMIDE CAPSULES
IP 2010
CAUTION -Temozolomide is cytotoxic; extra care required

to prevent inhaling particles and exposing the sIan to it.
Terazosin Hydrochloride contains not less than 99.0 per cent

and not more than 101.0 per cent ofCJ9H2sNs04,HCI, calculated
Identification
Description. A white or slightly yellow, crystalline powder.

Other tests. Complies with tests stated under capsules.
Test solution. Weigh accurately a quantity of mixed contents

of20 capsules containing 20 mg ofTemozolomide in 100.0 ml
of mobile phase.
temozolomide RS in the mobile phase.
octadecylsilane bonded to porous silica (5 ).tm), (such
as Hypersil BDS),
v/v acetic acid and 10 ml volumes of acetonitrile,
injection volume. 20 ).tl.
Calculate the content of C6H6N602 in the capsules.
Dose. 1 mg daily.
Identification
Compare the spectrum with that obtained with terazosin
hydrochloride RS or with the reference spectrum ofterazosin
hydrochloride.
Tests
Appearance ofsolution. A 1.0 per cent w/v solution in carbondioxide free water is clear (2.4.1) and not more intensely
pH (2.4.24). 3.0 to 5.0, determined in 2.0 per cent w/v solution
in carbon-dioxide free water.
Impurities Nand O. Determine by liquid chromatography
(2.4.14).

of water.
Reference solution (a). Dissolve 5 mg each of 2-chloro-6, 7dimethoxyquinazolin-4-amine RS (terazosin impurity A RS)
and 1-[[(2RS)-tetrahydrojilran-2-yl]carbonyl]piperazine RS
(terazosin impurity N RS) in acetonitrile, add 5 ml of the test
solution and dilute to 50 ml with acetonitrile. Dilute 10 mlof
this solution to 100 ml with the solvent mixture.
Terazosin Hydrochloride
------Morwc459~9
Terazosin Hydrochloride is (RS)-6, 7-dimethoxy-2-[4(tetrahydrofuran-2-carbonyl)piperazin-l-yl} quinazolin-4ylamine hydrochloride dihydrate.
octadecylsilane bonded to porous silica (5 ).tm),
- mobile phase: dissolve 2.8 g of sodium laurylsulphate
in 1000 mlof water and add 11 ml ofa solution containing
20.24 per cent w/v of triethylamine and 23.0 per cent
w/v of orthophosphoric acid and adjust the pH to 2.5
with orthophosphoric acid; mix 60 volumes of this
solution with 40 volumes of acetonitrile,
sp~ctrophotometer set at 210 nIll,
injection volume. 20 ).tl.
Inject reference solution (b). The relative retention time with
reference to terazosin for I,4-bis[(tetrahydrofuran-2yl)carbonyl]piperazine (terazosin impurity 0) is about 0.2, for
2194
IP 2010
TERBUTALINE SULPHATE
terazosin impurity N is about 0.3 and for terazosin impurity A

is about 0.4. The test is not valid unless the resolution between
the peaks due to terazosin impurity A and N is not less than
1.5.
of each peak corresponding to terazosin impurity Nand
terazosin impurity 0 is not more than the area ofthe peak due
to terazosin in the chromatogram obtained with reference
(2.4.14).

ml with the mobile phase. Dilute 5.0 ml ofthissolution to 100
Reference solution (b). Dissolve the contents of a vial of

terazosin for system suitability RS (containing terazosin
impurity A, terazosin impurity B (1-(4-hydroxy-6, 7dimethoxyquinazolin-2-yl)-4-[[(2RS)- tetrahydrofuran-2yl]carbonyl]piperazine), terazosin impurity C (6,7-dimethoxy2-(piperazin-l-yl)quinazolin-4-amine), terazosin impurity J (1(4-amino-6, 7-dimethoxyquinazolin-2-yl)-4-[(2RS)-2hydroxypentanoyl]piperazine), terazosin impurity K (prazosin)
and terazosin impurity M (1,4-bis(furan-2"ylcarbonyl)
piperazine) in 10 ml ofthe mobile phase.
Reference solution (c). Dissolve 5 mg of 1-(furan-2ylcarbonyl)piperazine RS (terazosin impurity L RS) in 100

ml ofthe mobile phase. Dilute 1.0 ml ofthis solution to 100 ml
Reference solution (d). To 5 mg of 2,2-(piperazine-1,4diyl)bis(6,7-dimethoxyquinazolin-4-amine) RS (terazosin

impurity E RS ) add 70 ml of methanol and 30 ml of water.
Inject the test solution, reference solution (a) and (c). Run the
of secondary peak corresponding to terazosin impurity A, C,
E and K is not more than 5 times the area ofthe principal peak
per cent). The area of secondary peak corresponding to
terazosin impurity L is not more than the area of the
corresponding peak in the chromatogram obtained with
reference solution (c) (0.1 per cent). The area of secondary
peak corresponding to terazosin impurity B, J, M is not more
obtained with reference solution (a) (0.1 per cent). Multiply
the peak areas of the impuirites by the correction factor for
calculating the contents, for impuirty C is 0.7 and for impuirty
M is 1.6. The area of any other secondary peak is not more
obtained with reference solution (a) (0.1 per cent); The sum of
the areas ofall other secondary peaks is not more than 5 times
with reference solution (a) (0.5 per cent). Ignore any peak with
an area less than 0.5 times the area ofthe principal peak in the
cent).
Assay. Weigh accurately about 0.3 g , dissolve in a mixture of
5 ml of 0.01 M hydrochloric acid and 50 ml of methanol.
Titrate with 0.1 M sodium hydroxide, determining the endpoint potentiometrically (2.4.25). Carry out a blank titration.
CI9Hz6ClNS04.
Allow to stand for at least 1 hour to dissolve the substance.

Chrmatographic system
mobile phase: mix 2 volumes of triethylamine, 350
volumes of acetonitrile, and 1650 volumes ofa solution
containing 0.6 per cent w/v of sodium citrate and 1.425
per cent w/v of anhydrous citric acid,
resolution between the peaks due to terazosin impurity Band
terazosin impurity J is not less than 1.5.
(ClzHI9N03)z,HzS04
Mol. Wt. 548.7
Terbutaline Sulphate is (RS)-2-(tert-butylamino)1-(3,5-dihydroxyphenyl)ethanol sulphate.
2195
IP 2010
TERBUTALINE SULPHATE
Terbutaline Sulphate contains not less than 98.0 per cent and
not more than 101.0 per cent of(C 12H I9N03h H 2S04 , calculated
on the dried basis.
RS (terbutaline impurity C RS) and 22.5 mg of terbutaline

sulphate RS in 50.0 ml of the mobile phase. Dilute 1.0 ml of
Reference solution (b). Dilute 1.0 ml of the test solution to'

Dose. Orally, upto 15 mg daily, in divided doses; by inhalation,

for an adult, one to two inhalations, each of 250 )..Lg, 3 or 4
times a day up to a maximum of24 inhalations in 24 hours; for
a child, one to two inhalations, each of250 )..Lg, 2 or 3 times a
day up to a maximum of 10 inhalations in 24 hours; by
subcutaneous injection, 250 to 500 )..Lg, 4 times a day.
Description. A white or almost white, clystalline powder;
Identification


Compare the spectrum with that obtained with terbutaline
sulphate RS or with the reference spectrum of terbutaline
sulphate.

absorption maxima at about 276 nm and 280 nrn, which may be
fused; absorbance at both 276 nrn and 280 nrn, 0.46 to 0.49.
D. A 2.0 per cent w/v solution in carbon dioxide-free water
gives reaction A of sulphates (2.3.1).
Tests
dioxide-fi-ee water is clear (2.4.1); absorbance ofa 2-cm layer
at 400 nm, not more than 0.11 (2.4.7).
Acidity. Dissolve 0.2 g in 10 ml of carbon dioxide-free water
and titrate with 0.01 M sodium hydroxide, using methyl red
solution as indicator. Not more than 1.2 ml of 0.01 M sodium
yellow.
tert-Butylamino-3,5-dihydroxyacetophenone. Absorbance of
a 2 per cent w/v solution in 0.01 M hydrochloric acid at
about 330 nm, not more than 0.50 (2.4.7).
)..Lm),
- mobile phase. dissolve 4.23 g of sodium
hexanesulphonate in 770 ml of 0.05 M ammonium
formate solution prepared by dissolving 3.15 g of
ammonium formate in about 980 ml of water, adjusted
to pH 3.0 by anhydrousformic acid and dilute to 1000
ml with water and add 230 ml of methanol,
- injection volume. 20 )..Ll.
resolution between the peaks due to terbutaline impurity C
and terbutaline is not less than 2.0.
of the peak due to terbutaline impurity C is not more than
twice area of the corresponding peak in the chromatogram
obtained with reference solution (a) (0.2 per cent), the area of
each peak dueto 3,5-dihydroxybenzoic acid (a-resorcylic acid)
(terbutaline impurity A), (4RS)-2-(l, I-dimethylethyl)-1,2,3,4tetrahydroisoquinoline-4,6,8-triol (terbutaline impurity B) and
2-[benzyl-(l, I-dimethylethyl)amino]-I-(3,5-dihydroxyphenyl)
ethanone (terbutaline impurity D) is not more than area ofthe
solution (b) (0.2 per cent). The sum ofall the secondary peaks
other than terbutaline impurity C is not more than twice the
cent).

(2.4.14).
Heavy metals (2.3.13). Mix 1.6 g with 0.6 g ofanhydrous sodium

sulphate and ignite without melting the sodium sulphate. Cool,
add 3 ml of 2 Mhydrochloric acid, boil and dilute to 50 ml
with water. Cool and filter, 25 ml ofthe filtrate complies with
Tesisoliliion. Dissol've 75 illg of tIie

examination 50.0 ml ofthe mobile phase.
sl.ibstance l.inder
Lossondrying(2.4.19);Not more than O.5percent,-determined

Reference solution (a). Dissolve 7.5 mg of 1-(3,5dihydroxyphenyl)-2 -[(1, 1~dimethylethyl)amino] ethanone

anhydrous glacial acetic acid with the aid of heat. Titrate
2196
TERBUTALINE INJECTION
IP 2010

(CI2HI9N03)2, H2S04,
Terbutaline Sulphate Inhalation; Terbutaline Inhalation
Aerosol; Terbutaline Sulphate Inhalation Aerosol
Terbutaline Inhalation is a suspension ofTerbutaline Sulphate,
as a superfine powder, in a suitable liquid in a suitable
pressurised container. It may contain suitable pharmaceutical
aids such as surfactants, stabilising agents, etc.
Terbutaline Inhalation delivers not less than 75.0 per cent and
not more than 125.0 per cent ofthe stated amount ofterbutaline
sulphate (CI2HI9N03)2,H2S04, per inhalation, by actuation of
the valve.
Use 35 ml of water and finally dilute to 50.0 ml with water.

Dilute a suitable volume ofthis solution with water to produce
a solution containing 50 Ilg ofTerbutaline Sulphate per m!. To
10.0 ml ofthis solution in a 50-ml volumetric flask add 35 ml of
buffer solution pH 9.5 and 1.0 ml of 4-aminoantipyrine
solution. Mix, add 1.0 ml of potassium jerricyanide solution
with vigorous swirlingofthe flask, dilute to volume with water
and mix. Exactly 75 seconds after the addition ofthe potassium
jerricyanide solution measure the absorbance ofthe resulting
solution at the maximum at about 550 nm (2.4.7), using as the
blank a solution prepared in the same manner using 10.0 ml of
water in place of the solution of the substance under
examination.
Calculate the content of (CI2HI9N03)z,H2S04 in the solution
from the absorbance obtained by repeating the operation using
a solution containing 100 Ilg of terbutaline sulphate RS in
place of the solution of the substance under examination.
Calculate the amount of (CI2HI9N03)2,H2S04 delivered per
actuation of the valve.

Determine the content of active ingredient a second and third

time by repeating the procedure on the middle ten and on the
last ten successive combined actuations of the valve. For
each of the three determinations the average content of
(C12H19N03)2,H2S04 delivered per actuation ofthe valve meets
the requirements.

25 volumes of cyclohexane and 5 volumes ofjormic acid.

exceeding 30.
Test solution. Remove the actuator from the pressurised

container, shake the container for about 30 seconds and place
it in an inverted position in a small beaker containing 5 ml of
water. Discharge a sufficient number of deliveries containing
5 mg ofTerbutaline Sulphate, under the surface ofthe solvent.
Labelling. The label states the amount of active ingredient

delivered per inhalation.
Usual strength. 250 Ilg in each metered dose.
Identification
Rejerence solution. A 0.1 per cent w/v solution ofterbutaline

dry the plate in air, spray with a 2 per cent wiv solution of
4-aminoantipyrine in methanol. Examine in daylight and in
the chromatogram obtained with reference solution.
Terbutaline Sulphate Injection
Terbutaline Injection is a sterile solution of Terbutaline
Sulphate in Water for Injections.
Terbutaline Injection contains not less than 90.0 per cent and
sulphate (CI2HI9N03)2, H 2S04
Tests
Usual strength. 500 Ilg per m!.

Preparations (Pressurised metered-dose Preparations).
Identification
Follow the procedure described under Assay wherever the

amount of active substance is to be determined in any test.
Assay. Carry out the test for Content of active ingredient
(Pressurised metered-dose Preparations).
Determine by thin-layer chromatography (2.4. I7), coating the


25 volumes of cyclohexane and 5 volumes ofjormic acid.
Test solution. Use the injection.
2197
TERBUTALINE INJECTION
IP 2010
Reference solution. A 0.1 per cent w/v solution of terbutaline

sulphate RS in saline solution.
4-aminoantipyrine in methanol. Dry the plate in air and spray

with a freshly prepared 8.0 per cent w/v solution ofpotassium
ferricyanide in a mixture of 4 volumes of strong ammonia
solution and 1 volume of water. The principal spot in the
Tests
pH (2.4.24). 3.0 to 5.0.

preparations (Injections).
Assay. Measure accurately a volume containing about 5 mg
of Terbutaline Sulphate and add sufficient water to produce
50.0 m!. To 5.0 ml add 35 ml of a buffer solution prepared by
dissolving 36.3 g of tris (hydroxymethyl) aminomethane in
900 ml of water, adjusting the pH to between 9.4 and 9.6 and
adding sufficient water to produce 1000 m!. Add 1.0 ml of a
freshly prepared 2.0 per cent w/v solution of
4-aminoantipyrine, mix and add 1.0 ml of a freshly prepared
8.0 per cent w/v solution of potassium ferricyanide with
vigorous swirling and sufficient of the buffer solution to
produce 50.0 m!. Exactly 75 seconds after the addition ofthe
potassium ferricyanide solution measure the absorbance of
the resulting solution at the maximum at about 550 nm (2.4.7),
for 10 minutes, dilute to 100 ml with 0.1 M sodium hydroxide

and filter. Dilute 20 ml ofthe filtrate to 50 ml with 0.1 M sodium
hydroxide.
When examined in the range 230 urn to 360 nm (2.4.7), the
296 urn.


25 volumes of cyclohexane and 5 volumes offormic acid.
containing 10 mg ofTerbutaline Sulphate with 4 ml ofa mixture
of equal volumes of ethanol (95 per cent) and water for
10 minutes, centrifuge and use the clear supernatant liquid.
terbutaline sulphate RS in water.
dry the plate in air, allow to stand for a few minutes in an
atmosphere saturated with diethylamine and spray with
diazotised nitroaniline solution. The principal spot in the
that in the chromatogram obtained with reference solution (a)
and the principal spot in the chromatogram obtained with
reference solution (b) appears as a single compact spot.
Tests
Calculate the content of (CI2HI9N03)2, H 2S04 from the

absorbance obtained by repeating the operation using a 0.01
per cent w/v solution of terbutaline sulphate RS and
beginning at the words "To 5.0 ml add 35 ml... ...".

the medium if necessary, at the maximum at about 550 urn
(2.4.7). Calculate the content of (ClzHI9N03)z.HzS04 in the
known concentration of terbutaline RS in the same medium.
Labelling. The label states that the injection should not be

used" if the solution is discoloured.
Terbutaline Tablets
Apparatus No.2,
Terbutaline Sulphate Tablets

(CI2HI9N03)2.HZS04.
Terbutaline Tablets contain not less than 90.0 per cent and
sulphate (CI2HI9N03)z, H2S04.

Tablets.
Usual strengths. 2.5 mg; 5 mg.

Identification

ofTerbutaline Sulphate with 50 ml of 0.1 M sodium hydroxide
Powder one tablet, transfer to a 25-ml volumetric flask, add

1~.lTIl of 0, OJ Ml1ydrQcl}lorJc gciciand ~Qak:e f()r lQ l11illlJtes,
Dilute to volume with 0.01 M hydrochloric acid and filter,
rejecting the first 5 ml of the filtrate. Dilute, if necessary, a
suitable volume of the filtrate with 0.01 M hydrochloric acid
to produce a solution containing 0.01 per cent w/v solution of
2198
IP20l0
TESTOSTERONE PROPIONATE
Terbutaline Sulphate. Carry out the method as described under

Assay beginning at the words "To 5.0 ml add 35 ml ofa buffer
solution....". Calculate the content of (CI2HI9N03h H2S04 in
the tablet from the absorbance obtained by carrying out the
Assay simultaneously using terbutaline sulphate RS.
Description. A white or almost white powder or colourless

crystals; odourless.
Identification
TestA may be omitted iftests Band Care carried out. Tests B


quantity ofthe powder containing about 5 mg ofTerbutaline
Sulphate, transfer to a 50-ml volumetric flask, add 30 ml of
0.01 M hydrochloric acid and shake for 10 minutes. Dilute to
volume with 0.01 M hydrochloric acid and filter, rejecting the
first 5 ml ofthe filtrate. To 5.0 mladd35 ml ofa buffer solution
prepared by dissolving 36.3 g of tris (hydroxymethyl)
aminomethane in 900 ml ofwater, adjusting the pH to between
9.4 and 9.6 and adding sufficient water to produce 1000 ml.
Add 1.0 ml of a freshly prepared 2.0 per cent w/v solution of
4-aminoantipyrine, mix and add 1.0 ml of a freshly prepared
8.0 per cent w/v solution of potassium ferricyanide with
vigorous swirling and sufficient of the buffer solution to
produce 50.0 ml. Exactly 75 seconds after the addition of the
potassium ferricyanide solution measure the absorbance of
the resulting solution at the maximum at about 550 urn (2.4.7),

Compare the spectrum with that obtained with testosterone
propionate RS or with the reference spectrum of testosterone
propionate.
Calculate the content of (C 12H I9N0 3h, H 2S0 4 from the

absorbance obtained by carrying out the Assay simultaneously
using terbutaline sulphate RS.

examination in methanol and dilute to 50 ml with the same
solvent.
Reference solution (a). Dissolve 2 mg of the substance under

examination and 2 mg oftestosterone acetate RS in methanol
and dilute to 50 ml with the same solvent.
Reference solution (b).

100 m1 with methanol.
C. Melting range. 119to 123 (2.4.21).
Tests
in a 1.0 per cent w/v solution in ethanol.
Related substances. Detelmine by. liquid chromatography
(2.4.14).
Dilute 1 ml of the test solution to
octadecylsilane bonded to porous silica (5 !lm),
injection volume. 20 !ll.
o
~2H3203
Testosterone Propionate is
propionate.
B. In the test for Related substances the principal peak in the

the peak due to testosterone propionate in the chromatogram
Mol. Wt. 344.5

3-oxoandrost-4-en-17~-yl
Testosterone Propionate contains not less than 97.0 per cent

and not more than 103.0. per cent ofC 22 H320 3, calculated on
the dried basis.
Category. Androgen; anabolic steroid.
Dose. By intramuscular injection, 5 to 25 mg, once or twice
weeldy.
Inject the test solution and the reference solutions. Continue

the chromatography for twice the retention time for testosterone propionate.
The relative retention time of about four impurities with
reference to testosterone propionate (retention time, about
9 minutes) range from 0.5 to about 1.4.
with reference solution (a) the resolution between the peaks
due to testosterone and testosterone acetate is not less than
4.0.
2199
TESTOSTERONE PROPIONATE INJECTION.
IP 2010

of any peak other than the principal peak is not more than
obtained with reference solution (b) (0.5 per cent); the sum of
the areas of any secondary peaks is not greater than the area
area less than 0.05 times the area ofthe principal peak in the
cent).
ethanol to produce 250.0 ml, mix, dilute 5.0 ml to 50.0 ml with
the same solvent and measure the absorbance ofthe resulting
solution at the maximum at about 241 urn (2.4.7).
Calculate the content of C22 H 320 3 taking 490 as the specific

Testosterone Propionate Injection is a sterile solution of
Testosterone Propionate in Ethyl Oleate or any other suitable
ester, in a suitable fixed oil or in any mixture ofthese.
Testosterone Propionate Injection contains not less than
amount of testosterone propionate, C22H 32 0 3
Usual strength. 25 mgper ml; 50 mgper ml.
Solvent mixture. A mixture of 90 volumes of light petroleum

(40 to 60) and 10 volumes of liquid paraffin.
Mobile phase. A mixture of 30 volumes of water and
solvent mixture.
testosterone propionate RS in the solvent mixture.
Place the dry plate in a tame containing a shallow layer of the
was done.
Apply to the plate 2 J!l of each solution. Allow the mobile
compact spot A.
Tests
Identification
Dissolve a volume containing 50 mg ofTestosterone Propionate
in 8 ml of light petroleum (40 to 60) and extract with three
quantities, each of 8 ml, of a mixture of 7 volumes.of glacial
acetic acid and 3 volumes of water. Wash the combined
extracts with 10 ml of lightpetroleum (40 to 60), dilute with
water until the solution becomes turbid, allow to stand for
2 hours in ice and filter. The precipitate, after washing with
water and drying at 105, complies with the following tests.

Compare the spectrum with that obtained with testosterone
propionate RS or with the reference spectrum oftestosterone
propionate.

0.1 g ofTestosterone Propionate add sufficient chloroform to
produce 100.0 ml and mix. Dilute 3.0 ml to 50.0 ml with
chloroform and to 5.0 ml ofthe solution add lOml of isoniazid
solution and sufficient methanol to produce 20.0 ml. Allow to
stand for 45 minutes and measure the absorbance of the
resulting solution at the maximum at about 380 urn (2.4.7),
using as the blank a solution prepared by treating 5 ml of
chloroform in the same manner.
Calculate the content of C22H3203 from the absorbance
obta.ined by repeating the operation usingaQ.@_6.p_eccent
w/v solution of testosterone propionate RS in chloroform
and beginning at the words "to 5.0 ml of the solution.....".


2200
IP 2010
TETRACYCLINE
Tetracycline
OH 0

w/v of 4-epitetracycline hydrochloride RS and 0.01 per cent
w/v of tetracycline hydrochloride RS in the diluting solvent.
Mol. Wt. 444.5

Tetracycline is (4S,4aS,5aS,6S,12aS)-4-dimethylamino1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxy6-methyl-1, 11-dioxonaphthacene-2-carboxamide. It contains
a variable quantity of water.
Tetracycline contains not less than 88.0 per cent ofC2zHz4NzOs,
Dose. Orally, 250 mg every 6 hours, increased in severe
infections to 500 mg every 6 to 8 hours.
Identification
A. In the test for Assay, the principal peak in the chromatogram
to tetracycline hydrochloride in the chromatogram obtained
B. To about 2 mg add 5 ml of sulphuric acid; a reddish violet

colour develops. Add the solution to 2.5 ml of water; the
C. Dissolve about 10 mg in a mixture of 1 ml of 2 M nitric acid
and 5 ml of water, shake well and add 1 ml of silver nitrate
solution. Any opalescence produced is not more intense than
that in a solution prepared in the same manner omitting the
Tests
suspension in carbon dioxide-free water.
Specific optical rotation (2.4.22).-260 to -280, determined at
20 in a 1.0 per cent w/v solution in 0.1 M hydrochloric acid.
(2.4.14)

examination in 100 rnl ofa mixture of68 rnl of 0.1 M ammonium
oxalate and 27 ml of dimethylformamide (diluting solvent).
4-epitetracycline hydrochloride RS in the diluting solvent.
mobile phase: a mixture of 680 ml of 0.1 M ammonium
oxalate, 270 ml of dimethylformamide and 50 ml of
0.2 M dibasic ammonium phosphate, the pH of the
mixture being adjusted, if necessary, to 7.6 to 7.7 with
3 M ammonia or 3 M phosphoric acid,
The test is not valid unless the resolution between the two
solution (b) is not less than 1.5. In the chromatogram obtained
with the test solution, the area of any peak corresponding to
4-epitetracycline is not greater than the area of the principal
(a) and the total area of all the peaks other than the principal
peak is not greater than 5.0 per cent.
heavy metals, Method B (50 ppm). Use 2.5 rnl ofleadstandard
Test solution. Weigh accl,lfately about 50 mg ofthe substance

under examination and dissolve in 100.0 ml ofa mixture of
68 ml of 0.1 M ammonium oxalate and 27 m1 of
dimethylformamide (diluting solvent).
tetracycline hydrochloride RS in the diluting solvent.
w/v of tetracycline hydrochloride RS inthe diluting solvent.
- mobile phase: a mixture of 680 ml of 0.1 M ammonium
oxalate, 270 ml of dimethylformamide and 50 ml of
0.2 M dibasic ammonium phosphate, the pH of the
mixture being adjusted, if necessary, to 7.6 to 7.7 with
3 M ammonia or 3 M phosphoric acid,
2201
TETRACYCLINE HYDROCHLORIDE
IF 2010
solution (b) is not less than 1.5.
Calculate the content ofCzzHz4NzOs.
1mg ofC22Hz~zOs,HCI is equivalent to 0.92 mg ofCzzHz4NzOs.
Storage. Store protected fi'om light and moistUTe.
Specific optical rotation (2.4.22). -239 to -255, detennined

at20 in a 1.0 per centw/v solution in 0.1 Mhydrochloric acid.
(2.4.14).

examination in 100 ml ofa mixture of68 ml of 0.1 M ammonium
oxalate and 27 ml of dimethylformamide (diluting solvent).
Reference solution (a). A 0.0025 per cent Mv solution of
4-epitetracycline hydrochloride RS in the diluting solvent.

w/v of tetracycline hydrochloi'ide RS in the diluting solvent.
, Hel
Mol. Wt. 480.9

Tetracycline Hydrochloride is (4S,4aS,5aS,6S, 12aS)4- dimethylamino-1 ,4,4a,5,5a,6,11,12a-octahydro3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxonaphthacene-2- carboxamide hydrochloride.
Tetracycline Hydrochloride contains not less than 95.0 per
cent and not more than 100.5 per cent of CzzHz4NzOs, HCI,
Dose. Orally, 250 mg every 6 hOUTS, increased in severe
infections to 500 mg every 6 to 8 hOUTS; by intramuscular
injection, 100 mg every 8 to 12 hOUTS, increased in severe
infections to every 4 to 6 hOUTS; by intravenous infusion, 500
mg every 12 hOUTS; maximum 2 g daily.
mobile phase: 680 ml of 0.1 M ammonium oxalate,
270 ml of dimethylformamide and 50 ml of 0.2 M dibasic
ammonium phosphate, the pH of the mixtUTe being
adjusted, ifnecessary, to 7.6 to 7.7 with 3 M ammonia or
3 M phosphoric acid,
with the test solution the area of any peak corresponding to
4-epitetracycline is not greater than the area of the principal
(a) and the total area of all peaks other than the principal peak
heavy metals, Method B (50 ppm). Use 2.5 ml ofleadstandard
Identification
on 1.0 g by drying in an oven at 60 over phosphorus pentoxide
at a pressure not exceeding 0.7 kPa for 3 hOUTS.
B. To about 2 mg add 5 ml of sulphuric acid; a reddish violet

colOUT develops. Add the solution to 2.5 ml of water; the
COIOUT changes to yellow.
Test solution. Weigh accUTately about 50 mg ofthe substance

under examination and dissolve in about 50 ml ofthe diluting
solvent described in the test for related substances and further
add sufficient diluting solvent to produce 100.0 ml.
Tests
pH (2.4.24). 1.8 to 3.0, determined in a LOper cent w/v
suspension in carbon dioxide-ji-ee water.

w/v of tetracycline hydrochloride RS in the diluting solvent.
2202
IP 2010
TETRACYCLINE CAPSULES
octylsilane bonded to porous silica (5 to 10 /.un),
ammonium oxalate, 270 volumes of dimethylformamide
and 50 volumes of 0.2 M dibasic ammonium phosphate,
the pH ofthe mixture being adjusted, ifn:ecessary, to 7.6
to 7.7 with 3 M ammonia or 3 M phosphoric acid,
Calculate the content ofC22H24N20s, HCl.
Tetracycline Hydrochloride intended for use in the
appropriate procedure for removal of bacterial endotoxins

per mg.
Tetracycline Hydrochloride intended for use in the
appropriate sterilisation procedure complies with the

as to exclude microorganisms.
Tetracycline Hydrochloride Capsules
Tetracycline Capsules contain not less than 90.0 per cent and
tetracycline hydrochloride, C22H24N20s, HCl.
Usual strength. 250 mg; 500 mg.
Identification
B. To a quantity of the mixed contents of 20 capsules

containing about 10 mg of Tetracycline Hydrochloride, add
20 ml of warm ethanol (95 per cent), allow to stand for
20 minutes, filter and evaporate the filtrate to dryness on a
water-bath. To 0.5 mg of the residue add 2 ml of sulphuric
acid; a reddish violet colour develops. Add the solution to 2.5
ml of water; the colour changes to yellow.
C. The residue obtained in test B gives reaction A of chlorides
(2.3.1).
Tests
(2.4.14).
Test solution. Shake a quantity of the mixed contents of
20 capsules containing about 25 mg of Tetracycline
Hydrochloride with 80 ml of 0.01 M methanolic hydrochloric
acid for 10 minutes, dilute to 100 ml with the same solvent, mix
and filter if necessary.
4-epitetracycline hydrochloride RS in 0.01 M methanolic
hydrochloric acid.
w/v each of 4-epitetracycline hydrochloride RS and
tetracycline hydrochloride RS in 0.01 M methanolic
hydrochloric acid.
- column. temperature 40,
- mobile phase: a mixture of 5 volumes of dimethylformamide and 95 volumes of 0.1 M oxalic acid, the
pH of the mixture being adjusted to 3.9 with
triethylamine,
with the test solution the area of any peak corresponding to
4-epitetracycline hydrochloride is not greater than the area of
solution (a) and the total area of all the peaks other than the
principal peak is not greater than 10.0 per cent.
Apparatus No.1,
Medium. 900 ml of water freshly prepared by distillation,
2203
TETRACYCLINE CAPSULES
IP 2010

a membrane filter disc having an average pore diameter not
greater than 1.0 /lm, rejecting the first few ml of the filtrate.
Measure the absorbance of the resulting solution, suitably
Calculate the content ofC22H24N20g, HCl in the medium from
concentration of tetracycline hydrochloride RS.
C22H24N20g, HCl.
.
on 1.0 g of the contents of the capsules by drying in an oven
at 60 at a pressure not exceeding 0.7 kPa for 3 hours.

contents of 20 capsules containing about 50 mg of
Tetracycline Hydrochloride, shake with about 50 ml of
0.01. M methanolic hydrochloric acid and dilute with the
same solvent to produce 100.0 ml, mix and filter. Discard the
first few ml ofthe filtrate.
tetracycline hydrochloride RS in 0.01 M methanolic
hydrochloric acid.
w/v of tetracycline hydrochloride RS in 0.01 M methanolic
hydrochloric acid.
octylsilane bonded to porous silica (5 tolO /lm),
ammonium oxalate and 27 volumes of dimethylformamide and 5 volumes of 0.2 M dibasic ammonium
phosphate, the pH of the mixture being adjusted,
if necessary, to 7.6 to 7.7 with 3 M ammonia or
Tetracycline Hydrochloride Eye Ointment

Tetracycline Ointment contains not less than 90.0 per cent
tetracycline hydrochloride, C22H24N20g, HCl.
Usual strengths: 0.5 per cent w/w; 1 per cent w/w.
Identification
In the test for Assay, the principal peak in the chromatogram
Tests
Calculate the content ofC22H24N20g, HCl in the capsules.

2.0 g dissolved in a mixture of 2 volumes of carbon
tetrachloride, 2 volumes of chloroform and 1 volume of
methanol.
Ointments.
Test solution. Weigh accurately a quantity containing about

25 mg of Tetracycline Hydrochloride and transfer to a
separating funnel with the aid of 15 ml of cyclohexane. Add
15 ml ofa mixture of68 volumes of 0.1 M ammonium oxalate
and 27 volumes of dimethylformamide (diluting solvent) and
shake well. Collect the lower layer in a 50-ml volumetric flask.
Repeat the extraction with two further quantities, each of
15 ml, of the diluting solvent, combining the extracts in the
same 50-ml volumetric flask. Add sufficient diluting solvent to
produce 50.0 ml, mix and filter.
Reference solution. A 0.05 per cent w/v solution of tetracycline
.
hydrochloride RS in the diluting solvent.
octylsilane bonded to porous silica (5 tolO /lm),
- mobile phase: a mixture of 6~ volumes of 0.1 M
ammonium oxalate and 27 volumes of dimethylformamide and 5 volumes of 0.2 M dibasic ammonium
phosphate, the pH of the mixture being adjusted, if
necessary, to 7.6 to 7.7 with 3 M ammonia or
Calculate the content ofC22H24N20g, HCI in the eye ointment.
2204
IP 2010
THEOPHYLLINE
Theophylline

.
C7HsN40 2
C7HsN402,H20

Mol. Wt. 198.2 (hydrate)
Theophylline is 1,3-dimethyl-3,7-dihydro-1H-purine-2,
6-dione. It contains one molecule of water or is anhydrous.
Theophylline contains not less than 98.0 per cent and not
more than 101.0 per cent ofC7H sN 40 2, calculated on the dried
basis.
Category. Xanthine bronchodilator.
Dose. By intravenous infusion, 2.5 to 5.0 mg of anhydrous
theophylline per kg body weight over a period of20 minutes.
Description. Awhite, crystalline powder; odourless.
Identification

Compare the spectrum with that obtained with theophylline
RS or with the reference spectrum oftheophylline.
B. Dissolve about 10 mg in 10 ml of water, add 0.5 ml ofa 5 per

cent w/v solution of mercuric acetate and allow to stand; a
white, crystalline precipitate is produced.
C. The melting range, after drying at 105,270 to 274 (2.4.21).
D. Gives the reaction ofxanthines (2.3.1 ).
Tests
Appearance of solution. Dissolve 0.5 gin 75 ml of carbon
dioxide-free water with heating; the resulting solution
(solution A), is clear (2.4.1), and colourless (2.4.1).
Acidity. To 50 ml ofsolution Aadd 0.1 rnl ofmethyl redsolution.
The solution is red and not more than 1.0 ml of 0.01 Msodium
yellow.
solution in 0.1 M hydrochloric acid at the maximum at about
270 nm, not less than 0.53.
(2.4.14).
Reference solution (b). Dissolve 10 mg of theobromine in the

mobile phase, add 5 ml ofthe test solution and dilute to 100 ml
with the mobile phase. Dilute 5 ml of this solution to 50.0 ml
octadecylsilane bonded to porous silica (7 /-Lm),
and 93 volumes of0.14 per cent w/v solution of sodium
acetate containing 0.5 per cent v/v solution of glacial
acetic acid,
- injection volume. 20 /-Ll.
resolution between the peaks. due to theobromine and
theophylline is not less than 2.0. The relative retention time
with reference to theophylline for theophylline impurity C is
about 0.3, for theophylline impurity B is about 0.4, for
theophylline impurity D is about 0.5 and for theophylline
impurity Ais about 2.5.
peak. In the chromatogram obtained with the test solution,
the area of any secondary peak is not more than the area of
solution (aHO.1 per cent). The sum ofall the secondary peaks
cent) Ignore any peak with an area less than 0.5 times the area
Loss on drying (2.4.19). Not more than 0.5 per cent (for the
anhydrous form) and 8.0 per cent to 9.5 per cent (for the
hydrated form), determined on 1.0 g by drying in an oven at
105.
gently warm the mixture on a water-bath until complete solution
is effected. Cool, add 20.0 ml of 0.1 M silver nitrate and 1.0 ml
of bromothymol solution and titrate with 0.1 M sodium
hydroxide until a blue colour is obtained..
2205
IP 2010
THEOPHYLLINE INJECTION

C7H gN 40 Z'
Theophylline in Dextrose Injection
Theophylline Injection is a sterile solution of Theophylline
and Dextrose in Water for Injections.
Theophylline Injection contains not less than 92.5 per cent
anhydrous theophylline, C 7H gN 40 Z, and not less than 95.0 per
dextrose, C6H 1Z0 6, HzO.
Usual strengths. Anhydrous theophylline, 0.4, 0.8, 1.6,2,3.2,
4 mg per ml in 5 per cent w/v Dextrose.
Identification
Assay. For theophylline - Dilute a suitable volume of the

injection with sufficient 0.1 M sodium hydroxide to produce a
solution containing 0.008 per cent w/v of anhydrous
theophylline. Measure the absorbance of the resulting
blank a solution prepared in the same manner omitting the
Calculate the content of C 7H gN 4 0 Z from the absorbance
obtained by repeating the operation using theophylline RS in
For dextrose - Transfer an accurately measured volume of
the injection containing about 5 mg of Dextrose to a 100-ml
volumetric flask, add 0.2 ml of 6 M ammonia, dilute to volume
with water and mix. Determine the optical rotation of the
resulting solution in a suitable polarimeter tube at 25 (2.4.22).
The observed rotation, in degrees multiplied by 1.042571,
represents the weight, in g, of C6HIZ06,HzO in the volume of
the injection taken for the assay, where A is the ratio 200 divided
by the length, in mm, ofthe polarimeter tube employed.

solution obtained in the Assay for theophylline shows an
absorption maximum at about 274 nm.
Labelling. The label states the strength in terms ofthe amounts

of anhydrous theophylline and Dextrose.
B. Add 0.2 ml of the injection to 5 ml of potassium cupritartrate solution and heat to boiling; a red to orange precipitate
is formed.
Thiabendazole
Tests
Tiabendazole
pH (2.4.24). 3.5 to 6.5.

5-Hydroxymethylfurfural and Related substances. Use a glass
chromatographic column (66 cm x 11 mm) with a sealed-in,
coarse-porosity sintered disc or a glass wool plug and fitted
with a stopcock. Mix 8 g of a 20- to 50-mesh styrenedivinyl~
benzene anion-exchange resin in the hydroxide form with
25 ml of water, allow to settle and decant the supernatant
liquid until a slurry of resin remains. Pour the slurry into the
column and allow to settle as a homogeneous bed having a
bed volume ofabout 15 ml. Wash the resin bed at a flow rate of
about 3 rn1 per minute with 100 ml ofa5.7 percentw/v solution
of ammonium carbonate followed by washing with water
until the eluate has a pH of 7.
Dilute an accurately measured volume, of the injection
containing 1.0 g of Dextrose, C6H I2 0 6,HzO, to 250.0 ml with
water. Pass this solution through the resin bed in the column
at a flow rate of about 3.5 ml per minute, discarding the first
50 ml of the eluate. Measure the absorbance of the eluate at
284'nm; usingwGteYas'theblafiK;'tne-absorbanceisnotfuofe
than 0.25 (2.4.7).
C IOH7N3S
Mol. Wt. 201.3
Thiabendazole is 2-(1 ,3-thiazol-4-yl)-IH-benzimidazole.

Thiabendazole contains not less than 98.0 per cent and not
more than 101.0 per cent of C IOH 7N 3S, calculated on the
anhydrous basis.
Dose. In the treatment of nematode infestation, 1.5 g twice
daily for three days.
Identification
B;CwidflmajJ oeomittecliftesrATscafriea out:'
~
__
..... .. .....
-_
..
... ".".
__ ...
...

Compare the spectrum with that obtained with thiabendazole
RS or with the reference spectrum of thiabendazole.
2206
IP 2010
THIABENDAZOLE TABLETS

absorption maxima at about 243 nm and 302 urn;ratio ofthe
243 nm, 1.8 to 2.1.
chromatogram obtained with the test solution (b) corresponds
(c).

1.0 g.
CIOH7N3S.
D. Dissolve 5 mg in 5 ml of 0.1 M hydrochloric acid, add 3 mg

of 4-phenylenediamine dihydrochloride and shake until
dissolved. Add 0.1 g of zinc powder, mix, allow to stand for
2 minutes and add 10 ml offen-ic ammonium sulphate solution;
a deep blue or bluish violet colour is produced.
Tiabendazole Tablets
Tests
Appearance of solution. Add 5 ml of methanol to 0.5 g in a
flask fitted with a ground-glass stopper, stir for 5 minutes,
with a magnetic stirrer and filter through a sintered-glass filter
(1.6 Ilm to 4 Ilm). The solution is not more intensely coloured
Mobile phase. A mixture of 62.5 volumes of toluene,

25 volumes of glacial acetic acid, 10 volumes of acetone and
2.5 volumes of water.
methanol.
Reference solution (a). Dilute 1 ml oftest solution (b) to 10 ml
with methanol.
Reference solution (b). Dilute 1 ml oftest solution (b) to 25 ml
with methanol.
Reference solution (c). Dissolve 20 mg of thiabendazole RS
Thiabendazole Tablets contain not less than 95.0 per cent and
thiabendazole, C IO H7N3 S.
Identification
A. When examined in the range 230 urn to 360 nm (2.4.7), the
maximum at about 302 nm.
30 mg of Thiabendazole in 5 ml of 0.1 M hydrochloric acid,
add 3 mg of 4-phenylenediamine dihydrochloride and shake
until dissolved. Add 0.1 g of zinc powder, mix, allow to stand
for 2 minutes and add 10 ml of ferric ammonium sulphate
solution; a deep bluish violet colour is produced.
Tests
Disintegration. The test does not apply.
Thiabendazole, add 75 ml of 0.1 M hydrochloric acid, warm
on a water-bath for 15 minutes, shaking occasionally, cool,
dilute to 100.0 ml with 0.1 M hydrochloric acid and filter.
Dilute 5.0 ml ofthe filtrate to 1000.0 ml with 0.1 Mhydrochloric
acid and measure the absorbance of the resulting solution at
the maximum at about 302 urn (2.4.7).
Calculate the content ofC IOH 7N 3S taking 1230 as the specific
Labelling. The label states that the tablets should be chewed
before swallowing.
2207
THIACETAZONE
IP 2010
Thiacetazone
Mobile phase. Ethyl acetate.

Reference solution. Dissolve with the aid of heat 0.016 g of
4-acetamidobenzalazine RS in 150 ml of methanol, cool and
dilute to 200 ml with methanol and further dilute 5 ml ofthis
solution to 50 ml with methanol.
C IOH I2N40S
Mol. Wt. 236.3
Thiacetazone is 4-acetamidobenzaldehyde
thiosemicarbazone.
Thiacetazone contains not less than 98.0 per cent and not
more than 102.0 per cent of C IOH I2N 40S, calculated on the
dried basis.

dry the plate in air, spray with 2 M nitric acid and within
2 minutes examine in ultraviolet light at 254 nIn. Any secondary
not more intense thl:ln the spot in the chromatogram obtained
Dose. 150 mg daily.
Description. Pale yellow crystals or a crystalline powder;

almost odourless.
Identification

methanol by heating at 60 in a water-bath, add slowly 20 ml
ofhot methanolic silver nitrate solution, maintain the solution
at 60 until the precipitate coagulates and leaves a clear
supematant liquid. Cool, filter through a sintered-glass crucible
(porosity No.4), wash the residue with methanol until the
washings are free from silver nitrate and dry to constant weight
at 105.


Compare the spectrum with that obtained with thiacetazone
RS or with the reference spectrum of thiacetazone.
an absorption maximum at about 328 nIn absorbance at about
328 nIn, about 0.55.
C. Boil I0 mg with 5 ml of 1 M hydrochloric acid for 3 minutes,
cool and add sufficient water to produce 200 ml. Mix 5 ml of
this solution with 0.25 ml of sodium nitrite solution and add
the mixture to 0.5 ml of 2-naphthol solution; a red colour is
produced.

Thiacetazone and Isoniazid Tablets contain one part by weight
of Thiacetazone and two parts by weight ofIsoniazid.
Tests
Thiosemicarbazide. To 2.0 g, finely powdered, add sufficient
water to produce 50 ml, shake, allow to stand for I hour with
occasional shaking and filter, discarding the first few ml ofthe
filtrate. Acidify 25 ml ofthe clear filtrate with dilute sulphuric
acid, add 0.1 ml of o-phenanthroline-ferrous complex solution
and titrate with 0.1 M eerie ammonium sulphate to a blue
end-pointwhich persists for I minute; not more than 0.8 ml is
required.
I g ofresidue is equivalent to 0.4606 g ofC IOH I2N 40S.
----- --_._-- -,-,-,..
-----.~-,,--,,--'''--,~'"
4-acetamidobenzalazine. Determine by thin-layer

GF254.
Thiacetazone and Isoniazid Tablets contain not less than

amounts ofthiacetazone, C IOH I2N 40S, and isoniazid, C6H7N 30 2
Dose. Thiacetazone, 150 mg and Isoniazid, 300 mg daily, in
divided doses.
Usual strengths. Thiacetazone, 37.5 mg and Isoniazid, 75 mg;
Thiacetazone, 75 mg and Isoniazid, 150 mg; Thiacetazone, 150
mg and Isoniazid, 300 mg.
2208
IP 2010
THIAMINE HYDROCHLORIDE
Identification
A. Extract a quantity ofthe powdered tablets containing about

30 mg of Thiacetazone with 70 ml of ethanol (95 per cent)
with the aid of heat for I5-minutes with occasional shaking.
Cool, dilute to 100 ml with ethanol (95 per cent) and filter.
Dilute 1 ml to 100 ml with ethanol (95 per cent). The solution
Aneurine Hydrochloride; Vitamin B 1
When examined in the range 230 nm to 360 nm (2.4.7), a

243 run, 1.8 to 2.1.
B. Shake a quantity of the powdered tablets containing 1 mg
ofIsoniazid with 50 ml of ethanol (95 per cent) and filter. To
5 ml ofthe filtrate add 0.1 g of borax and 5 ml of a 5 per cent
w/v solution of l-chloro-2,4-dinitrobenzene in ethanol
(95 per cent), evaporate to dryness on a water-bath and
continue heating for a further 10 minutes. To the residue add
10 ml of methanol and mix; a reddish purple colour is produced.
Tests
Disintegration (2.5.1). Not more than 30 minutes.
C 1zH 17ClN40S, HCl
Mol. Wt. 337.3
Thiamine Hydrochloride is 3-[(4-amino-2-methylpyrimidin5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium chloride

hydrochloride.
Thiamine Hydrochloride contains not less than 98.5 per cent
and not more than 101.5 per cent of C 12H 17CIN4 0S, HCl,
Dose. Prophylactic, orally, 2 to 5 mg once daily; therapeutic,
orally or by subcutaneous or intramuscular injection, 25 to
100 mg daily. In multivitamin preparations, prophylactic, orally,
I to 2 mg daily; therapeutic, orally, 4.5 to 10 mg daily.
small colourless crystals; odour, slight and characteristic.

Assay. For thiacetazone - Weigh and finely powder
20 tablets. Weigh accurately a quantity of the powder
containing about 30 mg ofThiacetazone, add 70 ml of ethanol
(95 per cent) and heat on a water-bath for 15 minutes with
intermittent shaking. Cool, add sufficient ethanol (95 per cent)
to produce 100.0 ml and filter. To 1.0 ml of the filtrate add
sufficient ethanol (95 per cent) to produce 100.0 ml and
maximum at about 328 nm (2.4.7), using ethanol (95 per cent)
as the blank.
Calculate the content of C IO H 1ZN 40S from the absorbance
obtained by repeating the operation using thiacetazone RS in
place of the tablets under examination.
For isoniazid- Weigh accurately a quantity ofthe powdered

tablets containing about 0.2 g of Isoniazid, dissolve as
completely as possible in 100 ml of water and filter. Wash the
residue with water, combine the filtrate and washings and
dilute to 250.0 ml with water. To 50.0 rn1 ofthe resulting solution
add 50 ml of water, 20 ml of hydrochloric acid and 0.2 g of
potassium bromide. Titrate with 0.0167 M potassium bromate,
I ml of 0.0167 M potassium bromate is equivalent to
0.003429 g ofC6H7N 30 z.
CI, HCI
Identification
(2.4.6). Compare the spectrum with that obtained with thiamine
hydrochloride RS or with the reference spectrum of thiamine
hydrochloride.
B. Dissolve about 20 mg in 10 ml of water, add 1 ml of
2 M acetic acid and 1.6 ml of 1 M sodium hydroxide, heat on
a water-bath for 30 minutes and allow to cool. Add 5 ml of
2 M sodium hydroxide, 10 ml of potassium ferricyanide
solution and 10 ml of I-butanol and shake vigorously for
2 minutes. The upper layer exhibits an intense light blue
fluorescence, particularly in ultraviolet light at 365 run. Repeat
the test but adding 0.9 ml of 1 M sodium hydroxide and 0.2 g
of sodium sulphite in place of the 1.6 ml of 1 M sodium
hydroxide; practically no fluorescence is produced.
Tests
Appearance of solution. A 5.0 per cent w/v solution is dear
YS70rGYS6(2.4.1).
2209
IP 2010
THIAMINE HYDROCHLORIDE

(2.4.14).
Solvent mixture. 5 volumes of glacial acetic acid and

95 volumes ofwater.
examination in 15.0 ml of the solution mixture and dilute to
Reference solution (a). Dissolve 5 mg each ofthe substance
under examination and thioxothiamine RS (thiamine impurity
A RS) in 4 ml ofthe solvent mixture and dilute to 25.0 ml with
water. Dilute 5.0 ml ofthe solution to 25.0 ml with water.
50.0 ml with water. Dilute 5.0 ml ofthis solution to 25.0 ml with
water.
(7Ilm),
mobile phase: A. 0.38 per cent w/v solution of sodium
hexanesulphonate, adjusted to pH 3.1 with
B. methanol,
below,
Time
(in min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
0-25
90 -"170
10 -"130
25 - 33
70 -"150
30 -"150
33 -40
50
50
40-45
50 -"190
50 -"110
Nitrates. To 2 ml of a 2.0 per cent w/v solution add 2 ml of
sulphuric acid, cool and superimpose 2 ml offerrous sulphate
solution; no brown ring is produced at the junction ofthe two
layers.

anhydrousformic acid, add 65 ml ofanhydrous glacial acetic
acid and 10 ml of mercuric acetate solution, with stirring.
C1zH 17CIN40S, HCl.
Storage. Store protected from light and moisture, non-metallic
containers.
Thiamine Injection
Thiamine Hydrochloride Injection; Aneurine Hydrochloride Injection; Vitamin B 1 Injection
Thiamine Injection is a sterile solution of Thiamine Hydrochloride in Water for Injection.
Thiamine Injection contains not less than 95.0 per cent and
not more than 110.0 per cent ofthe stated amount of thiamine
hydrochloride, C 1zH 17CIN40S, HCl.
Usualstrength.100mgpermi.
Identification
resolution between the peaks due to thiamine impurity A and
thiamine is not less than 1.6.
(0.4 per cent). The sum of all the secondary peaks is not more
than2.5times the area ofthe principalpeak inthe chromatogram
peak in the chromatogram obtained with reference
A. To a volume containing 20 mg ofThiamine Hydrochloride

in 10 ml of water, add 1 ml of 2 M acetic acid and 1.6 ml of
1 M sodium hydroxide, heat on a water-bath for 30 minutes
and allow to cool. Add 5 ml of2 M sodium hydroxide, 10 ml of
potassium ferricyanide solution and 10 ml of I-butanol and
shake vigorously for 2 minutes. The upper layer exhibits an
intense light blue fluorescence, particularly in ultraviolet light
at 365 nm. Repeat the test but adding 0.9 m1 of 1 M sodium
hydroxide and 0.2 g ofsodium sulphite in place of the 1.6 ml
of 1 MsoClTlini liyCl,;oxICle; practically no flu()res~cence is
prOduced.
B. To a mixture of0.1 ml ofnitrobenzene and 0.2 ml ofsulphuric

acidadd a volume containing 5 mg ofThiamine Hydrochloride.
2210
IP 2010
THIAMINE TABLETS
Allow to stand for 10 minutes, cool in ice and add slowly with
stirring 5 ml of water followed by 5 ml of 10M sodium
hydroxide. Add 5 ml of acetone and allow to stand; no violet
colour is produced in the upper layer.
Tests
pH (2.4.24).2.5 to 4.5.

0.1 g of Thiamine Hydrochloride to 100.0 ml with
0.1 M hydrochloric acid and further dilute 5.0 ml to 100.0 ml
with water.
Reference solution. A 0.005 per cent w/v solution of thiamine
mononitrate RS in 0.005 M hydrochloric acid.
- mobile phase: a solution prepared by dissolving 1 g of
sodium heptanesulphonate in a mixture of 180 ml of
methanol and. 10 ml of triethylamine, diluting to 1000 ml
with water and adjusting the pH to 3.2 with

fluorescence, particularly in ultraviolet light at 365 nm. Repeat
the test but adding 0.9 ml of 1 M sodium hydroxide and 0.2 g
of sodium sulphite in place of the 1.6 ml of 1 M sodium
B. To a mixture of0.1 ml of nitrobenzene and 0.2 ml ofsulphuric

acid add the powdered tablets containing 5 mg of Thiamine
Hydrochloride. Allow to stand for 10 minutes, cool in ice and
add slowly with stirring 5 ml of water followed by 5 ml of
10M sodium hydroxide. Add 5 ml of acetone and allow to
stand; no violet colour is produced in the upper layer.
C. The powdered tablets give the reactions ofchlorides (2.3.1).
Tests
Finely crush one tablet, add 20 ml of ethanol (95 per cent),
stir the mixture for 30 minutes and centrifuge. Repeat the
extraction with three further quantities, each of 15 ml, of
ethanol (95 per cent). Combine the extracts, add sufficient
ethanol (95 per cent) to produce 100.0 ml and mix; Dilute a
suitable volume of the resulting solution containing 1 mg of
Thiamine Hydrochloride with sufficient ethanol (95 per cent)
to produce 100.0 m!. Measure the absorbance ofthe resulting
solution at the maximum at about 233 nm (2.4.7).
Calculate the content ofC 1zH 17C1N40S, HCI in the tablet taking
Calculate the content ofC ,zH 17ClN40S.

1 mg ofC 12H 17Ns0 4S is equivalent to 1.030 mg ofC 12H 17C1N40S,
HCl.
Thiamine Tablets
Thiamine Hydrochloride Tablets; Anemine Hydrochloride
Tablets; Vitamin ~1 Tablets
Thiamine Tablets contain not less than 92.5 per cent and not
more than 110.0 per cent of the stated amount of thiamine
hydrochloride, C 1zH 17CIN40S, HCl.
Identification
20 mg ofThiamine Hydrochloride as completely as possible in
10 ml of water and 2 ml of 1 M acetic acid and filter. Add 5 ml
of 2 M sodium hydroxide, 10 ml of potassium ferricyanide
solution and 10 ml of 1-butanol and shake vigorously for
Test solution. For tablets containing less than 10 mg of

Thiamine Hydrochloride, add 5 ml of 0.1 Mhydrochloric acid
and 50 ml of water to a quantity of the powdered tablets
containing 6 mg of Thiamine Hydrochloride, shake for
20 minutes, dilute to 100.0 ml with water and filter. For tablets
containing 10 mg or more of Thiamine Hydrochloride, add
50 ml of 0.1 M hydrochloric acid and 500 ml of water to a
quantity ofthe powdered tablets containing 60 mg ofThiamine
Hydrochloride, shake for 20 minutes, dilute to 1000.0 ml with
water and filter.
Reference solution. A 0.006 per cent w/v solution of thiamine
mononitrate RS in 0.005 M hydrochloric acid.
- mobile phase: a solution prepared by dissolving 1 g of
sodium heptanesulphonate in a mixture of 180 ml of
methanol and 10 ml of triethylamine, diluting to 1000 ml
with water and adjusting the pH to 3.2 with
2211
THIAMINE MONONITRATE
IP20l0

injection volume. 20 f..tl.
0.2 g of sodium sulphite in place ofthe 1.6 ml of I M sodium

C. Gives reaction A ofnitrates (2.3.1).
Calculate the content ofC 1zH 17ClNPS in the tablets.

1mg OfCIZH17Ns04S is equivalent to 1.030 mg ofC 1zH 17ClN40S,
HCI.
Storage. Store protected from light and moisture in non-metallic
containers.
Tests
(2.4.14).
Solvent mixture. 5 volumes of glacial acetic acid and 95

volumes of water.
Thiamine Nitrate
Tes.t solution. Dissolve 0.35 g of the substance under

examination in 15.0 ml of the solution mixture and dilute to
ClzH17Ns04S
Mol. Wt. 327.4
Reference solution (a). Dissolve 5 mg each of the substance

under examination and thioxothiamine RS (thiamine impurity
A RS) in 4 ml ofthe solvent mixture and dilute to 25.0 ml with
water. Dilute 5.0 ml ofthe solution to 25.0 ml with water.
Thiamine Mononitrate is 3-[(4-amino-2-methylpyrimidin5-yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium nitrate.

Thiamine Mononitrate contains not less than 98.0 per cent

and not more than 102.0 per cent of ClzHI7Ns04S, calculated
on the dried basis.
(5f..tm),
mobile phase: A. 0.38 per cent w/v solution of sodium
B. methanol,
below,
flow rate. 1 ml per minute, .
injection volume. 25 pI.

Dose. Prophylactic, 2 to 5 mg daily; therapeutic, 25 to 100 mg
daily. In multivitamin oral preparations, prophylactic,
1 to 2 mg daily; therapeutic, 4.5 to 10 mg daily.
small, colourless crystals.
Identification
Time

Compare the spectrum with that obtained with thiamine
mononitrate RS or with the reference spectrum of thiamine
mononitrate.
B. Dissolve about 20 mg in 10 ml of water, add 1 ml of

2 M acetic acid and 1.6 ml of I M sodium hydroxide, heat on
a water-bath for 30 minutes ~nd allow to cool. Add 5 ml of
2 M sodium hydroxide, 10mi of potassium,ferxicyanide
solution and 10 ml of I-butanol and shake vigorously for
fluorescence, particularly in ultraviolet light at 365 nm. Repeat
the test but adding 0.9 ml of I M sodium hydroxide and
(min.)
Mobile phase A
(per cent v/v)
0-25
90
25-33
70
33-40
40-45
50
Mobile phase B
(per cent v/v)
~70
10
~30
~50
50
30 ~50
. 50
~90
50
~IO
Inject reference solution (a). The testis not valid unless the
res61Uti6iibet:We'en tliepeiik:fdue t6tliiiimiiie impuritY Aiiiid
thiamine is not less than 1.6.
2212
IP 2010
THIOCOLCHICOSIDE
(1.0 per cent). The sum ofall the secondary peaks is not more
Chlorides (2.3.12). 1.0 g complies with the limit test for chlorides
(250 ppm).
anhydrousformic acid, add 70 ml of acetic anhydride. Titrate
ClzHI7Ns04S.
Storage. Store protected from light and moisture, non-metallic
containers.
Identification
Compare the. spectrum with that obtained with
thiocolchicoside RS or with the reference spectrum of
thiocolchicoside.
B. When examined in the range of220 run to 440 nm (2.4.7), a

0.0015 per cent w/v solution in water shows absorption maxima
at the same wavelength as obtained with the reference
solution.
Tests
in carbon-dioxide free water.
Specific optical rotation (2.4.22). - 550 to - 580 at 23,
determined on 0.5 per cent w/v solution in water.
(2.4.14).

examination in 50.0 ml of methanol.
colchicoside RS in methanol.
thiocolchicoside RS in methanol.
Reference solution (c). Dilute 1.0 m1 each ofreference solution
(a) and (b) to 100.0 m1 with methanol.
silica (5 flm),
mobile phase: A. n- heptane containing 2 per cent acetic
Thiocolchicoside
acid,
OH
~.
0 HsCO
HO
HO
OH
?"
~
B. chloroform,
C. methanol,
below,
HsCO
o
SCH s
Time
Mobile
Mobile
Mobile
phase B
phase C
phase A
(in min.) (per cent v/v) (per cent v/v) (per cent v/v)
Mol.Wt. 563.5
Thiocolchicoside is 2-demethoxy-2-g1ucosidoxythiocolchicine.
Thiocolchicoside contains not less than 98.0 per cent and not
more than 102.0 per cent of CZ7H33NOIOS, calculated on the
dried basis.
Description. A yellow crystalline powder.
Category. Muscle relaxant.
Dose. 4 mg to 8 mg daily.
86
10
50
65
35
51
86
86
10
10
4
4
60
theoretical plates is not less than 80000, the tailing factor is
not more than 1.5 and the relative standard deviation for
replicate injections is not more than 2.0 per cent for the peak
due to thiocolchicoside.
2213
THIOCOLCHICOSIDE
IP 2010
The relative retention time with reference to thiocolchicoside

for colchicine is about 0.55, for N~ deacetyl N- formyl
thiocolchicoside is about 1.05 and for colchicoside is
about 1.10.
Inject the test solution and reference solution (c). In the
chromatogram obtained with the test solution the area ofeach
peak corresponding to N-deacetyl N-formyl thiocolchicoside
and colchicoside is not more than the area of corresponding
(c) (0.5 per cent) and the sum ofthe areas ofall other secondary
peaks is not more than the area ofpeak due to thiocolchicoside
in the chromatogram obtained with reference solution (c)
(0.5 per cent).
than 2.0 per cent.
.
Calculate content ofC27H33NOIOS.
Storage: Store protected from light and at a temperature not
exceeding 30.
Thiocolchicoside Capsules contain not less than 90.0 per cent
thiocolchicoside, C27H33NOIOS.
Dose. 4 mg; 8 mg.
Microbial contamination (2.2.9). Total microbial count not more

than 1000 CFU per g, total yeast and mould count not more
than 100 CFU per g, 1 g is free from Escherichia coli,
Salmonella, Staphylococcus aureus, Pseudomonas
aeruginosa and Enterobacteriaceae.


Units per mg ofThiocolchicoside.
B. Examine by thin-layer chromatography (2.4.17), coating the

Mobile phase. A mixture of 70 volumes of ethyl acetate, 20

volumes of ethanol and 10 volumes of water.
Test solution. Dissolve 0.1 g of substance under examination

in 100.0 ml of water. Dilute 10.0 ml ofthis solution to 100.0 ml
with watel:
thiocolchicoside RS in water. Dilute 10.0 ml ofthis solution to
100.0ml with watel:
mobile phase: A. watel;
B. acetonitrile,
below,
Time
(in min.)
Flow
(rnlImin.)
1.0
1.0
1.0
10
11
U
___. 12_
17
18
20
22
Mobile Phase A Mobile Phase B

(per cent lv/v)
(per cent v/v)
10
10
25
25
90
90
75
1~
J--,-~._
_72
1.8
1.8
1.8
1.0
70
70
90
90
------~
30
30
10
10
Identification

Apply to the plate 10 ml of each solution. After development,
dry the plate in a current ofair and examine in ultraviolet light
at 254 nm. The principal spot in the chromatogram obtained
with the test solution corresponds to the chromatogram
Tests
Apparatus No.1,
Detelmine by liquid chromatography (2.4.14).
Test solution. Use the filtrate.

Reference solution, A 0,-001 per cent w/v solution of
thiocolchicoside RS in water.
Use the chromatographic condition as described under Assay.
Calculate the content ofC27H33NOIOS.
2214
IP 2010
THIOMERSAL

C:17H33NO lOS.
(2.4.14).
Test solution. Disperse the content of the capsules containing

about 50 mg ofThiocolchicoside in 50.0 ml of methanol.
colchicoside RS in methanol.
Reference solution (c). Dilute 1.0 ml each ofreference solution
(a) and (b) to 100.0 ml with methanol.
silica (5 /-tm),
- mobile phase: A. n- heptane containing 2 per cent acetic
acid,
B. chloroform,
C. methanol,
below,
- injection volume. 20 /-tl.

Assay. Determine by liquidchromatography (2.4.14).
Test solution. Shake a quantity of the content of 20 capsules

containing about 10 mg ofThocolchicoside with 100.0 nil of
water.
thiocolchicoside RS in water.
octylsilane bonded to porous silica (5 /-tm),
mobile phase: A. water,
B. acetonitrile,
below,
- injection volume. 20 /-tl.
Mobile Phase A Mobile Phase B
(per cent v/v)
(per cent/v/v)
Time
(in min.)
Flow
(ml/min.)
om
1.0
90
10
10
Lo
90
10
11
1.0
75
25
12
1.8
75
25
15
1.8
75
25
Time
17
1.8
70
30
18
1.8
70
30
86
10
20
1.8
10
50
65
35
22
1.0
90 .
90
51
86
86
10
10
4
4
Mobile
Mobile
Mobile
phase A
phase C
phase B
(in min.) (per cent v/v) (per cent v/v) (per cent v/v)
60
theoretical plate is not less than 80000, the tailing factor is not
injections is not more than 2.0 per cent for the peak due to
thiocolchicoside.
The relative retention time with reference to thiocolchicoside
for colchicine is about 0.55, for N- deacetyl N- formyl
thiocolchicoside is about 1.05 and for colchicoside is about
1.10.
chromatogram obtained with the test solution the area ofeach
peak corresponding to N-deacetyl N-fonnyl thiocolchicoside
and colchicoside is not more than the area of corresponding
peak in the chromatogram obtained' with reference solution
(c) (0.5 percent) and the sum ofthe areas ofall other secondary
peaks is not more than the area ofpeak due to thiocolchicoside
in the chromatogram obtained with reference solution (c) (0.5
per cent).
10
relative standard deVIation for replicate injections is not more
than 2.0 per cent.
Calculate the content ofC 27H 33NO IO S in the capsules.
Thiomersal
Thimerosal
SHgCH 2CH 3
~COONa
V
C9H9HgNa02S
Mol. Wt. 404.8
Thiomersal is the sodium salt of [(2-carboxyphenyl)thio]

ethylmercury.
2215
IF 2010
THIOMERSAL
Thiomersal contains not less than 97.0 per cent and not more
than 101.0 per cent ofC9H9HgNaOzS, calculated on the dried
basis.
Category. Antiseptic; pharmaceutical aid (antimicrobial
preservative).
Description. A light cream, crystalline powder; odour, slight
and characteristic.
Assay. Weigh accurately about 0.5 g, transfer to a 100-mllongnecked flask, add 5 ml of sulphuric acid and heat gently until
charring occurs; continue to heat and add hydrogen peroxide
solution (l00 vol) dropwise, until the mixture is colourless.
Dilute with water, evaporate until slight fuming occurs, dilute
to 10 ml with water, cool and titrate with 0.1 M ammonium
indicator.
Identification
1 ml of 0.1 M ammonium thiocyanate is equivalent to

0.02024 g ofC;H9HgNaOzS.
A. Dissolve 0.1 g in 10 ml of water and add 2 ml of silver

nitrate solution; a pale yellow precipitate is produced.
B. Dissolve 0.5 g in 10 ml of water and add 2 ml of2 Mhydrochloric acid; a white precipitate is produced 'which, after
washing with water and drying over phosphorus pentoxide
at a pressure not exceeding 0.7 kPa, melts at about 110
(2.4.21).
Thiopentone Sodium
Thiopental Sodium
Tests
Ether-soluble matter. Shake 0.5 g with 20 ml of ether for
10 minutes, filter and evaporate to dryness. The residue, after
drying over phosphoruspentoxide at a pressure not exceeding
0.7 lePa for 24 hours, weighs not more thEm 3 mg.
Inorganic mercury compounds. Not more than 0.7 per cent,

Label five 10-ml volumetric flasks A, B, C, D and E. Place 5 rn1
examination in each offlasks A, B, C and D. To each offlasks
C and D add 0.5 ml ofa 0.0095 per cent w/v solution of mercuric
chloride. Add sufficient water to flasks A and C to produce
10.0 ml and add sufficient of a freshly prepared 33.2 per cent
w/v solution ofpotassium iodide to flasks Band D to produce
10.0 ml. Place 5 ml ofthe potassium iodide solution in flask E
and add sufficient water to produce 10.0 ml. Measure the
absorbances of each of the solutions (Aa, Ab, Ac, Ad, Ae) at
about 323 nm (2.4.7), using water as the blank.
Mol. Wt. 264.3

Thiopentone Sodium is a mixture ofsodium (RS)-5-ethyl5-(1-methylbutyl)-2-thiobarbiturate and anhydrous sodium
carbonate.
Thiopentone Sodium contains not less than 84.0 per cent and
not more than 87.0 per cent ofCjjHjsNzOzS and not less than
10.2 per cent and not more than 11.2 per cent of Na, both
Dose. By intravenous injection, initially, 100 to 500 mg, repeated
if necessary after 20 to 30 seconds; maximum dose, 2 g.'
Description. A yellowish white powder; odour, faintly
resembling garlic; hygroscopic.
Calculate the content of inorganic mercury compounds,

expressed as Hg, in the substance under examination from the
expression 0.7019 (Ab -Aa -Ae)/(Aa + Ad -Ab -Ac), where
the figure 0.7019 is a constant obtained from the formula
Identification
atomic weight of mercury

concentration of HgCl z
-----=-------=--x --------=--=molecular weight of HgCl z
100
A. AcidifY 10 rn1 ofa 10 per cent w/v solution in carbon dioxidefree water with 2 M hydrochloric acid; the solution
effervesces. Shake the solution with 20 ml of ether, separate
the ether layer, wash with 10 ml of water and dry over
anhydrous sodium sulphate. Filter, evaporate the filtrate to
dryness and dry the residue at 105.

Tests Band D may be omitted if tests A, C and E are carried
out.
2216
IP 2010
THIOPENTONE INJECTION

obtained with thiopentone RS or with the reference spectrum
of thiopentone.
B. Complies with the test for identification of barbiturates

Reference solution. Dissolve 85 mg of thiopentone RS in
10 ml of2 M sodium hydroxide and dilute to 100 ml with water.
C. Acidify 5 ml of a 5 per cent w/v solution with dilute acetic
acid and filter. Wash the precipitate with water, recrystallise
from water and dry at 700; the crystals melt at about 1600
(2.4.21).
D. Dissolve 1 mg of the crystals obtained in test A in 1 ml of
0.1 M sodium hydroxide. Add about 1 mg of sodium
nitroprusside and, after 15 minutes, 1 ml of dilute hydrochloric
acid; a reddish violet colour is produced.
E. Gives the reactions of sodium salts (2.3.1).
Tests
dioxide-free water (solution A)is clear (2.4.1), and not more
intensely coloured than reference solution GYS3 (2.4.1).
Related substances (2.3.4). Complies with the test, but using
water as the solvent for the test solution and the reference
solution. After development, examine the plate in ultraviolet
light at 254 om but do not spray it with the diphenylcarbazonemercury reagent.
Chlorides (2.3.12). To 10 ml ofsolutionAadd 30 ml of water
and 10 ml of 2 M nitric acid. Shake successively with three
quantities, each of25 ml, of ether, discard the ether lay~rs and
heat the aqueous solution on water-bath to remove any
residual ether. 30 ml of the aqueous layer complies with the
on 0.5 g by drying in an oven at 1000 at a pressure of 1.5 to
Assay. For thiopentone - Weigh accurately about 0.15 g,
dissolve in 5 ml of water, add 2 ml of 1 M sulphuric acid and
extract with four quantities, each of 10 ml, of chloroform. Filter
the combined chloroform extracts, evaporate the filtrate to
dryness on a water-bath and dissolve the residue in 30 ml of
dimethylformamide, previously neutralised with 0.1 M lithium
methoxide. Titrate immediately with 0.1 Mlithium methoxide,
using 1 drop of a 0.2 per cent w/v solution of thymol blue in
methanol as indicator, until a blue colour is obtained. Protect
the solution from absorption of carbon dioxide during the
titration.
1 ml of 0.1 M lithium methoxide is equivalent to 0.02423 g of

CIIHISN202S.
For sodium - Weigh accurately about 0.4 g, dissolve in 30 ml

of water, add 1 drop of methyl red solution and titrate with
0.05 M sulphuric acid until the yellow colour changes to red.
Boil gently for 2 minutes, cool and, ifnecessary, continue the
titration with 0.05 M sulphuric acid until the red colour is
restored.
Na.
Thiopentone Sodium Injection; Thiopental Injection
Thiopentone Injection is a sterile material consisting of
Thiopentone Sodium with or without auxiliary agents. It is
filled in a sealed container.

Thiopentone Injection contains thiopentone, CIIHlSN202S that
is not less than 77.0 per cent and not more than 92.0 per cent
and sodium, Na that is not less than 9.4 per cent and not more
than 11.8 per cent ofthe stated amount ofthiopentone sodium.

Usual strengths. 250mg; 500 mg; 1 g; 5 g.
Description. A yellowish white powder; hygroscopic.
Identification
out.
A. Acidify 10 ml ofa 10 per cent w/v solution in carbon dioxidefree water with 2 M hydrochloric acid; the solution
effervesces. Shake the solution with 20 ml of ether, separate
the ether layer, wash with 10 ml of water and dry over
2217
IP 2010
THIOPENTONE INJECTION
anhydrous sodium sulphate. Filter, evaporate the filtrate to

dryness and dry the residue at 105.
obtained with thiopentone RS or with the reference spectrum
of thiopentone.
B. .Complies with the test for identification of barbiturates
Test solution. 0.1 per cent w/v solution of the substance

Reference solution. Dissolve 85 mg of thiopentone RS in
10 ml of2 M sodium hydroxide and dilute to 100 ml with water.
C. Acidify 5 ml ofa 5 per cent w/v solution with dilute acetic
acid and filter. Wash the precipitate with water, recrystallise
from water and dry at 70; the crystals melt at about 160
(2.4.21).
For sodium - Mixed the contents of 10 containers and weigh

accurately about 0.4 g of the contents, dissolve in 30 ml of
water, add 1 drop of methyl red solution and titrate with
0.05 M sulphuric acid until the yellow colour changes to red.
Boil gently for 2 minutes, cool and, if necessary, continue the
titration with 0.05 M sulphuric acid until the red colour is
restored
Na.
Labelling. The label states the amount of active ingredient in
terms of Thiopentone Sodium.
Thiotepa
D. Dissolve 1 mg of the crystals obtained in test A in 1 mlof

0.1 M sodium hydroxide. Add about 1 mg of sodium
nitroprusside and, after 15 minutes, 1 ml of dilute hydrochloric
acid; a reddish violet colour is produced.
E. Gives the reactions of sodium salts (2.3.1).
[:::N-~-N::J
I
D
C6H I2N3PS
Mol. Wt. 189.2
Tests
Thiotepa is tris(l-aziridinyl)phosphine sulphide.

dioxide-free water (solution A) is clear (2.4.1), and not more
intensely coloured than reference solution GYS3 (2.4.1).
Thiotepa contains not less than 97.0 per cent and not more
than 102.0 per cent ofC 6H I2N 3PS, calculated on the anhydrous
basis.
Related substances (2.3.4). Complies with the test, but using

water as the solvent for the test solution and the reference
solution. After development, examine the plate in ultraviolet
light at 254 urn but do not spray it with the diphenylcarbazonemercUlY reagent.
Dose. By injection, 300 to 400
intervals of 1 to 4 weeks.
on 0.5g by drying in an oven at 100 at a pressure of 1.5 to
Identification
titrati611.
1 ml of 0.1 M lithium methoxide is equivalent to 0.02423 g of
CIIHISN202S.
__
'
'''_,
'
,.".
'
_.,
'
,_.
__._,
__"._,.,
__.,._ _'.' ',_..' ..'.'_'_..
'
__"__'''.'
'
__
"
'
of carbon dioxide dl.ifiiig:the
B. Determine on 20 mg by the oxygen-flask method (2.3.34),

using 5 ml of 1.25 M sodium hydroxide as the absorbing
liquid. When the process is complete, dilute to 25 ml with
water (solution A). To 5 ml ofsolutionAadd 0.1 mlofhydrogen
peroxide solution (100 vol) and 1 ml of 1 M hydrochloric
acid, mix and add 0.05 ml of barium chloride solution; a
turbidity is produced.
~
abso~rptioii

Compare the spectrum with that obtained with thiotepa RS or
with the reference spectrum of thiotepa.
the solution from
per kg of body weight at
Description. Fine, white, crystalline flakes; odourless or almost

odourless.
'
Assay. For thiopentone - Mixed the contents of! 0 containers

and weigh accurately about 0.15 g, of the contents, dissolve
in 5 ml of water, add 2 ml of 1 M sulphuric acid and extract
with four quantities, each of 10 ml, of chloroform. Filter the
combined chloroform extracts, evaporate the filtrate to dryness
on a water-bath and dissolve the residue in 30 mlof
dimethylformamide, previously neutralised with 0.1 M lithium
methoxide. Titrate immediately with 0.1 M lithium methoxide,
using 1 drop of a 0.2 per cent w/v solution of thymol blue in
methanol as indicator, until a blue colour is obtained. Protect
~g
C. To 2ml ofsolutionAadd40 mlofwater and 4 ml ofammonium

molybdate-sulphuric acid solution, mix, add 0.1 g of
L-ascorbic acid and boil for 1 minute; a blue colour is
produced.
2218
IP 2010
THIOTEPA INJECTION
Tests
and titrate immediately with 0.1 M hydrochloric acid, using

methyl orange solution as indicator, until a faint red colour
persists for 10 seconds. Stopper the flask, aUow to stand for
30 minutes and titrate with 0.1 M sodium hydroxide using
phenolphthalein solution as indicator. Subtract the volume
of 0.1 M sodium hydroxide used from the volume of 0.1 M
hydrochloric acid used. Repeat the operation without the
substance under examination. The difference between the
titrations represents the amount of hydrochloric acid required.

(2.4.1).
(2.4.14).

examination 100.0 ml ofthe water.
Reference solution (a). Dissolve 0.35 mg of the substance
under examination in 100.0 ml ofthe water.
Reference solution (b). Dissolve 10 mg of the substance
under examination in 2 ml of methanol in a ground-glassstoppered tube, add 50 III of a 0.1 per cent v/v solution of
orthophosphoric acid, stopper the tube and heat in a water
bath at.65 for 50 seconds (generation of methoxythiotepa).
Allow the solution to cool and add 1 ml of methanol.
.
Reference solution (c). Dissolve 15 mg of the substance
under examination in 10 ml of water, add I g of sodium
chloride, boil in a water bath for 10 minutes and cool
(generation of chloro- adduct).
1 ml of 0.1 M hydrochloric acid is equivalent to 0.006307 g of

C6H I2N3PS.
Storage. Store protected from light and moisture. At higher
temperatures it tends to polymerise with loss of activity.
Thiotepa Injection
Thiotepa Injection is a sterile material consisting of Thiotepa
with or without auxiliary agents. It is filledin a sealed container.
(5 11m) (Such as Nucleosil CI8),
- mobile phase: a mixture of IS volumes of acetonitrile.
and 85 volumes of 0.1 M phosphate buffer pH 7.0,
- flow rate. 1 mlperminute,
.
Thiotepa Injection contains not less than 90.0 per cent and
Inject reference solution (b) and (c). The test is not valid unless not more than 110.0 per cent ofthe stated amount ofthiotepa,
the resolution between the two principal peaks is not less C6HI2N3PS.
than 3. The relative retention time with reference to thiotepa The contents of the sealed container comply with the
for methoxythiotepa is about 1.3 and for chloro-adduct requirements stated under Parenteral Preparations
(thiotepa impurity A) is about 3.75.
of any peak corresponding to the chioro-adduct is not more
chromatogram obtained with reference solution (a.) (0.15 per
cent). The area of any other secondary peak is not more than
with reference solution (a) (0.1 per cent) and the sum of the
areas of all the secondary peaks is not more than twice the
Assay. Weigh accurately about 0.2 g into a stoppered flask,
add 50 ml ofa 20 per cent wlv solution of sodium thiosulphate

Description. A white powder.
Identification
A. Determine on 20 mg by the oxygen-flask method (2.3.34),
using 5 ml of 1.25 M sodium hydroxide as the absorbing
liquid. When the process is complete, dilute to 25 ml with
water (solution A). To 5 rnl ofsolution A add 0.1 ml of hydrogen
peroxide solution (l00 vol) and 1 ml of 1 M hydrochloric
acid, mix and add 0.05 ml of barium chloride solution; a
turbidity is produced.
B. To 2 ml ofsolutionAadd40rnl ofwaterand4mlofammonium
molybdate-sulphuric acid solution, mix, add 0.1 g of
L~ascorbic acid and boil for 1 minute; a blue colour is
produced.
2219
THYMOL
IP 2010
Tests
Appearance ofsolution. Dissolve a quantity containing 15 mg
ofThiotepa in 4 ml ofwater; the solution is clear (2.4.1).
pH (2.4.24).5.5 to 7.5, determined in a 1.0 per centw/v solution

Units per mg of Thiotepa.

Test solution. Dilute a suitable quantity of the injection
containing 0.15 g ofThiotepa in 100 ml ofwater.
Reference solution. A 0.15 per cent w/v solution of thiotepa
RS in water.
- injection volume. 20 ~l.
Calculate the content ofC 6H 12N 3PS in the injection.
Storage. Store protected from light. If solid matter separates

from the constituted injection, the solution should not be
used.

Compare the spectrum with that obtained with thymol RS or
with the reference spectrum ofthymol.
B. Dissolve 0.2 g with heating in 2 ml of2 M sodium hydroxide,
add 0.2 ml of chloroform and heat on a water-bath; a violet
colour develops.
C. Dissolve about 2 mg in 1 ml ofanhydrous acetic acid, add

0.15 ml ofsulphuric acid and 0.05 ml of nitric acid; a bluish
green colour develops.
D. Melting range (2.4.21).48 to 52.
Tests
Appearance ofsolution. Dissolve 1.0 g in 10 ml of 2 M sodium
hydroxide. The solution is not more opalescent than
opalescence standard OS4 (2.4.1), and not more intensely
coloured than reference solution RS6 (2.4.1).
Acidity. To 1.0 g in a glass-stoppered flask add 20 ml ofwater,

boil until dissolution is complete, cool, stopper the flask and
shake vigorously for 1 minute. Add a few crystals of the
substance under examination to induce crystallisation, shake
vigorously for 1 minute and filter. To 5 ml of the filtrate add
0.05 ml of methyl red solution and 0.05 ml of 0.01 M sodium
hydroxide; the solution is yellow.

(2.4.13).
Test solution. Dissolve 0.1 g in sufficient ethanol (95 per
cent) to produce 10 ml.
100 ml with ethanol (95 percent).
Thymol

10 ml with ethanol (95 per cent).
10 ml with ethanol (95 per cent).
Mol. Wt. 150.2

Thymol is 2-isopropyl-5-methylphenol.
- a glass column 4 m x 2 mm, packed with diatomaceous
support (125 to 180 mesh) impregnated with a mixture
suitable for the separation of free fatty acids (such as
FFAP),
Thymol contains not less than 99.0 per cent and not more
than 101.0 per cent ofC IOH I4 0.
- temperature:
column 80 for 2 minutes, increase @ 8 to 240 stand for
15 minutes,
Category. Pharmaceutical aid (antimicrobial preservative;

antiseptic).
Description. Colourless crystals; odour, characteristic.
Inject test solution, reference solution (a),'{b) and (c):
Identification
Intbe chiomatogramobta.ined with tbetest solutIon the sum

of the areas of any secondary peaks is not more than the area
2220
IP 2010
THYROXINE SODIUM
reference solution (a). Ignore any peak with an area less than
that of the principal peak in the chromatogram obtained with
Residue on evaporation. Evaporate 2.0 g on a water-bath and
heat at 105 for I hour. The residue weighs not more than
1.0 mg (0.05 percent).
Assay. Weigh accurately about 0.1 g, transfer to an iodine
flask and dissolve in 25 ml of 1 M sodium hydroxide. Add
20 ml of hot dilute hydrochloric acid and immediately titrate
with 0.05 M bromine to within 1 to 2 ml ofthe calculated endpoint. Warm the solution to about 75, add 0.1 m1 of methyl
orange solution and shake vigorously. If the solution is red,
continue the titration, dropwise and with shaking until the red
colour is discharged. Repeat the alternate addition of 0.05 M
bromine and methyl orange solution until the red colour is
discharged after the addition of the methyl orange solution.
1 n:l of 0.05 Mbromine is equivalentto 0.003755 gofC IO H I4 0.
Thyroxine Sodium
Levothyroxine Sodium; L-Thyroxine Sodium

325 nm, 0.73 to 0.79.
B. In the test for Liothyronine, the principal spot in the

(b).
C. To about 50 mg in a porcelain dish add a few drops of
sulphuric acid (96 per cent w/w); violet vapors are evolved.
D. To 20 mg add 2 ml of 1 M sulphuric acid. Heat on a waterbath followed by heating carefully over a naked flame,
increasing the temperature to about 600. Continue ignition
until most of the black particles have disappeared. Dissolve
the residue in 2 ml of water; the solution gives reaction A of
sodium salts (2.3.1).
Tests
Appearance of solution. Dissolve 0.5 g in 23 ml of a gently
boiling mixture of 4 volumes of ethanol (95 per cent) and
1 volume of 1 M hydrochloric acid. Cool and dilute to 25 ml
with the same mixture of solvents (solution A). The freshly
prepared solution is not more intensely coloured than reference
Specific optical rotation (2.4.22).+16.0 to +20.0, determined
in solution A.
Liothyronine. Determine by thin-layer chromatography
(2.4.17), coating the plate with a slurry oBO g ofsilica gel H in
60 ml of a 0.75 per cent w/v solution of soluble starch.

strong ammonia solution.
solution.
ClsHIO!4NNa04,xH20
Thyroxine Sodium is sodium Q4-(4-hydroxy-3,5-diiodophenyl)-3,5-diiodo-L-tyrosinate and contains a variable

quantity of water of crystallisation.
Thyroxine Sodium contains not less than 97.0 per cent and
not more than 101.0 per cent ofClsHIOI4NNa04, calculated on
the dried basis.
Category. Thyroid hormone.

w/v of the substance under examination and 0.01 per cent
w/v of the liothyronine RS in the solvent mixture..
levothyroxine sodium RS in the solvent mixture.
liothyronine RS in the solvent mixture.
Dose. 50 to 300 p.g daily.

Description. A white or slightly brownish yellow powder,
slightly coloured, crystalline powder.
Identification

dry the plate in air, spray lightly with ferric chlorideferricyanide-arsenite solution. Any spot corresponding to
liothyronine in the chromatogram obtained with the test
obtained with reference solution (c). The test is not valid unless
2221
THYROXINE SODIUM
IP 2010
the chromatogram obtained with reference solution (a) shows

Loss on drying (2.4.19). 6.0to 12.0 per cent, determined on


examination in 40 ml of 0.1 M sodium hydroxide. Shake for
about 30 minutes, add 40 ml of methanol, cool, mix well and
dilute to 100 ml with a mixture of equal volumes of 0.1 M
sodium hydroxide and methanol. Dilute 5.0 ml ofthe solution
levothyroxine sodium RS in a mixture of equal volumes of
0.1 M sodium hydroxide and methanol.
a stainless steel column 25 cm x 4.6 rom, packed with a
stationary phase consisting of porous silica particles
(5 to 10 jlm) to which nitrile groups are chemically
bonded,
mobile phase: a mixture of65 volumes of water, 1 volume
of orthophosphoric acid and 35 volumes of acetonitrile,
injection volume. 20 jll.
than 2.0 per cent.
Calculate the content OfC1SHIOI4NNa04.
Thyroxine Tablets
Thyroxine Sodium Tablets; Levothyroxine Tablets;
Levothyroxine Sodium Tablets; L-Thyroxine Sodium
Tablets
Thyroxine Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of anhydrous
thyroxine sodium, ClsHIOI4NNa04.
Usual strength. 50 jlg.

Identification
A.. To aquantity ot'the powdered tablets cOJ:ltalJ:liIigS66 ~gof

cllihydroL/s ihyl'oxil1e sodium a.dd a.roix.tureof3 rol of ethanol
(50 per cent) and 0.2 ml of hydrochloric acid, boil gently for
30 seconds, cool, filter, add 0.1 mlofa 10 per cent w/v solution
of sodium nitrite and boil; a yellow colour is produced. Cool

and make alkaline with 5 M ammonia; the solution becomes
orange.
R In the Assay, the principal peak in the chromatogram

to thyroxine sodium in the chromatogram obtained with the
reference solution.
Tests
Tablets. using the following solutions.
Test solution. To one tablet, add 10 ml of 0.1 M sodium
hydroxide and shake for about 30 minutes. Add 10 ml of
methanol, cool and dilute with sufficient ofa mixture ofequal
volumes of 0.1 M sodium hydroxide and methanol to produce
25.0 ml, mix well and filter. Dilute, ifnecessary, with the same
solvent mixture to produce a solution containing 0.0002 per
cent w/v ofThyroxine Sodium.
Follow the procedure described under Assay.
Calculate the content ofC IsH IOI4NNa04 in the tablet.

Test solution. Weigh and powder 20 or more tablets. Weigh

accurately a quantity of the powder containing about 1 mg of
Thyroxine Sodium, add 40 ml of 0.1 M sodium hydroxide and
shake for about 30 minutes. Add 40 ml of methanol, cool, mix
well and dilute with sufficient of a mixture ofequal volumes of
0.1 M sodium hydroxide and methanol to produce 100.0 ml,
mix well and filter.
stationary phase consisting of porous silica particles
(5 to 10 jlm) to which nitrile groups are chemically
bonded,
mobile phase: a mixture of65 volumes ofwatel; 1 volume
of orthophosphoric acid and 35 volumes of acetonitrile,
injection volume: 20 ill.
than 2.0 per cent.
2222
IP 2010
TlCARClLLIN AND CLAVULANlC ACID INJECTION
Calculate the content of CisHlOI4NNa04 in the tablets.
Sodium phosphate bufferpH4.3. Dissolve 13.8 g ofmonobasic

sodium phosphate in 900 ml ofwater, adjust the pH to 4.3 with
orthophosphoric acid or 10M sodium hydroxide, dilute to
1000 ml with water.

equivalent amount of anhydrous thyroxine sodium.
Sodium phosphate bufferpH 6.4. Dissolve 6.9 g ofmonobasic

sodium phosphate in 900 ml ofwater, adjust the pH to 6.4 with
10Msodium hydroxide, dilute 1000 ml with water.
Ticarcillin and Clavulanic Acid

Injection
Clavulanate lithium stock solution. A 0.06 per cent w/v

solution ofclavulanate lithium RS in sodium phosphate buffer
pH 6.4.
Clavulanic Acid and Ticarcillin Injection

Ticarcillin and Clavulanic Acid Injection is a sterile iso-osmotic
solution ofTicarcillin Monosodium and Clavulanate Potassium
in Water for Injections. It contains one or more suitable
buffering agents and a tonicity adjusting agent.
Ticarcillin and Clavulanic Acid Injection contains not less
stated amount of ticarcillin, CIsHl6N206S2 and not less than
amount ofclavulanic acid, CSH9NO s.
Ticarcillin and Clavulanic Acid for Injection

Ticarcillin and Clavulanic acid for Injection is a sterile, dry
mixture ofTicarcillin Monosodium and Clavulanate Potassium.
Test solution. Dissolve a quantity ofpowder in sufficient water

and dilute with sodium phosphate btiffer pH 6.4 to obtain a
0.09 per cent w/v solution ofTicarcillin.
Reference solution. Transfer about 100 mg of ticarcillin
monosodium monohydrate RS to a 100-ml volumetric flask
and add 150/J ml of clavulanate lithium stock solution (J is
the ratio ofthe stated amount ofticarcillin to the stated amount
of clavulanic acid in the powder) and dilute to 100.0 ml with
sodium phosphate btiffer pH 6.4.
octadecylsilane chemically bonded to porous silica (5
11m),
mobile phase: a mixture of 95 volumes of sodium
phosphate btifferpH 4.3 and 5 volumes of acetonitrile,

clarity of solution and particulate matter stated under
Usual Strength. Ticarcillin 3 g and Clavullanic acid 100 mg.
In the Assay, the principal peak due to ticarcillin in the

column efficiency of the principal peaks is not less than 1000
theoretical plates; the tailing factor for the principal peaks are
not more than 2.0; the resolution between the peaks due to
ticarcillin and clavulanic acid is not less than 5.0; and the
ticarcillin for clavulanic acid is about 0.2.
Tests
pH (2.4.24).5.5 to 7.5.
Calculate the content of ClsHl6N206S2 and CSH9NO s in the

injection.
Identification

Unit per mg ofticarcillin.
Storage. Store in single dose or multiple dose containers.

Water (2.3.43). Not more than 4.2 per cent.
Preparations (Powders for Injections).
Labelling. The label states (l) that the injection is to be thawed

just prior to use; (2) conditions for proper storage of the
resultant solution; (3) directs that the solution is not to be
refrozen.
2223
TIMOLOL MALEATE
IP 2010
Timolol Maleate
Tests
(eOOH
'~
eOOH
Specific optical rotation (2.4.22). -5.7 to -6.20, determined in

a 10.0 per cent w/v solution in 1 M hydrochloric aeid.
Mol. Wt. 432.5

Timolol Maleate is (S)-I-tert-butylamino-3-( 4-morpholino1,2,5-thiadiazol-3-yloxy)propan-2-01 hydrogen maleate.
Timolol Maleate contains not less than 98.5 per cent and not
more than 101.0 per cent ofC13H24N403S,C4H404, calculated
on the dried basis.
Category. Beta-adrenoceptor antagonist (Antiglaucoma).
Dose. In hypertension, initially 5 mg twice daily or 10 mg once
daily; maximum 60 mg daily. In angina, initially 5 mg 2 to 3
times daily; maintenance dose, 15 to 45 mg daily. For
prophylaxis after infarction, initially 5 mg twice daily, started 1
to 4 weeks after infarction. For migraine prophylaxis, 10 to 20
mgdaily.
colourless crystals.
Identification



solution.
Reference solution (b). A 0.1 per cent w/v solution of timolol
maleate RS in methanol.
dry the plate in air and examine in ultraviolet light at 254 nm
and then e)(:.pose to iodine vapour for 2 hours and examine in
daylight. By both methods of visualisation, any secondary
spot in the chromatogram obtained with test solution (a) is
A. Determine by infrared absorption spectrophotometrY (2.4.6).

Compare the spectrum with that obtained with timolol maleate
RS or with the reference spectrum oftimolol maleate.
B. In the test for Related substances, after exposure to iodine

vapour, the principal spot in the chromatogram obtained with
test solution (b) corresponds to that in the chromatogram

C. Triturate 0.1 g with a mixture of! ml of2 Msodium hydroxide

and 3 ml of water and shake with three quantities, each of5 ml,
of ether. To 0.1 ml ofthe aqueous layer add a solution of 10 mg
of resorcinol in 3 ml of sulphuric acid and heat on a waterbath for 15 minutes; no violet-red colour is produced. Neutralise
the remainder of the aqueous layer with 1 M sulphuric acid,
add 1 mlofbrominewater, heat on a water-bath for 15 minutes,
then heat to boiling and cool. To 0.2 m1 ofthis solution add a
solution of 10 mg of resorcinol in 3 ml of sulphuric acid and
heat on a water-bath for 15 minutes; a violet-red colour is
produced. Add 0.2 ml ofa 10 per cent w/v solution ofpotassium
bromide and heat on a water-bath for 5 minutes; the colour
changes to violet-blue.

CI3H24N403S, C~04.
Timolol Eye Drops

Timolol Maleate Eye Drops
Timolol Eye Drops are a sterile solution ofTimolol Maleate;: in
Purified Water. They may contain suitable antimicrobial
preservatives, buffering agents, stabilisers and suitable
substances to increase the viscosity of the solution.
2224
IP 2010
TIMOLOL TABLETS
Timolol Eye Drops contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of timolol,
C13H24N403S.
Usual strengths. The equivalent of 0.25 per cent w/v and 0.5
per cent w/v oftimolol.
peak, other than the peak corresponding to maleic acid, is not

greater than the area ofthe peak in the chromatogram obtained
with reference solution (a) and not more than two such peaks
have an area greater than that of the peak obtained with
refer.ence solution (b).
Identification
A. Add a volume containing 50 mg of timolol to an equal
volume of carbonate buffer pH 9.7 and extract with two
quantities, each of 40 ml, of dichloromethane. Reserve the
aqueous layer for test B. Dry the combined dichloromethane
extracts with anhydrous sodium sulphate and evaporate to
dryness. Dry the residue at 60 at a pressure of 2 kPa for
15 minutes.
obtained with timolol RS or with the reference spectrum of
timolol.
B. Filter the aqueous layer reserved in test A and evaporate to

about I ml. Add I ml of bromine solution, heat in a water-bath
for 10 minutes, boil, cool and add 0.1 ml of the solution to a
solution of I 0 mg resorcinol in 3 ml ofsulphuric acid; a bluish
black colour is produced on heating in a water-bath for
15 minutes.
Assay. To a volume containing about 25 mg of timolol add

water to produce 50.0 ml and mix. To 5.0 ml add 15 ml of
carbonate buffer pH 9.7 and extract with three quantities,
each of20 ml, and one quantity of I 0 ml of toluene. Wash each
extract successively with the same I O-ml volume of carbonate
bufjer pH 9. 7. Combine the toluene extracts and extract with
four quantities, each of 20 ml, of 0.05 M sulphuric acid.
Combine the extracts, dilute to 100.0 ml, filter and measure the
(2.4.7), using as the blank a solution prepared by treating
5.0 ml of water in the same manner beginning at the words
"add 15 ml of carbonate bufferpH 9.7...".
Calculate the content ofCI3H24N403S taking 279 as the specific

equivalent amount oftimolol; (2) the names and concentration
of any added antimicrobial preservatives.
Tests
pH (2.4.24).6.5 to 7.5.
Timolol Tablets
(2.4.14).
Timolol Maleate Tablets
Test solution. Use the undiluted eye drops.
Tirnolol Tablets contain not less than 90.0 percent and not
more than 110.0 per cent ofthe stated amount oftimolol maleate,
Reference solution (a). Dilute I volume of the eye drops to

250 volumes with the mobile phase.
Reference solution (b). Dilute I volume of the eye drops to
Reference solution (c). A 0.3 per cent w/v solution of maleic
acid in the mobile phase.
mobile phase: a mixture of 57.5 volumes of methanol
and 42.5 volumes of 0.02 M sodium octanesulphonate,
adjusted to pH 3.0 with glacial acetic acid,
Record the chromatogram of the test solution for 4 times the
obtained with the test solution the area of any secondary
CI3H24N403S,C~404'
Identification
Timolol Maleate add 20 ml of carbonate buffer pH 9. 7 and
extract with two quantities, each of40 ml, of dichloromethane.
Reserve the aqueous layer for test B. Dry the extracts with
anhydrous sodium sulphate, evaporate to dryness using a
rotary evaporator, dry the residue at 60 at a pressure of2 kPa
for 15 minutes.
obtained with timolol RS or with the reference spectrum of
timolol.
B. Filter the aqueous layer reserved in test A and evaporate to

about I ml. Add I ml of bromine water, heat on a water-bath
2225
TIMOLOL TABLETS
IP 2010
for 15 minutes, then heat to boiling and cool. Add 0.1 ml ofthis
solution to a solution of 10 mg of resorcinol in 3 ml ofsulphuric
acid and heat on a water-bath for 15 minutes; a bluish black
colour is produced.
maximum at about 295 nm (2.4.7), using as the blank a solution

prepared by treating a mixture of 5 ml of water and 15 ml of
carbonate buffer pH 9. 7 in the same manner beginning at the
words "and extract with three quantities,..."
Tests
Calculate the content of CI3H24N403S,C4H404 taking 204 as


(2.4.14).

containing 0.1 g ofTimolol Maleate with 10 ml of the mobile
phase for 10 minutes and filter.
Tinidazole

mobile phase: a mixture of 57.5 volumes of methanol
and 42.5 volumes of 0.02 M sodium octanesulphonate,
adjusted to pH 3.0 with glacial acetic acid,
Run the chromatogram of the test solution for 4 times the
peak, other than the peak corresponding to maleic acid, is not
more than the area ofthe peak in the chromatogram obtained
with reference solution (a) and not more than two such peaks
have an area more than that ofthe peak obtained with reference
solution (b).
Tablets.
To one tablet, add 25.0 ml of 0.05 M sulphuric acid, shake for
20 minutes and complete the Assay beginning at the words
"and centrifuge....".
quantity of the powder containing about 15 mg of Timolol
Maleate, add 25.0 ml of 0.05 M sulphuric acid, shake for
20 minutes and centrifilge until clear. Add 5.0 ml ofthe resulting
supernatant liquid to 15 ml of carbonate bujfer pH 9.7 and
extract with three quantities, each of 20 ml, and one quantity
of 1 0 ml of toluene. Wash each extract successively with.the
same 10-ml volume of carbonate bujferpH 9. 7, combiue the
toluene extracts and extract with four quantities, each of20 ml,
of 0.05 M sulphuric acid. Combine the acid extracts, dilute to
100.0 ml, filter and measure the absorbance ofthe filtrate at the
Mol. Wt. 247.3

Tinidazole is 1-[2-(ethylsulphonyl)ethyl]-2-methyl5-nitroimidazole.
Tinidazole contains not less than 98.0 per cent and not more
than 100.5 per cent of CSHI3N304S, calculated on the dried
basis.
Dose. For trichomoniasis, 2 g as a single dose and repeat after
3 to 5 days rest, ifnecessary. For giardiasis, 150mgtwice daily
for seven days or as a single dose of 2 g. For intestinal
amoebiasis, 600 mg as a single dose daily for 2 to 6 days. For
extraintestinal amoebiais, 2 g as a single daily dose for 3 days.
Description. Pale yellow crystals or a crystalline powder;
odour, slight and characteristic.
Identification

Compare the spectrum with that obtained with tinidazole RS
or with the reference spectrum oftinidazole.

0.00 I per cent w/v solution in methanol shows an absorption
maximum at about 31 0 nm; absorbance at about 310 nm, about
0.35.
C. To about 5 mg, add 5 ml of 0.1 M hydrochloric acid, 50 mg
of zinc powder, 4 ml ofhydrochlQric.JJcidandal1QwJQ s.tand
for 30 minutes. Add 4 ml of a I per cent w/v solution of
vanillin, heat on a boiling water-bath for 20 minutes, allow to
cool to room temperature and dilute to 20 ml with water; a
greenish yellow colour is produced.
2226
IP 2010
TIOTROPIUM BROMIDE MONOHYDRATE
Tests
room temperature and dilute to 20 ml with water; a greenish

yellow colour is produced.


5 volumes of methanol and 5 volumes of diethylamine.
examination in 10 ml ofa mixture ofequal volumes ofchlorofonl1
and methanol.
substance under examination in a mixture ofequal volumes of
chloroform and methanol.
chromatogram obtained with the reference solution:
C. Melting range (2.4.21). 125 to 128.
Tests
quantity ofthe powder containing about 0.15 g ofTinidazole,
add 20 ml of methanol, shake well and add sufficient methanol
to produce 100.0 ml. Mix well and filter. Dilute 10.0 ml ofthe
solution to 100.0 ml with methanol and further dilute 10.0 ml of
this solution to 100.0 ml with methanol. Measure the
310nm(2.4.7).
Calculate the content ofCgHl3N304S taking 356 as the specific

acid, using 0.15 ml of nile blue A solution as indicator. Carry
:~f~3k

CgH 13N 30 4S.
~1
~s
o
Tinidazole Tablets
OH
~ ~
CI9H22BrN04S2, H20
Tinidazote Tablets contain not less than 95.0 per cent and not
more than 105.0 per cent of the stated amount oftinidazole,
CgH 13N30 4 S.
Identification
maximum at about 31 0 nm.
Extract a quantity of the powdered tablets containing 0.1 g of
Tinidazole with i 0 ml of methanol, filter and evaporate the
tests.
B. To about 5 mg add 5 ml of 0.1 M hydrochloric acid, 50 mg
of zinc powder, 4 ml of hydrochloric acid and allow to stand
for 30 minutes. Add 4 ml ofa 1per cent w/v solution of vanillin,
heat on a boiling water-bath for 20 minutes, allow to cool to
Mol. Wt. 490.2
Tiotropium Bromide Monohydrate is 6/3,7{3-epoxy-3{3-hydroxy8-methyl-1 cxH-5cxH-tropanium bromide monohydrate.

Tiotropium Bromide Monohydrate contains not less than
98.0 per cent and not more than 102.0 per cent oftiotropium
bromide, CI9H22N04S2Br, calculated on the anhydrous basis.
Category. Bronchodilator.
Identification
A. Detennine by infrared absorption spectrophotometry
tiotropium bromide monohydrate RS or with the reference
spectrum oftiotropium bromide monohydrate.
obtained with test solution corresponds to the principal peak
in the chromatogram obtained with the reference solution.
2227
TIOTROPIUM BROMIDE MONOHYDRATE
IP 2010
Tests
(2.4.14).

Reference solution. A 0.002 per cent w/v solution oftiotropium
bromide monohydrate RS in the mobile phase.
octadecylsilane bonded to porous silica (5 ,"un),
3.85 g of ammonium acetate in 1000 ml of water and
adjusting the pH to 5.5 with dilute acetic acid
(10 per cent vlv),
- injection volume. 20 J..L1.
tailing factor is not more than 2.0 and the column efficiency is
Inject the test solution. Any individual impurity is not more
than 0.5 per cent and the sum of all the impurities is not more
than 1.0 per cent.

the resulting solution to 50.0 ml with the mobile phase.
Reference solution. A 0.002 per cent w/v solution oftiotropium
bromide monohydrate RS in the mobile phase.
octadecylsilane bonded to porous silica (5 J..Lm),
55 volumes ofa buffer solution prepared by dissolving
3.85 g of ammonium acetate in 1000 ml of water and
adjusting the pH to 55 with dilute acetic acid ( 10 per
Calculate the content ofCI9HzzN04Sz.Br.
Tiotropium Bromide Powder for

Inhalation
Tiotropium Powder for Inhalation consists of Tiotropium
Bromide in microfine powder either alone or admixed with
Lactose in a pre-metered unit for use in a suitable powder
inhaler.
Tiotropium Powder for Inhalation contains not less than
amount oftiotropium, CI9H22N04Sz per unit dose.
Identification
Tests
Preparations (Powders for Inhalation).
Solvent mixture. 90 volumes of 0.05 per cent v/v orthophosphoric acid and 10 volumes of acetonitrile.
Test solution. Dissolve a quantity of the mixed contents of
20 capsules in sufficient ofthe solvent mixture to get a solution
containing 1.8 J..Lg per ml ofTiotropium.
Reference solution. A solution of tiotropium bromide RS
equivalent to tiotropium 1.8 J..Lg per ml in the solvent mixture.
a stainless steel column 15 cm x 4.6 lnm, packed with

octadecylsilane bonded to porous silica (5 J..Lm),
prepared by dissolving 2 ml triethylamine in 1000 ml of
flow rate. 1 nil per minute,

injection volume. 20 J..L1.
2228
TITANIUM DIOXIDE
IP 2010
water and adjusting the pH to 2.5 with orthophosphoric

acid, and 20 volumes of acetonitrile,
- inject 200 fll.
Calculate the content of C19H22N04Sz per unit.
Storage. Store protected from moisture, at temperature not
exceeding 30.
Labelling. The label states the quantity of active ingredient
per pre-metered .unit.
Titanium Dioxide
Mol. Wt. 79.9
Titanium Dioxide contains not less than 98.0 per cent and not
more than 100.5 per cent ofTiOz.
Category. Pharmaceutical aid and topical protectant.
Description. A white or almost white, infusible powder;
odourless.
Identification
A. When strongly heated it becomes pale yellow; the colour

is discharged on cooling.
B. To 0.5 g, add 5 g ofanhydrous sodium sulphate and 10 ml
ofwater and mix. Add 10 ml ofsulphuric acid and boil gently
until clear; cool, add slowly 30 ml ofa 25 per cent v/v solution
ofsulphuric acid and dilute with water to 100 ml (solution A).
To 5 ml of solution A add 0.1 ml ofstrong hydrogen peroxide
solution; an orange-red colour is produced.
C. To 5 ml of solution A add 0.5 g of zinc, in granules; after

45 minutes a violet-blue colour is produced.
Tests
Appearance of solution. Solution A is not more opalescent
than opalescence standard OS2 (1.4.1), and colourless (2.4.1).
Acidity or alkalinity. Shake 5.0 g with 50 ml ofcarbon dioxidefree water for 5 minutes and centrifuge until a clear solution is
obtained. To 10 ml ofthe solution add 0.1 ml of bromothymol
blue solution. Not more than 1.0 ml of either 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change
Water-soluble substances. Not more than 0.5 per cent,

determined by the following method. Boil 10.0 g for 5 minutes
with 150 ml ofwater containing 0.5 g ofammonium sulphate.
Cool, dilute to 200 ml with water and filter until a clear solution
is obtained. Evaporate 100 ml of the filtrate to dryness ignite
and weigh.
Arsenic (2.3.10). To 0.2 g in a 100 ml Kjeldahl flask, add 2 g of
anhydrous sodium sulphate, 7 ml of sulphuric acid and 5 ml
of nitric acid. Heat gently until a clear solution is obtained,
cool, add 10 ml of water, cool again and add 5 g of hydrazine
reducing mixture and 10 ml ofhydrochloric acid. Immediately
attach an air condenser and distil into 15 ml of cooled water
until a total volume 000 ml is obtained. Rinse the condenser
with 5 ml of water and dilute the combined distillate and
rinsings to 40 ml with water. 20 ml of the resulting solution
complies with the limit test for arsenic. Use a mixture of0.5 ml
of arsenic standardsolution (l ppm As) and 24.5 ml ofwater
to prepare the standard (5 ppm).
Barium. Shake 20.0 g with 30 ml of hydrochloric acid, add
100 ml of distilled water and boil. Filter while hot through a
hardened filter paper until a clear filtrate is obtained. Wash the
filter. with 60 ml of distilled water and dilute the combined
filtrate and washings to 200 ml with distilled water. To 10 ml of
this solution add 1 ml of 1 M sulphuric acid. After 30'minutes
any opalescence is not more intense than that of a mixture of
10 ml of the test solution and 1 ml of distilled water.
Heavy metals (2.3.13). Dilute 10 ml ofthe solution prepared in
the test for Barium to 20 ml with water. 12 ml ofthe solution
(20 ppm).
Iron. To 8 ml of solution A, add 4 ml of water, mix and add
0.05 ml ofbromine water, allow to stand for 5 minutes, remove
the excess of bromine with a current of air and add 3 ml of
potassium thiocyanate solution. Any colour in the solution
is not more intense than that in a standard prepared at the
same time and in the same manner using a mixture of4 ml of
iron standard solution (2 ppm Fe) and 8 ml of a 20 per cent
w/v solution ofsulphuric acid (200 ppm).
Assay. Weigh accurately about 0.5 g, transfer to a 300 ml
Kjeldahl flask, add 5 g of anhydrous sodium sulphate and
10 ml ofwater, mix and add 10 ml ofsulphuric acid. Boil gently
until clear, cool, add slowly 40 ml of cooled sulphuric acid
(25 per cent), cool again and dilute with water to 100.0 ml
(solution B). To 300 g ofzinc, in granules, add 300 ml ofa2 per
cent w/v solution of mercuric nitrate and 2 ml of nitric acid,
shake for 10 minutes and wash with water. Pack the zinc
amalgam into a glass tube'(400 mm x 20 mm) fitted with a tap
and a filter plate. Pass through the column, at a rate of about
3 ml per minute, 100 ml of 1 M sulphuric acid followed by
100 ml of water, ensuring that the amalgam is covered with
liquid throughout. Pass slowly through the column, at a rate
2229
IP 2010
TIZANIDINE HYDROCHLORIDE
of about 3 ml per minute, 200 mlof 0.5 M sulphuric acid

followed by 100 ml of water. Collect the combined eluates in a
500-ml conical flask containing 50 ml of a 15 per cent w/v
solution of ferric ammonium sulphate in sulphuric acid
(25 per cent) and titrate immediately with 0.1 M eerie
ammonium nitrate usingferroin solution as indicator until a
greenish colour is obtained (nl ml). Pass slowly through the
column 100 ml of 0.5 Msulphuric acid followed by 20.0 ml of
solution B, wash with 100 ml of 0.5 M sulphuric acid followed
by 100 ml of water. Collect the combined eluates in a 500-ml
conical flask containing 50 ml ofa 15 percentw/v solution of
ferric ammonium sulphate in sulphuric acid (25 per cent),
rinse the lower end of the column with water and titrate
immediately with 0.1 M eerie ammonium nitrate usingferroin
solution as indicator until a greenish colour is obtained
(n2 ml). Calculate the percentage content of Ti0 2 from the
expression 3.99(n2 - nl)/w
Where, w is the weight, in g, of the substance under examination used in the preparation of solution A.
Storage. Store protected from moisture; Avoid contact with
aluminium.
Tests
pH (2.4.24). 3.5 to 5.3, determined on 5.0 per cent w/v solution
in water.
(2.4.14).

tizanidine hydrochloride RS in the mobile phase.
Reference solution (b). Dilute I ml ofreference solution (a) to
column temperature 50 0 ,
mobile phase: a mixture of 80 volumes of buffer solution
prepared by dissolving 3.5 g of sodium-I-pentane
sulphonate in 1000 ml of water, adjust the pH to 3.0 with
12 per cent orthophosphoric acid solution or 1 M
sodium hydroxide and 20 volumes of acetonitrile,
tailing factor is not more than 2.0 and the column efficiency is
,Hel
Mol. Wt. 290.2

Tizanidine Hydrochloride is 5-chloro-N-(2-imidazolin-2-yl)2,1 ,3-benzothiadiazol-4-yl amine.
Tizanidine Hydrochloride contains not less than 98.0 per cent
and not more than 102.0 per cent ofCgHgClNsS.HCI, calculated
on the dried basis.
Category. a-adrenergic agonist.
Dose. 2 mg three times a day.

not more than the area of the peak in the cl:rromatogram
Heavy metals (2.3.13). Dissolve 1.0 g in 20 rnl ofwater. 12 rnl of
this solution complies with limit test for heavy metals, Method
D(20ppm).
Total Chloride. 11.9 per centto 12.5 per cent.
Description. A white to yellowish white, crystalline powder.
Identification
Compare the spectrum with that obtained with tizanidine
hydrochloride RS.
C. Gives reaction A for chlorides (2.3.1).
Weigh accurately about 0.5 g and dissolve in 50 ml of water.

Titrate with 0.1 M silver nitrate. Determine the end point
I ml of 0.1 M silver nitrate is equivalent to 0.003545 g of
on I g by drying in an oven at 105 0 for 3 hours.
2230
IP 2010
TlZANIDINE TABLETS

the solution to 50.0 ml with the same solvent.
Reference solution. A 0.01 per cent w/v solution of tizanidine
prepared by dissolving 6.8 g of monobasicpotassium
phosphate in 1000 ml of water adjusting the pH to 7.5
with 5.3 M potassium hydroxide, and 50 volumes of
acetonitrile,
Calculate the content of C9HsClNsS in the medium from the

absorbance obtained from a solution of known concentration
of tizanidine Hydrochloride RS in the same medium.
C9HsClNsS.
Tablets.
under Assay.
Test solution. Cmsh one tablet and disperse in 50 ml phosphate

bufferpH 6. 6, dilute to 100.0 ml with acetonitrile and filter.
Reference solution. Weigh accurately 10 mg of tizanidine
hydrochloride RS, dissolve in 25 mlphosphate bufferpH 6.6
and dilute to 50.0 ml with acetonitrile. Dilute 5.0 ml of this
solution to 50.0 ml with acetonitrile.
Other tests. Comply with the tests stated underTablets.
Calculate the content ofC 9H sClNsS.HCl.
Test solution. Weigh'and powder 20tablets. Weigh accurately

a quantity of the powdered tabl.et containing about 20 mg of
Tizanidine, disperse in 50 ml phosphate buffer pH 6.6 and
dilute to 100.0 ml with acetonitrile and filter.
Tizanidine Tablets
Calculate the content ofC 9H sClNsS.
Reference solution. Weigh accurately '10 mg of

tizanidine hydrochloride RS, dissolve in 25 ml phosphate
buffer pH 6. 6 and dilute to 50.0 ml with acetonitrile.
Tizanidine Hydrochloride Tablets

Tizanidine Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount oftizanidine,
C9HsClNsS.
mobile phase: a mixture of 80 volumes of phosphate
Dose. 2 mg; 4 mg.
Identification
than 2.0 per cent.
Tests
Apparatus No.1,
Medium: 900 ml of 0.1 M hydrochloric acid,
Calculate the content ofC 9HsClNsS.

the absorbance ofthe filtrate, suitably diluted with dissolution
medium ifnecessary, at the maximum at about 320 nm (2.4.7).

Labelling. The label states the strength in, terms of the
equivalent amount ofTizanidine.
2231
IP 2010
TOBRAMYCIN
cent) and heat at 105 for 10 minutes. The principal spot in the
that in the chromatogram obtainedwith reference solution (a).
reference solution (b) shows three clearly separated principal
spots.
Tobramycin
OH
NH 2
B. Dissolve about 5 mg in 5 ml of water, add 5 ml ofa 0.1 per

cent w/v solution of ninhydrin in ethanol (95 per cent) and
heat in a water-bath for 3 minutes; a violet-blue colour
develops.
HO
NH 2
Tests
NH 2

solution.
Mol. Wt. 467.3

2-Methyl-l-propanol. Not more than 1.0 per cent w/w,
determined by gas chromatography (2.4.13).
Tobramycin is 6-0-(3-amino-3-deoxy-a-D-glucopyranosyl)2-deoxy-4-0-(2,6-diamino-2,3,6-trideoxy-a -D-ribohexopyranosyl)-D-streptamine, an antimicrobial substance

produced by Streptomyces tenebrarius or by any other
means.
Test solution (a). A 10 per cent w/v solution ofthe substance

under examination in wate!:
Tobramycin has potency not less than 930 Units per mg,
calculated on the anhydrous and 2-methyl-l-propanol-free
basis.
Dose. By intramuscular or intravenous injection, 3 to 5 mg per
kg daily, in divided doses.
Identification
the plate with silica gel H.

chloroform.
tobramycin RS in water.
w/v each of kanamycin sulphate RS, neomycin sulphate RS
and tobramycin RS in water.
Apply to the plate 5 I!l of each solution. After development,
dry the plateiilwaim arr,spiay witha mlxtllieofequal volumes
of a 46 per cent w/v solution of sulphuric acid and a 0.2 per
cent w/v solution of 1,3-naphthalenediol in ethanol (95 per
Test solution (b). A solution containing 10 per cent w/v of the

substance under examination and 0.2 per cent v/v of
2-propanol (internal standard).
2-methyl-l-propanol and 0.2 per cent v/v of the internal
standard.
a glass column 1.5 m x 4 mm, packed with porous polymer
beads (80 to 100 mesh) (such as Porapak Q),
- temperature. column 165,
Calculate the percentage w/w of 2-methyl-l-propanol.
Mobile phase. A mixture of equal volumes of strong ammonia

solution, 2-butanone and ethanol (95 per cent).
examination in 100 ml of 0.02 M ammonia.
substance under examination in 0.02 M ammonia.
dry the plate in warm air, heat at 110 for 10 minutes and spray
t~e hotplate with a so!~i~!J:J~r_eEl3:r~_~_~~_~i~!~IY_~~X()r~_~~e
by diluting sodium hypochlorite solution (3 per cent CI)
with water cOiltainO.5 percent Vi/v
available chlorine.
Dry in a current of cold air until a sprayed area of the plate
below the line of application gives at most a very faint blue
2232
fo
of
IP 2010
TOBRAMYCIN INJECTION
colour with a drop of potassium iodide and starch solution;

avoid prolonged exposure to cold air. Spray the plate with
potassium iodide and starch solution. Any secondary spot
Sulphated ash (2.3.18). Not more than OJ per cent.
OJ g.
Method B (2.2.10).
Tobramycin intendedfor use in the manufacture ofparenteral

preparations without a further appropriate procedure for
per mg oftobramycin.
Tobramycin intendedfor use in the manufacture ofparenteral

preparations or eye drops without a further appropriate
additional requirements.

chloroform.
water to produce a solution containing 0.4 per cent w/v
solution oftobramycin.

tobramycin RS in water.
w/v each of kanamycin sulphate RS, neomycin sulphate RS
and tobramycin RS in water.
dry the plate in wann air, spray with a mixture ofequal volumes
of a 46 per cent w/v solution of sulphuric acid and a 0.2 per
cent w/v solution of 1,3-naphthalenediol in ethanol (95 per
cent) and heat at 105 for 10 minutes. The principal spot in the
reference solution (b) shows three clearly separated principal
spots.
B. Gives the reactions ofsulphates (2.3.1).

Storage. Store protected from moisture. Ifthe material is sterile,
as to exclude micro-organisms.
Labelling. The label states (1) the number ofUnits per ri:J.g; (2)
where applicable, that it is sterile; (3) where applicable, that it
is free from bacterial endotoxins and depressor substances.
Tests
pH (2.4.24). 3.5 to 6.0.
(2.4.17), coating the plate with silica gel H
.
Mobile phase. A mixture ofequal volumes ofstrong ammonia

solution, 2-butanone and ethanol (95 per cent).
0.01 M ammonia to obtain a solution containing 40 mg of
Tobramycin in 4 ml. Shake with 10 ml of ether and use the
aqueous layer.
Tobramycin Sulphate Injection

Tobramycin Injection is a sterile solution of Tobramycin in
Water for Injections containing sufficient Sulphuric Acid to
adjust the pH to 3.5 to 6.0.
Tobramycin Injection contains not less than 90.0 per cent and
tobramycin, ClsH37NsOg.
Description. A colourless solution.
Identification

substance under examination in 0.02 M ammonia.
dry the plate in warm air, heat at 110 for 10 minutes and spray
the hot plate with a solution prepared immediately before use
by diluting sodium hypochlorite solution (3 per cent CI)
with water to contain 0.5 per cent w/v of available chlorine.
Dry in a current of cold air until a sprayed area of the plate
below the line of application gives at most a very faint blue
colour with a drop of potassium iodide and starch solution;
avoid prolonged exposure to cold air. Spray the plate with
potassium iodide and starch solution. Any secondary spot
2233
TOCOPHERYLACETATE
IP 2010
Bacterial endotoxins (2.23). Not more than 2.0 Endotoxin

Units per mg oftobramycin.

the plate with silica gel HF254.


20 volumes of ether.

Method B (2.2.10).

examination in 100 ml of cyclohexane.
Calculate the content of tobramycin in the injection, taking

each 1000 Units found to be equivalentto 1 mg oftobnimycin.

examination in 2 ml of 5 M ethanolic sulphuric acid, heat on
a water-bath for 5 minutes, cool, add 2 ml of water and 2 ml of
cyclohexane and shake for 1 minute; use the upper layer.
The upper fiducial limits of error is not less than 97.0 per cent
and not more than 110.0 per cent of the stated potency.

CMocopheryl acetate RS in' cyclohexane.
Tocopheryl Acetate.
(X- Tocopheryl Acetate; (X-Tocopherol Acetate; Vitamin
E Acetate
Mol. Wt. 472.8

Tocopheryl Acetate is (2RS,4'RS,8'RS)-6-acetoxy2,5,7,8- tetramethyl-2-(4', 8', 12'-trimethyltridecyl)chroman(all-rac-a-tocopherol acetate).
Tocopheryl Acetate contains not less than 96.0 per cent and
not more than 102.0 per cent ofC31Hs203.
Reference solution (b). Prepare in the same maimer as test

solution (b) but using 10 mg of a-tocopheryl acetate RS in
The principal spot in the chromatogram obtained with test
solution (a) correspondsto that in the chromatogram obtained
withTeference solution (a). There are two spots in each ofthe
chromatograms obtained with test solution (b) and reference
solution (b). The spots of higher R f value are due to
a-tocopheryl acetate and correspond to that in the
chromatogram obtained with reference solution (a). The spots
oflower Rf value are due to a-tocopheryl. Spray the plate with
a mixture of 1 volume of hydrochloric acid, 4 volumes of a
0.25 per cent w/v solution of ferric chloride in ethanol
(95 per cent) and 4 volumes of a 1.0 per cent w/v solution of
1,10-phenanthroline hydrochloride in ethanol (95 per cent).
In the' chromatograms obtained with test solution (b) and
reference solution (b) the spot oflower R f value a-tocopheryl
is orange.
Category. Vitamin E supplement.

Dose. Prophylactic, 5 to 10 mg; therapeutic, to be determined
by the physician according to the needs of the patient.
Description. A clear, slightly greenish yellow, viscous, oily
liquid.
Identification
arid C may be omitted if test A is carried out.
Compare the spectrum with that obtained with a-tocopheryl
acetate RS.
B.W1;J.en examinSldin the range 230 om to 360 om (2.4.7), I:!
0.01 per cent w/v solution in ethanol exhibits a maximum at
about 284 nm, a shoulder at about278 nm and a minimum at
about 254 nm.
Tests
Free tocopherol. Not more than 2.0 per cent, determined by
the following method. Weigh accurately about 0.5 g, dissolve
in 100 ml of 0.25 M ethanolic sulphuric acid, add 20 ml of
water and 0.1 ml of a 0.25 per cent w/v solution of
diphenylamine in sulphuric acid and titrate with 0.01 M eerie
ammonium nitrate until a bluecolour is produced that persists
for at least 5 seconds. Repeat the operation without the
required.
1 ml of 0.01 M eerie ammonium nitrate is equivalent to

0.002154 g oftocopherol.
2234
IP 2010
TOLAZAMIDE TABLETS
absorbances at the maxima are about 0.78, aboutO.83 and

about 0.62 respectively.
Tests

ethanol (95 per cent), add 20 ml of 2.5 M ethanolic sulphuric
acid and heat on a water-bath under a reflux condenser for
3 hours. Cool, transfer the solution quantitatively to a 200-ml
volumetric flask, rinse the apparatus with ethanol (95 per
cent) and add the rinsings to the flask. Make up to volume
with ethanol (95 per cent) and mix. To 25.0 ml ofthe resulting
solution in a flask add 25 ml of 0.25 M ethanolic sulphuric
acid, 10 ml of water and titrate with.0.01 M eerie ammonium
nitrate using 0.1 ml of diphenylamine as indicator, until a blue
colour persisting for at least 5 seconds is obtained. Repeat
the operation without the substance under examination. The
eerie ammonium nitrate required.
1 ml of 0.01 M eerie ammonium nitrate is equivalent to
0.002364 g ofC31H5203.
Tolazamide

Mobile phase.A mixture of200 volumes of chloroform, 100

volumes of methanol, 60 volumes of cyclohexane and 23
volumes of 13.5 M ammonia.
Reference solution. A 0.01 per cent w/vsolution of toluenep-sulphonamide in acetone.
Apply to the plate 10 III ofeach solution. After removal ofthe
plate, dry in a current of cold air, heat at 110 for 10 minutes,
place the hot plate in a tank of chlorine gas prepared by the
addition of hydrochloric acid to a 5 per cent w/v solution of
potassium permanganate contained in a beaker placed in the
tank and allow to stand for 2 minutes. Dry it in a CillTent of
cold air until an area ofthe plate below the line of application
gives at most a very faint blue colour with a 0.5 per cent w/v
solution of potassium iodide in starch mucilage; avoid
prolonged exposure to cold air. Spray the plate with a 0.5 per
cent w/v solution of potassium iodide in starch mucilage.
Ct.#2tN303S
Mol. Wt. 311.4
Tolazamide is l-perhydroazepin-l-yl-3-tolyl-psulphonylurea
Tolazamide contains not less than 98.0 per cent and not more
than 101.0 per cent of C14H2tN303S, calculated on the dried
basis.
0.7kPa.
butan-2-one with the aid of gentle heat. Allow to cool, add 30
ml of ethanol (95 per cent) and titrate with 0.1 M sodium
hydroxide using phenolphthalein solution as indicator.
Ct.#21 N 30 3S.
Category. Hypoglycaemic.
Identification
Compare the spectrum with that obtained with tolazamide RS
or with the reference spectrum oftolazamide.
B. When examined in the range 230 to 350 nm (2.4.7), a 0.04 per
cent w/v solution in ethanol (95 per cent) exhibits maxima at
256 mu, 263 nm and 275 nm and a shoulder at 268 nm. The
Tolazamide Tablets
Tolazamide Tablets contain not less than 95.0 per cent and not
more than 105.0 per cent ofthe stated amount oftolazamide,
Ct.#2t NP3 S,
2235
lP 2010
TOLAZAMIDE TABLETS
Identification
Tolbutamide

0.25 g ofTolazamide with 50 m1 of acetone and filter. Evaporate
the filtrate to dryness and dry the residue at 60 0 at a pressure
of 2 kPa for 3 hours. On the residue, determine by infrared
with that obtained with tolazamide RS or with the reference
spectrum oftolazamide.
Mol. Wt. 270.4
Tests
Tolbutamide is 3-butyl-1-[(4-methylphenyl)sulphonyl]urea.
Related substances. Determine by tliin-layer chromatography

Mobile phase. A mixture of 23 volumes of 13.5 M ammonia,

60 volumes of cyclohexane , 100 volumes of methanol and
200 volumes of chloroform.
containing about 0.2 g ofTolazamide with 10 ml of acetone
and filter.
Reference solution. A 0.0 1 per cent w/v solution of toluene-psulphonamide in acetone.
dry the plate in a current of cold air, heat at 1100 for 10 minutes,
place the hot plate in a tank of chlorine gas, prepared by the
addition of hydrochloric acid to a 5 per cent w/v solution of
potassium permanganate contained in a beaker placed in the
tanle and allow to stand for 2 minutes. Dry the plate in a
current of cold air until an area of the plate below the line of
application gives at most a very faint blue colour with a 0.5 per
cent w/v solution of potassium iodide in starch mucilage
avoid prolonged exposure to cold air. Spray the plate with a
0.5 per cent w/v solution of potassium iodide in starch
mucilage. Any secondary spot in the chromatogram obtained
cent).
Assay. Weigh and powder 20 tablets. Shake a quantity of the
powdered tablets containing about 0.5 g of Tolazamide with
50 ml of chloroform, filter, wash the residue with chloroform
and evaporate the combined filtrate and washings to dryness.
Dissolve the residue in 20 ml of butan-2-one with the aid of
gentle heat. Allow to cool, add 30 ml of ethanol (95 per cent)
and titrate the resulting solution with 0;1 Msodiumhydroxide
using phenolphthalein solution as indicator.
CIJIzIN303S,
Tolbutamide contains not less than 99.0 per cent and not more
than 101.0 per cent ofCI2HlsNz03S, calculated on the dried
basis.
Category. Hypoglycaemic.
Dose. 500 mg to 1.5 g daily as a single dose with breakfast or
in divided doses.
Description. A white, crystalline powder; almost odourless.
Identification

Compare the spectrum with that obtained with tolbutamide
RS or with the reference spectrum oftolbutamide.
B. Dissolve 25 mg in sufficient methanol to produce 100.0 ml.
resulting solution shows absorption maxima at about 258 nm,
263 nm and 275 nm and a shoulder at about 268 nm. Dilute the
solution with methanol to produce a 0.001 per cent w/v
solution. When examined in the range 220 nm to 235 nm, the
228 nm; absorbance at about 228 nm, about 0.50.
C. Boil 0.1 gwith 8 ml ofa 50 per centv/v solution ofsulphuric
acid under a reflux condenser for 30 minutes. Make the solution
strongly allcaline with sodium hydroxide solution and steam
distil for 30 minutes, receiving the distillate in 20 ml of 0.1 M
hydrochloric acid. To 1 ml of the solution containing the
distillate add 0.1 g of sodium acetate and 10 mlof buffer
solution pH 9.4. Cool in an ice-bath for 10 mmutes, add 1 mlof
diazotised nitroaniline solution, set aside for 20 minutes and
add dropwise 1 ml of sodium hydroxide solution; an orangered colour is produced.
Do Boil 0.1 gwith 8 ml ofa 50 per centv/vsolution of sulphuric
acid under a reflux condenser for 30 minutes. Cool in an icebath; a crystalline precipitate of 4-toluenesulphonylamide is
formed, which after recrystallisation from hot water and dry'ing
at105melts at 135 to 140 (2.4.21).
2236
IP 2010
TOLBUTAMIDE TABLETS
Tests

C12HlsN203S.
Appearance of solution. A 2.0 per cent w/v solution in 1 M

sodium hydroxide is clear (2.4.1), and colourless (2.4.1).
pH (2.4.24). 4.5 to 5.5, determined in a solution prepared by

dissolving 2.0 g in 50 ml of carbon dioxide-fi-ee water by
heating at 70 for 5 minutes, cooling rapidly and filtering.
Tolbutamide Tablets
Non-sulphonyl urea. Dissolve 0.5 gin 1 ml of dilute ammonia

solution and 9 ml of water; not more than a faint opalescence
is produced.

8 volumes of methanol and 2 volumes of anhydrous formic
acid.
4-toluenesulphonamide in acetone.
Reference solution (b). A mixture of equal volumes of the
test solution and reference solution (a).
Tolbutamide Tablets contain not less than 95.0 per cent and
tolbutamide, C12HlsN203S.
Identification
Extract a quantity of the powdered tablets containing 1 g of
Tolbutamide with 10 ml of chloroform, filter, evaporate the
filtrate to dryness, scratching the sides of the vessel, if
necessary, to induce crystallisation, and dry the residue at
105 for 30 minutes. The residue complies with the following
tests.
Compare the spectrum with that obtained with tolbutamide
RS or with the reference spectrum of tolbutamide.
B. Boil 0.1 g ofresidue with 8 ml of a 50 per cent v/v solution

of sulphuric acid under a reflux condenser for 30 minutes.
Cool in an ice-bath; a crystalline precipitate of
4-toluenesulphonylamide is formed, which after
recrystallisation from hot water and drying at 105 melts at
135 to 140.
Apply to the plate 5 III of each of test solution and reference

solution (a) and 10 III of reference solution (b). After
development, dry the plate in a current ofwarm air and heat at
110 for 10 minutes. While still hot, place the plate in a
chromatographic tame with an evaporating dish containing a
5 per cent w/v solution of potassium permanganate, add an
equal volume of hydrochloric acid and close the tank. Leave
the plate in the tank for 2 minutes, then place it in a current of
cold air until the excess of chlorine is removed and an area of
coating below the line of application gives only a very faint
blue colour with potassium iodide and starch solution; avoid
prolonged exposure to cold air. Spray the plate with potassium
iodide and starch solution and allow to stand for 5 minutes.
Reference solution (aJ. A 0.015 per cent w/v solution of

4ctoluenesulphonamide in acetone.
Reference solution (b). A mixture of equal volumes of the

test solution and reference solution (a).
Apply to the plate 5 III of each of test solution and reference

solution (a) and 10 III of reference solution (b). After
development, dry the plate in a current ofwarm air and heat at
110 for 10 minutes. While still hot, place the plate in a
chromatographic tame with an evaporating dish containing a
5 per cent w/v solution of potassium permanganate, add an
equal volume of hydrochloric acid and close the tame. Leave
the plate in the tame for 2 minutes, then place it in a current of
.Loss on drying (2.4.19). Not more than 0.5 per cent, determined
of 40 ml of ethanol (95 per cent) and 20 ml of water. Titrate
with 0.1 M sodium hydroxide usingphenolphthalein solution
as indicator.
Tests

8 volumes of methanol and 2 volumes of anhydrous formic
acid.
.
containing 0.5 g of Tolbutamide with 10 ml of acetone and
filter.
2237
TOLBUTAMIDE TABLETS
IP 2010
cold air until the excess of chlorine is removed and an area of

coating below the line of application gives only a very faint
blue colour with potassium iodide and starch solution; avoid
prolonged exposure to cold air. Spray the plate with potassium
iodide and starch solution and allow to stand for 5 minutes.
Description. A white or yellowish white powder.
Identification

Compare the spectrum with that obtained with tolnaftate RS
or with the reference spectrum of toInaftate.
B. Determine by thin-layer chromatography (2.4.17), as
The principal spot in the chromatogram obtained with test
Apparatus No.1,
solution (b) corresponds to that in the chromatogram obtained
Medium. 900 ml of a solution containing 2.04 per cent wlv of
disodium hydrogen phosphate and 0.135 per cent wlv of
C. Mix about 1mg with 0.5 mlofsulphuric acid. Add 0.05 ml of
potassium dihydrogen phosphate,
formaldehyde
solution. A greenish-blue colour develops.
D. Melting range (2.4.21).109 to 112.
the absorbance of the filtrate suitably diluted if necessary, at Tests
CizHISNz03S in the medium taking 417 as the specific Appearance ofsolution. A 5.0 per cent wIv solution in acetone
is clear (2.4.1) and not more intensely coloured than reference
ClzHISNz03S,
Assay. Weigh and powder 20 tablets. Weigh accurately a examination in 2 ml ofacetone.
quantity ofthe powder containing about 0.5 g ofTolbutamide,
add 50 ml of ethanol (95 per cent), previously neutralised to Testsolution (b). Dilute 0.5 ml oftest soh,ltion (a) to 10 ml with
phenolphthalein solution, warm to dissolve, add 25 ml of acetone.
water and titrate with 0.1 M sodium hydroxide using Reference solution (a). Dissolve 25 mg of toInaftate RS in 10
ml of acetone.
phenolphthalein solution as indicator.
CI2HISNz03S,
Reference solution (b). Dilute 1 ml oftest solution (b) to 10 ml

with acetone.
Reference solution (c). Dissolve 50 mg of{3-naphthol in 1 ml

of test solution (a) and dilute to 10 ml with acetone.
Tolnaftate
H3C

phase to rise 12 em. Dry the plate in current ofair and examine
under ultraviolet light at 254 nm. Any secondary spot in the
reference solution (b) (0.5 per cent). The test is not valid unless
the chromatogram obtained with reference solution (c) shows
vJtro
~
CH3
C I9H 17NOS
Mol.Wt. 307.4
Tolnaftate is O-naphthalen-2-yl methyl(3-methylphenyl)thiocarbamate.
TOlllliftlite. fOIltainsIl()t le.stJ:1aIl 9T0 IJe.r cent aIld ll()tIl1o~e.

than 103.0 per cent of C I9H 17NOS, calculated on the dried
basis.
Category. Antifungal.

Loss ondrying (2.4.19). Not more than 0.5 per cent, determined
()Ill ,0 g by drying in vacuUll1 at 60 aJ a pressure. not exceeding
Assay. Dissolve 50 mg in 250 rn1 ofmethanol. Dilute 2 rn1 ofthe
solution to 50 ml with methanol. Measure the absorbance at
the maximum at 257 nm (2.4.7).
2238
IP 2010
TOLNAFTATE TOPICAL POWDER
Calculate the content ofC 19H I7NOS taking 720 as the specific
absorbance at 257 run.
Usualstrength. 1 per cent w/w.
Identification

plate with silica GF254.
Tolnaftate Cream
Mobile phase. Toluene.
Tolnaftate Cream contains not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of tolnaftate,
C I9H17NOS.
Test solution. Evaporate 10 ml ofthe SolutionAon steam bath

to dryness and dissolve the residue in 1.0 ml of ethanol (95
per cent).
Reference solution. A 0.1 per cent w/v solution of tolnaftate
Identification

Test solution. Evaporate 10 ml ofthe SolutionAon steam bath
to dryness and dissolve the residue in 1.0 ml of ethanol (95
per cent).
Reference solution. A 0,1 per cent w/v solution of tolnaftate
Tests
Assay. Transfer an accurately weighed quantity of the cream
containing about 10 mg ofToInaftate to a separator containing
75 ml of chloroform. Wash the chloroform solution
successively with two 25 ml portions of 0.1 M hydrochloric
acid and 25 ml of water. Filter the chloroform layer through a
chloroform-washed cotton pledget into a 100-ml volumetric
flask. Add chloroform to volume and mix (Solution A). Dilute
5.0 ml of the solution with chloroform to 50 ml and mix.
maximum at about 258 run (2.4.7), using 0.001 per cent w/v
solution of tolnaftate RS in chloroform.
Calculate the content ofC ,9H 17NOS in the cream.

Tests
Other tests. Complies with the tests stated under Gels.
Assay. Transfer an accurately weighed quantity of the gel
containing about 10 mg ofToInaftate to a separator containing
75 ml of chloroform. Wash the chloroform solution
successively with two 25 ml portions of 0.1 M hydrochloric
acid and 25 ml of water. Dilute to 100 ml with chloroform and
mix (Solution A). Dilute 5.0 ml ofthe solution with chloroform
to 50 ml and mix. Measure the absorbance of the resulting
solution at the maximum at about 258 run (2.4.7), using 0.001
per cent w/v solution of tolnaftate RS in chloroform.
Calculate the content OfC I9H17NOS inthe gel.
Tolnaftate Topical Powder

Tolnaftate Topical Powder contains not .less than 90.0 per
C I9H 17NOS.
Identification
Tolnaftate Gel
Tolnaftate Gel contains not less than 90.0 per cent and not
more than 11 0.0 per cent of the stated amount of tolnaftate,
C I9H 17NOS.
Test solution. Evaporate the 5.0 ml portion of the methanol

solution reserved in the Assay, on a steam bath just to dryness
and dissolve the residue in 1.0 ml of alcohol.
2239
TOLNAFTATE TOPICAL POWDER
IP 2010


plate with silica gel GF25i
Tests
Test solution. Evaporate 25 ml of solution A on steam bath to

dryness and dissolve the residue in 1.0 ml of the ethanol (95
per cent).

progesterone in methanol.
Test solution. Transfer an accurately weighed quantity of the
topical powder containing about 5.0 mg oftolnaftate to a screwcapped, 50 ml centrifuge tube. Add 25 ml of methanol, place
the cap on the tube and rotate on a rotating device for 10
minutes, and centrifuge at about 2000 rpm for 5 minutes, filter
the supernatant and transfer 20 ml of the filtrate to 50-ml
volumetric flask, retaining the remaining portion ofthe filtrate
for identification test. Add 5.0 ml ofinternal standard solution
to the flask and dilute to volume with methanol and mix.
RS in methanol. To 20 ml ofthis solution add 5.0 ml ofinternal
standard solution and dilute to 50.0 ml with methanol.
octadecylsilane bonded to pbrous silica (10 Ilm),
and 1 volume of water,
resolution between the tolnaftate and internal standard peak
is not less than 3.0 and the relative standard deviation for
replicate injections is not more than 3.0 per cent. The relative
retention time of progesterone and tolnaftate are about 0.7
minute and 1.0 minute respectively.
Identification

Tests
Assay. Take an accurately measured volume containing about
10 mg ofToInaftate, add 50 ml of chloroform and extract with
50 ml of 0.1 M sodium hydroxide. Filter the chloroform layer
through a chloroform-washed cotton pledget into a 250- ml
volumetric flask and extract the aqueous layer with two 45 ml
portions of chloroform, filtering each into the flask. Add
chloroform to volume and mix (Solution A). Dilute 25 mlof
this solution with chloroform to 100 ml and mix. Measure the
258 urn (2.4.7), using 0.001 per cent w/v solution of tolnaftate
RS in chloroform.
Calculate the content ofC I9H 17NOS in topical solution.
0HH H3 C
CH3
y
~~NyCH3

~
Calculate the content ofC I9H 17NOS in the powder.
CH3
HOXCOOH
HOOC
OH

Mol Wt. 475.6
Tolterodine Tartrate is 2-[(1R)-3-[bis(l-methylethyl)amino]-1phenylpropyl]-4-methylphenol tartrate.
Tolnaftate Topical Solution

Tolnaftate Topical Solution contains not less than 90.0 per
tolnaftate, C I9 H 17NOS.
Tolterodine Tartrate contains not less than 98.0 per cent and
not more than 102.0 per cent ofC22H3INO,C4H606,calculated
on the dried basis.
2240
IP 2010
TOLTERODINE TARTRATE
Identification
Compare the spectrum with that obtained with tolterodine
tartrate RS or with the reference spectrum of t6/terodine
tartrate.
Tests
Specific optical rotation (2.4.22). +33 to +38, detennined in
1.0 per cent w/v solution in methanol.
(2.4.14).
Solvent mixture. 10 volumes ofmobile phase A and 10 volumes

ofmobile phase B.
examination in 10 ml of water and dilute to 50.0 ml with the
solvent mixture.
Reference solution (a). Dissolve about 10 mg of tolterodine
tartrate RS in 20 ml of water and dilute to 100.0 ml with the
solvent mixture. Dilute 5.0 ml ofthis solution to 100.0 ml with
Reference solution (b). A 0.03 per cent w/v solution oftartaric
acid in the solvent mixture.
as Inertsil),
mobile phase: A. a 0.05 M potassium dihydrogen
orthophosphate, adjusted to pH 3.5 with
B. acetonitrile,
below,
Time
Mobile Phase A
Mobile Phase B
(in min)
(per cent v/v)
(per cent v/v)
0-5
65
35
5-20
65~50
35
~50
20-40
50~30
50
~70
40-41
30~65
70~35
41-50
65
35
cent). Ignore the peak due to tartaric acid cOlTesponding to
solution (b).
Solvent mixture. 10 volumes ofmobile pqase A and 10 volumes

ofmobile phase B.
examination in 20 ml of water, sonicate and dilute to 100.0 ml
with the solvent mixture. Dilute 5.0 ml ofthis solution to 50.0
Reference solution (a). Dissolve about 50 mg of tolterodine
tartrate RS in 20.0 ml of water, sonicate and dilute to 100.0 ml
with the solvent mixture. Dilute 5.0 ml ofthis solution to 50.0
Reference solution (b). A 0.03per cent w/v solution oftartaric
acid in the solvent mixture.
as Inertsil),
mobile phase: A. a 0.05 M potassium dihydrogen
orthophosphate, adjusted to pH 3.5 with
B. acetonitrile,
below,
flow rate. 1 ml per minute, .
than 5.0 per cent.
2241
Time
(in min)
Mobile Phase A
(per cent v/v)
Mobile Phase B
(per cent v/v)
0-5
65
35
~50
35~50
20-21
50~65
50~35
21-25
65
35
5-20
65
TOPlRAMATE
IP 2010
Inject reference solution (a) and (b). The test is not valid
unless the theoretical plates of the principal peak is not less
than 2000 and the tailing factor is not more than 2.0. The
than 2.0 per cent.
Calculate the content ofCzzH31NO,C4H606.
octadecylsilane bonded to porous silica (5 pm),
refractive index detector,
detector cell temperature 50,
Inject the reference solution. Run the chromatogram for 40
minutes. The retention times of topiramate impurity A and
impurity B are about 2.9 and 4.5 minutes and the relative
retention times with respect to topiramate are 0.54 and 0.84
respectively. The test is not valid unless the resolution between
topiramate impurity A and topiramate impurity B is not less
than 3.
Topiramate
Mol. Wt. 339.4
Inject the test solution and the reference solution. For the
calculation ofcontent, multiply the peak areas ofthe following
impurities by the corresponding correction factor: topiramate
impurity A = 1.09; topiramate impurity B = 0.94
.
Topiramate is 2,3 :4,5-di-O-l-isopropylidene-~-D Not more than 0.15 percent each ofimpurity Aand impurity
fructopyranose sulphamate.
B, and not more than 0.1 per cent of any other impurity is
Topiramate contains not less than 98.0 per cent and not more found.
than 102.0 per cent ofClzHzlNOsS, calculated on the anhydrous Heavy metals (2.3.13). 1.0 g complies with the limit test for
basis.
Category. Anti-convulsant.
Dose. 25 mg daily.
Water (2.3.43). Not more than LOper cent, deterrninedon 1.0 g.
Identification
Test solution. Weigh accurately about 0.1 g of the substance

under examination and dissolve in 10 ml ofthe mobile phase.

Compare the spectrum with that obtained with topiramate RS
or with the reference spectrum of topiramate.
Reference solution. Weigh accurately ;1bout 0.1 g of topiramate

RS and dissolve in 10.0 ml of the mobile phase.
B. Melting range (2.4.21). 120 to 130.
Tests
Specific optical rotation (2.4.22). -28.0 to -36.0, detennined
in a 0.4 per cent w/v solution in methanol at 20.
(2.4.14).
.

under examination and dissolve in 10 ml of a mixture of 50
volumes of water and 50 volumes of acetonitrile.
Reference solution. Weigh accurately about 10 mg each of
(2;3;'4; 5-Bis-O-(l-methylethylidene)-beta-D-fructopyranose
RS (topiramate impurity A . RS) and2,3-0-(lmethylethylidene)-beta-D-fructopyranose sulphamate RS
(topiramate impurity B RS) and dissolve in sufficient mobile
phase to produce 10.0 ml.
column temperature. 50
refractive index detector (Detector cell temperature: 50)
Inject the reference solution. The relative standard deviation
Ca.lculate the coIiteIit ofClzHzlNOsS
exceeding 30.
2242
IP 2010
TOPOTECAN HYDROCHLORIDE

Topiramate Tablets
Topiramate Tablets contain not less than 90.0 per cent and
topiramate, C12H21NOsS.
Identification
A. Weigh a quantity ofthe powdered tablets containing 1 g of
Topiramate, disperse in 30 ml of dichloromethane, centrifuge
at 2000 rpm for 10 minutes and filter. Evaporate the filtrate to
dryness. The residue complies with the following test.
Compare the spectrum with that obtained with topiramate RS
or with the reference spectrum oftopiramate.
Tests
(2.4.14).
Test solution. To a quantity ofthe powdered tablets containing

100 mg ofTopiramate, add sufficient mobile phase to produce
100 ml, mix and centrifuge. Use the supernatant liquid.
Reference solution. A 0.1 per cent w/v solution of topiramine
as Inertsil ODS 3),
.
- flow rate. 1 ml perminute,
- refractive index detector (Detector cell temperature 50),

chromatographic conditions described in the test for Related
substances.
dissolution medium.
of topiramate RS in the dissolution medium and dilute with
the dissolution medium to obtain a solution having a lchown
concentration similar to the expected concentration of the
test solution.

C12H21NOsS.
Testsolution. Weigh and powder 20 tablets. Weigh accurately
a quantity of the powder containing. about! OOmg of
Topiramate, disperse in 100.0 ml with the mobile phase and
centrifuge. Use the supernatant liquid.
Reference solution. A 0.1 per cent w/v solution of topiramate

Calculate the content OfC12H21NOsS in the tablets.
exceeding 30.

for the replicate injections is not more than 2.0 per cent. The
retention time oftopiramate is about 5 minutes. The test is not
valid unless the column efficiency is not less than 3000
theoretical plates and the tailing factor is notmore than 2.0
Inject the test solution. Determine the amount of related
substances by the area normalization method. The content of
any individual impurity is not more than 1.0 per cent and the
sum of all impurities is not more than 2.0 per cent.
Apparatus No. 1,
HO
, Hel
o
C23H23N30s.HCI
Mol.Wt. 457.9
Topotecan Hydrochloride is (48)-10-[(dimethylamino) methyl]4-ethyl-4,9-dihydroxy-lH-pyrano[3' ,4' :6,7]indolizino[1 ,2b]quinoline-3,14(4H, 12H)-dione hydrochloride.

2243
TOPOTECAN HYDROCHLORIDE
IP 2010
Topotecan Hydrochloride contains not less than 98.0 per cent

and not more than 102.0 per cent ofC23H23N30s,HCl, calculated
Description. A light yellow to greenish yellow powder.
CAUTION - Topotecan Hydrochloride is cytotoxic; extra

care is required to prevent inhaling particles and exposing
the skin to it.
Identification
Compare the spectrum with that obtained with topotecan
hydrochloride RS or with the reference spectrum oftopotecan
hydrochloride.
camptothecin is about 1.87 and 2.62 with respect to topotecan

hydrochloride and the relative response factors for 10-hydroxy
camptothecin and camptothecin are about 0.79 and 0.85 with
respect to topotecan hydrochloride.
chromatogram obtained with the t~st solution, the area of any
(b) (0.15 per cent) and the sum ofthe areas ofall the secondary
Water (2.3.43). Not more than 13.0 per cent, detennined on 0.1 g.
per mg of topotecan hydrochloride.
Specific optical rotation (2.4.22). + 30.0 to + 38.0, detennined

Microbial contamination (2.2.9). Total viable aerobic count,

not more than 100 cfu per g. It also meets the requirements of
the tests for the absence of Staphylococcus aureus,
Pseudomonas aeruginosa, Salmonella species, and
Escherichia coli.
Total chloride. 7.6 per cent to 8.1 per cent.
Weigh accurately about 0.5 g, dissolve in 10 ml of methanol,

add 20 ml of water and 20 ml of glacial acetic acid. Titrate
with 0.1 M silver nitrate solution using eosin yellow solution
as indicator. Colour changes from orange to dark pink.
Solvent mixtur~. A mixture of30 volumes of acetonitrile and

70 volumes of a buffer solution, prepared by diluting 1 ml of
trifluoroacetic acid to 1000 ml with water.
Tests
1 ml of 0.1 Msilver nitrate is equivalent to 0.003545 g ofCI.


(2.4.14).
Referencesolution. A 0.04 per cent w/v solution of topatecan

hydrochloride RS in the solvent mixture.
Solvent mixture. A mixture of 30 volumes of acetonitrile and

70 volumes of a buffer solution prepared by diluting 1 ml of
trifluoroacetic acid in 1000 ml of water.
mobile phase: A. a solution of 1 ml of trifluoroacetic
acid in 1000 ml of water,
B. acetonitrile,
below,
Time
Mobile phase A
Mobile phase B
(in min.)
(per cent v/v)
(per cent v/v)
85
15
28
70
30
30
38
70
43
85
15
48
85
15

topotecan hydrochloride RS in the solvent mixture.
Reference solution (b). Dilute 1 ml of the reference solution
(a) to 100 ml with the solvent mixture.
w/v each of 10-hydroxy comptothecin RS and camptothecin
RS inN,N'-dimethylformamide. Add 1.0 ml ofthis solution to
a 0.04 per cent w/v solution of topotecan hydrochloride RS
-in the solvent mixture.
relative retention time for the 10-hydroxy camptothecin and
2244
IP 2010
TRAMADOL HYDROCHLORIDE

22 volumes of acetonitrile and 1 volume of
1 M hydrochloric acid,
than 2.0 per cent.
Calculate the content ofC23H23N30s,HCI.
2 to 8.
relative standard deviation is not more than 2.0 per cent.
Calculate the content ofC23H23N30s.
Topotecan Injection

exceeding 2 to 8.
Topotecan Hydrochloride Injection

Topotecan Injection is a sterile, stabilised solution of
Topotecan Hydrochloride in Water for Injection.
Topotecan Injection contains not less than 90.0 per cent and
not more than 110.0 per cent of the stated amount of the
topotecan, C23H23N30S.
Tramadol Hydrochloride
Usual strength. 1 mg per mi.

Description. A clear, light yellow solution.
Hel
Identification
B. It gives the reaction of chlorides (2.3.1).
Mol. Wt. 299.8
Tests
Tramadol Hyrochloride is (lRS,2RS)-2-[(dimethylamino)methyl]-I-(3-methoxyphenyl)cyclohexanol hydrochloride.
pH (2.4.24). 2.5 to 3.5.

Unit per mg of topotecan.
Tramadol Hydrochloride contains not less than 99.0 per cent

and not more than 101.0 per cent of C16H26ClN02, calculated
Category. Analgesic.
Identification
Test solution. Accurately measure the volume of injection

containing 2 mg ofTopotecan, dilute to 50.0 ml with mobile
phase.
Test B may be omitted if tests A and C are carried out. Test A

may be omitted if tests Band C are carried out.
Reference solution. Dissolve 10 mg of topotecan

hydrochloride RS in 5 ml of water and dilute to 25.0 ml with
mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with
mobile phase.

Compare the spectrum with that obtained with tramadol
hydrochloride RS or with the reference spectrum oftramadoI
hydrochloride.
octadecylsilane bonded to poro.us silica
(5 11m),
B. In the test for impurity E, the principal spot in the

the principal spot in the chromatogram obtained with reference
solution (a).
2245
IP 2010
TRAMADOL HYDROCHLORIDE
C. Gives reaction (a) ofchlorides (2.3.1).
Reference solution (b). Dissolve 5 mg of tramadol impurity A

RS in 4.0 m1 ofthe test solution and dilute to 100.0 ml with the
mobile phase.
Tests
Appearance of solution. A 5 per cent w/v solutionis clear
(2.4.1) and colourless (2.4.1).
Acidity. To 10 ml of 5 per cent w/v solution, add 0.2 ml of
methyl red solution and 0.2 ml of 0.01 M hydrochloric acid.
The solution is red. Not more than O.4ml of 0.01 M sodium
hydroxide is required to change the colour of the indicator to
yellow.
Specific optical rotation (2.4.22). - 0.1 00 to + 0.1 00 , determined
on 5.0 per cent w/v solution.
Impurity E. Determine by thin-layer chromatography (2.4.17),
coating the plate with silica gel GF254.
Mobile phase. A mixture of 1 volume of concentrated

ammonia, 19 volumes of 2.,.propanol and 80 volu!iles \If
toluene.
Test solution (a). Dissolve 0.1 g of the substances under
methanol.
tramadol hydrochloride RS in methanol.
Reference solution (b). A 0.1 per cent w/v solution of (2RS)2-[(dimethylamino)methyl]cyclohexanone RS (tramadol
impurity E RS) in methanol. Dilute 1 ml ofthis solution to 10
ml with methanol.
(1 RS,2SR)-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)
cyclohexanol RS (tramadol impurity A RS) in reference
solution (a).
Saturate the plate for 20 minutes with concentrated ammonia.
Apply to the plate 10 J.tl of each solution. Allow the mobile
phase to rise 15 cm. Dry the plate in air, expose the plate to
iodine vapour for 1 hour, and examine in ultraviolet light at 254
mn. The chromatogram obtained with reference solution (c)
shows 2 clearly separated spots. In the chromatogram obtained
with test solution (a), any secondary spot corresponding to
tramadol impurity E is not more intense than the spot in the
cent).
(2.4.14).
- a stainless steel column 25 cm ?I- 4.0 rmn packed with
endcapped octylsilane bonded to porous silica (5 J.tm),
- mobile phase: a mixture of29.5 volumes of acetonitrile
and 70.5 volumes ofa mixture of0.2 m1 of trifluoroacetic
acid and 100 ml of water,
- flow rate. 1 m1 per minute,
- injection volume. 20 J.tl.
resolution between the peaks due to tramadol impurity A and
tramadol is not less than 2.0. The relative retention time with
reference to tramadol fortramadol impurity Ais about 0.85.
IyUect the test solution and reference solution (a). Run the
of the peak due to tramadol impurity A is not more than the
reference solution (a) (0.2 per cent), the area of any other
solution (a) (0.1 per cent). The sum of all the secondary peaks
of the principal peak in the chromatogram obtaiiled with
Assay. Dissolve 0.18 gin 25 ml of anhydrous acetic acid and
add 10 ml of acetic anhydride. Titrate with 0.1 M perchloric
C16Hz6ClNOz.
Tramadol Capsules

examination in 1OO.Omlofthe mobile phase.
Tramadol Hydrochloride Capsules
Reference solution (a). Dilute 2.0ml of the test solution to

10.0 m1 with the mobile phase. Dilute 1.0 m1 ofthis solution to
Tramadol Capsules contain not less than 95.0 per cent and
not more than 105.0 per cent ofthe stated amount oftramado1
hydrochloride, CI6HzsNOz,HCl.
2246
IP 2010
TRANDOLAPRlL
Identification
Mobile phase. A mixture of 1 volume of anhydrous formic

acid, 50 volumes of acetone and 50 volumes of methanol.
Test solution. Disperse the contents of capsules containing
about 50 mg ofTramadol Hydrochloride in 25 ml of methanol
and filter through a glass fiber filter (Such as Whatmann OF/
A).
Reference solution. A 0.2 per cent w/v solution of tramadol
dry the plate in air and expose to iodine vapour until spots
appear and examine in daylight. The principal spot in the
spot obtained with the reference solution.
tramadol hydrochloride is not less than 3.0.
chromatogram four times the retention time of the principal
area of peak corresponding to tramadol impurity Ais not more
secondary peak other than tramadol impurity A is not more
obtained with reference solution (a) (0.2 per cent) and the sum
of the areas of all other secondary peaks is not more than 5
times the area of the principal peak in the chromatogram
.obtained with reference solution (a) (1.0 per cent).


Test solution. Dissolve a quantity of the mixed contents of20

capsules containing about 50 mg ofTramadol Hydrochloride
in 150 ml of the mobile phase with the aid of ultrasound and
dilute to 200 ml with the mobile phase, filter.
Tests

tramadol hydrochloride RS in the mobile phase.

(2.4.14).
Test solution. Disperse the contents of capsules containing

about 0.5 g ofTramadol Hydrochloride in 80 ml ofthe mobile
phase with the aid ofultrasound and dilute to 100 ml with the
mobile phase, filter through a glass fiber filter (Such as
Whatmann OF/A).
ml with the mobile phase. Dilute 1 ml ofthis solution to 10 ml
(1RS, 2SR)- 2-[(dimethylamino)methyl]-1-(3 -methoxyphenyl)
cyclohexanol RS ( tramadol impurity A RS) in the mobile
phase.
w/v each of tramadol hydrochloride RS and tramadol
.
impurity A RS in the mobile phase.
- a stainless steel column 25 cm x 4.6 mrn packed with
- mobile phase: a mixture of I volume of trifluoroacetic
acid, 30 volumes of acetonitrile and 69 volumes of
water,
- spectrophotometer set at 271 11ill,

w/v each of tramadol hydrochloride RS and tramadol
impurity A RS in the mobile phase.
substances.
tramadol hydrochloride is not less than 3.0.
Calculate the content ofC,6H2sN02,HCl in the Capsules.
Trandolapril
C24H34N20s
Mol. Wt. 430.5
Trandolapril is (2S,3aR,7as)-1 [(5)-2-[[(8)-1-(ethoxycarbonyl)3-phenylpropyl]amino]-1-oxo-propyl]octahydro-1H-indole-2carboxylic acid.
2247
TRANDOLAPRlL
IP 2010
Trandolapril contains not less than 98.5 per cent and not more
than 101.5 per cent ofC24H34N20s, calculated on the dried basis.
on 1.0 g by drying at 50 for 3 hours under vacuum.
Description. A white to offwhite crystalline powder.

Identification
Compare the spectrum with that obtained with trandolapril
RS or with the reference spectrum oftrandolapril.
Tests
Specific optical rotation (2.4.22). -16.so to -18.5, determined
on 2.0 per cent w/v solution in ethanol (95 per cent).
(2.4.14).

trandolapril RS in the mobile phase.
- a stainless steel column 25 cm x 4.6 mID, packed with
octylsilane bonded to porous silica (5 !-lm),
prepared by dissolving 6.71 g of monobasic potassium
orthophosphate in 1000 ml of water, 35 volumes of
acetonitrile and 5 volumes of methanol, adjusted to
-

octadecylsilane bonded to porous silica (5 !-lm) (Such
as Inertsil CI8),
orthophosphate in 1000 ml of water, 43 volumes of
acetonitrile and 5 volumes of methanol, adjusted to
injection volume. 20 !-ll.
Trandolapril Tablets contain not less than 90.0 per cent and
not more than 110.0 per cent of the stated amounts of
trandolapril, C24H34N20s.
injection volume. 20 !-ll.

relative standard deviation for replicates injections is not more
than 5.0 per cent.
Inject the reference solution and the test solution. In the
heavy metals, Method B(20 ppm).

Identification
B. When examined in the range of200 nm to 400 nm (2.4.7), a

an absorption maximum as obtained with trandolapril RS of
the same concentration.
Tests
Apparatus No.1,
2248
TRANDOLAPRIL TABLETS
IP 2010

the chromatogram obtained with the reference solution (2.0

per cent).

dissolution medium.

Tablets.

of trandolapril RS in the mobile phase and dilute with
dissolution medium to obtain a solution having a known
Detennine by liquid chromatography (2.4.14), as described

under Assay with the following solutions.
Use chromatographic system as described under Assay using

100 III injection volume.
Test solution. Disperse one tablet in the mobile phase, dilute

to obtain a solution containing 0.005 per cent w/v oftrandolapril
in the mobile phase and filter.
than 2.0 per cent.

of trandolapril RS in the mobile phase and dilute to obtain a
solution having a known concentration similar to the test
solution.
Calculate the content of CZ4H34NzOs in the tablet.


0.5 g.
CZ4H34NzOs.

(2.4.14).
NOTE-Use fi-eshly prepared solutions.
Test solution. Weigh and disperse 10 intact tablets in 100.0 ml

with the mobile phase, filter. Dilute 5.0 ml of this solution to

containing about 10 mg ofTrandolapril in 10.0 ml ofthe mobile
phase.
octylsilane bonded to porous silica (5 11m) (Such as
ACEC8),
orthophosphate anhydrous in 1000 ml of wate]; 35
volumes of acetonitrile and 5 volumes of methanol,
adjusted to pH 4.0 with orthophosphoric acid,
for replicate injections is not more than 5.0 per cent. .
peaks is not more than twice the area ofthe principal peak in

as Inertsil ODS~3),
orthophosphate anhydrous in 1000 ml of water, 43
volumes of acetonitrile and 5 volumes of methanol,
adjusted to pH 4.0 with orthophosphoric acid,
theoretical plates of the principal peak is not less than 2000,
the tailing factor is not more than 2.0 and the relative standard
Calculate the content of CZ4H34NzOs in the tablet.
Labelling. The label states the strength in terms ofthe amount
of Trandolapril.
2249
TRAVOPROST
IP 2010
(0.5 per cellt). The sum ofthe areas of all the secondary peaks
is not more than 12 times the area ofthe principal peak in the
cent).
"
Travoprost
Water (2.3.43). Not more than LO per cent, determined on

0.1 g.
Mol. Wt. 500.6

examination in acetonitrile and dilute to 100.0 m1 with water.
Travoprost is (52)-7-[(lR,2R,3R,5S)- 3,5-dihydroxy-2-[(lE,3R)3-hydroxy-4-[3-(trifluromethyl)phenoxy]-1-buteny1]cyclopentyl]-5-heptenoic acid 1-methylethyl ester.
Reference solution. Dissolve 10 mg of travoprost RS in

acetonitrile and dilute to 10.0 m1 with watel:
Travoprost contains not less than 96.0 per cent and not more
than 102.0 per cent ofC26H3sF306, calculated on the anhydrous
basis.
Category. Antiglaucoma.
Description. A colourless to yellowish oil.
Identification
Compare the spectrum with that obtained with travoprost RS
or with the reference spectrum of travoprost.
Tests
octadecylsilane bonded to porous silica (5 Ilm), (Such
as Hypersil ODS),
solution prepared by diluting 1 ml of orthophosphoric
acidto 1000 m1 with water, adjusted to pH 5.0 with 1 M
sodium hydroxide, 30 volumes of acetonitrile and 4
volumes of propanol-2-ol,
B. a mixture of80 volumes of acetonitrile,
4 volumes ofpropanol-2-o1 and 16 volumes ofwatel;
below,
Mobile phase B
(per cent V N)
Appearance of solution. A 1.0 per cent w/v solution in ethanol

(95 per cent) is not more intensely coloured than reference
Time
(in min)
Mobile phase A
(per cent v/v)
100
Specific optical rotation (2.4.22). +52 to +58, determined on

2.0 per cent. w/v solution in ethanol (95 per cent) at20.
5-50
80
20
50-70
. 70-80
80--70
0
20--7100
80-82
0--7100
100--70
82-90
100
Related substances. Not more than 3.5 per cent of5,6-transtravoprost.


examination in acetonitrile and dilute to 100.0 ml with water.
Reference solution. Dissolve 10 mg of travoprost RS in
acetonitrile and dilute to 100.0 rn1 with watel: Dilute 1.0 ml of
the solution to 20.0 ml with watel:
100
resolution between the peaks due to travoprost and 5-transtravoprost is not less than 1.5, the theoretical plates is not
than 2.0 per cent.
Inject thereferences6Iiiti6n. The reliitiveretenli6iifiilleWith

reference t6 travopr6st f6r 5;6;trans:trav6pmstis ab6ut L06:

Storage. Store protected from moisture, at a temperature

between 2 and 8.
Calculate the content ofC26H3sF306"
2250
TRAVOPROST EYE DROPS
IP 2010
Travoprost Eye drops

Travoprost Eye Drops contains not less than 90.0 per cent
travoprost, C26H3sF306.

the chromatogram obtained with the reference solution (6.0
per cent).
Drops.
Identification
peak inthe chromatogram obtained with the reference solution.
Solvent mixture. Equal volumes of water and acetonitrile.

Test solution. Dilute a suitable volume of eye drops containing

0.004 per cent w/v oftravoprost in the solvent mixture.
Mobile phase. A mixture of 50 volumes of benzene and 40

volumes of dioxane.
Reference solution. Dissolve 20 mg of travoprost RS with

10.0 ml of acetonitrile, sonicate and dilute to 20.0 ml with
water. Dilute 1.0 ml ofthis solution to 25.0 ml with water.
Test solution. Extract eye drops containing about 0.8 mg of

Travoprost with 20.0 ml of chloroform in a separating funnel.
Collect the chloroform layer in a 250 ml beaker. Give three
washings with chloroform. Evaporate the chloroform layer to
dryness and add 3.0 ml of methanol.
Reference solution. Dissolve 20 mg of travoprost RS in 10 ml
of acetonitrile, sonicate and dilute to 20.0 ml with water. Dilute
5.0 ml ofthis solution to 20.0 ml with methanol.
Apply to the plate 20 !J.l of each solution. Allow the mobile
phase to rise 8 cm. Dry the plate and spray with 10 per cent
w/v solution of phosphomolybdic acid in ethanol, heat the
plate at 1l0 for 15 minutes and examine immediately. The
solution corresponds to the spot obtained with the reference
solution.
Tests
octadecy1silane bonded to porous silica (5 !J.m) (Such
as Hypersil ODS),
sample temperature. 5,
mobile phase: A. a mixture oBO volumes of acetonitrile,
4 volumes of propan-2-o1 and 66 volumes of buffer
solution prepared by diluting 1.0 ml of orthophosphoric
acid to 1000.0 ml with water, adjusted to pH 5.0 with
1 M sodium hydroxide and filter,
B. a mixture of80 volmnes of acetonitrile,
4 volumes ofpropan-2-o1 and 16 volumes of water,
flow rate. Iml per minute,
injection volume. 100 !J.l.
pH (2.4.24). 5.7 to 6.3.

(2.4.14).
Test solution. Dilute the eye drops to obtain a solution

containing 0.004 per cent w/v oftravoprost.
Reference solution. Dissolve 20 mg of travoprost RS in 10.0
ml of acetonitrile, sonicate and dilute to 20.0 ml with water.
Dilute 1.0 m1 of this solution to 25.0 m1 with water. Further,
dilute 1.0 ml ofthis solution to 100.0 ml with water.
.secondary peak is not more than 4 times the area ofthe principal
Time
(in mill)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
0- 5
100-780
0-720
5-50
80
20
50-70
80-70
20-7100
70-80
100
80 -85
0-7100
100-70
Calculate the content ofC26H3sF306.
2251
TRIAMCINOLONE
IP 2010
Triamcinolone
-
o
Mol. Wt. 394.4
Triamcinolone is 9cx-fluoro-ll {3, 16~ 17 ~21-tetrahydroxy
pregna-l,4-diene-3,20-dione.
Triamcinolone contains not less than 97.0 per cent and not
more than 103.0 per cent ofCz1Hz7F06, calculated on the dried
basis.
Category. Corticosteroid.
Identification
Compare the spectrum with that obtained with triamcinolone
RS or with the reference spectrum of triamcinolone.
B. When examined in the range 220 nm to 360 nrn (2.4.7), a
maximum at about 238 mn; absorbance at about 238 nrn, about
0.76.
C. Dissolve 1 mg in 6 ml of ethanol (95 per cent), add 5 ml of
a 1 per cent w/v solution of butylated hydroxytoluene in
ethanol (95 per cent) and 5 ml of 1 M sodium hydroxide and
heat on a water-bath under a reflux condenser for 20 minutes;
a pinkish lavender colour is produced.
Tests
in a 1.0 per cent w/v solution in dimethylformamide.
base-deactivated end-capped octadecylsilane bonded

to porous silica (5 /-lm),
mobile phase: a mixture prepared by mixing 525 ml of
methanol with 400 ml of water, allowing to equilibrate,
adjusting the volume to 1000.0 ml with water and mixing
again,
spectrophotometer set at 238 nrn,
Inject the test solution and the reference solution. Continue

the chromatography for 4.5 times the retention time of
triamcinolone (about 11 minutes).
of any peak other than the principal peak is not more than the
the reference solution (l per cent); not more than two such
peaks have an area greater than half the area of the principal
(0.5 per cent); the sum of the areas of all the peaks other than
the principal peak is not more than twice the area ofthe principal
(2 per cent). Ignore any peak with an area less than 0.05 times
with the reference solution (0.05 per cent).
ethanol (95 per cent) to produce 100.0 ml and mix. Dilute
2.0 ml to 50.0 ml with ethanol (95 per cent) and measure the
238 nrn (2.4.7).
Calculate the content ofCzIHz7F06 taking 380 as the specific

(2.4.14).
Prepare the following solutions immediately before use and

protect fi'OIn light.
examination in a mixture of equal volumes of methanol and
water and dilute to 10 ml with the same solvent mixture.
Reje,;ence solutio'i. DilliteTinCoffhefesfs-ollifion fo~rOO iiil
Triamcinolone Tablets contain not less than 90.0 per cent and
triamcinolone, CZIHz7F06.
Q!!ahtrengths.. 2_mg;A_mg;BJJJ.g.__ .
with a mixture of equal voluIlles of methanol and water.
Identification
- a stainless steel column 25 cm x 4.6mm, packed with
A. Dissolve 1 mg ofthe residue obtained in the test for Related

substances in 6 ml of ethanol (95 percent), add 5 ml ofa 1 per
2252
IP 2010
TRIAMCINOLONE ACETONIDE
cent w/v solution of butylated hydroxytoluene in ethanol

(95 per cent) and 5 ml of 1 M sodium hydroxide and heat on
a water-bath under a reflux condenser for 20 minutes; a pinkish
lavender colour is produced.

Tablets.
B. In the Assay, the chromatogram obtained with the principal

peak obtained with the test solution corresponds to the peak
due to h'iamcinolone in the chromatogram obtained with
Crush one tablet to a fine powder, add 100 ml of ethanol

(95 per cent) and shake for 10 minutes. Filter, dilute 2.0 ml of
the filtrate with sufficient ethanol (95 per cent) to produce a
solution containing 0.002 per cent w/v of Triamcinolone and
maximum at about 238 urn (2.4.7).
Tests
Calculate the content of CZIHz7F06 in the tablet taking 380 as


(2.4.14).

containing 15 mg of Triamcinolone with 15 ml of ethanol for
15 minutes, filter under reduced pressure through a fine filter
paper (such as Whahnan No 42) and evaporate the filtrate to
dryness using a rotary evaporator. Reserve 1 mg ofthe residue
for Identification testA. Dissolve the remainder ofthe residue
Reference splution (b). Dilute 5 ml ofthe test solution to 50 ml
with methanol. Dilute 5 ml of the resulting solution to 50 ml

a quantity ofthe powder containing 2.5 mg ofTriamcinolone,
shake with 5 ml of methanol and 20 ml ofa mixture of5 volumes
of methanol and 3 volumes of water. Shake for 15 minutes, mix
with the aid of ulh'asound for 10 minutes, centrifuge and use
the supernatant liquid.
Reference solution (a). Prepare in the same manner as the test
solution but add 5 ml of a 0.06 per cent w/v solution of
testosterone (internal standard) in methanol (solution A) in
place of the 5 mlofmethanol.
Reference solution (b). Add 5 ml of solution A to 5 ml of a
0.08 per cent w/v solution of triamcinolone RS in methanol
and add 15 ml of methanol (50 per cent).
mobile phase: a mixture of methanol and water, adjusted
so that the retention time of triamcinolone is about
5 minutes (approximately equal volumes of methanol
and water),
30 volumes of water and 0.1 volume of glacial acetic
acid,
Inject reference solution (b) and record the chromatogram for

4 times the retention time of the triamcinolone peak.
The test is not valid unless the column efficiency detennined

from the principal peak in the chromatogram obtained with
reference solution (a) is at least 10,000 theoretical plates per
metre.
of any secondary peak is not more than twice the area of the
solution (b) (2 per cent) and not more than one such peak has
an area more than that ofthe principal peak in the chromatogram
obtained with reference solution (b) (1 per cent). The sum of
the areas of any such peaks is not more than the area of the
solution (a) (4 per cent).
Calculate the content ofCzIHz7F06 in the tablets.
Triamcinolone Acetonide
o
CZJf31F06
Mol. Wt. 434.5
Triamcinolone Acetonide is 9a- fluoro-I1 ~,21-dihydroxy
16a,17a-isopropylidenedioxy-l ,4-pregnadiene-3,20-dione.
2253
TRIAMCINOLONE ACETONIDE
IF 2010
Triamcinolone Acetonide contains not less than 96.0 per cent

and not more than 104.0 per cent ofCz4H3IF06, calculated on
examination and again heat in a naked flame until white fumes

appear; the solution does not wet the sides of the tube and
does not pour easily from the tube.
Category. Corticosteroid.
Tests
Dose. By deep intramuscular injection, 40 mg to 100 mg.

Identification
Compare the spectrum with that obtained with triamcinolone
acetonide RS.

56 volumes of chlor(}form and 29 volumes of toluene.
Reference solution (a). Dissolve 25 mg of triamcinolone
acetonide RS in 10 ml ofthe solvent mixture.
Place the dry plate in a tan1e containing a shallow layer of the
top, remove the plate from the tan1e and allow the solvent to
was done.
Apply to the plate 5 /-ll of each solution. Allow the mobile
the hot plate with ethanolic sulphuric acid (20 per cent vlv).
in daylight and in ultraviolet light at 365 mn. The principal
compact spot.
e. Heat 0.5 ml of chromic-sulphuric acid in a test-tube (5 cm x

about 6 mm) in a naked flame until white fumes are evolved;
the solution wets the sides of the tube readily and there is no
greasiness. Add about 2 mg of the substance under
Light absorption (2.4.7). When examined in the range 230 run

to 360 mn, a 0.001 per cent w/v solution in ethanol (95 per
cent) shows an absorption maximum at about 239 nm;
absorbance at about 239 nm, 0.34 to 0.37.
Specific optical rotation (2,4,22). +100 to +107, determined
(2.4.14).
NOTE - CarlY out the test protectedii"om light.

examination in 7 ml of methanol and dilute to 10 ml with water.
acetonide RS and 2 mg of triamcinolone in the mobile phase
and dilute to 100 ml with the mobile phase.
octadecylsilane bonded to porous silica (5 /-lm),
mobile phase: a mixture prepared by mixing 525 ml of
methanol with 400 ml of water, allowing to equilibrate,
adjusting the volume to 1000.0 ml with water and mixing
again,
Equilibrate the column with the mobile phase for about
10 minutes.
Inject reference solution (b). Adjust the sensitivity of the
system so that the height of the principal peak is at least
50 per cent ofthe full scale of the recorder.
Inject reference solution (a). The retention times are;
triamcinolone, about 5 minutes and triamcinolone acetonide
about 17 minutes. The test is not valid unless the resolution
between the peaks corresponding to triamcinolone and
triamcinolone acetonide is at least 15; if necessary, adjust the
concentration of methanol in the mobile phase.
Inject the test solution and reference solution (b). Continue
3.5 timesJhe retentiontimeofthe
principal peak in the chromatogram obtained with the test
solution. In the chromatogram obtained with the test solution
the area of any peak other than the principal peak is not more
tbe~bromJ:ltogmpby for
2254
IP 20ID
TRIAMCINOLONE ACETONIDE INJECTION

(0.25 per cent); the sum ofthe areas ofall the peaks other than
the principal peak is not greater than half the area of the
solution (b) (0.5 per cent). Ignore any peak due to the solvent
and any peak with an area less than 0.05 times the area ofthe
solution (b).
Water (2.3.43). Not more than 2.0 per cent, detennined on 0.2 g.
ethanol to produce 100.0 ml and mix. Dilute 5.0 ml to 100.0 ml
with ethanol (95 per cent) and measure the absorbance of
the resulting solution at the maximum at about 239 nm (2.4.7).
Calculate the content ofC24H31F06 taking 354 as the specific
Triamcinolone Acetonide Injection

Triamcinolone Acetonide Injection is a sterile suspension of
Triamcinolone Acetonide in very fine particles in Water for
Injections containing suitable dispersing agents.
Triamcinolone Acetonide Injection contains not less than
amount oftriamcinolone acetonide, C24H31F06.
Identification
Extract a volume containing about 50 mg of Triamcinolone
Acetonide with two quantities, each of 10 ml, ofperoxide-free
ether and discard the ether extracts. Filter the aqueous layer
through a sintered-glass filter, wash the residue with four
quantities, each of5 ml ofwater and dry at 105 for 1 hour. The
residue complies with the following tests.

Compare the spectnim with that obtained with triamcinolone
acetonide RS or with the reference spectrum oftriamcinolone
acetonide.

acetonide RS in 10 ml ofthe solvent mixture.
Place the dry plate in a tanle containing a shallow layer of the
was done.
phase to lise 12 cm. Dry the plate in a current ofwarm air, allow
compact spot.
e. Heat 0.5 ml of chromic-sulphuric acid in a test-tube (5 cm x

about 6 mm) in a naked flame until white fumes are evolved;
the solution wets the sides of the tube readily and there is no
greasiness. Add about 2 mg of the substance under
examination and again heat in a naked flame until white fumes
appear; the solution does not wet the sides of the tube and
does not pour easily from the tube.
Tests
pH (2.4.24).5.0 to 7.5.
Test solution. Mix an accurately measured volume of the

injection, diluted with methanol, with sufficient of a solution
ofprednisolone RS (internal standard) with methanol to obtain
a final concentration of 0.02 per cent w/v of triamcinolone
acetonide and 0.01 per cent w/v of prednisolone, centrifuge


w/v of triamcinolone acetonide RS and 0.01 per cent w/v of
prednisolone RS in methanol.

56 volumes of chloroform and 29 volumes of toluene.
Reference solution (b). Prepare in the same manner as test

solution but omitting the internal standard.
2255
TRIAMTERENE
IP 2010
Adjust the flow rate of the mobile phase such that the
separation ofthe triamcinolone acetonide and internal standard
is optimised with a retention time of about 15 minutes for
triamcinolone acetonide.

solution.

10 volumes of 18 M ammonia and 10 volumes of methanol.
Test solution. Dissolve 0.1 g of the substance under examination in 20 ml of dimethyl sulphoxide and dilute 2 ml of the
resulting solution to 50 ml with methanol.
detectable and examine in ultraviolet light at 365 urn. Any
Calculate the content of CZ4H31F06 in the injection.

Triamterene

Triamterene is 2,4,7-triamino-6-phenylpteridine.

anhydrous formic acid, add 100 ml of anhydrous glacial
acetic acid. Titrate with 0.1 M perchloric acid, determining
titration.
Triamterene contains not less than 99.0 per cent and not more
than 101.0 per cent ofC 1ZH lI N 7, calculated on the dried basis.

ClzHIIN7.
Category. Potassium sparing diuretic.
Mol. Wt. 253.3

Description. A yellow, crystalline powder; odourless.
Triamterene Capsules
Identification
A. When examined in the range 250 mn to 380 nm (2.4.6), a
0.001 per cent w/v solution in a mixture of9 volumes of ethanol
(95 per cent) and 1 volume of 1 M hydrochloric acid shows
absorption maxima at about 262 urn and 360 urn, and a shoulder
at about 285 nm.
Triamterene Capsules contain not less than 95.0 per cent and
triamterene, ClzHIIN7'
Identification
B. A 0.1 per cent w/v solution in anhydrousformic acid, when

examined in ultraviolet light at 365 nm shows an intense blue
fluorescence. Solutions in other acids also exhibit a blue
fluorescence.
The final solution obtained in the Assay has a bluish

fluorescence and when examined in the range 250 nm to
380 nm (2.4.7), shows an absorption maximum at about
360nm.
Tests
Tests
Acidity. Boil 1.0 g with 20 ml of water for 5 minutes, cool, filter

and wash the filter with three quantities, each of 10 ml, of
water. Combine the filtrate and washings and add 0.3 ml of
phenolphthalein solution. Not more than 1.5 ml of 0.01 M
Related substances. Deteiil'iine by thin~layeichromatograpliy


10 volumes of 18 M ammonia and 10 volumes of methanol.
2256
IP 2010
TRICHLOROMONOFLUOROMETHANE
Test solution. Dissolve a quantity of the contents of the

capsules containing 0.1 g ofTriamterene in sufficient dimethyl
sulphoxide to produce 20 ml and dilute 2 ml to 50 ml with
methanol.
Tests
Weight per ml (2.4.29). 1.037 to 1.045.

Acidity. Dissolve 32.0 g in 30 ml of ethanol (95 per cent),
previously neutralised to bromothymol blue, add bromothymol
blue solution and titrate with 0.1 M sodium hydroxide to a
faint blue endpoint; not more than 1.0 ml is required.

detectable and examine in ultraviolet light at 365 nm. Any
Assay. Weigh accurately a quantity of the contents of

20 capsules containing about 0.1 g ofTriamterene, dissolve in
50 ml ofa mixture ofequal volumes ofglacial acetic acid and
water with the aid of gentle heat, cool and add sufficient
100.0 ml with 1 M acetic acid and measure the absorbance of
the resulting solution at the maximum at about 360 urn (2.4.7).
Test solution. Dissolve about 300 mg of substance under

examination in 10 ml of toluene.
Calculate the content of C l2 H Il N 7 from the absorbance

obtained by repeating the operation using triamterene RS in
place of the contents of the capsules.
Tributyl Citrate
Water (2.3.43). Not more than 0.2 percent.

tributyl citrate RS and acetyltributyl citrate RS in toluene.
- a glass or stainless steel column 30 m x 0.32 mm, packed
with bonded with a 0.5-llm layer ofphase 042,
- temperature:
column. 80 to 220 from 0.5 to 10 minutes,
inlet port. 85 to 250 from 0.5 to 10 minutes (increased
@ 20 per minute) and detector 275,
- flow rate. 2.3 ml per minute ofthe nitrogen as carrier gas.
Inject 1 III ofthe reference solution. The test is not valid unless
the relative retention times with reference to acetyltributyl
citrate for tributyl citrate is about 0.9, the resolution between
the peaks corresponding to tributyl citrate and acetyltributyl
citrate is not less than 1.5 and the relative standard deviation
Inject 1 III ofthe test solution and the reference solution.
Calculate the content C 1s H32 0 7

Mol. Wt. 360.5
Tributyl Citrate contains not less than 99.0 per cent of
C 1sH 32 0 7, calculated on the anhydrous basis.
CI
I
CI-C-F
I
CI
Category. Excipient.
Identification
Compare the spectrum with that obtained with tributyl citrate
RS or with the reference spectrum oftributyl citrate.
CC~F
Mol. Wt. 137.4
Trichloromonofluoromethane contains not less than 99.6 per

cent and not more than 100.0 per cent ofCChF, calculated on
Category. Pharmaceutical aid (Excipient).
2257
TRICHLOROMONOFLUOROMETHANE
IP 2010
Identification
Determine by infrared absorption spectrophotometry (2.4.6),
compare the spectrum with the obtained with
trichloromonofluromethane RS.
Tests
Boiling temperature. Transfer a 100 ml sample at about 24 to
a tared, pear-shaped, 100-ml centrifuge tube containing a few
boiling stones, and weigh. Suspend a thermometer in the liquid,
and place the tube in a medium maintained at a temperature of
32 above the expected boiling temperature. When the
thennometer reading becomes constant, record as the boiling
temperature the thelmometer reading after at least 5per cent of
the substance under ex,amination has distilled. Retain the
remainder of the substance for the determination of HighBoiling Residues.
Water (2.3.43). Not more than 0.001 per cent (Method 3) with
the following modifications (a) provide the closed-system
titrating vessel with an opening through which passes a
coarse-porosity gas dispersion tube connected to a sample
cylinder; (b) dilute the reagent with anhydrous methanol to
give a water equivalence factor ofbetween 0.2 and 1.0 mg per
ml, age this diluted solution for not less than 16 hours before
sanitation; (c) obtain a 100 g sample as directed under inhalation
preparation, and introduce the same into the titration vessel
through the gas dispersion tube at a rate of about 100 ml of
gas per minute.
High-boiling residues. Not more than 0.01 per cent, Allow 85
ml ofthe sample to distill as directed in the test for Approximate
Boiling Temperature, and transfer the centrifuge tube
containing the remaining 15 ml of substance to a medium
maintained at a temperature 10 above the boiling temperature.
After 30 minutes, remove the tube from the water-bath, blot
dry, and weigh. Calculate the weight ofthe residue.
temperature:
column 70 to 170 @10 0 per minute and 170 for 5
minutes,
a flame ionisation detector,
flow rate. 20 ml per minute ofthe nitrogen as carrier gas
Head-space conditions which may be used:
bath temperature 100,
valve/loop temperature 105,
sampling time is 3 seconds.
The relative retention time with reference to
trichloromonofluoromethane for dichlorodifluoromethaneis
about 0.5 and for dichlorotetrafluoroethane is about 0.8. The
sum ofthese two peak areas is not more than 0.2 per cent of
the total of all peak areas and the sum ofthe areas of all peaks
other than that for trichloromonofluoromethane is not more
than 0.4 per cent of the total of all peak areas.
Inject the test solution and reference solution. The test is not
valid unless the resolution between the peaks due
dichlorotetrafluoroethane and trichloromonofluoromethane is
not less than 2.0.
Calculate the content of CCI3F.
Storage. Store protected from moisture and prevent exposure
to excessive heat.
Triethyl Citrate
Inorganic chlorides. Place 5 ml of anhydrous methanol in a

test tube, add 3 drops of a saturated solution of silver nitrate
in anhydrous methanol, shake, and add 7 g of
Trichloromonofluoromethane; no opalescence or turbidity is
produced.
Mol. Wt.276.3
Test solution. Introduce the liquid phase of Trichloromonofluoromethane into an evacuated headspace vial
Triethyl Citrate is triethyl 2-hydroxypropane-l, 2, 3tricarboxylate.
Reference solution. Introduce a liquid-phase mixture of

dichlorodifluoromethane, dichlorotetrafluoroethane, and
trichloromonofluoromethane into an evacuated headspace yjal.
Triethyl Citrate contains not less than 98.5 per cent and not
more than 101.0 per cent of ClzHzo07, calculated on the
anhydrous basis.
Category. Excipient.
- a steel column 1.8 m x 2 mm, packed with support S12,

containing 1 per cent phase G25,
.Description. A clear, viscous, colourless or almost colourless,

hygroscopic liquid.
2258
IP 2010
TRIFLUOPERAZINE HYDROCHLORIDE
Identification

Compare the spectrum with that obtained with triethyl citrate
RS or with the reference spectrum oftriethyl citrate.

solution. Ignore anypeak with an area less than 0.04 per cent
B. To 0.5 ml add 5 ml of ethanol (95 per cent) and 4 ml of

dilute sodium hydroxide solution. Boil under reflux for about
10 minutes, 2 ml ofthe solution gives the reactions of citrates
(2.3.1).
Heavy metals (2.3.13). Dissolve 4.0 g in 8 ml of ethanol (95 per

cent) and dilute to 20 ml with water. 12 ml of the resulting
solution complies with the limit test for the heavy metals,
method D (5 ppm). Prepare the standard solution using lead
standard solution (l ppm Pb) obtained by diluting lead
standard solution (l00 ppm Pb) with a mixture of equal
volumes of ethanol (95 per cent) and wate/:
C. It gives the reactions of esters (2.3.1).

Tests
LOg.
Appearance of solution. The substance under examination is

clear (2.4.1) and not more intensely coloured than reference
Acidity. Dilute 109 with 10 rnl ofpreviously neutralized ethanol
(95 per cent). Add 0.5 ml of bromothymol blue solution. Not
more than 0.3 ml of 0.1 M sodium hydroxide is required to
change the color of the indicator to blue.
Assay. To 1.5 g, add 25 ml of 2-propanol, 50 rnl of water, 25.0

ml of 1 M sodium hydroxide and a few glass beads into a 250ml borosilicate-glass flask fitted with a reflux condenser. Heat
under a reflux condenser for 1 hour and allow to cool. Titrate
with 1 M hydrochloric acid, using 1 ml of phenolphthalein
ClzHzo07.

(2.4.13).
Test solution. Disperse 1.0 ml of the substance under

examination in 50.0 ml of dichloromethane.
Reference solution. Disperse 1.0 ml of the substance under

examination and 0.5 ml of methyl tridecanoate in 50.0 ml of
dichloromethane.
- a fused silica column 30 m x 0.32 mrn, packed with poly
(dimethyl) siloxane (5 /lm),
temperature:
column.200,
injection port and detector at 220,
a flame ionization detector,
linear velocity about 26 cm per second using nitrogen
as carrier gas
Inject 1 III ofthe reference solution. The test is notvalid unless
the resolution between the peaks due to triethyl citrate and
methyl tridecanoate is not less than 1.5.
Inject 1 /ll each ofthe test solution and the reference solution.
Run the chromatogram twice the retention time ofthe prinCipal
the area of"any secondary peak is not more than 0.2 per cent
with the reference solution. The sum of the areas of all the
secondary peaks is not more than 0.5 per centthe area of the
sn
Y"N~Nl
~N'CH
"
3
,2HCI
CF 3
CzIHz~3N3S,2HCl
Mol. Wt. 480.4
Trifluoperazine hydrochloride is 10-[3-(4-methylpiperazin-1yl)propyl]-2-trifluoromethylphenothiazine dihydrochloride.

Trifluoperazine Hydrochloride contains not less than 99.0 per
cent and not more than 101.0 per cent ofCzIHz4F3N3S, 2HCl,
Dose. As antipsychotic, orally, the equivalent of2 to 30 mg of
trifluoperazine daily, in divided doses; by intramuscular
injection, the equivalent ofl to 3 mg oftrifluoperazine daily, in
divided doses. As antiemetic, orally, the equivalent of 2 to 4
mg of trifluoperazine daily; by intramuscular injection, the
equivalent of 1 to 3 mg of trifluoperazine daily, in divided
doses.
2259
TRIFLUOPERAZINE HYDROCHLORIDE
IP 2010
Description. A white to pale yellow, crystalline powder;

odourless or almost odourless; slightly hygroscopic.
NOTE - In the following procedures, the test and standard

specimens and the solutions containing them should be
protected by carrying out the tests without delay and in
subdued light.
Identification
A. Dissolve 20 mg in 10 ml ofwater, make the solution alkaline
to litmus paper with 5 M sodium hydroxide and extract with
two quantities, each of20 ml, of light petroleum (60 to 80).
Combine the extracts, wash with 10 ml of water, shake with 5 g
of anhydrous sodium sulphate, filter and evaporate the filtrate
carefully to dryness.
obtained with trifluoperazine hydrochloride RS or with the
reference spectrum oftrifluoperazine hydrochloride.
B. Complies with the test for identification ofphenothiazines

(2.3.3), using as reference solution a 0.2 per cent w/v solution
of trifluoperazine hydrochloride RS in chloroform.
0.01 per cent w/v solution in 0.1 M hydrochloric acid
measured immediately after preparation shows an absorption
maximum at about 305 nm. Dilute 10 ml of the solution to
100 ml with 0.1 M hydrochloric acid. When examined in the
range 230 nm to 280 nm, the resulting solution shows an
absorption maximum at about 256 nm; absorbance at about
256 nm, about 0.65.
Trifluoperazine Hydrochloride Injection
Trifluoperazine Injection is a sterile solution ofTrifluoperazine
Trifluoperazine Injection contains not less than 90.0 per cent
trifluoperazine, C2IH2<tF3N3S,
Usual strength. The equivalent of 1mg oftrifluoperazine perml.,

subdued light.
Identification
When examined in the range 230 nm to 360 nm (2.4.7), the final
solution obtained in Assay shows an absorption maximum at
about 256 nm.
Tests
pH (2.4.24). 4.0 to 5.0.
Assay. To an accurately measured volume of the injection

containing about 50 mg of trifluoperazine add 10 ml of 2 M
sulphuric acid and extract with three quantities, each of25 ml,
of carbon tetrachloride. Discard the carbon tetrachloride
extract after each extraction. Add 10 ml of strong ammonia
solution and extract with five quantities, each of 50 ml, of
cyclohexane. Extract the combined cyclohexane extracts with
five quantities, each of 50 ml, of 0.1 M hydrochloric acid.
Dilute the combined acid extracts to 500.0 ml and mix. Measure
about 256 nm (2.4.7).

substances in phenothiazines (2.3.5), using mobile phase A.
Calculate the content ofC21H24F3N3S taking 743 as the specific

absorbance at 256 11)11.
0.7kPa.

equivalent amount oftrifluoperazine per ml.
D. Dissolve 5 mg in 2 ml of sulphuric acid and allow to stand

for 5 minutes; an orange colour is produced.
E. A 10 per cent w/v solution gives the reactions of chlorides
(2.3.1).
Tests

anhydrous glacial acetic acid add 10 ml of mercuric acetate Trifluoperazine Tablets
solution. Titrate with 0.1 M perchloric acid, using crystal
vialelsalm/on as~indic~ator.~Cany~ouCablaiilnitfation.~~-~-~~ TrifluoperazineHydrochloride~Tablets'
1 .tril of 0.1 Mperchl()ric~ acid is equivalent to 0.02402
C2IH2<tF3N3S,2HCl.
g of
Trifluoperazine Tablets contain not less than 92.5 per cent

and not more than 107.5 per cent of trifluoperazine,
C21H24F3N3S, The tablets are coated.
2260
IP 2010
TRIFLUPROMAZINE HYDROCHLORIDE
Usual strengths. The equivalent of 1 mg and 5 mg of
trifluoperazine.
protected by canying out the tests without delay and in
subdued light.
Triflupromazine Hydrochloride
Flupromazine Hydrochloride
Identification
of trifluoperazine with 30 ml of I M hydrochloric acid for
10 minutes, filter, make the filtrate alkaline to litmus paper with
5 M sodium hydroxide and extract with two quantities, each
of20 ml, of lightpetroleum (60 to 80). Combine the extracts,
wash with 10 ml of water, shake with 5 g of anhydrous sodium
sulphate, filter and evaporate the filtrate carefully to cJ.r:Yness.
obtained with trifluoperazine hydrochloride RS or with the
reference spectrum oftrifluoperazine hydrochloride.
oftrifluoperazine with 5 ml ofacetone, filter and evaporate the
filtrate to dryness. Add 2 ml of sulphuric acid to the residue
and allow to stand for 5 minutes; an orange colour is produced.
Tests
Hel
MoL Wt. 388.9

Triflupromazine Hydrochloride is 10-[3-(dimethylamino)propyl)]-2-trifluoromethylphenothiazine hydrochloride.
Triflupromazine Hydrochloride contains not less than
97.0percent and not more than 103.0 per centofClsHl9F3N2S,
HCI, calculated on the dried basis.
Dose. As antipsychotic, orally, 30 to 150 mg oftriflupromazine
daily, in divided doses; by intramuscular injection, 10 mg

repeated 4-hourly, ifnecessary; by intravenous injection, 1 to
3 mg repeated 4-hourly, if necessary. As antiemetic, 20 to 30
mg oftriflupromazine daily; by intramuscular injection 1 to 3
mg.

Tablets.
Description. A white to pale yellowish brown, crystalline
Place one tablet in a 100-ml volumetric flask, add 50 ml of a

mixture of 19 volumes of water and 1 volume of hydrochloric
acid, shake until the tablet has completely disintegrated, dilute
to volume with the same mixture, mix and filter, rejecting the
first few ml ofthe filtrate. Dilute suitably, ifnecessary with the
same solvent mixture and measure the absorbance of the

subdued light.

quantity ofthe powder containing about 5 mg oftrifluoperazine,
shake for 15 minutes with 400 ml ofa m~ture of 19 volumes of
water and 1 volume of hydrochloric acid, dilute to 500.0 ml
with the same solvent mixture, mix and filter. Measure the
(2.4.7).

Storage. Store protected i'om light and moisture.
equivalent amount of trifluoperazine.
powder; odour slight and characteristic.
Identification
A. Dissolve 20 mg in 10 ml of water, make the solution alkaline
two quantities, each of20 ml, of light petroleum (60 to 80).
Combine the extracts, wash with 10 ml of water, shake with 5 g
of anhydrous sodium sulphate, filter and evaporate the filtrate
carefully to dryness.
The residue complies with the following test. Determine by
spectrum with that obtained with trijlupromazine hydrochloride RS or with the reference spectrum oftriflupromazine
hydrochloride.
B. Complies with the test for identification ofphenothiazines
(2.3.3), using as reference solution a 0.2 per cent w/v solution
of triflupromazine hydrochloride RS in chloroform.
0.00 I per cent w/v solution in 0.1 M hydrochloric acid shows
2261
IP 2010
TRIFLUPROMAZINE INJECTION
an absorption maximum atabout 256 nm and a less well-defined

D. A 10 per cent wlv solution gives the reactions of chlorides
(2.3.1).
Tests
substances in phenothiazines (2.3.5), using mobile phase A.
anhydrous glacial acetic acid add 10 ml of mercuric acetate

solution. Titrate with 0.1 M perchloric acid, using clystal
ClsHI9F3N2S, HCI.
Tests
pH (2.4.24).3.5 to 5.2.
Assay. To an accurately measured volume of the injection
containing about 20 mg ofTriflupromazine Hydrochloride add
sufficient ofa mixture ofl9 volumes of water and I volume of
hydrochloric acid to produce 100.0 ml and mix. Dilute 5.0 ml
of the solution to 100.0 ml with the same mixture and mix.
maximmn at about 256 nrtJ. (2.4.7).
Calculate the content of ClsHI9F3N2S, HCI taking 700 as the
Triflupromazine Hydrochloride Tablets; Flupromazine
Hydrochloride Tablets; Flupromazine Tablets
Triflupromazine Hydrochloride Injection; Flupromazine
Hydrochloride Injection; Flupromazine Injection
Triflupromazine Injection is a sterile solution ofTriflupromazine
.
Triflupromazine Injection contains not less than 90.0 per cent
triflupromazine hydrochloride, ClsHI9F3N2S, HCI.
Usual strength. 10 mg per mI.
Description. A clear, almost colourless solution.

subdued light.
Identification
A. To an appropriate quantity of the injection add sufficient
0.1 M hydrochloric acid to produce a solution containing
0.001 per cent wIv solution ofTriflupromazine Hydrochloride.
256nm.
B. To a volume containing about 100 mg of TrifluprOluazine
Hydrochloride add 5 ml of 8 M nitric acid and mix; a pink to
amber colour develops which quicldy turns dark brown and
then changes to a clear solution having a yellow tint.
Triflupromazine Tablets contain not less than 90.0 per cent

triflupromazine hydrocWoride, ClsHI9F3N2S, HCI.

protected by canying out the tests without delay and in
subdued light.
Identification
of Triflupromazine Hydrochloride with 30 ml of 1 M
hydrochloric acid for 10 minutes, filter, malce the filtrate al1caline
two quantities, each of 20 ml, of light petroleum (boiling
range 600 to 800) . Combine the extracts, wash with 10 ml of
water, shake with 5 g of anhydrous sodium sulphate, filter and
evaporate the filtrate carefully to dryness.
obtained with trijlupromazine hydrochloride RS or with the
reference spectrum oftriflupromazine hydrochloride.
B. Extract a quantity of the powdered tablets containing 5 mg
ofTriflupromazine Hydrochloride with 5 ml of acetone, filter
and evaporate the filtrate to dryness. .
- - _. --
When examIned in the range 230 nm to 360 11m (2A.7),

0.00 I per cent wlv solution in 0.1 M hydrochloric acid shows
an absorption maximum at about 256 nm.
2262
IP 2010
TRIMETAZIDINE HYDROCHLORIDE
Tests
Tests
Uniformity of content. (For tablets containing 10 mg or less.
Appearance of solution. A 10 per cent w/v solution is clear

(2.4.1) and not more intensely coloured than reference solution
BYS6(2.4.l).

Crush one tablet and transfer to a 1OO-ml volumetric flask with
the aid of a mixture of 19 volumes of water and 1 volume of
hydrochloric acid. Shake well, dilute to volume withthe same
mixture and complete the procedure described under the Assay
beginning at the words "mix and filter, ...".

(2.4.14).
ml with water. Dilute 5.0 ml of this solution to 100.0 ml with
Calculate the content ofClsH19F3NzS, HCl in the tablet.

water.

Triflupromazine Hydrochloride and shake for 15 minutes with
200 ml of a mixture of 19 volumes of water and 1 volume of
hydrochloric acid. Dilute to 500.0 ml with the same solvent
mixture, mix and filter, rejecting the first few ml ofthe filtrate.
Dilute 10.0 ml to 100.0 ml with the same solvent mixture.
Measure the absorbance ofthe filtrate at the maximum at about
256 urn (2.4.7).
- a stainless steel columnl5 cm x 4.6 mm packed with
mobile phase: A. a mixture of35.7 volumes of methanol
and 64.3 volumes of a 0.29 per cent w/v solution of
sodium heptanesulphonate, adjusted to pH 3.0 with
B. methanol,

below,
Time
Mobile phase A
Mobile phase B
(per cent v/v)
(min.)
(per cent v/v)
5~25
0-50
95~75
25~5
50-52
75~95
Calculate the content ofClsH19F3NzS, HCl taking 700as the

specific absorbance at 256 urn.
CIJfzzNz03,2HCl
Mol. Wt. 339.3
Trimetazidine Hydrochloride is 1-[(2,3,4-trimethoxyphenyl)methyl]piperazine dihydrochloride.

Trimetazidine Hydrochloride contains not less than 98.5 per
cent and not more than 101.5 per cent, calculated on the dried
basis.
Category. Antiischemic metabolic agent.
Identification
Compare the spectrum with that obtained with trimetazidine
dihydrochloride RS or with the reference spectrum of
trimetazidine dihydrochloride.
B. Gives reaction (a) for chlorides (2.3.1).
trimetazidine for (2,3,4-trimethoxyphenyl)methanol
(trimetazidine impurity D) is about 0.2, for 2,3,4trimethoxybenzaldehyde (trimetazidine impurity C) is about
0.4, for ethyl 4-(2,3,4-trimethoxybenzyl)piperazine-lcarboxylate (trimetazidine impurity H) is about 0.6, for 1-(3,4,5trimethoxybenzyl)piperazine (trimetazidine impurity A) and
N-methyltrimetazidine (trimetazidine impurity I) is about 0.9,
for 1-(2,4,5-trimethoxybenzyl)piperazine (trimetazidine impurity
E) is about 0.95, for 1-(2,4,6-trimethoxybenzyl) piperazine
(trimetazidine impurity F) is about 1.4 and for 1,4-bis(2,3,4trimethoxybenzyl) piperazine (trimetazidine impurity B) is about
1.8.
chromatogram obtained with the test solution, the area of
each peak due to trimetazidine impurity A, B, C, D, E, F, H, I is
not more than the area of the principal peak in the
cent), the area of any other secondary peak is not more than
2263
TRIFLUPROMAZINE HYDROCHLORIDE
IP 2010

with the reference solution (0.1 per cent). The sum ofareas of
all the secondary peaks is not more than twice the area ofthe
solution (0.2 per cent). Ignore any peak with an area less than
Impurity G. Determine by thin-layer chromatography (2.4.17),

ammonia and 80 volumes of ethanol (95 per cent).
Reference solution. Dissolve 22.6 mg of piperazine hydrate
(impurity G) in 100 m1 of methanol. Dilute 10 m1 ofthis solution
Apply to the plate 10 f.l.l of each solution. Allow the mobile
phase to rise 8 cm. Dry the plate at 105 0 for 30 minutes and
spray with iodoplatinate reagent. Any secondary spot
corresponding to trimetazidine impurity G is not more intense
than the spot in the chromatogram obtained with the reference
on 1.0 g by drying in an oven at 105 0 over diphosphorus
pentoxide at a pressure not exceeding 15 kPa.
Assay. Dissolve 0.12 g in 50.0 ml of water. Add 1 mlofnitric
acid and titrate with 0.1 M silver nitrate, determining the endpointpotentiometrically (2.4.25). Carry out a blank titration.
I ml of 0.1 M silver nitrate is equivalent to 0.01696 g of
ClJIz4ClzNz03'
Trimethoprim contains not less than 98.5 per cent and not
more than 101.0 per cent of CJ4HJ8N403, calculated on the
dried basis.
Description. A white or yellowish white powder; odourless or
almost odourless.
Identification

. and C may be omitted if test A is carried out.
A. Determine by infrared absorption spectrophotometry (2.4 .6).
Compare the spectrum with that obtained with trimethoprim
RS or with the reference spectrum oftrimethoprim.
B. Dissolve 25 mg in 25 ml of ethanol (95 per cent) and dilute

2.0 ml to 100 ml with 0.1 M sodium hydroxide.
When examined in the range 230 urn to 360 nm (2.4.7), the
287 urn; absorbance at about 287 urn, about 0.49.
C. Dissolve about 25 mg in 5 ml of 0.005 M sulphuric acid,
with heating ifnecessary, add 2 ml ofa 1.6 per cent wlv solution
ofpotassium permanganate in 0.1 M sodium hydroxide. Heat
to boiling and to the hot solution add 0.4 ml offormaldehyde
solution. Mix, add I ml of 0.5 M sulphuric acid, mix and heat
to boiling. Cool and filter. Add 2 ml of chloroform to the filtrate
and shake vigorously. The chloroform layer exhibits a green
fluorescence when examined in ultraviolet light at 365 urn.
Tests
Appearance ofsolution. A 5.0 per cent wIv solution in a mixture
of 10 volumes of chloroform, 9 volumes of methanol and
2 volumes of water is not more intensely coloured than

10 volumes of methanol, 5 volumes of water and 2 volumes of
anhydrous formic acid.
Trimethoprim

examination in 10 m1 ofa mixture of 10 volumes of chloroform,
9 volumes of methanol and 2 volumes of water.
Reference solution. A 0.008 per cent wlv solution of the
ch7orojoriii;9 v61urnes6fmelhCiiWland2v61iiiiies6f water:

Mol. Wt. 290.3
Trimethoprim is 5-(3,4,5-trimethoxybenzyl)pyrimidine2,4-diamine.
Apply to the plate 5 f.l.l of each solution. Allow the mobile

phase to rise 17 cm. Dry the plate in a current of cold air for
5 minutes and examine in ultraviolet light at 254 urn. Place an
2264
IP 20ID
TRIMETHOPRIM AND SULPHAMETHOXAZOLE ORAL SUSPENSION
evaporating dish containing a mixture of 2 volumes of a

1.5 per cent w/v solution of potassium permanganate,
1 volume of 7 M hydrochloric acid and 1 volume of water at
the bottom of a closed tank and allow to stand for 15 minutes.
Place the dried plate in the closed tank and expose to the
chlorine vapour for 5 minutes. Remove the plate from the tank
and remove the chlorine in a current of cold air until an area
below the line of application does not give a blue colour on
the addition of 0.05 ml of potassium iodide and starch
solution. Spray the plate with potassium iodide and starch
solution and examine in daylight. By both methods of
visualisation any secondary spot in the chromatogram
Loss on drying (2.4.19). Notmorethan 1.0 percent, determined
acid, determining the end-point potentiometrically (2.425).
CIJIISN403.
Usual strengths. Trimethoprim 40 mg and Sulpha- methoxazole

200 mg in 5 ml; Trimethoprim 80 mg and Sulphamethoxazole
400 mg in 5 ml.
Identification
.

2 volumes of methanol and 1 volume of dimethylformamide.
Test solution. Add 20 ml of methanol (or a suitable volume of
methanol to yield 0.4 per cent w/v solution ofTrimethoprim)
to 5 ml of the suspension, mix, shake with 10 g of anhydrous
sodium sulphate, centrifuge and use the supernatant liquid.
sulphamethoxazole RS in methanol.
trimethoprim RS in methanol.
iodobismuthate solution. One of the principal spots in the
and the other corresponds to that in the chromatogram
obtained with reference solution (b)
Tests
pH (2.4.24). 5.0 to 6.5.
Trimethoprim and Sulphamethoxazole

Oral Suspension
Other tests. Complies with the tests stated under Oral liquids.
Sulphamethoxazole and Trimethoprim Oral Suspension;

Co-trimoxazole Oral Suspension; Co-trimoxazole Mixture
Trimethoprim and Sulphamethoxazole Oral Suspension is a
suspension of Trimethoprim and Sulphamethoxazole in a
suitable flavoured vel1icle. It contains 5 parts of
Sulphamethoxazole for 1 part, by weight, ofTrimethoprim.
Trimethoprim and Sulphamethoxazole Oral Suspension
110.0 per cent of the stated amounts of trimethoprim,
C14HISN403, and sulphamethoxazole, CIOHIIN303S.
Assay. For trimethoprim

Weigh accurately about 4 g, add
30 ml of 0.1 M sodium hydroxide, shake and extract with four
quantities, each of50 ml, of chloroform,washing each extract
with the same two quantities, each of 10 ml, of 0.1 M sodium
hydroxide. Reserve the combined aqueous solution and
washings for the Assay for sulphamethoxazole. Extract the
combined chloroform extracts with four quantities, each of
50 ml, of 1 M acetic acid. Wash the combined extracts with
. 5 ml of chloroform and dilute the aqueous extracts to 250.0 ml
with 1 M acetic acid. To 10.0 ml ofthis solution add 10mi of
1 M acetic acid and sufficient water to produce 100.0 ml, mix
maxirninn at about 271 nm(2.4.7).
Dose. For children upto 1 year, Trimethoprim, 40 mg and
Sulphamethoxazole, 200 mg daily, in divided doses. For children
1 to 5 years of age, Trimethoprim, 80 mg and Sulphamethoxazole, 400 mg daily, in divided doses. For adults,
Trimethoprim, 160 to 480 mg and Sulphamethoxazole, 800 mg
to 2.4 g daily, in divided doses.
Calculate the content ofC,4H,sN403 taking 204 as the specific

absorbance at 271 nm. Detennine the weight per ml of the
suspension (2.4.29), and calculate the content ofC,4H,sN403,
weight in volume.
For sulphamethoxazole - Carry out thefollowing procedure

protected fi'om light.
2265
TRIMETHOPRIM AND SULPHAMETHOXAZOLE ORAL SUSPENSION
Dilute the combined aqueous solution reserved in the Assay

for ~ethoprim to 250.0 m1 with water, filter and dilute 5.0 m1
ofthe filtrate to 200.0 ml with water (solution A). To 2.0 ml of
solution A add 0.5 ml of 4 M hydrochloric acid and 1 ml of a
0.1 per cent w/v solution ofsodium nitrite and allow to stand
for 2 minutes. Add 1 ml of a 0.5 per cent w/v solution of
ammonium sulphamate and allow to stand for 3 minutes. Add
1 ml of a 0.1 per cent w/v solution of N- (l-napthyl)
ethylenediamine dihydrochloride and allow to stand for
10 minutes. Dilute the solution to 25.0 ml with water and
measure the absorbance of the resulting solution at about
538 Dill (2.4.7), using as the blank a solution prepared in the
same manner but using 2 ml of water in place of solution A.
Weigh accurately about 0.25 g of sulphamethoxazole RS,
dissolve in 50 ml of 0.1 M sodium hydroxide and dilute to
250.0 ml with water. Dilute 5.0 ml ofthe resulting solution to
200.0 ml with water (solution B). Repeat the procedure using
2.0 ml ofsolution B and beginning atthe words "add 0.5 mlof
4 M hydrochloric acid......".
Calculate the content ofCJOHIIN303S from the values of the
absorbances obtained. Calculate the content ofC JOH lI N 30 3S,
weight in volume from the weight per m1 determined in the
Assay for trimethoprim.
Storage. Store protected from light and moisture. The
suspension should not be allowed to freeze.
Labelling. The label states (1) the content of Trimethoprim
and ofSulphamethoxazole in each 5 ml ofthe suspension; (2)
that the contents should be shaken before use; (3) that a
suspension containing 40 mg ofTrimethoprim and 200 mg of
Sulphamethoxazole in 5 ml is meant for paediatric use.
IP 2010
Trimethoprim, 160 to 480 mg and Sulphamethoxazole, 800 mg

to 2.4 g daily, in divided doses.
Usual strengths. Trimethoprim 20 mg and Sulpha- methoxazole
100 mg; Trimethoprim 80 mg and Sulphamethoxazole 400 mg;
Trimethoprim 160 mg and Sulphamethoxazole 800 mg.
Identification
Trimethoprim add 30 ml of0.1 M sodium hydroxide and extract
with two quantities, each of 50ml, of chloroform. Wash the
combined chloroform extracts with two quantities, each of
10 ml, of 0.1 M sodium hydroxide and then with 10 ml of
water. Combine the aqueous extract and washings (solution
A) and reserve for test B. Shake with 5 g ofanhydrous sodium
sulphate, filter and evaporate to dryness using a rotary
evaporator.
spectrum with that obtained with trimethoprim RS or with the
reference spectrum oftrimethoprim.
B. Filter solution A, add, dropwise, sufficient 2 M hydrochloric acid to the filtrate to make it just acidic and extract
with 50 ml of ether. Wash the ether layer with 10 ml ofwater,
shake with 5 g of anhydrous sodium sulphate, filter and
evaporate the filtrate to dryness using a rotary evaporator.
Dissolve the residue in the minimum volume of a 5 per cent
w/v solution of sodium carbonate, add 1 M hydrochloric
acid dropwise until precipitation is complete and filter. Wash
the residue sparingly with water and dry at 105.
spectrum with that obtained with sulphamethoxazole RS or
with the reference spectrum ofsulphamethoxazole.

Tablets

Sulphamethoxazole and Trimethoprim Tablets;

Co-trimoxazole Tablets

2 volumes of methanol and 1 volume of dimethylformamide.
Trimethoprim and Sulphamethoxazole Tablets contain 5 parts

ofSulphamethoxazole for 1 part, by weight, ofTrimethoprim.

containing 0.4 g ofSulphamethoxazole with 20 m1 ofmethanol
and filter.
Trimethoprim and Sulphamethoxazole Tablets contain not less

stated amounts of trimethoprim, C14HISN403, and sulphamethoxazole, CJOHIIN303S.
.

sulphamethoxazole RS in methanol.

trimethoprim RS in methanol.
Dose. For children upto 1 year, Trimethoprirn,40 rngand

Sulphamethoxazole, 200 mg daily, in divided doses. For children
1 to 5 years of age, Trimethoprim, 80 mg and Sulphamethoxazole, 400 mg daily, in divided doses. For adults,
Apply to the plate 5 lliof {Jach solution. After development,

iodobismuthate solution. One of the principal spots in the
2266
TRIMETHOPRIM TABLETS
IP 2010

and the other corresponds to that in the chromatogram
B. Shake a quantity of the powdered tablets containing 0.1 g

of Trimethoprim with 60 ml of 0.1 M hydrochloric acid for
20 minutes, add sufficient 0.1 Mhydrochloric acidto produce
Tests

287nm.
100 ml, filter and dilute 5 ml of the filtrate to 250 ml with
Assay. For trimethoprzm - Weigh accurately a quantity of

the powdered tablets containing about 50 mg ofTrimethoprim,
add 30 ml of 0.1 M sodium hydroxide and extract with four
quantities, each of50 ml, of chloroform, washing each extract
hydroxide. Combine the chloroform extracts and extract with
four quantities, each of 50 ml, of 1 M acetic acid. Wash the
combined extracts with 5 ml of chloroform and dilute the
aqueous extracts to 250.0 ml with 1 M acetic acid. To 10.0 ml
of the solution add 10 ml of 1 M acetic acid and sufficient
water to produce 100.0 ml, mix and measure the absorbance of
the resulting solution at the maximum at about 271 nm (2.4.7).
Calculate the content OfCl4HlSN403 taking 204 as the specific
Weigh and powder 20 tablets.
Weigh accurately a quantity of the powder containing about
0.2 g ofSulphamethoxazole, dissolve as completely as possible
in 60 ml of water and 10 ml of hydrochloric acid. Add 3 g of
potassium bromide, cool in ice and carry out the nitrite titration
(2.3.31).
For sulphamethoxazole -

CIOHIIN303S.
Labelling. The label states the quantities of Trimethoprim
and of Sulphamethoxazole in each tablet.
Tests
(2.4.14).
containing 0.2 g of Trimethoprim with 50 ml of the mobile
phase, filter and use the filtrate.

- a stainless steel column 20 cm x 5 rom, packed with
- mobile phase: 0.14 per cent w/v solution of sodium
perchlorate in methanol (60 per cent) adjusted to
pH 3.1 with 0.1 M hydrochloric acid,
trimethoprim in the chromatogram obtained with reference
solution, should be at least 8,000 theoretical plates per
metre.
In the chromatogram obtained with the test solution the sum
reference solution.
Trimethoprim Tablets
Trimethoprim Tablets contain not less than 95.0 per cent and
not more than 105.0 per cent of the stated amount of Other tests. Comply with the tests stated under Tablets.
trirnethoprim, Cl~ISN403'
quantity ofthe powder containing about 0.1 g ofTrimethoprim,
add 100 ml ofglacial acetic acid, shake for 20 minutes, dilute
Identification
to 200.0 ml with glacial acetic acid and filter. To 5.0 ml ofthe
A. Shake a quantity of the powdered tablets containing 0.1 g filtrate add 15 ml of glacial acetic acid and dilute to 100.0 ml
ofTrimethoprim with 10 ml of chloroform, filter and evaporate with water. Measure the absorbance ofthe resulting solution
atthe maximum at about 271 nm (2.4.7).
the filtrate to dryness.
obtained with trimethoprim RS or with the reference spectrum
oftrirnethoprim.
Calculate the content OfCl4HlSN403 taking 204 as the specific

2267
TRlPROLIDINE HYDROCHLORIDE
IP 2010
H
y

Z-triprolidine RS in methanol.
corresponding to Z-triprolidine is not more intense than the
Nf)
CI9HzzNz,HCl,HzO
Mol. Wt. 332.9
Triprolidine Hydrochloride is (E)-2-(3-pyrrolidin-l-yl1-(p-tolyl)prop-l-enyl)pyridine hydrochloride monohydrate.

Triprolidine Hydrochloride contains not less than 98.0 per
cent and not more than 101.0 per cent of C19HzzNz, HCl,
Category. Histamine HI-receptor antagonist.
Description. A white, crystalline powder; almost odourless.
Identification
Compare the spectrum with that obtained with triprolidine
hydrochloride RS or with the reference spectrum oftriprolidine
hydrochloride.
0.001 per cent wIv solution shows absorption maxima at about
230 nm and 276 nm; absorbance at about 230 nm, about
0.50 and at about 276 nm, about 0.25.

290 nm, about 0.6.
D. Dissolve 0.1 g in 2 ml of 2 M hydrochloric acid and add
0.5 ml of potassium mercuri-iodide solution; a pale yellow
E. A 5 per cent w/v solution gives the reactions of chlorides
(2.3.1).
Tests
Mobile phase. A mixture of equal volumes of 2-butanone and

dimethylformamide.
Testsolution. Dissolve 0.1 g of the substance
nation in 10 ml of methanol.
of 50 ml of anhydrous glacial acetic acid and 0.5 ml of acetic
anhydride. Titrate with 0.1 M perchloric acid, using crystal
1 ml of 0.1 M perchloric acid is equivalent to
CI9HzzNz,HCl.
0~01574
g of
Triprolidine Hydrochloride Tablets
Triprolidine Tablets contain not less than 90.0 per cent and
not more than 110.0 per cent ofthe stated amount oftriprolidine
hydrochloride, CI9HzzNz,HCl,HzO.
Identification
10 mg ofTriprolidine Hydrochloride with ether, filter, discard
the ether extract and dry the residue. Extract the residue with
chloroform, filter and evaporate the filtrate to dryness. Add
0.1 ml of ether, stir and allow to evaporate.
obtained with triprolidine hydrochloride RS or with the
reference spectrum oftriprolidine hydrochloride.
underexami~
Tests


2268
TRISODIUM EDETATE CONCENTRATE FOR INJECTION
IP 2010
Mobile phase. A mixture of equal volumes of 2-butanone and

dimethylformamide.
Test solution. Extract a quantity ofpowdered tablets containing
10 mg of Triprolidine Hydrochloride with methanol, filter,
evaporate to dryness and dissolve the residue in 1 ml of
methanol.
Z-triprolidine RS in methanol.
Trisodium Edetate Concentrate for

Injection
Trisodium Edetate Concentrate for Injection is a sterile
solution in Water for Injections containing 20 per cent w/v
solution of trisodium edetate prepared by the interaction of
Disodium Edetate and Sodium Hydroxide. It should be diluted
with a suitable diluent in accordance with the manufacturer's
instructions.

triprolidine hydrochloride RS in methanol.
Trisodium Edetate Concentrate for Injection contains not less

than 19.0 per cent w/v and not more than 21.0 per cent w/v
solution oftrisodium edetate, CIOHI3N2Na30g.

triprolidine hydrochloride RS in methanol.
Category. Used in the treatment ofhypercalcaemia.
Dose. By slow intravenous infusion, upto 70 mg per kg daily

Apply to the plate 5 JlI of each solution. After development, over 2 to 3 hours. For topical use in the eye, dilute 1 ml to 50 ml
dry the plate in air and examine in ultraviolet light at 254 nm. In . with Water for InjeGtion.
Description. A colourless solution.
corresponding to Z-triprolidine is not more intense than the
spot in the chromatogram obtained with reference solution (a) Identification
and any other secondary spot is not more intense than the
spot in the chromatogram obtained with reference solution A. Dissolve 2 g in 25 ml of water, add 6 ml of lead nitrate
solution, shake and add 3 ml of potassium iodide solution;
(c).
no yellow precipitate is produced. Make alkaline to red litmus
Uniformity of content. (For tablets containing 10 mg or less). paper with 2 M ammonia and add 5 ml of ammonium oxalate
solution; no precipitate is produced.
Powder one tablet, weigh accurately a quantity ofthe powder
containing 7.5 mg ofTriprolidine Hydrochloride and carry out
the Assay beginning at the words "add 15 ml of water....".
Calculate the content ofC,9H22N2,HCl,H20 in the tablet.
C. Evaporate to dryness and ignite. The residue gives the


Assay. Weigh and. powder 20 tablets. Weigh accurately a
quantity ofthe powder containing about 7.5 mg ofTriprolidine
Hydrochloride, add 15 ml of water and 1 g of sodium chloride,
shake for 2 to 3 minutes and add sufficient 5 M sodium
hydroxide to make it alkaline. Extract with four quantities,
each of 20 ml, of ether and wash the combined extracts with
two quantities, each of5 ml, ofa mixture ofequal volumes ofa
saturated solution of sodium chloride and water. Extract the
ether solution with 20 ml of 0.1 M hydrochloric acid, wash
the ether with two quantities, each of 5 ml, of water and add
the washings to the acid extract. Heat on a water-bath for
30 minutes, cool and add sufficient water to produce 50.0 ml.
Dilute 10.0 ml to 100.0 nil with 0.1 M hydrochloric acid.
maximum at about 290 nm (2.4.7).
Calculate the content ofC19H22N2, HCl, H 20 taking 290 as the
B. Dissolve 0.5 gin 10 ml of water, add 0.5 ml ofa 10 percent

w/v solution of calcium chloride, make alkaline to red litmus
paper with 2 M ammonia and add 3 ml of ammonium oxalate
solution; no precipitate is produced.
Tests
pH (2.4.24). 7.0 to 8.0.
Pyrogens (2.2.8). Complies with the test, using per kg of the
rabbit's weight 5 ml of a solution prepared in the following
manner. To 1 volume ofthe injection add 2.5 volumes of calcium
gluconate solution. Dilute the resulting solution with
sufficient water for injection to give a final concentration of
5.0 per cent w/v solution of trisodium edetate.
Pryparations (Concentrated Solutions for Injection)..
Assay. Dilute 10.0 ml to 100.0 ml with water and use this
solution to titrate a mixture of25.0 ml of 0.05 M magnesium
sulphate and 10 ml of ammonia bufferpH 1 0.9 using mordant
black II mixed triturate as indicator.
1 ml of 0.05 Mmagnesium sulphate is equivalentto 0.01791 g
ofC,oHI3N2Na30g.
2269
TROPICAMIDE
IP 2010
Storage. Store in hermetically-sealed, lead-free glass

containers.

Labelling. The label states (1) the strength in terms of

anhydrous trisodium edetate contained in a suitable dosevolume; (2) 'Trisodium Edetate Concentrate for Injection'; (3)
that the solution must be dilutedwith either Sodium Chloride
Intravenous Infusion or Dextrose Intravenous Infusion before
administration.
Reference solution (b).A 0.002 per cent w/v solution of the

Tropicamide


Mol. Wt. 284.4

Tropicamide is (RS)-N-ethyl-3-hydroxy-2-phenyl-N-(pyrid4-ylmethyl)propionamide

anhydrous glacial acetic acid Titrate with 0.1 M perchloric
Tropicamide contains not less than 99.0 per cent and not more
than 101.0 per cent of C17HzoNzOz, calculated on the dried
basis.
C17HzoNzOz
Category. Mydriatic; cycloplegic.

Identification
Compare the spectrum with that obtained with tropicamide
RS or with the reference spectrum of tropicamide.

C. Dissolve 5 mg in 3 ml ofa mixture of9 ml ofacetic anhydride,
I ml of 6 M acetic acid and 0.1 g of citric acid and heat on a
water-bath for 5 to 10 minutes; a reddish yellow colour is
produced.
Tests
S volumesofmethiiiiol-ana-O:YvolUiiie--ofstFiTiTg-ammonfa
solution.
Test solution. Dissolve 0.1 g of the substance under examination in 10 ml of chloroform.

Tropicamide Eye Drops are a sterile solution of Tropicamide
in Purified Water. They may contain stabilisers, suitable
antimicrobial agents and suitable substances to increase the
viscosity of the solution.
Tropicamide Eye Drops contain not less than 95.0 per cent
tropicamide, C17HzoNzOz.
Usual strengths. 0.5 per cent w/v; 1.0 per cent w/v.
Identification
A. Shake a volume containing 20 mg of Tropicamide with
10 ml of chloroform, dry the chloroform layer over anhydrous
sodium sulphate, filter and evaporate the filtrate to dryness.
Dissolve the residue in minimum quantity of chloroform, add
dropwise to finely powdered potassium bromide JR, mix and
dry at 60.
obtained with tropicamide RS or with the reference SPf~ctrum
6ftropiCamide.
B. When examined in the range 230nm to 360 nm (2.4.7), the
2270
IP 2010
TROXIDONE
Tests
Troxidone contains not less than 98.0 per cent and not more
than 102.0 per cent ofC6H9N0 3, calculated on the dried basis.
pH (2.4.24). 4.0 to 5.8.

5 volumes of methanol and 0.5 volume of strong ammonia
solution.
Test solution. A volume ofthe eye drops containing 0.2 mg of
Tropicamide.
Dose. For a child, 250 mg to 500 mg daily, in divided doses; for
an adult, I to 2 g daily, in divided doses.
Description. Colourless or almost colourless crystals; odour,
slightly camphoraceous.
Identification
Reference solution (a). Dilute I volume of the eye drops to


Reference solution (b). Dilute I volume of the eye drops to


Compare the spectrum with that obtained with troxidone RS.

B. To 2 ml ofa 5.0 per cent w/v solution in carbon dioxide-free

water (solution A) add I ml of barium hydroxide solution; a
white precipitate is produced which dissolves on the addition

of 1 ml of 2 M hydrochloric acid.
C. Dissolve 0.3 g in a mixture of 5 ml of ethanolic potassium
hydroxide solution and 5 ml of ethanol (95 per cent) and
allow to stand for 10 minutes. Add 0.05 rn1 ofphenolphthalein
solution and carefully add hydrochloric acid until the solution
Assay. To a volume containing about 30 mg ofTropicamide is neutral. Evaporate to dryness on a water-bath, shake the
add sufficient water to produce 100.0 ml. To 10.0 ml of the residue with four quantities, each of 5 ml, of ether, filter the
resulting solution add 2 ml of a 10 per cent w/v solution of combined ether extracts and evaporate the filtrate to dryness.
anhydrous sodium carbonate and extract with four quantities, The residue, aft.er recrystallisation from 5 ml of toluene and
each of20 ml, of chloroform. Wash the combined chloroform drying, melts at about 80 (2.4.21).
extracts with 25 ml of phosphate buffer pH 6.5. Wash the
aqueous layer with 10 ml of chloroform, combine the Tests
chloroform extracts and shake with four quantities, each of
20 ml, of 0.5 M sulphuric acid. Combine the acid extracts,
colourless (2.4.1).
dilute to 100.0 ml with 0.5 M sulphuric acid and measure the
absorbance of the resulting solution at the maximum at about Acidity or alkalinity. To 10 ml of solution A add 0.1 ml of
methyl red solution. Not more than 0.1 ml of 0.01 M
254 nm (2.4.7).
hydrochloric
acid or 0.01 M sodium hydroxide is required to
Calculate the content ofC17H2oN202 taking 172 as the specific
change
the
colour
of the solution.
Storage. Store iJ;l a refrigerator (8 to 15). It should not be
allowed to freeze.
on 1.0 g by drying over anhydrous silica gel for 6 hours.
Troxidone
Trimethadione
H3 C
Assay. Determine by gas chromatography(2.4.13).
C}:T
o
Test solution. Weigh accurately 0.1 g of the substance under

examination and dissolve in sufficient solutions prepared by
dissolving 0.125 g of 1-decanol (internal standard) in sufficient
ethanol to produce 25.0 ml (solution B).
CJf9N03
Mol. Wt. 143.1
Troxidone is 3,5,5-trimethyloxazolidine-2,4-dione.
Reference solution. Dissolve 0.1 g of troxidone RS in sufficient
H3
CH 3
solution B to produce 10.0 rnl.
2271
IP 2010
TROXIDONE CAPSULES
- a stainless steel column 0.75 m x 3 mm, packed with
porous polymer beads (120 to 150 /lm),
- temperature:
column 210,
Inject 1 /ll of the test solution and the reference solution.
Calculate the content ofC6HgN0 3.
Tests
Test solution. To a quantity of the contents of the capsules
containing about LOg ofTroxidone add 25 ml ofa 0.5 per cent
w/v solution of l-decanol (internal standard) in ethanol, shake
for 30 minutes, add sufficient of internal standard solution to
produce 100.0 ml, mix and centrifuge. Use the supernatant
liquid.
Reference solution. Dissolve 0.1 g of troxidone RS in sufficient
solution B to produce 10.0 ml.
Troxidone Capsules
Trimethadione Capsules
Troxidone Capsules contain not less than 95.0 per cent and
not more than 105.0 per cent ofthe stated amount oftroxidone,
C6HgN03
a stainless steel column 0.75 m x 3 mm, packed with
porous polymer beads (120 to 150 /lm),
- temperature:
column21Q0 ,
inlet port at 240 and detector at 270 ,
- flow rate. 30 ml per minute oftIle carrier gas.
Calculate the content of C6HgN0 3in the capsules.
Identification
To a quantity ofthe contents of the capsules containing 1 g of

Troxidone add 25 mlof ether, set aside in a stoppered flask for
20 minutes, decant the ether through a filter, and ifan insoluble
residue remains, digest it with another 10-ml portion of ether
as before, and filter into the first ether filtrate. Evaporate the
ether extracts to dryness with the aid of a current of air and
dry the residue at a pressure of2 kPa for 2 hours. The residue


Compare the spectrum with .that obtained with troxidone RS
or with the reference spectrum oftroxidone.
B. To 2 ml ofa 5.0 per cent w/v solution in carbon dioxide-free
water (solution A) add 1 ml of barium hydroxide solution; a
white precipitate is produced which dissolves bnthe addition
of 1 ml of 2 M hydrochloric acid.
C. Dissolve 0.3 g in a mixture of 5 ml of ethanolic potassium
hydroxide solution and 5 ml of ethanol (95 pel~ cent) and
allow to stand for 19 minutes. Add 0.05 ml ofphenolphthalein
solution mid carefully add hydrochloric acid untilihe solution
is~neiltrar. . EvaliorateJQ ~dryIless . ollJI. :Wflt(.)r::b.!lth,.sllake 1M
residue with four quantities, each Of 5. 1111, ()fet~el;, filter tl1e
combined ether extracts and evaporate the filtrate to dryness.
The residue, after recrystallisationfrorn 5 ml of toluene and
drying, melts at about 80 (2.4.21).
C37IillClNz06,HCl,5HzO
Mol. Wt. 771.7
Tubocurarine Chloride is 7' ,12' -dihydroxy-6,6' -dimethoxy2,2' ,2' -trimethyltubocuraranium chloride hydrochloride
pentahydrate.
Tubocurarine Chloride contains riot less than 98.0 per cent
and not more than 102.0 per cent of C37H41CINz06,HCl,
calculated on the anhydrous-basis.
Dose. By intramuscular or intravenous injection, 100 to 300 /lg
per kg body weight, total dose not exceeding 40 mg.
2272
IP 2010
TUBOCURARINE INJECTION
Description. A white or yellowish white, crystalline powder.
Identification
out.
Compare the spectrum with that obtained with tubocurarine
chloride RS or with the reference spectrum of tubocurarine
chloride.
0.005 per cent w/v solution shows an absorption maximum at
about 280 nm and a minimum at about 255 run, 0.56 to 0.62,
C. To I ml of a 2.5 per cent w/v solution add 0.2 ml offerric
chloride solution and heat in a water-bath for I minute; a
green colour is produced; 1 ml of water treated in the same
manner gives a brown colour.
D. To 20 ml of a 0.05 per cent w/v solution add 0.2 ml of
sulphuric acid and 2 ml of a I per cent w/v solution of
potassium iodate, mix and warm on a water-bath for 30 minutes;
a yellow colour is produced.
E. Gives reaction A ofchlorides .and the reactions of allcaloids
(2.3.1).
Tests
Appearance of solution. Dissolve 0.5 g in sufficient carbon
dioxide-free water to produce 50.0 ml (solution A). Solution A
is clear (2.4.1), and not more intensely coloured than reference
Mobile phase. The lower layer of a mixture of equal volumes

of chloroform, methanol and a 12.5 pei cent w/v solution of
trichloroacetic acid.
examination in 10 ml ofwater.
Reference solution (a). A 0.0375 per cent w/v solution ofthe
Reference solution (b). AO.01875 per cent w/v solution ofthe
Apply to the plate 5 J..lI of each solution. After development,
dry the plate in a current ofcold air and spray with a mixture of
1 volume of potassium ferricyanide solution, 1 volume of
water and 2 volumes of ferric chloride solution, prepared
immediate,ly before use. Any secondary spot in the
onO.2g.
water to produce 500.0 ml and measure the absorbance ofthe
Calculate the content ofC37H4 1CIN20 6 ,HCI from the absorbance
obtained by repeating the operation using 25 mg, accurately
weighed, oftubocurarine chloride RS in place ofthe substance
under examination.
pH (2.4.24). 4.0 to 6.0, determined in Solution A.

in solution A 3 hours after preparation.
Chloroform-soluble substances. Not more than 2 per cent,

determined by the following method. Dissolve 0.25 gin 150 ml
of water contained in a separating funnel with a grease-free
stopcock. Add 5 ml of a saturated solution of sodium
bicarbonate and extract with three quantities, each of 20 ml,
of chloroform. Wash the combined chloroform extracts with
10 ml ofwater, filter the chloroform solution into a tared beaker
and wash the filter with two successive quantities, each of
5 ml, of chloroform. Add the washings to the filtrate. Remove
the chloroform on a water-bath, dry the residue at 105 for
I hour, cool and weigh. The residue does not dissolve in 10 ml
of water but dissolves on the addition of 1 ml of 2 M
hydrochloric acid.
Identification

A. Mix a volume containing 15 mg ofTubocurarine Chloride

with 5 ml of acetone, evaporate the liquid at a pressure of
Tubocurarine Chloride Injection

Tubocurarine Injection is a sterile solution of Tubocurarine
Chloride in Water for Injections. It may contain suitable
.
buffering agents.
Tubocurarine Injection contains not less than 95.0 per cent
tubocurarine chloride, C37~1 CIN20 6 ,HCI,5H20.
Description. A colourless or faintly coloured solut\on.
2273
IP 2010
TUBOCURARINE INJECTION
2 kPa and add successive quantities of 2 ml of acetone,

evaporating each quantity at a pressure of 2 kPa until a dry
residue is obtained.
obtained with tubocurarine chloride RS or with the reference
spectrum of tubocurarine chloride.
B. Dilute I ml to 30 ml with water. To 1 ml of the resulting
solution add 0.5 m1 of mercuric nitrate solution; a cherry-red
colour slowly develops.
Tests
pH (2.424). 4.0 to 6.0.
Optical rotation (2.422).+0.172 to +0.206 for each mg of
tubocurarine chloride, C37H41CIN206,HCI,5H20 per ml stated
on the label.
Mobile phase. The lower layer of a mixture of equal volumes
of chloroform, methanol and a 12.5 per cent w/v solution of
trichloroacetic acid.
Test solution. A volume of the injection containing 10 mg of
Tubocurarine Chloride diluted to 1 ml.
Reference solution (a). Dilute 3 volumes of test solution to

Reference solution (b). Dilute I volume of reference solution
(a) to 2 volumes with water.
dry the plate in a current ofcold air and spray with a mixture of
1 volume of potassium ferricyanide solution, 1 volume of
water and 2 volumes of ferric chloride solution, prepared
immediately before use. Any secondary spot in the
Assay. Dilute a volume containing about 50 mg ofTubocurarine
Chloride to 1000.0 m1 with water and measure the absorbance
Calculate the content OfC37~1 ClN20 6,HCI,5H20 taking 105 as
2274
MONOGRAPHS
u
Undecenoic Acid
2277
Urea
2277
Urea Cream
2278
Urokinase
2279
2275
IP 2010
UREA
o
Assay. Weigh accurately about 0.75 g, dissolve in lO ml of

ethanol (95 per cent) and titrate with 0.5 M sodium hydroxide
using 0.1 ml of dilute phenolphthalein solution as indicator.
Undecenoic Acid
Undecylenic Acid

Cll H200 2.
H2C~COOH
C,JI200 2
Mol. Wt. 184.3
Undecenoic Acid is 10-undecenoic acid.

Undecenoic Acid contains not less than 97.0 per cent and not
more than 102.0 per cent ofC ll H200 2
Urea
Category. Antifungal (topical).

Description. A white or very pale yellow, crystalline mass or
colourless or pale yellow liquid; odour, characteristic.
CHiN20
Mol. Wt. 60.1
Identification
Urea is the diamide ofcarbonic acid.
A. Dissolve 0.1 g in a mixture of2 ml of 1 M sulphuric acid and

5 ml of glacial acetic acid and add dropwise 0.25 mlof
potassium permanganate solution; the colour of the
permanganate solution is discharged.
Urea contains not less than 99.0 per cent and not more than
101.0 per cent ofCH4N 20, calculated on the dried basis.
B. Boil 2 g under a reflux condenser with 2 ml offreshly distilled

aniline for 10 minutes, allow to cool, add 30 ml of ether and
extract with three quantities, each of20 ml, of2 Mhydrochloric
acid and then with 20 ml of water. Evaporate the organic layer
to dryness on a water-bath. The residue, after recrystallising
twice from ethanol (70 per cent) and drying over phosphorus
pentoxide at a pressure of 1.5 to 2.5 kPa for 3 hours melts at
66 to 68.
Tests

Peroxide value (2.3.35). Not more than 10.
Iodine value (2.3.28). 131 to 140.

Fixed and mineral oils. Boil 1.0 g with 25 ml ofwater and 5 ml
of sodium carbonate solution for 3 minutes. The hot solution
is not more opalescent than opalescence standard OS2 (2.4.1).

Water-soluble acids. Shake 2.0 g with 20 ml ofwarm water,
allow to separate and filter .the aqueous layer through a

moistened filter paper. To 5 ml of the filtrate add 0.01 mlof
dilute phenolphthalein solution. Not more than 0.1 mlof
the solution.
Dose. 5 to 15 g.
Description. A white, crystalline powder or transparent
crystals; odourless or almost odourless, but may gradually

develop a slight odour of ammonia upon long standing;
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6):

Compare the spectrum with that obtained with urea RS or
with the reference spectrum of urea.
Congealing range (2.4.10). 21to 24.
Category. Keratolytic.
B. Heat 0.5 g in a test-tube; itliquefies, and ammonia is evolved

which is recognised by its characteristic odour. Heat further
until the liquid is turbid, cool and dissolve in 10 ml of water.
Add 1 ml of a 10 per cent w/v solution of sodium hydroxide
and 0.05 ml of coppersulphate solution; a reddish violet colour
is produced.
C. Dissolve 0.1 gin 1 ml of water and add 1 ml of nitric acid;
a white, crystalline precipitate is produced.
Tests
colourless (2.4.1).
Alkalinity. To 10 ml ofa 5.0 per cent w/v solution (solution A)
add 0.1 ml of methyl red solution and 0.4 ml of 0.01 M
hydrochloric acid; the resulting solution is red to orange.
2277
IP 2010
UREA
Biuret. Not more than 0.1 per cent, determined by the following
method. To 10 rnl of a 20 per cent w/v solution add 5 ml of
water, 0.5 m1 ofa 0.5 per cent w/v solution of copper sulphate
and 0.5 ml of 10Msodium hydroxide and allow to stand for
5 minutes. Any reddish violet colour obtained is not more
intense than that in a standard prepared at the same time and
in the same manner using lOrnl ofaO.02 percentw/v solution
of biuret in place of the substance under examination.
Ethanol-insoluble matter. Not more than 0.04 per cent,
determined by the following method. Dissolve 5.0 g in 50 ml of
warm ethanol (95 per cent), filter through a tared filter, wash
the filter with 20 ml ofwarm ethanol (95 per cent) and dry at
105 for 1 hour.
Heavy metals (2.3.13). Dissolve 1.0 g in 20 rnl ofwater and 5 rnl
of 0.1 M hydrQchloric acid. The solution complies with the
limit test for heavy metals, Method A (20 ppm).
Assay. Weigh accurately about 0.5 g, dissolve in sufficient of
a 10 per cent v/v solution of sulphuric acid to produce
100.0 ml and mix. Place 5.0 ml of the resulting solution in a
long-necked flask, add 10 rnl ofsulphuric acid and heat gently
until evolution ofgas ceases. Boil gently for 10 minutes, cool,
cautiously add 40 ml of water, cool again and place in a steamdistillation apparatus. Add 50 ml of 10Msodium hydroxide
and distil immediately by passing steam through the mixture.
Continue the distillation for 1 hour, collecting the distillate in
40 ml of a 4 per cent w/v solution of boric acid. Titrate with
0.1 M hydrochloric acid, using 0.25 ml of methyl redmethylene blue solution as indicator. Carry out a blank
titration.
1 ml of 0.1 Mhydrochloric acid is equivalent to 0.003003 g of
c:H4NzO.
Urea Cream
Urea Cream contains Urea in a suitable basis.
Urea Cream contains not less than 90.0 per cent and not more
than 110.0 per cent ofthe stated amount of urea, CH4N zO.
Usualstrength. 10 per cent w/w.
Identification
a mixture of 99 volumes of ethanol and 1 volume of strong

ammonia solution.
Test solution. Disperse with heating a quantity of the cream

containing 50 mg of Urea in 1 m1 of water, cool, add 4 ml of
acetone, mix and filter.
Referenc'e solution (a). Dissolve 50 mg of urea RS in 1 ml of
water and add 4 ml of acetone.
dry the plate in air, spray with a solution containing 0.5 per
cent w/v solution of 4-dimethylaminobenzaldehyde and
0.5 per cent v/v of sulphuric acid in ethanol. The principal
reference solution (a). The chromatogram obtained with
reference solution (b) shows a single, compact spot.
B. To a quantity containing 0.1 g of Urea add 50 ml of water
and heat until dispersed, cool in ice and filter through glass
wool. Adjust the pH to 6.0 to 7.0 using 0.1 M hydrochloric
acid or 0.1 M sodium hydroxide as necessary. To 5 ml add
5 ml ofa 0.1 per cent w/v suspension of urease-active meal
and allow to stand for 30 minutes in a stoppered flask at 37.
When the resulting solution is heated in a water-bath a vapour
is produced that turns moist red litmus paper blue.
Tests
Assay. Weigh accurately a quantity containing about 42 mg
ofUrea, shake with 150 rnl ofhot water for 20 minutes, allow to
cool and dilute to 500.0 ml with water. Filter through a fine
glass microfibrefilter paper or by any other means, transfer
1.0 ml ofthe filtrate to a 100-rnl volumetric flask, add 2 rnl ofa
0.1 per cent w/v suspension of urease-active meal, stopper
the flask and allow to stand for 15 minutes at 37. Immediately
add 25 ml of a solution containing 12 g of sodium salicylate
and 0.24 g of sodium nitroprusside in 200 ml and 25 ml of a
solution prepared by diluting a volume of sodium hypochlorite
solution containing 0.66 g of available chlorine with 0.2 M
sodium hydroxide to 1000 ml. Mix well, allow to stand at 37
for 5 minutes and dilute to 100.0 m1 with water. Measure the
665 nm (2.4.17), using as the blank a solution prepared in the
same manner but using 1~ 0 rnl of water in place ofl.0 ml ofthe
filtrate.
Calculate the content ofCH4N zO from the absorbance obtained
A. Deferrn.ille by tllill-layer C:1l.I'ornatograplly (2.4:17), coatlng by llsiJ.ig 42 IIlg of urea RS in place of the sllbstan-ce-Ui.ii:lef
exa.mina.tion.
Mobile phase. For the first development use 2,2,4-trimethylpentane. Dry the plate in air. For the second development use
Storage. Store in accordance with the instructions of the

manufacturer.
2278
IP 2010
UROKINASE
Urokinase
Urokinase is an enzyme, obtained from human urine that
activates plasminogen. It consists ofa mixture oflow molecular
weight and high molecular weight forms, the high molecular
weight form being predominant. The molecular weights ofthe
low and high molecular weight forms are 33,000 and 54,000
respectively.
It is prepared in conditions designed to minimise microbial

and viral contamination. In particular, adequate measures, such
as heating the substance in solution at 60 for 10 hours, are
taken to inactivate viruses.
Apply 1 ml ofthe test solution to the column, rinse twice with

. O.5-ml portions of the buffer and then carry out the elution.
The eluate may be collected in I-ml :Ii-actions. Plot the individual
absorbances on a graph. Draw perpendicular lines towards
the axis of the abscissae from the minima before the high
molecular weight peak, between the high and the low molecular
weight peaks, and after the low molecular weight peak.
Combine separately the high and low molecular weight
fractions and for each combined fraction determine the activity
by the method described under Assay. The ratio ofthe activity
in the combined high molecular weight fraction to that in the
combined low molecular weight fraction is not less than 2.0.
Category. Fibrinolytic enzyme.
Total protein. Determine by Method C for the determination

of nitrogen (2.3.30), using 10 mg of the substance under
examination and multiplying the result by 6.25 to obtain the
content of protein.
Dose. By instillation into arteriovenous shunt and for

intraocular administration, 5000 to 37,500 Units in 2 to 3 ml of
Sodium Chloride Injection.
Hepatitis-B surface antigen. Examine by a suitably sensitive

method such as radio-immunoassay; hepatitis-B surface
antigen is not detected.
Description. A white or almost white, amorphous powder.
Abnormal toxicity (2.2.1). Complies with the test, using a

solution containing 2,500 Units in 0.5 ml of saline solution.
Identification
Thromboplastic contaminants. Dissolve suitable quantities

of the substance under examination in barbitone bziffer
pH 7.4 to obtain solutions containing 5000, 2500, 1250, 625
and 312 Units per ml. Into each of six haemolysis tubes, 1 cm
in internal diameter, place 0.1 ml ofcitrated rabbit plasma. Add
0.1 ml of one of each of the solutions of the substance under
examination to each offive ofthe tubes and 0.1 ml of barbitone
bziffer pH 7.4 to the sixth (control). Incubate the six tubes at
25 O,SOfor 5 minutes and then add 0.1 ml ofa 0.3675 percent
w/v solution of calcium chloride. Using a stop-watch,
measure the clotting time for each solution and the control.
Plot the shortening ofthe recalcification time (control clotting
time minus clotting time for each solution) against log
concentration. Extrapolate the best-fitting straight line through
the five points until it reaches the log concentration axis. The
urokinase activity at the intersection point represents the limit
concentration for clotting activity (zero clotting activity). The
zero clotting activity is not less than 150 Units per ml.
Urokinase contains not less than 70,000 Units of urokinase

activity per mg of protein.
A. Place 0.5 ml ofcitrated human plasma and 0.5 ml ofcitrated

bovine plasma in two separate haemolysis tubes maintained
in a water-bath at 37. To each tube add 0.1 ml ofa solution of
the substance under examination containing 1000 Units per
ml in phosphate buffer pH 7.4 and 0.1 ml of a solution of
thrombin containing 20 Units per ml in phosphate buffer
pH 7.4 and shake immediately; in both tubes, a clot forms and
lyses within 30 minutes.
B. Carry out a suitable immunodiffusion test.
Tests
Appearance ofsolution.AO.1 percentw/v solution in water is
clear (2.4.1), and colourless (2.4.1).
Molecular fractions. Determine by size-exclusion
Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of 0.02 M phosphate buffer pH 8.0.
a column 90 cm x 16 mm, packed with a cross-linked
dextran suitable for fractionation ofproteins in the range
of molecular weight from 4000 to 150,000 (such as
Sephadex 0-100), at 5,
- mobile phase: 1.75 per cent w/v solution of sodium
chloride in 0.02 M phosphate bziffer pH 8.0,
- flow rate. 6 ml per hour,
- spectrophotometer set at 280 nm.
Vasoactive substances. Anesthetise a rabbit by intraperitoneal

injection of 0.15 g of phenobarbitone sodium per kg of body
weight. Dissolve in normal saline solution a sufficient
quantity ofthe substance under examination to give a solution
containing 40,000 Units per m!. Administer by intravenous
infusion at a rate of 1 ml per minute a sufficient volume ofthe
solution of the substance under examination such that the
dose is 20,000 Units per kg ofbody weight. Measure the arterial
pressure and heart rate at intervals of 15 minutes for 5 hours
after the infusion. No significant and lasting alterations in
arterial pressure or heart rate are produced, except those arising
from the effects of the anaesthetic.
2279
IP 2010
UROKJNASE
Assay. The potency ofurokinase is determined by comparing

its ability to activate human plasminogen to form plasmin with
that of the Standard Preparation. The plasmin generated is
determined by measurement of the time taken to lyse a fibrin
clot under the conditions of a suitable method of Assay.
The Standard Preparation is the 1st International Reference
Preparation for Urokinase, human, established in 1968,
consisting of partially purified freeze-dried urokinase from
human urine with lactose (supplied in ampoules containing
4800 Units ofurokinase activity) or another suitable preparation
the activity of which has been determined in relation to the
International Reference Preparation.
containing 3 per cent w/v solution of bovine albumin and

0.1 ml of a solution containing 20 Units of thrombin per ml.
Place the tubes in a water-bath at 37 and allow to stand for
2 minutes to attain temperature equilibrium. Using an automatic
pipette, introduce into the bottom ofthe first tube 0.5 ml of a
1.0 per cent w/v solution of bovine euglobulin fraction
ensuring mixing. At 5-second intervals introduce successively
into the remaining tubes 0.5 ml ofa 1.0 per cent w/v solution of
bovine euglobulin fraction. Using a stop-watch, measure for
each tube the time in seconds that elapses between the
addition of the euglobulin and the lysis of the clot.
Using the logarithms of the lysis times, calculate the result of
The estimated potency is not less than 90.0 per cent and not
more than 111.0 per cent of the stated potency.
Method
Unless otherwise prescribed, use phosphate btiffer pH 7.4
containing 3 per cent w/v solution of bovine albumin for the
preparation of solutions and dilutions.
Prepare a solution of the Standard Preparation containing
1000 Units ofurokinase activity per ml and prepare a solution
of the preparation under examination expected to have the
same concentration; keep the solutions in ice and use within
6 hours. Prepare three 1.5-fold serial dilutions ofthe solution
ofthe Standard Preparation so that the longest clot-lysis time
is less than 20 minutes and the shortest clot-lysis time is greater
than 3 minutes. Prepare three similar dilutions ofthe solution
of the preparation under examination. Keep the solutions in
ice and use within 1 hour. Using 24 tubes 8 mm in diameter,
label the tubes SJ, S2, S3 for the dilutions of the Standard
Preparation and T I, T2, T3for the dilutions of the preparation
urider examination, allocating four tubes to each dilution. Place
the tubes in ice. Into each tube introduce 0.2 ml of the
appropriate dilution, 0.2 ml of phosphate btiffer pH 7.4
The fiducial limits oferror are not less than 80 per cent and not
more than 125 per cent of the stated potency.
Urokinase intendedfor use in the manufacture ofparenteral

preparations or ophthalmic preparations complies with the
following additional requirements.
Unit per Unit ofurokinase activity.
The containers should be sterile, tamper-evident and sealed
so as to exclude micro-organisms.
Labelling. The label states (1) the number ofUnits ofuroldnase
activity in the container; (2) the number ofUnits ofuroldnase
activity per mg of protein; (3) the storage conditions; (4)
whether or not it is intended for use in the manufacture of
parenteral preparations or ophthalmic preparations.
2280
MONOGRAPHS
v
2283
2283
2284
2285
2286
2287
2287
2289
2291
2291
2292
2294
2294
2295
2296
Valproate Injection
Valproic acid
Valproic Acid Capsules
ValproicAcid Oral Solution
Valsartan
Valsartan Tablets
Valsmian and Hydrochlorothiazide Tablets
Vancomycin Hydrochloride
Vancomycin Capsules
Vancomycin Intravenous Infusion
Vancomycin Oral Solution
Vanillin
Vasopressin Injection
Verapamil Hydrochloride
Verapamil Injection
2297
2298
Verapamil Tablets
Vmblastine Sulphate
2299
2300
2302
2303
2304
2305
2306
2306
2307
2308
2308
Vmblastine Injection
Vmcristine Sulphate
Vmcristine Injection
Vmorelbine Tartrate
Vmorelbine Injection
Vitamin A Concentrate Oil
Vitamin A Concentrate Powder
Water-Miscible Vitamin AConcentrate
Vitamins A and D Capsules
ConcentratedVitamin D Solution
Concentrated Vitamins A and D Solution
2281
VALPROIC ACID
IP 2010
Valproate Injection
Valproate Sodium Injection
Valproate Injection is a sterile solution of sodium valproate in
Water for Injections with one or more suitable buffering or
sequestering agents.
Inject 2111 ofreference solution (b). The test in not valid unless
the resolution between the peaks due to valproic acid and
biphenyl peaks is not less than 3.0 and the relative standard
Inject 2 III of the test solution and reference solution (a).
Calculate the content of C SH I6 0 2 in the injection.
Valproate Injection contains not less than 90.0 per cent and
not more than 110.0 per cent ofthe stated amount ofvalproic
acid, C SH I6 0 2
Storage. Store at a temperature not exceeding 30 and in single

dose containers, preferably of Type 1 glass.
Labelling. The label states the amount of any buffering or

sequestering agents used.
Identification
Valproic Acid
B. Gives the reaction of sodium salts (2.3.1).
Tests
pH (2.4.24).7.0 to 9.0.
Mol. Wt. 144.2

Units per ml.
Valproic acid is 2-propylpentanoic acid.
Valproic acid contains not less than 99.0 per cent and not
more than 101.0 percent of C SH I6 0 2

Description. A colourless or very slightly yellow liquid, slightly

viscous.

biphenyl in dichloromethane.
Identification

400 mg ofvalproic acid with 20 ml of5 per cent v/v solution of
hydrochloric acid and add 50 ml ofinternal standard solution.
Shake for 1 hour, ifemulsion fonus, stir with a glass rod. Take
5 ml of the organic layer and dilute to 50 ml with
dichloromethane.
Reference solution (a). A 0.8 per cent w/v solution ofvalproic
acid RS in internal standard solution.
to 50 m1 with dichloromethane.
phase G34 on 80 to 100 mesh support S lA,
temperature:
column. 155,
injection port at 275 and detector at 300,
- a flame ionization detector,
- flow rate. 20 ml per minute using nitrogen as the carrier
gas.

Compare the spectrum with that obtained with valproic acid
RS or with the reference spectrum ofva1proic acid.
B. Detenuine by thin-layer chromatography (2.4.17), coating

Mobile phase. A mixture of50 volumes of ether and 50 volumes

of dichloromethane.
Reference solution. A 1.0 per cent w/v solution of valproic
acid RS in methanol.
Apply 2 III ofeach solution. Allow the mobile phase to rise 15
cm. Dry the plate in air and spray with bromocresol green
2283
VALPROIC ACID
IP 2010
C. To 1 ml add 3.0 m1 ofdilute sodium hydroxide solution. Add

3.0 ml of water and 1.0 m1 of a 10 per cent w/v solution of
cobalt nitrate. A violet precipitate is formed, filter it. The
precipitate dissolves in di~hloromethane.
Tests
Appearance of solution. A20.0 per cent w/v solution in dilute
sodium hydroxide is clear (2.4.1) and not more intensely

Assay. Weigh accurately about O.lg and dissolve in 25 m! of
ethanol (95 per cent). Add 2 ml of water. Titrate with 0.1 M
CgH I60 2

(2.4.13).

butyric acid in heptane.
Valproic Acid Capsules contain not less than 90.0 per cent
valproic acid, CgH I6 0 2

under examination in the internal standard solution. Dilute 1.0
ml ofthis solution to 10 ml with heptane.
Reference solution. Dissolve 20 mg of the substance under
examination and 20 mg of 2-(l-methylethyl)pentanoic acid
RS (valproic acid impurity C RS) in 10 ml of heptane. Dilute
1.0 ml ofthis solution to 10 ml with heptane.
- a fused-silica capillary column 30 m x 0.53 mm, coated
with wide-bore rnacrogol20000 2-nitroterephthalate (0.5
11m),
- temperature.
column 130 from 0-10 minutes and 130 - 190 from 1030 minutes
- inlet port and detector 220,
- flow rate. 8 ml per minute ofthe carrier gas (helium).
Inject I III ofthe reference solution. The test is not valid unless
the resolution between the peaks due to valproic acid impurity
C and valproic acid is not less than 3.0.
Inject I III of the test solution and the reference solution. In
the chromatogram obtained with the test solution the area of
peak due to any impurity, for each impurity is not more than
0.1 times the area ofthe peak due to the internal standard (0.1
per cent). The sum of areas of all the secondary peaks is not
more than 0.3 times the area of the peak due to the internal
standard (0.3 per cent). Ignore any peak with an area less than
0.01 times the area of the peak due to the internal standard
(0.01 percent).
Heavy metals (2.3.13). Dissolve 2.0 g in 20 ml ethanol (80per
c::enJL!!'DJ1.J!~e.J 2.ml
of.Jhes.olutioD...J'he.f.e.s.uhings.olution
complies with the limit test for heavy metals, Method D (20
ppm). Prepare the standard using lead standard solution (2
ppm Pb) obtained by diluting lead standard solution (100
ppm Pb) with ethanol (80 per cent).
Identification
A. In the Assay, the retention time ratio of the principal peak
to the internal standard peak in the chromatogram obtained
with the test solution and reference solution, are not more
than 2.0 per cent.
B. Disperse a quantity of the content of capsules containing
about 250 mg of valproic acid, add 20 ml of 1 M sodium
hydroxide, shake and allow the layers to separate. Filter the
aqueous layer, add 4 ml of hydrochloric acid, mix and extract
with 40 ml of n-heptane. Filter the n-heptane layer through
glass wool and evaporate the solvent completely on a steam
bath with the aid of a current of air. Transfer 2 drops of the
residue to a test tube containing 0.5 ml each of2 per cent v/v
ofpotassiunm iodide solution and 4 per cent v/v ofpotassium
iodate solution and mix, a yellow colour is produced.
Tests
Apparatus No. I,
Medium. 900 ml ofa 0.5 per cent w/v solution ofsodium lauryl
sulphate in simulated intestinal fluid prepared by dissolving
6.8 g of monobasic sodium phosphate in 250 ml water, mix and
77 ml of 0.2 M sodium hydroxide, add 500 ml of water. Adjust
the pH to 6.8 with 0.2 M sodium hydroxide or 0.2 M
hydrochloric acid dilute to 1000 ml with water adjust to pH
7.5 with 5 M sodium hydroxide,
DeterrlJ.ille by gas cll1:omat()grapl1y (2.4.13) as described under
Assay, using the following solutions.
Test solution. To 10 ml ofthe filtrate, add about 3.0 g ofsodium

chloride, and mix on a vortex mixer for 5 minutes. Add about
2284
VALPROIC ACID ORAL SOLUTION
IP 2010
1.0 ml of6 M hydrochloric acid and 5.0 ml ofintemal standard

solution, shake and allow to separate, remove the n-heptane
layer and filter. Discard the aqueous layer.
Reference solution. Weigh accurately a quantity of valproic

acid RS and dilute with the dissolution medium to obtain a
solution having a concentration similar to that of the test
solution. Take 10 ml of this solution and proceed as in test
solution beginning with the words "add about 3.0 g........".
Calculate the content ofC gH I6 0 2 in the capsule.
D. Not less than 85 per cent of the stated amount ofC gH 160 2
Assay. Detennine by gas chromatography (2.4.13).
Test solution. Take 20 capsules in a container and add 150 ml

of dichloromethane, cool in a solid carbon dioxide-acetone
mixture until the contents have solidified. If necessary,
transfer the mixture of capsules and dichloromethane to a
blender jar and blend until all the solids are reduced to fine
particles. Transfer the mixture to a 500-ml flask, add n-heptane
to volume, mix and allow the solids to settle. Transfer an
accurately measured volume ofthis solution, containing about
250 mg ofvalproic acid, to a 100 ml flask, dilute with n-heptane
to volume and mix. Transfer 5.0 ml to a container equipped
with a closure. Add 2.0 ml of internal standard solution, close
the container and mix.
acid RS in n-heptane. Transfer 5.0 ml to a container equipped
with a closure. Add 2.0 ml of internal standard solution, close
the container and mix.
biphenyl in n-heptane.
a glass column 1.8 rn x 2mm, packed with 10 per cent
phase 034 on 80 to 100 mesh support SlA,
temperature:
column. 150,
injection port and detector. 250,
flow rate. 40 ml per minute using nitrogen as carrier gas.
Inject 2 f.Ll ofthe reference solution. The test is not valid unless
the resolution between the peak due to valproic acid and
biphenyl is not less than 3.0 and the relative standard deviation
for replicate injections is not more than 2.0 per cent. The
relative retention time with reference to valproic acid for
biphenyl is about 2.0.
Inject 2 f.Ll of the test solution and the reference solution.
Calculate the content of C gH I6 0 2 in the capsules.
Storage. Store protect from moisture, at a temperature not
exceeding 30.
Valproic Acid Oral Solution

Valproic Acid Oral Solution contains not less than 90.0 per
valproic acid, CgH I6 0 2
Identification
A. In the Assay, the retention time ratio of the principal peak

to the internal standard peak in the chromatogram obtained
with the test solution and reference solution is not more than
2.0 per cent.
B. Dissolve a volume of solution containing about 250 mg of .
valproic acid in 40 ml of water and 2.0 ml of hydrochloric
acid. Mix and extract with 40 ml of n-heptane. Filter the nheptane layer through glass wool into a beaker, and evaporate
the solvent completely on a steam bath with the aid ofa current
of air. Transfer 2 drops ofthe residue to a test tube containing
0.5 ml each 2 per cent v/v ofpotassium iodide solution and 4
per cent v/v solution ofpotassium iodate solution and mix; a
Tests
pH (2.4.24).7.0 to 8.0.
Test solution. Dissolve a volume of solution containing about

250 mg of valproic acid in 40 ml of water, add 2.0 ml of
hydrochloric acid and extract with 80 ml of n-heptane until
the aqueous layer is clear. Filter the n-heptane layer through
glass wool with small portions of n-heptane, add the rinsings
to the flask, dilute to 100.0 ml with n-heptane and mix.. Transfer
5.0 ml to a container equipped with a closure. Add 2.0 ml ofthe
internal standard solution, close the container and mix.
acid RS in n-heptane. Transfer 5.0 ml to a container equipped
with a closure. Add 2.0 ml of internal standard solution and
mix.
biphenyl in n-heptane.
glass column 1.8 m x 2 mrn, packed with 10 per cent
phase 034 on 80 to 1010 mesh support SlA,
temperature:
column. 150,
injection port and detector at 250,
flow rate. 40 ml per minute using nitrogen as carrier gas.
2285
VALSARTAN
IP 2010
Inject 2 III ofthe reference solution. The test is not valid unless
the resolution between valproic acid and biphenyl is not less
injections is not more than 2.0 per cent. The relative retention
time with reference to valproic acid for biphenyl is about 2.0.
Inject 2 III each ofthe test solution and the reference solution.
Calculate the content of C SH I6 0 2in oral solution.
Valsartan
H3 C
CH 3
H3C~NXCOOH f'J=tj
~

valsartan RS in the mobile phase.
Reterence solution (b). Dilute 1 ml ofreference solution (a) to
as Nucleosil),
volumes of acetonitrile and 0.1 volume of glacial
acetic acid,
i~ection volume. 10 Ill.

HN ..-;N
Mol. Wt. 435.5

Valsartan is N-pentanonyl-N-[2' -(lH-tetrazol-5-yl)biphenyl4-ylmethyl]-L-valine.
Valsartan contains not less than 98.0 per cent and not more
than 102.0 per cent ofC24H29Ns03, calculated on the anhydrous
basis.
Dose. 80-160 mg daily.
chromatogram three times the retention time of the principal
the area of any secondary peak is not more than 0.2 times the
solution (b) (0.2 per cent) and the sum of areas of all the
(b)(0.5 per cent).

Water (2.3.43). Not more than 2.0 per cent, detennined on 1 g.
Identification

Compare the spectrum with that obtained with valsartan RS
or with the reference spectrum of vaIsartan.
0.001 percentw/v solution in 0.1 Mhydrochloricacidexhibits
a maximum at about 248 nm.
Tests

Reference solution. A 0.005 per cent w/v solution ofvalsal'tan
as Purospher 18e),
Specfiic optical rotation (2.4.22). ---- 60 to ----~67;deterliimea ill

1.0 pet cent 'ii/v sohition in 111ethanol.
.
(2.4.14).
2286
_~~Y9Jume~_Q.fQ..~{Qlll([ileJ!J1dj1Lv_olum5).Qf glClI:1CllClf:.rgLc.
acid,
VALSARTAN AND HYDROCHLOROTHIAZIDE TABLETS
IP 2010

valsartan RS in the mobile phase.
Reference solution (b). Dilute 1.0 ml of reference solution (a)

Calculate the content ofCz4Hz9Ns03.

Valsartan Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of valsartan,
CZ4Hz9Ns03.
.

secondary peaks is not more than the area of the principal
(b) (1.0 per cent).
Identification
Valsartan Tablets

ofValsartan with 40 ml of methanol, filter and evaporate the
filtrate to dryness. On the residue, determine by infrared
with that obtained from valsartan RS or with the reference
spectrum of valsartan.
Tests
Apparatus No.1,
(2.4.7). Calculate the content of CZ4Hz9Ns03 in the medium
concentration of valsartan RS prepared by dissolving in
minimum amount of methanol and diluted with the dissolution
medium.
Cz~z9Ns03'
Related Substances. Determine by liquid chromatography

(2.4.14).
containing 25 mg ofValsartan with 15 Jill of mobile phase and
dilute to 25.0 ml with the mobile phase, filter.

a quantity ofpowder containing 50 mg ofValsartan, disperse
in 25 ml ofthe mobile phase and dilute to 100.0 ml with the
mobile phase and filter. Dilute 5.0 ml ofthis solution to 50.0 ml
Reference solution. A 0.005 per cent w/v solution of vaIsartan
- mobile phase: a mixture of 50 volumes of water, 50
volumes of acetonitrile and 0.1 volume ofglacial acetic
acid,
Calculate the content ofCz4Hz9Ns03 in the tablets.
Valsartan and Hydrochlorothiazide

Tablets
Valsartan and Hydrochlorothiazide Tablets contain not less
stated amount of valsartan, CZ4Hz9Ns03 and
hydrochlorothiazide, C7HsCIN304SZ.
2287
VALSARTAN AND HYDROCHLOROTHIAZIDE TABLETS
IP 2010
Usual Streugth . Valsartan 80 mg and Hydrochlorothiazide

12.5 mg; Valsartan 60 mg and Hydrochlorothiazide 12.5 mg.
Identification

volumes of dehydrated alcohol and 10 volumes of a 25 per
cent w/v solution of ammonium hydroxide.
containing about 20 mg of valsartan in 100 ml of acetone,
sonicate for 15 minutes and filter.
each of valsal'tan RS and hydrochlorothiazide RS in acetone.
Apply to the plate 2 j..ll of each solution. Allow the mobile
phase to rise 7 cm. Dry the plate in a current of warm air and
examine in ultraviolet light at 254 nm. The principal spot in the
B. In the Assay, the retention time ofthe principal peak in the
Tests

the medium ifnecessary, at the maximum at about 250 nm for
valsartan and 272 urn for hydrochlorothiazide (2.4.7). Calculate
the content of CZ4Hz9Ns03 and C7HgCIN304SZ in the medium
concentration of valsartan RS and hydrochlorothiazide RS
in the same medium.
Calculate the content of valsartan dissolved by the formula:
xlZ,SOO
(AI percelltV2sUIlnl X Alpe1'ce11l H 272l1m ) -(AI percentV2721lnl X Al percent H 2SO /lm )
Calculate the content of hydrochlorothiazide dissolved by

the formula:
(AT! x Alpercell/V"'"m) -(AT2x AlpercentV"'"m)
xgO,OOO
(AI percent H 272nm X Al percent V2S0mn) - (AI percent H2S0nnl X AI percent V21211m )
where,
ATI
AT2
absorbance of the test solution at 272

urn,
= absorbance of the test solution at 250
=
lllll,
D. Not less than 80 per cent ofthe stated amount ofCz4Hz9Ns03

and C7HgCIN304SZ'
(2.4.14), as described under Assay using the following
solutions.
Solvent mixture. Equal volumes of acetonitrile and water.

containing about 62.5 mg ofHydrochlorothiazide with 5.0 ml
of water and 100 ml ofsolvent mixture, sonicate for 15 minutes
and shake for 30 minutes. Dilute to 250 ml with the solvent
mixture, centrifuge and dilute 25.0 ml ofthe supematantto200
w/v of benzothiadiazine impurity A RS, 0.006 per cent w/v of
hydrochlorothiazide RS, 0.008 per cent w/v of valsartan RS
and 0.02 per cent w/v of valsartan impurity B RS in the
solvent mixture.
Apparatus No.1,
(ATZx AI per cellI H"'"m) - (AT! x AI percent H",,,,,,)
Al percent Vm,llll = absorptivity (l per cent, 0.2 cm, 272 urn)

ofvaIsartan in medium,
Al per cent V250 11111 = absorptivity (1 per cent, 0.2 cm, 250 urn)
ofvalsartan in medium,
Al per cent H mlllll = absorptivity (l percent, 0.2 cm, 272 urn)
ofhydrochlorothiazide in medium,
Al per cent H 250 11/11 = absorptivity (l per cent, 0.2 cm, 250 urn)
ofhydrochlorothiazide in medium,
Reference solution (c). Dilute 10 ml of reference solution (b)

Inject reference solution (b) and (c). The test is not valid unless
in the chromatogram obtained with reference solution (b), the
resolution between va1sartan impurity Band valsartan and
between benzothiadiazine impurity A and hydrochlorothiazide
is not less than 1.4. In the chromatogram obtained with
reference solution (c) the relative standard deviation for
replicate injections is not more than 10 per cent.
Inject the test solution, reference solution (a), (b) and (c). The
area of the peak due to benzothiadiazine impurity A is not
more than 1.0 per cent; the area of any secondary peak other
than valsartan impurity A is not more than 0.2 per cent; and
the sum of areas of all the secondary peaks other than
valsartan impurity A is not more than 1.3 per cent. (Valsartan
impurity A is the enantiomer of valsartan and coelutes with
valsartan in this test.)
Unifofinitj of content. Comply with the tests shitedTiiider

Tablets.
under Assay using the following solution as the test solution.
2288
VANCOMYCIN HYDROCHLORIDE
IP 2010
Solvent mi.;r:ture. Equal volumes of acetonitrile and water.

Test solution. Disperse 1 tablet with 5.0 ml of water and about
100 ml ofsolvent mixture, sonicate for 15 minutes and dilute to
200 ml with the solvent mixture. Centrifuge a portion of this
solution and dilute a volume of the clear supernatant with the
solvent mixture to obtain a 0.02 per cent w/v solution of
valsartan.
Calculate the content ofC24H29Ns03 and C7HgCIN304S2 in the

tablet.
than 2.0 per cent.
Calculate the content ofC24H29Ns03 and C7HgCIN304S2 in the
tablet.

Solvent mixture. Equal volumes of acetonitrile and water.
containing about 62.5 mg ofHydrochlorothiazide with 5.0 ml
of water and 100 ml ofsolvent mixture, sonicate for 15 minutes
and shake for 30 minutes. Dilute to 250 ml with the solvent
mixture, centrifuge and dilute 25.0 ml ofthe supernatantto 200
ml with the solvent mixture. Dilute a suitable volume of this
solution with the solvent mixture to obtain a 0.02 per cent w/v
solution of VaIsartan.
Reference solution. Transfer about 12.5 mg of
hydrochlorothiazide RS to a 200 ml volumetric flask and add
about 12.5 J mg of vaIsartan RS, J being the ratio ofthe stated
amount, in mg, of valsartan to the stated amount, in mg, of
hydrochlorothiazide per tablet. Add about 100 ml of solvent
mixture, sonicate for 15 minutes, dilute to volume and mix.
Transfer 25 ml of this solution to a 50 ml flask, dilute with
solvent mixture to volume and mix. Dilute a suitable volume of
this solution with the solvent mixture to obtain a 0.02 per cent
w/v solution of valsartan RS.
octadecylsilane bonded to porous silica
(5Ilm),
- mobile phase: A. a mixture of 90 volumes of water,
10 volumes of acetonitrile and 0.1 volume of
trifluroacetic acid,
B. a mixture of90 volumes of acetonitrile,
10 volumes of water and 0.1 volume of trifluroacetic
acid,
below,
Time
Mobile phase A
Mobile phase B
(per cent v/v)
(in min.)
(per cent v/v)
0-25
90 -710
10-790
25-27
10-790
90-710
27-40
90
10
, Hel
Mol.Wt. 1485.7
Vancomycin Hydrochloride is a mixture ofrelated
glycopeptides, consisting principally of the
monohydrochloride of(3S,6R,7R,22R,23S,26S,30aS,36R,
38aR)-3-(2-amino-2-oxoethyl)-44-[[2-0-(3-amino-2,3,6trideoxy-3-C-methyl-{3-L-lyxo-hexopyranosyl)-{3-Dglucopyranosyl]oxy]-10,19-dichloro-7,22,28,30,32pentahydroxy-6-[[(2R)-4-methyl-2(methylamino)pentanoyl]amino]-2,5,24,38,39-pentaoxo2,3,4,5,6,7,23,24,25,26,36,37,38,38a-tetradecahydro-22H8,11: 18,21-dietheno-23,36-(iminomethano)-13,16:31,35dimetheno-lH, 13H-[1 ,6,9]oxadiazacyclohexadecinol[4,5-m]
[10,2,16]benzoxadiazacyclotetracosine-26-carboxylic acid
(vancomycin B).
2289
VANCOMYCIN HYDROCHLORIDE
IP 2010
It is produced by certain strains of Amycolatopsis orientalis

or obtained by any other means.
Time
(in min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
Vancomycin Hydrochloride contains not less than 1050 ill

per mg ofC66H7sClzNg024, RCI, calculated on the anhydrous
basis and not less than 93.0 per cent of vancomycin B.
0-13
100
13-22
100-70
0-7100
22-26
100
26-27
100
Description. A white or almost white hygroscopic powder.
Identification
A. In the test for Vancomycin B, the principal peak in the
chromatogram obtained with test solution (a) corresponds to
Inject test solution (b), (c) and the reference solution. The test
is not valid unless the resolution between the 2 principal peaks
not less than 5.0, signa1-to-noise ratio for the principal peak in
the chromatogram obtained with test solution (c) is not less
than 5.0 and tailing factor for the peak due to vancomycin in
the chromatogram obtained with the test solution (b) is not
more than 1.6.
Tests
Inject test solution (b) and the reference solution.
Appearance ofsolution. Al 0.0 per cent w/v solution in water

is clear (2.4.1), absorbance ofthe solution at 450 TIm (2.4.7) is
not more than 0.1.
Calculate the content of vancomycin B hydrochloride.

in carbon-dioxide ji-ee water.
Vancomycin B. Determine by liquid chromatography (2.4.14).
NOTE-Use freshly prepared solutions.
examination in 100 m1 ofthe mobile phase A.
Test solution (b). Dilute 2.0 ml oftest solution (a) to 50 m1 with
mobile phase A.
Test solution (c). Dilute 0.5 ml oftest solution (b) to 20 ml with
mobile phase A.
Reference solution. A 0.5 per cent w/v solution of vancomycin
hydrochloride RS in water. Heat at 65 for 24 hours, allow to
cool.
- mobile phase: A. a lllixture of 1 volume of
tetrahydrofilran, 7 volumes of acetonitrile and 92
volumes ofbuffer solution prepared by diluting I ml of
triethylamine to 499 ml with watel; adjust the pH to 3.2
B. a mixture of I volume of
tetrahydrofilran, 29 volumes of acetonitrile and
70 volumes ofbuffer solution,
. . . JdiI!~atgmdientpmgm111J1le_usiI!gJhe_c_oI!ditioI!sgi_v_eI!
bel()~,


(2.4.14) as described under Vancomycin B with the following
modifications.
Inject test solution (a), (b) and (c). In the chromatogram
peak due to N-demethylvancomycin B (Vancomycin
Hydrochloride impurity A), (4S, 7R,8R,23R,24S,27 S,
31aSa,37R,39aR)-45-[[2-0-(3-amino-2,3,6-trideoxy-3-C-methyla-L-Iyxo-hexopyranosyl)-~ -D-glucopyranosyl]oxy]-11,20dichloro-8,23,29,31 ,33-pentahydroxy-7-[[(2R)-4-methyl-2(methylamino)pentanoyl]amino]-2,6,25,39,40-pentaoxo1,2,3,4,5,6,7,8,24,25,26,27,37,38,39,39a-hexadecahydro-23H9,12: 19,22-dietheno-24,37-(iminomethano)-14,17:32,36dimetheno-14H-[ 1,6,1 O]oxadiazacycloheptadecino[4,5m][10,2,16]benzoxadiazacyclotetracosine-4,27-dicarboxylic
acid ([ ~ ASp3] vancomycin B) (Vancomycin Hydrochloride
impurity B), aglucovancomycin B (Vancomycin Hydrochloride
impurity C) and desvancosaminylvancomycin B (Vancomycin
Hydrochloride impurity D) is not more than 4.0 per cent. The
sum ofarea ofall the secondary peaks is not more than 7.0 per
cent. Ignore any peak with an area less than the area of the
principal peak in the chromatogram obtained with test solution
(cHO.1 percent).

Unit per mg.
Vancomycin Hydrochloride intended for use in the

manufacture ofparenteral preparations without a filrther
appropriate sterilization procedure complies with the
2290
VANCOMYCIN INTRAVENOUS INFUSION
IP 2010

0.5 g.
Method B (2.2.10) using vancomycin hydrochloride RS.
If it is intended for use in the manufacture of parenteral
preparations, the container should be sterile, tamper-proof
and sealed so as to exclude micro-organisms.
Vancomycin Capsules
Vancomycin Hydrochloride Capsules
Vancomycin Capsules contain not less than 90.0 per cent and
not more than 115.0 per Ctlnt of the stated amount of
vancomycin, C66H7sC12N9024.

C66H7sClzN9024.
Method A (2.2.10) on a solution prepared in the following
manner.
Place not less than 5 capsules in a glass blender jar and blend
at high speed for 3 to 5 minutes with a sufficient volume of
Buffer No.3 to yield a stock solution having a convenient
concentration of vancomycin. Dilute an accurately measured
volume'ofthis stock solution with Buffer No.3 to obtain a test
dilution having a concentration assumed to be equal to the
median dose level of the standard.
Identification
Mobile phase. A mixture of 6 volumes of butyl alcohol, 4

voli.nnes of water and 3 volumes of pyridine.
Vancomycin Intravenous Infusion is a sterile solution of

Vancomycin Hydrochloride in Water for Injections. It is
prepared by dissolving Vancomycin Hydrochloride for
Intravenous Infusion in Water for Injections and then diluting
with the requisite volume of a suitable diluent in accordance
with the manufacturer's instructions.
Test solution. Disperse the content of capsules containing

about 100 mg ofvancomycin in 100.0 ml ofwater.
The intravenous infilsion complies with the tests stated under

Parenteral Preparations.

Storage. Vancomycin Intravenous Infusion should be used

immediately after preparation but, in any case, within the period
recommended by the manufacturer when prepared and stored
strictly in accordance with the manufacturer's instructions.
Determine by paper chromatography (2.4.15), using a suitable

sheet of chromatographic filter paper.
Apply to the plate 5 l.d of each solution. Develop by

descending chromatography for about 7 hours. After
development, dry the paper and place it on an inoculated agar
surface of sufficient area to accommodate the paper and
prepared for vancomycin assay described in microbiological
assay ofantibiotics (2.2.10) except to use Medium B. Remove
the paper from the agar surface after 30 minutes and incubate
the agar medium at 37 for 18 hours; clear zones of inhibition
are produced at corresponding positions on the two
chromatograms.
Tests
Apparatus No.2,
Carry out the method as described under Assay.
Usual strengths. 500 mg; 1 g.
Vancomycin Hydrochloride for Intravenous

Infusion
Vancomycin Hydrochloride for Intravenous Infusion is a sterile
material consisting of Vancomycin Hydrochloride with or
without excipients. It is filled in a sealed container.

Vancomycin Hydrochloride for Intravenous Infusion contains
not less than 88.0 per cent ofVancomycin B.
Identification
A. In the test for vancomycin B, the retention time of the
2291
VANCOMYCIN INTRAVENOUS INFUSION
IP 2010

Tests
pH (2.4.24).2.5 to 4.5, determined on a 5.0 per centw/v solution
ofVancomycin Hydrochloride.
Appearance of solution. A 10.0 per cent w/v solution of
Vancomycin Hydrochloride is clear (2.4.1) and the absorbance
ofthe solution at 450 urn (2.4.7) is not more than 0.1.
NOTE-Use the solutions within 4 hours ofpreparation.

Test solution (a). Dissolve a quantity of the substance under
examination in sufficient mobile phase A to produce a solution
containing 2,000 ill ofvancomycin per ml.
Test solution (b). Dilute I ml oftest solution (a) to 25 ml with

mobile phase A.
Test solution (c). Dilute 1 ml oftest solution (b) to 40 ml with

mobile phase A.

vancomycin hydrochloride RS in water. Heat at 65 for
24 hours and allow to cool.
(5 Ilm) (Such as Hypersil ODS),
mobile phase: A. a mixture of 1 volumes of
92 volumes ofbuffer solution prepared by diluting I ml
of triethylamine to 500 ml with water, adjust the pH to
B. a mixture of I volume of
70 volumes of buffer solution,
below,
Time
(in min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
0-13
100
13-21
100-*>
0-400
21-25
100
25-35
100
solution (c), signal-to- noise ratio of the principal peak is not

less than 5; the tailing factor of the vancomycin peak in the
chromatogram obtained with test solution (b) is not more than
1.6 and the resolution between the two principal peaks in the
chromatogram obtained with the reference solution is not less
than 5.0.
(2.4.14), using test solutions (a), (b) and (c) as described under
Vancomycin B.
Chromatographic system as described under Vancomycin B.
In the chromatogram obtained with test solution (a) calculate
the content of each impurity.
The content of any impurity is not more than 4.0 per cent and
the sum of the contents of any such impurities is not more
than 12.0 per cent. Ignore any peak with an area less than that
of the principal peak in the chromatogram obtained with test
0.5g.
sealed container in tris-chloride bufferpH 7.4 prepared using
water BET to give a solution containing 9000 Units of
vancomycin per ml. Carry out the test on the resulting solution;
the maximum allowable endotoxin concentration ofthe solution
is 2.5 Units of endotoxins per ml. Carry out the test using the
maximum valid dilution of the prepared solution calculated
from the declared sensitivity of the lysate used in the test.
Assay. Determine the weight ofthe contents often containers
as described in the test for Uniformity of weight under
Parenteral Preparations (Powders for Injection).
Determine on the mixed contents of ten containers by the
microbiological assay ofantibiotics, Method B (2.2.10).
cent of the stated number of Units.
Labelling. The label states (1) the total number of Units of
vancomycin in the container and (2) the number of Units of
vancomycin per mg.

Vancomycin Oral Solution is a solution of Vancomycin
Hydrochloride in a suitable flavoured vehicle. It is prepared
by dissolvingVancofiiyciiiHydrochlofidefofOfalSolutioiiifi
the requisite amount of a suitable diluent.
Inject the reference solution, test solution (b) and (c). The test
is not valid unless in the chromatogram obtained with test
The oral solution complies with the tests stated under Oral
Liquids.
2292
IP 2010
VANCOMYCIN ORAL SOLUTION
mobile phase: A. a mixture of4 volumes oftriethylamine

diluted with 1996 volumes of water, adjusted to pH 3.2
with orthophosphoric acid (solution A), 10 volumes
of tetrahydrofitran and 70 volumes of acetonitrile to
920 volumes of solution A,
B. a mixture of 10 volumes of
tetrahydrojitran, 290 volumes of acetonitrile and 700
volumes of solution A,
below,
Storage. Vancomycin Oral Solution should be used

recommended by the manufacturer when prepared and stored
strictly in accordance with the manufacturer's instructions.
Vancomycin Hydrochloride for Oral Solution

Vancomycin Hydrochloride for Oral Solution is a dry powder
consisting of Vancomycin Hydrochloride with or without
excipients. It is filled in a sealed container.

requirements stated under Powders and Granules for Oral
Solutions and Oral Suspensions stated under Oral Liquids
and with the following requirements.
Vancomycin Hydrochloride for Oral Solution contains not less
than 88.0 per cent ofVancomycin B.
Identification
A. In the test for Vancomycin B, the retention time of the
Time
(in min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
0-13
13 -21
100
100-70
21-25
0-7100
100
25-35
100
Tests
Inject each solution. The test is not valid unless in the

chromatogram obtained with test solution (c) signal-to- noise
ratio of the principal peak is not less than 5.0. The tailing
factor of the principal peak in the chromatogram obtained
with test solution (b) is not more than 1.6; and the resolution
between the two principal peaks in the chromatogram obtained
with the reference solution is not less than 5.0.
pH (2.4.24).2.5 to 4.5, determined on a 5.0 per cent w/v solution

ofVancomycin HydrocWoride.
(2.4.14), using test solution (a), (b) and (c) as described under

(2.4.1) and the absorbance at 450 urn (2.4.7) is not more than

Vancomycin B.
Use chromatographic system as described under Vancomycin
0.1.
B.
Inject test solution (c) and the reference solution. The area of
any secondary peak is not more than 4.0 per cent and the sum
of the areas of all the secondary peaks is not more than 12.0
per cent. Ignore any peak with an area less than the area ofthe
solution (c)(0.1 per cent).
NOTE-Use the solutions within 4 hours ofpreparation.

Test solution (a). Dissolve a quantity ofthe powder in mobile
phase A to produce a solution containing 2,000 IU of
vancomycin per ml.
mobile phase A.
Test solution (c). Dilute 1 ml oftest solution (b) to 40 ml with

mobile phase A.
Assay. Determine on the mixed contents often containers by

the microbiological assay ofantibiotics, Method A or Method

hydrochloride RS in water. Heat at 65 for 24 hours and allow
to cool.
/lm) (Such as HypersiIODS),
0.5g.
B(2.2.10).
cent of the stated number of Units.
Labelling. The label states (1) the total number of Units of
vancomycin in the container and (2) the number of Units of
vancomycin per mg.
2293
VANILLIN
IP 2010
Vanillin
Reference solution (b). A 0.2 per cent w/v solution of vanillin

RS in methanol.
CHO
yOCH 3
OH
CSHS0 3
Mol. Wt. 152.2
Vanillin is 4-hydroxy-3-methoxybenzaldehyde.
Vanillin contains not less than 99.0 percent and not more than
10 1.0 per cent of CSH S0 3, calculated on the dried basis.
Category. Pharmaceutical aid (flavouring agent).
Description. A white or slightly yellow powder or crystalline
needles; odour, characteristic of vanilla.
Identification
Compare the spectrum with that obtained with vanillin RS.
(b). Examine the chromatograms in daylight after spraying.
C. To 5 ml ofa saturated solution add 0.2 ml offerric chloride

solution; a blue colour is produced. Heat to 800 ; the solution
becomes brown and a white precipitate is produced on cooling.

on 1.0 g by drying over phosphorus pentoxide for 4 hours.
ethanol (95 per cent), add 60 ml ofcarbon dioxide-Fee water.
Titrate with 0.1 M sodium hydroxide, determining the endpoint potentiometrically(2.4.25).
I ml of 0.1 M sodium hydroxide is equivalent to 0.01521 g of
CSH S0 3
Vasopressin Injection is a sterile aqueous solution containing
the water soluble pressor principle obtained from the posterior
lobe of the pituitary of healthy oxen or other mammals or by
synthesis.
Vasopressin Injection contains a pressor activity equivalent
of the stated number of Units.
Tests
Appearance ofsolution. A 5.0 per cent w/v solution in ethanol
(95 per cent) is clear (2.4.1) and not more intensely coloured
than reference solution BS6{2.4.1).
Mobile phase. A mixture of98.5 volumes ofdichloromethane,
1 volume of methanol and 0.5 volume of anhydrous acetic
acid.
Test solidioli (b). Dissolve O:Z g of
exatl1inatiol1 i111 00 illl ofmethdl1ol.
Use an unsaturated tame and allow the mobile phase to rise

10 cm. Apply to the plate 5 III of each solution. After
development, dry the plate in cold air and examine in ultraviolet
light at 254 urn. Any secondary spot in the chromatogram
obtained with test solution (a) is not more intense than the
(a). Spray with dinitrophenylhydrazine-aceto-hydrochloric
solution and examine in daylight. Any secondary spot in the
the substance-iiiider

Usual strength. 20 Units per ml.

Description. A clear, colourless or practically colourless liquid;
Identification
obtained with test solution corresponds to the peak in the
B. Inject into the vein ofa mammal anaesthetised by a general
anaesthetic or by destruction of the brain; it causes a rise of
blood pressure.
Tests
pH (2.4.24). 2.5 to 4.5.
2294
IP 2010
VERAPAMIL HYDROCHLORIDE

per Unit of vasopressin.
mobile phase: a mixture of87 volumes of0.66 per cent

w/v solution of dibasic ammonium phosphate, adjusted
acetonitrile, filter through 0.45 /lm nylon membrane,
- spectrophometer set at 220 nm,

Vasopressin injection containing Vasopressin ofnatural origin
obtained by extraction and purification complies with the
Oxytocin impurity. Not more than 1.2 Units per ml.

the same concentration in /lg ofoxytocin as that stated on the
label ofthe injection.
- a stainless steel column 10 Cln x 4.6 mm, packed with
(5 /lm) (Such as Nucleosil C18),
- mobile phase: a mixture of85 volumes ofa 0.2 per cent
of acetonitrile,

Storage. Store at the temperature between 2 to 8.
Labelling. The label states (1) the number of Units of the
vasopressor activityper ml; (2) either the animal source ofthe
vasopressin or that it is synthetic.
Verapamil Chloride; Iproveratril Hydrochloride
H3CO~
H3CO
~
H
~OCH3
theoretical plates is not less than 50,000.
~I
N HC
3
CN
, HCI
OCH 3
CH 3
Mol. Wt. 491.1
Calculate the content of C13 H66N 12012S2 in the injection.


vasopressin RS in a known volume of solvent mixture. If
/lm),
Verapamil Hydrochloride is (RS)-2-(3,4-dimethoxyphenyl)-5[[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino]-2-(1methylethyl)pentanenitrile hydrochloride.

Verapamil Hydrochloride contains not less than 99.0 per cent
and not more than 101.0 per cent of~7H38N204, HCl, calculated
on the dried basis.
Category. Calcium channel blocker.
Dose. Orally, as antiarrhythmic, 40 to 120 mg thrice daily; as
antianginal, 80 to 120 mg thrice daily; as antihypertensive, 240
to 480 mg daily, in 2 to 3 divided doses; by slow intravenous
injection, 5 to 10 mg.
Identification

2295
VERAPAMIL HYDROCHLORIDE
IP 2010
Time
Compare the spectrum with that obtained with verapamil

hydrochloride RS or with the reference spectrum ofverapamil
hydrochloride.
(min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
0-22
63
37
22-27
63-735
37 -765
35
65
35-36
35-763
65-737
36-50
63
37

0.002 per cent w Iv solution in 0.01 M hydrochloric acid shows
absorption maxima at about 229 nm and 278 nm and there may
be a shoulder at about 282 nm. The ratio ofthe absorbance at
the maximum at about 278 nm to that at the maximum at about
229 nm is 0.35 to 0.39.
C. In test A for Related substances, the principal spot in the
chromatogram obtained with test solution (b) conesponds to
Tests
dioxide-free water prepared with the aid of gentle heat is clear
pH (2.4.24).4.5 to 6.0, detennined in a 5.0 per cent w/v solution

prepared with the aid of gentle heat.
(2.4.14).
Solvent mixture. 63 volmnes ofmobile phase A and 37 volumes

ofmobile phase B.
Reference solution (a). Dissolve 5 mg each of of verapamil
hydrochloride RS and (2RS)-2-(3,4-dimethoxyphenyl)-2-[2[[2-(3, 4-dimethoxy-phenyl)ethyl](methyl)aminoJethyl] -3methylbutanenitrile RS (verapamil impurity A RS) in 20.0 ml
ofthe solvent mixture. Dilute 1 ml ofthis solution to 10 ml with
. 27 -35
resolution between the peaks due to verapamil and verapamil
impurity A is not less than 5.0.
per cent). The sum ofall the secondary peaks is not more than
3 times the area of the principal peak in the chromatogram
solution. Titrate with 0.1 M perchloric acid, detennining
titration.
CnH38N204, HCl.

palmitamidopropylsilane bonded to porous silica (5 /-lm),
mobile phase: A. a 0.7 per cent w/v solution of
dipotassium hydrogen phosphate adjusted to pH 7.2
B. acetonitrile,
a linear gradientprogrammeusingthe conditions given
below,
Verapamil Injection
Verapamil Hydrochloride Injection; Verapamil Chloride
Injection; Iproveratril Hydrochloride Injection
Verapamil Injection is a sterile solution ofVel'apami1
Verapamil Injection contains not less than 90;0 percent and
not more than 110.0 per cent ofthe stated amount ofverapamil
hydrochloride, C 27H 38N 20 4, HCl.
Usual strength. 2.5 mg per ml.
2296
VERAPAMIL TABLETS
IP 2010
Identification
A. Dilute a volume containing 10 mg of Verapamil
Hydrochloride to 5 ml with 0.1 M hydrochloric acid, extract
with 5 ml of ether, discard the ether extract and make the
aqueous layer just alkaline with 2 M potassium carbonate.
Extract with 5 ml of ether, filter the ether layer through
anhydrous sodium sulphate and evaporate to dryness. The
residue complies with the following test.
reference spectrum ofverapamil.
B. To a volume conta~ning 5 mg of Vel'apami1Hydrochloride
add 0.2 rnl ofa 5 per cent w/v solution of mercuric chloride; a
C. To a volume containing 5 mg ofVerapamil Hydrochloride
add 0.5 ml of 3 M sulphuric acid and 0.2 ml of dilute potassium
permanganate solution; a violet precipitate is produced which
quickly dissolves to produce a very pale yellow solution.
B. Carry out test A but using a mixture of 85 volumes of

cyclohexane and 15 volumes of diethylamine as the mobile
phase and applying separately to the plate 30 Itl of each 6fthe
test solution and the reference solution.

Assay. Dilute an accurately measured volume containing 5 mg
of Verapamil Hydrochloride to 100.0 ml with 0.01 M
hydrochloric acid and measure the absorbance ofthe resulting
content of CZ7H3sNz04, HCl taking 118 as the specific
Verapamil Tablets
Verapamil Hydrochloride Tablets; Verapamil CWoride
Tablets; Iproveratril Hydrochloride Tablets
Verapamil Tablets contain not less than 92.5 per cent and not
more than 107.5 per cent of the stated amount of verapamil
hydrochloride, CZ7H3sNz04' HCl. The tablets may be coated.
Tests
pH (2.4.24).4.5 to 6.0.
chromatography (2.4.17), coating the plate with silica gel G

of methanol, 5 volumes of glacial acetic acid and 5 volumes
of acetone.
Test solution. Evaporate a volume containing 5 mg ofVerapamil
Hydrochloride carefully to dryness on a water-bath in a current
of nitrogen and dissolve the residue as completely as possible
in 0.25 ml of chloroform.
100 volumes with chlorojorm and dilute 1 volume of the
resulting solution to 10 volumes with chloroform.
Apply to the plate 30 Itl of each solution. After development,
Dry the plate at 110 for 30 minutes and allow to stand until the
odour ofsolvent is no longer detectable. Spray with a solution
prepared by dissolving 5 g of ferric chloride hexahydrate
and 2 g of iodine in sufficient ofa mixture ofequal volumes of
acetone and a 20 per cent w/v solution of (+ )-tartaric acid to
produce 100 ml, applying a total of 15 to 20 ml ofthe reagent
for a plate (20 cm x 20 cm), and examine immediately. Any
solution is not more intense than the principal spot in the
chromatogram obtained with the reference solution. Ignore
. any spot remaining on the line of application.
Usual strengths.40 mg; 80 mg; 120 mg; 160 mg.
Identification
ofVerapamil Hydrochloride with 25 ml of 0.1 Mhydrochloric
acid, filter, extract the filtrate with 25 ml of ether, discard the
ether extract and make the aqueous layer just allmline with
2 M potassium carbonate. Extract with 25 ml of ether, filter the
ether layer through anhydrous sodium sulphate and
evaporate to dryness. The residue complies with the following
tests.
reference spectrum ofverapamil.

ofVerapamil Hydrochloride with 10 ml of dichloromethane,
filter, evaporate the filtrate to dryness and dissolve the residue
in 10 ml of water (solution A). To 2 ml ofthe resulting solution
add 0.2 ml ofa 5 per cent w/v solution of mercuric chloride; a
C. To 2 ml of solution A obtained in test B add 0.5 ml of 3 M
sulphuric acid and 0.2 ml of dilute potassium permanganate
solution; a violet precipitate is produced which quickly
dissolves to produce a very pale yellow solution.
2297
VERAPAMIL TABLETS
IP 2010
Tests
Vinblastine Sulphate

(2.4.14).

containing about 0.24 g ofVerapamil Hydrochloride with 100.0
ml ofthe mobile phase.
Reference solution (a). Dilute 1 ml ofthe test solution to 100.0
ml with the mobile phase. Further dilute 1.0 ml of this solution

w/v each of verapamil hydrochloride RS and (2RS)-2-(3,4di methoxyphenyl) -2- [2 - [[2 - (3, 4 -dime thoxyphenyl)ethylj(methyl)aminoJethylj-3-methylbutanenitrile
RS (verapamil impurity A RS) in the mobile phase.
as Hypersil ODS),
- mobile phase: a mixture of 1 volume of n-heptylamine,
4.7 volumes of glacial acetic acid, 58 volumes of
acetonitrile and 137 volumes of0.01 Msodiumacetate,
resolution between the peaks due to verapamil and verapamil
impurity A is not less than 2.0.
(a) (0.1 per cent) and the sum ofthe areas of all the secondary
peaks is not more than three times the area of the principal
(a) (0.3 per cent). Ignore any peak with an area less than 0.5
quantity of the powder containing 0.1 g of Verapamil
Hydrochloride, shake with 150 ml of 0.1 M hydrochloric acid
for 10 minutes, add sufficient 0.1 M hydrochloric acid to
~~produce 200.0ml and filter. Dilute 10.OmLoLthe filtrate to
100.0 ml with water and measure the absorbance of the
Calculate the content of C27H3sN204, HCl taking 118 as the
Mol. Wt. 909.1

Vinblastine Sulphate is methyl (3aR,4R,5S,5aR, IObR,13aR)4-acetoxy-3a-ethyl-9-[(5S,7R,9S)-5-ethyl-5-hydroxy9-methoxycarbonyl-l,4,5,6,7,8,9,10-octahydro-2H-3,
7-methanoazacycloundecino[(5,4-b)indol-9-yl]5-hydroxy-8-methoxy-6-methyl-3a,4,5,5a,6, 11,12,13aoctahydro-lH-il1dolizino[8,I-cd]carbazole-5-carboxylate
sulphate.
Vinblastine Sulphate contains not less than 95.0 per cent and
not more than 104.0 per cent ofC46HssN409, H2S04, calculated
on the dried basis.
Dose. By intravenous injection, according to the needs ofthe
patient.
Description. A white or yellowish, amorphous or crystalline
powder; very hygroscopic.
CAUTION~ Handle Vinblastine Sulphate with great care
since it is a potent cytotoxic agent. Avoid contact with eyes;
irritant to tissues.
Identification
Compare the spectrum with that obtained with vinblastine
sulphate RS.
to vinblastine sulphate in the chromatogram obtained with
C. A 10 per cent w/v solution gives the reaction ofsulphates
(2.3.1).
Tests
pH (2.4.24).3.5 to 5.0, determined in a 0.15 percentw/v solution.
2298
VINBLASTINE INJECTION
IP 2010
Related substances. In the Assay, the area of any secondary

peak in the chromatogram obtained with the test solution is
not greater than that ofthe principal peak in the chromatogram
obtained with reference solution (c) and the sum of the areas
of any such peaks is not greater than 2.5 times the area of the
solution (c). Ignore any peak with an area less than that
of the peak in the chromatogram obtained with reference
solution (d).
determined by Method B, on an appropriately calibrated
instrument using about 3.0 mg, accurately weighed. Heat the
substance under examination at the rate of 5 per minute
between ambient temperature and 200 in a current ofnitrogen
for chromatography with a flow rate of40 ml per minute. From
the thermogram, determine the accumulated loss in weight
between ambient temperature and a point on the plateau before
decomposition is indicated (at about 160).

w/v each of vinblastine sulphate RS and vincristine sulphate
RSin watel:
to-noise ratio of the peak in the chromatogram obtained with

reference solution (d) is at least 5.
Calculate the percentage content of C46H5sN409, H 2S04,
Vinblastine Sulphate intendedfor use in the manufacture of

parenteral preparations without a further appropriate
procedure for the removal ofbacterial endotoxins complies
Units per mg.
Vinblastine Sulphate intended for use in the manufacture of

parenteral preparations without a fitrther appropriate
Storage. Store protected from light and moisture in a deep
freezer (Below -18). If the material is intended for use in the
manufacture ofparenteral preparations, it should be stored in
sterile, tamper-evident glass containers and sealed so as to
exclude micro-organisms.
suitable for use in the manufacture ofparenteral preparations.

vinblastine sulphate RS in watel:
vinblastine sulphate RS in watel:
vinblastine sulphate RS in water.
octylsilane bonded to porous silica (5 /lm), (b) a guard
column packed with a suitable silica gel placed between
the pump and the injection device,
38 volumes ofa 1.5 per cent v/v solution of diethylamine
adjusted to pH 7.5 with phosphoric acid and 12 volumes
of acetonitrile,
NOTE - Store all solutions in ice before use.

Inject each solution and record the chromatograms for 3 times
the retention time of the peak due to vinblastine.
The assay is not valid unless the resolution between the peaks
due to vincristine and vinblastine in the chromatogram
obtained with ref~rence solution (a) is at least 4 and the signal-
Vinblastine Injection
Vinblastine Sulphate Injection
Vinblastine Injection is a sterile material consisting of
Vinblastine Sulphate with or without auxiliary substances. It
is filled in a sealed container.
sealed container in the requisite amount of sterile 0.9 per cent
w/v solution ofSodium Chloride, immediately before use.

Vinblastine Injection contains not less than 90.0 per cent and
not more than 11 0.0 per cent of the stated amount of
vinblastine sulphate, C46H5sN409,H2S04.

2299
VINBLASTINE INJECTION
IP 2010
Identification
CAUTION - Handle Vinblastine Injection with great care
at about 267 nm.

weight of dried vinblastine sulphate contained in it; (2) the
names ofauxiliary substances, if any; (3) that the contents are
to be used by intravenous injection only; (4) the storage
conditions.
Vincristine Sulphate

C. To 1 ml add 0.2 ml of a freshly prepared 1 per cent w/v
solution of vanillin in hydrochloric acid; a pink colour is
produced in about 1 minute (distinction from Vincristine
Sulphate).
Tests
pH (2.4.24).3.5 to 5.0, deterrnined in a 0.15 per cent w/v solution
of the dried contents.

of chloroform and 6 volumes of diethylamine.
Test solution. Dissolve a quantity ofthe contents ofa container
in sufficient methanol to produce a solution containing the
equivalent of 1 per cent w/v of dried vinblastine sulphate.
Mol. Wt. 923.1

Vinblastine Sulphate is methyl (3aR,4R,5S,5aR, 1ObR, 13aR)
4-acetoxy-3a-ethyl-9-[(5S,7R,9S)- 5-ethyl-5-hydroxy9-methoxycarbonyl-l,4,5,6,7,8,9,10-octahydro-2H-3,
7- methanoazacycloundecino[(5,4-b)indol-9-yl]-6-formyl-5hydroxy- 8-methoxy-3a,4,5,5a,6,11,12,13a-octahydroIH- indolizino[8,I-cd]carbazole-5-carboxylate sulphate.
Vincristine Sulphate contains not less than 95.0 per cent and
.
Reference solution (a). A 1 per cent w/v solution of vinblastine not more than 104.0 per cent ofC46Hs6N401O,HzS04, calculated
on the dried basis.

vincristine sulphate RS in methanol.
Units per mg of the dried contents.
Assay. Weigh the contents of 20 sealed containers. Weigh
accurately a quantity of the mixed contents containing about
20 mg ofdried vinblastine sulphate and dissolve it in 100.0 ml
of methanol. Dilute 10.0 ml to 100.0 ml with methanol and
C<ji;HssN<j09,HzSO<j taking 185 as thespecificaosoroance -at
267nm.
Storage. Store in sealed containers in a deep freezer (Below
-18).
Dose. By intravenous injection, according to the needs of the
patient.
Description. A white to slightly yellowish, amorphous or
crystalline powder; very hygroscopic.
CAUTION- Handle Vincristine Sulphate with great care

Identification
Compare the spectrum with that obtained with vincristine
sulphate RS.

fo viriciistirie- stiTphafe-iilfhe-chfomafogram-obtafnecfwitIi
C. A 10 per cent w/v solution gives the reaction for sulphates
(2.3.1).
2300
IP 2010
VINCRISTINE SULPHATE
Tests
reference solution (d).
Appearance ofsolution. A 0.5 per cent w/v in carbon dioxidefree water is clear (2.4.1), and not more intensely coloured
pH (2.4.24).3.5 to 4.5. Determined in a 0.1 per centw/v solution.

(2.4.14).
NOTE- Keep the solutions in iced water before use.

determined by Method B, on an appropriately calibrated
instrument using about 3.0 mg, accurately weighed. Heat the
substance under examination at the rate of 50 per minute
between ambient temperature and 2000 in current of nitrogen
for chromatography with a flow rate of40 ml per minute.
Test solution. Dissolve about 50.0 mg of the substance under

examination in 10.0 ml of carbon dioxide-free water. Dilute 1.0
ml ofthis solution to 5.0 ml with water.

vincristine sulphate RS in water.

w/v each of vinblastine sulphate RS and vincristine sulphate
RSin wate1:

vinblastine sulphate RS in reference solution (a).
Reference solution (c). Dilute 1.0 ml of the test solution to
50.0 ml with water.
Reference solution (d). Dilute 1.0 ml of reference solution (c)
mobile phase: A. a 1.5 per cent v/v solution of
diethylamine, adjusted to pH 7.5 Witl;l orthophosphoric
acid
B. methanol,
-
a linear gradient programme using the condition given

below,
(min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
0-12
38
62
Time
12-27
38-8
62-92
27-29
8-38
92-62
29-34
38
62
Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of water.

vincristine sulphate RS in wate1:
vincristine sulphate RS in wate1:
- a stainless steel column 25 cm x 4.6mm, packed with
octylsilane bonded to porous silica (5 Ilm), (b) a guard
column packed with a suitable silica gel placed between
the pump and the injection device,
30 volumes ofa 1.5 per cent v/v solution of diethylamine
adjusted to pH 7.5 with phosphoric acid,
NOTE - Store all solutions in ice before use.

Inject each solution and record the chromatograms for 3 times
the retention time of the peak due to vincristine.
resolution between the peaks due to vincristine and vinblastine
is not less than 4.
in the chromatogram obtained with reference solution (c) (2.0
.not more than 2.5 times the area of the principal peak in the
The assay is not valid unless the resolution between the peaks
obtained with reference solution (a) is at least 4 and the signalto-noise ratio in the peak in the chromatogram obtained with
reference solution (d) is at least 5.
Calculate the content ofC46Hs6N401O,H2S04'
Vincristine Sulphate intended for use in the manufacture of

procedure for the removal of bacterial endotoxins complies
Units per mg.
2301
VINCRISTINE INJECTION
IP 2010
Vincristine Sulphate intended for use in the mamifacture of

Storage. Store protected from light in a deep freezer (Below
-18). If the material is intended for use in the manufacture of
parenteral preparations, it should be stored in sterile, tamperevident glass containers and sealed so as to exclude microorganisms.
suitable for use in the manufacture ofparenteral preparations.
the combined chloroform solutions to dryness at 40. Add

0.2 ml ofa freshly prepared 1 per cent w/v solution of vanillin
in hydrochloriC acid to the residue; an orange colour is
produced in about 1 minute (distinction from vinblastine
sulphate).
Tests
Appearance of solution. A solution prepared by dissolving
the contents of a sealed container in 10 ml of carbon dioxidefree water is clear (2.4.1).
pH (2.4.24). 3.5 to 5.0, determined in a solution containing
0.15 per cent w/v solution of the dried contents.
Related substances. Deteremine by liquid chromatography
(2.4.14).
Vincristine Injection
NOTE-Keep the solutions in ice before use.
Vincristine Sulphate Injection
Test solution. Dissolve the contents of a sealed container in

water to produce a solution containing about 0.1 per cent wi
Vrncristine Injection is a sterile material consisting ofVmcristine

Sulphate with or without auxiliary substances. It is filled in a
sealed container.
sealed container in the requisite amount of sterile 0.9 per cent
w/v solution ofSodium Chloride, immediately before use.

after preparation but, in any case, within the period recommended by the manufacturer.
Vincristine Injection contains not less than 90;0 per cent and
not more than 110.0 per cent ofthe stated amount ofvincristine
sulphate, C46Hs6N401O,HzS04'
Usual strengths. The equivalent of 1 mg, 2 mg and 5 mg of
dried vincristine sulphate.

v of anhydrous vincristine sulphate.
Reference solution (a). A solution containing 0.1 per cent wi

veach of vincristine sulphate RS and vinblastine sulphate
RSin water.
a stainless steel colunrn 25 em x 4.6 mm packed with
endcapped octylsilane bonded to porous silica (5 /lm),
(Such as Zorbax C8),
mobile phase: a mixture oBO volumes of a 1.5 per cent
v/v solution of diethylamine adjusted to pH 7.5 with
orthophosphoric acid and 70 volumes of methanol,
Inject reference solution (a). Run the chromatogram 3 times
the retention time of the principal peak.
Identification
CAUTION - Handle Vincristine Injection with great care
A. In the test for Related substances, the principal peak in the
-1:I1arffitl:fecnromatograrh oommedWitnreference solUtion (or
B. Shake a quantlt,Ycontalning Tmg o{Cifledvincristlne

sulphate with 3 ml of chloroform, filter and wash the filter with
2 ml of chloroform. Reserve the residue for test D. Evaporate
obtained with reference solution (a) is not less than 4 and
signal-to-noise ratio in the principal peak in the chromatogram
obtained with reference solution (d) is not less than 5.
~-,~-"",,-,,~-"."'
..
'.'"~~'''''--''''''''''-~''''''~'''''
"
,,'
""
~"~'~",,,,,,,-,,,-,,~~~,,,,~.,,,,-,,-,,-,-,,,-,~._,,,,,,,._~,,,,,-,,,,._,,,,'"~~".,''-''''~-'''''
Inject the test solution, reference solution (c) and (d). In the
secondary peak is not more than the ania ofthe principal peak
2302
VINORELBINE TARTRATE
IP 2010
(2.0 per cent) and th~ sum of the areas of all the secondary
peaks is not more than 2.5 times the area ofthe principal peak
(5.0 percent). Ignore any peak with an area less than the ofthe
solution (d) (0.1 per cent).
Uniformity of content. The content of anhydrous vincristine
sulphate in each of 10 individual containers as determined in
the Assay is not less than 90.0 per cent and not more than
110.0 per cent ofthe average except that in one container the
content may be not less than 80.0 per cent and not more than
120.0 per cent ofthe average..
Units per mg of dried vincristine sulphate.
Assay. Dissolve the contents ofa sealed container in a suitable
volume of methanol to produce a solution containing about
0.005 per cent w/v ofanhydrous vincristine sulphate. Measure
297 nm (2.4.7). Calculate the content of C46Hs6N401O,H2S04
Repeat the procedure with a further nine containers.
Calculate the average content of C46Hs6N401O,H2S04 in the
sealed container.
Vinorelbine Tartrate contains not less than 98.0 per cent and
not more than 102.0 per cent ofC4sHs4N40s.2C4H606, calculated
Description. A white to yellow or light brown amorphous
powder.
CAUTION - Vinorelbine Tartrate is cytotoxic; extra care

required to prevent inhaling particles and exposing the skin
to it.
Identification
A. Dissolve 10 mgin 5 ml of water, add 0.5 ml of5 Msodium
hydroxide, and extract with 5 ml of methylene chloride. Filter
the organic layer through anhydrous sodium sulphate, and
evaporate to dryness.
Determine by Infrared absorption spectrophotometry (2.4.6).
Compare the spectrum with that obtained with vinorelbine
tartrate RS treated in the same manner.
B. In the test for Related substances, the principal peakin the

chromatogram of the test solution corresponds to that due to
vinorelbine tartarate in the chromatogram obtained with the
refrence solution (a).
Storage. Store in sealed containers in a deep freezer (Below

-18).
C. It gives the reactions for tartrate (2.3.1).

weight of dried vincristine sulphate contained in it; (2) the
names of auxilary substances, if any; (3) that the contents are
to be used by intravenous injection only; (4) the storage
conditions.
Tests
(2.4.1).
Light absorption (2.4.7). The absorbance of 1.0 per cent w/v
solution, at about 420 urn is not more than 0.03.
pH (2.4.24).3.3 to 3.8, determined on a 1.0 per cent w/v solution.
Vinorelbine Tartrate

(2.4.14).

H ,oH
COOH
,2HOOCXyOH

vinorelbine tartrate RS in the mobile phase.
Reference solution (b). Dilute 1 ml of the reference solution
(a) to 100 ml with the mobile phase.

C4sHs4N40S, (C~606)2
Mol Wt. 1079.1
Vinorelbine Tartrate is 3' ,4' -didehydro-4' -deoxy-C' norvincaleukoblastine ditartrate.
Vinorelbine Tartrate is the ditartrate salt ofvinorelbine,a

semisynthetic Vinca alkaloid; structurally relate to
vinblastine.

2303
VINORELBINE TARTRATE
IP 2010

(0.3 per cent) and the sum of areas of all the secondary
peaks is not more than the area of the peak in the
(1.0 per cent). Ignore any secondary peak having area less
than 0.1 per cent.
Vinorelbine Injection contains not less than 90.0 per cent and
vinorelbine, C4sHs4N40g.
Identification
per mg of vinorelbine base.

Description. A clear, colourless to pale yellow solution.

Microbial contamination (2.2.9). Total viable aerobic count,

not more than 100 cfu per g, total combined molds and yeast
does not exceed 50 cfu per g. It also meets the requirement of
the tests for the absence of Staphylococcus aureus,
Pseudomonas aeruginosa, Salmonella species, Escherichia
coli, Enterobacteria and Closteridia.

solution containing 0.01 per cent w/v ofVinorelbine Tartrate,
exhibits the maxima at about 267 nm.
Light absorption. The absorbance of the injection at about

420 nm (2.4.7), is not more than 0.06.

examination in 25.0 ml ofmobile phase.
Reference solution. A 0.14 per cent w/v solution ofvinorelbine
tartrate RS in mobile phase.
mobile phase: 62 volumes of methanol containing
1.22 g of sodium I-decane sulphonate and 38 volumes
of phosphate buffer solution, prepared by dissolving
6.9 g of monobasic sodiumphosphate in 900 ml of water,
adjust the pH to 4.2 with orthophosphoric acid and
dilute to 1000 ml with water,
than 2.0 per cent.
Calculate the content ofC4sHs4N40g.2C4H606.
Storage. Store protected from light, in a deep freezer (below 18).
Tests
pH (2.4.24).3.0 to 3.8.

(2.4.14).
Test solution. Accurately measured volume of injection

containing 10 mg ofVrnorelbine, dilute to 10 ml with the mobile
phase.
vinorelbine tartrate RS in the mobile phase.
Reference solution (b). Dilute 1 m1 of the reference solution
(a) to 100 ml with the mobile phase.
chromatograms three times the retention time ofthe peak due
to vinorelbine. In the chromatogram obtained with the test
solution, the area of any secondary peak is not more than
with reference solution (b) (0.2 per cent) and the sum ofareas
of all the secondary peaks is not more than twice the area of
(b) (2.0 per cent).
Preparation (Injections).
Yiuordbixte Illle_cJion
. Bacterialendotoxins(2.2.3). Notmore than 3.0 EndotoxinDnit

per mg ofvinorelbinetart.rate.
Vinorelbine Tartrate Injection

Vinorelbine Injection is a sterile solution ofvinorelbine tartrate
in Water for Injection.

2304
VITAMIN A CONCENTRATE OIL
IP 2010
Test solution. Accurately measured volume of injection

containing 10 mg ofVinorelbine, diluted to 10.0 ml with water.

Reference solution. A 0.14 per cent w/v solution ofvinorelbine

tartrate RS in water.

mobile phase: a mixture of 62 volumes of methanol
containing 1.22 g of sodiuml-decane sulphonate and
38 volumes of phosphate buffer solution prepared by
dissolving 6.9 g of monobasic sodium phosphate in
900 ml of watel; adjusted the pH to 4.2 with orthophosphoric acid and dilute to 1000 ml with water,
spectrophotometer set at 267 TIm,
Test solution. Prepare a solution containing 5 Units per fll of

the substance under examination in cyclohexane.
relative standard deviation for replicate injection is not more
than 2.0 per cent.
Reference solution (a). Prepare a solution containing

5 Units per fll of retinyl acetate RS in cyclohexane.
Reference solution (b). Prepare a solution containing
5 Units per fll of retinyl propionate RS in cyclohexane.
Reference solution (c). Prepare a solution containing
5 Units per fll of retinyl palmitate RS in cyclohexane.
dry the plate in air, spray with antimony trichloride solution.
The principal spot or spots in the chromatogram obtained
with the test solution correspond to one or more of the spots
in the chromatograms obtained with reference solutions (a),
(b) and (c).
Calculate the content ofC4sHs4N40g.
C. Dissolve a quantity containing 10 to 15 Units in 1 mlof

chloroform and add 5 ml of antimony trichloride solution; a
transient bright blue colour is produced immediately.
Storage. Store protected from light, in a single-dose container,

in a refrigerator (2 to 8) ; do not freeze.
Tests
Peroxides. Add 0.3 g to 25.0 ml ofa mixture of6 volumes of

toluene and 4: volumes of methanol (solution A). Add in a
test-tube, in the following order, mixing after each addition,
0.3 ml ofa 1.8 percent w/v solution of ammonium thiocyanate,
Vitamin A Concentrate Oil consists of an ester or a mixture of 10.0 ml of methanol, 0.3 ml offerrous sulphate solution and
esters ofretinol (as acetate, propionate or palmitate) prepared 15.0 ml of toluene and add 1.0 ml of solution A. The colour
by synthesis. It may be diluted with a suitable vegetable oil. It produced after 5 minutes is not more intense than that obtained
may contain suitable stabilising agents such as antioxidants. in a solution prepared at the same time and in the same manner
Vitamin A Concentrate Oil contains not less than 500,000 Units but using a solution prepared in the following manner in place
of Vitamin A per g, and not less than 95.0 per cent and not of solution A. Add 1.0 ml of a 27.0 per cent w/v solution of
more than 110.0 per cent of the stated number of Units of ferriC chloride hexahydrate to 99 ml ofa mixture of6 volumes
of toluene and 4 volumes of methanol and dilute 2.0 ml to
Vitamin A per g.
100.0 ml with the same solvent mixture.
Category. Antixerophthalmic vitamin.
Dose. Prophylactic, 5000 Units ofVitamin A, daily; therapeutic, Assay. Carry out the assay ofvitamin A, Method A (2.3.41).
Synthetic Vitamin A Concentrate (Oily Form); Synthetic

Retinol Concentrate (Oily Form).
10,000 to 200,000 Units ofVitamin A, daily.

Description. A yellow to brownish yellow, oily liquid; odour,
faint and characteristic.
Identification
A. When examined in the range 230 TIm to 360 TIm (2.4.7), a
solution in 2-propanol containing 10 to 15 Units per ml shows
an absorption maximum at about 325 TIm to 327 nm.
Storage. Store protected from light, in well-filled containers.

Once the container has been opened, its contents should be
used as soon as possible; any part ofthe contents not used at
once should be protected by an atmosphere of an inert gas.
Labelling. The label states (1) the number ofUnits ofVitamin
A per g; (2) the name ofthe ester or esters; (3) the name(s) of
any added stabilising agent(s); (4) the method of restoring
the solution if partial crystallisation has occurred.
2305
VITAMIN A CONCENTRATE POWDER
IP 2010
Reference solution (c). Prepare a solution containing 5 Units

per III of retinyl palmitate RS in cyclohexane.

Synthetic Vitamin A Concentrate (Powder Form);
Synthetic Retinol Concentrate (Powder Form).
Vitamin A Concentrate Powder consists ofan ester or a mixture
ofesters ofretinol (as acetate, propionate or palmitate) prepared
by synthesis and dispersed in a matrix of Gelatin, Acacia or
any other suitable material. It may contain suitable stabilising
agents such as antioxidants.
Vitamin A Concentrate Powder contains not less than
250,000 Units of Vitamin A per g, and not less than 95.0 per
cent and not more than 115.0 per cent ofthe stated number of
Units ofVitaminA per g.

(b) and (c).
C. Dilute 2 ml of sblution A to 50 ml with n-pentane and
evaporate 1 ml of the solution to dryness in a current of
nitrogen. Dissolve the residue in 1 ml of chloroform and add
5 ml of antimony trichloride solution; a transient bright blue
colour is produced irilrnediately.
Tests
Dose. Prophylactic, 5000 Units ofVitamin A, daily; therapeutic,

10,000 to 200,000 Units ofVitamin A, daily. In multi-vitamin
oral preparations, prophylactic, 1600 to 2500 Units ofVitamin
A; therapeutic, 5000 to 10,000 Units ofVitamin A.
Description. A yellowish powder usually in the form ofpellets
or particles ofalmost uniform size.
Identification
To a quantity containing 50,000 Units ofVitamin A, add 1.5 ml
of 2 M ammonia previously heated to 60 and heat in a waterbath at 60, shaking occasionally. After 10 minutes add 40 ml
of ethanol, dilute to 200 ml with ether and shake. Allow to
stand for a few minutes and use the supernatant liquid
(solution A) for the following tests.
Certain samples may not react sufficiently during the course
of the above treatment In such cases the volume of solution
A used in the following tests should be increased which may
be as much as lO-fold.
Related substances and degradation products. Using the

relative absorbances obtained in the Assay, the ratio
A 30o/Am is not more than 0.612 and the sum of the ratios
A 30o /A m and A 350 /A m is not more than 1.054, where
A 300 , Am and A 350 are the absorbances measured at about
300 nm, 325 urn and 350 urn respectively.
Assay. Carry out the assay of vitamin A, Method B (2.3.41),
adding 5 ml of water to the mixture for saponification, using
2-propanol as the blank and taking 0.612 as the maximum
value of the ratio A 300/ Am.
Storage. Store protected from light. Once the container has

been opened, its contents should be used as soon as possible;
any part of the contents not used at once should be protected
by an atmosphere of an inert gas.
Labelling. The label states (1) the number ofUnits ofVitamin
A per g; (2) the name ofthe ester or esters; (3) the name ofthe
principal excipient or excipients used; (4) the name of any
added stabilising agents.
A. When examined in the range 230 urn to 360 urn (2.4.7), a

solution in2-propanol containing 10 to 15 Units perml shows
an absorption maximum at about 325 urn to 327 urn.

Water-Miscible Vitamin A Concentrate
Mobile phase. A mixtu+e of 80 volumes of cyclohexane and

Synthetic Vitamin A Concentrate (Water-dispersible

Form); Synthetic Retinol Concentrate (Water-dispersible
Form).
Testsolution. Evaporate 10 ml of solution A to dryness in a

current of nitrogen and dissolve the residue in 0.5 ml of
cyclohexane.
Reference solution (aJ .Prepareasolution-containingSUnits
per III of retinyl acetate RS in cyclohexane.
Water-miscible Vitamin AConcentrate consists of an ester or

a mixture ofesters ofretinol (as acetate, propionate or palmitate)
prepared by synthesis to which suitable solubilisers have
lJeenadded: lfifiaycolltainSuitaole-stalJilising agentssuch
as antimicrobial preservatives and antioxidants.
Reference solution (b). Prepare 5 Units per III of retinyl

propionate RS in cyclohexane.
Water-miscible Vitamin A Concentrate contains not less than

100,000 Units of Vitamin A per g, and not less than 95.0 per
2306
VITAMINS A AND D CAPSULES
IP 20ID
cent and not more than 115.0 per cent of the stated number of
Units ofVitamin A per g.
Dose. Prophylactic, 5000 Units ofVitamin A, daily; therapeutic,
10,000 to 200,000 Units ofVitamin A, daily.. In multi-vitamin
oral preparations, prophylactic, 1600 to 2500 Units ofVitamin
A; therapeutic, 5000 to 10,000 Units ofVitamin A.
Description. A yellow or yellowish liquid of variable
opalescence and viscosity; odour, characteristic. Highly
concentrated solutions may become cloudy at low
temperatures or gel at room temperature.
Identification
To a quantity containing about 10,000 Units ofVitamin A, add
5 ml of water and homogenise. Add 5 ml of ethanol (95 per
cent) and 20 ml of n-pentane and shake vigorously for
30 seconds. Allow to stand for a few minutes and use the
supernatant liquid (solution A) for the f?llowing tests.
A. Dilute solution A with sufficient 2-propanol so that the

absorbance at the wavelength of maximum absorption is
0.3 to 0.7 (2.4.7).
When examined in the range 230 run to 360 urn (2.4.7), the
solution shows an absorption maximum at 325 to 327 nm.

Test solution. Evaporate 10 ml of solution A to dryness in a
current of nitrogen and dissolve the residue in 0.5 ml of
cyclohexane.
Reference solution (a). Prepare a solution containing 5 Units
per III of retinyl acetate RS in cyclohexane.
Reference solution (b). Prepare a solution containing 5 Units
per III of retinyl propionate RS in cyclohexane.
Reference solution (c). Prepare a solution containing 5 Units
per III of retinyl palmitate RS in cyclohexane.
(b) and (c).
C. Evaporate 0.1 ml of solution A to dryness in a current of
nitrogen, dissolve the residue in 1 ml of chloroform and add
5 ml of antimony trichloride solution; a transient bright blue
colour is produced immediately.
Tests
Water miscibility. Mix about 1 g with 10 ml of water previously
heated to 50 and cool to 20. Immediately after cooling, a
uniform, slightly opalescent and slightly yellow dispersion is
obtained.
Related substances and degradation products. Using the
relative absorbances obtained in the Assay, the ratio
A 3001A325 is not more than 0.618 and the sum of the ratios
A 3001A 325 and A 3soIA325 is not more than 1.060, where A300, A325
and A 3S0 are the absorbances measured at about 300 urn, 325
urn and 350 nm respectively.
Assay. Carry out the assay of vitamin A, Method B (2.3.41),
using 2-propanol as the blank and taking 0.618 as the maximum
value of the ratio A 30o /A 325
Storage. Store protected from light at the temperature stated
on the label. Once the container has been opened, its contents
should be used as soon as possible; any part of the contents
not used at once should be protected by an atmosphere of an
inert gas.
Labelling. The label states (1 )the number ofUnits ofVitamin
A per g; (2) the name ofthe ester or esters; (3) the name ofthe
principal excipient or excipientsused; (4) the temperature at
which it should be stored.
Vitamins A.and D Capsules

Vitamins A and D Capsules contain Vitamin A Oil and a source
of vitamin D such as Cholecalciferol or Ergocalciferblin an
edible vegetable oil.
Vitamins A and D Capsules contain not less than 90.0 per cent
of the stated number of Units of vitamin A and vitamin D.
Category. Antixerophthalmic and antirachitic vitamins.
Dose. Prophylactic, 1600 to 2500 Units ofVitamin A and 100 to
200 Units ofvitamin D once a day; therapeutic, 5000 to 10,000
Units ofVitamin A and 400 to 1000 Units ofvitamin D once a
day.
Tests
Assay. For vitamin A - Weigh accurately a portion of the
mixed contents of20 capsules containing about 500 Units of
Vitamin A and carry out the assay of vitamin A, Method A
(2.3.41).
.
For vitamin D - Weigh accurately a portion of the mixed

contents of20 capsules containing about 5000 Units ofvitamin
D and carry out the assay ofvitamin D (2.3.42).
2307
CONCENTRATED VITAMIN D SOLUTION
IP 2010
Labelling. The label states the number of Units of vitamin A

and vitamin D per capsule.
Labelling. The label states (1) the number ofUnits ofvitamin

D per g; (2) the storage conditions; (3) the nature and
concentration of any stabilising agent added.
Concentrated Vitamin D Solution

Concentrated Vitamin D Solution is a solution of
Cholecalciferol or Ergocalciferol in an edible vegetable oil. It
may contain suitable stabilising agents such as antioxidants.
Concentrated Vitamin D Solution contains not less than 10,000
Units ofvitamin D and not less than 90.0 per cent ofthe stated
number ofUnits ofvitamin D.
Category. Antirachitic vitamin.
Dose. Prophylactic, 400 to 1000 Units daily; therapeutic, 5000
to 50,000 Units daily.
Description. A pale yellow to yellow, oily liquid; odour, faint
but not rancid.
Concentrated Vitamins A and D

Solution
Concentrated Vitamins A and D Solution is a solution of
Vitamin A Oil and a source ofvitamin D such as Cholecalciferol
or Ergocalciferol in an edible vegetable oil. It may contain
suitable stabilising agents such as antioxidants.
Concentrated Vitamins A and D Solution contains not less
than 50,000 Units ofvitamin A and 5000 Units ofvitamin D per
g, and not less than 90.0 per cent and not more than 110.0 per
cent ofthe stated number ofUnits ofVitamin A and vitamin D
perg.
Category. Antixerophthalmic and antirachitic vitamins.
Identification
Dissolve a quantity containing about 1000 Units ofvitamin D
in I ml of chloroform and add 10 ml of antimony trichloride
solution; a pinkish red colour appears at once.
Dose. 2500 to 25,000 Units ofVitamin A and 250 to 2500 Units

ofvitamin D daily.
Tests
Tests
Assay. For vitamin A

MethodA (2.3.41).
Assay. Carry out the assay ofvitamin D (2.3.42).
For vitamin D - Carry out the assay ofvitamin D (2.3.42).
Storage. Store protected from light, in well-filled containers at

a temperature of 6 to 15. The contents ofan opened container
should be used as soon as possible.
Carry out the assay of vitamin A,
Labelling. The label states (1) the number ofUnits ofvitamin

A and vitamin D per gram; (2) the storage conditions.
2308
MONOGRAPHS
w
2311
2312
2313
2314
2315
2315
2316
2317
2318
Warfarin Sodimn
Warfarin Sodimn Clathrate
Warfarin Tablets
Purified Water
Water for Injections
Water for Injections in Bulle
Sterile Water for Injections
Wool Fat
Hydrous Wool Fat
2309
IP 2010
WARFARIN SODIUM
Phenolic ketones. Absorbance ofa 12.5 per cent w/v solution

in a 5.0 per cent w/v solution of sodium hydroxide at the
maximum at about 385 urn, measured within 15 minutes of
preparation, is not more than 0.20 (2.4.7).
Warfarin Sodium

(2.4.14).
wate/:
o
Mol. Wt. 330.3
Warfarin Sodium is sodium 2-oxo-3-[(1RS)-3-oxo-l-phenylbutyl]-2H-l-benzopyran-4-0Iate.
Warfarin Sodium contains not less than 98.0 per cent and not
more than 102.0 per cent of CI9HlSNa04, calculated on the
anhydrous basis.

Reference solution (a). Dissolve 2 mg each of of 4hydroxycoumarin (warfarin impurity B) and benzalacetone
(warfarin impurity C) in 25 ml of methanol and dilute to 100 ml
with wate/:
100.0 rnl with the solvent mixture. Dilute 1.0 ml ofthis solution
Dose. 10 to 15 mg.
Reference solution (c). A 0.08 per cent w/v solution of wa/1arin

sodium RS in the solvent mixture.
Description. A white powder; hygroscopic.
Identification
out.
A. Dissolve 1 gin 25 ml of water, add 2 ml of 2 M hydrochloric

acid and filter. Wash the residue with water and dry over
phosphorus pentoxide.
obtained with warfarin sodium RS or with the reference
spectrum ofwarfarin sodium.
solution (c).
C. Dissolve 1 g in 10 ml of water, add 5 ml of nitric acid and
filter. To the filtrate add 2 ml ofpotassium dichromate solution,
shake for 5 minutes and allow to stand for 20 minutes; the
solution i.s not greenish blue when compared with a blank.
D. The residue obtained in test A, after washing with water
and drying at 105, melts at 159 0 to 163 0 (2.4.21).
E. The filtrate obtained in testA gives the reactions of sodium
salts (2.3.1).
Tests
pH (2.4.24). 7.6 to 8.6, determined ina 1.0 per centw/v solution.
- a stainless steel COIUlllil 25 cm x 4.0 mm, packed with
spherical nitrile silica gel (5 /lm),
mobile phase: a mixture of 1 volume of glacial acetic
acid, 25 volumes of acetonitrile and 75 volumes of water,
resolution between the peaks due to warfarin impurity Band
warfarin impurity C is not less than 2.0. The relative retention
time with reference to warfarin for warfarin impurity B is about
0.4 and for warfarin impurity C is about 0.6.
chromatogram twice the retention time ofthe principal peak.
ofthe peak due to warfarin impurity B and warfarin impurity C
is not more than the area of the principal peak in the
cent) and the area of any other secondary peak is not more
obtained with reference solution (b) (0.1 per cent). The sum of
all the secondary peaks is not more than 3 times the area ofthe
cent).
0.75g.
2311
IP 2010
WARFARIN SODIUM CLATHRATE

0.01 M sodium hydroxide to produce 100.0 m1. Dilute 10.0 ml
to 100.Oml with 0.01 Msodium hydroxide and dilute 5.0 ml to
50.0 ml with 0.01 M sodium hydroxide. Measure the
308 run (2.4.7). Calculate the content ofCI9HIsNa04 taking 431
Tests
Phenolic ketones. Absorbance ofa 12.5 per cent w/v solution
in a 5.0 per cent w/v solution of sodium hydroxide at the
maximum at about 385 nm, measured within 15 minutes of
preparation, is not more than 0.20 (2.4.7).
(2.4.14).
Warfarin Sodium Clathrate

Warfarin Sodium Clathrate is a clathrate form of Warfarin
Sodium consisting principally of Warfarin Sodium and
Isopropyl Alcohol in a 2: 1 molecular ratio.

wate/:
Warfarin Sodium Clathrate contains not less than 97.0 per

cent and more than 102.0 per cent ofCI9HIsNa04 calculated on
the anhydrous, isopropyl alcohol-free basis and not less than
8.0 per cent and not more than 8.5 per cent ofisopropy alcohol.
Reference solution (a). Dissolve 2 mg each of 4hydroxycoumarin (warfarin impurity B) and benzalacetone
(warfarin impurity C) in 25 ml of methanol and dilute to 100 ml
with water.
Reference solution (b). Dilute 1.0 ml ofthe test solution to

Dose. 10 to 15 mg.
Description. A white crystalline powder; hygroscopic.
Rejerencesolution (c). AO.08 percentw/v solution ofwaifarin

sodium RS in the solvent mixture.
Identification
Test A may be omitted if test B, C, D and E are carried out.
Tests B ai7d D may be omitted if tests A, C and E are carried
out.
A. Dissolve about 1 g in 25 ml of water, add 2 ml of 2 M

hydrochloric acid and filter. Wash the precipitate 5 to 6 times
with water. Dry the residue over phosphorus pentoxide.
obtained with waifarin sodium RS or with the reference
solution (c).
C. Dissolve 1 g in 10 ml of water, add 5ml of nitric acid and
filter. To the filtrate, add 2 ml ofpotassium dichromate solution,
shake for 5 minutes and allow to stand for 20 minutes; the
solution is not greenish-blue when compared with a blank.
...... ~D: Tlfefesidue-615tainedinlestA, aftefwasningwithwater

and drying at105 melts at 159 to 163.
E. The filtrate obtained in testA gives the reactions of sodium

salts (2.3.1).
spherical nitrile silica gel (5 ).un),
resolution between the peaks due to warfarin impurity Band
warfarin impurity C is not less than 2.0. The relative retention
time with reference to warfarin for warfarin impurity B is about
0.4 and for warfarin sodium impurity C is about 0.6.
chromatogram twice the retention time ofthe principal peak.
ofthe peak due to warfarin impurity B and warfarin impurity C
is not more than the area of the principal peak in the
cent) and the area of any other secondary peak is not more
.. thl:!nthe.l:!rl:1!Qf tl1l:principl:!l Peak in tl1e CJ:JIQ!1!!ltograJ;!1
obta-ined \\lith. referencesolutio.n(~) (O.lpl;lr cl;lnt). The sulIlo.f
all the secondary peaks is not more than 3 times the area ofthe
2312
WARFARIN TABLETS
IP 2010
0.75g.
Isopropyl alcohol. Detennine by gas chromatography (2.4.13).

examination in sufficient water to produce 10 ml.
w/v of the substance under examination and 0.5 per cent v/v
of propan-1-o1 (internal standard).
Reference solution (b). A solution containing 0.5 per cent v/v
each of propan-2-o1 and the internal standard.
a glass column 1.5 m X 4 mm, packed with porous polymer
beads (125 to 150 mm) (Such as Porapak Q),
temperature:
column 150,
inlet port at 180 and detector at 200 0 ,
- flow rate. 40 Jill per minute ofthe carrier gas.
The column temperature may be varied so that the resolution,
R, between propan-l-ol and propanol-2 01 is not less than 2.0,
the tailing factor. T, for the propan-2-01 is not less than 2.0 the
tailing factor T, for the propan-2-01 peak is not more than 1.5
and the relative standard deviation ofthe ratio ofthe area due
to the peak ofproptl1101-2-01 to that due to propan-l-ol for five
replicate injections of reference solution (b) is not more than
2.0 per cent.
Calculate the content of isopropyl alcohol.
0.01 M sodium hydroxide to produce 100.0 ml. Dilute 10.0 ml
to 100.0 ml with 0.01 M sodium hydroxide and dilute 5.0 ml to
50.0 ml with 0.01 M sodium hydroXide. Measure the
308 urn (2.4.7). Calculate the content ofCI9HIsNa04 taking 431
~arfarin
1rablets
Warfarin Sodium Tablets

Warfarin Tablets contain not less than 92.5 per cent and not
more than 107.5 per cent of the stated amount of warfarin
sodium, C,9H,sNa04.
Usual strengths. I mg; 3 mg; 5 mg; 10 mg.
Identification
A. Extract a quantity ofthe powdered tablets containing 0.1 g
of Warfarin Sodium with 30 ml of water, add 0.1 ml of 2 M
hydrochloric acid, filter, wash the precipitate with water and
dry. Wann the residue gently with 3 ml of ethanol (95 per
cent), filter and add the filtrate to 25 .ml of water containing
0.1 ml of 2 M hydrochloric acid. Filter, wash the precipitate
with water and dry it at 105.
obtained with waljarin sodium RS or with the reference
B. The final residue obtained in test A melts at about 159
(2.4.21).
Tests

acid.
containing 40 mg ofWarfarin Sodium with 30 ml of water for
15 minutes, add 0.1 ml of hydrochloric acid and extract with
three quantities, each of 10 ml, of chloroform, drying each
extract with anhydrous sodium sulphate. Evaporate the
combined extracts at a temperature not exceeding 40 and
dissolve the residue in 2 ml of acetone.
.Reference solution (a). Dilute I volume of the test solution to
(E)-4-phenylbut-3-en-2-one in acetone.
4-hydroxycoumarin in acetone.
dry the plate in air and examine immediately in visible light
noting the position ofany coloured spots and then examine in
ultraviolet light at 254 urn, ignoring any spot that was noted in
visible light. Any spots corresponding to (E)-4-phenylbut3-en-2-one and 4-hydroxycoumarin in the chromatogram
obtained with the test solution are not more intense than the
spots in the chromatograms obtained with reference
solutions (b) and (c) respectively and any other secondary
Tablets.
2313
WARFARIN TABLETS
IP 2010
Purified Water
Testsolution. Shake one tablet with 10 ml of 0.01 M sodium

hydroxide for 15 minutes, add 10 ml ofa 2 per cent v/v solution
ofglacial acetic acid in acetonitrile, centrifuge for lOminutes
45 volumes of water and 1 volume of glacial aceti~
acid,
spectrophotometer set at 283 11m,
Detennine the content ofC I9H 1sNa04 in the tablet.
Apparatus No.1,
Medium. 900 ml of a 0.68 per cent w/v solution ofpotassium
dihydrogen phosphate with the pH adjusted to 6.8 by the
addition of 1 M sodium hydroxide,
For tablets containing 2 mg or less of warfarin sodium, use
three tablets for each test; for tablets containing more than
2 mg of warfarin sodium, use a single tablet for each test.
the absorbance of a layer of suitable thickness of the filtrate,
suitably diluted if necessary, at the maxima at about 307 nm
and 360 nm (2.4.7), and calculate the difference between the
two absorbances (DA). Calculate the total content ofwarfarin
sodium, CI9HlsNa04, in the medium taking 428 as the specific
absorbance value of DA.
CI9H,sNa04.
quantity of the powder containing about 20 mg of Warfarin
Sodium ~n,d shake with 250.0 ml of 0.01 M sodium hydroxide
for 15 rrunutes and filter. To 20.0 ml ofthe filtrate add 0.15 ml of
hydrochloric acid and extract with three quantities, each of
15 ml, ofchloroform. Extract the combined chlorofonn layers
with three quantities, each of20 ml, of0.01 M sodium hydroxide.
Dilute the combined aqueous layers to 100.0 ml with
0.01 M sodium hydroxide, filter and measure the absorbance
oftnetesaltingsolationattnelffaximartfatabout307tuu (2.4:7)~
Calculate the content ofC1ijHisNaO" taking 431 as the specific
Mol. Wt. 18.0

Purified Water is prepared by distillation, by means of ion
exchange or by any other appropriate means from suitable
potable water that complies with all relevant statutory
regulations.
During production and subsequent storage, it is recommended
that adequate measures are taken to ensure that the microbial
quality is controlled and monitored. Appropriate alert and
action limits are set so as to detect adverse trends. Under
controlled conditions, an appropriate action limit is a total
viable count (2.2.9) of 100 micro-organisms per ml, determined
by membrane filtration. In addition, the test for oxidisable
substances (given below) is carried out. The adequacy of
these measures may be detennined by carrying out the test
for conductivity (2.4.9) off-line or on-line.
Category. Phannaceutical aid (solvent).
Description. A clear, colourless and odourless liquid.
Tests
Acidity or alkalinity. To 10 ml, freshly boiled and cooled in a
borosilicate glass flask, add 0.05 ml of methyl red solution;
the resulting solution is not red. To 10 ml add 0.1 ml of
bromothymol blue solution; the resulting solution is not blue.
Ammonium. To 20 ml add 1 ml ofalkalinepotassium mercuriiodide solution, and allow to stand for 5 minutes. When
viewed vertically the solution is not more intensely coloured
than a solution prepared at the same time by adding 1 ml of
alkaline potassium mercuri-iodide solution to a mixture of
4.0 ml of ammonium standard solution (l ppm NH4) and
16.0 ml ofammonia-free water (0.2 ppm).
Calcium and magnesium. To.l 00 ml add 2 ml of ammonia
bufferpH 10.0, 50 mg of mordant black II mixture and 0.5 ml
of 0.01 M disodium edetate; a pure blue colour is produced.
Heavy metals (2.3.13). Evaporate 150 ml to 15 ml on a waterbath; 12 ml of the solution complies with the limit test for
heavy metals, Method D (0.1 ppm). Use leadstandardsolution
(l ppm Pb) to prepare the standard.
Chlorides. To 10 ml add 1 ml of 2 M nitric acid and
0.2 ml of 0.1 M silver nitrate; the appearance of the solution
does not change for at least 15 minutes.
Nitrates. To 5 ml in a test-tube immersed in ice add 0.4 ml ofa
10 per cent w/v solution of potassium chloride, 0.1 ml of
diphenylamine solutionand, . dropwisewithshaking,5_mLof
sJA./phur}c:a,cid. 'TIans{erthe ttlbc:: tq 11. Wl1tc::r::batb at 50 and
allow to stand for 15 minutes. Any blue colour in th~~~luti~~
is not more intense than that in a solution prepared at the
same time and in the same manner using a mixture of4.5 ml of
2314
WATER FOR INJECTIONS IN BULK
IP 2010
nitrate-fi"ee water and 0.5 ml of nitrate standard solution

(2 ppm N0 3) (0.2 ppm).
stored in conditions designed to prevent the growth ofmicroorganisms and to avoid any other contamination.
Sulphates. To 10 ml add 0.1 ml of 2 M hydrochloric acid and

0.1 ml of barium chloride solution. The appearance of the
solution does not change for at least 1 hour.
During production and subsequent storage, it is recommended

that adequate measures are taken to ensure that the microbial
quality is controlled and monitored. Appropriate action limit
of total viable count (2.2.9) of 10 micro-organisms per ml,
detennined by membrane filtration using at least 200 ml of
water for injection in bulle. Appropriate alert and action limits
are set so as to detect adverse trends. The adequacy of these
measures is determined by the following tests that may be
done off-line or on-line.
Oxidisable substances. To 100 ml add 10 ml of 1 M sulphuric

acid and 0.1 ml of 0.02 M potassium permanganate and boil
for 5 minutes; the solution remains faintly pink.
Residue on evaporation. Evaporate 100 ml to dryness on a
water-bath and dry to constant weight at 105. The residue
weighs not more than I mg (0.001 per cent).
Purified Water intendedfor use in the manufacture ofdialysis

solutions and also without a fitrther procedure for the
additional requirements. l!l
Aluminium (2.3.8). Not more than 10 ppb, determined using
the following solutions.
Test solution. To 400 ml of the water under examination, add

10 ml of acetate buffer solution pH 6. aand 100 ml of ~istilled
water.
Reference solution. Mix 2 ml of aluminium standardsolution
(2 ppm AI), 10 ml of acetate buffer solution pH 6. and 98 ml
of distilled water.
Blank solution. Mix 10 ml of acetate btifJer solution pH 6.0

and 100 ml of distilled water.
Unit per ml.

H20
Mol. Wt. 18.0
Water for Injections is water intended for use in the

preparations of medicines for parenteral administration when
water is used as a vehicle (Water for Injections in bulk) and for
dissolving or diluting substances or preparations for injectable
preparations (Sterile Water for Injections).
Water for Injections in Bulk

Production
Water for Injections in bulk is obtained by distilling potable
water or Purified Water from a neutral glass, quartz or suitable
metal still fitted with an effective device for preventing the
entrainment of droplets; the still must be suitably maintained
to ensure the production ofapyrogenic water. The first portion
ofthe distillate is discarded and the remainder is collected and
Total organic carbon (2.4.30). Not more than 0.5 mg per litre.
Conductivity (2.4.9). Meets the requirements ofthe test.
Description. A clear, colourless and o"dourless liquid.
Tests
Acidity or alkalinity. To 10 ml, freshly boiled and cooled in a
borosilicate glass flask, add 0.05 ml of methyl red solution;
the resulting solution is not red. To 10 ml add 0.1 ml of
bromothymol blue solution; the resulting solution is not blue.
Ammonium. To 20 ml, add 1ml of alkalinepotassium mercuriiodide solution and allow to stand for 5 minutes. When viewed
vertically the solution is not more intensely coloured than a
solution prepared at the same time by adding 1 ml of alkaline
potassium mercuri-iodide solution to a mixture of 4.0 ml of
ammonium standard solution (1 ppm NH4) and 16.0 ml of
ammonia-free water (0.2 ppm).
Calcium and magnesium. To 100 ml, add 2 ml of ammonia
btifJerpH 10.0,50 mg ofmordant black II mixture and 0.5 ml of
0.01 M disodium edetate; a pure blue colour is produced.
Heavy metals (2.3 .13). Evaporate 150 ml to 15 ml on a waterbath. 12 ml of the solution complies with the limit test for
(1 ppm Pb) to prepare the standard.
Chlorides. To 10 ml, add 1 ml of 2 M nitric acid and
0.2 ml of 0.1 M silver nitrate; the appearance of the solution
does not change for at least 15 minutes.
diphenylamine solution and, dropwise with shaking, 5 ml of
sulphuric acid. Transfer the tube to a water-bath at 50 and
allow to stand for 15 minutes. Any blue colour in the solution
is not more intense than that in a solution prepared at the
same time and in the same manner using a mixture of4.5 mlof
nitrate-free water and 0.5 ml of nitrate standard solution
(2 ppm N0 3) (0.2 ppm).
Sulphates. To 10 ml, add 0.1 ml of2 M hydrochloric acid and
2315
IP 2010
WATER FOR INJECTIONS IN BULK
Aluminium (2.3.8) For waterfor injections intendedfor use

in the manufacture of dialysis solutions.
Not more than 10 ppb, determined using the following
solutions.
Calcium and magnesium. Tol 00 ml, add 2 ml of ammonia

bufferpH 10.0,50 mg of mordant blackII mixture and 0.5 ml
of 0.01 M disodium edetate; a pure blue colour is produced.
Test solution. To 400 ml of the water under examination add

10 m1 of acetate buffer solution pH 6. 0 and 100 ml of distilled
water.
Heavy metals (2.3.13). Evaporate 150 ml to 15 ml on a waterbath. 12 ml of the solution complies with the limit test for
(1 ppm Pb) to prepare the standard.
Reference solution. Mix 2 rnl of aluminium standardsolution

(2 ppm Al), 10 ml of acetate buffer solution pH 6.0 and 98 ml
of distilled water.
Chlorides.To 10 ml, add 1 ml of 2 Mnitric acid and 0.2 ml of

0.1 ivJ silver nitrate; the appearance of the solution does not
change for at least 15 minutes.
Blank solution. Mix 10 ml of acetate buffer solution pH 6.0

and 100 ml of distilled water.
Unitperml.
Storage. Store in containers designed to prevent the growth
ofmicro-organisms.
Labelling. The label on the container in which the bulk has
been distributed states that the contents haVeliot been
sterilised.

Sterile Water for Injections is Water for Injections in bulle that
has been distributed in suitable containers of glass or any
other material, sealed and sterilised by heat under conditions
that ensure that the water complies with the test for bacterial
endotoxins. It is free from any added substances. Each
container contains a sufficient quantity ofWater for Injections
to permit the withdrawal ofthe nominal volume.
Description. A clear, colourless liquid; odourless.
Tests
Appearance ofsolution. When examined in suitable conditions
ofvisibility, it is clear (2.4.1) colourless (2.4.1) and practically
free from suspended particles.
Acidity or alkalinity. To 20 ml, add 0.05 ml of phenol red
solution. Ifthe solution is yellow, it becomes red on the addition
of0.1 rnl of O. 01 M sodium hydroxide; ifred, it becomes yellow
on the addition of 0.15 ml of 0.01 M hydrochloric acid.
For containers with a nominal volume oflOO ml or less, 15 ml

complies with the limit test for chlorides (2.3.12) (0.5 ppm),
using a standard solution prepared by mixing 1.5 ml of chloride
standard solution (5 ppm Cl) and 13.5 ml of water.

diphenylamine solution and, dropwise with shaking, 5 ml of
sulphuric acid. Transfer the tube to a water-bath at 50 and
allow to stand for 15 minutes. Any blue colour in the solution
is not more intense than that in a solutioIl prepared at the
same time and in the same manner using a mixture of4.5 mlof
nitrate-free water and 0.5 ml of nitrate standard solution
(2 ppm N03) (0.2 ppm).
Sulphates. To 10 ml, add 0.1 ml of2 Mhydrochloric acid and
Oxidisable substances. Boil 100 rnl with 10 rnl of1 M sulphuric
acid, add 0.4 ml of 0.02 M potassium permanganate (for Sterile
Water for Injection in containers with fill volume ofless than
50 ml) or 0.2 ml of 0.02 Mpotassiumpermanganate (for Sterile
Water for Injection in containers with fill volume of 50 ml or
more) and boil for 5 minutes. Ifa precipitate fonus, cool in an
ice-bath to room temperature and filter through a sintered
glass filter (porosity No.3). The pink colour of the solution
does not disappear completely.
Residue on evaporation. Evaporate 100 ml to dryness on a
water-bath and dry the residue to constant weight at 105. For
containers with a nominal volume of 10 ml or less, the residue
weighs not more than 4 mg (0.004 per cent) and for containers
with a nominal volume greater than 10 ml, the residue weighs
not more than 3 mg (0.003 per cent).
Particulate contamination (2.5.9). Complies with the
requirements of Method 1 or Method 2.
Ammonium. To 20 m1, add 1 rnl ofalkalinepotassium mercuriiodide solution and allow to stand for 5 minutes. When viewed Bacterial endotoxills (2.2.3). Not more than 0.25 Endotoxin
.vertically the. solution is. not mcireintenselycolouredthana -Unitper ml.--------------------------------.
soilltion prepared at the same time by adding 1. ml of alkaline
Sterility (2.2;11 ).Complies with the test for sterility;
potassium mercuri-iodide solution to a mixture of 4.0 ml of
ammonium standard solution (l ppm NH,J and 16.0 ml of Storage. Store in single dose containers ofnot larger than one
litre size.
ammonia-free water (0.2 ppm).
c
2316
WOOL FAT
IP 2010
Wool Fat
hydroxide and boil; the vapours do not turn red litmus paper
blue.
Anhydrous Lanolin
Wool Fat is purified, anhydrous, waxy material obtained from
the wool of sheep. It may contain Butylated Hydroxytoluene
as an antioxidant.
Category. Pharmaceutical aid (absorbent ointment base).
Description. A pale yellow, unctuous substance; odour,
characteristic.
Identification
A. To a solution of 0.5 g in 5 ml of chloroform add I ml of
acetic anhydride and 0.1 ml of sulphuric acid; a green colour
develops.
B. To a solution of 50 mg in 5 ml of chloroform add 5 ml of

sulphuric acid and shake; a red colour is produced and a
strong fluorescence appears in the lower layer.
Tests
Melting range (2.4.21). 34 to 44, determined by Method IV.
To fill the metal cup, melt the substance under examination on
a water-bath, cool to about 50, pour into the cup and allow to
stand at 15 to 20 for 24 hours.
Acid value (2.3.23). Not more than 1.0, determined on 5.0 g
dissolved in 25 ml ofthe prescribed mixture of solvents.
Chlorides. Boil 1.0 g with 20 ml of ethanol (90 per cent) under

a reflux condenser for 5 minutes, cool, add 40 ml of water and
0.5 ml of nitric acid and filter. To the filtrate add 0.15 ml of a
I per cent w/v solution of silver nitrate in ethanol (90 per
cent). After 5 minutes, protected from light; any opalescence
produced is not more intense than that obtained by adding
0.15 ml ofa I per cent w/v solution of silver nitrate in ethanol
(90 per cent) to a mixture of 0.2 ml of 0.02 M hydrochloric
acid, 20 ml of ethanol (90 per cent), 40 ml of water and 0.5 ml
of nitric acid (150 ppm).
Paraffins. Prepare an alumina column 23 cm x 2 cm by adding
a slurry of anhydrous aluminium oxide and light petroleum
(40 to 60) to a glass tube fitted with a tap and containing
the light petroleum; the tap and absorbent cotton plugs should
be free from grease. Allow to settle and reduce the depth of
the solvent above the column to about 4 cm. Dissolve 3.0 g of
the substance under examination in 50 ml of warm light
petroleum (40 to 60), cool, pass the solution through the
column at a rate on ml per minute and wash with 250 ml ofthe
light petroleum. Distil the combined eluate and washings to
low bulle, evaporate to dryness on a water-bath and heat the
residue at 105 for periods of 10 minutes until the difference
between two successive weighings is not greater than 1 mg;
Butylated hydroxytoluene (ifpresent). Not more than 200 ppm,
detennined by gas chromatography (2.4.13).

Saponification value. (2.3.37). 90 to 105. Heat for 4 hours.
Water-absorption capacity. Weigh 10.0 g into a mortar. Add
water in quantities of 0.2 to 0.5 ml from a burette and stir
vigorously, incorporating all the water before proceeding to
the next addition. The end-point is reached when visible
droplets r~main that cannot be incorporated; not less than
20 ml ofwater is absorbed.
Water-soluble acidic or alkaline substances. Shake
vigorously 5.0 g, previously melted on a water-bath, for
2 minutes with 75 ml of water previously heated to 90 to 95.
Allow to cool and filter through filter paper previously washed
with water. To 60 ml ofthe filtrate, which may not be clear, add
0.25 ml of bromothymol blue solution. Not more than 0.2 ml of
0.02 M hydrochloric acid or 0.15 ml of 0.02 M sodium
hydroxide is required to change the colour of the solution.
Water-solubleoxidisable substances. To 10 ml ofthe filtrate
obtained in the test for Water-soluble acidic or alkaline
substances add 1 ml of 1 M sulphuric acid and 0.1 ml of
0.02 M potassium permanganate; the solution is not
completely decolorised within 10 minutes.
Ammonia. To 10 ml ofthe filtrate obtained in the test for Watersoluble acidic or alkaline substances, add 1 ml of 1 M sodium
Test solution (a). A 10 per cent w/v solution of the substance

under examination in carbon disulphide.
Test solution (b). A solution containing 10 per cent w/v ofthe
substance under examination and 0.002 per cent w/v of methyl
n-decanoate (internal standard) in carbon disulphide.
each of butylated hydroxytoluene and the internal standard.
a glass column 1.5 m x 4 mm, packed with silanised
diatomaceous support (80 to 100 mesh) (such as
Diatomite) impregnated with 10 percent w/w ofsilicone
gum rubber (methyl) (such as SE-30),
temperature:
column 150,
- flow rate. 40 ml per minute ofthe caITier gas.
Calculate the content of butylated hydroxytoluene in the
substance under examination from the heights or areas of the
peaks due to butylated hydroxytoluene and the internal
standard in the chromatograms obtained with test solution
(b) and the reference solution.
2317
HYDROUS WOOL FAT
IP 2010

residue at 105 for periods of 10 minutes until the difference

between two successive weighings is not greater than 1 mg;
Labelling. The label states the proportion of any butylated

hydroxytoluene present.
Saponification value (2.3.37). 67 to 79. Heat for 4 hours.
Hydrous Wool Fat

Hydrous Wool Fat is a mixture of75 per cent w/w ofWool Fat
and 25 per cent w/w ofPurified Water.
Category. Phannaceutical aid (water-in-oil emulsion ointment
base).
Description. A pale yellow, unctuous substance; odour, faint
and characteristic. On heating, it separates at first into two
layers; with continued heating with stirring, water is driven
off and the residue which is transparent while wann, cools to
fonn a yellowish, tenacious, soft mass.
Identification
A. To a solution of 0.5 g in 5 ml of chloroform add 1 ml of
acetic anhydride and 0.1 ml of sulphuric acid; a green colour
develops.
B. To a solution of 50 mg in 5 ml of chloroform add 5 ml of
sulphuric acid and shake; a red colour is produced and a
strong fluorescence appears in the lower layer.
Tests
Melting range (2.4.21). 34 to 44, detennined by Method IV
To fill the metal cup, melt the substance under examination on
a water-bath, cool to about 50, pour into the cup and allow to
stand at 15 to 20 for 24 hours.
Acid value (2.3.23). Not more than 1.0, detennined on 5.0 g
dissolved in 25 ml ofthe prescribed mixture of solvents.
Paraffins. Prepare an alumina column 23 cm x 2 cm by adding
a slurry of anhydrous aluminium oxide and light petroleum
(40 to 60) to a glass tube fitted with a tap and containing
the light petroleum; the tap and absorbent cotton plugs should
be free from grease. Allow to settle and reduce the depth of
the solvent above the column to about 4 cm. Dissolve 3 g of
the substance under examination in 50 ml of warm light
petroleum (40 to 60); cool, pass the solution through the
column at a rate 00 ml per minute and wash with 250 ml ofthe
lightpetroleum.Distil the combined eluate and washings to
low bulk, evaporate to dryness on a water~bath and heat the
Water-absorption capacity. Weigh 10.0 g of the residue

obtained in the test for Wool fat content into a mortar. Add
water in quantities of 0.2 to 0.5 ml from a burette and stir
vigorously, incorporating all the water before proceeding to
the next addition. The end-point is reached when visible
droplets remain that cannot be incorporated; not less than
20 ml ofwater is absorbed.
Water-soluble acidic or alkaline substances. Shake
vigorously 6.7 g, previously melted on a water-bath,
for 2 minutes with 75 ml of water previously heated to 90 to
95, Allow to cool and filter through filter paper previously
washed with water. To 60 ml ofthe filtrate, which may not be
clear, add 0.25 ml of bromothymol blue solution. Not more
than 0.2 ml of 0.02 M hydrochloric acid or 0.15 ml of
the solution.
Water-soluble oxidisable substances. 10 ml of the filtrate
obtained in the test for Water-soluble acidic or alkaline
substances, add 1 ml of 1 M sulphuric acid and 0.1 ml of
0.02 M potassium permanganate; the solution is not
completely decolorised within 10 minutes.
Ammonia. To 10 ml ofthe filtrate obtained in the test for Watersoluble acidic or alkaline substances add 1 ml of 1 M sodium
hydroxide and boil; the vapours do not turn red litmus paper
blue.
Chlorides. Boil 1.0 g with 20 ml of ethanol (90 per cent) under
a reflux condenser for 5 minutes, cool, add 40 ml of water and
0.5 ml of nitric acid and filter. To the filtrate, add 0.15 ml of
1.0 per cent w/v solution of silver nitrate in ethanol (90 per
cent). After 5 minutes, protected from light, any opalescence
produced is not more intense than that obtained by adding
0.15 ml ofa 1 per cent w/v solution of silver nitrate in ethanol
(90 per cent) to a mixture of 0.2 ml of 0.02 M hydrochloric
acid, 20 ml of ethanol (90 per cent), 40 ml of water and 0.5 ml
of nitric acid (150 ppm).
Wool fat content. 72.5 to 77.5 per cent.
Weigh accurately about 30 g in a tared porcelain dish
containing a glass rod, heat on a water-bath with continuous
stirring to constant weight and weigh the residue.
2318
MONOGRAPHS
x
XanthanGum
2321
Xylometazoline Hydrochloride
2322
Xylometazoline Nasal Drops
2323
Xylose
2324
2319
XANTHAN GUM
IP 2010
2-Propanol. Not more than 750 ppm
Xanthan Gum
Xanthan Gum is high-molecular-mass anionic polysaccharide
produced by fermentation ofcarbohydrates withXanthomonas
campestris. It consists ofa principal chain ofb(1-lJ1.)-linked
o-glucose units with trisaccharide side chains, on alternating
anhydroglucose units, consisting of 1 glucuronic acid unit
included between 2 mannose units. Most ofthe terminal units
contain a pyruvate moiety and the mannose unit adjacent to
the principal chain may be acetylated at C-6.
Xanthan Gum has a relative molecular mass ofapproximately
1 x 106 . It exists as the sodium, potassium or calcium salt.
Xanthan Gum contains not less than 1.5 per cent ofpyruvoyl
groups, (C 3H30 2 ; Mol. Wt. 71.1), calculate on the dried basis.
Category. Pharmaceutical aid (Excipient).
Description. A white or yellowish-white, free-flowing powder.
Identification
A. Suspend 1 g in 15 ml of 0.1 M hydrochloric acid in a flask,
close the flask with a fermentation bulb containing barium
hydroxide solution and heat carefully for 5 minutes. The
barium hydroxide solution shows a white turbidity.
B. To.300 m1 of water, previously heated to 80 and stirred

rapidly with a mechanical stirrer in a 400 ml beaker, add, at the
point ofmaximum agitation, a dry blend of 1.5 g of carob bean
gum and 1.5 g of the substance under examination. Stir until
the mixture forms a solution, and then continue stirring for 30
minutes or longer. Do not allow the water temperature to drop
below 60 during stirring. Discontinue stirring and allow the
mixture to stand for at least 2 hours. A firm rubbery gel forms
after the temperature drops below 40 but no such gel forms in
a I per cent control solution of the sample prepared in the
same manner but omitting the carob bean gum.
Tests
pH (2.4.24). 6.0 to 8.0 determined in I per cent w/v solution.
Viscosity (2.4.28). Not less than 600 mPas.
Add 3.0 g within 45-90 seconds into 250 ml of a 1.2 per cent
w/v solution ofpotassium chloride in a 500 ml beaker stirring
with a low-pitch propeller-type stirrer rotating at 800 rpm.
When adding the substance take care that agglomerates are
destroyed. Add an additional quantity of 44 ml of water, to
rinse any adhering residue from the walls of the beaker. Stir
the preparation at 800 rpm for 2 hours whilst maintaining the
temperature at 24 1. Determine the viscosity within 15 min
at 24 I using a rotating viscosimeter set at 60 rpm and
equipped with rotating spindle 12.7 rom in diameter and
1.6 rom high which is attached to a shaft 3.2 mm in diameter.
The distance from the top of the cylinder to the lower tip of
the shaft being 25.4 mm, and the immersion depth being
50.0mm.
Determine by gas chromatography (2.4.13).

Internal standard solution. Dilute 0.50 g of 2-methyl-2propanol to 500 ml with water.
Test solution. To 200 ml of water in a IOOO-mi round bottomed
flask, add 5.0 g of the substance under examination and I ml
ofa 1.0 per cent w/v emulsion of dimeticone in liqzdd paraffin,
stopper the flask and shake for I hour. Distil about 90.0 ml, mix
the distillate with 4.0 ml ofthe internal standard solution and
dilute to 100.0 ml with water.
Reference solution. Dilute a suitable quantity of 2-propanol,
accurately weighed, with water to obtain a solution having a
Imown concentration of 2-propanol of about I mg per ml. To
4.0 ml of this solution add 4.0 ml of the internal standard
solution and dilute to 100.0 ml with water.
- a glass or stainless steel column 1.8 m x 4 mm, packed
with styrene-divinylbenzene copolymer (149-177 /lm),
- temperahlre:
column. 165,
injection port and detector 200,
- flow rate. 30 ml per minute ofnitrogen as carrier gas.
Inject 5 /ll of the reference solution. The test is not valid
unless the relative retention times with reference to 2-propanol
for 2-methyl-2-propanol is about 1.5.
Other polysaccharides. Determine by thin-layer
chromatography (2.4.17), coating the plate with silica gel.
Mobile phase. A mixture of 10 volumes of 1.6 per cent w/v

solution of sodium dihydrogen phosphate, 40 volumes of
butanol and 50 volumes of acetone.
Test solution. To 10 mg ofthe substance under examination in
a thick-walled centrifuge test tube add 2ml of a 23 per cent
w/v solution of trifluoroacetic acid, shake vigorously to
dissolve the forming gel, stopper the test tube, and heat the
mixture at 120 for 1 hour. Centrifuge the hydrolysate, transfer
the clear supernatant liquid carefully into a 50 ml flask, add
10 ml of water and evaporate the solution to dryness under
reduced pressure. Take up the residue thus obtained in 10 ml
of water and evaporate to dryness under reduced pressure.
Wash 3 times with 20 ml of methanol and evaporate under
reduced pressure. To the resulting clear film which has no
odour ofacetic acid, add 0.1 ml of water and 1 m10fmethanol.
Centrifuge to separate the amorphous precipitate. Dilute the
supernatant liquid, if necessary, to 1 ml with methanol.
Reference solution. Dissolve 10 mg of glucose and 10 mg of
mannose in 2 ml of water and dilute to 10 ml with methanol.
2321
XANTHAN GUM
IP 2010
Apply to the plate 5 JlI of each solution as bands 10 mm by 2

nun. Allow the mobile phase to rise 15 cm. Dry the plate in air
and spray with 2 per cent w/v solution of diphenylamine in
methanol to which added 0.5 ml of aniline and 2.5 ml of
orthophosphoric acid. Heat the plate at 1200 for 5 minutes
and examine in day light. The principal spot in the
chromatogram obtained with the test solution shows 2 spots
cOlTesponding to the spots due to glucose and mannose in
the chromatogram obtained with the reference solution. In
addition, 1 weak reddish and 2 faint bluish-grey bands may be
visible just above the starting line. 1 or 2 bluish-grey bands
may also be seen in the upper quarter of the chromatogram.
No other bands are visible.
Xylometazoline Hydrochloride is 2-(4-tert-butyl2,6-dimethylbenzyl)-2-inlidazoline hydrochloride.

the reference solution shows two clearly separated greyishbrown spots due to glucose and mannose in the middle third.
Xylometazoline Hydrochloride contains not less than 99.0 per

cent and not more than 101.0 per cent of C16H24N2' HCl,
Total ash (2.3.19). Not less than 6.5 and not more than 16.0per
cent, determined on 1.0 g.
determined on 1.0 g by drying in an oven at 105 0 for 2.5 hours.
Microbial contamination (2.2.9). Total microbial count is not
more than 103 bacteria and 102fungi per gram, determined by
plate count. 1 g is free from Escherichia coli.
Assay.

examination containing 120 mg ofthe dried substance in 20 ml
of water.
Reference solution. Dissolve 45 mg ofpyruvic acid in 500 ml
of water.
Place 10.0 ml of the test solution in a 50 ml round-bottomed
flask, add 20.0 ml of 0.1 M hydrochloric acid and weigh. Boil
on a water-bath under a reflux condenser for 3 hours. Weigh
and adjust to the initial mass with water. In a separating funnel
mix 2.0 ml ofthe solution with 1.0 ml ofdinitrophenylhydrazinehydrochloric solution. Allow to stand for 5 minutes and add
5.0 ml of ethyl acetate. Shake and allow the solids to settle.
Collect the upper layer and shake with three quantities, each
of 5.0 ml, of sodium carbonate solution. Combine the aqueous
layers and dilute to 50.0 ml with sodium carbonate solution,
mix. Treat 10.0 ml of the reference solution at the same time
and in the same manner as for the test solution.
Immediately measure the absorbance of the two solutions at
the maximumat about375nm (2.4.7), using sodium carbonate
solution asthe COmpensation liquid. The absorbance of the
test solution is not less than that of the reference solution,
which corresponds to a content of pyruvic acid of not less
than 1.5 per cent.
CI6H2~2,HCI
Mol. Wt. 280.8
Category. Sympathomimetic amine.

Identification
Compare the spectrum with that obtained with xylometC17oline
xylometazoline hydrochloride.
an absorption maximum at about 265 urn and a minimum at
about 257 nm with two inflections at about 270 nm and
C. To 1 ml ofa 0.05 per cent w/v solution, add 0.2 ml ofa 5 per
cent w/v solution of sodium nitropnlsside and 0.1 ml of 5 M
sodium hydroxide, allow to stand for 10 minutes and add 2 ml
of sodium bicarbonate solution; a violet colour is produced.
Tests
pH (2.4.24).5.0 to 6.6, determined in a 5.0 per centw/vsolution.
N-(2-Aminoethyl)-4-tert-butyl-2, 6-xylylacetamide. Determine
by thin-layer chromatography (2.4.17), coating the plate with
silica gel HF254.

exaiiiiiiafioniiiTOillI6f mi!lhanoZ:-~----------- - - - --,------- Reference solution .. A 0.01 per cent w/v solution of
N-(2-aminoethyl)-4-tert-butyl-2,6-xylylacetamide RS in
methanol.
2322
IP 2010
XYLOMETAZOLINE NASAL DROPS

dry the plate in air, spray with a solution containing 0.3 g of
ninhydrin in a mixture of 100 ml of 1-butanol and 3 ml of
glacial acetic acid. Heat at 1000 for 10 minutes, allow to cool,
and spray with dilute potassium iodobismuthate solution.
Any spot corresponding to N-(2-aminoethyl)-4-tert-butyl2,6-xylyl-acetamide in the chromatogram obtained with the
(2.4.14).

examination in 50.0 ml of watel: Allow to stand for 1 hour
before injection.
100.0 ml with water. Dilute 2.0 ml ofthis solution to 100.0 ml
with water.
Reference solution (b). Dissolve 5 mg each of N-(2aminoethyl)-2-[4-(1, 1-dimethylethyl)-2, 6- dimethylphenyl}
acetamide RS (xylometazoline impurity A RS) and the
substance under examination in 50.0 of watel: Dilute 10.0 ml of
this solution to 50.0 ml with water.
to 50.0 ml with watel:
(5Ilm),
mobile phase: A. a 0.14 per cent w/v solution of
potassium dihydrogen phosphate adjusted to pH 3.0
B. acetonitrile,
below,
Time
(min.)
0-5
5-20
20-35
35-37
37-47
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
70
30
70 ~15
30~85
15
85
15 ~70
85~30
70
30
Inject the test solution, reference solution (a) and (c). In the
peak due to xylometazoline impurity A is not more than the
with reference solution (c) (0.2 per cent) and the area of any
other secondary peak is not more than the area ofthe principal
(a) (0.1 per cent). The sum of all the secondary peaks is not
Iron (2.4.14). Moisten the residue obtained in the test for
Sulphated ash with 5 ml of hydrochloric acid, evaporate to
dryness and dissolve in sufficient water to produce 50 ml.
10 ml of the resulting solution complies with the limit test for
iron (50 ppm).
Sulphates (2.3.17). O. 75 g complies with the limit test for
solution. Titrate with 0.1 M perchloric acid, using
titration.
C 16Hz4Nz, HCl.

Xylometazoline Hydrochloride Nasal Drops
Xylometazoline Nasal Drops are a solution ofXylometazoline
Hydrochloride in Purified Water.
Xylometazoline Nasal Drops contain not less than 90.0 per
xylometazoline hydrochloride, C16Hz4Nz, HCl.
resolution between the peaks due to xylometazpline and
xylometazoline impurity A is not less than 2.5. The relative
retention time with reference to xylometazoline for
xylometazoline impurity A is about 0.79.
Identification
A. To a volume containing 50 mg of Xylometazoline
Hydrochloride add 5 ml of 1 M sodium hydroxide, extract with
10 ml of dichloromethane, evaporate to dryness and dissolve
the residue in 0.5 ml of dichloromethane.
2323
XYLOMETAZOLINE NASAL DROPS
IP 2010

Compare the spectrum with that obtained with xylometazoline
reference spectrum ofxylometazoline.
B. To a volume containing 0.5 mg of Xylometazoline
HydrocWoride add 0.2 m1 ofa 5 per cent w/v solution ofsodium
nitroprusside and 0.1 ml of 5 M sodium hydroxide, allow to
stand for 10 minutes and add 1 inl of sodium bicarbonate
solution; a violet colour is produced.
Tests
pH (2.4.24).5.6 to 6.6.
N-(2..,Aminoethyl)-4-tert-butyl-2,6-xylylacetamide. Determine
silica gel HF254.

Test solution. Add a volume containing 10 mg of
Xylometazoline Hydrochloride to 30 ml of water, add 5 mlof
5 M sodium hydroxide, mix, extract with three quantities, each
of20 ml, of dichloromethane, evaporate the combined extracts
to dryness and dissolve the residue in 1 ml of dichloromethane.
the residue in 10.0 ml of 0.01 Mhydrochloric acid. To 2.0 ml

of this solution add 3 ml of water, 2.5 ml of 1 M sodium
hydroxide and 2.5 ml of a 5 per cent w/v solution of sodium
nitroprusside, mix and allow to stand protected from light for
10 minutes. Add 10 ml of a freshly prepared 8.3 per cent w/v
solution of sodium bicarbonate, dilute to 100.0 ml with water,
allow to stand protected from light for 10 minutes and measure
about 560 nm (2.4.7), using as blank a solution prepared by
treating 5 ml of water and 2.5 ml of 1 M sodium hydroxide in
the same manner beginning at the words "and 2.5 m1 ofa 5 per
cent w/v solution of sodium nitroprusside,.....".
Calculate the content of C16H24N2, HCl from the absorbance
obtained by repeating the operation using a 0.1 per cent w/v
solution of xylometazoline hydrochloride RS in place of the
nasal drops.
Xylose
D-Xylose; D-Xylopyranose
~O~OH
Reference solution. A 0.03 per cent vi/v solution of

N-(2-aminoethyl)-4-tert-butyl-2,6-xylylacetamide RS in
dichloromethane.
illy the plate in air, spray with a solution containing 0.3 g of
ninhydrin in a mixture of 100 ml of I-butanol and 3 ml of
glacial acetic acid. Heat at 100 for 10 minutes, allow to cool,
and spray with dilute potassium iodobismuthate solution.
Any spot corresponding to N-(2-aminoethylj::4-tert-butyl2,6-xylylacetamide in the chromatogram obtained with test
Other tests. Comply with the tests stated under Nasal
Preparations.
Assay. To a, volume containing 10 mg of Xylometazoline
Hydrochloride add 5 ml of water, 10 ml of 2 M hydrochloric
acid and 10 ml of dichloromethane and shake for 1 minute.
Discard the dichloromethane layer and repeat the extraction
with two further quantities, each of 10 ml, of dichloromethane.
Add to the aqueous extract 10 ml of 5 M sodium hydroxide
and 10 ml of dichloromethane, shake for 1 minute and allow to
.~t~.I2fI.I!lt~,. f il!{l! tl1~Qi(;111QrQ1!1{ltl1ll.11_{l~x:trll.~t1lrQl1gl1.g!ll.~i3.~Q9!

and repeat the extraction with four further quantities, each of
10 ml, of dichloromethane. Evaporate the combined
dichloromethane extracts almost to dryness on a water-bath,
remove the final traces ofsolvent in a current ofair and dissolve
H6~
OH
Mol. Wt. 150.1

Xylose contains not less than 98.0 per cent and not more
than 102.0 per cent of CSHIOOS, calculated on the dried basis.
Category. Diagnostic aid in intestinal malfunction.
Description. Colourless needles or a white, crystalline powder.
Identification
the plate with a suspension of silica gel G in a 0.3 per cent
w/v solution of sodium acetate to fonn a uniform layer 0.5 mm
thiclc
Mobile phase. A mixture ono volumes ofglacial acetic acid,

60 volumes of chloroform and 10 volumes of water.
examination in 10 m1 of water.
Referencesolution{a):k5:0percentw/vsolutionufxylose
RS in water.
2324
XYLOSE
IP 2010
Apply to the plate 2 III of each solution. Develop the plate in

a continuous elution tank for about 4 hours. Dry the plate in
warm air, spray with a solution in acetone containing 1 per
cent w/vsolution of diphenylamine, 1 per cent v/v of aniline
and 1 per cent v/v ofphosphoric acid and heat for 10 minutes
at 1300. The principal spot in the chromatogram obtained with
the test solution corresponds to that in the chromatogram
on 1.0 g by drying in an oven at 1000at a pressure not exceeding
0.7kPa.
B. When heated with potassium cupri-tartrate solution it

produces a copious precipitate of cuprous oxide.
Assay. Carry out the follOWing procedure keeping strict

control oftime between steps.
Tests
Appearance ofsolution. A 10.0 per cent w Iv solution in carbon
dioxide-ji-ee water is clear (2.4.1), and colourless (2.4.1).
Acidity. Dissolve 5.0 g in 50 ml of carbon dioxide-ji-ee water.
Not more than 0.2 ml of 0.1 M sodium hydroxide is required to
neutralise the solution using dilute phenolphthalein solution
as indicator.
Specific optical rotation (2.4.22). +l8S to +19S, determined
at 200 in a 10.0 per cent w/v solution containing 0.4 per cent
v/v of 5 M ammonia.
Arsenic (2.3.10). Dissolve 10.0 g ill 50 ml of water and add
10 ml of stannated hydrochloric acid AsT. The resulting
solution complies with the limit test for arsenic (l ppm).
Iron (2.3.14). A solution of 4.0 g in 25 ml of water complies

with the limit test for iron (l0 ppm).
Chlorides (2.3.12). Dissolve 3.0 g in 20 ml of water. 5 mlofthe
(330 ppm).
Weigh accurately about 1.0 g of the substance under

examination, dissolve in a saturated solution of benzoic acid
in a 1OO-ml volumetric flask and dilute to volume with the same
solvent. Dilute 1.0 ml ofthis solution to 100.0 rn1 with the same
solvent. To 1.0 ml of the resulting solution, in two different
test-tubes, add 5 ml of 4-bromoaniline solution into each
tube and mix. Loosely stopper one tube, place in a water-bath
maintained at 700for 10 minutes, remove, cool rapidly to room
temperature and mix. Keep the tube in the dark for 70 minutes
and measure the absorbance at the maximum at about 520 urn
(2.4.7), using the untreated solution in the second test tube as
the blank. Simultaneously, carry out the operation using
1.0 ml ofa 0.01 per centw/v solution ofxylose RS ill a saturated
solution of benzoic acid beginning at the words "add 5 ml of
4-bromoaniline solution.....".
Calculate the content ofCsHIQOs.
2325
MONOGRAPHS
z
Zidovudine
2329
Zidovudine Capsules
2330
Zidovudine fujection
2331
Zidovudine Oral Solution
2332
Zidovudine Tablets
2333
Zidovudine, Lamivudine and Nevirapine Tablets
2334
Zinc Chloride
2335
Zinc Oxide
2336
Zinc Oxide Cream
2336
Zinc Stearate
2337
Zinc Sulphate
2337
Zinc Sulphate Eye Drops
2338
Zinc Undecenoate
2338
Zinc Undecenoate Ointment
2339
Zoledronic Acid
2339
Zoledronic Acid fujection
2340
Zolpidem Tartrate
2341
Zolpidem Tablets
2342
2327
IP 20ID
ZIDOVUDINE
Zidovudine
Dry the plate in air and examine in ultraviolet light at 254 om.
Any secondary spots observed in the chromatogram obtained
with the test solution correspond to those of the principal
spots in the chromatogram obtained with the reference
solution. No secondary spot in the chromatogram obtained
with the test solution is more intense than the principal spot
(0.5 per cent).
Mol. Wt. 267.2

Zidovudine is 1-(3-azido-2,3-dideoxy-{3-o-ribofuranosyl)5-methylpyrimidine-2,4(lH,3H)-dione.
Zidovudine contains not less than 97.0 per cent and not more
than 102.0 per cent ofC IOH 13Ns0 4, calculated on the anhydrous
basis.
Spray the plate with a mixture of0.5 g of carbazole in 95 m10f

ethanol (95 per cent) and 5 ml of sulphuric acid, heat for
10 minutes at 120 and compare the intensities ofany secondary
spots observed in the chromatogram obtained with the test
solution with those ofthe principal spots in the chromatogram
obtained with the reference solution. No spot corresponding
to triphenylmethanol (Rr value about 2.3 relative to the Rr of
zidovudine) is more intense than the corresponding spot in
the chromatogram obtained with the reference solution
(0.5 per cent). No secondary spot in the chromatogram
obtained with the test solution is more intense than the
principal spot in the chromatogram obtained with the reference
solution.
B. Deterinine by liquid chromatography (2.4.14).
Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of methanol.
Identification
Compare the spectrum with that obtained with zidovudine RS
or with the reference spectrum ofzidovudine.
to zidovudine in the chromatogram obtained with reference
solution (a).
C. Melting range (2.4.21). 122 to 125,
Tests
Specific optical rotation (2.4.22). +60.so to +63.0, determined
in a 1.0 per cent w/v solution in ethanol (95 per cent).
GF254.

zidovudilJe RS in methanol.
w/v of zidovudine RS and 0.001 per cent w/v each of
zidovudine-related compound B RS and zidovudine- related
compound C RS in methanol.
octadecylsilane' bonded to porous silica (5 /lm),
mobile phase: A. water
B. methanol,
below,
spectrophotometer set at 265 om,'

Time
(in min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
90
10
15
35
65

each of zidovudine RS and triphenylmethanol in methanol.
30
35
65
35
95
Apply to the plate 10 /ll of each solution. Allow the mobile

phase to rise to about three-fourths of the height of the plate.
40
95
45
90
10

2329
ZIDOVUDINE
IP 2010
resolution between the peaks due to zidovudine and
zidovudine-related compound B is not less than 1.5 and the
tailing factor for zidovudine is not more than 1.5.
The area of the peak corresponding to zidovudine- related
compound B is not greater than 1.0 per cent and of that to
zidovudine-related compound C is not greater than 2.0 per
cent.
The sum of the percentages of related substances by tests A
and B is not greater than 3.0 per cent.
Zidovudine Capsules
Zidovudine Capsules contain not less than 90.0 per cent and
zidovudine, C IOH J3N s0 4
Identification
A. Transfer a quantity of the mixed contents of the capsules

containing about 15 mg ofZidovudine to a 1OO-ml volumetric
flask. Add about 80 ml ofa mixture of75 volumes of methanol
and 25 volumes of water, shake for 10 minutes and dilute to
volume with the same solvent mixture, mix and filter. Dilute
10 ml ofthe filtrate to 100 ml with the same solvent mixture.
265nm.
Test solution. Weigh accurately about 100 mg ofthe substance

under examination, dissolve in a suitable quantity of methanoI
in a 50-ml volumetric flask and make up to volume with the
mobile phase. Dilute 5.0 ml ofthis solution to 50.0 ml with the
mobile phase.

solution (c).

zidovudine RS, dissolve in a suitable quantity of methanol in
a 50-ml volumetric flask and make up to volume with the mobile
phase (solution A). Dilute 5.0 ml ofsolutionAto 50.0 ml with
the mobile phase.
Tests
Reference solution (b). Transfer 2.0 ml ofa 0.005 percentw/v

solution of zidovudine-related compound B RS in the mobile
phase to a 50-ml volumetric flask, add 5.0 ml ofsolution A and
make up to volume with the mobile phase.
zidovudine-related compound B is not less than 2.0.
.......... Separately-inject the test solution-andreferencesolution(a}
and measure the responses for the principal peak.
Calculate the content ofC IOHJ3N s0 4

(2.4.14).

contents of20 capsules containing about 100 mg ofZidovudine
and transfer to a 100-ml volumetric flask. Add about 60 ml of
the mobile phase, mix with the aid ofultrasound for 10 minutes,
dilute to volume with the mobile phase, mix and filter. Dilute
5.0 ml ofthe filtrate to 50.0 ml with the mobile phase.
zidovudine RS and transfer to a 100-ml volumetric flask.
Dissolve in about 60 ml of the mobile phase and dilute to
volume with the mobile phase.
thymine and transfer to a 200-ml volumetric flask, dissolve
and make up to volume with methanol.
Reference solution (c). Transfer 10.0 ml of the reference
solution (a) and 2.0 ml of reference solution (b) to a 100-ml
volumetric flask and make up to the volume with the mobile
phase.
. a.stainless.. steeLcolumn 25_cm.~ . 4.6.mm,pJtc.kedJYith
Qctadecylsilal1e b()nded t() porous.sili(;fl. (5 IllTI)'
2330
IP 2010
ZIDOVUDINE INJECTION
solution and reference solution (c) and measure the responses

for the principal peak.

relative retention times are about 0.2 for thymine and 1.0 for
zidovudine, the resolution between zidovudine and thymine
is not less than 5.0, the tailing factor is not more than 2.0 and
and measure the responses for the thymine peak. The content
of thymine in the capsules should not be more than 3.0 per
cent.
Calculate the content ofCIOHI3Ns04 in the capsules.

Zidovudine Injection
Zidovudine Injection is a sterile solution of Zidovudine in
.'
Water for Injections.
Zidovudine Injection contains not less than 90.0 per cent and
CIOH 13N s0 4
Usual strengths. 10 mg per ml.
Apparatus No.1,
Medium. 900 ml ofwate];

not greater than 1.0 flm, rejecting the first few ml ofthe filtrate
and dilute a suitable volume of the filtrate, if necessary with
water.
Reference solution. A known quantity of zidovudine RS is
dissolved in l.ml ofmethanol and suitably diluted with water
to obtain a solution having a similar concentration as that of
the test solution.
- mobile phase: a mixture of20 volumes ofmethanol and
80 volumes of wate];
than 2.0 for zidovudine peak and the relative standard deviation
Inject the test solution and the reference solution and measure
the peak responses of the major peak.
Calculate the content ofCIOHI3Ns04 in the medium.
C IOH 13N s0 4.
described under Related substances. Inject separately the test
Identification
0.0015 per cent w/v solution in a mixture of75 volumes of
methanol and 25 volumes ofwater shows absorption maxima
similar to those obtained with a solution of zidovudine RS of
the same concentration.

Tests
pH (2.4.24). 3.5 to 7.0, in a mixture containing a volume of
injection containing 150 mg ofzidovudine and 5 ml of0.12 M
potassium chloride.
(2.4.14).
injection containing 25 mg of Zidovudine, to 25 ml with
methanol.
zidovudine RS in methanol.
w/v of zidovudine RS and 0.001 per cent w/v of zidovudine
related compound C (thymine).
octadecylsilane bonded to porous silica ( 5 flm),
- mobile phase: a mixture of 80 volumes of water and 20
volumes of methanol,
2331
ZIDOVUDlNE ORAL SOLUTION
IP 2010
resolution between zidovudine and thymine is not less than
4.0, the tailing factor is not more than 2.0 and the relative
2.0 per cent.
peak corresponding to thymine is not greater than twice the
Unit per mg ofzidovudine.
Assay. Detennine by liquid chromatography (2.4.14), as given
under the test for Related substances.
Calculate the content ofC IOH 13N s0 4 in the injection.

Zidovudine Oral Solution is a solution of Zidovudine in a
suitable flavoured vehicle.
Zidovudine Oral Solution contains not less than 90.0 per cent
zidovudine, C IOH 13N s0 4
Identification
Mobile phase. A mixture of 40 volumes of i-butanol,
30 volumes of heptane, 30 volumes of acetone and 10 volumes
methanol to obtain a solution containing 5 mg of zidovudine
perml.
Reference solution. A 0.5 per cent w/v solution of zidovudine
RS in a mixture of75 volumes of methanol and 25 volumes of
water.
:6jJPJytQ!!l~pI~teJ .. l!.l()f.t:l.a:~!ls()I!1!i()Il,Af1:~r_Q~y~I().PJll~l1t,

solution (c).
Tests
pH (2.4.24). 3.0 to 4.0, determined in a mixture containing a
volume of the preparation under examination containing
150 mg ofzidovudine and 5 ml of 0.i2 M potassium chloride.
(2.4.14).
Test solution. Transfer an accurately measured volume ofthe

preparation under examination containing about 100 mg of
zidovudine to a 100-ml volumetric flask, dissolve and dilute to
volume with the mobile phase and mix. Dilute 5.0 m1 of this
zidovudine RS and transfer to a 100-ml volumetric flask, add
about 50 ml ofthe mobi1ephase, mix with the aid ofultrasound
to dissolve, dilute to volume with the mobile phase and mix.
thymine RS and transfer to a 200-ml volumetric flask, add about
150 ml of the mobile phase, mix with the aid of ultrasound to
dissolve, dilute to volume with the mobile phase and mix.
Reference solution (c). Transfer 10.0 ml of the reference
solution (a) and 2.0 ml of reference solution (b) to a lOO-ml
volumetric flask and make up to the volume with the mobile
phase.
octadecylsilanebonded to porous silica (5 /-lm),
mobile phase: a mixture of90 volumes of 0.04 M sodium
acetate, 9 volumes of methanol, 1 volume of acetonitrile
and 0.2 volume of glacial acetic acid,
relative retention times are about 0.12 for thymine and 1.0 for
zidovudine, the resolution between zidovudine and thymine
is not less than 4.0, the taiiing factor is not more than 2.0 and
atRlm:easure the responses for the thymifiep-eak The-content
of thymine in the capsules should not be more than 3:0 per
cent.
2332
IP 2010
ZlDOVUDINE TABLETS
mobile phase: a mixture of90 ml of methanol and 40 ml

of acetonitrile and a buffer prepared by dissolving
3.0 g of sodium acetate and 3.0 g of sodium
l-octanesulphonate in 900 ml of water and adjusting
the pH to 5.3 with glacial acetic acid,

described under Related substances. Inject the test solution
and reference solution (c) and measure the responses for the
major peak.
Calculate the content of CIOHI3Ns04 in the preparation under
examination.

than 2.0 for zidovudine peak and the relative standard deviation
Zidovudine Tablets
Zidovudine Tablets contain not less than 90.0 per cent and
zidovudine, C IOH 13 N s0 4.
the peak responses of the major peak.
Calculate the content ofC IO H 13 N s0 4 in the medium.
C IOH 13N s0 4
Identification
to Zidovudine in the chromatogram obtained with reference
solution (a).
B. Remove the coating from a few tablets and crush them in a
mortar so that no large pieces remain.
Related substances. Determine by liquid dU'omatography

(2.4.14).
Test solution. Disperse the powdered tablets containing about

1500 mg ofZidovudine in 50 ml of water, shake for 30 minutes.
Add about 150 ml of methanol, sonicate for 10 minutes and
dilute to 500.0 ml with wate/: Dilute 4.0 ml ofthis solution to
100.0 ml with water, filter.
On the powder determine by infrared absorption

obtained with zidovudine RS or with the reference spectrum
of zidovudine.
Reference solution (a). A 0.01 per cent w/v solution of 3'chloro-3 '-deoxythymidine (zidovudine impurity B RS) in
methanol.
Tests
Reference solution (b). A 0.02 per cent w/v solution of thymine

RS (zidovudine impurity C RS) in methanol.
Reference solution (c). Dissolve about 30 mg of zidovudine

RS in 3.0 ml of methanol, add 2.5 ml ofreference solution (a),
Apparatus No.1,
5.0 ml of reference solution (b) and dilute to 250.0 ml with
water.

not greater than 1.0 flm, rejecting the first few ml ofthe filtrate
water.

Reference solution. A known quantity of zidovudine RS is
dissolved in 1 ml of methanol and suitably diluted with water
to obtain a solution having a similar concentration as that of
the test solution.
a stainless steel column 15 cm x 4.6 rum, packed with
octadecylsilane bonded to porous silica (5 flm) (Such
as Hypersil BDS C 18),
mobile phase: dissolve 3.0 g of sodium acetate and
1.3 g of sodium l-octanesulphonate in 900 ml of water.
Add 90 ml of methanol and 40 ml of acetonitrile,
adjusted to pH 5.3 with glacial acetic acid, filter,
zidovudine impurity B is'not less than 2.5, the tailing factor of
the principal peak is not more than 2.0 and the relative standard
The relative retention time with reference to zidovudine for
2333
ZIDOVUDINE, LAMIVUDINE AND NEVIRAPINE TABLETS
IP 2010
zidovudine impurity C is about 0.17 and for zidovudine impurity

B is about 1.2.
octylsilane bonded to porous silica (5 /lm) (Such as
Hypersil C8),
mobile phase: A. 0.1 M ammonium acetate,
B. acetonitrile,
below,
Inject reference solution (c) and the test solution. In the

chromatogram obtained the test solution, the area ofthe peak
due to zidovudine impurity C is not more than 1.5 per cent,
multiplying with response factor of 1.7 and the area of other
secondary peaks is not more than 0.2 per cent. The sum of
areas of all the secondary peaks is not more than 2.0 per cent.
described under Related substances. Separately inject the test
solution and reference solution (c) and measure the peak
responses for the major peak.
Time 0.1 M ammonium acetate acetonitrile

(in min.)
(per cent v/v)
(per cent v/v)
95
5
95
5
5
25
20
80
30
20
80
31
95
5
35
95
5
Calculate the content OfClOHl3Ns04 in the tablets.

Zidovudine, Lamivudine and

Nevirapine Tablets
Zidovudine, Lamivudine and Nevirapine Tablets contain not
less than 90.0 percent and not more than 110.0 per cent ofthe
stated amounts of zidovudine, CloH13Ns04, lamivudine,
CSHIIN303S andnevirapine, C 1sH 14N 40.
Usual strength. Zidovudine, 300 mg, Lamivudine, 150 mg and
Nevirapine , 200 mg.
column efficiency determined from the zidovudine, lamivudine
and nevirapine peaks is not less than 3000 theoretical plates
and the tailing factor for the same peaks is not more than 2.0.
chromatogram obtained with water for any extraneous peaks
and ignore the corresponding peaks observed in the
A. In the Assay, the three principal peaks in the chromatogram

obtained with the test solution correspond to the peaks due
to zidovudine, lamivudine and nevirapine in the chromatogram

with the test solution corresponding to a relative retention
time of 0.35 should not be more than 1.0 per cent. Any other
secondary peak observed in the chromatogram obtained with
the test solution should not be more than 0.5 per cent and the
sum of the areas of all the secondary peaks should not be
more than 2.5 per cent when calculated by percentage area
normalisation.
Tests

(2.4.14).
Apparatus No.1,
Identification

tablets containing about 100 mg of Zidovudine and transfer
to a 200-ml volumetric flask. Add about 150 ml of water, mix
with the aid of ultrasound for 10 minutes, dilute to volume
with water, mix and filter.
Reier.en~~_sQ1J-'JigJ1,Wejgb_!l_cJ<ltratelY_j~12Q1tLlJ1! Lmg-9J
zirjovu(jin!} R., 50 mg oflamivudine R. and5 IIlg ofnevirapine

RS and transfer to a 200-ml volumetric flask. Add about 20 ml
ofmethanol, mix with the aid ofultrasound to dissolve, dilute
to volume with water and mix.

not greater than 1.0 /lm, rejecting the fIrst few ml ofthe filtrate
water.
Hetermine-byliquidchromatography(2;4;14);
zidovudine RS, 150 mg of lamivudine RS and 200 mg of
2334
ZINC CHLORIDE
IP 2010
nevirapine RS and transfer to a 100 ml volumetric flask. Add

about 20 ml of methanol, mix with the aid of ultrasound to
dissolve and dilute to volume with a solvent mixture of equal
volumes of methanol and water. Dilute 5.0 Ji11 ofthis solution
to 50.0 ml with 0.1 M hydrochloric acid.
- a stainless steel column 15 cm X 4.6 mm, packed with
as Hypersil BDS C18),
0.68 g ofpotassium dihydrogen phosphate and 1.0 g of
sodium octanesulphonate in 1000.0 ml of water to which
1 ml of triethylamine is added and the pH of which is
adjusted to 2.5 with phosphoric acid,
Inject the reference solution. The tailing factor for the individual
zidovudine, lamivudine and nevirapine peaks is not more than
of all the analyte peaks is not more than 2.0 per cent.
the peak responses of the major peaks due to zidovudine,
lamivudine and nevirapine.
Calculate the contents of C IOH 13N s0 4, C sH\\N 30 3 S and
C\SH\4N40 in the medium.
C IOH 13Ns0 4, CsH\\N30 3S and C\SH\4N40.
replicate injections of all the analyte peaks is not more than

2.0 per cent.
the responses for the major peaks.
Calculate the contents of C IOH 13N s0 4, C SH 1\N 30 3 S and
C\SH I4N 40 in the tablets.
Zinc Chloride
ZnCh
Mol. Wt. 136.3
Zinc Chloride contains not less than 95.0 per cent and not
more than 100.5 per cent ofZnCh.
Descl"iption. A white or practically white, crystalline powder;
odourless; very deliquescent.
Identification
A. To 2 g add 38 ml of carbon dioxide-free water prepared
from distilled water and add 2 M hydrochloric acid dropwise
until solution is complete and dilute to 40 ml with carbon
dioxide-free water prepared from distilled water (solution
A). Solution A gives the reaction of zinc salts (2.3.1).
B. A 5 per cent w/v solution in 2 M nitric acid gives the
reactions ofchlorides (2.3.1).
Tests
dissolving 1.0 g in 9 ml of freshly boiled and cooled water,
ignoring any slight turbidity.

Zidovudine and transfer to a 200-ml volumetric flask. Add
10 minutes, dilute to volume with water, mix and filter. Further
dilute 10.0 ml ofthe filtrate to 25.0 ml with water.
zidovudine RS, 50 mg of lamivudine RS and 65 mg of nevirapine
RS and transfer to a 200-ml volumetric flask. Add about 20 ml
of methanol, mix with the aid ofultrasound to dissolve, dilute
to volume with water and mix. Further dilute 10.0 ml of this
solution to 25.0 ml with water.
Follow the chromatographic procedure described under
Dissolution.
Inject the reference solution. The tailing factor for the
individual peaks due to zidovudine, lamivudine and nevirapine
is not more than 2.0 and the relative standard deviation for
Aluminium, calcium, heavy metals, iron and magnesium. To

8 ml of solution A add 2 ml of strong ammonia solution and
shake; the solution is clear (2.4.1), and colourless (2.4.1). Add
1 ml of a 9 per cent w/v solution of disodium hydrogen
phosphate; the resulting solution remains clear for at least
5 minutes. Add 0.2 ml of sodium sulphide solution; a white
precipitate is produced and the supernatant liquid remains
colourless.
Ammonium salts. To 5 ml of a 10 per cent w/v solution add
1 M sodium hydroxide until the precipitate fIrst formed is
redissolved and then warm the solution; no odour ofammonia
is perceptible.
Oxychlorides. Dissolve 1.5 gin 1.5 ml of carbon dioxide-free
water; the solution is not more opalescent than opalescence
standard OS2 (2.4.1). Add 7.5 ml of ethanol (95 percent); the
solution may become cloudy within 10 minutes but becomes
clear on the addition of 0.2 ml of 2 M hydrochloric acid.
2335
ZINC OXIDE
IP 2010
Sulphates (2.3.17).15 ml ofsolution A complies with the limit

test for su1phates (225 ppm).
solution AsT. The resulting solution complies with the limit

test for arsenic (5 ppm).

water, add 3 g ofammonium chloride and add sufficient water
to produce 250.0 ml. To 25.0 ml ofthe resulting solution add
100 ml of water and 10 ml of strong ammonia-ammonium
chloride solution. Titrate with 0.1 M disodium edetate, using
eriochrome black T solution as indicator until a deep blue
colour is obtained.
Iron (2.3.14). Dissolve 0.1 gin a mixture of5 ml ofwater and

1 ml ofhydrochloric acid and dilute to 40 ml with water. The
resulting solution complies with the limit test for iron
(400 ppm). Use 0.5 ml of thioglycollic acid in the test.
Lead. Dissolve 2 g in a mixture of 20 ml water and 5 ml of
glacial acetic acid and add 0.25 ml of potassium chromate
solution; the solution remains clear.
1 ml of 0.1 M disodium edetate is equivalent to 0.01363 g of

ZnCh.
Loss on ignition (2.4.20). Not more than 1.0 per cent, determined
on 2.0 g by igniting at 500.
Storage. Store protected from moisture, in non-metallic

containers.
Assay. Dissolve 0.15 gin 10 ml of2 M acetic acid and dilute to

50 ml with water. To the resulting solution add about 50 mg of
xylenol orange triturate and sufficient hexamine to produce
violet-pink colour. Add a further 2 g of hexamine and titrate
with 0.1 M disodium edetate until the solution becomes yellow.
ZnO.
Zinc Oxide
ZnO
Mol. Wt. 81.4
Zinc Oxide contains not less than 99.0 per cent and not
more than 100.5 per cent of ZnO, calculated on the ignited
basis.
Category. Mild astringent; topical protectant.
Zinc Oxide Cream
Description. A soft, white or faintly yellowish white

amorphous powder, free from grittiness. It gradually absorbs
carbon dioxide from air.
Zinc Cream
Identification
Zinc Oxide Cream contains not less than 30.0 per cent and not
more than 34.0 per cent w/w ofzinc oxide, ZnO.
A. It becomes yellow when strongly heated; the yellow colour

disappears on cooling.
B. Dissolve 0.1 gin 1.5 ml of2 M hydrochloric acid and dilute
to 5 ml with water. The solution gives the reaction ofzinc salts
(2.3.1).
Zinc Oxide Cream contains 32 per cent w/v ofZinc Oxide in a

suitable water-in-oil emulsified base.
Identification
The residue obtained in the Assay is yellow when hot and
white when cool.
Tests
Tests
Alkalinity. Shake 1.0 g with 10 ml ofboiling water, add 0.1 ml
ofphenolphthalein solution and filter. Ifthe filtrate is red, not
more than 0.3 ml of 0.1 M hydrochloric acid is required to
discharge the colour.
Carbonate and substances insoluble in acids. Dissolve 1.0 g
in 15 ml of 2 M hydrochloric acid; no effervescence is
produced and the solution is not more opalescent than
olfalescef1cestaf1dataOS2-(2.4-n;~af1dc~ol(jatless-(2.4;1):~
. . ._.~
Arsenic (2.3.10). Dissolve 2.0 g in 15 ml of brominated
hydrochloric acid AsT and 45 ml of water and remove the
excess of bromine with a few drops of stannous chloride
Assay. Weigh accurately about 0.5 g in a porcelain dish, heat

gently over a small flame until the base is completely volatilised
or charred. Increase the heat until all the carbon is removed.
Dissolve the residue in 10 ml of 2 M acetic acid and add
sufficient water to produce 50 ml. To the resulting solution
add about 50 mg of xylenol orange triturate and sufficient
hexamine to produce violet-pink colour. Add a further 2 g of
nexamineand titrate with O:IM disodiilmedeti:tte~untilthe
solution becomes yellow.
1 ml of 0.1 M disodiumedetate is equivalent to 0.008138 g of
ZnO.
2336
IP 2010
ZINC SULPHATE
Zinc Stearate
arsenic (2 ppm).
Mol. Wt. 632.3
Zinc Stearate consists mainly of zinc stearate but many

contain variable proportions ofzinc palmitate (C 1sH 3I COO)Z
Zn, and zinc oleate (C I7 H33 COO)ZZn.
Zinc Stearate contains not less than 10.0 per cent and not
more than 12.0 per cent ofzinc, Zn.
Category. Pharmaceutical aid (dusting powder).
Description. A [me, white, bulky, amorphous powder, free from
grittiness; odour, faint and characteristic.
Identification
A. To 5.0 g add 50 ml of ether and 40 ml of a 7.5 per cent v/v
solution of nitric acid in distilled water and heat under a
reflux condenser until dissolution is complete. Allow to cool,
separate the aqueous layer and shake the ether layer with two
quantities, each of 4 ml, of distilled water. Combine the
washings with the aqueous layer, wash with 15 ml of ether
and heat on a water-bath until ether is completely eliminated.
Allow to cool and dilute to 50.0 ml with distilled water (solution
A). Evaporate the ether layer to dryness and dry the residue
at 105. The freezing point ofthe residue is notlower than 53
(2.4.11).
B. Neutralise 5 ml ofsolution A to red litmus paper with 10 M

sodium hydroxide. The solution gives the reactions of zinc
salts (2.3.1).
Heavy metals (2.3 .13). Heat 5.0 g with 40 ml of2 M acetic acid
and allow to cool. Filter, wash the residue with two successive
quantities, each of5 ml, ofwarm water and dilute the.combined
filtrate and washings to 100.0 rnl with water. 12 rnl ofthe solution
(20 ppm). Use 1.0 ml oflead standardsolution (10 ppm Pb) to
prepare the standard.
Chlorides (2.3.12).10 ml ofsolution A complies with the limit
Sulphates (2.3.17). 2.5 ml ofsolution A complies with the limit
Assay. Weigh accurately about 1.0 g and boil with 50 ml of
2 M acetic acid until the fatty acid layer which separates is
clear, adding more water ifnecessary to maintain the original
volume. Cool, filter and wash the filter and the flask thoroughly
with water until the last washing is not acidic to blue litmus
paper. To the combined filtrate and washings add about
50 mg of xylenol orange triturate and sufficient hexamine to
produce violet-pink colour. Add a further 2 g ofhexamine and
titrate with 0.1 M disodium edetate until the colour changes
to yellow.
Zn.
Zinc Sulphate
Tests
Appearance of solution. Solution A is not more intensely
Acidity or alkalinity. Shake 1.0 g with 5 ml ofethanol (95 per
cent) and add 20 ml ofcarbon dioxide-free water and 0.1 mlof
phenol red solution. Not more than 0.3 ml of 0.1 M
hydrochloric acid or 0.1 ml of 0.1 M sodium hydroxide is
Alkalis and alkaline earths. Add 1.0 g to a mixture of25 ml of
water and 5 ml of hydrochloric acid, boil, filter immediately
and wash with 25 ml of hot water. Add dilute ammonia
solution to make the filtrate just alkaline and then add
ammonium sulphide solution in excess to precipitate the zinc
as zinc sulphide completely. Filter, add 0.5 ml of sulphuric
acid to the filtrate, evaporate to dryness and ignite to constant
weight; the residue weighs not more than 20 mg.
Arsenic (2.3.1 0). Mix 5.0 g with 10 ml ofbromine solution and
evaporate to dryness on a water-bath. Ignite gently, dissolve
the cooled residue, ignoring any carbon, in 50 ml ofwater and
14 ml of brominated hydrochloric acid AsT and remove the
excess of bromine with 2 ml of stannous chloride solution
ZnS04,7HzO
Mol. Wt. 287.5
Zinc Sulphate contains not less than 99.0 per cent and not
more than 104.0 per cent ofZnS04, 7H zO.
Category. Astringent.
Description. Colourless, transparent crystals or a white,
crystalline powder; odourless; efflorescent.
Identification
Dissolve 2.5 g in sufficient carbon dioxide-free water to
produce 50 ml (solution A). Solution A gives the reactions of
zinc salts and sulphates (2.3.1).
Tests
colourless (2.4.1).
Arsenic (2.3.1 0). Dissolve 1.0 g in 50 nil ofwater and add 10 ml
2337
ZINC SULPHATE EYE DROPS
IP 2010
Iron (2.3.14). Dissolve 0.4 g in 20 m1 of water. The resulting

2 M acetic acid and dilute to 50 ml with water. To the resulting
solution add about 50 mg of xylenol orange triturate and
sufficient hexamine to produce violet-pink colour. Further,
add 2 g of hexamine and titTate with 0.1 M disodium edetate
until the colour changes to yellow.
1 ml 0/0.1 M disodium edetate is equivalent to 0.02875 g of
ZnS04,7HzO.
Storage. Store protected from moisture, in non-metallic
containers.

Zinc Sulphate Eye Drops are a sterile solution containing
0.25 pet cent \>.iN of Zinc Sulphate ifiPtirified Water.
Zinc Sulphate Eye Drops contain not less than 0.22 per cent
and not more than 0.28 per cent w/v of zinc sulphate, ZnS04,
7HzO.
Zinc Undecenoate contains not less than 98.0 per cent and
not more than 102.0 per cent ofCzzH3s04Zn, calculated on the
dried basis.
Category. Antifungal (topical).
Description. A white or almost white, fine powder.
Identification
A. To 2.5 g add 10 ml of water and 10 ml of1 M sulphuric acid
and extract with two quantities, each of 10 ml, ofether. Reserve
the aqueous layer for test C. Wash the combined ether extracts
with water and evaporate to dryness. To the residue add 2 ml
of freshly distilled aniline and boil under a reflux condenser
for 10 minutes, cool and add 30 ml of ether. Extract with three
quantities, each of 20 ml, of 2 M hydrochloric acid and then
with 20 ml of water. Evaporate the ether extract to dryness on
a water-bath. The residue, after recrystallising twice from
ethanol (70 per cent) and drying at a pressure not exceeding
2 kPa for 3 hours, melts at about 67 (2.4.21).
B. Dissolve 0.1 g in a mixture of2 mLof1 Msulphuric acidand

5 ml of glacial acetic acid and add, dropwise, 0.25 ml of
potassium permanganate solution; the pink colour of
permanganate is discharged.
Usual strenghts. 200 mg; 500 mg; 1 g.
C. A mixture of 1 ml ofthe aqueous layer reserved in test A and

4 ml of water gives the reaction of zinc salts (2.3.1).
Identification
Tests
Give the reactions of zinc salts and of sulphates (2.3.1).
Alkalinity. Mix 1.0 g with 5 ml of ethanol (95 per cent) and

0.5 ml of phenol red solution, add 50 ml of carbon dioxidefree water and examine immediately; no reddish colour is
produced.
Tests
Assay. To 5.0 rnl add 50 ml of water and 5 ml ofammonia bliffer
mordant black II solution as indicator.
ZnS0 4,7HzO.
Storage. Store in containers ofglass or any other non-metallic
material and sealed so as to exclude micro-organisms.
Allmlis and alkaline earths. Add 1.0 g to a mixture of25 mlof

water and 5 ml of hydrochloric acid, boil, filter immediately
and wash with 25 ml of hot water. Add dilute ammonia
solution to make the filtrate just alkaline and then add
ammonium sulphate solution in excess to precipitate the zinc
as zinc sulphide completely. Filter, add 0.5 ml of sulphuric
acid to the filtrate, evaporate to dryness and ignite to constant
weight; the residue weighs not more than 20 mg.
Sulphates (2.3.17). To 0.25 g add a mixture ofl 0 rnl of distilled
water and 5 ml of 2 M hydrochloric acid. Cool, filter and
dilute to 20 ml with distilled water. The resulting solution
complies with the limit test for sulphates (600 ppm).
Zinc Undecenoate
Degree of unsaturation. Dissolve 0.1 g in a mixture of5 mlof

2Mhydrochloricacid and 30 ml of glCic.:ial aceticacid and
wTth~0~05Mbro/;ZineusiniO.05ml ofethoXychrysoidine
hydrochloride solution, added towards the end ClfthetihitiClll,
as indicator. Not less than 9.1 ml and not more than 9.4 ml of
0.05 M bromine is required to discharge the red colour.
titrate
CZZH3S04Zn
Mol. Wt. 431.9
Zinc Undecenoate is zinc di(undec-l O-enoate).
2338
IP 2010
ZOLEDRONIC ACID
Assay. Weigh accurately about 0.35 g, add 25 m1 of2 M acetic
acid, heat to boiling, cool and dilute to 50 ml with water. Add
about 50 mg of xylenol orange triturate and sufficient
hexamine to produce a violet-pink colour. Add a further 2 g of
hexamine and titrate with 0.1 M disodium edetate until the
C22H3S04Zn.
Storage. Store protected from light and moisture..
small separator and drain the carbon tetrachloride layer into

a 100-ml volumetric flask. Rinse the beaker with three
quantities, each of 5 ml, of carbon tetrachloride and transfer
the rinsings to the volumetric flask, dilute to volume with
carbon tetrachloride and mix. Evaporate 50.0 ml ofthe resulting
solution to about 5 ml, add 100 ml of ethanol (95per cent),
previously neutralised, and 0.15 ml of phenolphthalein
solution and titrate the total undecenoic acid with
CIIH2002.
Calculate the content of free undecenoic acid from the

difference between the total undecenoic acid and the
undecenoic acid equivalent to the determined zinc
undecenoate (the content of zinc undecenoate multiplied by
0.8533 gives the equivalent ofundecenoic acid).
Zinc Undecylenate Ointment
Zinc Undecenoate Ointment contains 20 per cent w/v of Zinc

Undecenoate in a suitable ointment basis.
Zinc Undecenoate Ointment contains not less than 18.0 per
cent and not more than 22.0 per cent of zinc undecenoate,
C22H3S04Zn, and not less than 4.5 per cent and not more than
5,5 per cent of free undecenoic acid, CIIH2002.
Zoledronic acid
Tests
Assay. For zinc undecenoate - Weigh accurately about
2.0 g, add 20 ml of dilute hydrochloric acid and boil under a
reflux condenser for at least 20 minutes or until the fatty layer
is clear. Filter while hot and wash the residue with hot water.
Cool the combined filtrate and washings, neutralise to litmus
paper with dilute ammonia solution, add 3 ml of dilute
hydrochloric acid and 5 g of hexamine and titrate with
0.05 M disodium edetate using xylenol orange solution as
indicator, until the colour changes to yellow.
1 mlof 0.05 M disodium edetate is equivalent to 0.02160 g of
C22H3S04Zn.
For ji-ee undecenoic acid

Weigh accurately about 5.0 g,
add 100 ml of dilute hydrochloric acid and heat to 70 with
constant stirring. Cool and transfer to a separator with the aid
of four quantities, each of 25 ml of ether and add the rinsings
to the mixture in the separator. Dilute the aqueous phase to
300 ml, saturate it with sodium chloride and shake the mixture.
Transfer the aqueous layer to a second separator and extract
with another 100 ml of ether. Wash the combined ether extracts
with successive quantities, each of 10 ml, of water until the
washings are free from chloride. Transfer the ether solution to
a beaker and evaporate on a water-bath to about 5 ml. Add
20 ml of carbon tetrachloride, mix, transfer the mixture to a
Mol. Wt. 272.1

ZoledronicAcid is [1-hydroxy-2-(lH-imidazol-lyl)ethylidene]diphosphonic acid.
Zoledronic Acid contains not less than 98.0 per cent and not
more than 102.0 per cent of CsHIQN20 7P2, calculated on the
dried basis.
Category. Bone resorption inhibitor.
Description. A white to almost white crystalline powder.
Identification
Compare the spectmm with that obtained with zoledronic
acidRS or with the reference spectrum ofzoledronic acid.
B. In the assay, the principal peak in the chromatogram
Tests
2339
ZOLEDRONIC ACID
IP 2010

(2.4.14).
Zoledronic Acid Injection
Test solution. Dissolve 50 mg of the substance uJider

examination in 50.0 m1 ofthe mobile phase.
Zoledronic Acid Injection is prepared by dissolving the

contents of a sealed container containing Zoledronic Acid
with or without auxiliary substances in the requisite a.mount
of water for injection as directed on the label.

a stainless steel column 25 em x 4 mm, packed with
as Hypersil BDS),
- mobile phase: mix 1 ml of triethylamine with 500 ml of
water, adjust the pH to 3.2 with orthophosphoric acid,
the tailil1g factor is not more than 2.0.
In the chromatogram obtained with the test solution, area of
any secondary peale is not more than the area ofthe principal
(1.0 per cent). The sum of areas of all the secondary peaks is

Zoledronic Acid Injection contains not less than 90.0 per cent
and not more than 110.0 per cent ofstated amount ofzoledronic
acid, CSHION207P2.

(Powders for Injection) and with thefollowing requirements:
Identification
Tests
pH (2.4.24).4.5 to 7.0, determined in a solution constituted as
directed on the label, in Water for Injections.
Loss on drying (2.4.19).5.0 to 8.0 per cent, determined on

1.0 g by drying in an oven at 105 for 6 hours.
Other tests. Complies with the tests stated under Parental


Units per mg of Zoledronic acid.

examination in 50.0 ml ofthe mobile phase. Dilute 20 ml ofthe
Reference solution. A 0.02 per cent w/v solution ofzoledronic
acid RS in the mobile phase.
substances.
than 2.0 per cent.
Calculate-the-content-ofCsHloN2Q7P2.--------------------------Storage. Store protected froIIllight a.nd IIloisture.

Test solution. Dissolve the contents of sealed container in

sufficient mobile phase to give a concentration 0.16 mg per m!.
Reference solution. A 0.0 16 per cent w/v solution ofzoledronic
acid RS in the mobile phase.
as Hypersil BDS),
rnobilephase:.. .mix..l..ml_oftr.iethylaminewith..5_0.0mlo f
water, adjusted to pH: 3.2 with orthophosphoric acid,
2340
IP 2010
ZOLPIDEM TARTRATE
than 2.0 per cent.
Reference solution (a). A 0.01 per cent w/v solution of N,Ndimethyl- 2-[7-methyl-2-(4-methylphenyl) imidazo[1 ,2-aJ
pyridin-3-yl acetamide RS (zolpidem impurity A RS) in the
mobile phase.
zolpidem tartrate RS in the mobile phase.
Calculate the content ofCsHIONz07Pz in the Injection.

Storage. Store the sealed container at a temperature not
exceeding 30. Use the prepared solution within the period
recommended by the manufacturer and stored strictly in
accordance with the instructions of the manufacturer.
Reference solution (c). Dilute 1.0 ml ofthe reference solution

(b) to 100.0 ml with the mobile phase.
Reference solution (d). To 2.0 ml of reference solution (b),
add 10.0 ml ofreference solution (a).
octadecylsilane bonded to porous silica (4 J.tm),
- mobile phase: 18 volumes of acetonitrile, 23 volumes
of methanol and 59 volumes of 0.56 per cent w/v
solution of orthophosphoric acid adjusted to pH 5.5
with triethylamine,
- injection volume. 20 J.tl.
Zolpidem Tartrate
Mol. Wt. 765.0

Zolpidem Tartrate is bis[N,N-dimethyl-2-[6-methyl-2-(4methylphenyl)imidazo[1,2-a]pyridine-3-yl]acetamide] tartrate.
Zolpidem Tartrate contains not less than 98.5 per cent and not
more than 101.0 percent Of(CI9HzIN30)Z' C 4H 60 6, calculated
Category. Sedatives.
Identification
A. Detenmne by infi:ared absorption spectrophotometry (2.4.6).
Compare the spectrum with that obtained with zolpidem
tartrate RS or with the reference spectrum ofzolpidem tartrate.
B. Al 0 per cent w/v solution in methanol gives reaction (c) of
tartrates (2.3.1).
Inject reference solution (d). Adjust the sensitivity of the

system so that the height ofthe peak due to zolpidem impurity
A is at least 50 per cent of the full scale of the recorder. The
test is not valid unless the resolution between the peaks due
to zolpidem impurity A and zolpidem tartrate is at least 2.0.
.Inject the test solution and reference solution (c). In the
obtained with reference solution (c) (1.0 per cent). Ignore any
peak with a relative retention time of0.16 to the zolpidem peak,
corresponding to tartaric acid and any secondary peak
with an area less than 0.05 times the area of the peak in
the chromatogram obtained with reference solution (c)
(0.05 per cent).
Tests

0.5g.
Appearance ofsolution. Dissolve 1.0 g ofthe substance under

examination in 100 ml ofa 0.5 per centw/v solution of tartaric
acid. The solution is clear (2.4.1), and not more intensely
coloured than reference solution YS6 or BYS6 (2.4.1).

20 ml of anhydrous acetic acid and 20 ml of acetic anhydride.
Titrate with 0.1 M perchloric acid, detennining the end point
potentiometrically (2.4.25). Carry out a blank titration

(2.4.14).

(CI9HzIN30)Z, C~606'


2341
ZOLPIDEM TABLETS
IP 2010

containing 25 mg ofZolpidem Tartrate with 25 ml ofthe mobile
phase and dilute to 50.0 ml with the same solvent and filter.
Zolpidem Tablets
Zolpidem Tartrate Tablets
Zolpidem Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of zolpidem
tartrate, C42RtsN60S'
Reference solution (a). A 0.01 per cent w/v solution of N,Ndimethyl- 2-[7-methyl-2-(4-methylphenyl) imidazo[1,2-a]
pyridin-3-yl acetamide RS (zolpidem impurity A RS) in the
mobile phase.
zolpidem tartrate RS in the mobile phase.
Identification
Shake a quantity ofthe powdered tablets containing 0.1 g of
Zolpidem Tartrate with 40 m1 of methanol, filter and evaporate
test.

Compare the spectrum with that obtained with zolpidem
tartrate RS or with the reference spectrum ofzolpidem tartrate.
obtained with the test~olutiQ!1 QOITesPQIlclS to the R~al.<: in the
Tests
Reference solution (c). Dilute 1.0 ml of the reference solution

(b) to 100.0 ml with the mobile phase.
Reference solution (d). To 2.0 ml of reference solution (b),
add 10.0 ml ofreference solution (a).
octadecylsilane bonded to porous silica (4 J!m),
- mobile phase: 18 volumes of acetonitrile, 23 volumes
of methanol and 59 volumes of0.56 per cent w/v solution
of orthophosphoric acid adjusted to pH 5.5 with
triethylamine,
- injection volume. 20 J!l.
Inject reference solution (d). Adjust the sensitivity of the
system so that the height ofthe peak-due to zolpidem impurity
A is at least 50 per cent of the full scale of the recorder. The
test is not valid unless the resolution between the peaks due
to zolpidem impurity A and zolpidem tartrate is at least 2.0.
Apparatus No.1,
Medium. 900 ml of 0.01 Mhydrochloric acid,

dissolution medium.
Reference solution. Dissolve 0.01 g'of zolpidem tartrate RS
in sufficient methanol to produce 100 ml. Dilute the resulting
solution with the dissolution medium to obtain a solution
containing 0.0005 per cent w/v ofzolpidem tartrate.
Related substances.
than 2.0 per cent.

peaks is not more than twice the area of the peak in the
cent). Ignore any peak with a relative retention time of0.16 to
the zolpidem peak, corresponding to tartaric acid and any
secondary peak with an area less than 0.05 times the area of
(c) (0.05 per cent).
Tablets.
Determine by liquid chromatography (2.4.14), as described in
the Assay, using the following solution as the test solution.

Calculate the content ofC42H4sN60s.
D;-Not-lessthan-7-5-per cent-of-the--statedamount-of
C42RtsN60SRelated substances. Determine by liquid chromatography
(2.4.14).
Test solution. Disperse one tablet in the mobile phase, mix and
dilutetoobtaina-solution-containing-0;005per-cent-w/vof
zolpidem tartrate and filter.
2342
ZOLPIDEM TABLETS
IP 2010

a quantity ofpowder containing 25 mg ofZolpidem Tartrate,
disperse in 25 ml ofthe mobile phase and dilute to 50.0 ml with
same solvent and filter. Dilute 5.0 ml ofthe solution to 50.0 ml
Reference solution. A 0.005 per cent w/v solution ofzolpidem

tartrateRS in the mobile phase.
Calculate the content of C42H4SN60S in the tablets.

Related substances.

2343
MONOGRAPHS
VACCINES AND IMMUNOSERAFORHUMAN USE

General Requirements
2347
2349
Vaccines
Antisera
Monographs
Adsorbed Diphtheria, Tetanus and Hepatitis B (rDNA) Vaccine

Adsorbed Diphtheria, Tetanus, Pertussis (Acellular Component) and
Haemophilus Type b Conjugate Vaecine
Hepatitis B (rDNA) Vaccine
Adsorbed Diphtheria, Tetanus, Pertussis (Acellular Component),
Inactivated Poliomyelitis Vaccine and Haemophilus Type b Conjugate Vaccine
Inactivated Poliomyelitis Vaccine
Adsorbed Diphtheria, Tetanus, Pertussis and Poliomyelitis (Inactivated) Vaccine
Adsorbed Diphtheria, Tetanus, Pertussis, Poliomyelitis (Inactivated) and
Haemophilus Type b Conjugate Vaccine
Adsorbed Pertussis Vaccine (Acellular Component)
Adsorbed Pertussis Vaccine (Acellular, Co-purified)
Bacillus Calmette-Guerin Vaccine (Freeze-Dried)
DiphtheriaAntitoxin
Diphtheria and Tetanus Vaccine (Adsorbed)
Diphtheria and Tetanus Vaccine (Adsorbed) for Adults andAdolescents
Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed)
Diphtheria, Tetanus, Pertussis (Whole Cell), Hepatitis B (rDNA) and
Haemophilus Type b Conjugate Vaccine (Adsorbed)
Diphtheria, Tetanus, Pertussis (Whole Cell) Hepatitis B (rDNA)
Vaccine (Adsorbed)
Diphtheria, Tetanus, Pertussis (Whole Cell) and Haemophilus Type b
Conjugate Vaccine (Adsorbed)
Diphtheria Vaccine (Adsorbed)
Gas-Gangrene Antitoxin (Oedematiens)
Gas-Gangrene Antitoxin (perfringens)
Gas-Gangrene Antitoxin (Septicum)
2345
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2359
2361
2364
2366
2369
2371
2374
2375
2376
2378
2379
2382
2384
2387
2391
2392
2393
MONOGRAPHS
Mixed Gas-Gangrene Antitoxin

Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine (Adsorbed)
Hepatitis B Vaccine (rDNA)
Inactivated Hepatitis AVaccine (Adsorbed)
InactivatedHepatitis B Vaccine
Inactivated Influenza Vaccine (SplitVIrion)
Inactivated Influenza Vaccine (Surface Antigen)
Inactivated InfluenzaVaccine (Whole VIrion)
Japanese Encephalitis Vaccine (Human)
Measles and Rubella Vaccine (Live)
Measles Vaccine (Live)
Measles, Mumps and Rubella Vaccine (Live)
Meningococcal Polysaccharide Vaccine
Mumps Vaccine (Live)
Pertussis Vaccine
Plague Vaccine
Pneumococcal Polysaccharide Vaccine
Poliomyelitis Vaccine (Inactivated)
Poliomyelitis Vaccine, Live (Oral)
RabiesAntiserum
Rabies Vaccine, Human
Rubella Vaccine (Live)
Snake VenomAntiserum
TetanusAntitoxin
Tetanus Vaccine (Adsorbed)
Tick-borne Encephalitis Vaccine (Inactivated)
Tuberculin Purified Protein Derivative
Typhoid (Strain Ty 21 a) Vaccine, Live (Oral)
Typhoid Polysaccharide Vaccine
Typhoid ParatyphoidAVaccine
Typhoid Vaccine
Typhoid Vaccine (Freeze Dried)
Iypbm; Ya,GGiJ:l~
.VaricellaVaccine (Live)
Viper Venom
Yellow Fever Vaccine (Live)
2346
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2395
2398
2399
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2408
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IP 2010
VACCINES: GENERAL REQUIREMENTS
Vaccines : General Requirements

Vaccines are preparations of antigenic substances that are
administered for the purpose of inducing in the recipient a
specific and active immunity against the infective agent or
toxin produced by it.
Vaccines may contain living micro-organisms suitably treated
to attenuate their virulence but retain their immunogenicity or
they may consist of pathogenic organisms which have been
killed or inactivated. Some vaccines consist of antigenic
fractions or substances produced by the same pathogenic
organisms but rendered harmless whilst retaining their
immunogenicity. Vaccines may b~ prepared from one species
only or from a mixture oftwo or more species.
Vaccines may be prepared by the method described in the
individuill monographs or by the general methods given below
or in any other manner provided the identity ofthe antigens is
maintained and the vaccines are free from microbial
contamination and extraneous agents. Suitable adjuvants may
be added during the preparation but streptomycin, penicillin
or other p-Iactam antibiotics may not be added at any stage of
manufacture or in the final vaccine. A suita.ble microbicide
may be added to sterile and inactivated vaccines. The final
products are distributed aseptically into sterile containers
which are then sealed to exclude extraneous micro-organisms.
Unless otherwise indicated in the monograph, thefinal vaccine
may be filled into single dose or multiple dose containers but
vaccines in multiple dose containers must invariably contain
a microbicide.
Bacterial Vaccines. Bacterial vaccines are either sterile
suspensions of live or killed bacteria or sterile extracts of
derivatives ofbacteria. They may be simple vaccines prepared
from one species or may be mixed vaccines prepared by
blending two or more simple vaccines from different species
or strains.
Bacterial vaccines may be prepared from cultures grown on
suitable solid or liquid media. The whole culture or parts of it
may be used in preparing the vaccine. The identity, antigenic
potency and purity ofeach bacterial culture must be carefully
controlled.
Vaccines containing killed organisms may be prepared by
killing the organisms by chemical or physical means provided
the immunogenicity of the vaccine is preserved. Vaccines
containing living bacteria may be prepared from str~ins which
are avirulent for humans but can stimulate the production of
antibodies active against pathogenic strains of the same
species. The final vaccines must be free from any substance
known to cause toxic, allergic or other undesirable
immunological reactions in humans.
Bacterial vaccines are suspensions of varying degrees of
opacity in colourless or slightly coloured liquids or they may
be freeze-dried so that the water content is not more than 3.0

per cent w/w unless otherwise stated in the individual
monograph. They may be standardized in tenns ofinteropacity
units or, where appropriate, by numbers of living or killed
bacteria detennined by direct cell count or by viable count.
Bacterial toxoids. Bacterial toxoids are toxins or material
derived therefrom, the toxicity ofwhich has been reduced to a
very low level or completely eliminated by,chemical or physical
means without destroying their immunizing potency. The toxins
are obtained from selected strains ofspecific micro-organisms,
grown in media free from ingredients known to cause toxic,
allergic or other undesirable immunological reactions in
humans. Toxoids produced by the action offonnaldehyde are
lmown as formol toxoids.
Bacterial toxoids may be liquid or may be prepared by adsorbing
on mineral carriers such as aluminium phosphate, aluminium
hydroxide or any other suitable adsorbent; the adsorbed
product may be separated, washed and suspended in a saline
or other appropriate solution isotonic with blood.
Bacterial toxoids are clear or slightly opalescent liquids,
colourless or slightly yellow. Adsorbed toxoids may be white
or greyish white suspensions or pale-yellow liquids with a
sediment at the bottom of the container. Freeze-dried
preparations are greyish white or yellowish white powders or
pellets.
Viral and rickettsial vaccines. Viral and rickettsial vaccines
are suspensions of viruses or rickettsiae and are prepared
from infected tissues or blood obtained from artificially infected
animals, from cultures in fertile eggs, or from cell or tissue
culture. Viral vaccines may be live or killed and'they may be
freeze-dried. Live vaccines are usually prepared using
attenuated strains of the specific organisms. Killed vaccines
may be inactivated by suitable chemical or physical means.
Mixed Vaccines. Mixed vaccines are mixtures oftwo or more
vaccines. A suitable antibacterial substance may be added to
inactivated or live viral and rickettsial vaccines provided that
it has no action against the specific organisms.
Production
General provisions. Requirements for production including
in-process testing are included in individual monographs.
Where justified and authorized, certain tests may be omitted
where it can be demonstrated, for example by validation
studies, that the production process consistently ensures
compliance with the test.
Unless otherwise justified and authorized, vaccines are
produced using a seed-lot system. The methods ofpreparation
are designed to maintain adequate immunogenic properties,
to render the preparation harmless and to prevent
contamination with extraneous agents.
2347
IP 2010
VACCINES: GENERAL REQUIREMENTS
Unless otherwise justified and authorized, in the production

of a final lot ofvaccine, the number ofpassages of a virus, or
the number of subcultures of a bacterium, from the master
seed lot shall not exceed that used for production ofthe vaccine
shown in clinic~.l studies to be satisfactory with respect to
safety and efficacy.
Control eggs. For live vaccines produced in SPF eggs, control

eggs are incubated and tested as prescribed in the monograph.
Purification. Where applicable, validated purification
procedures may be applied.
Inactivation. Inactivated vaccines are produced using a

validated inactivation process whose effectiveness and
Vaccines are as far as possible free from ingredients Imown to consistency have been demonstrated. Where there are
cause toxic, allergic or other undesirable reactions in man. recognised potential contaminants of a harvest, for example
Suitable additives, including stabilizers and adjuvants may be in vaccines produced in eggs from healthy, non-SPF flocks,
incorporated. Penicillin and streptomycin are neither used at . the inactivation process is also validated with respect to the
any stage of production nor added to the final product; potential contaminants. A test for inactivation is carried out
however, master seed lots prepared with media containing as soon as possible after the inactivation process, unless
penicillin Or streptomycin may, where justified and authorized, otherwise justified and authorised.
be used for production.
Intermediates. Where applicable, the stability ofintermediates
Substrates for propagation. Substrates for propagation in given storage conditions shall be evaluated and a period of
comply with the relevant requirements ofthe Pharmacopoeia validity .established.
or in the absence of such requirements with those of the
competent authority. Processing of cell banks and subsequent Final bulk. The final bulk is prepared by aseptically blending
cell cultures is done under aseptic conditions in an area where the ingredients of the vaccine.
no other cells are being handled. Serum and trypsin used in Adsorbents. Vaccines rtlay be adsorbed on aluminium
the preparation of cell suspensions shall be shown to be fi:ee hydroxide, aluminium phosphate, calcium phosphate or other
from extraneous agents.
suitable adsorbent; the adsorbents are prepared in special
Seed lot. The strain of bacterium or virus used in a master
seed lot is identified by historical records that include
information on the origin of the strain and its subsequent
manipulation. No micro-organism other than the seed strain
shall be present in a seed lot.
Culture media. Culture media are as far as possible free from
ingredients Imown to cause toxic, allergic or other undesirable
reactions in man; ifinclusion of such ingredients is necessary,
it shall be demonstrated that the amount present in the final
lot is reduced to such a level as to render the product safe.
Approved animal (but not human) serum may be used in the
growth medium for cell cultures but the medium used for
maintaining cell growth during virus multiplication shall not
contain serum, unless otherwise stated. Cell culture media
may contain a pH indicator such as phenol red and approved
antibiotics at the lowest effective concentration although it is
preferable to have a medium free from antibiotics during
production.
Propagation and harvest. The seed cultures are propagated
and harvested under defined conditions. The purity of the
harvest is verified by suitable tests as defined in the
monograph.
conditions which confer the appropriate physical form and

adsorptive properties.
Antimicrobial preservative. A suitable antimicrobial
preservative may be included in sterile and inactivated
vaccines and is invariably added if these preparations are
issued in multidose containers, unless otherwise stated. If an
antimicrobial preservative is used, it shall be shown that it
does not impair the safety or efficacy of the vaccine and its
effectiveness throughout the period of validity shall be
demonstrated.
Final lot. For vaccines for parenteral administration, the final
lot is prepared by aseptically distributing the final bulle into
sterile tamper-proofcontainers which, after freeze-drying where
applicable, are closed so as to exclude contamination. For
vaccines for administration by a non-parenteral route, the final
lot is prepared by distributing the final bulle under suitable
conditions into sterile, tamper-proof containers.
Stability. Maintenance of potency ofthe final lot throughout
the period of validity shall be demonstrated by validation
studies; the loss of potency in the recommended storage
conditions is assessed and excessive loss even within the
limits of acceptable potency may indicate that the vaccine is
unacceptable.
Control cells. For vaccines produced in cell cultures, control

cells. are maintained and tested as prescribed. In order to
Degree of adsorption. During development of an adsorbed
conditions that are rigorously identical with those used for

the production cell cultures, including use ofthe same batches
of media and media changes.
consistency testing. A release specification for the degree of

adsorption is established in the light of results found for
batches used in clinical testing. From the stability data
provIde av~aHdcontror these ce1fsmustbemalntalnedin vaccIne; the-degreeof adsorptlon!seva1uatedaspartoftiie
2348
ANTISERA
IP 2010
generated for the vaccine it must be shown that at the end of

the period ofvalidity the degree of adsorption will not be less
than for batches used in clinical testing.
Antisera
Tests
Antisera are native (unconcentrated) sera or preparations from

native sera containing specific immunoglobulins that have
prophylactic or therapeutic action when injected into persons
exposed to or suffering from a disease caused by a specific
micro-organism.
Vaccines, reconstituted where necessary, comply with the

following requirements unless otherwise stated in the
individual monograph.
Phenol (Ifpresent) (2.3.36). Not more than 0.25 per cent w/v.
Thiomersal (Ifpresent) (2.3.48). Between 0.005 per cent w/v
and 0.02 per cent w/v.
Free formaldehyde (Ifpresent) (2.3.20). Maximum 0.2 gil.
Aluminium (Ifpresent) (2.3.9). Not more than 1.25 mg per
dose.
Sterility (2.2.11). Unless otherwise stated all vaccines comply
with tests for sterility, except that for living bacterial vaccines,
growth of the organism from which the vaccine was prepared
is permitted (sterility means absence of bacterial and fungal
contaminants except where specified in the individual
monograph).
Abnormal toxicity (2.2.1). Unless otherwise stated, all vaccines
comply with the test for abnormal toxicity, Method B. In
vaccines containing phenol as preservative, the test in mice
may be inappropriate.
NOTE - The statements given in this general chapter is

intended to be read in conjunction with the monographs on
the individual vaccine in. this Pharmacop0tf,ia which refer to
preparations for human use; they do not necessarily apply
to vaccines for use in veterinmy practice.
Storage. Liquid vaccines must be stored at a temperature
between 20 and 80 and should not be allowed to freeze unless
otherwise specified in the individual monograph. Freeze~dried
preparations must be stored at temperatures between 20 and
80 and for long term storage a temperature of -200 protected.
At higher temperatures vaccines deteriorate rapidly.
Labelling. The label states (1) for liquid vaccines, the total
number of ml in the container and, for freeze dried vaccines,
the number of doses in the container; (2) unless otherwise
indicated the minimum number ofUnits per dose or perml or,
for viral vaccines, the minimum viral titre; (3) the dose and
route of administration; (4) the name and proportion of any
antibacterial preservative or other auxiliary substances added
to the vaccine; (5) the date after which the vaccine is not
intended to be used; (6) the conditions under which it should
be stored; (7) for freeze dried vaccines, the liquid to be used
for reconstitution and its volume; (8) that the vaccine should
be used immediately after reconstitution; (9) unless otherwise
directed, that the vaccine should be shaken well before use;
(10) any contraindication to the use of the vaccine.
Immunosera
Antisera are prepared by injecting antigens which are

preparations of cultures of the specific organisms or venoms
or their products into healthy humans or animals such as
horses so as to produce in them antibodies which are normally
associated with the globulin fraction of serum. Antigens
commonly used for this purpose are toxins, toxoids, venoms,
bacterial and viral vaccines. Non-lethal amounts of the toxin
or the corresponding toxoid/vaccine are injected in gradually
increasing doses into animals. Specific antitoxins are formed
in the serum and the animals become actively immune. During
the process of immunisation, the animals should not be treated
with penicillin. When a satisfactory degree ofthe immunity is
produced, larger volumes of blood are withdrawn from the
animals and the plasma or serum is processed to :produce
specificantjsera. The globulins may be obtained from the
immune serum by enzyme treatment and fractional precipitation
or by other physical or chemical methods.
Antiviral sera with the exception of Rabies Antiserum are
usually obtained from the plasma or serum ofhuman patients
who have recovered from the viral disease or who have been
artificially immunised. Rabies Antiserum is obtained from
animals by injecting gradually increasing doses of a rabies
vaccine, a killed vaccine being used first and when some
immunity is established living virus being used as an antigen.
When sufficient virus-neutralising titre is reached the blood
is collected and processed.
Antisera in liquid form are distributed under aseptic conditions
into sterile containers which are then sealed so as to exclude
micro-organisms. A suitable antibacterial substance may be
added and is invariably added when the final product is filled
in multiple dose containers. The produCt may be freeze-dried
by a procedure which reduces the water content of the final
product to less than 1.0 per cent w/w. Antisera are almost
colourless or very faintly yellow liquids free from turbidity.
Freeze-dried antisera consist ofwhite or pale-yellow powders
or friable masses which dissolve in water to form colourless or
pale yellow liquids having the same characteristics as the
corresponding liquid preparations.
Tests
The following tests refer to liquid antisera and to the
reconstituted freeze-dried preparations.
2349
ADSORBED DIPHTHERIA, TETANUS AND HEPATITIS B (rDNA) VACCINE
pH (2.4.24).6.0 to 7.0.
Total protein. Not more than 17.0 per cent w/v, determined by
carrying out the determination ofnitrogen, Method C (2.3.30)
and multiplying the result by 6.25.
Foreign proteins. When examined by precipitation reactions
with specific antisera, they areshown to consist exclusively
of protein of the decla.red animal species.
Phenol (2.3.36) (ifpresent). Not more than 0.25 per cent w/v.
Sterility (2.2.11). Comply with the tests for sterility.
Abnormal toxicity (2.2.1). Comply with the test for abnormal
toxicity. In antisera containing phenol as preservative, the
test in mice may be inappropriate.
NOTE - The statements given in this general monograph

are intended to be read in cOlyunction with the monographs
on the individual antiserum in the Pharmacopoeia which
refer to preparations for human use; they do not necessarily
apply to antisera for use in veterinary practice.
Storage. Antisera should be stored at a temperature between

2 and 8 and should not be allowed to freeze unless otherwise
stated in the individual monograph.
Labelling. The label states the name and quantity of
antibacterial substance added, if any.
Adsorbed Diphtheria, Tetanus and

Diphtheria, Tetanus and Hepatitis B (rDNA) Vaccine
(Adsorbed) is a combined vaccine composed of: diphtheria
formol toxoid; tetanus formol toxoid; hepatitis B surface
antigen (HBsAg); a mineral adsorbent such as aluminium
hydroxide or hydrated aluminium phosphate.
The formol toxoids are prepared from the. toxins produced by
the growth of COlynebacterium diphtheriae and Clostridium
.
tetani, respectively.
HBsAg is a component protein ofhepatitis B virus; the antigen
is obtained by recombinant DNA technology.
Production
General provisions
The production method shall have been shown to yield
consistently 'vaccines comparable with the vaccine ofproven
clinical efficacy and safety in man;
The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
toxicity for antisera and vaccines, and with the following test
for specific toxicity ofthe diphtheria and tetanus components:
IP 2010
inject subcutaneously 5 times the single human dose stated

on the label into each of5 healthy guinea-pigs, each weighing
between 250 and 350 g, that have not previously been treated
with any material that will interfere with the test. Ifwithin 42
days ofthe injection any ofthe animals shows signs ofor dies
from diphtheria toxaemia or tetanus, the vaccine does not
comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal
dies in the second test, the vaccine does not comply with the
test.
The content of bacterial endotoxins in the bulle purified
diphtheria toxoid and tetanus toxoid is determined to monitor
the purification procedure and to limit the amount in the final
vaccine. For each .component, the content of bacterial
endotoxin is less than the limit approved for the particular
vaccine and in any case the contents are such that the final
vaccine contains less than 100 ill per single human dose.
Reference vaccine(s)
Provided valid assays can be performed, monocomponent

reference vaccines may be used for the aSsays on the combined
vaccine. Ifthis is not possible because of interaction between
the components of the combined vaccine or because of the
difference in composition between monocomponent reference
vaccine and the test vaccine, a batch of combined vaccine
shown to be effective in clinical trials or a batch representative
thereof is used as a reference vaccine. For the preparation of
a representative batch, strict adherence to the production
process used for the batch tested in clinical trials is necessary.
The reference vaccine may be stabilised by a method that has
been shown to have no effect on the assay procedure.
Production of the components
The production of the components complies with the
requirements of the monographs on Diphtheria Vaccine
(Adsorbed), Tetanus Vaccine (Adsorbed) and Hepatitis B
Vaccine (rDNA).
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption, separately
or together, of suitable quantities of bulle purified diphtheria
toxoid, tetanus toxoid and HBsAg onto a mineral carrier such
as aluminium hydroxide or hydrated aluminium phosphate.
Suitable antimicrobial preservatives may be added.
Only a final bulle vaccine that complies with the following
requirements may be used in the preparation ofthe final lot.
Antimicrobial preservative. Where applicaqle, determine the
!l.!U~-':lI1_t ()r!lnctil1?-icro~_~!l1 I'~~erv_a~~~~:t!l.slli~!l~l~<::_~~I1?-~<::!ll
method. The content is not less than 85.0 per cent and not
greater thanll5.0per cent of the intended amount.
Sterility (2.2.11). Carry out test for sterility using 10 ml ofbulk
for each sterility medium.
2350
IP 2010
ADTP (ACELLULAR COMPONENT) AND HAEMOPHILUS TYPE b CONJUGATE VACCINE
FINAL LOT
Only a final lot that is satisfactory with respect to the test for
osmolality and with respect to each ofthe requirements given
below under Identification, Tests and Assay may be released
for use.
Provided the test for antimicrobial preservative and the assays
for the diphtheria and tetanus components have been carried
out with satisfactory results on the final bulle vaccine, they
may be omitted on the final lot.
Pyrogens (2.2.8). Complies with the test for pyrogens. Inject

the equivalent of 1 human dose into each rabbit.
Assay
Diphtheria component
Carry out one of the prescribed methods for the assay as
stated under Diphtheria Vaccine (Adsorbed).
The lower confidence limit (P = 0.95) ofthe estimated potency
is not less than 30 ill per single human dose.
Tetanus component
Provided the content of free formaldehyde has been

detennined on the bulk purified antigens or on the final bulle
and it has been shown that the content in the final lot will not
exceed 0.2 gil, the test for free fonnaldehyde may be omitted
on the final lot.
Cany out one of the prescribed methods for the assay as

stated under Tetanus Vaccine (Adsorbed).
If an in vivo assay is used for the hepatitis B component,

provided it has been carried out with satisfactory results on
the final bulk vaccine, it may be omitted on the final lot.
Hepatitis B component
Osmolality (2.4.23). The osmolality of the vaccine is within

the limits approved for the particular preparation.
Identification
A. Diphtheria toxoid is identified by a suitable irnmunochemical

method (2.2.14). The following method, applicable to certain
vaccines, is given as an example. Dissolve in the vaccine under
examination sufficient sodium citrate to give a 10 per cent
w/v solution. Maintain at 37 for about 16 hours and centrifuge
until a clear supernatant is obtained. The clear supernatant
liquid reacts with a suitable diphtheria antitoxin, giving a
precipitate.
B. Tetanus toxoid is identified by a suitable immunochemical
vaccines, is given as an example. The clear supernatant liquid
obtained during identification test A reacts with a suitable
tetanus antitoxin, giving a precipitate.
C. The assay or, where applicable, the electrophoretic profile,
serves also to identifY the hepatitis B component of the
vaccine.
Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose,
if aluminium hydroxide or hydrated aluminium phosphate is
used as the adsorbent.
Free formaldehyde (2.3.20). Maximum 0.2 gil.
Antimicrobial preservative. Where applicable, detennine the
amount of antimicrobial preservative by a suitable chemical
greater than 115.0 per cent ofthe intended amount.

It complies with the assay of Hepatitis B Vaccine.

Labelling. The label states (1) the minimum number of
International Units of diphthetia and tetanus toxoid per single
human dose; (2) the amount ofHBsAg per single human dose;
(3) the type of cells used for production of the HBsAg
component; (4) where applicable, that the vaccine is intended
for primary vaccination of children and is not necessarily
suitable for reinforcing doses or for administration to adults;
(5) the name and the amount of the adsorbent; (6) that the
vaccine must be shaken before use; (7) that the vaccine is not
to be frozen.
Adsorbed Diphtheria, Tetanus,

Pertussis (Acellular Component) and
Haemophilus Type b Conjugate
Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component) and
Haemophilus Type b Conjugate Vaccine (Adsorbed) is a
combined vaccine composed of: diphtheria formol toxoid;
tetanus formol toxoid; individually purified antigenic
components of Bordetella pertussis; polyribosylribitol
phosphate (PRP) covalently bound to a carrier protein; a
mineral absorbent such as aluminium hydroxide or hydrated
aluminium phosphate. The product may be presented with
the haemophilus type b component in a separate container,
the contents of which are mixed with the,ofh~Lcomponents
immediately before use.
The fOlmol toxoids are prepared from the toxins producedoy
the growth of Corynebacterium diphtheriae and Clostridium
tetani respectively.
The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties produced by
2351
expression ofa genetically modified form ofthe corresponding

gene. Pertussis toxoid is prepared from pertussis toxin by a
method that renders the toxin harmless while maintaining
adequate immunogenic properties and avoiding reversion to
toxin. The acellular pertussis component may also contain
filamentous haemagglutinin, pertactin (a 69 lilia outermembrane protein) and other defined components' of
B. pertussis such as fimbrial-2 and fimbrial-3 antigens. The
latter two antigens may be copurified. The antigenic
composition and characteristics are based on evidence of
protection and freedom from unexpected reactions in the target
group for which the vaccine is intended.
PRP is a linear copolymer composed of repeated units of
3 -[3- D-ri bofurano sy 1-( 1 ~ 1)-ri b i tol- 5 -phosphate
[(CIOHI9 0 ,l),J, with a defined molecular size and derived from
a suitable strain of Haemophilus injluenzae type b. The carrier
protein, when conjugated to PRP, is capable of inducing a
T-cell-dependent B-cell immune response to the
polysaccharide.
IP 2010
Reference vaccine

reference vaccines may be used for the assays on the combined
vaccine. If this is not possible because of interaction between
the components ofthe combined vaccine or because of the
difference in composition between mOriocomponent reference
The production of the components complies with the tests of
the monographs on Diphtheria Vaccine (Adsorbed), Tetanus
Vaccine (Adsorbed), Pertussis Vaccine (Acellular Component,
Adsorbed) and Haemophilus Type b Conjugate Vaccine.
Production
FINAL BULK VACCINE
General provisions
consistently the vaccines comparable with the vaccine of
proven clinical efficacy and safety in man.
If the vaccine is presented with the haemophilus component
in a separate vial, as part of consistency studies the assays of
the diphtheria, tetanus and pertussis components are carried
out on a suitable number of batches of vaccine reconstituted
for use. For subsequent routine control, the assays of these
components may be carried out without mixing with the
haemophilus component.
The content ofbacterial endotoxins in bulle purified diphtheria
toxoid, tetanus toxoid, pertussis components and PRP
conjugate is determined to monitor the purification procedure
and to limit the amount in the final vaccine. For each
component, the content of bacterial endotoxins is less than
the limit approved for the particular vaccine; ifthe vaccine is
presented with the haemophilus component in a separate
container, the contents ofthe diphtheria, tetanus and pertussis
antigens are in any case such that the final vial for these
components contains less than 100 IV per single human dose.
Different methods of preparation may be used: a fmal bulle

vaccine may be prepared by adsorption, separately or together,
ofsuitable quantities ofbulle purified diphtheria toxoid, tetanus
toxoid, acellular pertussis components and PRP conjugate
onto a mineral carrier such as aluminium hydroxide or hydrated
aluminium phosphate; or 2 final bulles may be prepared and
filled separately, one containing the diphtheria, tetanus and
pertussis components, the other containing the haemophilus
component, which may be freeze-dried. Suitable antimicrobial
preservatives may be added.
requirements may be used in thepreparation ofthe final lot.
Antimicrobial preservative. Where applicable, determine the
Sterility (2.2.11). Carry out test for sterility using 10 ml ofbulle
FINAL LOT

toxicity for antisera and vaccines.
osmolality shown below and with respect to each of the
requirements given below under Identification, Tests and
Assay may be released for use.
During de'vel,opIIlelllt
necessary, it shall be demonstrated by tests in animals that
the vaccine induces a T-cell dependent B-cell immune response
toPRP.
irreversibility ofpertussis toxoid and antimicrobial preservative

and the assays have been carried out with satisfactory results
on the final bulk vaccine, they may be omitted on the final lot.
t~sts.f()r, !l~sellS:e. ()f..re:s.i~ll,aLpe.t:J:lls.sis.t()(il1?
2352
IP 2010
Provided the free fonnaldehyde content has been detennined

on the bulle purified antigens or the final bulk and it has been
shown that the content in the final lot will not exceed 0.2 gil,
the test for free fonnaldehyde may be omitted on the final lot.
Ifthe haemophilus component is freeze-dried, some tests may

be carried out on the freeze-dried product rather than on the
bulle conjugate where the freeze-drying process may affect
the component under test.
Osmolality (2.4.23). The osmolality of the vaccine,

reconstituted where applicable, is within the limits approved
for the particular preparation.
Absence ofresidual pertussis toxin and irreversibility

of pertussis toxoid
pH (2.4.24). The pH ofthe vaccine, reconstituted ifnecessary,

is within the range approved for the particular product.
Free PRP. Unbound PRP is determined after removal of the
conjugate, for example by anion-exchange, size-exclusion or
hydrophobic chromatography (2.4.16), ultrafiltration or other
validated methods. The amount offree PRP is not greater than
that approved for the particular product.
Identification
in a separate vial: identification tests A, Band C are carried
out using the vial containing the diphtheria, tetanus and
pertussis components; identification test D is carTied out on
the vial containing the haemophilus components.
A. Diphtheria toxoid is identified by a suitable imrnunochemical
wlv solution. Maintain at 37 for about 16 hours and centrifuge
reacts with a suitable diphtheria antitoxin, giving a precipitate.
vaccines, is given as an example. The clear superna tan t
obtained as described in Identification test A reacts with a
suitable tetanus antitoxin, giving a precipitate.
C. The pertussis components are identified by a suitable
immunochemical method (2.2.14). The following method,
applicable to certain vaccines, is given as an example. The
clear supernatant obtained as described in Identification
test A reacts with a specific antisera to the pertussis
components of the vaccine.
D. The haemophilus component is identified by a suitable
imrntinochemical method (2.2.14) for PRP.
Tests
If the product is presented with the haemophilus component
in a separate contairter: the tests for absence of residual
pertussis toxin, irreversibility ofpertussis toxoid, aluminium,
free fonnaldehyde, antimicrobial preservative and sterility are
carried out on the container with the diphtheria, tetanus and
pertussis components; the tests for PRP content, water (where
applicable), sterility and pyrogens are carried out on the
container with the haemophilus component.
This test is not necessaJyfor the product obtained by genetic

modification. Use 3 groups each of not less than 5 histaminesensitive mice. Inject intraperitoneally into each mouse ofthe
first group twice the single human dose ofthe vaccine stored
at 2 to 8. Inject intraperitoneally into each mouse of the
second group twice the single human dose of the vaccine
incubated at 37 for 4 weeks. Inject diluent into the third group
ofmice. After 5 days, inject into each mouse 2 mg ofhistamine
base intraperitoneally in a volume not exceeding 0.5 ml and
observe for 24 hours. The test is invalid if 1 or more control
mice die following histamine challenge. The vaccine complies
with the test if no animal in the first or second group dies
following histamine challenge. If 1 mouse dies in either or
both of the first and second groups, the test may be repeated
with the same number ofmice or with a greater number and the
results ofvalid tests combined; the vaccine complies with the
test if, in both ofthe groups given the vaccine, not more than
5.0 per cent ofthe total number ofmice die following histamine
challenge.
The histamine sensitivity ofthe strain ofmice used is verified
at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in
phosphate-buffered saline solution containing 0.2 per cent
wlv of gelatin and challenge with histamine as above; the
strain is suitable if more than 50 per cent of the animals are
sensitised by 50 ng of pertussis toxin and none of the control
animals injected with only diluent and challenged similarly
with histamine shows symptoms of sensitisation.
PRP. Minimum 80.0 per cent ofthe amount ofPRP stated on

the label. PRP is detennined either by assay ofribose (2.7.1) or
phosphorus (2.7.1), by an immunochemical method (2.2.14) or
by anion-exchange liquid chromatography with pulsedamperometric detection.
Free formaldehyde (2.3.20). Maximwn 0.2 gil.
greater than 115.0 per cent of the intended amount.
Water (2.3.43). Maximum 3.0 per cent for the freeze-dried
2353
ADTP (ACELLULAR COMPONENT) AND HAEMOPHILUS TYPE b CONWGATE VACCINE

per kg ofthe rabbit's mass a quantity ofthe vaccine equivalent
to: 1 mg ofPRP for a vaccine with diphtheria toxoid or CRM
197 diphtheria protein as carrier; 0.1 mg ofPRP for a vaccine
with tetanus toxoid as carrier; 0.025 mg ofPRP for a vaccine
with OMP as carrier.
Assay
is not less than the minimum potency stated on the label.
Unless otherwise justified and authorised, the minimum
potency stated on the label is 30 IU per single human dose.
Tetanus component
is not less than 40 IU per single human dose. ._
Pertussis component
The vaccine complies with the assay as the stated Adsorbed
Pertussis Vaccine (Acellular Component).
International Units of diphtheria and tetanus toxoid per single
human dose; (2) the names and amounts of the pertussis
components per single human dose; (3) the number of
micrograms ofPRP per single human dose; (4) the type and
nominal amount of carrier protein per single human dose; (5)
where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
reinforcing doses or for administration to adults; (6) the name
and the amount ofthe adsorbent; (7) that the vaccine must be
shaken before use; (8) that the vaccine is not to be frozen;
(9) where applicable, that the vaccine contains a pertussis
toxin-like protein produced by genetic modification.

Diphtheria, Tetanus; Pertussis (Acellular Component) and
Hepatitis B (rDNA) Vaccine (Adsorbed) is a combined vaccine
composed_of:diphtheriaforrnoLtoxoid; teJanus_formolJoxoid;
individuaUy PllriJ'ii::<:ll!l1t.igi::l1icc()lllPQlleI1ts .of 13grq?tejla.
pertussis; hepatitis B surface antigen; a mineral adsorbent
such as aluminium. hydroxide or hydrated aluminium
phosphate.
IP 2010
The formol toxoids are prepared from the toxins produced by

tetani, respectively.
The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties, produced by
method that renders the latter harmless while maintaining
adequate immunogenic propelties and avoiding reversion to
toxin. The vaccine may also contain filamentous
haemagglutinin, peltactin (a 691cDa outer-membrane protein)
and other defined components ofB. pertussis such as fimbrial2 and fimbrial-3 antigens. The latter 2 antigens may be
copurified. The antigenic composition and characteristics are
based on evidence ofprotection and freedom from unexpected
reactions in the target group for which the vaccine is intended.
Hepatitis B surface antigen is a component protein ofhepatitis
B virus; the antigen is obtained by recombinant DNA
technology.
Production
General provisions
consistently vaccines comparable with the vaccine of proven
clinical efficacy and safety in man.
The content of bacterial endotoxins in the bulk purified
diphtheria toxoid, tetanus toxoid and pertussis components
is determined to monitor the purification procedure and to
limit the amount in the final vaccine. For each component, the
content ofbacterial endotoxins is less than the limit approved
for the particular vaccine.
ProductionoHbecomponents
(Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine
2354
IP 2010
ADTP (ACELLULAR COMPONENT) AND HEPATITIS B (rDNA) VACCINE
(Acellular Component, Adsorbed) and Hepatitis B Vaccine

(rDNA).
FINAL BULK VACCINE
The final bull( vaccine is prepared by adsorption, separately
or together, of suitable quantities of bulk purified diphtheria
toxoid, tetanus toxoid, acellular pertussis components and
hepatitis B surface antigen onto a mineral carrier such as
aluminium hydroxide or hydrated aluminium phosphate.
Suitable antimicrobial preservatives may be added.
Only a final bulk vaccine that complies with the following
requirements may be used in the preparation of the final lot.
vaccines, is given as an example. The clear supernatant

obtained as described in identification test A reacts with a
immunochemical method (2.2.14). The following method;
clear supernatant obtained as described in identification test
A reacts with a specific antisera to the pertussis components
of the vaccine.
D. The assay or, where applicable, the electrophoretic profile,
serves also to identify the hepatitis B component of the
vaccine.

Tests
Sterility (2.2.11). Carry out test for sterility using 10 ml ofbull(

This test is not necessmyfor the product obtained by genetic

modification. Use 3 groups each ofnot less than 5 histaminesensitive mice. Inject intraperitoneally into each mouse ofthe
first group twice the single human dose of the vaccine stored
test if, in both of the groups given the vaccine, not more than
5 per cent ofthe total number ofmice die following histamine
challenge.
FINAL LOT
for use.
Provided the tests for absence of residual pertussis toxin,
irrever:sibility ofpertussis toxoid and antimicrobial preservative
and the assays for the diphtheria, tetanus and pertussis
components have been carried out with satisfactory results
on the final bull( vaccine, they may be omitted on the final lot.
Provided the content of free formaldehyde has been
determined on the bull( purified antigens or on the final bull<
exceed 0.2 gil, the test for free formaldehyde may be omitted
on the final lot.
.
If an in vivo assay is used for the hepatitis B component,
provided it has been carried out with satisfactory results on
the final bull( vaccine, it may be omitted on the final lot.

Identification
A. Diphtheria toxoid is identified by a suitable irnmunochemical
Absence of residual pertussis toxin and iueversibility

of pertussis toxoid

at suitable intervals as follows: inject intravenously threefold
dilutions of a reference pertussis toxin preparation in
wlv of gelatin and challenge with histamine as above; the
strain is suitable if more than 50.0 per cent of the animals are
sensitised by 50 ng of pertussis toxin and none ofthe control

2355
ADTP (ACELLULAR COMPONENT) AND HEPATITIS B (rDNA) VACCINE

Sterility ( 2.2.11). Complies with the test for sterility.
the equivalent of 1 human dose into each rabbit.
Assay

potency stated on the label is 30 IV per single human dose.
Tetanus component

is not less than 40 IV per single human dose.
Pertussis component
The vaccine complies with the assay as stated under Adsorbed

The vaccine complies with the assay as stated under Hepatitis

B Vaccine (rDNA).
International Units ofdiphtheria and tetanus toxoid per single
components per single human dose; (3) the amount ofHBsAg
per single human dose; (4) the type ofcells used for production
of the hepatitis B component; (5) where applicable, that the
vaccine is intended for primary vaccination of children and is
not necessarily suitable for reinforcing doses or for
administration to adults; (6) the name and the amount of the
adsorbent; (7) that the vaccine must be shaken before use;
(8) that the vaccine is not to be frozen (9) where applicable,
that the vaccine contains a pertussis toxin-like protein
produced by genetic modification.

Pertussis (Acellular Component),
Inactivated Poliomyelitis Vaccine and
Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component),
Inactivated Poliomyelitis Vaccine and Haemophilus Type b
Conjugate Vaccine (Adsorbed) is a combined vaccine
IP 2010
composed of: diphtheria fonnol toxoid; tetanus fonnol toxoid;

individually purified antigenic components of Bordetella
pertussis; suitable strain of human polioviruses 1,2, and 3
grown in suitable cell cultures and inactivated by validated
method; polyribosylribitol phosphate (PRP) covalently bound
to a carrier protein; a mineral absorbent such as alumipium
hydroxide or hydrated aluminium phosphate. The product may
be presented with the haemophilus type b component in a
separate container, the contents of which are mixed with the
other components immediately before use.
the growth of COIynebacterium diphtheriae and Clostridium
expression ofa genetically modified fonn ofthe corresponding
toxin. The acellular pertussis component may also contain
filamentous haemagglutinin, pertactin (a 69 lcDa outermembrane protein) and other defined components of B.
pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter
2 antigens may be co-purified. The antigenic composition and
characteristics are based on evidence of protection and
freedom from unexpected reactions in the target group for
which the vaccine is intended.
3-13-D-ri bofuranosy 1-( 1-71 )-ri bito 1- 5 -phosphate
[(CIOHI9 0 IZP\], with a defined molecular size and derived from
a suitable strain ofHaemophilus injluenzae type b. The carrier
protein, when conjugated to PRP, is capable of inducing a
T-cell-dependent B-cell immune response to the
polysaccharide.
Production
General provisions
The content ofbacterial endotoxins in bulk purified diphtheria
toxoid, tetanus toxoid, pertussis components, purified,
inactivated monovalent poliovirus harvests and bulk PRP
conjugate is determined to monitor the purification procedure
and to limit
the amount in the final vaccine. For each
I
component, the content of bacterial endotoxins is less than
th-limitapprov-d forJhe particular vaccine and, ih any case,
the contents aresuch that the finalvact;:inepontains less than
100 IV per single human dose.
product, iftested, would comply with the following test. Inject
2356
IP 2010
ADTP (ACELL. COMP.), INACTIVATED POLIOMYELITIS VACCINE AND HAEMOPHILUS TYPE b CONJUGATE VACCINE
subcutaneously 5 times the single human dose stated on the

label into each of 5 healthy guinea-pigs, each weighing
with any material that will interfere with the test. If within
42 days of the injection any of the animals shows signs of or
dies from diphtheria, toxaemia or tetanus, the vaccine does
not comply with the test. Ifmore than 1 animal dies from nonspecific causes, repeat the test once; if more than I animal
test.
During development studies and wherever revalidation is
toPRP.
As part of consistency studies the assays of the diphtheria,
tetanus, pertussis and poliomyelitis components are carried
'
Refe,~ence
of a trivalent pool of such monovalent harvests. Suitable

antimicrobial preservatives may be added.
The final bulle of the haemophilus component is prepared by
dilution ofthe bulk conjugate to the final concentration with a
suitable diluent. A stabiliser may be added.
Bovine serum albumin. Determine on the poliomyelitis

components by a suitable immunochemical method (2.2.14)
during preparation of the final bulle vaccine, before addition
of the adsorbent, the amount ofbovine serum albumin is such
that the content in the final vaccine will not be more than 50
ng per single human dose.
Sterility (2.2.11). Carry out test for sterility using 10 ml ofbullc
vaccine(s)

Production ofthe components

(Acellular Component, Adsorbed), Poliomyelitis Vaccine
(Inactivated) and Haemophilus Type b Conjugate Vaccine.
FINALBULKVACClNE
The final bulk of the diphtheria, tetanus, pertussis and
poliomyelitis components is prepared by adsorption,
separately or together, of suitable quantities of bulk purified
diphtheria toxoid, bulle purified tetanus toxoid and bulle purified
acellular pertussis components onto a mineral carrier such as
aluminium hydroxide or hydrated aluminium phosphate and
admixture of suitable quantities of purified, monovalent
harvests ofhuman polioviruses 1,2 and 3 or a suitable quantity
FINAL LOT
The final bulk of the haemophilus component is fi:eeze-dried.
Provided that the test for absence of residual pertussis toxin
and irreversibility ofpertussis toxoid, the test for antimicrobial
preservative and the assay have been carried out with
satisfactOly results on the final bulle vaccine, they may be
omitted on the final lot.
Provided that the free formaldehyde content has been
determined on the bulk purified antigens and the purified
monovalent harvests or the trivalent pool of polioviruses or
the final bulle and it has been shown that the content in the
final lot will not exceed 0.2 gil, the test for free formaldehyde
Provided that the in vivo assay for the poliomyelitis component
has been carried out with satisfactory results on the final bulle
vaccine, it may be omitted on the final lot.
Free PRP
Unbound PRP is determined on the haemophilus component
after removal ofthe conjugate, for example by anion-exchange,
2357
ADTP (ACELL. COMP.), INACTIVATED POLIOMYELITIS VACCINE AND HAEMOPHILUS TYPE b CONJUGATE VACCINE
IP 2010
size-exclusion or hydrophobic chromatography (2.4.16), sensitive mice. Inject intraperitoneally into each mouse ofthe
ultrafiltration or other validated methods. The amount offree .first group twice the single human dose ofthe vaccine stored
PRP is not greater than that approved for the particular product. at 2 to 8. Inject intraperitoneally into each mouse of the
Identification
ofmice. After 5 days, inject into each mouse 2 mg Ofhistamine
Identification tests A, B, C and D are carried out using the vial
containing the diphtheria, tetanus, pertussis and poliomyelitis
components; identification test E is carried out on the vial
containing the haemophilus component.
A. Diphtheria toxoid is identified by a suitable immunochemical following histamine challenge. If 1 mouse dies in either or
method (2.2.14). The following method, applicable to certain both of the first and second groups, the test may be repeated
vaccines, is given as an example. Dissolve in the vaccine under with the same number ofmice or with a greater number and the
examination sufficient sodiuin citrate to give a 10 per cent results ofvalid tests combined; the vaccine complies with the
w/v solution. Maintain at 37 for about 16 hours and centrifuge test if, in both ofthe groups given the vaccine, not more than
until a clear supernatant is obtained. The clear supernatant 5 per cent ofthe total number ofmice die following histamine
reacts with a suitable diphtheria antitoxin, giving a precipitate. challenge.
w/v of gelatin and challenge with histamine as above; the
C. The pertussis components are identified by suitable strain is suitable if more than 50.0 per cent of the animals are
immunochernical methods (2.2.14). The following method, sensitised by 50 ng of pertussis toxin and none ofthe control
clear supernatant obtained during identification test A reacts with histamine show symptoms of sensitisation.
with specific antisera to the pertussis components of the
vaccine.
D. The vaccine is shown to contain human polioviruses 1,2 the label. PRP is detennined either by assay of ribose ( 2.7.1)
and 3 by a suitable immunochemical method (2.2.14), such as or phosphorus (2.7.1), by an immunochemical method (2.2.14)
detennination ofD-antigen by enzyme-linkedimmunosorbent or by anion-exchange liquid chromatography with pulsedamperometric detection.
assay (ELISA).
E. The haemophilus component is identified by a suitable
immunochemical method (2.2.14) for PRP.
Tests
The testsfor absence ofresidualpertussis toxin, irreversibility

of pertussis toxoid, aluminium, free formaldehyde,
antimicrobial preservative and sterility are carried out on
the container with the diphtheria, tetanus, pertussis and
poliomyelitis components; the tests for PRP content, wate]~
sterility and pyrogens are carried out on the container with
the haemophilus component.
Some tests for the haemophilus component may be carried
out on the fi-eeze-dried product rather than on the bulk
conjugate where the fi-eeze-drying process may affect the
component under test.
Absence of resldual pertuSSiS toxin and irreverSibility

of pertussis toxoid
This test is not necessaryfor the product obtained by genetic

modification. Use 3 groups each ofnot fewer than 5 histamine-

Water (2.3.43). Maximum 3.0 per cent for the haemophilus
component.
Sterility (2.2.11 ). Complies with the test for sterility.
J,lyrogens (2.2.8). Complies with the test for pyrogens. Inject
per leg. Qftll~l'fl1:>1:>it 'sl11assa qllfllltitYQftlle vflcci!l.~t::q1Ji"fll~ll.t
to: 1 mg ofPRP for a vaccine with diphtheriatoxoid orCRM:
197 diphtheria protein as canier; 0.1 mg ofPRP for a vaccine
with OMP as a carrier.
2358
IP 2010
ADTP (ACELLULAR COMPONENT) AND INACTIVATED POLIOMYELITIS VACCINE
Assay
Unless otherwise justified and authorised, the lower
confidence limit (P = 0.95) ofthe estimated potency is not less
than 30 IU per single human dose.
Tetanus component
is not less than 40 IU per single human dose.
Pertussis component
It complies with the assay as stated under Adsorbed Pertussis
Vaccine (Acellular Component).
Poliomyelitis component
D-antigen content
As a measure of consistency of production, determine the
D-antigen content for human polioviruses 1, 2 and 3 by a
suitable immunochemical method (2.2.14) using a reference
preparation calibrated in units of D-antigen. For each type,
the content, expressed with reference to the amount of
D-antigen stated on the label, is within the limits approved for
the particular product. Poliomyelitis vaccine (inactivated)
reference preparation is calibrated in Units and intended for
use in the assay ofD-antigen. The Unit and the International
Unit are equivalent.
In vivo test
The vaccine complies with the in vivo assay as stated under
Inactivated Poliomyelitis Vaccine.

International Units of diphtheria and tetanus toxoid per single
human dose (2) the names and amounts of the pertussis
components per single human dose; (3) the nominal amount
of poliovirus of each type (1, 2 and 3), expressed in units of
D-antigen per single human dose; (4) the type of cells used
for production ofthe poliomyelitis component; (5) the number
ofmicrograms ofPRP per single human dose; (6) the type and
nominal amount of carrier protein per single human dose;
(7) where applicable, that the vaccine is intended for primary
and the amount of the adsorbent; (9) that the vaccine must be

Inactivated Poliomyelitis Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component) and
Poliomyelitis (Inactivated) Vaccine is a combined vaccine
containing: diphtheria formol toxoid; tetanus formol toxoid;
individually purified antigenic components of Bordetella
pertussis; suitable strains of human polioviruses 1, 2 and 3
grown in suitable cell cultures and inactivated by a validated
method; a mineral adsorbent such as aluminium hydroxide or
hydrated aluminium phosphate.
haemagglutinin, 'pertactin (a 69lcDa outer-membrane protein)
Production
General provisions
consistently vaccines comparable with the vaccine ofproven
The content ofbacterial endotoxins in bulk purified diphtheria
toxoid, tetanus toxoid, pertussis components and purified,
inactivated monovalent poliovirus harvests is determined to
monitor the purification procedure and to limit the amount in
the final vaccine. For each component, the content ofbacterial
endotoxins is less than the limit approved for the particular
vaccine and, in any case, the contents are such that the final
vaccine contains less than 100 IU per single human dose,
2359
ADTP (ACELLULAR COMPONENT) AND INACTIVATED POLIOMYELITIS VACCINE
the components of the combined vaccine ,or because of the

vaccine and the test vacCine, a batch of combined vaccine
process used for the batch tested in cliniCal trials is necessary.
(Acellular Component, Adsorbed) and Poliomyelitis Vaccine
(Inactivated).
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption onto a mineral
carrier such as aluminium hydroxide or hydrated aluminium
phosphate, separately or together, of suitable quantities of
bulk purified diphtheria toxoid, tetanus toxoid, acellular
pertussis components and admixture of suitable quantities of
purified monovalent harvests of human polioviruses 1,2 and
3 or a suitable quantity of a trivalent pool of such purified
monovalent harvests. Suitable antimicrobial preservatives may
be added.
after virus harvest and before addition ofthe adsorbent in the
preparation of the final bulle vaccine, the amount of bovine
serum albumin is such that the content in the final vaccine will
be not more than 50 ng per single human dose.
Antimicrobial preservative, Where applicable, determine the
IP 2010
components have been carried out with satisfactory results

on the final bulle vaccine, they may be omitted on the final lot.
Provided the free formaldehyde content has been determined
on the bulk purified antigens or on the final bulle and it has
been shown that the content in the final lot will not exceed
0.2 gil, the test for free formaldehyde may be omitted on the
final lot.
Provided that the determination ofD-antigen content has been
carried out with satisfactory results during preparation ofthe
final bulk before addition ofthe adsorbent, it may be omitted
on the final lot.
has been carried out with satisfactory results on the final bulk
Identification
A. Diphtheria toxoid is identified by a suitable immunochemical
wlv solution. Maintain at 37 for about 16 h and centrifuge
obtained as described in Identification test A reacts with a
immunochemical method (2.2.14). The following method,
clear supernatant obtained as described in Identification test
A reacts with a specific antisera to the pertussis components
of the vaccine.

D. The vaccine is shown to contain human polioviruses 1,2

and 3 by a suitable immunochemical method (2.2.14) such as
the determination of D-antigen by enzyme-linked immunosorbent assay (ELISA).
FINAL LOT
Tests
Absence of residual pertussis toxin and irreversibility

of pertussis toxoid
Provided the tests for absence of residual pertussis toxin,

irreversibility ofpertussis toxoid and antimicrobial preservative
and the assays for the diphtheria, tetanus and pertussis
This test is not necessaryfor theproc/uct obtained by genetic

modification. Use 3 groups each of not less than 5 histaminesensitive mice. Inject intraperitoneally into each mouse ofthe
first group twiCe the single human dose ofthe vaccine stored
2360
IP 2010
ADSOR)3ED DIPHTHERIA, TETANUS, PERTUSSIS AND POLIOMYELITIS (INACTIVATED) VACCINE

test if, in both of the groups given the vaccine, not more than
5.0 per cent ofthe total number ofmice die following histamine
challenge.
at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in
strain is suitable ifmore than 50.0 per cent of the animals are
sensitised by 50 ng ofpertussis toxin and none ofthe control
Aluminium (2.3.9). Maximum 1.25 mg per single human dose

Assay
Pertussis component
The vaccine complies with the assay as stated under Adsorbed
D-antigen content
As a measure of consistency of production, determine the Dantigen content for human polioviruses 1,2 and 3 by a suitable
immunochemical method (2.2.14) following desorption using
a reference preparation calibrated in units of D-antigen. For
each type, the content, expressed with reference to the amount
ofD-antigen stated on the label; is within the limits approved
for the particular product. Poliomyelitis vaccine (inactivated)
reference preparation is calibrated in Units and intended for
use in the assay ofD-antigen. The Unit and the International
Unit are equivalent.
In vivo test
Inactivated Poliomyelitis Vaccine.
Labelling. The label complies with the requirements stated
under Vaccine and also states (1) the minimum number of
components per single human dose; (3) the nominal amount
of poliovirus of each type (1, 2 and 3), expressed in units of
D-antigen per single human dose; (4) the type of cells used
for production ofthe poliomyelitis component; (5) the number
ofmicrograms ofPRP per single human dose; (6) the type and
nominal amount of carrier protein per single human dose;
(7) where applicable, that the vaccine is intended for primary
and the amount ofthe adsorbent; (9) that the vaccine must be
potency stated on the label is 30 ill per single human dose.
Tetanus component

Pertussis and Poliomyelitis
(Inactivated) Vaccine
Diphtheria, Tetanus, Pertussis and Poliomyelitis (Inactivated)
Vaccine (Adsorbed) is a combined vaccine containing:
diphtheria formol toxoid; tetanus formol toxoid; an inactivated
suspension of Bordetella pertussis; suitable strains of human
polioviruses 1, 2 and 3 grown in suitable cell cultures and
inactivated by a validated method; mineral adsorbent such
as aluminium hydroxide or hydrated aluminium phosphate.
2361
ADSORBED DIPHTHERIA, TETANUS, PERTUSSIS AND POLIOMYELITIS (INACTIVATED) VACCINE

the growth of COlynebacterium diphtheriae and qostridium
'
Production
IP 2010
suspension of B. pertussis and purified monovalent harvests

of human polioviruses 1,2 and 3 or a suitable quantity of a
trivalent pool of such purified monovalent harvests. Suitable
General provisions
for specific toxicity ofthe diphtheria and tetanus components
: inject subcutaneously 5 times the single human dose stated
with any material that will interfere with the test. If within 42
test.
Specific toxicity
Use not less than 5 healthy mice each weighing between
14 and 16 g for the vaccine group and for the saline control.
Use mice ofthe same sex or distribute males and females equally
between the groups. Allow the animals access to food and
water for at least 2 hours before injection and durIng the test.
Inject each mouse ofthe vaccine group intraperitoneally with
0.5 ml, containing a quantity of the vaccine equivalent to not
less than halfthe single human dose. Inject each mouse ofthe
control group with 0.5 ml of a 0.9 per cent sterile solution of
sodium chloride, preferably containing the same amount of
antimicrobial preservative as that injected with the vaccine.
Weigh the groups of mice immediately before the injection
and 72 hours and 7 days after the injection. The vaccine
complies with the test if: (a) at the end of 72 hours the total
mass of the group of vaccinated mice is not less than that
preceding the injection; (b) at the end of 7 days the average
increase in mass per vaccinated mouse' is not less than
60 per cent of that per control mouse; and (c) not more than
5 per cent of the vaccinated mice die during the test. The test
may be repeated and the results of the tests combined.
during preparation of the final bulle vaccine; before addition
ofthe adsorbent, the amount ofbovine serum albumin is such
that the content in the final vaccine will be not more than 50
ng per single human dose.

(Adsorbed) and Poliomyelitis Vaccine (Inactivated).
FINAL LOT
FINAL BULK VACCINE
for use.
ThefinaLbulkvaccinejsprepared.by adsorption onto. amineral

Qarril:lr. such as ah,uninium hydroxide or hyclnl.ted aluminium
phosphate, separately or together, of suitable quantities of
bulle purified diphtheria toxoid and bulle purified tetanus toxoid
and admixture of suitable quantities of an inactivated
Provided thatthe tests for specifictoxicity and antimicrobial

preservative, and the assays focthediphtheria,Jeta.l1usa.l1cl
pertussis components have been carried out with satisfactory
results on the final bulle vaccine, they may be omitted on the
final lot.
2362
IP 2010
ADSORBED DIPHTHERIA, TETANUS, PERTUSSIS AND POLIOMYELITIS (INACTIVATED) VACCINE

determined on the bulk purified antigens, the inactivated
B. pertussis suspension and the purified monov~lent harvests
or the trivalent pool ofpolioviruses or on the final bulle and it
has been shown that the content in the final lot will not exceed
final lot.
has been carried out with satisfactory ~esults on the final bulle
Identification
examination sufficient sodium citrate to give aiD per cent
wIv solution. Maintain at 37 for about 16 hours and centrifuge
Assay
Tetanus component
If the test is carried out in guinea pigs, the lower confidence
limit (P = 0.95) ofthe estimated potency is not less than 40 IU
per single human dose; if the test is carried out in mice, the
lower confidence limit (P = 0.95) ofthe estimated potency is
not less than 60 IU per single human dose.
Pertussis component
Carry out the assay as stated under Pertussis Vaccine.
The estimated potency is not less than 4 IU per single human
dose and the lower confidence limit (P = 0.95) ofthe estimated
potency is not less than 2 IU per single human dose.
B. Tetanus toxoid is identified by a suitable immunochemical Poliomyelitis component

vaccines, is given as an example. The clear supernatant D-antigen content
.As a measure of consistency of production, determine the Dtetanus antitoxin, giving a precipitate.
antigen content for human polioviruses 1,2 and 3 by a suitable
C. The centrifugation residue obtained in identification A may immunochemical method (2.2.14) using a reference preparation
be used. Other suitable methods for separating the bacteria calibrated in Units ofD-antigen. For each type, the content,
from the adsorbent may also be used. Identify pertussis expressed with reference to the amount of D-antigen stated
vaccine by agglutination ofthe bacteria from the resuspended on the label, is within the limits approved for the particular
precipitate by antisera specific to B. pertussis or by the assay product. Poliomyelitis vaccine (inactivated) refei-ence
of the pertussis component prescribed under Assay.
preparation is calibrated in Units and is intended for use in
the assay ofD-antigen. The Unit and the IU are equivalent.
D. The vaccine is shown to contain humanpolioviruses 1,2
and 3 by a suitable immunochemical method (2.2.14) such as In vivo test
the determination ofD-antigen by enzyme-linked immunoThe vaccine complies with the in vivo assay as stated under
sorbent assay (ELISA).
Poliomyelitis Vaccine (Inactivated).
Tests
Antimicrohial preservative. Where applicable, determine the

human dose; (2) the minimum number ofInternational Units
of pertussis vaccine per single human dose; (3) the nominal
amount of poliovirus of each type (1, 2 and 3), expressed in
units ofD-antigen per single human dose; (4) the type ofcells
used for production ofthe poliomyelitis component; (5) where
applicable, that the vaccine is intended for primary vaccination
'ofchildren and is not necessarily suitable for reinforcing doses
or for administration to adults; (6) the name and the amount of
the adsorbent; (7) that the vaccine must be shaken before
use; (8) that the vaccine is not to be frozen.
2363
ADTP, POLIOMYELITIS (INACTIVATED) AND HAEMOPHILUS TYPE b CONJUGATE VACCINE
IF 2010
As part of consistency studies the assays of the diphtheria,

tetanus, pertussis and poliomyelitis components are carried

Pertussis, Poliomyelitis (Inactivated)
and Haemophilus Type b Conjugate
Vaccine
Diphtheria, Tetanus, Pertussis, Poliomyelitis (Inactivated) and
Haemophilus Type b Conjugate Vaccine (Adsorbed) is a
combined vaccine composed of: diphtheria formol toxoid;
tetanus formol toxoid; an inactivated suspension ofBordetella
pertussis; suitable strains of human polioviruses 1, 2 and 3
grown in suitable cell cultures and inactivated by a suitable
method; polyribosylribitol phosphate (PRP) covalently bound
to a carrier protein; a mineral adsorbent such as aluminium
hydroxide or hydrated aluminium phosphate. The product is
presented with the haemophilus component in a separate
container, the contents of which are mixed with the other
components immediately before use.
The formo1 toxoids are prepared from the toxins produced by
3 - ~-D-ri bofuranosy 1- (1 ~ 1)-ri bi to 1- 5 -phosphate
[(CIOH1Pll)n]' with a defined molecular size and derived from
a suitable strain of Haemophilus injluenzae type b. The carrier
protein, when conjugated to PRP, is capable of inducing a Tcell-dependent B-ce1l immune response to the polysaccharide.
Production of components
(Adsorbed), Poliomyelitis Vaccine (Inactivated) and
Haemophilus Type b Conjugate Vaccine.
FINAL BULK VACCINE
Production
General provisions
for specific toxicity ofthe diphtheria and tetanus components:
on the label into each of 5 healthy guinea-pigs, each weighing
test.
During development studies and wherever revalidation is
toPRP.
The final bulk of the diphtheria, tetanus, pertussis and

poliomyelitis components is prepared by adsorption,
separately or together, of suitable quantities of bulk purified
diphtheria toxoid, and bulk purified tetanus toxoid onto a
mineral carrier such as aluminium hydroxide or hydrated
aluminium phosphate and admixture of suitable quantities of
an inactivated suspension of B. pertussis and of purified,
monovalent harvests of human polioviruses 1, 2 and 3 or a
suitable quantity of a trivalent pool of such monovalent
harvests. Suitable antimicrobial preservatives may be added.
The final bulk ofthe haemophilus component is prepared by
dilution ofthe bulle conjugate to the final concentration with a
suitable diluent. A stabiliser may be added.
Only final bulk that complies with the following requirements
may be used in the preparation of the final lot.
Specific toxicity
Use not less than 5 healthy mice each weighing between 14
andJ 6-i~, for the-.Yaccine grQ!JP andJ.QLthe salin(;t~QIlJ:rol.JJse
Illice()f t~~saIlle s~x ()~.<ii.str:i1:>llte I118cles al1clfeIll~les .eqll~llY
water for at least 2 hours before injection and during the test.
2364
IP 2010
ADTP, POLIOMYELITIS (INACTIVATED) AND HAEMOPHILUS TYPE b CONJUGATE VACCINE

control group with 0.5 ml of a 0.9 per cent sterile solution of
sodium chloride, preferably containing the same amount of
antimicrobial preservative as that injected with the vaccine.
Weigh the groups of mice immediately before the injection
and 72 hours and 7 days after the injection. The vaccine
complies with the test if (a) at the end of 72 hours the total
mass of the group of vaccinated mice is not less than that
preceding the injection; (b) at the end of 7 days the average
increase in mass per vaccinated mouse is not less than
60 per cent of that per control mouse; and (c) not more than
5 per cent of the vaccinated mice die during the test. The test
may be repeated and the results of the tests combined.
during preparation of the final bulle vaccine, before addition
ofthe adsorbent, the amount ofbovine serum albumin is such
that the content in the final vaccine will not be more than
50 ng per single human dose.
FINAL LOT
The final bulle ofthe haemophilus component is freeze-dried.
FreePRP
Unbound PRP is determined on the haemophilus component
after removal ofthe conjugate, for example by anion-exchange,
size-exclusion or hydrophobic chromatography (2.4.16),
ultrafiltration or other validated methods. The amount offree
PRP is not greater than that approved for the particular product.
Identification
Identification tests A, B, C and D are carried out using the
vial containing the diphtheria, tetanus, pertussis and
poliomyelitis components; identification test E is carried
out on the vial containing the haemophilus component.
A. Diphtheria toxoid is identified by a suitable immunochernical
vaccines, is given as an example. The clear sup ern a tan t
C. The centrifugationresidue obtained in identification A may
be used. Other suitable methods for separating the bacteria
from the adsorbent may also be used. Identify pertussis
vaccine by agglutination ofthe bacteria from the resuspended
precipitate by antisera specific to B. pertussis or by the assay
of the pertussis component prescribed under Assay.
D. The vaccine is shown to contain human polioviruses 1,2
and 3 by a suitable immunochemical method (2.2.14), such as
determination ofD-antigenby enzyme linked immunosorbent
assay (ELISA).
Provided that the tests for specific toxicity and antimicrobial

preservative, and the assays for the diphtheria, tetanus and
pertussis components have been carried out with satisfactory
final lot.
E. The haemophilus component is identified by a suitable

immunochemical method (2.2.14) for PRP.

determined on the bulle purified antigens, the inactivated B.
pertussis suspension and. the purified monovalent harvests
or the trivalent pool ofpolioviruses or on the final bulle and it
has been shown that the content in the final lot will not exceed
final lot.
The tests for speCific toxicity, aluminium, free formaldehyde,

antimicrobial preservative and sterility are carried out on
the container with diphtheria, tetanus, pertussis and
poliomyelitis components; the tests for PRP content, wate/;
sterility and pyrogens are carried out on the container with
the haemophilus component.

Tests
Some tests for the haemophilus component may be carried

out on the freeze-dried product rather than on the bulk
conjugate where theji-eeze-drying process may affect the
component under test.
the label. PRP is determined either by assay ofribose (2.7.1) or
2365
ADTP, POLIOMYELITIS (INACTIVATED) AND HAEMOPHILUS TYPE b CONJUGATE VACCINE9
phosphorus (2.7.1), by an immunochemical method (2.2.14)or

by anion-exchange liquid chromatography with pulsedamperometric detection.
if aluminiUlnhydroxide or hydrated aluminiumphosphate is
.
Water (2.3.43). Maximum 3.0 per cent for the haemophilus
component.
Sterility (2.2. n). Complies with the test for sterility.
per kg ofthe rabbit's mass a quantity ofthe vaccine equivalent
to 1 mg ofPRP for a vaccine with diphtheria toxoid or CRM
197 diphtheria protein as carrier; 0.1 mg ofPRP for a vaccine
with OMP as carrier.
Assay
IP 2010
expressed with reference to the amount of D-antigenstated

on the label, is within the limits approved for the particular
product. Poliomyelitis vaccine (inactivated) reference
preparation is calibrated in Units and intended for use in the
assay of D-antigen. The Unit and the ill are equivalent.
In vivo test
Poliomyelitis Vaccine (Inactivated).
human dose; (2) the minimum number ofIntemational Units
of pertussis vaccine per single human dose; (3) the nominal
amount of poliovirus of each type (1, 2 and 3), expressed in
UnitsofD-antigen per single human dose; (4) the type ofcells
used for production of the poliomyelitis component; (5) the
number ofmicrograms ofPRP per single human dose; (6) the
type and nominal amount of carrier protein per single human
dose; (7) where applicable, that the vaccine is intended for
primary vaccination ofchildren and is not necessarily suitable
for reinforcing doses or for administration to adults; (8) the
name and the amount of the adsorbent; (9) that the vaccine
must be shaken before use; (10) that the vaccine is not to be
frozen.
Adsorbed Pertu.ssis Vaccine (Acellu.lar

Component)
Pertussis Vaccine (Acellular Component, Adsorbed) is a

preparation of individually prepared and purified antigenic
components of Bordetella pertussis adsorbed on a mineral
carrier such as aluminium hydroxide or hydrated aluminium
phosphate.
The vaccine contains either pertussis toxoid or a pertussis
toxin, like protein free from toxic properties, produced by
method that renders the latter harmless while maintaining
haemagglutinin, pertactin (a 691cDa outer-membrane protein)
D-antigen content
Production
As a Illeasure of consistency of productio.n,deterIl1in~t~eDantigen content for human polioviruses 1,2 and 3 by a suitable
immunochemical method (2.2..1 4) using a reference preparation
calibrated in Units of D-antigen. For each type, the content,

Tetanus component
stated under Tetanus. Vaccine (Adsorbed).
If the test is carried out in guinea-pigs, the lower confidence
limit (P = 0.95) ofthe estimated potency is not less than 40 ill
per single human dose; if the test is carried out in mice, the
lower confidence limit (P = 0.95) ofthe estimated potency is
not less than 60 ill per single human dose.
Pertussis component
Carry o.ut the assay as stated under Pertussis Vaccine.
The estimated potency is not less than 4 ill per single human
dose and the lower confidence limit (P = 0.95) ofthe estimated
potency is not less than 2 IU per single human dose.
2366
IP 2010
ADSORBED PERTUSSIS VACCINE (ACELLULAR COMPONENT)
Reference vaccine
PURIFIED COMPONENTS
A batch ofvaccine shown to be effective in clinical trials or a

batch representative thereof is used as a reference vaccine.
For the preparation of a representative batch, strict adherence
to the production process used for the batch tested in clinical
trials is necessary. The reference vaccine is preferably
stabilised by a method that has been shown to have no
significant effect on the assay procedure when the stabilised
and non-stabilised batches are compared.
Production of each component is based on a seed-lot system.

The seed cultures from which toxin is prepared are managed
to conserve or where necessary restore toxinogenicity by
deliberate selection.
CHARACTERISATION OF COMPONENTS
During development of the vaccine, the production process
shall be validated to demonstrate that it yields consistently
individual components that comply with the following
requirements; after demonstration of consistency, the tests
need not be applied routinely to each batch.
Adenylate cyclase. Not more than 500 ng in the equivalent of
1 dose ofthe final vaccine, determined by immunoblot analysis
or another suitable method.
Tracheal cytotoxin. Not more than 2 pmol in the equivalent of
1 dose of the final vaccine, determined by a suitable method
such as a biological assay or liquid chromatography (2.4.14).
Absence ofresidual dermonecrotic toxin. Inject intradennally
into each of 3 unweaned mice, in a volume of 0.1 ml, the
amount of component or antigenic fraction equivalent to 1
dose of the final vaccine. Observe for 48 hours. No
dermonecrotic reaction is demonstrable.
Specific properties. The components of the vaccine are
analysed by one or more of the methods shown below in
order to detelmine their identity and specific properties (activity
per unit amount of protein) in comparison with reference
preparations.
Pertussis toxin
Chinese hamster ovary (CHO) cell-clustering effect and
haemagglutination as in vitro methods; lymphocytosispromoting activity, histamine-sensitising activity and insulin
secretory activity as in vivo methods. The toxin shows ADPribosyl transferase activity using transducin as the acceptor.
None of the media used at any stage contains blood or blood

products of human origin. Media used for the preparation of
seed lots and inocula may contain blood or blood products of
animal origin.
Pertussis toxin and, where applicable, filamentous
haemagglutinin and pertactin are purified and, after appropriate
characterisation, detoxified using suitable chemical reagents,
by a method that avoids reversion of the toxoid to toxin,
particularly on storage or exposure to heat. Other components
such as fimbrial-2 and fimbrial-3 antigens are purified either
separately or together, characterised and shown to be free
from toxic substances. The purification procedure is validated
to demonstrate appropriate clearance of substances used
during culture or purification.
The content of bacterial endotoxins is determined to monitor
vaccine. The limits applied for the individual components are
such that the final vaccine contains less than 100 IU per single
human dose.
Before detoxification, the purity of the components is
determined by a suitable method such as polyacrylamide gel
electrophoresis (pAGE) or liquid chromatography. SDS-PAGE
or irnmunoblot analysis with specific monoclonal or polyclonal
antibodies may be used to characterise subunits. Requirements
are established for each individual product.
Only purified components that comply with the following
requirements may be used in the preparation of the final bulle
vaccine.
Sterility (2.2.11). Carry out the test for sterility using for each
medium a quantity of purified component equivalent to not
less than 100 doses.
Absence of residual pertussis toxin
Pertussis toxoid
This test is not necessmyfor the product obtained by genetic

modification. Use a group of not fewer than 5 histaminese;nsitive mice each weighing between 18 and 26 g. Inject into
each mouse the equivalent of 1 human dose intravenously or
twice the human dose intraperitoneally, diluted to not more
than 0.5 ml with phosphate-bufferedsaline solution containing
0.2 per cent w/v of gelatin. Inject diluent into a second group
of control mice. After 5 days, inject 2 mg of histamine base
intraperitoneally in a volume not exceeding 0.5 ml and observe
for 24 hours. Ifno animal dies, the preparation complies with
the test.
The toxoid induces in animals production of antibodies

capable of inhibiting all the properties of pertussis toxin.

at suitable intervals as follows: inject three-fold dilutions ofa
Filamentous haemalfglunnin
Haemagglutination and inhibition by specific antibody.
Pertactin, fimbrial-2 and fimbrial-3 antigens. Reactivity with
specific antibody.
2367
ADSORBED PERTUSSIS VACCINE (ACELLULAR COMPONENT)
reference pertussis toxin preparation in phosphate-buffered

saline solution containing 0.2 per cent w/v of gelatin and
challenge with histamine as above; the strain is suitable if
more than 50 per cent ofthe animals are sensitised by 50 ng of
pertussis toxin and none of the control animals injected with
only diluent and challenged similarly with histamine show
symptoms of sensitisation.
A validated test based on the clustering effect ofthe toxin for
Chinese hamster ovary (CHO) cells may be used instead of
the test on mice.
Residual detoxifying agents and other reagents

, The content ofresidual detoxifying agents and other reagents
is determined and shown to be below approved limits unless
validation of the process has demonstrated acceptable
clearance.
Antigen content
Determine the antigen content by a suitable immunochemical
method (2.2.14) and protein nitrogen by sulphuric acid
digestion (2.2.30) or another suitable method. The ratio of
antigen content to protein nitrogen is within the limits
established for the product.
FINAL BULK VACCINE
The vaccine is prepared by adsorption of suitable quantities
ofpurified components, separately or together, onto aluminium
hydroxide or hydrated aluminium phosphate. A suitable
antimicrobial preservative may be added.

FINAL LOT
Only a final lot that is satisfactory with respect to each of the
Assay may be released for use. Provided that the tests for
absence of residual pertussis toxin and irreversibility of
pertussis toxoid, antimicrobial preservative, free formaldehyde
and the assay have been carried out with satisfactory results
on the final bulk vaccine, these tests may be omitted on the
IP 2010
sufficient sodium citrate to give a 10 per cent w/v solution;

maintain at 37 for about 16 hours and centrifuge until a clear
supernatant liquid is obtained. Examined by a suitabl,e
immunochemical method (2.2.14), the clear supematant liquid
reacts with specific antisera to the components stated on the
label.
Tests
Absence of residual pertussis toxin and irreversibility of
pertussis toxoid
This test is not necessaryfor the product obtained by genetic

modification. Use 3 groups each ofnot fewer than 5 histaminesensitive mice. Inject intraperitoneally into the first group twice
the single human dose ofthe vaccine stored at 2 to 8. Inject
intraperitoneally into the second group twice the single human
dose ofthe vaccine incubated at 37 for 4 weeks. Inject diluent
into the third group of mice. After 5 days, inject into each
mouse 2 mg of histamine base intraperitoneally in a volume
not exceeding 0.5 ml and observe for 24 hours. The test is
invalid if 1 or more control mice die following histamine
challenge. The vaccine complies with the test ifno animal in
the first or second group dies following histamine challenge.
If 1 m.ouse dies in either or both ofthe first and second groups,
the test may be repeated with the same number ofmice or with
a greater number and the results of valid tests combined; the
vaccine complies with the test if, in both of the groups given
the vaccine, not more than 5.0 per cent ofthe total number of
mice die following histamine challenge.
phosphate-buffered saline solution c'ontaining 0.2 per cent
strain is suitable if more than 50.0 per cent ofthe animals are
sensitised by 50 ng of pertussis toxin and none of the control
with histamine show symptoms of sensitisation.

Identification
Assay
Subject the vaccine to a suitable desorption procedure such

as the following: dissolve in the vaccine under examination
The capacity ofthe vaccine to induce the formation ofspecific

antibodies is compared with the same capacity of a reference
2368
IP 2010
ADSORBED PERTUSSIS VACCINE (ACELLULAR, CO-PURIFIED)
preparation examined in parallel; antibodies are determined

using suitable immunochemical methods (2.2.14) such as
enzyme-linked immunosorbent assay (ELISA). The test on
mice shown below uses a three-point model but, after
validation, for routine testing a single-dilution method may be
used.
Requirement
The capacity to induce antibodies is not significantly
(P = 0.95) less than that of the reference vaccine.
The following test model is given as an example of a method
that has been found to be satisfactory.
Selection and distribution of test animals
Use in the test healthy mice (for example, CDI strain) of the
same stock 4 to 8 weeks old. Distribute the animals in 6 groups
of a number appropriate to the requirements ofthe assay. Use
3 dilutions ofthe vaccine under examination and 3 dilutions of
a reference preparation and attribute each dilution to a group
of mice. Inject intraperitoneally or subcutaneously into each
mouse 0.5 ml of the dilution attributed to its group.
Collection of serum samples
4 to 5 weeks after vaccination, bleed the mice individually
under anaesthesia. Store the sera at -20 until tested for
antibody content.
Antibody determination
Assay the individual sera for content of specific antibodies to
each component using a validated method such as the ELISA
test shown below.
ELISA
Microtitre plates (polyvinyl chloride or polystyrene as
appropriate for the specific antigen) are coated with the purified
antigen at a concentration of 100 ng per well. After washing,
unreacted sites are blocked by incubating with a solution of
bovine serum albumin and then washed. Two-fold dilutions
of sera from mice immunised with test or reference vaccines
are made on the plates. After incubation at 22 to 25 for
1 hour, the plates are washed. A suitable solution of antimouse IgG enzyme conjugate is added to each well and
incubated at 22 to 25 for 1 hour. After washing, a substrate is
added from which the bound enzyme conjugate liberates a
chromophore which can be quantified by measurement of
absorbance. The test conditions are designed to obtain a linear
response for absorbance with respect to antibody content
over the range of measurement used and absorbance values
within the range 0.1 to 2.0.
A reference antiserum of assigned potency is used in the test
and serves as the basis for calculation of the antibody levels
in test sera. A standardised control serum is also included in
the test.
The test is not valid if (a) the value found for the control
serum differs by more than 2 standard deviations from the
assigned value; (b) the confidence interval of the potency
estimate is greater than 50.0 per cent to 200.0 per cent.
Calculation
The antibody titres in the sera of mice immunised with
reference and test vaccines are calculated and from the values
obtained the potency of the test vaccine in relation to the
reference vaccine is calculated by the usual statistical methods.
LabeUing. The label states (1) the names and amounts of the
components present in the vaccine; (2) where applicable, that
the vaccine contains a pertussis toxin-like protein produced
by genetic modification; (3) the name and amount of the
adsorbent; (4) that the vaccine must be shaken before use;
(5) that the vaccine is not to be frozen.
Adsorbed Pertussis Vaccine (Acellular,

Co-Purified)
Pertussis Vaccine (Acellular, Co-Purified, Adsorbed) is a
preparation of antigenic components of Bordetella pertussis
adsorbed on a mineral carrier such as aluminium hydroxide or
hydrated aluminium phosphate.
The vaccine contains an antigenic fraction purified without
separation ofthe individual components. The antigenic fraction
is treated by a method that transfonns pertussis toxin to toxoid,
rendering it hannless while maintaining adequate immunogenic
properties of all the components and avoiding reversion to
toxin. The antigenic fraction is composed ofpertussis toxoid,
filamentous haemagglutinin, pertactin (a 69 kDa outermembrane protein) and other defined components of
B. pertussis such as fimbrial-2 and fimbrial-3 antigens. It may
contain residual pertussis toxin up to a maximum level
approved by the competent authority. The antigenic
composition and characteristics are based on evidence of
protection and freedom from unexpected reactions in the target
group for which the vaccine is intended.
Production
General provisions
Reference vaccine. A batch of vaccine shown to be effective
in clinical trials or a batch representative thereof is used as a
reference vaccine. For the prepar~tion of a representative
batch, strict adherence to the production process usedfor the
batch tested in clinical trials is necessary. The reference vaccine
is preferably stabilised, by a method that has been shown to
have no significant effect on the assay procedure when the
stabilised and non-stabilised batches are compared.
2369
ADSORBED PERTUSSIS VACCINE (ACELLULAR, CO-PURIFIED)

CHARACTERISATION OF COMPONENTS
During development of the vaccine, the production process

shall be validated to demonstrate that it yields consistently
an antigenic fraction that complies with the following
requirements; after demonstration of consistency, the tests
need not be applied routinely to each batch.
Adenylate cyclase. Not more than 500 ng in the equivalent of
I dose ofthefinal vaccine, determined by immunoblot analysis
or another suitable method.
Tracheal cytotoxin. Not more than 2 pmol in the equivalent of
1 dose of the final vaccine, determined by a suitable method
such as a biological assay or liquid chromatography (2.4,14).
Absence of residual dermonecrotic toxin. Inject intradermally
into each of3 unweaned mice, in a volumeofO.1 ml, the amount
of antigenic fraction equivalentto I dose ofthe final vaccine.
Observe for 48 hours. No dermonecrotic reaction is
demonstrable.
Specific properties. The antigenic fraction is analyzed by one
or more ofthe methods shown below in order to determine the
. identity and specific properties (activity per unit amount of
protein) of its components in comparison with reference
preparations.
Pertussis toxin
Chinese hamster ovary (CRO) cell-clustering effect and

haemagglutination as in vitro methods; lymphocytosispromoting activity, histamine-sensitising activity and insulin
secretory activity as in vivo methods. The toxin shows
ADP-ribosyl transferase activity using transducin as the
acceptor.
Filamentous haemagglutinin
Raemagglutination and inhibition by specific antibody.

Pertactin, fimbrial-2 and fimbrial-3 antigens. Reactivity with
specific antibody.
Pertussis toxoid
The toxoid induces in animals the production of antibodies

capable of inhibiting all the properties of pertussis toxin.
PURIFIED ANTIGENIC FRACTION
Production of the antigenic fraction is based on a seed-lot

system. The seed cultures are managed to conserve or, where
necessary, restore toxinogenicity by deliberate selection.
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_-,
None of the lTIedia used atally stage contains blood or blood

products of human origin. Media used for the preparation of
seed batches and inocula may contain blood or blood products
ofanimal origin.
IP 2010
The antigenic fraction is purified and, after appropriate

characterisation, detoxified using suitable reagents by a
method that ensures minimal reversion of toxoid to toxin,
particularly on or exposure to heat. The purification procedure
is validated to demonstrate appropriate clearance of
substances used during culture or purification.
The content of bacterial endotoxins is determined to monitor
vaccine. The limits applied are such that the final vaccine
contains not more than 100 ill per single human dose.
Before detoxification, the purity of the antigenic fraction is
determined by a suitable method such as polyacrylamide gel
electrophoresis (PAGE) (2.4.12) or liquid chromatography
(2.4.14). SDS-PAGE or immunoblot analysis with specific
monoclonal or polyclonal antibodies may be used to
characterise subunits. Requirements are established for each
individual product.
Only a purified antigenic fraction that complies with the
following requirements may be used in the preparation ofthe
final bulk vaccine.
medium a quantity ofpurified antigenic fraction equivalent to
not less than 100 doses of the final vaccine.
Test for residual pertussis toxin. Use 3 groups of not fewer
than 5 histamine-sensitive mice each weighing between 18
and 26 g. Using phosphate-buffered saline containing 0.2 per
cent of gelatin, prepare a series of dilutions of the purified
antigenic fraction that have been shown to yield a graded
response and attribute each dilution to a separate group of
mice. Inject intraperitoneally into each mouse the dilution
attributed to its group. Inject diluent into a fourth group of
control mice. After 5 days, inject intraperitoneally into each
mouse 1 mg of histamine base in a volume not exceeding
0.5 mL Record the number ofanimals that die within 24 hours
of histamine challenge. Calculate the weight or volume of a
preparation that sensitises 50.0 per cent ofthe mice injected
using a suitable statistical method such as probit analysis.
The residual activity of pertussis toxin does not exceed that
of batches shown to be safe in clinical studies.

at suitable intervals as follows: inject threefold dilutions of a
reference pertussis toxin preparation in phosphate-buffered
saline solution containing 0.2 per cent w/v of gelatin and
challenge with histamine as described above; the strain is
suitable if more than 50 per cent of the animals are sensitised
by 50 ng of pertussis toxin and none of the control animals
injected with only diluent and challenged similarly with
histalJlil1e showsymptoills of sellsitisation.
A validated test based on the clustering effect ofthe toxin for
Chinese hamster ovary (CRO) cells may be used instead of
the test on mice.
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IP 2010
BACILLUS CALMETTE-GUERIN VACCINE (FREEZE-DRIED)
Residual detoxifying agents and other reagents. The content

ofresidual detoxifYing agents and other reagents is determined
and shown to be below approved limits unless validation of
the process has demonstrated acceptable clearance.
saline containing 0.2 per cent w/v of gelatin, prepare a series

of dilutions of the vaccine under examination that have been
shown to yield a graded response and attribute each dilution
to a separate group of mice. Inject intraperitoneally into each
mouse the dilution attributed to its group. Inject diluent into
a fourth group of control mice. After 5 days, inject
intraperitoneally into each mouse 1 mg of histamine base in a
volume not exceeding 0.5 ml. Note the number ofanimals that
die within 24 hours ofhistamine challenge. Calculate the weight
or volume of a preparation that sensitises 50 per cent of the
mice injected using a suitable statistical method such as probit
analysis. The residual activity of pertussis toxin does not
exceed that of batches shown to be safe in clinical studies.
Antigen content. Determine the complete quantitative antigen

composition of the antigenic fraction by suitable
immunochemical methods (2.2.14) and protein nitrogen by
sulphuric acid digestion or another suitable method. The ratio
oftotal antigen content to protein nitrogen is within the limits
established for the product.
FINAL BULK VACCINE
The vaccine is prepared by adsorption of a suitable quantity
ofthe antigenic fraction onto aluminium hydroxide or hydrated
aluminium phosphate. A suitable antimicrobial preservative
may be added.
Sterility (2.2.11). Carry out test for sterility using 10 ml of bulle
Reversibility oftoxoid. Carry out the testfor residual pertussis

toxin described above using the vaccine incubated at 37for
4 weeks in parallel with a sample stored at 2 to 8. The degree
of reversibility does not exceed that of batches shown to be
safe in clinical studies.
method. The content is not less than 85.0 per cent andnot
Aluminium (2.3 .9). Maximum 1.25 mg per single human dose,
FINAL LOT

Provided that the tests for residual pertussis toxin, reversibility
of toxoid, antimicrobial preservative, free formaldehyde and
the assay have been carried out with satisfactory results on
the final bulle vaccine, these tests may be omitted on the final
lot.
Identification
Subject the vaccine to a suitable desorption procedure such
as the following: dissolve in the vaccine under examination
sufficient sodium citrate to give a 10 per cent w/v solution;
maintain at 37 for about 16 hours and centrifuge until a clear
supernatant is obtained. Examine by a suitable immunochemical
method (2.2.14), the clear supernatant reacts with specific
antisera to the components in the vaccine.
Tests
Test for residual pertussis toxin. Use 3 groups of not fewer
than 5 histamine-sensitive mice (see under Production) each
weighing between 18 and 26 g. Using phosphate-buffered
Assay
The vaccine complies with the assay as stated underAdsorbed
Labelling. The label states (1) the names and amounts of the
antigenic components present in the vaccine; (2) the maximum
amount of residual pertussis toxin present in the vaccine;
(3) the maximum degree ofreversion oftoxoid to toxin during
the period of validity; (4) the name and amount of the
adsorbent; (5) that the vaccine must be shaken before use; (6)
that the vaccine is not to be frozen.
Bacillus Calmette-Guerin Vaccine

(Freeze-Dried)
Freeze-dried BCG Vaccine is a preparation of live bacteria
derived from a culture of the bacillus ofCalmette and Guerin
(Mycobacterium bovis BCG) capacity of which to protect
against tUberculosis has been established.
Vaccine complies with the requirements stated under Vaccines
with the following modifications.
2371
IP 2010
Production
Identification
General provisions
The bacteria in the working seed lot are identified as

Mycobacterium bovis BCG using microbiological techniques,
which may be supplemented by molecular biology techniques
(for example, nucleic acid amplification and restrictionfragment-length polymorphism).

product, if tested, would comply with the tests for safety and
efficacy.
BCG vaccine shall be produced by a staffconsisting ofhealthy
persons who do not work with other infectious agents; in
particular they shall not work with virulent strains of
Mycobacterium tuberculosis, during the course ofproduction
cycle nor shall they be exposed to a known risk oftuberculosis
infection. BCG vaccine is susceptible to sunlight: the
procedures for the preparation of the vaccine shall be so
designed that all cultures and vaccines are protected from
direct sunlight and from ultraviolet light at all stages of
manufacture, testing and storage.
Sterility (2.2.11). Complies with the test for sterility, carried

out using 10 ml for each medium. The working seed lot
complies with the test for sterility exceptfor the presence of
mycobacteria.
Production of the vaccine is based on a seed-lot system. The

production method shall have been shown to yield
consistently B.CG vaccines that induce adequate sensitivity
to tuberculin in man, that have acceptable protective potency
in animals and are safe. The vaccine is prepared from cultures
which are derived from the master seed lot by as few
subcultures as possible and in any case not more than 12
subcultures e.g. Ifthe secondary seed lot is 4 culture passages
removed from the primmy seed lot, the number of passages
from the secondary seed lot must not exceed 8.
The bacteria are grown in a suitable medium for not more than
21 days by surface or submerged culture. The culture medium
shall contain no substances known to cause toxic or allergic
reactions in human beings or to cause the bacteria to becol11e
virulent for guinea-pigs. The culture is harvested and
suspended in a sterile liquid medium that protects the viability
of the vaccine as determined by a suitable method of viable
count.
The capacity of the working seed lot to induce sensitivity to

tuberculin in guinea-pigs is demonstrated.
Ifa bioluminescence test or other biochemical method is used
instead of viable count, the method is validated against the
viable count for each stage of the process at which it is used.
Virulent mycobacteria
Examine the working seed lot as prescribed under Tests, using
10 guinea pigs.
PROPAGATION AND HARVEST
Test for purity. Purity is checked by acid fast staining.

FINAL BULK VACCINE
The final bulle vaccine is prepared from a single harvest or by
pooling a number of single harvests. A stabiliser may be
added; if the stabiliser interferes with the determination of
bacterial concentration on the final bulk vaccine, the
determination is carried out before addition of the stabiliser.
Only final bulk vaccine that complies with the following
SEED LOT
The strain used to establish the master seed lot is chosen for
and maintained to preserve its stability, its capacity to sensitise
man and guinea-pigs to tuberculin and to protect animals
against tuberculosis, and its relative absence ofpathogenicity
for man and laboratory animals. The strain used shall be
identified by historical records that include information on its
origin and subsequent manipulation.
A suitable batch ofvaccine is prepared from the first working
seed lot and is reserved for use as the comparison! in-house
reference vaccine. When a new working seed lot is
established, a suitable test for delayed hypersensitivity in
guinea-pigs is carried out on a batch ofvaccine prepared from
thenewwofk.ingseealot; th-vac-cinis snown fo -be not

significantly different in activity from. the com.parison vaccine.
Only a working seed lot that complies with the following
requirements may be used for propagation.
Virulent mycobacteria. Examine as prescribed under Tests.

Sterility (2.2.11). Complies with the test for sterility using
10 ml for each medium except for the presence ofmycobacteria.
Count of viable units
Determine the number of viable units per ml by viable count
on solid medium using a method suitable for the vaccine under
examination or by determination of adenosine triphosphate
by a bioluminescence reaction. Carry out the test in parallel
on a reference preparation of the same strain.
Bacterial concentration
Determine the total bacterial concentration by a suitable
method, either directly by determining the mass ofthe microorganisms, or indirectly by an opacity method that has been
2372
IP 2010
calibrated in relation to the mass of the organisms; if the

bacterial concentration is determined before addition of a
stabiliser, the concentration in the final bulk vaccine is
established by calculation. The total bacterial concentration
is within the limits approved for the particular product by
National Regulatory Authority.
Inject subcutaneously or intramuscularly into each of6 guineapigs,each weighing between 250 and 400 g and having received
no treatment likely to interfere with the test, a quantity of
vaccine equivalent to at least 50 human doses. Observe the
animals for at least 42 days. At the end of this period, kill the
guinea-pigs and examine by autopsy for signs of infection
with tuberculosis, ignoring any minor reactions at the site of
injection. Animals that die during the observation period are
also examined for signs oftuberculosis. The vaccine complies
with the test if none of the guinea-pigs shows signs of
tuberculosis and if not more than one animal dies during the
observation period. If 2 animals die during this period and
autopsy does not reveal signs of tuberculo~is repeat the test
on 6 other guinea-pigs. The vaccine complies with the test if
not more than one animal dies during the 42 days following
the injection and autopsy does not reveal any sign of
tuberculosis.
The ratio of the count of viable units to the total bacterial

concentration is not less than that approved for the particular
product by National Regulatory Authority.
FINAL LOT
The final bulle vaccine is distributed into sterile containers
and freeze-dried to a moisture content favourable to the
stability ofthe vaccine; the containers are closed either under
vacuum or under a gas that is not deleterious to the vaccine.
Except where the filled and closed containers are stored at a
temperature of_20 0 or lower, the expiry date is not later than 4
years from the date of harvest.
Only a final lot that complies with the following requirement
for count of viable units and with each of the requirements
given below under Identification, Tests and Assay may be
released for use. Provided the test for virulent mycobacteria
vaccine, it may be omitted on the final lot. Provided the test
for excessive dermal reactivity has been carried out with
satisfactory results on the working seed lot and on 5
consecutive final lots produced from it, the test may be omitted
on the final lot.
Sterility (2.2.11). The reconstituted vaccine complies with the

test for sterility except for the presence of mycobacteria.
Excessive dermal
reacti~ity
Use 6 healthy white or pale-coloured guinea-pigs, each

weighing not less than 250 g and having received no treatment
likely to interfere with the test. Inject intradennally into each
guinea-pig, according to a randomised plan, 0.1 ml of the
reconstituted vaccine and of 2 tenfold serial dilutions of the
vaccine and identical doses of the comparison vaccine.
Observe the lesions formed at the site of the injection for 4
weeks. The vaccine complies with the test if the reaction it
produces is not markedly different froin that produced by the
comparison vaccine.
Count of viable units

Determine the number of viable units per ml of the
reconstituted vaccine by viable count on solid medium using
a method suitable for the vaccine under examination or by
determination ofadenosine triphosphate by a bioluminescence
reaction. The ratio of the count of viable units after freezedrying to that before is not less than that approved for the
particular product.
Identification
BCGvaccine is identified by microscopic examination ofthe
bacilli in stained smears demonstrating their acid-fast property
and by the characteristic appearance of colonies grown on
solid medium. Alternatively, molecular biology techniques (like
nucleic acid amplification) may be used.
Tests
Virulent mycobacteria
Ifthis test is satisfactory at final bulle stage it can be omitted at
the final lot.
Temperature stability
Maintain samples ofthe freeze-dried vaccine at 37 0 for 4 weeks.
Determine the number of viable units in the heated vaccine
and in unheated vaccine as described below. The number of
viable units in the heated vaccine is not less than 20.0 per cent
of that in unheated vaccine.
Water (2.3.43). Not more than 3.0 per cent, determined by the
semi-micro determination ofwater.
Assay
Determine the number of viable units in the reconstituted
vaccine by viable count on solid medium or using a suitable
validated biochemical method for the vaccine under
examination. The number is within the range stated on the
label. Determine the number ofviable units in the comparison
vaccine in parallel.
Labelling. The label states (l) the minimum and maximum
number of viable units per ml in the reconstituted vaccine;
(2) that the vaccine must be protected from direct sunlight;
2373
IP 2010
DIPHTHERIA ANTITOXIN
(3) that the vaccine is to be used immediately after broaching

the container; (4) the age group for which the vaccine is
intended; (5) the dose for each age group; (6) follow
instructions as mentioned in the product insert/leaflet.
Diphtheria Antitoxin
Diphtheria Antitoxin is a preparation containing the specific
antitoxic globulins or their derivatives obtained by purification
of hyperimmune serum or plasma of healthy horses or other
suitable animals and having the specific activity ofneutralising
the toxin fonned by Corynebacterium diphtheriae. The liquid
preparation may contain a suitable antimicrobial preservative.
Diphtheria Antitoxin has a potency ofnot less than 1000 Units
per ml when obtained from horse serum and not less than 500
Units per ml when obtained from other animals.
Description. A clear, colourless or pale yellow liquid or a freezedried, cream-coloured powder or pellet.
Identification
Specifically neutralises and renders the toxin fonned by C.
diphtheriae hannless to susceptible animals or by any other
suitable in-vitro test.
Tests
Potency. Carry out the biological assay ofdiphtheria antitoxin
described below.
Biological Assay of Diphtheria Antitoxin

The potency of diphtheria an.titoxin is determined by
comparing the dose necessary to protect guinea-pigs or
rabbits against the erythrogenic effects of a fixed dose of the
Standard Preparation ofdiphtheria antitoxin necessary to give
the same protection. For this purpose, a suitable preparation
of diphtheria toxin is required to be used as a test toxin. The
test dose ofthe toxin is detennined in relation to the Standard
Preparation. The potency ofthe preparation under examination
is then detennined in relation to the Standard Preparation using
the test toxin.
The Standard Preparation is the 1st International Standard for
Diphtheria antitoxin, equine, established in 1934, consisting
ofthe dried hyperimmune horse serum and glycerin, or another
suitable preparation the potency ofwhich has been detennined
in relation to the International Standard.
Suggested Method
Test toxin. Prepare diphtheria toxin by filtering through
bacteria-prooffilter the medium in which a toxigenic strain of
C. diphtheriae has grown. Store at a temperature of 2 and 8.
Selection of test toxin.. In selecting a toxin for use as the test

toxin detennine the following.
Lr/l00 dose -This is the smallest quantity ofthe toxin which,

when mixed with 0.01 Unit of antitoxin and injected
intracutaneously into guinea-pigs or rabbits causes a
characteristic reaction at the site of the injection within 48
hours.
Minimal reacting dose - This is the smallest quantity of
toxin which, when injected intracutaneously into guinea-pigs
or rabbits, causes a characteristic reaction at the site of
injection within 48 hours.
A suitable toxin is one which contains at least 200 minimal
reacting doses in the Lrll 00 dose. The test toxin is allowed to
stand for some months before being used for the assay of
samples ofantitoxin. During this time its toxicity declines and
the Lrll 00 dose may be slightly increased. When experiment
shows that the Lrll 00 dose is constant, the test toxin is ready
for use and may be used for a long period. Determine the
minimal reacting dose and the Lrll 00 dose at frequent intervals:
Store the test toxin in the dark at a temperature between 0 and
50 . Maintain its sterility by the addition of toluene or other
antimicrobial preservative which does not cause a rapid decline
in specific toxicity.
Determination oftest dose oftoxin [Lr/l00 dose). Prepare a
solution ofthe Standard Preparation with saline solution such
that 1 rnl contains 0.1 Unit. Prepare mixtures such that 2.0 ml of
each mixture contains 1.0 ml of the dilution of the Standard
Preparation (0.1 Unit) and one of a series of graded volumes
of the test toxin. Dilute each mixture with saline solution to
the same final volume (2.0 ml). Allow the mixtures to stand at
room temperature, protected from light, for 15 to 60 minutes
and inject intracutaneously 0.2 ml of each mixture at suitably
spaced sites into the shaven or depilated flanks oftwo animals.
Observe the animals for 48 hours.
The test dose (Lrll 00) ofthe toxin is the amount present in 0.2
ml ofthat mixture which causes at the site ofinjection a small,
characteristic reaction in the skin of the guinea-pig or rabbit.
Mixtures containing larger amounts of toxin cause larger
reaction and necrosis and mixtures containing smaller amount
of toxin cause no reaction.
Determination ofpotency ofthe antitoxin. Dilute the test toxin
with saline solution so that 1.0 ml contains 10 times the test
dose. Prepare mixtures such that 2.0 ml ofeach mixture contains
1.0 ml ofthe dilution ofthe toxin and one ofa series ofgraded
volumes ofthe preparation under examination. PrePase:futi1:ler
mixtures such that 2.0 rnl ofeach contains 1.0 rnl ofthe solution
ofthe test toxin and 0.1 Unit of antitoxin. Dilute each mixture
with saline solution to the same final volume (2.0 ml). Allow
the mixtures to stand at room temperature, protected from
2374
DIPHTHERIA AND TETANUS VACCINE (ADSORBED)
IP 2010
light, for 15 to 60 minutes. Inject a dose of 0.2 ml of each

mixture into the animals under the conditions described in the
determination ofthe Lr/lOO dose ofthe toxin.
The mixture ofthe preparation under examination that contains
0.01 Unit ofantitoxin in 0.2 ml is the mixture that produces the
same degree oflocal reaction as that produced by the injection
into the same animals ofthe mixture ofthe Standard Preparation
that contains in 0.2 ml the test dose (Lr/l 00) of the toxin and
0.01 Unit ofantitoxin.
When at least four distinct tests are carried out by this method,
the limits of error have been estimated to be between 90 per
cent and III per cent.
Other tests. Complies with the tests stated under Antisera.
Storage. As stated under Antisera.

Labelling. The label states (1) the number ofUnits per ml; (2)
the species of animal from which the preparation has been
made; (3) the recommended dose (if space is inadequate, it
may be stated in the instruction leaflet); (4) the name and
proportion ofany added preservative; (5) that the preparation,
if liquid, should not be allowed to freeze; (6) that the
preparation, if dried, should be used immediately after
reconstitution in the stated quantity of the diluent supplied
by the manufacturer.
adsorbent. Animals that die shall be autopsied and examined

for symptoms of diphtheria intoxication (red adrenals). The
bulle purified toxoid shall pass the test ifno guinea-pig shows
symptoms ofspecific intoxication within six weeks ofinjection
and ifat least 80 per cent ofthe animals survive the test period.
The guinea-pigs shall not have been used previously for
experimental purposes.
Alternatively, a cell-culture test system may be used; in this
case, the sensitivity of the test shall have been demonstrated
to be not less than that of the guinea-pig test, and the test
procedures shall be approved by the National Regulatory
Authority.
Each bulk purified toxoid shall be tested to ensure that
reversion to toxicity cannot take place on storage. The bulle
purified toxoid shall be diluted in order to obtain the same
concentration and chemical environment as that present in
the final bulle vaccine, except for the presence of adjuvant.
To determine whether reversion has occurred, diluted toxoids
that have been stored at 37 for six weeks shall be tested. The
test employed shall be approved by the National Regulatory
Authority and should be sufficiently sensitive to detect very
small amounts oftoxin. No toxicity shall be detected.
Intradermal tests in guinea-pigs and cell-culture tests both
are considered to be suitable.
Antigenic purity
Diphtheria and Tetanus Vaccine

(Adsorbed)
Diphtheria and Tetanus Vaccine (Adsorbed) is a preparation
ofdiphtheria formol toxoid and tetanus formol toxoid adsorbed
on mineral carrier. The formol toxoids are prepared from the
toxins produced by the growth of Corynebacterium
diphtheriae and Clostridium tetani, respectively.
The specification for individual component used in formulation
is referred in the text of individual monograph.
Production
General provisions
Not less than 1500 Lfper mg ofprotein nitrogen for diphtheria

toxoid and not less than 1000 Lflmg of protein nitrogen for
tetanus toxoid.
FINAL BULK VACICNE
The final bulk vaccine is prepared by adsorption of suitable
quantities ofbulk purified diphtheria toxoid and tetanus toxoid
onto mineral carrier such as hydrated aluminium phosphate,
aluminium hydroxide; the resulting mixture is approximately
isotonic with blood. Suitable antimicrobial preservatives may
be added. Antimicrobial preservatives of the phenolic type
must not be used.
Bulk purified diphtheria and tetanus toxoids

The bulk purified diphtheria and tetanus toxoids are prepared
as described in the monographs on Diphtheria vaccine
(adsorbed) and Tetanus vaccine (adsorbed) and comply with
the requirements prescribed therein.
Sterility (2.2.11). Carry out test for sterility using 10 ml of
bulle for each sterility medium.
Identification
A. Dissolve sufficient sodium citrate in the vaccine under
examination to give a 10 per cent wIv concentration. Maintain
at 37 for about 16 hours and centrifuge. The clear supernatant
reacts with a suitable diphtheria antitoxin and yields a
precipitate.
Absence of toxin and irreversibility of toxoid

B. The clear supernatant obtained in testA reacts with a suitable
Inject subcutaneously into each of5 guinea-pigs at least 500 Lf
tetanus antitoxin and yields a precipitate.
ofthe non-incubated bulle purified toxoid in a volume of 1 ml,
using the same buffer solution as for the final vaccine, without pH (2.4.24).6.0 to 7.0.
2375
DIPHTHERIA AND TETANUS VACCINE (ADSORBED)
IP 2010
Specific toxicity. Use 5 normal, healthy guinea-pigs weighing

between 250 and 350 g which have been maintained for at
least 1 week on a uniform, unrestricted diet, and have not
been previously treated with any material that will interfere
with the test. Weigh the animals separately and record their
weights. Inject subcutaneously into each animal 5 times the
dose stated on the labe1. Weigh all the animals at weekly
intervals for 6 weeks. None of the animals shows any
symptoms of diphtheria or tetanus toxaemia or dies from
diphtheria within 42 days or loses weight at the end of the
test. Ifmore than one animal diys from non-specific causes or
loses weight, repeat the test. If an animal dies or loses weight
in the second test, the vaccine fails the test.
The clear supernatant liquid obtained during test A reacts

with a suitable tetanus antitoxin, giving a precipitate or visible
floccules.
Tests
toxicity
Aluminium (2.3 .9). Not more than 1.25 mg per single human
dose when hydrated aluminium phosphate or aluminium
hydroxide is used as the adsorbent.
Assay
pH (2.4.24). The pH ofthe vaccine is within the range approved

for the product (6.0 to 7.0).
Diphtheria toxoid
Complies with the test as stated under Diphtheria Vaccine

(Adsorbed).
Tetanus toxoid

greater than 115.0 per cent ofthe quantity stated on the label.
Complies with the test as stated under Tetanus Vaccine

(Adsorbed).
Assay

method. The amount is not less than 85.0 per cent and not
Carry out one of the described methods for the assay of
Diphtheria Vaccine (Adsorbed).

Viz a) Intradermal challenge method, b) Lethal challenge

method, c) Antibody induction method, d) Validated serological
assay in guinea pigs or mice as approved by National
Regulatory Authority.
FINAL LOT
Tetanus component
The final bulle vaccine is filled and stored aseptically into

sterile containers. The containers are closed so as to prevent
contamination.
Carry out one of the described methods for the assay of

Tetanus Vaccine (Adsorbed) Viz a) Antibody induction
method; b) Challenge method in guinea pigs/mice; c) Validated
serological assay in guinea pigs or mice as approved by
Free formaldehyde (2.3.20): Maximum 0.2 gil
Only a final lot that is satisfactory with respect to each ofthe

Assay may be released for use. Provided the tests for specific
toxicity, free formaldehyde and antimicrobial preservative and
the final bulle vaccine, they may be omitted on the final lot.
Identification
Labelling. The label states (1) the human dose; (2) the minimum
Lfunits per single human dose or the minimum International
Units per single human dose ifpotency test done by challenge
method; (3) the name and the amount of the adsorbent and
preservative; (4) that the vaccine must be shaken before use;
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14).

Dissolve in the vaccine under examination by adding sufficient
sodium citrate to give a 10 per cent w/v solution. Maintain at
37 for about 16 hours and centrifuge until a clear supernatant
liqUid is obtained. Thecleaf siipefriatanffea6ts with a suitable
diphtheria antitoxin, giving a precipitate or visible floccules.
B. Tetanus toxoid is identified by a suitable immuno-chemical

method (2.2.14).
Diphtheria and Tetanus Vaccine

(Adsorbed) for Adults and Adolescents
Diphtheria and Tetanus Vaccine (Adsorbed) for Adults and
Adolescents is a preparation of diphtheria formol toxoid and
tetanus formo1 toxoid adsorbed ona mineral carrier. The formol
toxoids are prepared from the toxins produced by the growth
2376
IP 2010
DIPHTHERIA AND TETANUS VACCINE (ADSORBED) FOR ADULTS AND ADOLESCENTS
of Corynebacterium diphtheriae and Clostridium tetani,

respectively.
Production
Sterility (2.2.11). Carry out the test for sterility using 10 ml for
each medium.
General provisions
FINAL LOT
Bulk purified diphtheria and tetanus toxoids
The final bulle vaccine is distributed aseptically into sterile,

tamper-proof containers. The containers are closed so as to
prevent contamination.
The bulle purified diphtheria and tetanus toxoids are prepared

as described in the monographs on Diphtheria vaccine
(adsorbed) and Tetanus vaccine (adsorbed) and comply with
the requirements prescribed therein.
FINAL BULK VACCINE
The vaccine is prepared by adsorption of suitable quantities
of bulle purified diphtheria toxoid and tetanus toxoid onto a
mineral carrier such as hydrated aluminium phosphate or
aluminimn hydroxide. Suitable antimicrobial preservatives may
be added. Certain antimicrobial preservatives, particularly
those of the phenolic type, adversely affect the antigenic
activity and must not be used.
Only final bulle vaccine that complies with the following
Identification
A. Dissolve sufficient sodium citrate in the vaccine under
examination to give a 10 per cent w/v concentration. Maintain
at 37 for about 16 hours and centrifuge. The clear supernatant
precipitate.
B. The clear supernatant obtained in testAreacts with a suitable
tetanus antitoxin and yields a precipitate.
pH (2.4.24 ). 6.0 to 7.0.
Specific toxicity
Inject subcutaneously 5 times the single human dose stated
with any material that will interfere with the test. Weigh the
animals separately and record their weights. Inject
subcutaneously into each animal 5 times the dose stated on
the label. Weigh all the animals at weekly intervals for 6 weeks.
None of the animals shows any symptoms of diphtheria or
tetanus toxaemia or dies from diphtheria within 42 days or
loses weight at the end of the test. If more than one animal
dies from non-specific causes or loses weight, repeat the test.
Ifan animal dies or loses weight in the second test, the vaccine
fails the test.

Assay may be released for use. Provided the tests for free
formaldehyde and antimicrobial preservative and the assay
have been carried out with satisfactory results on the final
bulle vaccine, they may be omitted on the final lot.
Identification
Ifa satisfactory result is not obtained with a vaccine adsorbed
on aluminium hydroxide, carry out the test as follows.
Centrifuge 15 ml ofthe vaccine under examination and suspend
the residue in 5 m1 ofa freshly prepared mixture of 1 volume of
a 56 gil solution of sodium edetate and 49 volumes of a 90 gl
I solution of disodium hydrogen phosphate. Maintain at 37
for not less than 6 hours and centrifuge. The clear supernatant
B. Tetanus toxoid is identified by a suitable immunochemica1
tetanus' antitoxin, giving a precipitate.
Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose.
pH (2.4.24).6.0 to 7.0.
toxicity.
2377
DIPHTHERIA, TETANUS AND PERTUSSIS VACCINE (ADSORBED)

Assay
IP 2010
FINAL BULK VACCINE
The fmal bulk vaccine is prepared by adsorption of suitable

quantities ofbulk purified diphtheria toxoid and tetanus toxoid
Carry out the prescribed method for assay of Diphtheria' onto hydrated aluminium phosphate or aluminium hydroxide
Vaccine by lethal challenge method described in the assay of and admixture of an appropriate quantity of a suspension of
inactivated B. pertussis. The B. pertussis concentration ofthe
final
bulle vaccine does not exceed that corresponding to an
opacity
of20 IOU per single human dose. Iftwo or more strains
of B. pertussis are used, the composition of consecutive lots
of the final bulle vaccine shall be consistent with respect to
Tetanus component
the proportion of each strain as measured in opacity units.
Suitable antimicrobial preservatives may be added to the bulk
vaccine. Antimicrobial preservatives particularly those of
The lower confidence limit (P = 0.95) ofthe estimated potency phenolic type which affect the antigenic activity must not be
used.
Labelling. The label states (l) the human dose; (2) the minimum
number of International Units of each component per single
human dose, ifpotency determined by challenge method; (3)
the name and the amount of the adsorbent and preservative;
(4) that the vaccine must be shaken before use; (5) that the
vaccine is not to be frozen.

method. The content is not less than 85.0 percent and not
more thanl15.0 per cent of the intended amount.
Diphtheria, Tetanus and Pertussis

Vaccine (Adsorbed)
FINAL LOT
Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed) is a

preparation ofdiphtheria formol toxoid, tetanus formol toxoid
adsorbed on mineral carrier and a suspension of killed
Bordetella pertussis organisms. The formal toxoids are
prepared from the toxins produced by the growth of
Corynebacterium diphtheriae and Clostridium tetani,
respectively. The Bordetella pertussis suspension is prepared
by growth of suitable strains in an appropriate medium, under
controlled conditions.
The specification for individual component used in formulation
is referred in the text of individual monograph.
Production
General Provisions
The production method must be validated to demonstrate
that the product if tested, would comply with the tests for
safety as described under monographs ofDiphtheria Vaccine,
Tetanus Vaccine (Adsorbed) and Pertussis Vaccine.
The hullcpmified_diphtheria_ancLtetanus.. . toxoids.-and
inactivated B. pertussis suspension are preparl'l<:l as <:lesGribl'ld
in the monograph on Diphtheria Vaccine (Adsorbed), Tetanus
Vaccine (Adsorbed) and Pertussis Vaccine respectively and
comply with the respective requirements.
The final bulk vaccirie is filled and stored aseptically into

sterile containers. The containers are closed so as to prevent
contamination.
toxicity of diphtheria, tetanus and pertussis components, free
formaldehyde, antimicrobial preservative and the Assay have
been carried out with satisfactory results on the final bulk
vaccine, they may be omitted on the final lot.
Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14).
Dissolve in the vaccine under examination by adding sufficient
sodium citrate to give a 10 per cent w/v solution. Maintain at
37 for about 16 hours and centrifuge until a clear supernatant
is obtained; reserve the precipitate for identification test C.
The clear supernatant reacts with a suitable diphtheria
antitoxin, giving a precipitate.
~l::'~1?~ (2.2.14).
The clear supernatant obtained during identification test A

reacts with a suitable tetanus antitoxin, giving a precipitate.
C. The pertussis component is identified by agglutination of
the bacteria from the resuspended centrifugation residue (see
2378
IP 2010
DTP (WHOLE CELL), HEPATITIS B (rDNA) AND HAEMOPHILUS TYPE b CONJUGATE VACCINE (ADSORBED)
identification test A; other suitable methods for separating

the bacteria from the adsorbent may also be used) by antisera
specific to B. pertussis or by the assay of the pertussis
component.
Aluminium (2.3 .9). Not more than 1.25 mg per single human
dose, when hydrated aluminium phosphate or aluminium
hydroxide is used as the adsorbent.
Tests

more than 115.0 per cent of the intended amount.
Abnormal toxicity (2.2.1). Each final lot shall be tested for

abnormal toxicity by injecting intraperitoneally one human
dose, but not more than 0.25 ml into each of the five mice
weighing between 17 to 22 g and at least one human dose but
not more than 1.0 ml into each ofthe two guinea pigs weighing
between 250 and 350 g. The preparation passes the test if
none of the animals dies or shows signs of ill health in 7 days
following the injection. If one ofthe animal dies or shows the
signs of ill health, repeat the test. The preparation passes the
test if none of the animals in the second group dies or shows
signs of ill health in the time interval specified.
Specific toxicity
Diphtheria and tetanus components. Inject subcutaneously

five times the single human dose stated on the label into each
of five healthy guinea-pigs, each weighing between 250 and
350 g, that have not previously been treated with any material
that will interfere with the test. The animals should be weighted
every week and observations be made. None of the animals
shows any symptoms of diphtheria or tetanus toxaemia or
dies from diphtheria within 42 days or loses weight at the end
of the test. If more than one animal dies from non-specific
cause or loses weight, repeat the test. If an animal dies or
loses weight in the second test, the vaccine fails the test.
Pertussis component. Use not less than 10 healthy mice each
weighing between 14 and 16 g for the vaccine group and for
the saline control. Use mice ofthe same sex or distribute males
and females equally between the groups. Allow the animals
access to food and water for at least 2 hours before injection
and during the test. Inject each mouse of the vaccine group
intraperitoneally with 0.5 ml, containing a quantity of the
vaccine equivalent to not less than half the single human
dose. Inject each mouse of the control group with 0.5 ml of a
0.9 per cent sterile solution of sodium chloride, preferably
containing the same amount of antimicrobial preservative as
that injected with the vaccine. Weigh the mice groups
immediately before the injection and 72 hours and 7 days after
the injection. The vaccine complies with the test if: (a) at the
end of 72 hours the total mass ofthe group ofvaccinated mice
is not less than that preceding the injection; (b) at the end of
7 days the average increase in mass per vaccinated mouse is
not less than 60 per cent ofthat per control mouse; and (c) not
more than 5 per cent ofvaccinated mice should die during the
test. The test may be repeated and the results of the tests
combined.
pH (2.4.24).6.0 to 7.0.
Assay
Carry out one of the methods for the assay as stated under
Tetanus component
Carry out one of the methods for the assay as stated under
Tetanus Vaccine (Adsorbed).
Iftest is carried out in Guinea pigs, the lower confidence limit
(P=0.95) of estimated potency is not less than 40 IUISingle
human dose; ifthe test is carried in mice, the lower confidence
limit (P=0.95) of estimated potency is not less than 60 lUI
Single human dose.
Pertussis component
Carry out the assay as stated under Pertussis Vaccine.
Labelling. The label states (l) in case done by challenge
method the minimum number of International Units; if units
(as applicable for each component) per single human dose;
(2) In case done by antibody induction method the minimum
number of International Units per single human dose of
pertussis component and minimum number ofLfofdiphtheria
toxoid and tetanus toxoid; (3) the name and the amount ofthe
adsorbent and preservative; (4) that the vaccine must be
(6) for vaccine contained in single-dose containers where the
space is too small to accommodate the full name ofthe vaccine,
the abbreviation 'DTP' may be used in the label and the
container provided that the same code is also stated in the
label on the package.
Diphtheria, Tetanus, Pertussis (Whole

Cell), Hepatitis B (rDNA) and
Vaccine (Adsorbed)
Diphtheria, Tetanus, Pertussis (Whole cell), Hepatitis B (rDNA)
and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a
combined vaccine composed of diphtheria formol toxoid
2379
containing not less than 1,500 Lf, (2.2.16) per mg of protein

nitrogen, purified tetanus formol toxoid containing not less
than 1,000 Lf, (2.2.16), per mg ofprotein nitrogen, hepatitis B
surface antigen and haemophilus type b conjugated to suitable
protein with a mineral adsorbent to which a suspension of
killed Bordetellapertussis has been added. Mineral adsorbent
is a suspension ofhydrated aluminium hydroxide,aluminium
phosphate or calcium. phosphate, in saline solution or other
appropriate solution isotonic with blood.
The formol toxoids are prepared from the toxin produced by
tetani, respectively, in suitable media. The toxins are converted
to toxoids by treatment with formaldehyde solution by
methods which avoid reversibility of the toxoids.
technology.
The polysaccharide, polyribosyl ribitol phosphate, PRP is a
lillear cop()lYlller c.Olnp()s~~ of repeated units of 3-~-D
ribofuranosyl-(1-.71 )-ribitol-5-phosphate [(CIOH,P,l)n]' with
a defined molecular size and derived from a suitable strain of
Haemophilus injluenzae type b. The canier protei'n, when
conjugated to PRP, is capable of inducing a T-cell dependent
B-cell immune response to the polysaccharide.
The product may be presented with the haemophilus
component in a separate container, the contents of which are
mixed with the other components immediately before or during
use.
The final product contains a suitable antimicrobial
preservative. The antigenic properties of the vaccine are
adversely affected by the presence of certain antimicrobial
preservatives particularly those ofthe phenolic type and some
of the quaternary ammonium type and must not be used.
Production
General provisions
in a separate vial, as part of consistency studies the assays of
the diphtheria, tetanus, pertussis and hepatitis Bare canied
as for use. For subsequent routine control, the assays ofthese
components may becanied out without mixing with the
liaemophiliis"
comiioneiil~
_._~
. _"._-_._-"',"-"'-, .,._-" .-,-,'",-".. . --.-.. .-.-.-.-

product, if tested, would comply with the following test for
specific toxicity of the diphtheria and tetanus component:
IP20lO
inject subcutaneously 5 times the single human ,dose. stated

on the label into each of 5 healthy guinea pigs, each weighing
days ofthe injection any ofthe animals shows signs of or dies
from diphtheria, toxemia or tetanus, the vaccine does not
shows signs of or dies in the second test, the vaccine does
not comply with the test.
The stability of the final lot and the relevant intennediates is
evaluated using one or more indicator tests. For the
haemophilus component, such tests may include determination
ofrnolecular size, determinatiol1offree PRP in the conjugate
and kinetics ofdepolymerisation. Taking accountofthe results
of the stability testing, release requirements are set for these
indicator tests to ensure that the vaccine will be satisfactory
at the end of the period of validity.
The reference vaccine may be stabilized by a method that has

(Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine,
Hepatitis B Vaccine (rDNA) and Haemophilus Type b
Conjugate Vaccine.
FINAL BULK VACCINE
Vaccine with all components in the same container

The final bulk is prepared by adsorption, separately or
together, of suitable quantities of bulle purified diphtheria
toxoid, bulle purified tetanus toxoid, bulle purified hepatitis B
surface antigen onto a mineral canier such as aluminium
hydroxide or hydrated aluminium phosphate, admixture ofan
appmpriate__ quantity_oLa_ suspensioll-oLinactiYated
B, p?,r(ussis component and admixture ofa sJlitaple quantity
of PRP conjugate; the resulting mixture is approximately
isotonic with blood. The B. pertussis concentration of the
final bulle vaccine does not exceed that conesponding to an
2380
IP 2010
opacity of20 ill per single human dose. If2 or more strains of
B. pertussis are used, the composition of consecutive lots of
the final bulle vaccine shall be consistent with respect to the
proportion ofeach strain as measured in opacity units. Suitable
Vaccine with the haemophilus component in a separate
container
together, of suitable quantities of bulk purified diphtheria
toxoid, bulk purified tetanus toxoid, bulk purified hepatitis B
surface antigen onto a mineral carrier such as aluminium
hydroxide or hydrated aluminium phosphate, admixture ofan
appropriate quantity of a suspension of inactivated B.
pertussis component and admixture of a suitable quantity of
PRP conjugate; the resulting mixture is approximately isotonic
with blood. The B. pertussis concentration of the final bulle
vaccine does not exceed that corresponding to an opacity of
20 ill per single human dose. If2 or more strains ofB. pertussis
are used, the composition of consecutive lots ofthe final bulle
vaccine shall be consistent with respect to the proportion of
each strain as measured in opacity units. The final bulle is
filled separately. Suitable antimicrobial preservatives may be
added. The final bulk of the haemophilus component is
prepared by dilution of the bulk conjugate to the final
concentration with a suitable diluent. A stabilizer may be added.
greater than 115.0 per cent ofthe intended content.
FreePRP
Unbound PRP is determined after removal of the conjugate,
for example by anion exchange, size exclusion or hydrophobic
chromatography (2.4.16), ultrafiltration or other validated
methods. The amount of free PRP is not greater than that
approved for the particular product.
Osmolality (2.4.23). The osmolality ofthe vaccine is within
pH (2.4.24). 6.0 to 7.0.
Description. Whitish turbid liquid in which the mineral carrier
tends to settle down slowly on keeping.
Identification
Tests A, B, C, D and E may be omitted if test F is carried out.

Test F may be omitted iftests A, B, C, D and E are carried out.
A. Diphtheria toxoid. Dissolve sufficient sodium citrate in
the vaccine under examination to give a 10 per cent w/v
concentration. Maintain at 37 for about 16 hours and
centrifuge. Reserve the residue for test C. The clear
supernatant reacts with a suitable diphtheria antitoxin and
yields a precipitate.
B. Tetanus toxoid. Tetanus toxoid is identified by suitable
immunochemical method. The clear supernatant obtained as
described in identification testA reacts with a suitable tetanus
antitoxin to give a positive reaction, when tested by a suitable
validated immunological method.
C. Pertussis component. To a suspension of the residue
obtained in test A in saline solution add a suitable Bordetella
pertussis antiserum; agglutination indicates presence of
pertussis component.
D. Hepatitis B swface antigen. The suspension ofthe residue
obtained in test A gives a positive reactions when tested by
suitable in-vitro assay.
FINAL LOT
Onlya final lot that is satisfactory with respect to the test for
osmolality and with respect to eachofthe requirements given
for use. Provided the tests for specific toxicity of diphtheria
toxoid, tetanus toxoid and pertussis component and
antimicrobial preservative and the assays for the diphtheria,
tetanus and pertussis components have been carried out with
satisfactory results on the final bulle vaccine, they may be
omitted on the final lot. Provided the content of free
formaldehyde has been determined on the bulk purified
antigens or on the final bulle and ithas been shown that the
content in the final lot will not exceed 0.2 gil, the test for free
formaldehyde may be omitted on the final lot. If an in vivo
assay is used for the hepatitis B component, provided it has
been carried out with satisfactory results on the final bulle
E. PRP. The suspension of the residue obtained in the test A

gives a positive reaction when tested by a suitable
immunochemical method for PRP.
F. The vaccine confers an active immunity in mjce and

bguinea-pigs when administered as directed in the test for
Assay.
Tests

in a separate container; the tests for specific toxicity of
diphtheria toxoid, tetanus toxoid and pertussis component,
aluminium, free formaldehyde, antimicrobial preservative
and sterility are carried out on the container with the
diphtheria, tetanus, pertussis and hepatitis B components;
the tests for PRP content, water (where applicable), sterility
2381
IF 2010
Assay
and pyrogens are carried out on the container with the Diphtheria toxoid (adsorbed)
Complies with the test as stated under Diphtheria and Tetanus
Ifthe haemophilus component isfreeze-dried, some tests may Vaccine (Adsorbed).
be carried out on the freeze-dried product rather than on the Tetanus toxoid (adsorbed)
bulk conjugate where the freeze-drying process may affect
Complies with the test as stated under assay ofTetanus Vaccine
(Adsorbed).
PRP. Not less than 80.0 per cent of the amount ofPRP stated
on the label. PRP is determined either by assay ofribose (2.7.1), Pertussis vaccine
or phosphorus (2.7.1), by an immunochemical method (2.2.14)
Complies with the test as stated under Diphtheria, Tetanus
or by anion exchange liquid chromatography with pulsed
and Pertussis Vaccine (Adsorbed).
amperometric detection.
Aluminium (2.3.9). Not more than 1.25 mg per single human Hepatitis B suiface antigen (adsorbed)
dose, ifaluminium hydroxide or hydrated aluminium phosphate Complies with the test as stated under Hepatitis B Vaccine
is used as the adsorbent.
(Adsorbed).
method. The content is not less than 85 per cent and is not
greater than 115 per cent of the quantity stated on the label.
abnormal toxicity by injecting intraperitoneally 0.25 ml ofthe
vaccine into each offive mice weighing between 17 and 22 g
and at least one human dose but not more than 1.0 ml into
each oftwo guinea pigs weighing between 250 and 350 g. The
preparation passes the test if none of the animals dies or
shows signs of ill health in seven days following the injection.
If one of the animals dies or shows signs of ill health, repeat
the test. The preparation passes the test if none of the animals
in the second group dies or shows signs of ill health in the
time interval specified.
Storage. When stored under the prescribed conditions the

vaccine may be expected to retain potency for not less than 2
years from the date on which the potency test forthe pertussis
component was started.
Labelling. The label states (l) the human dose; (2) Diphtheria
and Tetanus components; (a) in case done by challenge
method, the minimum number of International Units (as
applicable for each component) per single human dose; (b) in
case done by antibody induction, the minimum Lf units per
single human dose; (3) pertussis component - ill or IOU per
single human dose; (4) hepatitis B component - J.lg HBsAg
per single human dose; (5) haemophilus conjugate component
- mg PRP per single human dose; (6) the type and nominal
amount ofcarrier protein per single human dose; (7) the name
and amount of adsorbent and added preservative; (8) that the
vaccine must be shaken before use; (9) that the vaccine is not
to be frozen.
Pyrogens (2.2.8). This test is carried out for Haemophilus

injluenzae type b vaccine only if Haemophilus injluenzae
type b vaccine is presented as separate lyophilized vial. The Diphtheria, Tetanus, Pertussis (Whole
vaccine complies with the test for pyrogens. Inject per kg of Cell) and Hepatitis B (rDNA) Vaccine
the rabbit's mass a quantity ofthe vaccine equivalent to: I mg
(Adsorbed)
of PRP for a vaccine with diphtheria toxoid or CRM 197
diphtheria toxoid as carrier; 0.1 mg ofPRP for a vaccine with Diphtheria, Tetanus, Pertussis and Hepatitis B (rDNA) Vaccine
tetanus toxoid as carrier protein; 0.025 mg ofPRP for vaccine (Adsorbed) is a combined vaccine composed of diphtheria
with aMP as carrier.
formol toxoid containing not less than 1,500 Lf (2.2.16), per
mg of protein nitrogen, purified tetanus formol toxoid
Specific toxicity
containing not less than 1,000 Lf ( 2.2.16), per mg of protein
nitrogen and hepatitis B surface antigen with a mineral
Diphtheria and tetanus components
adsorbent-towhich-asuspension-ofkilledBordetellapertussis_c;~lIlPli~s",ith the t~sta~stat~dul1d.er piphtheriaand 'Tetanus has been added. MineraLadsorbenLis a suspension of
Vaccine (Adsorbed).
hydrated aluminium hydroxide, aluminium phosphate or
calcium
phosphate in saline solution or other appropriate
Pertussis component
solution isotonic with blood.
and Pertussis Vaccine (Adsorbed):
2382
~'_"__'~_'
_ _'_"_""'___' __ __.__ ,,_.._.,m_.,,_
._ _
,_ _.
,._"_......
.,
._ __ _ _ _
"._ _
,__,.__._._.
IP 2010
DIPHTHERIA, TETANUS, PERTUSSIS (WHOLE CELL), HEPATITIS B (rDNA) VACCINE (ADSORBED)
The formol toxoids are prepared from the toxin produced by

tetani, respectively, in suitable media. The toxins are converted
methods, which avoid reversibility of the toxoids.
technology.
The final product contains a suitable antimicrobial
preservative. The antigenic properties of the vaccine are
adversely affected by the presence of certain antimicrobial
of the quaternary ammonium type and must not be used.
Production
General provisions
on the label into each of 5 healthy guinea pigs, each weighing
with any material that will interfere with the test. If within
42 days of the injection any of the animals shows signs of or
dies from diphtheria, toxaemia or tetanus, the vaccine does
not comply with the test. Ifmore than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal
The stability of the final lot and the relevant intermediates is
evaluated using one or more indicator tests. Taking account
of the results of the stability testing, release requirements are
set for these indicator tests to ensure that the vaccine will be
satisfactory at the end of the period of validity.

thereof is used as a reference vaccine.


and Hepatitis B Vaccine (rDNA).
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption, separately
or together, of suitable quantities of bulle purified diphtheria
toxoid, tetanus toxoid, pertussis whole cell suspension and
hepatitis B surface antigen onto a mineral carrier such as
aluminium hydroxide or hydrated aluminium pqosphate.
Suitable antimicrobial preservatives may be added. Only a
final bulle vaccine that complies with the following requirements
may be used in the preparation ofthe final lot.

greater than 115.0 per cent of the intended content.
pH (2.4.24). 6.0 to 7.0.

FINAL LOT
for use. Provided the tests for specific toxicity of diphtheria
toxoid, tetanus toxoid and pertussis component and
antimicrobial preservative and the assays for the diphtheria,
tetanus and pertussis components have been carried out with
satisfactory results on the final bulle vaccine, they may be
omitted on the final lot. Provided the content of free
formaldehyde has been determined ori the bulk purified
antigens or on the final bulle and it has been shown that the
content in the final lot will not exceed 0.2g/l, the test for free
formaldehyde may be omitted on the final lot. If an in vivo
assay is used for the hepatitis B component, provided it has
been carried out with satisfactory results on the final bulle

Identification
Tests A, B, C andD may be omitted iftest E is carried out. Test
E may be omitted if tests A, B C and D are carried out.

2383
DIPHTHERIA, TETANUS, PERTUSSIS (WHOLE CELL), HEPATITIS B (rDNA) VACCINE (ADSORBED)

B. Tetanus toxoid. The clear supernatant obtained in test A
reacts with a suitable tetanus antitoxin and yields a precipitate.
obtained in testA in saline solution add a suitable B. pertussis
antiserum; agglutination indicates presence of pertussis
component.
D. HepafJitis B surface antigen. The suspension ofthe residue
obtained in test A gives a positive reactions when tested by
suitable in-vitro assay.
E. The vaccine confers an active immunity in mice and guineapigs when administered as directed under Assay.
Tests
pH (2.4.24).6.0 to 7.0.
Aluminium (2.3.9). Not more than 1.25 mg per single human
dose, ifaluminium hydroxide or hydrated aluminium phosphate
is used as the adsorbent.
IP 2010
Tetanus toxoid (adsorbed)

Complies with the test as stated under assay for Tetanus
Vaccine (Adsorbed).
Pertussis vaccine
Complies with the test as stated under Pertussis Vaccine
(Adsorbed).
Hepatitis B sw1ace antigen (adsorbed)

Complies with the test as stated under Hepatitis B Vaccine
(Adsorbed).
years from the date on which the potency test for the pertussis
Labelling. The label states (1) the human dose (ml); (2)
diphtheria and tetanus components, (a) in case done by
challenge method, the minimum number ofInternational Units
(as applicable for each component) per single human dose
and (b) in case done by antibody induction, the minimum Lf
units per single human dose; (3) pertussis component - ill or
IOU per single human dose; (4) hepatitis B component -!J.g
HBsAg per single human dose; (5) the name and amount of
adsorbent and added preservative; (6) that the vaccine must
be shaken before use; (7) that the vaccine is not to be frozen.

abnormal toxicity by injecting intraperitoneally one human
dose but not more than 0.25 m1 into each offive mice weighing
between 17 and 22 g and at least one human dose but not
more than 1.0 ml into each of two guinea pigs weighing
between 250 and 350 g. The preparation passes the test if
none of the animals dies or shows signs of ill health in seven
days following the injection. If one of the animals dies or
shows signs of ill health, repeat the test. The preparation
passes the test ifnone ofthe animals in the second group dies
or shows signs of ill health in the time interval specified.
Specific toxicity

Vaccine (Adsorbed).
Pertussis component
(Adsorbed).
Diphtheria, Tetanus, Pertussis (Whole

Cell) and Haemophilus Type b
Diphtheria, Tetanus, Pertussis and Haemophilus type b
Conjugate Vaccine (Adsorbed) is a combined vaccine
composed ofdiphtheria formol toxoid containing not less than
1,500 Lf, (2.2.16) per mg ofprotein nitrogen, purified tetanus
formol toxoid containing not less than 1,000 Lf, (2.2.16), per
mg of protein nitrogen, and Haemophilus type b conjugated
to suitable protein with a mineral adsorbent to which a
suspension of killed Bordetella pertussis has been added.
The mineral adsorbent is a suspension ofhydrated aluminium
hydroxide, aluminium phosphate or calcium phosphate, in
saline solution or other appropriate solution isotonic with
blood.
Diphtheria toxoid (adsorbed)
The fonnol toxoids are prepared from the toxin produced by

tetani, respectively in suitable media. The toxins are converted
methods which avoid reversibility ofthe toxoids; .

(Adsorbed).
The polysaccharide, polyribosyl ribitol phosphate, PRP is a

linear copolymer composed of repeated units of
Assay
2384
IP 2010
DTP (WHOLE CELL) AND HAEMOPHILUS TYPE b CONJUGATE VACCINE (ADSORBED)
3 - ~ - D-ri b ofurano s y 1- (1-71) -ri b i to 1- 5 -phosp hate Reference vaccine(s)

[(CIQH'Pll)n]' with a defined molecular size and derived from ' Provided valid assays can be performed, monocomponent
a suitable strain ofHaemophilus injluenzae type b. The carrier reference vaccines may be used for the assays on the combined
protein, when conjugated to PRP, is capable of inducing a T- vaccine. If this is not possible because of interaction between
cell dependent B-cell immune response to the polysaccharide. the components of the combined vaccine or because of the
The product may be presented with the haemophilus difference in composition between monocomponent reference
component in a separate container, the contents of which are vaccine and the test vaccine, a batch of combined vaccine
mixed with the other components immediately before or during shown to be effective in clinical trials or a batch representative
use.
The final product contains a suitable antimicrobial process used for the batch tested in clinical trials is necessary.
preservative. The antigenic properties of the vaccine are The reference vaccine may be stabilized by a method that has
adversely affected by the presence of certain antimicrobial been shown to have no effect on the assay procedure.
of the quaternary ammonium type must not be used.
Production
General provisions
in a separate vial, as part of consistency studies, the assays
of the diphtheria, tetanus and pertussis are carried out on a
suitable number of batches of vaccine reconstituted for use.
For subsequent routine control, the assays of these
on the label into each of5 healthy guinea pigs, each weighing
with any material that will interfere with the test. If within 42
days ofthe injection any ofthe animals shows signs of or dies
from diphtheria, toxemia or tetanus, the vaccine does not
The stability of the final lot and the relevant intermediates is
evaluated using one or more indicator tests. For the
haemophilus component, such tests may include determination
of molecular size, determination offree PRP in the conjugate
and kinetics ofdepolymerisation. Taldng account ofthe results
of the stability testing, release requirements are set for these

(Whole Cell) and Haemophilus injluenzae Type b Conjugate
Vaccine.
FINAL BULK VACCINE
Vaccine with all components in the same container

toxoid, bulle pmi.fied tetanus toxoid, onto a mineral carrier such
as aluminium hydroxide or hydrated aluminium phosphate,
admixture of an appropriate quantity of a suspension of
inactivated B. pertussis component and admixture ofa suitable
quantity of PRP conjugate; the resulting mixture is
approximately isotonic with blood. The B. pertussis
concentration of the final bulle vaccine does not exceed that
corresponding to an opacity of 20 ill per single human dose.
If2 or more strains ofB. pertussis are used, the compositionof
consecutive lots of the final bulle vaccine shall be consistent
with respect to the proportion of each strain as measured in
opacity units. Suitable antimicrobial preservatives may be
added.
Vaccine with thehaemophilus component in a separate

container
toxoid, bulle purified tetanus toxoid onto a mineral carrier such
as aluminium hydroxide or hydrated aluminium phosphate,
admixture of an appropriate quantity of a suspension of
inactivated B. pertussis component and admixture ofa suitable
quantity of PRP conjugate; the resulting mixture is
approximately isotonic with blood. The B. pertussis
concentration of the final bulle vaccine does not exceed that
corresponding to opacity of20 ill per single human dose. If 2
2385
IP 2010
DTP (WHOLE CELL) AND HAEMOPillLUS TYPE b CONJUGATE VACCINE (ADSORBED)
or more strains of B. pertussis are used, the composition of

consecutive lots of the final bulk vaccine shall be consistent
with respect to the proportion of each strain as measured in
opacity units. The final bulle is filled separately. Suitable
antimicrobial preservatives may be added. The final bulle of
the haemophilus component is prepared by dilution of the
bulle conjugate to the final concentration with a suitable diluent.
A stabilizer may be added.
greater than 115.0 percent of the intended content.
bulk for each sterility medium.

B. Tetanus toxoid. The clear supernatant obtained in test A
reacts with a suitable tetanus antitoxin and yields a precipitate.
obtained in test A in saline solution add a suitable Bordetella
pertussis antiserum; agglutination indicates presence of
pertussis component.
D. PRP. The suspension of the residue obtained in test A
gives a positive reaction when tested by a suitable
immunochemical method for PRP.
E. The vaccine confers an active immunity in mice and guinea
pigs when administered as directed in the test for Potency.
FINAL LOT
Tests
Where the haemophilus component is in a separate container,

the final bulk of the haemophilus component is freeze-dried.
for use. Provided the tests specific toxicity ofdiphtheria toxoid,
tetanus toxoid and pertussis component and antimicrobial
preservative and the assays for the diphtheria, tetanus and
pertussis components have been calTied out with satisfactory
results on the final bulk vaccine, they may be omitted on the
final lot. Provided the content of free formaldehyde has been
detennined on the bulle purified antigens or on the final bulle
exceed 0.2 gil, the test for free formaldehyde may be omitted
on the final lot.
conjugate, for example by anion exchange, size exclusion or
hydrophobic chromatography (2.4.16), ultrafiltration or other
validated methods. The amount offree PRP is not greater than

in a separate container; the tests for specific toxicity of
diphtheria toxoid, tetanus toxoid and pertussis component,
aluminium, free formaldehyde, antimicrobial preservative
and sterility are carried out on the container with the
diphtheria, tetanus and pertussis components; the tests for
PRP content, water (where applicable), sterility and
pyrogens are carried out on the container with the
Ifthe haemophilus component isfreeze-dried, some tests may

be carried out on the jioeeze-driedproduct rather than on the
bulk conjugate where the freeze-d,ying process may affect
PRP. Not less than 80.0 per cent ofthe amount ofPRP stated
on the label. PRP is determined either by as~ay ofribose (2.7.1 )
or phosphorus (2.7.1), by an immunochemical method (2.2.14)
or by anion exchange liquid chromatography with pulsed
amperometric detection.


pH (2.4.24). 6.0 to 7.0.


Production
test
Identification
Tests A, B, C andD may be omitted iftest E is carried out. Test
E may be omitted if tests A, B, C and D are carried out.

abnormal toxicity by injecting intraperitoneally a maximum of
one human dose, but not more than 0.25 rol into each of the
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IP 2010
DIPHTHERIA VACCINE (ADSORBED)
five mice weighing between 17 to 22 g and at least one human

dose but not more than 1.0 ml into each ofthe two guinea pigs
weighing between 250 and 350 g. The preparation passes the
test if none ofthe animals dies or shows signs of ill health in
7 days following the injection . If one of the animal dies or
shows the signs of ill health, repeat the test. The preparation
passes the test ifnone of the animals in the second group dies
or shows signs of ill health in the time interval specified.
Pyrogens (2.2.8). This test is carried out for Haemophilus
injluenzae type b vaccine only if Haemophilus injluenzae
type b vaccine is presented as separate lyophilized vial. The
vaccine complies with the test for pyrogens. Inject per kg of
the rabbit's mass a quantity ofthe vaccine equivalent to: 1 mg
of PRP for a vaccine with diphtheria toxoid or CRM 197
diphtheria toxoid as carrier; 0.1 mg ofPRP for a vaccine with
.tetanus toxoid as carrier protein; 0.025 mg ofPRP for vaccine
with aMP as carrier.
Specific toxicity
nominal amount of carrier protein per single human dose; (6)

the name and amount of adsorbent and added preservative;
(7) that the vaccine must be shaken before use; (9) that the
vaccine is not to be frozen.

Diphtheria Vaccine (Adsorbed) is a preparation of diphtheria
formol toxoid with a mineral adsorbent. The formol toxoid is
prepared from the toxin produced by the growth of
COIynebacterium diphtheriae.
Production
General provisions
The maximum number ofLfunits per single human dose
of diphtheria vaccine (adsorbed) is 30.
Bulk purified toxoid

Vaccine (Adsorbed).
For the production of diphtheria toxin, from which toxoid is

prepared, seed cultures are managed in a defined seed-lot
system in which toxinogenicity is conserved and, where
necessary, restored by deliberate reselection. A highly
toxinogenic strain of Corynebacterium diphtheriae with
lmown origin and history is grown in a suitable liquid medium.
At the end of cultivation, the purity of each culture is tested
and contaminated cultures are discarded. Toxin-containing
culture medium is separated aseptically from the bacterial mass
as soon as possible. The toxin content (Lf per ml) is checked
to monitor consistency ofproduction. Single harvests may be
pooled to prepare the bulle purified toxoid. The toxin is purified
to remove components likely to cause adverse reactions in
humans. The purified toxin is detoxified with formaldehyde by
a method that avoids destruction ofthe immunogenic potency
ofthe toxoid and reversion ofthe toxoid to toxin, particularly
on exposure to heat. Alternatively, purification may be carried
out after detoxification.
Pertussis component
and Pertussis Vaccine (Adsorbed).
Assay
Diphtheria toxoid (adsorbed)

(Adsorbed).
Tetanus toxoid (adsorbed)

Complies with the test as stated under assay of Tetanus
Vaccine (Adsorbed).
Pertussis vaccine
(Adsorbed).
years from the date on which the potency test for the pertussis
Labelling. The label states (1) the human dose (ml); (2)
Diphtheria and Tetanus components (a) in case done by
challenge method the minimum number ofintemational units
(as applicable for each component) per single human dose;
(b) in case done by antibody induction method, the minimum
Lfunits per single human dose; (3) pertussis component - ill
or IOU per single human dose; (4) haemophilus conjugate
component - mg PRP per single human dose; (5) the type and
Only bulle purified toxoid that complies with the following

requirements may be used in the preparation ofthe final bulle
vaccine.
Absence of toxin and irreversibility of toxoid
Inject subcutaneously into each of5 guinea-pigs at least 500 Lf
of the non-incubated bulk purified toxoid in a volume of
I ml, using the same buffer solution as for the final vaccine,
without adsorbent. Animals that die shall be autopsied and
examined for symptoms of diphtheria intoxication (red
adrenals). The bulle purified toxoid shall passthe test if no
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IP 2010
DIPHTHERIA VACClNE (ADSORBED)

guinea-pig shows symptoms of specific intoxication within
six weeks ofinjection and ifat least 80 per cent ofthe animals
survive the test period. The guinea-pigs shall not have been
used previously for experimental purposes.
Altematively, a cell-culture test system may be used; in this
case, the sensitivity of the test shall have been demonstrated
to be not less than that of the guinea-pig test, and the test
procedures shall be approved by the National Regulatory
Authority.
Each bulk purified toxoid shall be tested to ensure that
reversion to toxicity cannot take place on storage. The bulk
purified toxoid shall be diluted in order to obtain the same
concentration and chemical enviromnent as those present in
the final bulk vaccine, except for the presence of adjuvant.
To determine whether reversion has occurred, diluted toxoids
that have been stored at 37 for six weeks shall be tested: The
test employed shall be approved by the National Regulatory
.Authority and should be sufficiently sensitive to detect very
smaJI amounts oftoxin. No toxicity shall be detected.
Intradermal tests in guinea-pigs and cell-culture tests both
are considered to be suitable.
plates and incubate at 37 for 5 to 6 days. Cytotoxic effect is

judged to be present where there is complete metabolic
inhibition of the Vero cells, indicated by the pH indicator of
the medium. Confirm cytopathic effect by microscopic
examination or suitable staining such as MIT dye. The test is
invalid if5 x 10-5Lfperml ofreference diphtheria toxin in 100
Lfper m1 toxoid has no cytotoxic effect on Vero cells or ifthe
cytotoxic effect of this amount of toxin is not" neutralised in
the wells containing diphtheria antitoxin. The bulk purified
toxoid complies with the test if no toxicity neutralisable by
antitoxin is found in either sample.
Antigenic purity. Not less than 1,500 Lf per mg of protein
nitrogen.
FINAL BULK VAc:CINE
The final bulk vaccine is prepared by adsorption of a suitable
quantity ofbulk purified toxoid onto a mineral carrier such as
hydrated aluminium phosphate or aluminium hydroxide; the
resulting mixture is approximately isotonic with blood. Suitable
antimicrobial preservatives may be added. Certain
antimicrobial preservatives, particularly those ofthe phenolic
type, adversely affect the antigenic activity and must not be
used.
Cell culture method

Using the same buffer solution as for the final vaccine, without
adsorbent, prepare a solution ofbulk purified toxoid at 100 Lf
per m!. Divide the solution into 2 equal parts. Maintain I part
at 5 3 and the other at 37 for 6 weeks. Carry out a test in
Vero cells for active diphtheria toxin using 50 III per well of
both samples. The sample should not contain antimicrobial
preservatives and detoxifying agents should be detennined
to be below the concentration toxic to Vera cells. Non-specific
toxicity may be eliminated by dialysis.
Use freshly trypsinised Vero cells at a suitable concentration,
for example 2.5 x 105per ml and a reference diphtheria toxin
diluted in 100 Lfper ml diphtheria toxoid. A suitable reference
diphtheria toxin will contain either not less than 100 LD5iml
or 67 to 133 h"/100 in I Lfand 25,000 to 50,000 minimal reacting
doses for guinea-pig skin in I Lf (diphtheria toxin RP is
suitable for use as the reference toxin). Dilute the toxin in 100
Lflml diphtheria toxoid to a suitable concentration, for example
2 x 10-4 Lfperml. Prepare serial twofold dilutions ofthe diluted
diphtheria toxin and use undiluted test samples (50 III per
well). Distribute them in the wells of a sterile tissue culture
plate containing a medium suitable for Vera cells. To ascertain
that any cytotoxic effect noted is specific to diphtheria toxin,
prepare.inparalleLdilutions.where.the toxin.isneutralisedbya
suitable concentration of diphtheria antitoxin, .for example
100 IU/m!. Include control wells without toxoid or toxin and
with non-toxic toxoid at 100 Lfper ml on each plate to verify
normal cell growth. Add cell suspension to each well, seal the

Identification
Dissolve sufficient sodium citrate in the vaccine under
examination to give a 10 per cent w/v concentration. Maintain
at 37 for about 16 hours and centrifuge. The clear supematant
precipitate.
pH (2.4.24). 6.0 to 7.0.
Specific toxicity. Use 5 normal, healthy guinea-pigs weighing
between 250 and 350 g, which have been maintained for at
least 1 week on a uniform, unrestricted diet, and have not
been previously treated with any material that will interfere
with the test. Weigh the animals separately and record their
weights. Inject subcutaneously into each animal 5 times the
dose stated on the label. Weigh all the animals at weekly
intervals for 6 weeks. None of the animals show signs of or
dies from diphtheria toxaemia within 42 days or loses weight
at the end of the test. Ifmore than one animal dies from nonspecific causes or loses weight, repeat the test. If an animal
dies or loses weight in-the second test, the vaccine fails the
test.
Potency. Determine by any ofthe methods ofbiological assay
of Adsorbed Diphtheria Vaccine described.
2388
DIPHTHERIA VACCINE (ADSORBED)
IP 2010
Biological assay of adsorbed diphtheria vaccine

(a) Intradermal challenge method
The potency of adsorbed diphtheria vaccine is detennined by

comparing the dose necessary to protect guinea-pigs against
the erythrogenic effects ofa range ofintradermal injections of
diphtheria toxin with the dose of the Standard preparation of
adsorbed diphtheria toxoid necessary to give the same
protection. For this comparison, the Standard preparation of
adsorbed diphtheria toxoid and a suitable preparation of
diphtheria toxin, for use as a challenge toxin, are required.
Standard preparation
The Standard preparation is International standard of

Diphtheria toxoid, adsorbed, or another suitable preparation
the potency of which has been detennined in relation to the
International Standard.
Suggested method
Test animals. Use white guinea-pigs, weighing between 250
and 350 g, from the same stock. Distribute the guinea-pigs
into no fewer than six equal groups; use groups containing a
number of animals sufficient to obtain results that fulfill the
requirements for a valid Assay prescribed below. The guineapigs are all of the same sex or the males and females are
distributed equally among the groups. If the challenge toxin
to be used has not been shown to be stable or has not been
adequately standardized, include five guinea-pigs as
unvaccinated controls.
Selection of the challenge toxin. Select a preparation of
diphtheria toxin containing 67 to 133 Limes reactionis/I 00 (Lr/
100) in Limes flocculationis (Lf) and 25,000 to 50,000 minimal
reacting doses for guinea-pig skin in I Lf. If the challenge
toxin preparation has been shown to be stable, it is not
necessary to verif'y the activity for every assay.
Preparation ofthe challenge toxin solutions. Immediately prior
to use, dilute the challenge toxin with a suitable diluent to
obtain a challenge toxin solution containing about 512xI04 Lf
in 0.2 m!. Dilute a portion of this challenge toxin solution to
give a series of five 4-fold dilutions.
Determination of potency of the vaccine. Prepare in saline
solution dilutions of the vaccine under examination and of
the Standard preparation such that, for each, the dilutions
fonn a series differing by not more than 2.5 fold steps and in
which the dilutions of intermediate concentration, when
injected subcutaneously in 1.0 ml volumes into guinea-pigs,
result in an intradennal score ofapproximately three when the
animals are challenged. Allocate the dilutions, one to each of
the groups of guinea-pigs, and inject subcutaneously 1.0 ml
of each dilution into each guinea-pig in the group to which
that dilution is allocated. After 28 days shave both flanks of
each guinea-pig and inject each animal intradennally with 0.2
ml of the challenge toxin solution and with 0.2 ml ofeach of

the five dilutions thereof in such a way as to minimize
interference between adjacent sites. If necessaty, inject the
unvaccinated control guinea-pigs with dilutions containing
8x10-5 , 4x10-5, 2x 10-5 , Ix10-5 and 5xlO-6 Lfofthe challenge toxin.
Examine all the injection sites 48 hours after injection of the
challenge toxin and record the incidence ofspecific diphtheria
elythema. Record also the number of sites free from such
reactions as the intradermal challenge score. Tabulate the
intradermal challenge scores for all the animals receiving the
same dilution of vaccine and use those data with a suitable
transfonnation, such as (score? or arcsin [(score/6)2], to obtain
an estimate of the relative potency for each of the test
preparations by parallel-line quantitative analysis.
The test is not valid unless (a) for both the preparation under
examination and the Standard preparation, therriean score
obtained at the lowest dose level is more than three; (b) if
applicable, the toxin dilution that contains 4x105 Lf gives a
positive erythema in at least 80.0 per cent ofthe control guineapigs and the dilution that contains 2x I0-5 Lf gives no reaction
in at least 80 per cent of the guinea-pigs (if these criteria are
not met a different toxin has to be selected); (c) the fiducial
limits of the assay fall between 50.0 and 200.0 per cent ofthe
estimated potency; (d) the statistical analysis shows no
deviation from linearity and parallelism. The test may be
repeated but when more than one test is perfonned the results
ofall valid tests must be combined in the estimate ofpotency.
The lower fiducial limit oferror ofthe estimated potency is not
less than 30 Units per dose.
(b) Lethal challenge method
Test animals. Use healthy, white or light-coloured guineapigs from the same stock, weighing between 250 and 350 g.
Distribute them into six groups ofsixteen; and four groups of
four. The guinea-pigs should all be of the same sex or the
males and females should be distributed equally between the
six groups of sixteen.
Challenge toxin. Select a preparation of
containing not less than 100 LD50 in 1.0 m!.
diphthe~ia toxin

to use, prepare from the challenge toxin by dilution in
phosphate buffiredsalinepH 7.4, or normal saline a challenge
toxin containing approximately 100 LD 50 in 1.0 m!. Dilute
portions of this challenge toxin solution to 2LD50' I LD 50 and
Yz LD 50 in the same solution
Determination of potency of the vaccine. Prepare in saline
solution three dilutions of the vaccine under examination and
three dilutions ofthe Standard preparation such that for each,
the dilutions form a series differing by not more than 2.5 fold
steps and in which the dilutions ofintermediate concentration,
when injected subcutaneously in 1.0 ml volumes into guinea-
2389
IP 2010
DIPHTHERIA VACCINE (ADSORBED
pigs, protect approximately 50 per cent of the animals from

the lethal effects ofthe subcutaneous injection ofthe quantity
of diphtheria toxin prescribed for this test. Allocate the six
dilutions, one to each of the six groups of sixteen guineapigs, and inject subcutaneously 1.0 ml of each dilution into
each guinea-pig in the groups to which that dilution is
allocated. After 28 days inject subcutaneously into each animal
in the six groups of sixteen, 1.0 ml of the challenge toxin
solution. Allocate the challenge toxin solution and the three
dilutions made from it, one to eachofthe four groups of four
guinea-pigs and inject subcutaneously 1.0 ml of each toxin
solution into each guinea-pig in the group to which that
solution is allocated. Examine the guinea pigs twice in a day,
remove dead animals and loll the animals showing definite
signs of diphtheria. Count the number ofsurviving animals 5
days later and calculate the potency of the vaccine under
examination relative to the potency of the Standard
preparation on the basis ofthe number ofanimals that survive
in each ofthe six groups ofsixteen, using appropriate statistical
methods.
The test is not valid unless (a) for the vaccine under
examination and the Standard preparation the 50.0 per cent
protective doses lie between the largest and smallest doses
ofthe preparations given to the guinea-pigs; (b) the survivors
among the four groups of guinea-pigs injected with the
challenge toxin and its dilutions indicate that the challenge
was approximately 100 LD50 and; (c) statistical analysis shows
parallelism, linearity and a significant slope of the doseresponse lines. The test may be repeated any number oftimes
but when more than one test is performed the results of all
valid tests must be combined in the estimat~ of potency.
Estimated potency should not be less than 30 ill per single
human dose. If the lower limit of 95.0 per cent confidence
interval of estimated potency is less than 30 ill per single
human dose then the limits of the 95.0 per cent confidence
interval should be within 50 to 200 ofthe estimated potency.
(c) Antibody induction method

Inject subcutaneously on each of two occasions separated
by an interval of not more than 4 weeks, one-fiftieth of the
stated human dose diluted to 1 ml with saline solution, into
each of 10 normal, healthy guinea-pigs weighing between 250
and 350 g. Not earlier than 2 weeks and not later than 3 weeks
after the second injection, collect the serum from each animal
and determine the antitoxin content of the serum of each
animal, as described under Diphtheria Antitoxin or any other
method approved by National Regulatory Authority. The
geometric mean of the antitoxin contents shall be not less
than2.0 ITiilis permfw-lfureferel1cetofueDiphfuenaantitoXiii
stannard. ---

FINAL LOT
The final bulk vaccine is distributed aseptically into sterile,
requirements given below under, Identification, Tests and
Identification
Diphtheria toxoid is identified by a suitable immunochemical
method. The following method, applicable to certain vaccines,
is given as an example. Dissolve in the vaccine under
until a clear supernatant liquid is obtained. The clear
supernatant liquid reacts with a suitable diphtheria antitoxin,
giving a precipitate.
Tests
Aluminium (2.3.9). Maximum 1.25 mgper single human dose,
used as the absorbent.
toxicity.
pH (2.4.24). 6.0 to 7.0.
Assay
Carry -ouCone-ofllw- prescri15ecl metlfods-foY-tl:nnrssay-of
(d) Any other validated serological assay in guinea pigs or

mice as approved by National Regulatory Authority

is not less than 30 IV per single human dose.
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GAS-GANGRENE ANTITOXIN (OEDEMATIENS)
Labelling. The label states (I) the human dose; (2) the minimum
Lfunits per single human dose or the minimum International
Units per single human dose ifpotency test done by challenge
method; (3) the name and the amount of the adsorbent and
preservative; (4) that the vaccine must be shaken before use;
Gas-Gangrene Antitoxin
(Oedematiens)
Gas-gangrene Antitoxin (Novyi); Anti-gas-gangrene
(Oedematiens) Serum
Gas-gangrene Antitoxin (Oedematiens) is a preparation
containing the specific antitoxic globulins obtained by
purification ofhyperimmune serum ofhorses or other suitable
animals and having the specific activity of neutralising the
alpha toxin formed by Clostric/ium oedematiens (c. novyi).
Gas-gangrene Antitoxin (Oedematiens) reconstituted where
necessary as stated on the label complies with the following
tests.
Gas-Gangrene Antitoxin (Oedematiens) has a potency of not
less than 3750 Units per ml.
Description. A clear colourless or pale yellow liquid free from
suspended particles ora freeze-dried, cream coloured powder
or pellet for reconstitution with the diluent supplied by the
manufacturer.
Identification
Specifically neutralises and renders the alpha toxin formed by
Cl. oedematiens harmless to susceptible animals or may be
identified by any other suitable in-vitro test.
Tests
Potency. Carry out the biological assay of gas-gangrene
antitoxin (oedematiens).
Biological Assay of Gas-Gangrene Antitoxin

(Oedematiens)
The potency of the gas-gangrene antitoxin (oedematiens) is
determined by comparing the dose necessary to protect mice
or other suitable animals against the lethal effects of gasgangrene toxin (oedematiens) with the dose of the Standard
Preparation of gas-gangrene antitoxin (C. oedematiens)
necessary to give the same protection. For this purpose the
Standard Preparation of gas-gangrene toxin (c. oedematiens)
and a suitable preparation of gas-gangrene toxin
(oedematien),for use as a test toxin, are required. The test
dose of the toxin is determined in the relation to the Standard
Preparation and the potency of the preparation under

examination is then determined in relation to the Standard
preparation using the test toxin.
The Standard Preparation is a dried hyperimmune horse serum
or another suitable preparation of gas-gangrene antitoxin
(oedematiens), the potency of which has been determined in
relation to the International Standard. The Unit is the specific
neutralising activity for gas-gangrene toxin (oedematiens)
contained in such an amount of the Standard Preparation as
the Ministry of Health and Family Welfare, Government of
India may from time to time indicate as the quantity exactly
equivalent to the Unit accepted for international use.
Suggested Method
Test animals. Use healthy mice having body weights such
that the difference between the lightest and heaviest is not
more than 5 g.
Test toxin. Prepare gas-gangrene toxin (oedematiens) by
growing C. oedematiens in a liquid culture medium for about
5 days, filtering aseptically and precipitating with ammonium
sulphate. The resulting precipitate, which contains the toxin,
is collected, dried over phosphorus pentoxide at a pressure of
1.5 to 2.5 kPa, powdered and kept my.
Selection oftest toxin. Select toxin for use as the test toxin by
determining the following quantities.
L+ dose - This is the smallest quantity of the toxin which,

when mixed with I Unit ofantitoxin and injected intramuscularly
into mice, causes the death of the animals within 72 hours.
LDso - This is the smallest quantity of toxin which, when
injected intramuscularly into mice, causes the death of half
the animals within 72 hours.
A suitable toxin is one which has an L+ dose in 0.5 mg or less
and contains not less than 25 LD so in an L+ dose.
Determination of test dose of toxin (L+ dose). Prepare a
that 1.0 ml contains 12.5 Units. Weigh accurately a quantity of
the dried toxin and dissolve in saline solution so that 1.0 ml
contains a precise known amount such as 10 mg. Prepare
mixtures so that 2.0 ml of each mixture contains 0.8 ml ofthe
solution of the Standard Preparation (l0 Units) and one of a
series of graded volumes of the solution of the test toxin.
Dilute each mixture with saline solution to the same final
volume (2.0 ml). Allow the mixtures to stand at room
temperature, protected from light, for 60 minutes and, using
six mice for each mixture, inject intramuscularly into each mouse
a dose of 0.2 ml of each mixture. Observe the mice thereafter
for 72 hours.
2391
GAS-GANGRENE ANTITOXIN (OEDEMATIENS)
IP 2010
The test dose of toxin is the amount present in 0.2 ml of that

mixture which contains the smallest amount oftoxin sufficient
to cause the death within 72 hours of all six mice injected
with it.
with saline solution so that 1.0 ml contains 12.5 times the test
dose. Prepare mixtures such that 2.0 rn1 ofeach mixture contains
volumes ofthe preparation under examination. Prepare further
mixtures such that 2.0 ml ofeach contains 0.8 ml ofthe dilution
of the toxin and one of a series of graded volumes, of the
Standard Preparation diluted with saline solution so that the
central graded volume contains 10 Units. Dilute each mixture
light, for 60 minutes and then inject intramuscularly a dose of
0.2 rn1 ofeach mixture into each ofsix mice under the conditions
described for determination ofthe test dose oftoxin. Observe
the mice for 72 hours. Ifnone ofthe mice is lulled, 0.2 ml ofthe
mixture contains more than 1 Unit ofantitoxin. Ifall the mice
are lulled, 0.2 ml of the mixture contains less than 1 Unit of
antitoxin.
The mixture that contains the largest volume ofthe preparation
under examination that fails to protect the mice from death
contains 10 Units. The test is not valid unless all the mice
injected with mixtures containing 0.8 ml or less ofthe solution
of the Standard Preparation die and all those injected with
mixtures containing more survive.
Storage. Store protected from light at a temperature between
2 to 8. Liquid preparations should not be allowed to freeze.
Labelling. The label states (1) the number of Units per ml,
where appropriate; (2) the animal species from which the
preparation has been made; (3) the recommended dose; (4)
the name and concentration ofany antimicrobial preservative
added; (5) that in case of a liquid preparation, it should not to
Gas-Gangrene Antitoxin (Perfringens)

Anti-gas-gangrene (perfringens) Serum
Gas-gangrene Antitoxin (Perfringens) is a preparation
containing the specific antitoxic globulins obtained by
purification ofhyperimmune serum ofhorses or other suitable
animals and having the specific activity of neutralising the
alpha toxin formed by Clostridium perfringens.
Qal':g al1gr l:ll1 e .Antito)(il1(.(>l:lrfril1gl:l.l1s1rl:lC:Q}!~ti1:l11:l:lcl.""hl:lre
tests.
Gas-Gangrene Antitoxin (perfringens) has a potency of not
less than 1500 Units per ml.
Description. A clear, colourless or pale yellow liquid free from

suspended particles or a freeze- dried, cream coloured powder
manufacturer.
Identification
Specifically neutralises and renders the alpha toxin formed by
G. perfringens harmless to susceptible animals or may be
Tests
antitoxin (perfringens).

(Perfringens)
The potency of the gas-gangrene antitoxin (perfringens) is
or other suitable animals against the lethal effects of fixed
dose of the Standard Preparation of gas-gangrene toxin
(perfringens, type A) with the dose ofthe Standard Preparation
ofgas-gangrene antitoxin (perfringens) (G. perfringens alpha
antitoxin) necessary to give the same protection. For this
purpose the Standard Preparation of gas-gangrene antitoxin
(G. perfringens alpha antitoxin) and a suitable preparation of
gas-gangrene toxin (perfringens, j:ype A),for use as a test toxin,
are required. The test dose of the toxin is determined in the
relation to the Standard Preparation and the potency of the
preparation under examination is then determined in relation
to the Standard preparation using the test toxin.
The Standard Preparation is a dried 1:3 dilution of
hyperimmune horse serum or another suitable preparation of
gas-gangrene antitoxin (perfringens), the potency of which
has been determined in relation to the International Standard.
The Unit is the specific neutralising activity for gas-gangrene
toxin (perfringens) contained in such an amount of the
Standard Preparation as the Ministry of Health and Family
Welfare, Government ofIndia may from time to time indicate
as the quantity exactly equivalent to the Unit accepted for
international use.
Suggested Method
more than Sg.
Test toxin. Prepare gas-gangrene toxin (perfringens) by
growing G. perfringens, j:ype A, in a liquid culture medium for
about 5 days, filtering aseptically and precipitating with
2392
IP 2010
GAS-GANGRENE ANTITOXIN (SEPTICUM)
ammonium sulphate. The resulting precipitate, which contains

the toxin, is collected, dried over phosphoruspentoxide at a
pressure of 1.5 to 2.5 kPa,powdered and kept dry.
injected with mixtui'es containing 2.0 ml or less ofthe solution

detennining the following quantities.
L+ dose - This is the smallest quantity of the toxin which,

when mixed with 1 Unit ofantitoxin and injected intravenously
into mice, causes the death of the animals within 48 hours.
LD jO - This is the smallest quantity of toxin which, when
injected intravenously into mice, causes the death of half the
animals within 48 hours.
A suitable toxin is one which has an L+ dose in 4 mg or less,
Determination of test dose of toxin (L+dose). Prepare a

that 1.0 ml contains 5 Units. Weigh accurately a quantity of
the dried toxin and dissolve it in saline solution so that 1.0 ml
mixtures such that 5.0 ml ofeach mixture contains 2.0 ml ofthe
solution of the Standard Preparation (10 Units) and one of a
series of graded volumes of the solution of the toxin. Dilute
each mixture with saline solution to the same final volume (5.0
mI). Allow the mixtures to stand at room temperature, protected
from light, for 60 minutes and, using six mice for each mixture,
inject intravenously into each mouse a dose of 0.5 ml of each
mixture. Observe the mice thereafter for48 hours.
The test dose of toxin is the amount present in 0.5 mlofthat
. with it.

with saline solution so that 1.0 ml contains five times the test
of the toxin and one of a series of graded volumes of the
Standard Preparation diluted with saline solution so that the
light, for 60 minutes and then inject intravenously a dose of
0.5 ml ofeach mixture into each ofsix mice under the conditions
described for detennination ofthe test dose oftoxin. Observe
the mice for 48 hours. Ifnone ofthe mice is killed, 0.5 ml ofthe
mixture contains more than 1 Unit of antitoxin. Ifall mice are
killed, 0.5 ml ofthe mixture contains less than 1 Unit ofantitoxin.

the name and concentration ofany antimicrobial preservative
added; (5) that in case ofa liquid preparation, it should not be
allowed to freeze.
Gas-Gangrene Antitoxin (Septicum)

Anti-gas-gangrene (Septicum) Serum
Gas-gangrene Antitoxin (Septicum) is a preparation containing
the specific antitoxic globulins obtained by purification of
hyperimmune serum of horses or other suitable animals and
having the specific activity of neutralising the alpha toxin
fonned by Clostridium septicum.
Gas-gangrene Antitoxin (Septicum) reconstituted where
tests.
Gas-gangrene Antitoxin (Septicum) has a potency of not less
than 1500 Units perml.

suspended particles or a freeze-dried, cream coloured powder
manufacturer.
Identification
Specifically neutralises and renders the alpha toxin fonned by
C. septicum harmless to susceptible animals or may be
Tests
antitoxin (septicum).

(Septicum)
The potency of the gas-gangrene antitoxin (septicum) is
or other suitable animals against the lethal effects of gasgangrene toxin (septicum) with the dose of the Standard
Preparation of gas-gangrene antitoxin (c. septicum)
necessary to give the same protection. For this purpose the
2393
IP 2010
GAS-GANGRENE ANTITOXIN (SEPTICUM)
Standard Preparation of gas-gangrene antitoxin (c. septicum)

and a suitable preparation of gas-gangrene toxin (septicum),
for use as a test toxin, are required. The test dose ofthe toxin
is determined in the relation to the Standard Preparation and
the potency of the preparation under examination is then
determined in relation to the Standard preparation using the
test toxin.
from light, for 60 minutes and, using six mice for each mixture,
inject intravenously intoeach mouse a dose of 0.5 ml of each
mixture. Observe the mice thereafter for 72 hoUrs.

with saline solution so that 1.0 ml contains five times the test
of the toxin and one of a series of graded volumes of the
Standard Preparation diluted with,saline solution so that the
with saline solution to the same final volume (5.0 mI). Allow
light, for 60 minutes and then inject intravenously a dose of
0.5 ml ofeach mixture into each ofsix mice under the conditions
described for determination ofthe test dose of toxin. Observe
themice for 48 hours. Ifnone ofthe mice is killed, 0.5 mlofthe
mixture contains more than 1 Unit of antitoxin. If all the mice
are killed, 0.5 ml of the mixture contains less than 1 Unit of
antitoxin.
The Standard Preparation is a dried 1: 3 dilution of

hyperimmune horse serum of gas-gangrene antitoxin
(septicum) in phosphate-buffered saline or another suitable
preparation the potency of which has been determined in
relation to the International Standard. The Unit is the specific
neutralising activity for gas-gangrene toxin (septicum)
contained in such an amount of the Standard Preparation as
the Ministry of Health and Family Welfare, Government of
India may from time to time indicate as the quantity exactly
equivalent to the Unit accepted for international use.
Suggested Method
more than 5 g.
Test toxin. Prepare gas-gangrene toxin (septicum) by growing
C. septicum (Vibrion septique) in a liquid culture medium for
about 5 days, filtering aseptically and precipitating with
ammonium sulphate. The resulting precipitate, which contains
the toxin, is collected, dried over phosphorus pentoxide at a
pressure of 1.5 to 2.5 kPa, powdered and kept dry.
detennining the following quantities.
L+ dose - This is the smallest quantity oftoxin which, when

mixed with 1 Unit ofantitoxin and injected intravenously into
mice, causes the death of the animals within 72 hours.
LDso - This is the smallest quantity of the toxin which, when

injected intravenously into mice, causes the death of half the
A suitable toxin is one which has an L+ dose in 0.5 mg or less
Determination of test dose of toxin (L+dose). Prepare a
solution of the Standard Preparation in saline solution such
that 1.0 ml contains 5 Units. Weigh accurately a quantity of
the dried toxin and dissolve in saline solution so that 1.0 ml
mixtures suchthat-5.0-ml-ofeachmixturecontains 2.0-mlofthe
solution ofthe Standard Preparation (1 0 Units) and one of a
series of graded volumes of the solution of the toxin. Dilute
each mixture with saline solution to the same final volume (5.0
rnl). Allowthe mixtures to stand at room temperature, protected

with it.

injected with mixtures containing 2.0 ml or lessofthe solution
the name and concentration of any antimicrobial preservative
added; (5) that in case of a liquid preparation, it should not to
Mixed Gas-Gangrene Antitoxin

Mixed Gas-gangrene Antitoxin is prepared by mixing Gasgangrene Antitoxin (Oedematiens), Gas-gangrene Anti-toxin
(Perfringens) and Gas=gangrene AntItoxin (Septicl.lrnfin
appropriate quantities.
Mixed Gas-gangrene Antitoxin has a potency of not less than
1000 Units per ml ofGas-gangrene Antitoxin (Oedematiens),
2394
IP 2010
HAEMOPHILUS TYPE b CONJUGATE VACCINE
not less than 1000 Units per ml of Gas-gangrene Antitoxin

(Perfringens) and not less than 500 Units per ml of Gasgangrene Antitoxin (Septicum).
the stability testing, release requirements are set for these

Description. An almost colourless or very faintly yellow liquid,

free from turbidity.
SEED LOT
Identification
Specifically neutralises and renders the alpha-toxins formed
by C. oedematiens, C. perfringens and C. septiclim harmless
to susceptible animals.
Tests
The strain of H. inflllenzae type b used in preparing

Haemophilus type b conjugate vaccine shall be identified by
a record of its history, including the source from which it was
obtained and the tests made to determine the characteristics
of the strain. The strain shall have been shown to be capable
of producing Type b polysaccharide.
Storage. Store protected from light at a temperature 2 to 8. It

should not be allowed to freeze. Liquid preparations should
not be allowed to freeze.
The production of PRP and of the canier protein is based on

defined seed lot systems. Master seed lot and working seed
lot shall be properly characterized and defined. Cultures
derived from the working seed shall have the same
characteristics as ofthe master seed lot. The sample ofculture
of single harvests taken before killing shall be tested for
contamination by examination ofGram-stained smears and by
inoculation on suitable media.
Labelling. The label states the number of Units of each

component per m!.
H. Injlllenzae Type b Polysaccharide (pRP)
Potency. Carry out the biological assay for each component

as described in the relevant individual monographs.

Vaccine
H. inflllenzae Type b is grown in a liquid medium that does

not contain high-molecular-weight polysaccharides; if any
ingredient of the medium contains blood-group substances,
the process shall be validated to demonstrate that after the
purification step they are no longer detectable. The culture
may be inactivated. PRP is separated from the culture liquid
and purified by a suitable method. Volatile matter, including
water, In the purified polysaccharide is determined by methods
such as thermogravimetry, Karl Fischer or any other suitable
method. All chemical analysis shall be based on the dry weight
of the polysaccharide, in its salt form.
Haemophilus Type b Conjugate Vaccine is a liquid or freezedried preparation ofa polysaccharide, derived from a suitable
strain of Haemophillis inflllenzae type b, covalently bound
to a carrier protein. The polysaccharide, polyribosylribitol
phosphate, referred to as PRP, is a linear copolymer composed
of repeated units of 3-~-D-ribofuronosyl-(l-71)-ribitol-5phosphate [(C IOH tPtl)Il]' with a defined molecular size. The
carrier protein, when conjugated to PRP, is capable ofinducing
a T-cell-dependent B-cell immune response to the
polysaccharide.
Only those pools of PRP that comply with the following

requirements may be used in the preparation ofthe conjugate.
The partially purified PRP shall be stored frozen at or
below -20.
Production
Identification
General provisions
The PRP is identified by an immunochemical method (2.2,14)

or other suitable method (e.g. IH or 13C NMR spectroscopy).

consistently H. inflllenzae tYpe b conjugate vaccines of
adequate safety and immunogenicity in humans. The
production method is validated to demonstrate that the product,
iftested, would comply with the test for safety and efficacy of
Vaccines. The stability of the final lot and relevant
intermediates is evaluated using one or more indicator tests.
Such tests may include determination of molecular size,
determination of free PRP in the conjugate and the
immunogenicity test on mice. Taking account ofthe results of
Molecular size. The percentage ofPRP eluted before a given

K o value or within a range of K o values, is determined by gel
filtration or high performance size-exclusion chromatography
(HPSEC) (2.4.16), either alone or in combination with light
scattering and refractive index detectors (e.g. multiple angle
laser light scattering i.e. MALLS) or any other suitable method.
An acceptable value is established for the particular product
and each batch of PRP must be shown to comply with this
limit. Limits for currently approved products, using the
2395
IP 2010
Table 1 - Specifications for different components ofHaemophilus Type b ConjugateVaccine.

Polysaccharide
TypeofPRP
Nominal
amount
per dose
Carrier Protein
Conjugation
Type
Purity
Nominal
amount
per dose
Coupling
method
Procedure
Polysaccharide 25 flg
(size reduced)
K o 60;0 per cent:
0.6-0.7
Diphtheria
toxoid
>1500 Lfpermg
of protein nitrogen
18 flg
Cyanogen
bromide
activation
ofPRP
Activated diphtheria
toxoid (D-AH+),
cyanogen bromide
activated PRP
Polysaccharide:
PRP 2': 50.0 per
cent :::Ko: 0.30
Tetanus
toxoid
>1500 Lfpermg
of protein nitrogen
20flg
Carbodiimidemediated
coupling
ADH-activated PRP
(pRP-cov.-AH) +
tetanus toxoid +
EDAC
CRM197
diphtheria
protein
>90.0 per cent

diphtheria
protein
25flg
Reductive
amination one(step method) or
Direct coupling of
PRP to CRM 197
(cyanoboro-hydride
activated)
10flg
Polysaccharide 10 flg
(size reduced)
Dp=15-35 or 10-35
N-hydroxy~
succinimide
activation
Polysaccharide
(size-reduced)
K o.3-0.6
15 flg
Meningococcal
group B outer
membrane
protein (OMP)
Thioether
bond
Outer membrane
125 or
protein vesicles
250 flg
<8.0 per cent of
lipopolysaccharide
PRP activation by
CDIPRP-IM+
BuA2+ BrAc=
PRP-BuA2-BrAc+
OMP
Abbreviations:
ADH
BrAc
BuA2
CDl
adipic acid dihydrazide

bromoacetyl chloride
butane-l, 4-diarnide
carbonyldi-imidazole
Dp
EDAC
1M
Mw
degree ofpolymerization
l-ethyl-3-(3-dimethylaminopropyl) carbodimide
imidazoliuill
weight-average molecular weight.
Table 2 - Requirements on bulle conjugate
Test
Protein Carrier
SpecificatiOlis
Diphtheria toxoid
Free Polysaccharide (PRP)
<37.0 per cent
Unbound protein
Tetanus toxoid
CRM 197
OMP
<20.0 per cent
<25.0 per cent
<15.0 per cent
<5.0 per cent,

where applicable
<1.0 per cent,

where applicable
<1.0 per cent or

<2.0 per cent,
depending oh the
coupling method
Not applicable
PRP to protein ratio
1.25-1.75
0.30-0.55
0.3-0.7
0.05-0.1
Molecular size (Ko): Cross-
95.0 per cent <0.75
60.0 per cent <0.2
50.0 per cent
85.0 per cent < 0.25
chromatography R
Cross-linked agarose for
chromatography Rl
0.6-0.7
85.0 per cent <0.5
2396
IP 2010
indicated stationary phases, are shown for information in

Tables 1 and 2. Where applicable, the molecular-size
distribution is also determined after chemical modification of
the polysaccharide.
A validated determination of the degree ofpolymerization or
ofthe weight-average molecular weight and the dispersion of
molecular masses may be used instead ofthe determination of
molecular size distribution.
Ribose (2.7.1). Not less than 32.0 per cent, calculated with
reference to the dried substance, as estimated by Bial reaction
for pentose, using D-ribose as a standard or any other suitable
assay.
Phosphorus (2.7.1). 6.8 per cent to 9.0 per cent, calculated
with reference to the dried substance.
Protein (2.3.49). Not more than 1.0 per cent, calculated with
reference to the dried substance. Use sufficient PRP to allow
detection of 1 per cent ofprotein (e.g. a minimum of 1 mg of
PRP).
Nucleic acid (2.7.1). Not more than 1.0 per cent, calculated
with reference to the dried substance by spectroscopy or any
other suitable method.
Bacterial endotoxins (2.2.3 ). Not more than 25 ill ofendotoxin
per microgram ofPRP.
Residual reagents. Where applicable, tests are carried out to
determine residues of reagents used during inactivation and
purification. An acceptable value for each reagent is
established for the particular product and each batch of PRP
must be shown to comply with this limit. Where validation
studies have demonstrated removal of a residual reagent, the
test on PRP may be omitted.
Carrier protein
The carrier protein is chosen in a way so that when the PRP is
conjugated it is able to induce a T-cell-dependent immune
response. Currently approved carrier proteins and coupling
methods are listed for information in Table 1. The carrier
proteins are produced by culture of suitable microorganisms;
the bacterial purity of the culture is verified; the culture may
be inactivated; the carrier protein is purified by a suitable
method.
Only a carrier protein that complies with the following
requirements may be used in preparation of the conjugate.
Diphtheria toxoid. Diphtheria toxoid is produced as stated

under Diphtheria Vaccine (Adsorbed) and complies with the
requirementsprescribed there for bulk purified toxoid.
Tetanus toxoid. Tetanus toxoid is produced as stated under
Tetanus Vaccine (Adsorbed) and complies with the
requirements prescribed there for bulle purified toxoid except
that the antigenic purity is not less than 1500 Lf per mg of
protein nitrogen.
Diphtheria protein CRM 197. Suitable tests are carried out,
for validation or routinely, to demonstrate that the product is
non-toxic. The protein obtained contains not less than 90.0
per cent of diphtheria CRM 197 protein, when prepared by
liquid chromatography (2.4.14) or any other suitable method.
The carrier protein shall be characterized by a suitable chemical
or physicochemical method like SDS-PAGE, HPLC, isoelectric
focusing, amino acid sequencing, circular dichroism,
fluorescence spectroscopy, peptide mapping or mass
spectroscopy, as appropriate.
OMP (Meningococcal group B outer membrane protein
complex)
aMP complex of Neisseria meningitidis complies with the
following requirements for lipopolysaccharide and pyrogens.
Lipopolysaccharide. Not more than 8.0 per cent of

lipopolysaccharide, determined by a suitable method.
Pyrogens (2.2.8). Inject into each rabbit 0.25 jJ.g ofaMP per kg
body weight, for determining the pyrogenic effect.
Bulk conjugate
PRP is chemically.modified to enable conjugation; it is usually
partly depolymerised either before or during this procedure.
Reactive functional groups or spacers may be introduced into
the carrier protein or PRP prior to conjugation. The conjugate
is obtained by the covalent binding ofPRP and carrier protein.
Where applicable, unreacted but potentially reactogenic
functional groups are made unreactive by means of capping
agents; the conjugate is purified to remove reagents. Where
validation studies have demonstrated removal of a residual
reagent (eg. CN, Br etc.), the test on bulle conjugate may be
omitted.
Only a bulk conjugate that complies with the following

requirements may be used in preparation of the final bulk
vaccine. For each test and for each particular product, limits
The carrier protein is identified by a suitable immunochemical

method (2.2.14)..
of acceptance are established and each batch of conjugate

must be shown to comply with these limits. Limits applied to
cunently approved products for some of these tests are listed
for information in Table 2.
medium 10 ml or the equivalent of one hundred doses,
whichever is less.
PRP. The PRP content is determined by assay ofphosphorus

(2.7.1) or by assay ofribose (2.7.1) or by an immunochemical
method (2.2.14) or by any suitable method.
Identification
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HAEMOPHJLUS TYPE b CONJUGATE VACCINE
IP 2010
Protein (2.7.1). The protein content is determined by a suitable

chemical method.
PRP to protein ratio. Determine the ratio by calculation.
Identification
The vapcine is identified by a suitable irnmunochernical method
(2.2.14) for PRP.
Molecular size. Molecular-size distribution is determined by

gel filtration or size-exclusion chromatography (2.4.16), using
a gel matrix, appropriate to the expected size ofthe conjugate.
Tests

conjugate, for example by size-exclusion or hydrophobic
chromatography (2.4.16), ultra-filtration or other validated
methods.

toxicity.
Free carrier protein. Free carrier protein is determined by a

suitable method (which may include deriving the content by
calculation from the results of other tests). The amount is
within the limits approved for the particular product.

per kg ofthe.rabbit's mass a quantity ofthe vaccine equivalent
to 1 Ilg ofPRP for a vaccine with diphtheria toxoid or CRM 197
diphtheria protein as carrier; O.lllg ofPRP for a vaccine with
tetanus toxoid as carrier; 0.025 Ilg ofPRP for a vaccine with
OMP as carrier.
pH (2.4.24). The pH ofthe vaccine, reconstituted ifnecessary,
is within the range approved for the product.
Unreacted functional groups. No unreacted functional groups

are detectable in the bulk conjugate unless process validation
has shown that unreacted functional groups detectable at
this stage are removed during the subsequent manufacturing
process (for example, owing to short half"life);
Aluminium (2.3.9). Not more than 1.25 mg per single human

dose. When aluminium hydroxide or hydrated aluminium
phosphate is tised as the -adsorbent;
Residual reagents. Removal of residual reagents such as

cyanide, EDAC (ethyldimethylaminopropylcarbodimide) and
phenol is confirmed by suitable tests or by validation of the
purification process.

greater than 115.0 per cent of that stated on the label.
Sterility (2.2.11). Carry out the tests for sterility using for
each medium 10 ml or the equivalent of one hundred doses,
whichever is less.
FINAL BULK VACCINE
An adjuvant, an antimicrobial preservative and a stabilizer

may be added to the bulle conjugate before dilution to the final
concentration with a suitable diluent.
requirements may be used in preparation ofthe final lot.
FINAL LOT
The final lots are filled in suitable containers, under stringent
aseptic conditions.
Qply a filla11Qt tl1fl.t i!i !ill,ti!ifll,c:tQry witl1.!~!ill.el.c:t tO~fl.c:l!.Qft!lt;:
requirements given under Identification, Tests and Assay may
be released for use. Provided the test for antimicrobial
preservative has been carried out on the final bulk vaccine, it

PRP. Not less than 80.0 per cent and not greater than 120.0
per cent of the amount of PRP stated on the label as
determined by ribose assay (2.7.1) or by phosphorus assay
(2.7.1) or by an immunochemical method (2.2.14) or by any
other. suitable method like colorimetery or by anion exchange
liquid chromatography (2.4.14) with pulsed amperometric
detection.
conjugate for example by size-exclusion or hydrophobic
chromatography (2.4.16), ultra filtration or other validated
methods.
Labelling. The label states (l) the number of micrograms of
PRP per human dose; (2) the typeand nominal amount of
carrier protein per single human dose; (3) for vaccine contained
in single-dose containers where the space is too small to
accommodate the full name of the vaccine the abbreviation
'Hib' may be used in the label on the container provided that
the same code is also stated in the label on the package.
Hepatitis A (Inactivated) and Hepatitis

B (rDNA) Vaccine (Adsorbed)
Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine
(Adsorbed) is a suspension consisting of a suitable strain of
hepatitis A virus, grown in cell cultures and inactivated by a
2398
HEPATITIS B VACCINE (rDNA)
IP 2010
validated method, and ofhepatitis B surface antigen (HBsAg),

a component protein of hepatitis B virus obtained by
recombinant DNA technology; the antigens are adsorbed on
a mineral carrier such as aluminium hydroxide or hydrated
aluminium phosphate.
Production
General provisions
The two components are prepared as described in the
monographs on Hepatitis A Vaccine (Inactivated, Adsorbed)
and Hepatitis B Vaccine (rDNA) and comply with the
requirements prescribed therein. The production method is
validated to demonstrate that the product, if tested, would
comply with the test for abnormal toxicity for antisera and
vaccines.
Reference preparation
The reference preparation is part of a representative batch
shown to be at least as immunogenic in animals as a batch
that, in clinical studies in young, healthy adults, produces not
less than 95.0 per cent seroconversion, corresponding to a
level of neutralizing antibody recognized to be protective,
after a full-course primary immunization. For hepatitis A, an
antibody level not less than 20 mIU/ml determined by enzymelinked immunosorbent assay is recognized as being protective.
For hepatitis B, antibody level not less than 10 mill/ml against
HBsAg is recognized as being protective.
carried out with satisfactory results on the final bulle vaccine,

it may be omitted on the final lot.
Identification
The vaccine is shown to contain hepatitis A virus antigen and
hepatitis B surface antigen by suitable immunochemical
methods (2.2.14), using specific antibodies or by the mouse
immunogenicity tests described under assay.
Tests
Aluminium (2.3 .9). Maximum 1.25 mg per single human dose
Bacterial endotoxins (2.2.3). Less than 2 IV per human dose.
Assay
Hepatitis A component
Complies with the assay as stated under Inactivated Hepatitis
A Vaccine (Adsorbed).
FINAL BULK VACCINE

The final bulk vaccine is prepared from one or more inactivated
harvests of hepatitis A virus and one or more batches of
purified antigen ofHepatitis B (rDNA).
FINAL LOT
Only a final lot that complies with each of the requirements
released for use. Provided that the tests for free formaldehyde
(where applicable) and antimicrobial preservative content
(where applicable) have been carried out on the final bulle
vaccine with satisfactory results, they may be omitted on the
final lot. If the assay of the hepatitis A and/or the hepatitis B
component is carried out in vivo, then provided it has been
Complies with the-assay as stated under Hepatitis B Vaccine

(rDNA).
Labelling.The label states (1) the amount ofhepatitis A virus
antigen and hepatitis B surface antigen per container; (2) the
type of cells used for production of the vaccine; (3) the name
and amount of the adsorbent used; (4) that the vaccine must
be shaken before use; (5) that the vaccine must not be frozen.

Hepatitis B Vaccine (rDNA) is a non-infectious preparation
containing the purified major surface antigen of Hepatitis B
virus (HBsAg). This preparation is white or almost white
translucent liquid in which the mineral carrier tends to settle
down slowly on keeping but is free from foreign' particles/
floccules.
Production
General provisions
The antigen is manufactured by recombinant DNA technology
by culturing genetically engineered yeast cells or other suitable
2399
IP 2010
cell lines which carry the gene that codes for major surface
antigen ofthe Hepatitis-B virus as approved by the competent
authority. Several physico-chemical steps are employed to
purify the Hepatitis-B surface antigen (HBsAg). The vaccine
may contain the product of the S gene (major protein), a
combination of the S gene and pre-S2 gene products (middle
protein) or a combination ofS gene, the pre-S2 gene, and preSI gene products (large protein). The purity of the antigen is
determined by comparison with a reference preparation using
liquid chromatography or other suitable methods such as SDSPAGE with any suitable staining method. The purified antigen
is finally adsorbed on aluminium hydroxide or aluminium
phosphate.
The method used for production of the vaccine must have
been shown to yield a product consistently complying with
the requirements for immunogenicity and safety. It must also
have been shown to induce specific, protective antibodies in
human beings.
The production method must be validated to demonstrate
that the product if tested; would comply with the tests for
Reference preparation. A part of a batch shown to be at least
as immunogenic as a batch that was used in clinical studies
and approved by National Regulatory Authority and
detennined by any suitable method. For hepatitis B, antibody
level not less than 10 mIDIml against HBsAg is recognized as
being protective..
Characterisation ofthe substance

Development studies are carried out to characterize the
antigen. The complete protein, lipid and carbohydrate structure
ofthe antigen is established. The morphological characteristics
ofthe antigen particles are established by electron microscopy.
The buoyant density oftheantigen particles is determined by
a physico-chemical method, for example gradient
centrifugation. The antigenic epitopes are characterized. The
protein fraction ofthe antigen is characterized in terms ofthe
primary stmcture (for example, by detennination ofthe aminoacid composition, by partial amino-acid sequence analysis).
Total protein (2.3 .49). The total protein is determined by a

validated method. The content is within the limits approved
for the specific product.
Antigen content and identification. The quantity and
specificity of HBsAg is determined in comparison with the
International standard for HBsAg subtype ad or an in-house
reference, by a suitable immunochemical method such as
radioimmunoassay (RIA), enzyme-linked immunosorbent
assay (ELISA), immunoblot (preferably using a monoclonal
antibody directed against a protective epitope) or single radial
diffusion. The antigen/protein ratio is within the limits approved
The molecular weight of the major band in a sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
under reduced conditions corresponds to the value expected
from the gene sequence and possible glycosylation.
Antigenic purity. The purity of the antigen is determined by
comparison with a refereIice preparation using liquid
chromatography or other suitable methods such as SDS-PAGE
with staining. Asuitable method is sensitive enough to detect
a potential contaminant at a concentration of 1.0 per cent of
total protein. Not less than 95.0 per cent of the total protein
consists of hepatitis B surface antigen.
Composition. The content of proteins, lipids, nucleic acids
and carbohydrates is detelmined.
Host-cell and vector-derived DNA (2.2.15). Ifmammalian cells
are used for production, not more than 10 pg of DNA in the
quantity ofpurified antigen equivalent to a single human dose
of vaccine.
Caesium. If a caesium salt is used during production, a test
for residual caesium is carried out on the purified antigen. The
content is within the limits approved for the specific product.
Sterility (2.2.11). The purified antigen complies with the test
for sterility, carried out using 10 ml for each medium.
Additional tests on the purified antigen may be required
depending on the production method used: for example, a test
for residual animal semm where mammalian cells are used for
production or tests for residual chemicals used during
extraction and purification.

FINAL BULK VACCINE
Identity, microbial purity, plasmid retention and consistency
of yield are determined at suitable production stages. If
mammalian cells are used, tests for extraneous agents and
mycoplasmas are performed in accordance with tests for
extraneous agents in viral vaccines for human use.
PURIFIED ANTIGEN
Only a purified antigen that complies with the following
requirements may be used in the preparation ofthe final bulle.
An antimicrobial preservative and an adjuvant may be

included in the vaccine.
greater than 115.0 per cent. of the intended amount.
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IP 2010
Sterility (2.2.11). Carty out test for sterility using 10 ml ofbulk

FINAL LOT
released for use. Provided that the tests for free formaldehyde,
antimicrobial preservative content and the assay in animals,
where applicable, have been carried out on the final bulle
final lot. The estimate ofpotency shall be expected in terms of
Ilg/ml i.e not less than 20 Ilg/ml.
Identification
The assay or, where applicable, the electrophoretic profile,
also serves to identify the vaccine.
Tests
used as the adsorbant.
method. The content is not less than the minimum amount
shown to be effective and is not greater than 115.0 per cent of
that stated on the label.
old) that have not been previously treated with any material
that will interfere with the test will also be suitable for the test.
Use animals ofthe same sex in the test. Inject similar groups of
animals with the reference preparation ofHepatitis-B vaccine
(r DNA). One group of control animals remains unvaccinated
but is injected intraperitoneally with the same volume of the
diluent alone. Anaesthetize and bleed the animals 28 to 42
days later, keeping the individual sera separate. Assay the
individual sera for specific HBsAg antibody concentration
by a suitable immunochemical method such as ELISA or RIA.
Calculate the result of the assay by standard statistical
methods (5.7). From the distribution of reaction levels
measured on all the sera in the unvaccinated (control group),
the maximum reaction level that can be expected to occur in an
unvaccinated animal for that particular assay is determined.
Any response in the vaccinated animals that exceeds this
level is by definition seroconversion. The percentage of
animals showing seroconversion in each group is transformed
(for example, probit transformation) and a parallel line model,
using the log dose response curve, is applied to the data. The
potency of the preparation under examination relative to the
reference preparation is thus established. The test is not valid
unless (a) for both the test and reference vaccine, the EDsolies
between the smallest and the largest doses given to animals ;
(b) the statistical analysis shows no deviation from linearity
or parallelism; (c) the fiducial limits of the estimated relative
potency fall between 33.0 and 300.0 per cent ofthe estimated
potency.

the equivalent of one human dose into each rabbit.
Method B(ln vitro)
A validated test for bacterial endotoxins may be used instead

of the test for pyrogens.
The potency of the vaccine under examination is determined

by an in vitro method that has been validated against the
biological test.
Assay. The vaccine complies with the assay of Hepatitis-B

Vaccine (rDNA) described below.
Potency. The upper fiducial limit (P = 0.95) ofthe estimated
relative potency is not less than 1.0. The estimate of potency
shall be expressed in terms of Ilg/ml i.e, not less than 20 Ilg/ml.
Determine the potency either in animals (Method A) or by a
validated in vitro procedure (Method B) described below:
Method A (Biological)
The potency of the vaccine under examination is determined
in animals by comparing in given conditions its capacity to
induce specific anti-HBsAg antibodies in mice or guinea-pigs
with the same capacity as with the reference standard.
Inject intraperitoneally not less than three suitable dilutions
of the vaccine under examination diluted with adjuvant used
in the vaccine into groups ofa suitable strain ofmice, weighing
between 15 and 20 g (about 5 weeks old), of either sex
distributed randomly into several groups of mice. Healthy
guinea pigs weighing between 300 and 350 g (about 7 weeks
Enzyme Linked Immunosorbent Assay (ELISA) using

monoclonal antibodies specific for protection inducing
epitopes ofHBsAg have been shown to be suitable. Adequate
number of dilutions and replicates of the vaccine under
examination and the reference standard are employed in the
assay. The data obtained is analyzed by a parallel-line model
and may be suitably transformed for statistical evaluation.
Commercially available kits for measuring HBsAg in vitro may
be used provided they are validated to produce equally precise
and accurate results.
The test is not valid unless (a) the st\ltistical analysis shows
no deviation from linearity or parallelism; (b) the fiducial limits
ofthe estimated relative potency fall between 80.0 and 125.0
per cent of the estimated potency.
The acceptance criteria are approved for a given reference
preparation by the National Regulatory Authority in the light
of the validation data.
Labelling. The label states (a) the amount ofHBsAg per dose;
(b) the type ofcells used for production ofthe vaccine; (c) the
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IP 2010
INACTIVATED HEPATITIS A VACCINE (ADSORBED)
name and amount of the adjuvant; (d) that the vaccine must
be shaken before use; (e) that the vaccine mustnot be frozen.
Inactivated Hepatitis A Vaccine

(Adsorbed)
Hepatitis A Vaccine (Inactivated, Adsorbed) is a liquid
preparation of a suitable strain of hepatitis A virus grown in
cell cultures, inactivated by a validated method and adsorbed
on a mineral carrier. The vaccine is an opalescent suspension.
Virus concentration. The virus concentration ofeach master

and working seed lot is determined to monitor consistency of
production.
Extraneous agents (2.7.3). The master and working seed lots
comply with the requirements for seed lots for virus vaccines.
In addition, if primary monkey cells have been used for
isolation of the strain, measures are taken to ensure that the
strain is not contaminated with simian viruses such as simian
immunodeficiency virus and filoviruses.
The vaccine complies with the monograph on Vaccines.
Production
Production of the vaccine is based on a virus seed-lot system
and a cell-bank system. The production method shall have
been shown to yield consistently vaccines that comply with
the requirements for immunogenicity, safety and stability.
product, if tested, would comply with the test for safety and
efficacy.
Unless otherwise justified and authorised, the virus in the
final vaccine shall not have undergone more passages from
the master seed lot than were used to prepare the vaccine
shown in clinical studies to be satisfactory with respect to
as immunogenic as a batch that, in clinical studies in young
healthy adults, produced not less than 95.0 per cent
seroconversion, corresponding to a level of neutralising
antibody accepted to be protective, after a full-course of
primary immunisation is used as a reference preparation. An
antibody level of 20 mIU Iml determined by enzyme-linked
immunosorbent assay is recognised as being protective.
Substrate for virus propagation

The virus is propagated in a human diploid cell line or in a
continuous cell line approved by the competent authority.
SEED LOT
The strain ofhepatitis A virus used to prepare the master seed
lot shall be identified by historical records that include
manipulation.
Only a seed lot that complies with the following requirements
may be used for virus propagation.
Description. A._-clear, colourless or light coloured liquid.
-
_.~,~
Identification
Each master and working seed lot is identified as hepatitis A
virus using specific antibodies.
All processing ofthe cell bank and subsequent cell cultures is

done under aseptic conditions in an area where no other cells
are being handled. Animal serum (but not human serum) may
be used in the cell culture media. Serum and trypsin used in
the preparation of cell suspensions and media are shown to
be free from extraneous agents. The cell culture media may
contain a pH indicator, such as phenol red and approved
antibiotics at the lowest effective concentration. Not less than
500 ml ofthe cell cultures employed for vaccine production is
set aside as uninfected cell cultures (control cells). Multiple
harvests from the same production cell culture may be pooled
and considered as a single harvest.
Only a single harvest that complies with the following
requirements may be used in the preparation of the vaccine.
When the determination of the ratio of virus concentration to
antigen content has been carried out on a suitable number of
single harvests to demonstrate consistency, it may
subsequently be omitted as a routine test.
Identification
The test for antigen content also serves to identify the single
harvest.
Sterility (2.2.11). The single harvest complies with the test for
sterility, carried out using 10 ml for each medium.
Mycoplasmas (2.7.4). The single harvest complies with the
test for mycoplasmas carried out using Iml for each medium.
Control cells. The control cells ofthe production cell culture
comply with a test for identity and the requirements for
extraneous agents.
Antigen content. Determine the hepatitis A antigen content
by a suitable immunochemical method (2.2.14) to monitor
production consistency; the content is within the limits
Ratio--oLvh:us_concentration_toantigencontent.The
consistency of the ratio of the .concentration of infectious
virus, as determined by a suitable cell culture method, to
antigen content is established by validation on a suitable
number of single harvests.
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IP 2010
INACTIVATED HEPATITIS A VACCINE (ADSORBED)
PURIFICATION AND PURIFIED HARVEST
The harvest, which may be a pool of several single harvests,

is purified by validated methods. If continuous cell lines are
used for production, the purification process shall have been
shown to reduce consistently the level ofhost-cell DNA. Only
a purified harvest that complies with the following requirements
may be used in the preparation of the inactivated harvest.
Virus concentration. The concentration of infective virus in
the purified harvest is determined by a suitable cell culture
method to monitor production consistency and as a starting
point for monitoring the inactivation curve.
Antigen total protein ratio. Determine the hepatitis A virus
antigen content by a suitable immunochemical method (2.2.14).
Determine the total protein by a validated method. The ratio
of hepatitis A virus antigen content to total protein content is
Bovine serum albumin. Not more than 50 ng in the equivalent
of a single human dose, determined by a suitable
imrnunochemical method (2.2.14). Where appropriate in view
of the manufacturing process, other suitable protein markers
may be used to demonstrate effective purification.
Residual host-cell DNA (2.2.15). If a continuous celllineis
used for virus propagation, the content of residual host-cell
DNA, determined using a suitable method is not greater than
10 ng per single human dose.
Residual chemicals. If chemical substances are used during
the purification process, tests for these substances are carried
out on the purified harvest (or on the inactivated harvest),
unless validation of the process has demonstrated total
clearance. The concentration must not exceed the limits
INACTIVATION AND INACTIVATED HARVEST
Several purified harvests may be pooled before inactivation.

In order to avoid interference with the inactivation process,
virus aggregation must be prevented or aggregates must be
removed immediately before and/or during the inactivation
process. The virus suspension is inactivated by a validated
method; the method shall have been shown to be consistently
capable of inactivating hepatitis A virus without destroying
the antigenic and immunogenic activity; as part of the
validation studies, an inactivation curve is plotted representing
residual live virus concentration measured on at least three
occasions (for example, on days 0, 1 and 2 ofthe inactivation
process). Iffonnaldehyde is used for inactivation, the presence
of excess free formaldehyde is verified at the end of the
inactivation process.
Only an inactivated harvest that complies with the following
vaccine.
Inactivation. Carry out an amplification test for residual

infectious hepatitis A virus by inoculating a quantity of the
inactivated harvest equivalent to 5 per cent of the batch or if
the harvest contains the equivalent of 30,000 doses or mort<,
not less than 1,500 doses of vaccine into cell cultures of the
same type as those used for production of the vaccine and
incubating the cells for at least 28 days. Make two passages
and at the end of incubation carry out a test of suitable
sensitivity for residual infectious virus. No evidence of
hepatitis A virus multiplication is found in the samples taken
at the end of the inactivation process. Use infective virus
inocula concurrently as positive controls to demonstrate
cellular susceptibility and absence of interference.
Sterility (2.2.11). The inactivated viral harvest complies with
the test for sterility, carried out using i 0 ml for each medium.
Bacterial endotoxins (2.2.3). Not more than 2 ill ofendotoxin
in the equivalent of a single human dose.
Antigen content. Determine the hepatitis A virus antigen
content by a suitable immunochemical method (2.2.14).
Residual chemicals. As stated under Purification and Purified
Harvest.
FINAL BULK VACCINE

The final bulle vaccine is prepared from one or more inactivated
harvests. Approved adjuvants, stabilisers and antimicrobial
preservatives may be added.
FINAL LOT
The final bulle vaccine is distributed aseptically into sterile
containers. The containers are then closed so as to avoid
contamination.
(where applicable) and antimicrobial preservative content
(where applicable) and the assay have been carried out on the
final bulk vaccine with satisfactory results, these tests may be
omitted on the final lot. If the assay is carried out using mice
or other animals, then provided it has been carried with
satisfactory results on the final bulle vaccine, it may be omitted
on the final lot.
2403
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INACTNATED HEPATITIS A VACCINE (ADSORBED)
Identification
The vaccine is shown to contain hepatitis A virus antigen by
a suitable immunochemical method using specific antibodies
or by the mouse immunogenicity test described under Assay.
Tests
if hydrated aluminium phosphate or aluminium hydroxide is
Free formaldehyde (2.3.20). When formaldehyde has been
used for inactivation, the vaccine complies with the test
prescribed in Vaccines.
Calculations. Cany out the calculations by the usual statistical

methods (5.7) for an assay with a quantal response.
From the distribution of reaction levels measured on all the
sera in the unvaccinated group, determine the maximum
reaction level that can be expected to occur in an unvaccinated
animal for that particular assay. Any response in vaccinated
animals that exceeds this level is by definition a seroconversion.
Make a suitable transformation of the percentage of animals
showing seroconversion in each group (for example, a probit
transformation) and analyse the data according to a parallelline log dose-response model. Detennine the potency of the
test preparation relative to the reference preparation.

toxicity.
Validity conditions. The test is not valid unless (a) for both
the test and the reference vaccine, the ED so lies between the
smallest and the largest doses given to the animals; (b) the
statistical analysis shows no significant deviation from
linearity or parallelism; (c) the fiducial limits ofthe estimated
relative potency fall between 33.0 and 300.0 per cent of the
estimated potency.
Assay
Potency. The upper fiducial limit (P = 0.95) of the estimated

relative potency is not less than 1.0.
The vaccine complies with the assay of Hepatitis A vaccine.

The assay is carried out either in vivo, by comparing in given
condition the capacity to induce specific antibodies in mice
with the same capacity of a reference preparation or in vitro
by an immunochemical determination of antigen content
(2.2.14).
In vitro assay
Carry out an immunochemical determination ofantigen content
(2.2.14) with acceptance criteria validated against the in vivo
test. The acceptance criteria are approved for a given reference
preparation by the National Regulatory Authority in the light
of the validation data.
In vivo assay
The test on mice shown below is given as an example of a
method that has been found suitable for a given vaccine;
other validated methods may also be used.
Selection and distribution ofthe test animals. Healthy mice

from the same stock, about 5 weeks old and from a strain
shown to be suitable should be used in the test. Use animals
ofthe same sex. Distribute the animals in at least seven equal
groups of a number suitable for the requirements of the assay.
Determination ofpotency ofthe vaccine under examination.
Using a 0.9 per cent w/v solution of sodium chloride containing
the aluminium adjuvant used for the vaccine, prepare at least
three dilutions ofthe vaccine under examination and matching
dilutions of the reference preparation. Allocate the dilutions
one to each of the groups of animals and inject
subcutaneously not more than 0.5 ml of each dilution into
each animal in the group to which that dilution is allocated.
Maintain a group of unvaccinated controls, injected
Labelling. The label states (1) the biological origin ofthe cells
and; (2) the adjuvant used for the preparation of the vaccine.
Inactivated Hepatitis B Vaccine

Inactivated Hepatitis B Vaccine is a non-infectious inactivated
liquid preparation derived from the surface antigen ofHepatitis
B virus (HbsAg). This preparation is white or almost white
translucent liquid in which the mineral carrier tends to settle
down slowly on keeping but is free from foreign particles /
floccules.
Production
The antigen is harvested and purified from the plasma ofhmnan

carriers of Hepatitis B virus. The surface antigen contains all
the three antigen species (S, Pre-S 1, Pre-S2). The individual
donor plasma is shown by sensitive tests to be seronegative
subGutane0usly-withthesame-v01ume-0fdiluent~After-28-toformv~landHJ:V,,2. andforHCV..Theplasmapoolistestedfor
32 days, anaesthetise and bleed all animals, keeping the freedom from adventitious viruses and blood borne
individual sera separate. Assay the individual sera for specific transmissible pathogens by appropriate methods. The purified
antibodies against hepatitis A virus by a suitable antigen is further inactivated by a validated method, usually
with formalin or any other inactivating agent, to render the
inununochemical method (2.2.14).
2404
INACTIVATED HEPATITIS B VACCINE
lP 2010
hepatitis B virus harmless. The preparation is also tested for

the residual HBV DNA using a sensitive test approved by the
competent authority and the level is shown to be less than 1
pg HBV DNA per 50 doses.
The molecular weight of the major band in a sodium dodecyl

sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
under reduced conditions corresponds to the value expected
from the gene sequence and possible glycosylation.
The method used for production of the vaccine must have

been shown to yield a product consistently complying with
the requirements ofimmunogenicity, safety and stability. The
production method must also be validated to demonstrate
that the product, if tested, would comply with the tests for
Antigenic purity. The purity of the antigen is determined by

comparison with a reference preparation using liquid
chromatography or other suitable methods such as SDS-PAGE
with staining. A suitable method is sensitive enough to detect
a potential contaminant at a concentration of 1.0 per cent of
total protein. Not less than 95.0 per cent of the total protein
consists of hepatitis B surface antigen.

as immunogenic as a batch that produced in clinical studies in
young healthy adults not less than 95.0 per cent
seroconversion, corresponding to a level of neutralizing
antibody accepted to be protective (HbsAg antibody titre not
less than 10 mIU/rnl after a full course ofprimary immunization
determined by enzyme-linked immunosorbent assay (ELISA)
is used as a reference preparation.
Additional tests on the purified antigen may be required

depending on the production method used.
Characterisation of the substance
FINAL BULK VACCINE
Development studies are carried out to characterize the

antigen. The complete protein, lipid and carbohydrate structure
ofthe antigen is established. The morphological characteristics
ofthe antigen particles are established by electron microscopy.
The buoyant density ofthe antigen particles is determined by
a physico-chemical method (2.4.29), for example gradient
centrifugation. The antigenic epitopes are characterized. The
protein fraction ofthe antigen is characterized in terms of the
primary structure (for example, by determination ofthe aminoacid composition, by partial amino-acid sequence analysis).
An antimicrobial preservative and an adjuvant may be

included in the vaccine.
Composition. The content of proteins, lipids, nucleic acids

and carbohydrates is determined.
Sterility (2.2.11). The purified antigen complies with the test
for sterility carried out using 10 ml for each medium.


amount ofantimicrobial preservative by a suitable chemical or
physico-chemical method. The amount is not less than the
85.0 per cent and not greater than 115.0 per cent ofthat stated
on the label.
Sterility (2.2.11). Carry out test for sterility using J 0 ml of

Identity, microbial purity, plasmid retention and consistency

of yield are determined at suitable production stages.
FINAL LOT
PURIFIED ANTIGEN
Only a purified antigen that complies with the following
Total protein (2.3.49). The total protein is determined by a

validated method. The content is within the limits approved
Antigen content and identification. The quantity and
specificity of HBsAg is determined in comparison with the
International standard for HBsAg subtype ad or an in-house
reference, by a suitable immunochemical method such as radio
immunoassay (RIA), enzyme-linked immunosorbent assay
(ELISA), immunoblot (preferably using a monoclonal antibody
directed against a protective epitope) or single radial diffusion.
The antigen/protein ratio is within the limits approved for the
specific product.
Only a final lot that complies with each of the requirenlents

released for use. Provided that the tests for free formaldehyde,
antimicrobial preservative content and the assay in animals,
where applicable, have been carried out on the final bulle
final lot.
Identification
The assay or, where applicable, the electrophoretic profile,
also serves to identify the vaccine.
Tests
Aluminium (2.3.9). When hydrated aluminium phosphate or
aluminium hydroxide is used as the adsorbent, the vaccine
complies with the test prescribed in the monograph on
Vaccines.
2405
IP 2010
INACTIVATED INFLUENZA VACCINE (SPLIT VIRION)
Test for inactivating agent. The concentration of any

inactivating agent remaining in the final vaccine shall be
determined by methods approved by the competent authority.
The concentration shall not exceed a specified upper limit.
The origin and passage history of virus strains shall be

approved by the National Regulatory Authority.

Influenza virus seed to be used in the production ofvaccine is

propagated in fertilised eggs from chicken flocks free from
specified pathogens or in. suitable cell cultures, such as chickembryo fibroblasts or chick kidney cells obtained from chicken
flocks free from specified pathogens. For production, the virus
of each strain is grown in the allantoic cavity of eggs derived
from specific pathogen free flocks.

the equivalent of one human dose into each rabbit.
A validated test for bacterial endotoxins (2.2.3) may be used
instead of the test for pyrogens.
Assay
The upper fiducial limit (P = 0.95) of the estimated relative
potency is not less than 1.0.
Determine the potency by method A (I3iological)as ciescribed
under Hepatitis-B vaccine (rDNA).
Labelling. The label states (1) the amount ofHBsAg per dose;
(2) the name and amount of inactivating agent; (3) the name
and amount of the adjuvant; (4) that the vaccine must be
shaken before use; (5) that the vaccine must not be frozen.
SEED LOT
The production of vaccine is based on a seed-lot system.
Working seed lots represent not more than fifteen passages
from the approved reassorted virus or the approved virus
isolate. The final vaccine represents one passage from the
working seed lot. The haemagglutinin and neuraminidase
antigens ofeach seed lot are identified as originating from the
correct strain of influeni8.viriisbY suitable methods..
Only a working virus seed lot that complies with the following
requirements may.be used in the preparation ofthe monovalent
pooled harvest.
each medimn.
Mycoplasmas (2.7.4). Carry out the test for mycoplasmas using
lOml.
Inactivated Influenza Vaccine (Split

Virion)
Influenza Vaccine (Split Virion, Inactivated) is a sterile, aqueous

suspension of a strain or strains of influenza virus, type A or
B, or a mixture of strains ofthe two types grown individually
in eggs derived from specific pathogen free flock or cell
cultures, inactivated and treated so that the integrity of the
virus particles has been disrupted without diminishing the
antigenic properties ofthe haemagglutinin and n,euraminidase
antigens. The stated amount of haemagglutinin antigen for
each strain present in the vaccine is 15 /-lg per dose, unless
clinical evidence supports the use of a different amount.
Production
efficacy.
Choice of vaccine strain
The World Health. Organisation reviews the world
epidemiological situation annually and if necessary
recommends new strains corresponding to prevailing
epidemiological evidence.
An antimicrobial agent may be added to the inoculum. After

incubation at a controlled temperature, the allantoic fluids are
harvested and combined to form a monovalent pooled harvest.
An antimicrobial agent may be added at the time of harvest.
At no stage in the production, penicillin or streptomycin is
used.
MONOVALENT POOLED HARVEST
To limit the possibility ofcontamination, inactivation is initiated
as soon as possible after preparation. The virus is inactivated
by a method that has been demonstrated on three consecutive
batches to be consistently effective for the manufacturer. The
inactivation process shall have been shown to be capable of
inactivating the influenza virus without destroying its
antigenicity; the process should cause minimum alteration of
the haemagglutinin and neuraminidase antigens. The
inactivation.processshallalsohavebeenshownJobecapable
of inactivating avian leucosis viruses and mycoplasmas. If
the monovalent pooled harvest is stored after inactivation, it
is held at a temperature of5 30. Ifformaldehyde solution is
used, the concentration does not exceed 0.2g/l of
2406
IP 2010
INACTIVATED INFLUENZA VACCINE (SPLIT VIRION)
formaldehyde at any time during inactivation; if

betapropiolactone is used, the concentration does not exceed
0.1 per cent v/v at any time during inactivation.
Before or after the inactivation procedure, the monovalent
pooled harvest is concentrated and purified by high-speed
centrifugation or other suitable method and the virus particles
are disrupted into component subunits by the use of approved
procedures. For each new strain, a validation test is carried
out to show that the monovalent bulk consists predominantly
of disrupted virus particles.
Only a monovalent pooled harvest that complies with the
final bulle vaccine.
Haemagglutinin antigen. Determine the content of
haemagglutinin antigen by an immunodiffusion test (2.2.14),
by comparison with a haemagglutinin antigen reference
preparation or with an antigen preparation calibrated against
it. Carry out the test at 20 to 25.
For some vaccines, the physical form of the haemagglutinin

particles prevents quantitative determination by
immunodiffusion after inactivation of the virus. For these
vaccines, a determination of haemagglutinin antigen is made
on the monovalent pooled harvest before inactivation. The
production process is validated to demonstrate suitable
conservation of haemagglutinin antigen and a suitable tracer
is used for formulation, for example, protein content.
Neuraminidase antigen. The presence and type of
neuraminidase antigen are confirmed by suitable enzymatic or
immunological methods (2.2.14) on the first three monovalent
pooled harvests from each working seed lot.
each medium.
Viral inactivation. Carry out as described below under Tests.
Chemicals used for disruption. Tests are carried out on the
monovalent pooled harvest for the chemicals used for
disruption, the limits being approved by the National
Regulatory Authority.
FINAL BULK VACCINE

Appropriate quantities of the monovalent pooled harvests
are blended to make the final bulk vaccine.
FINAL LOT
requirements given below under Tests and Assay may be
released for use. Provided that the test for viral inactivation
has been performed with satisfactory results on each
monovalent pooled harvest and that the tests for free
formaldehyde, ovalbumin and total protein have been
performed with satisfactory results on the final bulk vaccine,
they may be omitted on the final lot.
Description. The vaccine is a slightly opalescent liquid.
Identification
The assay serves to confinn the antigenic specificity of the
vaccine.
Tests
Viral inactivation. Inoculate 0.2 ml of the vaccine into the
allantoic cavity of each of ten fertilised eggs and incubate at
33 to 37 for 3 days. The test is not valid unless at least eight
ofthe ten embryos survive. Harvest 0.5 ml of the allantoic
fluid from each surviving embryo and pool the fluids. Inoculate
0.2 ml ofthe pooled fluid into a further ten fertilised eggs and
incubate at 33 to 37 for 3 days. The test is not valid unless at
least eight of the ten embryoes survive. Harvest about 0; 1 ml
ofthe allantoic fluid from each surviving embryo and examine
each individual harvest for live virus by a haemagglutination
test. Ifhaemagglutination is found for any ofthe fluids, carry
out for that fluid a further passage in eggs and test for
haemagglutination; no haemagglutination occurs.
Total protein (2.3.49). Not more than six times the total
haemagglutinin content of the vaccine as determined in the
assay, but in any case, not more than 100 f!g of protein per
virus strain per human dose and not more than a total of 300
f!g of protein per human dose.
Ovalbumin. Not more than 1 f!g ofovalbumin per human dose,
determined by a suitable technique using a suitable reference
preparation of ovalbumin.


toxicity.
each sterility medium.

per human dose.
2407
IP 2010
INACTIVATED INFLUENZA VACCINE (SURFACE ANTIGEN)
Assay
Determine the content of haemagglutinin antigen by an
immunodiffusion test (2.2.14), by comparison with an
appropriate haemagglutinin antigen reference preparation.
Carry out the test at 20 to 25. The confidence interval (P =
0.95) ofthe assay is not greater than 80.0 per cent to 125.0 per
cent ofthe estimated content. The lower confidence limit (P =
0.95) of the estimate ofhaemagglutinin antigen content is not
less than 80.0 per cent of the amount stated on the label for
each strain.
For some vaccines, quantitative determination of
haemagglutinin antigen with respect to available reference
preparations is not possible. An immunological identification
of the haemagglutinin antigen and a semi-quantitative
detetmination are carried out instead by suitable methods.
under Vaccines and also states (a) that the vaccine has been
prepared on eggs; (b) the strain or strains of influenza virus
used to prepare the vaccine; (c) the method of inactivation;
(d) the haemagglutinin content in fJ.g per virus strain per dose;
(e) the season during which the vaccine is intended to protect.
specified pathogens or in suitable cell cultures, such as chickembryo fibroblasts or chick kidney cells obtained from chicken
from SPF flocks.
SEED LOT
The production of vaccine is based on a seed-lot system.
Working seed lots represent not more than fifteen passages
correct strain of influenza virus by suitable methods.
requirements may be used in the preparation ofthe monovalent
pooled harvest.
each medium.
lOmi.
Inactivated Influenza Vaccine (Surface

Antigen)
Influenza Vaccine (Surface Antigen, Inactivated) is a sterile,
aqueous suspension of a strain or strains of influenza virus,
type A or B, or a mixture of strains of the two types grown
individually in eggs derived from specific pathogen free flocks
or cell cultures, inactivated and treated so that the preparation
consists predominantly ofhaemagglutinin and neuraminidase
antigens, without diminishing the antigenic properties ofthese
antigens. The stated amount of haemagglutinin antigen for
each strain present in the vaccine is 15 fJ.g per dose, unless
clinical evidence supports the use of a different amount.
Production
efficacy.
The World Health Organisation reviews the world
epidemiological situation annually and if necessary
approve_db~Jhe competentauthority.__
_
Substrate for virus propagatioll

harvested and combined to form a monovalent pooled harvest.
At no stage in the production penicillin or streptomycin is
used.
inactivation process shall also have been shown to be capable
is held at a temperature of53. If formaldehyde solution is
used, the concentration does not exceed 0.2 gil of
0.1 per cent vlv at any time during inactivation.
Before or after the inactivation process, the wonovalent
p_o_oled_hanrest.is._concentrate&andpurifiedbyhigh~speed
c~ntrifugati()l1.gr. otlle! . sl!itll1:>le.l11etl1()Q, Yil:us. pr:lJiiclesare

disrupted into component subunits by approved procedures
and further purified so that the monovalent bulk consists
mainly of haemagglutinin and neuraminidase antigens.
2408
IP 2010
INACTIVATED INFLUENZA VACCINE (SURFACE ANTIGEN)

Description. The vaccine is a clear liquid.

it. Carry out the test at 20 0 to 25 0
.
Neuraminidase antigen. The presence and type of
neuraminidase antigen are confinued by suitable enzymatic or
Sterility (2.2.11). Carry out the test for sterility, using
10 ml for each medium.
Viral inactivation. Carry out the test described below under
Tests.
Purity. The purity of the monovalent pooled harvest is
examined by polyacrylamide gel electrophoresis or by other
approved techniques. Mainly haemagglutinin and
neuraminidase antigens shall be present.
Chemicals used for disruption and purification. Tests are
carried out on themonovalent pooled harvest for the chemicals
used for disruption and purification, the limits being approved
by the competent authority.
FINAL BULK VACCINE
each medium.
Identification
The assay serves to confinn the antigenic specificity of the
vaccine.
Tests
33 0 to 37 0 for 3 days. The test is not valid unless at least eight
of the ten embryos survive. Harvest 0.5 ml of the allantoic
0.2 ml of the pooled fluid into a further ten fertilised eggs and
incubate at 33 0 to 37 0 for 3 days. The test is not valid unless at
least eight ofthe ten embryos survive. Harvest about O.lml of
the allantoic fluid from each surviving embryo and examine
each indiviclual harvest for live virus by a haemagglutination
test. Ifhaemagglutination is found for any of the fluids, carry
Total protein (2.3.49). Not more than 40 Ilg of protein other
than haemagglutinin per virus strain per human dose and not
more than a total of120 Ilg ofprotein other than haemagglutinin
per human dose.
Ovalbumin. Not more than 11lg ofovalbumin per human dose,
detennined by a suitable technique using a suitable reference
Free formaldehyde (2.3.20). Complies with the test for free
fonnaldehyde as stated under General Requirements for
Vaccines for Human Use.
Abnormal toxicity (2.2.1). Complies with the test for abnonnal
toxicity.
FINAL LOT

per human dose.

Assay

Assay may be released for use. Provided that the test for viral
inactivation has been perfonned with satisfactory results on
each monovalent pooled harvest and that the tests for free
formaldehyde, ovalbumin and total protein have been
perfonned with satisfactory results on the final bulle vaccine,
Determine the content haemagglutinin antigen by an

appropriate haemagglutinin antigen reference preparation.
Carry out the test at 20 0 to 25 0 The confidence interval
(P = 0.95) ofthe assay is not greater than 80.0 per centto 125.0
per cent of the estimated content. The lower confidence limit
(P = 0.95) ofthe estimate ofhaemagglutinin antigen content is
not less than 80.0 per cent of the amount stated on the label
for each strain.
2409
IP 2010
INACTIVATED INFLUENZA VACCINE (WHOLE VIRION)
Labelling. The label states (1) that the vaccine has been
prepared on eggs; (2) the strain or strains of influenza virus
used to prepare the vaccine; (3) the method of inactivation;
(4) the haemagglutinin content in micrograms per virus strain
per dose; (5) the season during which the vaccine is intended
to protect.

correct strain of influenza virus by suitable methods.
pooled harvest.
each medium.
Inactivated Influenza Vaccine (Whole

Virion)
IOml.
Influenza Vaccine (Whole Virion, Inactivated) is a sterile,

aqueous suspension of a strain or strains of influenza virus,
type A or B, or a mixture of strains of the two types grown
individually in eggs derived from specific pathogen free flocks
or cell culture and inactivated in such a manner that their
antigenic properties are retained. The stated amount of
haemagglutinin antigen for each strain present in the vaccine
is 15 J.l.g per dose, unless clinical evidence supports the use of
a different amount.
Production
efficacy.
The World Health Organisation reviews the world
epidemiological situation annually and, if necessary,
approved by the competent authority.
specified pathogens or in suitable cell cultures, such as chickembryo fibroblasts or chick kidney cells obtained from chicken
from specific pathogen free flocks.
SEED LOT
The_productiolLoLvaccine_is_basecLolLa__see<hloLsystem.
Working Seed lots represent not more thanfifteell passages

harvested and combined to fonn a monovalent pooled harvest.
At no stage in the production is penicillin or streptomycin
used.
inactivation process shall also have been shown to be capable
is held at a temperature of 53 0. Iffonnaldehyde solution is
used, the concentration does not exceed 0.2 gil of
0.1 per cent vlv at any time during inactivation.
Before or after the inactivation process, the monovalent
pooled harvest is concentrated and purified by high-speed
centrifugation or other suitable method.
final bulk vaccine.
it. CafI}'_()l:!t th.e test at?.2_t()}5_:..
_
Neununinid_ase_antigen._ ThepJ'es_enG.eaod type oJ
neuraminidase antigen are confinned by suitable enzymatic or
2410
JAPANESE ENCEPHALITIS VACCINE (HUMAN)
IP 2010
each medium.
Viral inactivation. Carry out the test described below under
Tests.
assay, but in any case, not more than 100 /lg of protein per
virus strain per human dose and not more than a total of
300 /lg ofprotein per human dose.
FINAL BULK VACCINE
Ovalbumin. Not more than l/lg ofovalbumin per human dose,

determined by a suitable technique using a suitable reference



greater than 115.0 per cent of the quantity stated on the label.
toxicity.

Bacterial endotoxins (2.2.3). Not more than 100 IU ofendotoxlll

per human dose.
FINAL LOT
Assay
Determine the content of haemagglutinin antigen by an

Only a final lot that is satisfactory with respect to each ofthe appropriate haemagglutinin antigen reference preparation.
requirements given below under Tests and Assay may be Carry out the test at 20 to 25. The confidence interval
released for use. Provided that the test for viral inactivation (P = 0.95) oftheassay is not greater than 80.0 per centto 125.0
has been performed with satisfactory results on each' per cent of the estimated content. The lower confidence limit
monovalent pooled harvest and that the tests for free (P = 0.95) ofthe estimate ofhaemagglutinin antigen content is
formaldehyde, ovalbumin and total protein have been not less than 80.0 per cent of the amount stated on the label
performed with satisfactory results on the final bulle vaccine, for each strain.
Labelling. The label states (1) that the vaccine has been
prepared
on eggs; (2) the strain or strains of influenza virus
Description. The vaccine is a slightly opalescent liquid.
used to prepare the vaccine; (3) the method of inactivation;
Identification
(4) the haemagglutinin content in micrograms per virus strain
per
dose; (5) the season during which the vaccine is intended
The assay serves to confirm the antigenic specificity of the
to
protect.
vaccine.
Tests
33 to 37 for 3 days. The test is not valid unless at least eight
of the ten embryos survive. Harvest 0.5 ml of the allantoic
0.2 ml ofthe pooled fluid into a further ten fertilised eggs and
incubate at 33 to 37 for 3 days. The test is not valid unless at
least eight ofthe ten embryos survive. Harvest about 0.1 mlof
the allantoic fluid from each surviving embryo and examine
each individual harvest for live virus by a haemagglutination
test. Ifhaemagglutination is found for any ofthe fluids, carry
Total protein (2.3.49). Not more than six times the total
haemagglutinin content of the vaccine as determined in, the
Japanese Encephalitis Vaccine

(Human)
Japanese Encephalitis Vaccine for human use'is a liquid or
freeze dried preparation ofJapanese encephalitis virus grown
in approved substrate and inactivated by a validated method.
PrOduction
General provisions
The vaccine is produced on the basis of virus seed lot system
consistently the vaccines that comply with the tests for
immunogenicity, safety and stability. The production method
is validated to demonstrate that the product, if tested, would
comply with the test for safety and efficacy.
2411
JAPANESE ENCEPHALITIS VACCINE
(~
IP 2010

The virus is propagated in an approved cell substrate like a
Vero cell line.
SEED LOT
The strain of Japanese encephalitis virus used shall be
identified by historical records that include information on
the origin of the strain and its subsequent manipulation.
The National Regulatory Authority shall determine the
acceptable number ofpassages from the master virus seed lot
to produce working virus seed lots.
Only a working seed lot that complies with the following tests
Virus harvests that comply with the tests given under

Identification and Virus concentration are pooled in the
preparation of the inactivated viral harvest.
from which the single harvest is derived should comply with
the test for identification and with the tests for extraneous
agents (2.7.3).
Purification and inactivation
Each working seed lot is identified as Japanese encephalitis

virus using specific antibodies by an approved method.
The virus harvest may be concentrated and/or purified by

suitable methods; the virus harvest is inactivated by a validated
method at a fixed, well defined stage ofthe process which may
be before, during or after any concentration or purification.
The method shall have been shown to be capable of
inactivating Japanese encephalitis virus without destruction
of the immunogenic activity. If formalin is used, the
concentration shall at no time exceed 1:2000.
Virus concentration. The virus concentration ofeach working

seed lot is determined by a cell culture method using
immunofluorescence or any other approved method.
Only an inactivated viral suspension that complies with the

following tests may. be .used in the preparation. of the fmal
bulle vaccine.
Identification
Extraneous agents (2.7.3). The working seed lot complies with

the tests for the virus seed lots.
a) Mouse brain vaccine
The vaccine is prepared by using a seed-lot system. An
approved strain ofvirus is grown by inoculating intracerebrally
into healthy mice. Virus harvests are pooled, concentrated
and inactivated by addition of formalin or any other suitable
inactivating agent. It may contain a suitable preservative. The
vaccine may be issued in single or multidose containers.
b) Cell culture vaccine
All processing of the cell bank and subsequent cell cultures
are done under aseptic conditions in an area where no other
cells are handled. Approved animal (but not human) serum
may be used in the media, but the final medium for maintaining
cell growth during virus multiplication does not contain animal
serum; the media may contain human albumin. Serum proteins,
ifpresent are reduced to an acceptable level by suitable method
of purification. Serum and trypsin used in the preparation of
cell suspension and media are shown to be free from infectious
extraneous agents. The cell culture media may contain a pH
indicator such as phenol red. Not less than 500 ml of the cell
c;l,ltt':!!:fl se_lIlPlQYflcl fQr V3,c;c;illfl. prQclllctiQIl 3,!fJ. SfJt afjiclfJ .3,S
uninfected cell cultures (control cells). The virus suspension
is harvested on one or more occasions during incubation.
Multiple harvests from the same production cell culture may
be pooled and considered as a single harvest.
Inactivation. Inactivation is confirmed by carrying out an

amplification test for residual infectious Japanese encephalitis
virus. Inoculate a quantity of inactivated viral suspension
equivalent to not less than 25 doses into cell cultures of the
same type as those used for production of the vaccine. Make
a passage after 7 days. Maintain the cultures for a further
period of 14 days and then examine the cell cultures for
Japanese encephalitis virus using an immunofluorescence test.
No Japanese encephalitis virus is detected. Alternatively,S ml
ofeach culture fluid is pooled on day 14 and 21 and 0.03 ml is
inoculated intracerebrally into each of the 10 mice weighing
between 12 and 15 g. The mice are observed for 14 days for
symptoms caused by Japanese encephalitis virus, and mice
showing symptoms of Japanese encephalitis virus are
sacrificed and virus presence is confirmed by
immunofluorescence test. No Japanese encephalitis virus shall
be detected.
Residual host-cell DNA (2.2.15). The content ofresidual hostcell DNA, determined using a suitable method, should not be
greater than lOng per single human dose if cells are used in
the production.
FINAL BULK VACCINE
The final bulle vaccine is prepared from one or more inactivated
viral suspensions. An approved stabilizer may be added to
llla:inta:in-t1re activity of the product Thioff:fersa:tca:nl:5(~msed
as preservative;
Only a final bullc vaccine that complies with the following
2412
IP 2010
JAPANESE ENCEPHALITIS VACCINE (HUMAN)
Formaldehyde (2.3.20). Not more than 0.01 per centw/v.

FINAL LOT
containers. The containers are then sealed so as to prevent
contamination.
Only a final lot that complies with each of the tests given
under Identification, Tests and Assay may be released for
use. Provided that the test for inactivation has been carried
out with satisfactory results on the inactivated virus
suspension and the test for bovine serum albumin has been
these tests may be omitted on the final lot.
Identification
The vaccine is shown to contain Japanese encephalitis virus
antigen by a suitable immunochemical method using specific
antibodies, alternatively, the Assay also serves to identify the
vaccine.
Tests
Complies with the test for Inactivation under final Purification
and Inactivation.

Bacterial endotoxins (2.2.3). Less than 25 ill per single human
dose.
.
toxicity.
Bovine serum albumin (for cell culture vaccine). Not more
than 50 ng per single human dose, determined by a suitable
immunochernical method (2.2.14).
Residual host-cell DNA (for continuous cell line vaccines)
(2.2.15). Not more than 10 ng per single human dose.
Free formaldehyde (2.3.20). Not more than 0.01 per cent w/v.
Antimicrobial preservative. Determine the amount of
.antiniicrobial preservative by a suitable chemical method. The
content is not less than 85 per cent and is not greater than
115.0 per cent of that stated on the label.
Biological assay
Potency of Japanese encephalitis virus vaccine is determined
by titrating the neutralizing antibodies produced in the
immunized mice by plaque reduction method or serum

neutralization test (SNT) using appropriate cell culture.
The Standard preparation is a freeze-dried Japanese
encephalitis virus vaccine the potency of which has been
detennined in relation to the Japanese encephalitis reference
vaccine obtained from the National Institute ofHealth, Tokyo,
Japan.
Suggested method
Preparation of challenge virus suspension
The approved challenge virus strain is stored in freeze-dried
form or in liquid form stored below -70. Prepare a working
pool ofthe challenge virus strain by inoculating intracerebrally
0.03 ml of 100 fold dilution of the standard strain in Hanks'
balanced salt solution containing 5 per cent calf serum into a
suitable number of 2-day old suckling mice. Sacrifice the
animals after they show characteristic symptoms of
encephalitis and become moribund. Harvest their brains
aseptically, wash them in chilled sterile saline solution to
remove blood clots. Homogenize the brains with Hanks'
balanced salt solution containing 5 per cent calf serum to'
make a 10 per cent emulsion. Centrifuge the emulsion at
2000 g for 30 minutes. Dilute the supernatant with Hanks'
balanced salt solution containing 5 per cent calf serum so as
to contain about 200 Plaque-Forming Units (PFU) ofthe virus
per OA rnI.
Determination of potency
Prepare appropriate dilutions ofthe vaccine under examination
and of the Standard Preparation in a suitable medium. Inject
intraperitoneally in two doses of 0.5 ml each at 7-day interval
into at least 20 mice of 4 weeks of age. Bleed each mouse 7
days after the second injection, pool the separated serum
from each group and inactivate the sera by heating at 56 for
30 minutes. The inactivated sera may be stored at -20, if
necessary.
Dilute the sera appropriately, e.g. 1:40. 1: 160, 1:640 etc., mix
with an equal volume of the challenge virus suspension and
incubate at 37 for 90 minutes for neutralization. Inoculate the
mixture into cell cultures and overlay the infected cells with 1
per cent agar. After incubation for an appropriate time (about
48 hours), stain the cells and count the number of plaques
formed on the cultures to obtain the plaque reduction rates
for the vaccine under examination and the Standard
preparation. Calculate the neutralizing antibody titres for each
group using standard statistical methods (5.7). The test is not
valid unless (a) the mean number ofplaques obtained with the
Standard preparation is between 100 and 150 per dish and (b)
the potency of the vaccine under examination is not less than
that of the Standard preparation.
2413
IP 2010
MEASLES AND RUBELLA VACCINE (LIVE)
Labelling. The label states (1) the biological origin ofthe cells
used for the preparation of the vaccine; (2) the strain ofvirus
used.

Measles and Rubella Vaccine (Live) is a freeze-dried preparation
of suitable attenuated strains of measles virus and rubella
virus grown in suitable cell cultures.
The vaccine is reconstituted immediately before use to give a
clear liquid that may be coloured owing to the presence of a
pH indicator.
Production
Identification
When the vaccine reconstituted as stated on the label is mixed
with antibodies specific for measles virus and rubella virus, it
is no longer able to infect cell cultures susceptible to these
viruses. When the vaccine reconstituted as stated on the
label is mixed with quantities of specific antibodies sufficient
to neutralize anyone viral components, the second viral
component infects susceptible cell cultures.
Tests
Water (2.3.43). Not more than 3.0 per cent, determined by Karl
Fischer, semi-micro determination ofwater or by any suitable
validated method.
test for sterility.
General provisions
The two components are prepared as described in the
monographs on Measles vaccine (live) and Rubella vaccine
(live) and comply with the tests prescribed therein.
product~iftested;would-coiJjplywith the tesrforsafety and
efficacy.
Bovine serum albumin. Not more than 50 ng per single human

dose, determined by .a suitable immunochemicalmethod
(2.2.14).
'FINAL BULK VACCINE
Assay
Virus harvests for each component are pooled and clarified to

remove cells. A suitable stabilizer may be added and the pooled
harvests diluted as appropriate. Suitable quantities of the
pooled harvest for each component are mixed.
A. Mix the vaccine with a sufficient quantity of antibodies

specific for rubella virus.Titrate the vaccine for infective
measles virus at least in triplicate, using at least eight cell
cultures for each dilution 0.5 10glO step or by a method of
equal precision. Use an appropriate virus reference preparation
to validate each assay. The estimated measles virus
concentration is not less than that stated on the label; the
minimum measles virus concentration stated on the label is
not less than 1x 103 CCID50 per single human dose. The assay
is not valid ifthe confidence limits (P = 0.95) ofthe logarithm
of the virus concentration are greater than 0.3.
FlNALLOT
For each component, a minimum virus concentration for
release of the product is established such as to ensure, in the
light of stability data, that the minimum concentration stated
on the label will be present at the end ofthe period ofvalidity.
Only a final lot that complies with the tests for minimum virus
concentration of each component for release, with the
following test for thermal stability and with each of the tests
given below under Identification and Tests and Assay may be
released for use. Provided that the test for bovine serum albumin
has been carried out with satisfactory results on the final bulk
Thermal stability. Maintain samples ofthe final lot offreezedried vaccine in the dry state at 37 for 7 days. Determine the
..virus.. concentration.as.described_underAssayJn.parallelfor
the vaccine held at 37 for 7 days and for vaccine stored at 2
to 8. For each component, the virus concentration of the
heated vaccine is not more than 1.0 loglo lower than that ofthe
unheated vaccine.

toxicity.
Measles vaccine (Live) RS is suitable for use as a reference

preparation.
B. Titrate the vaccine for infective rubella virus at least in

triplicate, using at least eight cell cultures for each 0.5 10glO
dilution step or by a method of equal precision. Use an
appropriate virus reference preparation to validate each assay.
The estimated rubella virus concentration is not less than that
stated on the label; the minimum rubella virus concentration
stated on the label are not less than 1xl03 CCID50 per single
human dose. The assay is not valid if the confidence limits .
(P = 0.95) ofthe logarithm ofthe virus concentration are greater,
thanO.3.
1i1!!!~II{l~{lccine (Live)
RS issllitab1e for use asa reference
preparation.
Labelling. The label states (1) the strains of virus used in the
preparation ofthe vaccine; (2) the type and origin ofthe cells
used for the preparation ofthe vaccine; (3) the minimum virus
2414
MEASLES VACCINE (LIVE)
IP 20ID
concentration for each component ofthe vaccine; (4) the time

within which the vaccine must be used after reconstitution;
(5) that the vaccine must not be given to a pregnant woman
and that a woman should not become pregnant within two
months after having the vaccine.

the tests for seed lots.
Neurovirulence (2.7.5). The master/working seed lot complies
with the test for neurovirulence oflive virus vaccines. Macaca
and Cercopithecus monkeys susceptible to measles virus are
suitable for the test.
Measles Vaccine (Live) is a freeze-dried preparation of a

suitable attenuated strain of measles virus. The vaccine is
reconstituted immediately before use, as stated on the label,
to give a clear liquid that may be coloured owing to the
presence of a pH indicator.
Production
General provisions
The production ofvaccine is based on a virus seed-lot system
and, if the virus is propagated in human diploid cells, a cellbank system. Unless otherwise justified and authorized, the
virus in the final vaccine shall have undergone no more
passages from the master seed lot than were used to prepare
the vaccine shown in clinical studies to be satisfactory with
respect to abnormal toxicity and efficacy; even with authorized
exceptions, the number ofpassages beyond the level used for
clinical studies shall not exceed five.
product, if tested, would comply with the tests for abnormal
toxicity and efficacy.

The virus is propagated in human diploid cells or in cultures
of chick embryo cells derived from a chicken flock free from
specified pathogens.
SEED LOT
The strain of measles virus used in the production of measles
vaccine shall be identified by historical records that include
.manipulation. Virus seed lots are prepared in large quantities
and stored at temperatures below -20 0 iffreeze-dried, or below
-60 0 ifnot freeze-dried.
Only a seed lot that complies with the following tests may be
used for virus propagation.
Identification
The master and working seed lots are identified as measles
virus by serum neutralization in cell culture, using specific
antibodies.
Virus concentration. The virus concentration of the master
and working seed lots is determined to monitor consistency
of production.
All processing ofthe cell banle and subsequent cell cultures is

are handled. Suitable animal (but not human) serum may be
used in the growth mediLUTI, but the final medilUTI for maintaining
serum. Serum and trypsin used in the preparation of cell
suspensions and culture media are shown to be free from
extraneous agents. The cell culture medium may contain a pH
indicator such as phenol red and suitable antibiotics at the
lowest effective concentration. It is preferable to have a
substrate free from antibiotics during production. Not less
than 500 ml of the production cell culture is set aside as
uninfected cell cultures (control cells). The viral suspensions
are harvested at a time appropriate to the strain ofvirus being
used.
Only a single harvest that complies with the following tests
may be used in the preparation of the final bulle vaccine.
Identification
The single harvest contains virus that is identified as measles
virus by serum neutralisation in cell culture, using specific
antibody.
Virus concentration. The virus concentration in the single

harvest is determined as prescribed under Assay to monitor
consistency ofproduction and to determine the dilution to be
used for the final bulle vaccine.
Extraneous agents (2.7.3). Complies with the test for extraneous
agents.
Control cells. Ifhuman diploid cells are used for production,
the control cells comply with the test for identification and
extraneous agents.
FINAL BULK VACCINE
Virus harvests that comply with the above tests are pooled
and clarified to remove cells. A suitable stabilizer may be added
and the pooled harvests diluted as appropriate.
Sterility (2.2.11). The final bulle vaccine complies with the test
for sterility carried out using 10 ml for each medium.
2415
IP 2010
MEASLES VACCINE (LIVE)
FINAL LOT
A minimum virus concentration for release of the product is
established so as to ensure, in the light of stability data, that
the minimum concentration stated on the label will be present
at the end ofthe period of validity.
concentration for release, with the following requirement for
thermal stability and with each ofthe requirements given below
under Identification, Tests and Assay may be released for
use. Provided that the test for bovine serum albumin has been
carried out with satisfactory results on the final bulk vaccine,
virus concentration as described under Assay in parallel for
to 8. The virus concentration of the heated vaccine is not
more than 1.0 10glO lower than that of the unheated vaccine.
Identification
with specific measles antibodies, it is no longer able to infect
susceptible cell cultures.
concentration; (4) the time within which the vaccine must be

used after reconstitution.
Measles, Mumps and Rubella Vaccine

(Live)
Measles, Mumps and Rubella Vaccine (Live) is a freeze-dried
preparation of suitabie attenuated strains of measles virus,
mumps virus and rubella virus grown in suitable cell cultures.
The vaccine is reconstituted immediately before use to give a
clear liquid that may be coloured owing to the presence of a
pH indicator.
Production
General provisions
The three components are prepared as described in the
monographs on Measles Vaccine (Live), Mumps Vaccine (Live)
and Rubella Vaccine (Live) and comply with the tests
prescribed therein.
efficacy.
Tests
validated method.
Sterility ( 2.2.11). The reconstituted vaccine complies with
the test for sterility.
toxicity.
FINAL BULK VACCINE

Virus harvests for each component are pooled and clarified to
remove cells. A suitable stabilizer may be added and the pooled
harvests diluted as appropriate. Suitable quantities of the
pooled harvest for each component are mixed.

dose, determined by a suitable immunochemical method
(2.2.14).
each medium.
Assay
FINAL LOT
Titrate the vaccine for infective virus at least in triplicate,

using at least five cell cultures for each 0.5 log,o dilution step
or by a method of equal precision. Use an appropriate virus
reference preparation to validate each assay. The estimated
virus concentration is not less than that stated on the label;
the minimum virus concentration stated on the label is not
less than 1 x 103 CCID50 per human dose. The assay is not
valid ifthe confidence limits (P =:= 0.95) ofthe logarithm ofthe
virus concentration is greater than 0.3.
!lfeq[?.yq(::J:}I1?(LiJl?)RS. iil Jl1tl:ipl~ IQr.1JJ:l . .l:iil.. l:i I~lfl.r~!1<::fl
preparation.
Labelling. The label states (1) the strain of virus used for the
For each component, a minimum virus concentration for

release of the product is established such as to ensure, in the
light of stability data, that the minimum concentration stated
on the label will be present at the end ofthe period ofvalidity.
concentration of each component for release, with the
following requirement for thermal stability and with each of
the requirements given below under Identification and Tests
may be released for use. Provided that the test for bovine
serumalbumiIilias been -carrIed out WIth .satIsfactoryfesu1ts
on the final bulle va.ccine, itmay be omitted on the final lot.
2416
MENINGOCOCCAL POLYSACCHARIDE VACCINE
IP 2010

more than 1.0 loglo lower than that of the unheated vaccine.
Identification
with antibodies specific for measles virus, mumps virus and
rubella virus, it is no longer able to infect cell cultures
susceptible to these viruses. When the vaccine reconstituted
as stated on the label is mixed with quantities of specific
antibodies sufficient to neutralize any two viral components,
the third viral component infects susceptible cell cultures.
Tests
validated method.
test for sterility.

toxicity.

(2.2.14).
not valid ifthe confidence limits (P = 0.95) ofthe logarithm of

the virus concentration are greater than 0.3.
Mumps vaccine (Live) RS is suitable for use as a reference

preparation.
C. Mix the vaccine with a sufficient quantity of antibodies
specific for mumps virus and measles virus. Titrate the vaccine
for infective rubella virus at least in triplicate, using at least
eight cell cultures for each dilution 0.510g lO step or by a method
of equal precision. Use an appropriate virus reference
preparation to validate each assay. The estimated rubella virus
minimum rubella virus concentration stated on the label is not
less than 1x103 CCIDso per single human dose. The assay is
not valid ifthe confidence limits (P = 0.95) ofthe logarithm of
the virus concentration are greater than 0.3.
Rubella vaccine (Live) RS is suitable for use as a reference

preparation.
Labelling. The label states (1) the strains ofvirus used in the
preparation of the vaccine; (2) the type and origin of the cells
concentration for each component ofthe vaccine; (4) the time
within which the vaccine must be used after reconstitution;
(5) that the vaccine must not be given to a pregnant woman
and that a woman should not become pregnant within two
months after having the vaccine.
Assay
A. Mix the vaccine with a sufficient quantity of antibodies
specific for mumps virus and rubella virus.Titrate the vaccine
for infective measles virus at least in triplicate, using at least
eight cell cultures for each dilution 05 10glO step or by a method
preparation to validate each assay. The estimated measles
the minimum measles virus concentration stated on the label
is not less than 1xl0 3 CCIDso per single human dose. The
assay is not valid if the confidence limits (P = 0.95) of the
logarithm ofthe virus concentration are greater than 0.3.
Measles vaccine (Live) RS is suitable for use as a reference

preparation.
B. Mix the vaccine with a sufficient quantity of antibodies
specific for measles virus and rubella virus. Titrate the vaccine
for infective mumps virus at least in triplicate, using at least
eight cell cultures for each dilution 0.510g 10 step or by a method
preparation to validate each assay. The estimated mumps virus
minimum mumps virus concentration stated on the'label is not
less than 5 x 103 CCIDso per single human dose. The assay is
Meningococcal Polysaccharide
Vaccine
Meningococcal Polysaccharide Vaccine is a freeze-dried
preparation of one or more purified capsular polysaccharides
obtained from one or more suitable strains of Neisseria
meningitidis group A, group C, group Y and group W135 that
are capable ofconsistently producing polysaccharides known
to be safe and effective in man.
N. meningitidis group A polysaccharide consists of partly

O-acetylated repeating units ofN-acetylmannosamine, linked
with 1&n6 phosphodiester bonds.
N. meningitidis group C polysaccharide consists of partly
O-acetylated repeating units of sialic acid, linked with 2&n9
glycosidic bonds.
N. meningitidis group Y polysaccharide consists of partly
O-acetylated alternating units of sialic acid and D-glucose,
linked. with 2&n6 and 1&n4 glycosidic bonds.
N. meningitidis group W135 polysaccharide consists ofpartly
O-acetylated alternating units of sialic acid and D-galactose,
linked with 2&n6 and 1&n4 glycosidic bonds.
2417
IF 2010
Production
Production of the meningococcal polysaccharides is based

on a well defined seed-lot system. The method of production
shall have been shown to yield consistently meningococcal
polysaccharide vaccines of satisfactory immunogenicity and
.
safety for man.
proteins and lipopolysaccharides. The purification step

consists of ethanol precipitation of the polysaccharides or
purification with chloroform and n-butanol or by cold phenol
treatment, which are then dried and stored at or below -20.
The loss on drying is detennined by thennogravimetry, Karl
Fischer or any other suitable method and the value is used to
calculate the results ofthe other chemical tests with reference
to the dried substance.

product, if tested, would comply with the test of abnonnal
Only purified polysaccharides that tested comply with the

SEED LOT
Protein (2.7.1). Not more than 10 mg ofprotein per gram of

purified polysaccharide for group A and C organisms and less
than 50 mg ofprotein per gram ofpolysaccharide for group Y
and W135 calculated using bovine plasma albumin as a
reference or other methods approved by National Regulatory
Authority.
General provisions
The strains of N. meningitidis used for the master seed lots

shall be identified by historical records that include infonnation
on their origin and by their biochemical, serological,
physicochemical or molecular characteristics. Cultures from
the working seed lot shall have the same characteristics as the
strain that was used to prepare the master seed lot. The strains
have the following characteristics:
a) Colonies obtained from a culture are round, unifonn in
shape and smooth with a mucous, opalescent, greyish
appearance.
b) Gram staining reveals characteristic Gram-negative
diplococci in 'coffee-bean' arrangement.
c) The oxidase test is positive.
d) The culture utilizes glucose and maltose.
e) Suspensions of the culture agglutinate with specific
antisera oflmown titre.
The working seed lots are cultured on solid media that do not
contain blood-group substances or ingredients of mammalian
origin. The inoculum may undergo one or more subcultures in
liquid medium before being used for inoculating the final
medium. The liquid media used and the final medium are
semisynthetic and free from substances precipitated by
cetrimonium bromide (hexadecyltrimethylammonium
bromide) and do not contain blood-group substances or highmolecular-mass polysaccharides. The bacterial purity of the
culture is verified by microscopic examination ofGram-stained
smears and by inoculation into appropriate media. The cultures
are centrifuged and the polysaccharides precipitated from the
supernatant by addition of cetrimonium bromide. The
precipitate obtained is harvested and may be stored at or
below -20 awaiting further purification.
PURIFIEDPOINSACCHARIDES The polysaccharides are purified, after dissociation of the

complex of polysaccharide and cetrimonium bromide, using
suitable procedures to remove successively nucleic acids,
Nucleic acids (2.7.1). Not more than 10 mg ofnucleic acids per

gram ofpurified polysaccharide, calculated with reference to
the dried substance.
O-Acetyl groups (2.7.1). Not less than 2 mmol ofO-acetyl
groups per gram of purified polysaccharide for group A, not
less than 1.5 mmol per gram of polysaccharide for group C,
not less than 0.3 romol per gram ofpolysaccharide for groups
Y and W135, all calculated with reference to the dried
substance.
Phosphorus (2.7.1). Not less than 80 mg of phosphorus per
gram of group A purified polysaccharide, calculated with
reference to the dried substance.
Sialic acid (2.7.1). Not less than 800 mg ofsialic acid per gram
of group C polysaccharide and not less than 560 mg of sialic
acid per gram of purified polysaccharide for groups Y and
W135, all calculated with reference to the dried substance.
Use the following reference solutions:
Group C polysaccharide. A 150 mg/l solution of Nacetylneuraminic acid.

Group Ypolysaccharide. A solution containing 95 mg/l of Nacetylneuraminic acid and 55 mg/l of glucose.
Group W135 polysaccharide. A solution containing 95 mg/l
of N-acetylneuraminic acid and 55 mg/l of galactose.
Calcium. If a calcium salt is used during purification,
determination of calcium is carried out on the purified
polysaccharide by a suitable method; the content is within
the limits approved for the product.
Molecular size. Examinebygel.filtrationor.highp_erfonnance
size-ex_clusion. chrOInatography (HPSEC) (2.4.16), using
agarose for chromatography or cross-linked agarose for
chromatography either alone or in combination with light
scattering and refractive index detector (e.g. multiple angle
2418
IP 2010
LASER light scattering, MALLS) or any other suitable method.

Use a column 0.9 m x 15 mm equilibrated with a solvent having
an ionic strength of0.2 mollkg and a pH of7.0 to 7.5. Apply to
the column about 2.5 mg of polysaccharide in a volume of
about 1.5 ml and elute at about 20 mllh. Collect fractions of
about 2.5 ml and detennine the content of polysaccharide by
a suitable method.
At least 65.0 per cent ofgroup A polysaccharide, 75.0 per cent
of group C polysaccharide, 80.0 per cent of group Y
polysaccharide and 80.0 per cent of group W135
polysaccharide is eluted before a distribution coefficient (Ko)
of 0.50 is reached. In addition, the percentages eluted before
this distribution coefficient are within the limits approved for
the particular product.
Identification and serological specificity

The identity and serological specificity are detennined by a
suitable immunochemical method (2.2.14). Identity and purity
of each polysaccharide shall be confinned; it shall be shown
that there is not more than LO per cent mlm of groupheterologous N. meningitidis polysaccharide.
each of the rabbit with 1ml per kg body weight of solution
containing
a)
0.025 Ilg ofpolysaccharide for a monovalent vaccine,
b)
0.050 Ilg ofpolysaccharide for a bivalent vaccine,
c)
0.075 Ilg ofpolysaccharide for a trivalent vaccine,
d)
0.100 Ilg ofpolysaccharide for a tetravalent vaccine.
Tests
toxicity.
each of the rabbit with lml per kg body weight of solution
containing
a)
0.025 Ilg ofpolysaccharide for a monovalent vaccine,
b)
0.050 Ilg ofpolysaccharide for a bivalent vaccine,
c)
0.075 Ilg ofpolysaccharide for a trivalent vaccine,
d)
0.100 Ilg ofpolysaccharide for a tetravalent vaccine.
Water (2.3.43). Not more than 3.0 per cent, ofmoisture content
by thennogravimetery, Karl Fischer or any other suitable
method.
Molecular size. Examine by gel filtration or size-exclusion
chromatography (2.4.16). Use a column about 0.9 m long and
16 mm in internal diameter equilibrated with a solvent having
an ionic strength of0.2 mollkg and a pH of7.0 to 7.5. Apply to
the column about 2.5 mg of each polysaccharide in a volume
ofabout 1.5 ml and elute at about 20 ml/h. Collect fractions of
about 2.5 ml and detennine the content of polysaccharide by
a suitable method.
Use cross-linked agarose for chromatography and apply a
suitable immunochemical method (2.2.14) to establish the
elution pattern ofthe different polysaccharide(s). The vaccine
complies with the test if:
a)
FINAL BULK VACCINE

One or more purified polysaccharides of one or more N.
meningitidis groups are dissolved in a suitable solvent that
may contain a stabilizer.
Only a final bull( vaccine that complies with the following
requirement may be used in the preparation of the final lot.
FINAL LOT
b)
65.0 per cent of Group A polysaccharide is eluted before

KoofO.50,
75.0 per cent of Group C polysaccharide is eluted before
~of0.50,
c)
80.0 per cent ofGroup Y & W135 polysaccharide is eluted

before Ko ofO.50.
For a tetravalent vaccine (group A + group C + group Yand

group W135), use cross linked agarose for chromatography
R1 and apply a suitable immunochemical method (2.2.14) to
establish the elution pattern of the different polysaccharides.
The vaccine complies with the test ifKo for the principal peak
is
The final bull( vaccine is distributed aseptically into sterile

containers. The containers are then closed so as to avoid
contamination. Only a final lot that is satisfactory with respect
to each of the requirements prescribed below under
Identification, Tests and Assay may be released for use.
Identification
Assay
Carry out an identification test for each polysaccharide present

in the vaccine by a suitable immunochemical method (2.2.14).
Carry out an assay as stated under each polysaccharide

present in the vaccine.
a)
not greater than 0.70 for group A and group C

polysaccharide,
b)
not greater than 0.57 for group Y polysaccharide,
c)
not greater than 0.68 for group W135 polysaccharide.
2419
IP 2010
MUMPS VACCINE (LIVE) -
For a divalent vaccine (group A + group C), use measurement

of phosphorus (2.7.1) to determine the content of
polysaccharide A and measurement of sialic acid (2.7.1) to
detennine the content ofpolysaccharide C. To detennine sialic
acid, use as reference solution a 150 mg/l solution of Nacetylneuraminic acid.
For a tetravalent yaccine (group A + group C + group Y +
group W135) a suitable immunochemical method (2.2.14) is
used with a reference preparation of purified polysaccharide
for each group.
The vaccine contains not less than 70.0 per cent and not more
than 130.0 per cent of the quantity of each polysaccharide
Labelling. The label states (1) the group or groups of
polysaccharides (A, C, Y or W135) present in the vaccine;
(2) the number of Ilg of polysaccharide per human dose.

Mumps Vaccine (Live) is a freeze-dried preparation ofa suitable
attenuated strain ofmumps virus. The vaccine is reconstituted
immediately before use to give a clear liquid that may be
coloured owing to the presence of a pH indicator.
Production
General provisions
The production ofvaGcine is based on a virus seed-lot system
and, if the virus is propagated in human diploid cells, a cellbank system. The production method shall have been shown
to yield consistently live mumps vaccines of adequate
immunogenicity and safety in man.
final vaccine shall have undergone no more passages from
the master seed lot than were used to prepare the vaccine
safety and efficacy even with authorized exceptions, the
number ofpassages beyond the level used for clinical studies
shall not exceed five.
product, if tested, would comply with the tests for safety and
efficacy.
The virus is propagated in human diploid cells or in primary
cultures of chick embryo cells derived from a chicken flock
J!eeJ!0Il1 spe.cifi~~path.0gens.
and its subsequent manipulation. To avoid unnecessary use

ofmonkeys in the test for neurovirulence, Virus seed lots are
prepared in large quantities and stored attemperatures below
-20 0 iffreeze-dried, or below -60 0 ifnot freeze-dried.
Identification
The master and working seed lots are identified as mumps
antibodies.
.
and working seed lots is detennined to ensure consistency of
production.
and Cercopithecus monkeys are suitable for the test.
used in the growth media. Serum and trypsin used in the
preparation of cell suspensions and culture media are shown
to be free from extraneous agents. The cell culture medium
may contain a pH indicator such as phenol red and suitable
antibiotics at the lowest effective concentration. It is preferable
to have a substrate free from antibiotics during production.
Not less than 500 ml ofthe production cell culture is set aside
as uninfected cell culture (control cells). The viral suspensions
are harvested at a time appropriate to the strain ofvirus being
used.
requirements may be used in the preparation of the final bulk
vaccine.
Identification
The single harvest contains virus that is identified as mumps
antibodies.
harvest is detennined as prescribed under Assay to monitor
conslstencyofprocfiiCiiOn-ancrfodeteriiilneThediliition fO-be
SEED LOT
used fof the fmal bulk vaccine.
The strain ofmumps virus used shall be identified by historical

records that include infonnation on the origin of the strain
Sterility (2.2.11). Single harvest complies with sterility test

should be processed further.
2420
IP 2010
PERTUSSIS VACCINE
Control cells. The control cells comply with a test for

extraneous agents (2.7.3).
Single harvests that comply with the above tests are pooled
and clarified to remove cells. A suitable stabilisermay be added

less than 5 x 103 CCIDso per human dose. The assay is not
valid ifthe confidence limits (P = 0.95) ofthe logarithm ofthe

Mumps vaccine (Live) RS is suitable for use as a reference

preparation.
Sterility (2.2.11). The final bulle vaccine complies with the test
for sterility, carTied out using 10 ml for each medium.
preparation of the vaccine; (2) the type and origin ofthe cells
concentration and; (4) the time within which the vaccine must
be used after reconstitution.
FINAL BULK VACCINE
FINAL LOT
established such as to ensure, in the light of stability data,
that the minimum concentration stated on the label will be
present at the end of the period of validity.
, Only a fma110t that complies with the tests for minimum virus
under Identification and Tests and Assay may be released for
use. Provided that the test for bovine serum albumin has been
it may be omitted on the fmallot.
to go. The virus concentration of the vaccine exposed to 37
for 7 days is not more than 1.0 10glO lower than that of the
unheated vaccine.
Identification
When the vaccine is reconstituted as stated on the label is
mixed with specific mumps antibodies, it is no longerable to
infect susceptible cell cultures.
Tests
Water (2.3.43). Not more than 3.0 percent, determined by Karl
validatedmethod.
toxicity.
(2.2.14).
Assay
using at least five cel! cultures for each 0.5 10glO dilution step
Pertussis Vaccine
Pertussis Vaccine is a sterile saline suspension of inactivated
whole cells of one or more strains of Bordetella pertussis.
Production
General provisions
Inactivated B. pertllssis suspension
Production is based on a seed-lot system. One or more strains
ofB. pertussis with Imown origin and history are used. Strains,
culture medium and cultivation method are chosen in such a
way that agglutinogens 1, 2 and 3 are present in the final
vaccine. Each strain is grown for 24 to 72 hours in a liquid
medium or on a solid medium; the liquid medium used in the
final cultivation stage does not contain blood or blood
products. Human blood or blood products are not used in any
culture media. The bacteria are harvested, washed to remove
substances derived from the medium and suspended in a
0.9 per cent w/v solution of sodium chloride or other suitable
isotonic solution. The opacity ofthe suspension is deterrriined
not later than 2 weeks after harvest by comparison with the
reference preparation of Opacity and used as the basis of
calculation for subsequent stages in vaccine preparation.
Single harvests are not used for the final bulle vaccine unless
they have been shown to contain B. pertussis cells with the
same characteristics with regard to growth and agglutinogens,
as the parent strain and to be free from contaminating bacteria
and fungi. The bacteria are lalled and detoxified in controlled
conditions by means ofa suitable chemical agent or by heating
or by a combination of these methods. Freedom from live B.
pertussis is tested using a suitable culture medium. The
suspension is maintained at 5 3 for a suitable period to
diminish its toxicity.
2421
IP 2010
PERTUSSIS VACCINE
FINAL BULK VACCINE

Suitable quantities ofthe inactivated single harvests are pooled
to prepare the final bulk vaccine. Suitable antimicrobial
preservatives may be added. The bacterial concentration of
the final bulle vaccine does not exceed that con-esponding to
an ~pacity of20 ill per single human dose. If2 or more strains
of B. pertussis are used, the composition of consecutive lots
of the final bulle vaccine shall be consistent with respect to
the proportion of each strain as measured in opacity units.
requirements may be used in the preparation of the fmallot.
(b) at the end of 7 days the average increase in mass per

vaccinated mouse is not less than 60.0 per cent of that per
control mouse; and (c) not more than 5.0 per cent of the
vaccinated mice die during the test. The test may be repeated
and the results of the tests combined.
more than 115.0 per cent of the intended amount.
Assay
Carry out the assay of Pertussis Vaccine as described below:
Biological assay of pertussis vaccine

FINAL LOT
the assay have been can-ied out with satisfactory results on
Identification
Identify pertussis vaccine by agglutination of the bacteria in
the vaccine by antisera specific to B. pertussis.
Tests
Specific toxicity
Use not less than 10 healthy mice each weighing between 14
to 16 g for the vaccine group and for the saline control. Use
mice of the same sex or distribute males and females equally
water for at least 2 hours before injection and during the test.
control group with 0.5 ml ofa 0.9 per cent w/v sterile solution
of sodium chloride, preferably containing the same amount
ofantimicrobial-preseFVativeas-that-injected-with-the-vaccine.
Weigh the groups ofmice-immediately before the injection,
72 hours and 7 days after the injection. The vaccine complies
with the test if(a) at the end of 72 h the total mass_ofthe group
ofvaccinated mice is not less than that preceding the injection;
The potency of PertussisVaccine is detennined by comparison

of the dose necessary to protect mice against the effects of a
lethal dose of B. pertussis challenge culture, administered
intracerebrally, with the dose of a reference preparation,
calibrated in Internatiqnal Units, required to give the same
level of protection. For this comparison, the Standard
preparation of Pertussis vaccine & a suitable strain of B.
pertussis (e.g.18323, to be used as challenge strain), are
required.
Reference preparation
The reference preparation is an International standard of
Pertussis vaccine, consisting of a freeze dried vaccine or
another suitable preparation, calibrated in comparison to
International standard, from time to time. The International
Unit is the activity contained in a stated amount of the
International standard, which consists of a quantity of dried
pertussis vaccine. The equivalence in International Units of
the International Standard is stated by the World Health
Organization.
Use healthy mice of a suitable strain, weighing between 13
and 16 g from the same stock. Distribute the mice randomly, in
six to eight groups of not less than 16 and not more than 24
and four groups of 10. The mice should all be ofthe same sex
or the males and females should be distributeq equally between
the groups. Half of the groups of 16 to 24 should receive the
reference preparation and the other half should receive the
vaccine under examination. The four groups of 10 each should
be used for the LD 50 titration of challenge suspension.
Us_eat le.ast, threedill!tiOl1s.QftheI.ef.eLell~
Yflf(;il}.eJ:t!1<:li@~I
dilutions of the vaccine under examination. In each case the

dilutions are so selected that the dilution protecting 50 per
cent ofthe mice (ED 50) is as near as possible to middle ofthe
dilution range. For example, suggested ~ilutions are (1/8,1/40
2422
IP 2010
PLAGUE VACCINE
and 1/200 ofthe human dose ofthe vaccine under examination)

and (0.5 IV, 0.1 IV and 0.02 IV or any other suitable
standardized dilutions, ofthe reference preparation), each dose
being contained in a volume, not exceeding 0.5 ml. For each
dilution use 16 to 24 mice and inject intraperitoneally, into
each mouse one dose of the dilution.
Select a suitable strain ofB. pertussis (e.g. 18323), capable of
causing the death of mice within 14 days of intracerebral
injection. Make two subcultures after reviving the strain on a
suitable medium (e.g. E.G. medium) and suspend the harvested
growth in a solution containing 1 per cent w/v of casein
hydrolysate (e.g. casamino acid) and 0.85 per cent w/v of
sodium chloride and having a pH of 7.0 to 7.2 or in another
suitable solution. Determine the opacity ofthe suspension by
using 5th International reference preparation for opacity (10
aU) and/or spectrophotometrically. Alternatively, aliquots of
challenge suspension frozen in liquid nitrogen with a suitable
preservative like 10 per cent DMSO may be used, to avoid
heterogenicity. After 14 to 17 days of immunization, Inject
intracerebrally, a dose of 0.02 to 0.03 ml of the challenge
dilution randomly, into each immunized mouse. The challenge
should contain, approximately 1,00,000 organisms and 100 to
1000 LD50 per dose, in a volume ofnot more than 0.03 ml. In the
same way, inject 4 groups of 10 control mice each, for LD 50
titration of challenge preparation, prepared by a series of
dilutions, from the dilution selected for challenge. The
challenge should be completed within 2 to 2.5 hours of
preparation. Exclude any mouse from consideration, that dies
within 3 days ofchallenge. Count the number ofmice surviving
in each ofthe groups, after 14 days. On the basis ofthe numbers
of animals surviving in each of the groups of 16 to 24 mice,
calculate the potency of vaccine under examination, against
the potency of reference preparation. Seed a suitable highest
dilution of the challenge suspension, into each of two B.G
medium plates, before and after challenge. Incubate the plates
at 37 for 48 to 72 hours and calculate the number of colony
forming units (CFUs).
Calculate the potency of the vaccine by Probit analysis and
, LD 50 of the challenge suspension by Reed and Munch
Method.
The test is not valid unless: a) for both the vaccine under
examination and the reference preparation, the ED 50 (protective
dose), lies between the largest and the smallest doses given
to the mice; b) the number of animals, which die in the four
groups of 10 injected with the challenge suspension and its
dilutions indicate that the challenge dose contains 100 to 1000
LD 50 and 1 LD 50 contains not more than 300 colony forming
units; c) and the statistical analysis shows no deviation from
linearity or parallelism, in terms ofsignificance ofthe scope of
dose response curve; d) the vaccine passes the requirements
for potency, if the test results of a statistically valid assay
show that the estimated potency of the vaccine is not less
than 4.0 ru per single human dose and the lower fiducial limit
(P = 0.95) of estimated potency is not less than 2.0 ru.
The test may be repeated once, but when more than one test
is performed the results ofall valid tests must be combined, in
the estimate of potency.
International Units per single human dose; (2) that the vaccine
must be shaken before use; (3) that the vaccine is not to be
frozen.
Plague Vaccine
Fonnolised Plague Vaccine
Plague Vaccine is a sterile suspension of killed plague bacilli,
Yersinia pestis, ofthe 195/P strain grown in a suitable enriched
medium such as acid hydrolysate of casein, harvested and
killed by the addition offormaldehyde. It may contain a suitable
preservative.
Plague Vaccine contains in each ml of human dose not less
than 250 mouse median effective irnmunising doses (250 ED50)'
Description. A turbid, golden yellow or brownish liquid with
or without flakes or clumps.
Tests
pH (2.4.24). 6.8 to 7.4.
Sterility (2.2.11). Complies with the tests for sterility.
toxicity but injecting into each mouse 1.0 ml and into each
guinea-pig 3.0 ml.
Potency. Carry out the biological assay of plague vaccine
described below.
Biological Assay of Plague Vaccine

The potency of plague vaccine is estimated by determining
the dose necessary to protect mice against a lethal dose of a
virulent strain of Yersinia pestis.
Test animals. Use white mice, 6 to 7 weeks old, each weighing
between 20 and 28 g and of a strain susceptible to plague
infection. The selected strain of mice should be such that an
infective dose of 6 to 12 organisms of a virulent strain of Y.
pestis per mouse given subcutaneously produces a mortality
of not less than 80 per cent of the animals used. The animals
should be healthy and free from intercurrent infection with
organisms such as Salmonella.
Suggested Method
Selection of suitable virulent strain. A freeze-dried virulent
culture of Y. pestis, established to be suitable for challenge, is
2423
IP 2010
PLAGUE VACCINE
revived by subculturing 0.5 ml in 9.5 ml of nutrient broth

contained in another test-tube and incubating at 28 for exactly
48 hours. Such a culture should contain 300 to 600 million
organisms per ml. Make lO-fold dilutions in nutrient broth
and test for virulence. Use a 10-7 dilution containing 6 to 12
organisms in 0.2 ml as the test infective dose per animal. Those
strains which produce a mortality of 80 per cent among the
animals tested with this dose are considered to be virulent.
Standard challenge dose. FresWy reconstitute the freeze-dried
culture and dilute with nutrient broth to a strength such that
0.2 ml contains 60 to 120 organisms.
Measurement of protective power. Prepare a series of five
graded doses of the preparation under examination arranged
in such a manner that the 50 per cent protective dose (ED so)
lies about the middle of the selected series. For each dose a
batch of 16 mice is used. Inject subcutaneously the selected
dose in two equal parts with an interval of 7 days between
them. Seven days after the second half of the dose, inject
subcutaneously into each group ofmice the standard challenge
dose. At the same time inject into 10 control mice, 8 to 9 weeks
old and weighing between 28 and 30 g, the standard challenge
dose.
Observe the animals for 15 days and record the number of
deaths in each group. Carry out a post-mortem on the dead
animals and look for signs of plague in them. If plague
organisms are not seen, such deaths are excluded from the
calculation. The test is not valid unless the number of such
deaths is not more than 1. At the end of the period of
observation kill all the surviving animals and examine for signs
of plague. Calculate the median effective immunising dose,
ED so ' by standard statistical methods. The vaccine passes the
test ifit has an ED so of 0.004 ml or less per mouse.
Storage. Store at a temperature between 2 and 8 . The vaccine
should not be allowed to freeze. When stored under the
prescribed conditions the vaccine is expected to retain its
potency for not less than 3 years from the date of initiation of
the potency test.
Labelling. The label states (1) ED so unitage per dose; (2) the
name and proportion of any preservative added; (3) that the
contents should be shaken well before use; (4) that the vaccine
should not be allowed to freeze.
levels of specific antibodies in man. It contains upto 23

immunochemically different capsular polysaccharides listed
in the Table 1.
Production
General provisions
Production ofthe vaccine is based on a well defined seed-lot
system for each type. The production method shall have been
shown to yield consistently pneumococcal polysaccharide
vaccines of acceptable immunogenicity and safety in man.
product, if tested, would comply with the tests for abnormal
toxicity of vaccines for human use, modified as follows for
the test on guinea-pig; inject 10 human dose into each guineapig and observe for 12 days.
Monovalent bulk polysaccharides
The bacteria are grown in a suitable liquid medium that does
not contain blood-group substances or high-molecular-mass
polysaccharides. The bacterial purity ofthe culture is verified
and the culture is inactivated with phenol. Impurities are
removed by such techniques as fractional precipitation,
enzymatic digestion and ultrafiltration. The polysaccharide is
obtained by fractional precipitation, washed, and dried in a
vacuum to a residual moisture content shown to be favourable
to the stability of the polysaccharide. The residual moisture
content is determined by drying under reduced pressure over
diphosphorus pentoxide or by thermogravimetric analysis and
the value obtained is used to calculate the results of the tests
shown below with reference to the dried substance. The
monovalent bulk polysaccharide is stored at a suitable
temperature in conditions that avoid the uptake of moisture.
Only a monovalent bulk polysaccharide that complies with
the following requirements may be used in the preparation of
the final bulk vaccine. Percentage contents of components,
determined by the methods prescribed below, are shown in
the Table 1.
The purified polysaccharides comply with the following tests
as applicable:
Protein (2.7.1). Comply with the test for protein.
Nucleic acids (2.7.1). Comply with the test for nucleic acids.
Total nitrogen (2.3.30). Comply with the test for total nitrogen.
Pneumococcal Polysaccharide Vaccine consists of a mixture

Qfequal.. partsofpJU'ifiedcaps.ular polysaccharide ofvarious
serotype antigens prepared from suitableJ')Clt}:1()g~I!ic_.tl'llil1
of Streptococcus pneumoniae in different desired
combinations whose capsules have been shown to be made
up ofpolysaccharides that are capable ofinducing satisfactory
Phosphorus (2.7.1). Comply with the test for phosphorus.

Uronicacids.(2.7. L).Comply.with.the.test-for.uronic.acids..-Hexosamine (2.7.1). Comply with the test for hexosamine.
Methylpentoses (2.7.1). Comply with the test for
methylpentoses.
2424
PNEUMOCOCCAL POLYSACCHARIDE VACCINE
IP 2010
Table 1- Specifications on monovalent bulk polysaccharides (per cent contents):

Molecular
Type*
Proteins Nucleic
acids
1
2
3
4
5
:::;2
:::;2
:::;5
:::;3
:::;7.5
:::;2
:::;5
:::;2
:::;2
:::;2
:::;7
:::;3
:::;3
:::;5
:::;3
:::;2
:::;3
:::;2
:::;3
:::;2
6B
7F
8
9N
W
lOA
llA
l2F
14
l5B
l7Aor17F
l8C
19A
19F
20
22F
23F
33F
:::;2
:::;2
:::;2.5
:::;2
:::;2
:::;2
:::;2
:::;2
:::;2
:::;2
:::;2
:::;1
:::;2
:::;2
:::;2
:::;2
:::;2
:::;2
:::;2
:::;2
:::;2
:::;2
:::;2
:::;2
:::;2
:::;2
Total
3.5-6
0-1
0-1
4-6
2.5-6.0
0-2
1.5-4.0
0-1
2.2-4
0.5-3
0.5-3.5
0-2.5
3-5
1.5-4
1-3
0-1.5
0-1
0.6-3.5
1.4-3.5
0.5-2.5
0-2
0-1
0-2
Phosphorus Molecular size Ko

Nitrogen
CL-4B** CL-2B***
0-1.5
0-1.0
0-1.0
0-1.5
:::;2
2.5-5.0
0-1.0
0-1.0
0-1.0
0-1.0
1.5-3.5
2.0-5.0
0-1.0
0-1.0
2.0-4.5
0-3.5
2.4-4.9
3.0-7.0
3.0-5.5
1.5-4.0
0-1.0
3.0-4.5
0-1.0
Uronic
acids
MethylHexosammes pentoses
;;::45
;;:: 15
;;::40
:::;0.15
:::;0.15
:::;0.15
:::;0.15
:::;0.60
:::;0.50
;;::12
O-acetyl
Groups
;;:: 1.8
;;::38
;;::40
;;::20
;;:: 15
;;::13
:::;0.20
:::;0.15
:::;0.20
:::;0.45
:::;0.65
:::;0.40
;;::25
320
315
;;::28
;;::13
;;::12
;;::9
;;::25
;;::20
;;:: 15
:::;0.25
:::;0.30
:::;0.55
:::;0.45
:::;0.15
:::;0.45
:::;0.20
;;::12
;;::12.5
:::;0.60
:::;0.55
;;::20
;;:: 14
;;::20
;;::20
;;::12
;;:: 15
:::;0.15
;;::25
;;::37
:::;0.50
* The different types are indicated using the Danish nomenclature

** Cross linked agarose for chromatography R
*** Cross linked agarose for chromatography Rl
O-Acetyl groups (2.7.1). Comply with the test for O-acetyl
groups.
Sterility (2.2.11). Comply with the test for sterility.
Molecular size. Molecular size is determined by gel filtration
or high performance size-exclusion chromatography (HPSEC)

(2.4.16) using cross linkedAgarose for chromatography R or
chromatography Agarose for chromatograph Rl, either alone
or Multiple angle light laser scattering (MALLS) or any other
suitable method.
Identification
Confirm the identity of the monovalent bulle polysaccharide
by immunochemical method (2.2.14)(except for polysaccharides
7F,14and33F).
Specificity
For establishing the specificity, no reaction should occur,

when the antigens are tested against all the antisera specific
for the other polysaccharides of the vaccine, including factor
sera for distinguishing types within groups. The
polysaccharides are tested at a concentration of50 Ilg/ml using
a method capable ofdetecting 0.5 Ilg/ml.
FINAL BULK VACCINE
The final bulle vaccine is obtained by aseptically mixing the

different polysaccharide powders. The uniform mixture is
aseptically dissolved in a suitable isotonic solution so that
one human dose of 0.5 ml contains 25 Ilg of each
polysaccharide. An antimicrobial preservative may be added.
2425
IP 2010
PNEUMOCOCCAL POLYSACCHARIDE VACCINE
The solution is sterilized by filtration through a bacteriaretentive filter.

FINAL LOT
The final bulk vaccine is distributed and filled aseptically into
sterile containers (vials or ampoules). Only a final lot that is
satisfactory with respect to each of the requirements given
for use. Provided that the tests for phenol and for antimicrobial
preservative have been carried out with satisfactory results
on the final bulle vaccine, they may be omitted on the final lot.
When consistency of production has been established on a
suitable number of consecutive batches, the assay may be
replaced by a qualitative test that identifies each
polysaccharide, provided that an assay has been performed
on each monovalent bulk polysaccharide used in the
preparation ofthe final lot.
Identification
The assay also serves to identify the vaccine.
Tests
toxicity with the following modifications.
Inject 10 human doses each in two guinea pigs weighing
between 250 and 350 g by intraperitoneal route and observe
for 12 days,
(2.2.14), using antisera specific for each polysaccharide

contained in the vaccine, including factor sera for types within
groups, and purified polysaccharides of each type as
standards.
The vaccine contains not less than 70.0 per cent and not more
than 130.0 per cent ofthe quantity stated on the label for each
polysaccharide. The confidence interval (P = 0.95) ofthe assay
is not less than 80.0 and not more than 120.0 per cent of the
estimated content.
Labelling. The label states (1) the number of Ilg of each
polysaccharide per human dose; (2) the total amount of
polysaccharide in the container.

Poliomyelitis Vaccine (Inactivated) is a liquid preparation of
suitable strains of human polioviruses 1, 2 and 3 grown in
suitable cell cultures and inactivated by a validated method.
ProductiOl1.
General provisions
The production method should consistently yield vaccines
of acceptable safety and immunogenicity in man.
Production of the vaccine is based on a virus seed-lot system.
Cell lines are used according to a cell-bank system. Ifprimary,
secondary or tertiary monkey kidney cells are used, production
complies with the requirements indicated below.
the master seed lot than was used to prepare the vaccine
efficacy.

each of the rabbit with 1 ml of a dilution of the vaccine
containing 2.5 Ilgfml of each polysaccharide.
The virus is propagated in a human diploid cell line (2.7.2), in

a continuous cell line (2.7.2) or in primary, secondary or tertiary
monkey kidney cells.

Primary, secondmy or tertiary monkey kidney cells. The

following special requirements for the substrate for virus
propagation apply to primary, secondary or tertiary monkey
kidney cells.
Phenol (2.3.36). Not more than 2.5 gil.
Monkeys used in the preparation of kidney cell cultures for

production andcontrolofthe vaccine. The animals-used are
of a species approved by the competent authority, in good
health and, unless otherwise justified and authorised, have
not been previously employed for experimental purposes.
Kidney cells used for vaccine production and control are
llU(2:4:24). 45to-7.4: Assay

Determine the content of each polysaccharide by a suitable
biochemical, physicochemical or immunochemical method
2426
POLIOMYELITIS VACCINE (INACTIVATED)
IP 2010
derived from monitored, closed colonies of monkeys bred in

captivity, not from animals caught in the wild; a previously
approved seed lot prepared using virus passaged in cells from
wild monkeys may, subject to approval by the competent
authority, be used for vaccine production if historical data on
safety justify this.
Monitored, closed colonies of monkeys. The monkeys are

kept in groups in cages. Freedom from extraneous agents is
achieved by the use of animals maintained in closed colonies
that are subject to continuous and systematic veterinary and
laboratory monitoring for the presence of infectious agents.
The supplier ofanimals is certified by the competent authority.
Each monkey is tested serologically at regular intervals during
a quarantine period of not less than 6 weeks imposed before
entering the colony and then during its stay in the colony.
The monkeys used are shown to be tuberculin-negative
and free from antibodies to simian virus 40 (SV40) and
simian immunodeficiency virus. If Macaca spp. monkeys are
used for production, the monkeys are also shown to
be free from antibodies to herpesvirus B (Cercopithecine
helpesvirus 1) infection. Human herpesvirus 1 has been used
as an indicator for freedom from herpesvirus B antibodies on
account of the danger of handling herpesvirus B
(Cercopithecine herpesvirus 1).
Monkeys from which kidneys are to be removed are thoroughly
examined, particularly for evidence of tuberculosis and
herpesvirus B (Cercopithecine herpesvirus 1) infection. If a
monkey shows any pathological lesion relevant to the use of
its kidneys in the preparation of a seed lot or vaccine, it is not
to be used nor are any of the remaining monkeys of the group
concerned unless it is evident that their use will not impair the
safety of the product.
All the operations described in this section are conducted
outside the area where the vaccine is produced.
Monkey cell cultures for vaccine production. Kidneys that

show no pathological signs are used for preparing cell cultures.
Each group ofcell cultures derived from a single monkey forms
a separate production cell culture giving rise to a separate
single harvest.
The primary monkey kidney cell suspension complies with
the test for mycobacteria ; disrupt the cells before carrying
out the test.
If secondary or tertiary cells are used, it shall be demonstrated
by suitable validation tests that cell cultures beyond the
passage levelused for production are free from tumorigenicity.
SEED LOT
Each ofthe three strains ofpoliovirus used shall be identified
by historical records that include information on the origin of
the strain and its subsequent manipulation.

requirements may be used for virus propagation.
Identification
Each working seed lot is identified as human poliovirus 1,2 or
3 by virus neutralisation in cell culture using specific
antibodies.
Virus concentration. The virus concentration of each
working seed lot is determined to define the quantity
of virus to be used for inoculation of production cell
cultures.
the requirements for seed lots for virus vaccines. In addition,
if primary, secondary or tertiary monkey Iddney cells have
been used for isolation of the strain, measures are taken to
ensure that the strain is not contaminated with simian
viruses such as simian immunodeficiency virus, simian virus
40, filoviruses and herpesvirus B (Cercopithecine
helpesvirus 1). A working seed lot produced in primary,
secondary or tertiary monkey Iddney cells complies with the
requirements given below under Virus Propagation and
Harvest for single harvests produced in such cells.
All processing ofthe cell banle and subsequent cell cultures is
or viruses are being handled. Approved animal serum (but not
human serum) may be used in the cell culture media. Serum
and trypsin used in the preparation of cell suspensions and
media are shown to be free from extraneous agents. The cell
culture media may contain a pH indicator such as phenol red
and approved antibiotics at the lowest effective concentration.
Not less than 500 ml ofthe cell cultures employed for vaccine
production is set aside as uninfected cell cultures (control
cells); where continuous cell lines in a fermenter are used for
production, 200 x 106 cells are set aside to prepare control
cells; where primary, secondary or tertiary monleey Iddney
cells are used for production, a cell sample equivalent to at
least 500 ml of the cell suspension, at the concentration
employed for vaccine production, is taken to prepare control
cell cultures.
requirements may be used in the preparation of the vaccine.
The tests for Identification and Sterility may be carried out
instead on the purified, pooled monovalent harvest. After
demonstration of consistency of production at the stage of
the single harvest, the test for virus concentration may be
carried out instead on the purified, pooled monovalent harvest.
Control cells. The control cells of the production cell culture
comply with a test for Identification (if a cell-banle system is
2427
IP 2010
used for production) and with the requirements for extraneous

agents, where primary, secondary or tertiary monkey kidney
cells are used, the tests in cell cultures are carried out as
shown below under Test in Rabbit Kidney Cell Cultures and
Test in Cercopithecus Kidney Cell Cultures).
Test in rabbit kidney cell cultures. Test a sample of at least
10 ml ofthe pooled supernatant fluid from the control cultures
for the absence of herpesvirus B (Cercopithecine herpesvirus 1)
and other viruses in rabbit kidney cell cultures. The dilution
of supernatant in the nutrient medium is not greater than 1:4
and the area ofthe cell layer is at least 3 cm2 per m1 ofinoculum.
Set aside one or more containers of each batch of cells with
the same medium as non-inoculated control cells. Incubate
the cultures at 37 and observe for at least 2 weeks. The test is
not valid if more than 20 per cent of the control cells are
discarded for non-specific, accidental reasons.
Test in Cercopithecus kidney cell cultures. Test a sample of
at least 10 ml ofthe pooled supernatant fluid from the control
cultures for the absence of SV40 virus and other extraneous
agents by inoculation onto cell cultures prepared from the
kidneys of cercopithecus monkeys, or other cells shown to be
at least as sensitive for SV40, by the method described under
Test in Rabbit Kidney Cell Cultures. The test is not valid if
more than 20 per cent ofthe control cell cultures are discarded
for non-specific, accidental reasons.
Identification
The single harvest is identified as containing human poliovirus
1, 2 or 3 by virus neutralisation in cell cultures using specific
antibodies.
Virus concentration. The virus concentration of each single
harvest is determined by titration of infectious virus in cell
cultures.
demonstrated to be at least as susceptible for SV40. Incubate

the cultures at 37 and observe for 14 days. At the end ofthis
period, make at least one subculture of fluid in the same cell
culture system and observe both primary cultures and
subcultures for an additional 14 days.
PURIFICATION AND PURIFIED MONOVALENT
HARVEST
Several single harvest of the same type may be pooled and
may be concentrated. The monovalent harvest or pooled
monovalent harvest is purified by validated methods. If
continuous cell lines are used for production, the purification
process shall have been shown to reduce consistently the
content of substrate-cell DNA to not more than 500 pg per
single human dose.
Only a purified monovalent harvest that complies with the
following requirements may be used for the preparation ofthe
inactivated monovalent harvest.
Identification
The virus is identified by virus neutralisation in cell cultures
using specific antibodies or by determination ofD-antigen.
Virus concentration. The virus concentration is determined
by titration of infectious virus.
Specific activity. The ratio ofthe virus concentration or the Dantigen content, determined by a suitable immunochemical
method (2.2.14) to the total protein content (specific activity)
ofthe purified monovalent harvest is within the limits approved
for the particular product.
INACTIVATION AND INACTIVATED MONOVALENT
HARVEST
Several purified monovalent harvests of the same type may

be mixed before inactivation. To avoid failures in inactivation
caused by the presence ofvirus aggregates, fil1Tation is carried
out before and during inactivation; inactivation is started
Mycoplasmas (2.7.4). The single harvest complies with the
within a suitable period, preferably not more than 24 h and in
test for mycoplasmas, carried out using 10 ml.
any case not more than 72 h, ofthe prior filtration. The virus
Test in rabbit kidney cell cultures. Where primary, secondary suspension is inactivated by a validated method that has been
or tertiary monkey kidney cells are used for production, test a shown to inactivate poliovirus without destruction of
sample ofat least 10 ml ofthe single harvest for the absence of immunogenicity; during validation studies, an inactivation
herpesvirus B (Cercopithecine herpesvirus 1) and other viruses curve with at least four points (for example, time 0, 24, 48, and
in rabbit kidney cell cultures as described for the control cells. 96 h) is established showing the decrease in concentration of
Test in Cercopithecus Iddney cell cultures. Where primary, live virus with time. Ifformaldehyde is used for inactivation,
secondary or tertiary monkey kidney cells are used for the presence of an excess of formaldehyde at the end of the
inactivation period is verified.
_. __ _ _-,_._ __.E~~~~~ti~ll,_~~!a.~a.1!1J>It::()fa.tl~s!}Q
!Jl1of~~~siI!gl~.~~rye~t
for the absence of SV40 virus and other extraneous agents. Only an inactivated monovalent harvest that complies with
Neutralise fhe sample by -il high::iftreaiitiserl.lmagaiiisfthe the following requirements may be used in the preparation of
specific type of poliovirus. Test the sample in primary a trivalent pool of inactivated monovalent harvests or a final
cercopithecus kidney cell cultures or cells that have been bulle vaccine.
Sterility (2.2.11). The single harvest complies with the test for
sterility, carried out using 10 ml for each medium.
.......
2428
.....
..
IP 2010
Test for effective inactivation. After neutralisation of the

formaldehyde with sodium bisulphite (where applicable), verifY
the absence of residual live poliovirus by inoculation on
suitable cell cultures of two samples of each inactivated
monovalent harvest, corresponding to at least 1500 human
doses. Take one sample not later than three-quarters of the
way through the inactivation period and the other at the end.
Inoculate the samples in cell cultures such that the dilution of
vaccine in the nutrient medium is not greater then Y4 and the
area of the cell layer is at least 3 cm2 per ml of inoculum. Set
aside one or more containers with the same medium as noninoculated control cells. Observe the cell cultures for at least
3 weeks. Make not fewer than two passages from each
container, one at the end of the observation period and the
other 1 week before; for the passages, use cell culture
supernatant and inoculate as for the initial sample. Observe
the subcultures for at least 2 weeks. No sign of poliovirus
multiplication is present in the cell cultures. At the end of the
observation period, test the susceptibility of the cell culture
used by inoculation oflive poliovirus ofthe same type as that
present in the inactivated monovalent harvest.
Sterility (2.2.11). The inactivated monovalent harvest complies
with the test for sterility, carried out using 10 ml for each
medium.
D-antigen content. The content of D-antigen determined by a
suitable immunochemical method (2.2.14) is within the limits
approved for the particular preparation.
for inactivation is carried out on that pool rather than on the

final bulk vaccine.
FINAL LOT
and antimicrobial preservative and the in vivo assay have
been performed with satisfactory results on the final bulle
vaccine, they may be omitted on the final lot. Provided that
the test for bovine serum albumin has been performed with
satisfactory results on the trivalent pool of inactivated
monovalent harvests or on the final bulk vaccine, it may be
omitted on the final lot.
Identification
The vaccine is shown to contain human polioviruses 1, 2 and
3 by a suitable immunochemical method such as the
detennination of D-antigen by enzyme-linked immunosorbent
assay (ELISA).
Tests
Protein content (2.3.49). Not more than 10 Ilg of protein
nitrogen per human dose.
FINAL BULK VACCINE

The final bulle vaccine is prepared directly from the inactivated
monovalent harvests ofhuman polioviruses 1,2 and 3 or from
a trivalent pool of inactivated monovalent harvests. If a
trivalent pool of inactivated monovalent harvests is used, a
test for effective inactivation is carried out on this pool instead
of on the final bulk vaccine. A stabiliser and an antimicrobial
preservative may be added.

(2.2.14).

Assay

amount of antimicrobial preservative. by a suitable chemical
Inactivation. Before addition ofany antimicrobial preservative,
a sample ofat least 1500 ml or, for a purified and concentrated
vaccine, the equivalent of 1500 doses is tested for residual
live poliovirus in cell cultures, as described for the inactivated
monovalent harvest. Ifthe final bulk vaccine is prepared from
a trivalent pool of inactivated monovalent harvests, the test

Bacterial endotoxins (2.2.3). Not more than 5 ill per human
dose.
D-antigen content. As a measure ofconsistency ofproduction,

determine the D-antigen content for human polioviruses 1, 2
and 3 by a suitable immunochemical method (2.2.14) using an
appropriate reference preparation calibrated in D-antigen units.
For each type, the content, expressed with reference to the
amount of D-antigen stated on the label, is within the limits
In vivo test. The capacity ofthe vaccine toinduce the formation

of neutralizing antibodies is detennined in-vivo by one ofthe
following methods:
Test in chicks or guinea-pigs. Prepare a suitable series of at
least three dilutions ofthe vaccine under examination using a
suitable bufferedsaline solution. Inject 0.5 ml ofthe dilutions
2429
IP 2010
intramuscularly into groups of ten 3-week-old chickens or

groups of ten guinea-pigs, each weighing between 250 and
350 g, using a separate group for each dilution of vaccine.
Bleed the animals on the fifth or sixth day after the injection
and separate the sera. Examine the sera for the presence of
neutralising antibody, at a dilution of 1 in 4, to each of the
human polioviruses 1,2 and 3. Mix 100 CCID so ofvirus with
the dilution ofserum and incubate at 37 for 4 h 30 min to 6 h.
Keep at 5 3 for 12 to 18 h. Inoculate the mixtures into cell
cultures for the detection of unneutralised virus and read the
results up to 7 days after inoculation. For each group ofanimals,
note the number of sera which have neutralising antibody
and calculate the dilution of the vaccine giving an antibody
response in 50.0 per cent ofthe animals. Carry out in parallel a
control test using a suitable reference preparation.
The vaccine complies with the test if a dilution of 1 in 100 or
more produces an antibody response for each of the three
types of virus in 50.0 per cent of the animals.
geometric mean titres ofthe series on or more tests is used as

the cut-off value. For each of the 3 poliovirus types, the
potency of the vaccine is not significantly less than that of
the reference preparation. The test is not valid unless (1) for
both the test and reference vaccines the ED so lies between the
smallest and the largest doses given to the animals; (2) the
statistical analysis shows no significant deviation from
linearity or parallelism; (3) the fiducial limits ofthe estimated
relative potency fall between 25.0 per cent and 400.0 per cent
of the estimated potency.
Labelling. The label states (1) the types of poliovirus

contained in the vaccine; (2) the nominal amount of virus of
each type (1, 2 and 3), expressed in units of D-antigen per
single human dose; (3) the cell substrate used to prepare the
vaccine.
Test in rats. A suitable in vivo assay method consists of

intramuscular injection into the hind limb(s) ofnot fewer than Oral PoliomyelitisyacciIle.is a preparation ()fappr()v(;:dstraill.s
3 dilutions of the vaccine under examination and a reference of live attenuated poliovirus type 1, 2 or 3 grown in in vitro
cultures of approved cells, containing anyone type or any
vaccine, using for each dilution a group of 10 specific
combination ofthe three types of Sabin strains, prepared in a
pathogen-free rats of the suitable strain. Use of 4 dilutions is
fonn suitable for oral administration. The vaccine is a clear
often necessary to obtain valid results for all 3 serotypes. The
liquid that may be coloured owing to the presence of a pH
number of animals per group must be sufficient to obtain
indicator.
results that meet the validity criteria; groups of 10 rats are
usually sufficient although valid results may be obtained with Production
fewer animals per group. The weight ofindividual animal must
.General provisions
not vary by more than 10.0 per cent from the group mean. An
inoculum of 0.5 ml per rat is used. The dose range is chosen The vaccine strains and the production method should
such that a dose response to all 3 poliovirus types is obtained. consistently yield vaccines that are both immunogenic and
Bleed the animals after 20 to 22 days. Neutralising titres against safe inman.
all 3 polivirus types are measured separately using 100 CCID50
The production ofvaccine is based on a virus seed-lot system.
of the Sabin strains as challenge viruses. Vero or Hep2 as
Cell lines are used according to a cell-bank system. Ifprimary
indicator cells, and neutralization conditions of 3 h at 35 to
monl<ey
kidney cells are used, production complies with the
37 followed by 18 h at 2 to 8. Results are read
requirements
indicated below. Unless otherwise justified and
microscopically following fixation and staining after 7 days of
authorised,
the
virus in the final vaccine shall not have
incubation at 35. For a valid antibody assay, the titre of each
undergone
more
than two passages from the master seed lot.
challenge virus must be shown to be within the range of 10 to
1000 CCID so and the neutralizing antibody titre of a control Substrate for virus propagation
serum must be within 2 twofold dilutions of the geometric
The virus is propagated in human diploid cells (2.7.2) or in
mean titre of the serum. The potency is calculated by
continuous cell lines (2.7.2) or in primary monkey kidney cells
comparison of the preparation of responders for the vaccine
(including serially passaged cells from primary monkey kidney
under examination and the reference vaccine by the probit
cells). Continuous cell lines are approved by the competent
method or, after validation, using a parallel-line model. For the
authority.
probit method it is necessary to establish a cut-offneutralising
antibody titre for each poliovirus type to define a responder. Primary monkey kidney cells. The following special
requirements for the substrate for virus propagation apply
J:)ue JQ!Ilt~IaJ?Ql"CltQryYCll"iClti~n,.jJi~IlQtl?QssilJl(;:JQ_~flll~
cut-offvalues that could be applied by all laboratories. Rather, toprlma,ymonkeY7craiieycells~
the cut-off values are detennined for each laboratory based Monkeys usedfor preparation ofIddney cell cultures andfor
on a minimum series on tests with the reference vaccine. The testing ofvirus. If the vaccine is prepared in monkey lodney
mid-point on a log 2 scale of the minimum and maximum cell cultures, animals of a species approved by the competent
2430
IP 2010
POLIOMYELITIS VACCINE, LNE (ORAL)
authority, in good health, and not previously employed for

experimental purposes shall be used.
The monkeys shall be kept in well-constructed and adequately
ventilated animal rooms in cages spaced as far apart as
possible. Adequate precautions shall be taken to prevent
cross-infection between cages. Not more than two monkeys
shall be housed per cage and cage-mates shall not be
interchanged. The monkeys shall be kept in the country of
manufacture of the vaccine in quarantine groups for a period
of not less than 6 weeks before use. A quarantine group is a
colony of selected, healthy monkeys kept in one room, with
separate feeding and cleaning facilities, and having no contact
with other monkeys during the quarantine period. If at any
time dming the quarantine period the overall death rate of a
shipment consisting of one or more groups reaches 5 per cent
(excluding deaths from accidents or where the cause was
specifically determined not to be an infectious disease),
monkeys from that entire shipment shall continue in quarantine
from that time for a minimum of 6 weeks. The groups shall be
kept continuously in isolation, as in quarantine, even after
completion of the quarantine period, until the monkeys are
used. After the last monkey of a group has been taken, the
room that housed the group shall be thoroughly cleaned and
decontaminated before being used for a fresh group. Ifkidneys
from near-term monkeys are used, the mother is quarantined
for the term ofpregnancy.
Monkeys from which kidneys are to be removed shall be
anaesthetised and thoroughly examined, particularly for
evidence of tuberculosis and cercopithecid herpesvirus 1 (B
vims) infection.
Ifa monkey shows any pathological lesion relevant to the use
of its kidneys in the preparation of a seed lot or vaccine, it
shall not be used, nor shall any of the remaining monkeys of
the quarantine group concerned be used unless it is evident
that their use will not impair the safety ofthe product.
All the operations described in this section shall be conducted
outside the areas where the vaccine is produced.
The monkeys used shall be shown to be free from antibodies
to simian virus 40 (SV40) and simian immunodeficiency virus.
If Macaca spp. are used for production, the monkeys shall
also be shown to be free from antibodies to cercopithecid
herpesvims 1 (B virus). Human herpesvirus has been used as
an indicator for freedom from B virus antibodies on account
ofthe danger ofhandling cercopithecid herpesvirus 1 (B vims).
Monkey ladney cell culturesfor vaccine production. Kidneys
that show no pathological signs are used for preparing cell
cultures. If the monkeys are from a colony maintained for
vaccine production, serially passaged monkey kidney cell
cultmes from primary monkey kidney cells may be used for
vims propagation, otherwise the monkey kidney cells are not
propagated in series. Vims for the preparation of vaccine is

grown by aseptic methods in such cultures. Ifanimal semm is
used in the propagation ofthe cells, the maintenance medium
after vims inoculation shall contain no added serum.
Each group of cell cultures derived from a single monkey or
from fetuses from no more than ten near-term monkeys is
prepared and tested as an individual group.
SEED LOT
The strains ofpoliovirus used shall be identified by historical
records that include information on the origin and subsequent
manipulation of the strains.
Working seed lots are prepared by a single passage from a
master seed lot and at an approved passage level from the
original Sabin vims. Vims seed lots are prepared in large
quantities and stored at a temperature below -60.
Only a virus seed lot that complies with the following
requirements may be used for vims propagation.
Identification
Each working seed lot is identified as poliovirus of the given
type, using specific antibodies.
Virus concentration. Determined by the method described

below, the virus concentration is the basis for the quantity of
virus used in the nemovirulence test.
Extraneous agents (2.7.3). Ifthe working seed lot is produced
in human diploid cells (2.7.2) or in continuous cell lines (2.7.2)
it complies with the requirements for seed lots for virus
vaccines. If the working seed lot is produced in primary
monkey cells, it complies with the requirements given below
under Propagation and Harvest and Monovalent Pooled
Harvest and with the tests in adult mice, suclding mice and
guinea-pigs given under Tests for extraneous agents in viral
vaccines for human use.
Working seed lot shall be free from detectable DNA sequences
from simian virus 40 (SV40).
Neurovirulence (2.7.6). Each master and working seed lot

complies with the test for neurovimlence of poliomyelitis
vaccine (oral) in monkeys. Furthermore, the seed lot shall cease
to be used in vaccine production ifthe frequency offailme of
the monovalent pooled harvests produced from it is greater
than predicted statistically. This statistical prediction is
calculated after each test on the basis of all the monovalent
pooled harvests tested; it is equal to the probability of false
rejection on the occasion of a first test (i.e. 1 per cent), the
probability offalse rejection on retest being negligible. Ifthe
test is carried out only by the manufacturer, the test slides are
provided to the control authority for assessment.
2431
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POLIOMYELITIS VACCINE, LIVE (ORAL)
Genetic markers. Each working seed lot is tested for its

replicating properties at temperatures ranging from 36 to 40
as described under Monovalent Pooled Harvest.
All processing ofthe cell-banks and subsequent cell-cultures
is done under aseptic conditions in an area where no other
suspensions and media are shown to be free from live
extraneous agents. The cell-culture medium may contain a pH
indicator such as phenol red and approved antibiotics at the
than 5 per cent and not more than 1000 ml of the cell cultures
employed for vaccine production are set aside as uninfected
cell cultures (control cells); special requirements, given below,
apply to control cells when the vaccine is produced in primary
monkey cells The vilUs suspension is harvested not later than
4 days after vilUs inoculation. After inoculation of the
production cell culture with the vilUs working seed lot,
inoculated cells are maintained at a fixed temperature, shown
to be suitable, within the range 33 to 35; the temperature is
maintained constant to 0.5; control cell cultures are
maintained at 33 to 35 for the relevant incubation periods.
Only a single virus harvest that complies with the following
pooled harvest.
Virus concentration. The vilUs concentration of vilUs

harvests is determined as prescribed under Assay to monitor
used for the final bulk vaccine.
Extraneous agents (2.7.3 ). Complies with tests for extraneous
agents.
from which the vilUs harvest is derived comply with a test for
identity and with the requirements for extraneous agents or,
where primmy monkey cells are used, as shown below.
Primary monkey kidney cells. The following special
requirements apply to virus propagation and harvest in

primmy monkey Iddney cells.
Cell cultures. On the day of inoculation with virus seed, each
fetuses from not more than ten near-term monkeys is divided

into two equal portions. One portion of the pooled fluid is
tested in monkey Iddney cell cultures prepared from the same
species, but not the same animal, as that used for vaccine
production. The other portion of the pooled fluid is, where
necessary, tested in monkey Iddney cell cultures from another
species so that tests on the pooled fluids are done in cell
cultures from at least one species known to be sensitive to
SV40. The pooled fluid is inoculated into bottles ofthese cell
cultures in such a way that the dilution of the pooled fluid in
the nutrient medium does not exceed 1 in 4. The area ofthe cell
sheet is at least 3 cm2 per ml ofpooled fluid. At least one bottle
ofeach kind of cell culture remains uninoculated to serve as a
control. If the monkey species used for vaccine production is
known to be sensitive to SV40, a test in a second species is
not required. Animal serum may be used in the propagation of
the cells, provided that it does not contain SV40 antibody, but
the maintenance medium after inoculation of test material
contains no added serum except as described below.
The cultures are incubated at a temperature of 35 to 37 and
are observed for a total period of at least 4 weeks. During this
observation period and after not less than 2 weeks incubation,
at least one subculture of fluid is made from each of these
cultures in the same cell culture system. The subcultures are
also observed for at least 2 weeks.
Serum may be added to the original culture at the time of
subculturing, provided that the serum does not contain SV40
antibody.
Fluorescent-antibody techniques may be useful for detecting
SV40 virus and other viruses in the cells.
A further sample of at least 10 ml ofthe pooled fluid is tested
for cercopithecid herpesviIus I (B virus) and other viruses in
rabbit lddney cell cultures. Serum used in the nutrient medium
of these cultures shall have been shown to be free from
inhibitors ofB virus. Human herpesvirus has been used as an
indicator for freedom from B virus inhibitors on account ofthe
danger ofhandling cercopithecid herpesvirus I (B virus). The
sample is inoculated into bottles ofthese cell cultures in such
a way that the dilution of the pooled fluid in the nutrient
medium does not exceed 1 in 4. The area ofthe cell sheet is at
least 3 cm2 per ml ofpooled fluid. At least one bottle ofthe cell
cultures remains uninoculated to serve as a control.
The cultures are incubated at a temperature of 35 to 37
and observed for at least 2 weeks.
cell culture is examined for degeneration caused by an infective

agent. If, in this examination, evidence is found ofthe presence
il1 .a cell c;ulture oLal1y eXtran~()lls agent, the entire group of
cultures concerned shall be rejected.
A further sample of I0 rnl ofthe pooled fluid removed from the

cell cultures on the day of inoculation with the seed lot virus
i~te~e4J()E tl1.e prese llce ()f e)(tr8:Ileoll~ ageIlts]:>y inoculation
into human cell cultures sensitive to measles virus.
On the day of inoculation with the vilUs worldng seed lot, a

sample of at least 30 ml ofthe pooled fluid removed from the
cell cultures of the kidneys of each single monkey or from
The tests are not valid if more than 20 per cent ofthe culture
vessels have been discarded for non-specific accidental
reasons by the end of the respective test periods.
2432
IP 2010
If, in these tests, evidence is found of the presence of an

extraneous agent, the single harvest from the whole group of
cell cultures concerned is rejected.
If the presence of Cercopithecid helpesvirus J (B virus) is
demonstrated, the manufacture of oral poliomyelitis vaccine
shall be discontinued and the competent authority shall be
informed. Manufacturing shall not be resumed until a thorough
investigation has been completed and precautions have been
taken against any reappearance of the infection, and then
only with the approval of the competent authority.
Ifthese tests are not done immediately, the samples ofpooled
cell-culture fluid shall be kept at a temperature of -60 0 or below,
with the exception ofthe sample for the test for B virus, which
may be held at 4 0 , provided that the test is done not more than
7 days after it has been taken.
Control cell cultures. On the day of inoculation with the virus

working seed lot 25 per cent (but not more than 2500 ml) ofthe
cell suspension obtained from the kidneys of each single
monkey or from not more than ten near-term monkeys is taken
to prepare uninoculated control cell cultures. These control
cell cultures are incubated in the same conditions as the
inoculated cultures for at least 2 weeks and are examined during
this period for evidence of cytopathic changes. The tests are
not valid if more than 20 per cent of the control cell cultures
have been discarded for non-specific, accidental reasons. At
the end ofthe observation period, the control cell cultures are
examined for degeneration caused by an infectious agent. If
this examination or any of the tests required in this section
shows evidence of the presence in a control culture of any
extraneous agent, the poliovirus grown in the corresponding
inoculated cultures from the same group shall be rejected.
Tests for haemadsorbing viruses. At the time of harvest or
within 4 days of inoculation of the production cultures with
the virus working seed lot, a sample of4 per cent ofthe control
cell cultures is taken and tested for haemadsorbing viruses.
At the end of the observation period, the remaining control
cell cultures are similarly tested. The tests are made as described
in (2.7.3), Tests for extraneous agents in viral vaccines for
human use.
Tests for other extraneous agents. At the time of harvest, or
within 7 days of the day of inoculation of the production
cultures with the working seed lot, a sample ofat least 20 m1 of
the pooled fluid fi:om each group of control cultures is taken
and tested in two kinds of monkey kidney cell culture, as
described above.
At the end of the observation period for the original control
cell cultures, similar samples ofthe pooled fluid are taken and
the tests referred to in this section in the two kinds ofmonkey
kidney cell culture and in the rabbit cell cultures are repeated,
as described above under Cell cultures.
If the presence of Cercopithecid herpesvirus J (B virus) is

demonstrated, the production cell cultures shall not be used
and the measures concerning vaccine production described
above must be undertaken.
The fluids collected fi'om the control cell cultures at the time
ofvims harvest and at the end of the observation period may
be pooled before testing for extraneous agents. A sample of
2 per cent of the pooled fluid is tested in each of the cell
culture systems specified.
Single harvests
Tests jor neutralised single harvests in monkey kidney cell
cultures. A sample of at least 10 ml of each single harvest is
neutralised by a type-specific poliomyelitis antiserum prepared
in animals other than monkeys. In preparing antisera for this
purpose, the immunising antigens used shall be prepared in
non-simian cells.
Half of the neutralised suspension (corresponding to at least
5 ml ofsingle harvest) is tested in monkey kidney cell cultures
prepared from the same species, but not the same animal, as
that used for vaccine production. The other half of the
neutralised suspension is tested, if necessary, in monkey
kidney cell cultures from another species so that the tests on
the neutralised suspension are done in cell cultures from at
least one species known to be sensitive to SV40.
The neutralised suspensions are inoculated into bottles of
these cell cultures in such a way that the dilution of the
suspension in the nutrient medium does not exceed 1 in 4. The
area of the cell sheet is at least 3 cm2 per ml of neutralised
suspension. At least one bottle of each type of cell culture
remains uninoculated to serve as a control and is maintained
by nutrient medium containing the same concentration ofthe
specific antiserum used for neutralisation.
Animal serum may be used in the propagation of the cells,
provided that it does not contain SV40 antibody, but the
maintenance medium, after the inoculation ofthe test material,
contains no added serum other than the poliovirus neutralising
antiserum, except as described below.
The cultures are incubated at a temperature of35 to 37 0 and
observed for a total period of at least 4 weeks. During this
observation period and after not less than 2 weeks incubation,
at least one subculture of fluid is made from each of these
cultures in the same cell-culture system. The subcultures are
also observed for at least 2 weeks.
Serum may be added to the original cultures at the time of
subculturing, provided that the serum does not contain SV40
antibody.
Additional tests are made for extraneous agents on a further
sample of the neutralised single harvests by inoculation of
lO ml into human cell cultures sensitive to measles virus.
2433
IP 2010
Fluorescent-antibody techniques may be useful for detecting

SV40 virus and other viruses in the cells.
The tests are not valid if more than 20 per cent of the culture
vessels have been discarded for non-specific accidental
reasons by the end of the respective test periods.
If any cytopathic chauges occur in any of the cultures, the
causes of these change are investigated. If the cytopathic
changes are shown to be due to unneutralised poliovirus, the
test is repeated. Ifthere is evidence ofthe presence ofSV40 or
other extraneous agents attributable to the single harvest,
that single harvest is rejected.
Monovalent pooled harvests are prepared by pooling a number
of satisfactory single harvests of the same virus type.
Monovalent pooled harvests from continuous cell lines may
be purified. Each monovalent pooled harvest is filtered through
a bacteria-retentive filter.
final bulk vaccine.
Identification
Each monovalent pooled harvest is identified as poliovirus of
the given type, using specific antiserum.
Virus concentration
The virus concentration is determined by the method
described below and serves as the basis for calculating the
dilutions for preparation of the final bulle, for the quantity of
virus used in the neurovirulence test and to establish and
monitor production consistency.
Genetic markers. A ratio of the replication capacities of the
virus in the monovalent pooled harvest is obtained over a
temperature range between 36 and 40 in comparison with
the seed lot or a reference preparation for the marker tests and
with appropriate rct/40- and rct/40+ strains of poliovirus of
the same type. The incubation temperatures used in this test
are controlled to within 0.1 0. The monovalent pooled harvest
passes the test if, for both the virus in the harvest and the
appropriate reference material, the titre determined at 36 is at
least 5.0 log greater than that detennined at 40. If growth at
40 is so low that a valid comparison cannot be established, a
temperature in the region of39.0 to 39.5 is used, at which
temperature the reduction in titre of the reference material
must hI:: jnlh.t:: nlligl:: 3,tLJo5,Q IQgQLi~ Yl:ih!Sl.flt 36~~11J:
acceptable mininmm reduction is determined for each virus
strain at a given temperature. If the titres obtained for one or
more of the reference viruses are not concordant with the
expected values, the test must be repeated.
Neurovirulence (2.7.6). Each monovalent pooled harvest

complies with the test for neurovirulence of poliomyelitis
vaccine (oral). Ifthe test is carried out only by the manufacturer,
the test slides are provided to the competent authority for
assessment. The TgPVR21 transgenic mouse model provides
a suitable alternative to the monkey neurovirulence test for
neurovirulence testing of types 1, 2 or 3 vaccines once a
laboratory qualifies as being competent to perform the test
and the experience gained is to the satisfaction of the
competent authority. The test is carried out using a standard
operating procedure approved by the competent authority. A
suitable procedure (Neurovintlence test oftype 1,2 or 3 live
poliomyelitis vaccines (oral) in transgenic mice susceptible
to poliovirus) is available from WHO, Quality and Safety of
Biologicals, Geneva.
Primary monkey kidney cells. The following special
requirements apply to monovalent pooled harvests derived
from primmy monkey kidney cells.
Retroviruses. The monovalent pooled harvest is examined
using a reverse transcriptase assay. No indication of the
presence of retrovirus is found.
Test on rabbits. A sample ofthe monovalent pooled harvest is

tested for cercopithecid herpesvirus 1 (B virus) and other
viruses by injection ofnot less than 100 ml into not fewer than
10 healthy rabbits each weighing between 1.5 and 2.5 kg. Each
rabbit receives not less than 10 ml and not more than 20 ml, of
which 1 ml is given intradermally at multiple sites, and the
remainder subcutaneously. The rabbits are observed for at
least 3 weeks for death or signs of illness.
All rabbits that die after the first 24 h of the test and those
showing signs of illness are examined by autopsy, and the
brain and organs removed for detailed examination to establish
the cause of death.
The test is not valid ifmore than 20.0 per cent ofthe inoculated
rabbits show signs of intercurrent infection during the
observation period. The monovalent pooled harvest passes
the test if none of the rabbits shows evidence of infection
with B virus or with other extraneous agents or lesions of any
kind attributable to the bulk suspension.
If the presence of B virus is demonstrated, the measures
concerning vaccine production described above under Cell
cultures are taken.
Test on guinea pigs. Administer to not less than five guineapigs, each weighing between 350 and 450 g, 0.1 ml of the
monovalent pooled harvest by intracerebral injection and
0.5 ml by intraperitoneal injection. Measure the rectal
temperature of each animal on each working day for 6 weeks.
Afi1ie~end ofihe
observatIon perrodcarry~outaufopsyoii
each animal.
In addition, administer to not fewer than five guinea-pigs
0.5 ml by intraperitoneal injection and observe as described
2434
IP 2010
RABIES ANTISERUM
above for 2 to 3 weeks. At the end of the observation period,

carry out a passage from these animals to not fewer than five
guinea pigs using blood and a suspension of liver or spleen
tissue. Measure the rectal temperature of the latter guinea
pigs for 2 to 3 weeks. Examine by autopsy all animals that,
after the first day of the test, die or are lolled because they
show disease or show for three consecutive days a body
temperature higher than 39; carry out histological examination
to detect infection with Marburg virus; in addition, inject a
suspension of liver or spleen tissue or of blood
intraperitoneally into not fewer than three guinea pigs. If any
signs ofinfection with Marburg virus are noted, confilwatory
serological tests are carried out on the blood of the affected
animals. The monovalent pooled harvest complies with the
test if not fewer than 80.0 per cent ofthe guinea pigs survive
to the end ofthe observation period and remain in good health
and no animal shows signs of infection with filoviruses virus.
more than one poliovirus type, titrate each type separately,

using appropriate type-specific antiserum (or preferably a
monoclonal antibody) to neutralise each of the other types
present.
For a trivalent vaccine, the estimated mean virus titres must
be: not less than 1 x 106.0 infectious virus units (CCID so ) per
single human dose for type I; not less than 1 x IO s.oinfectious
virus units (CCID so) for type 2; and not less than I x 10s.8
infectious virus 'units (CCID so ) for type 3.
For monovalent or divalent vaccine, the minimum virus titres
are decided by the competent authority.
FINAL BULK VACCINE
Method. Groups of eight to twelve flat-bottomed wells in a

microtitre plate are inoculated with 0.05 ml of each of the
selected dilutions of virus followed by a suitable cell
suspension of the Hep-2 (Cincinnati) line. The plates are
incubated at a suitable temperature. Examine the cultures on
days 7 to 9.
The final bulk vaccine is prepared from one or more

satisfactory monovalent pooled harvests and may contain
more than one virus type. Suitable flavouring substances and
stabilisers may be added.
The assay is not valid if(a) the confidence interval (P = 0.95)

of the logarithm of the virus concentration is greater than
0.3; (b) the virus concentration of the reference preparation
differs by more than 0.5 log CCIDso from the assigned value.

requirement may be used in the preparation of the final lot.
FINAL LOT
Only a final lot that complies with the following requirement
for thennal stability and is satisfactory with respect to each of
the requirements given below under Identification, Tests and
Thermal stability. Expose samples of the final lot at 37 for
48 hours. Determine the total virus concentration as described
under Assay in parallel for the heated vaccine and for unheated
vaccine. The estimated difference between the total virus
concentration of the unheated and heated vaccines is not
greater than 0.5 loglo infectious virus units (CCIDso) per single
human dose.
Identification
The vaccine is shown to contain poliovirus ofeach type stated
on the label, using specific antibodies.
Tests
Labelling. The- label states (l) the types of poliovirus

contained in the vaccine; (2) the minimum amount ofvirus of
each type contained in one single human dose; (3) the cell
substrate used for the preparation of the vaccine; (4) that the
vaccine is not to be injected.
Rabies Antiserum
Antirabies Serum; Antirabic Serum
Rabies Antiserum is a preparation containing the specific
globulin or its derivatives obtained by purification of
hyperimmune serum or plasma of healthy horses or other
suitable animals having the specific activity of neutralising
the rabies virus. It may contain a suitable preservative.
Rabies Antiserum contains not less than 300 Units per m!.
suspended particles or cream-coloured powder or pellet to be
reconstituted with diluent supplied by the manufacturer.
Identification
Specifically neutralizes the standard strain of rabies virus
rendering it harmless to susceptible animals or by any other
suitable in vitro method.

Assay
Titrate for infectious virus at least in triplicate using the method
described below. Use an appropriate virus reference
preparation to validate each assay. If the vaccine contains
Tests
Potency. Carry out the biological assay of rabies antiserum
described below.
2435
IP 2010
RABIES ANTISERUM
Biological Assay of Rabies Antiserum

The potency of rabies antiserum is determined by comparing
the dose necessary to protect mice against a lethal intracerebral
dose ofrabies virus with the dose ofthe Standard Preparation
of rabies antiserum necessary to give the same protection.
The standard preparation is a dried serum or other suitable
preparation the potency of which has been detennined in
relation to the International Standard.
Suggested Method
Test animals. Use healthy mice of either sex from the same
stock weighing between 10 and 14 g, but animals ofthe same
sex should be used in any given test.
Test virus. Any suitable strain ofrabies virus ofknown potency
such as the CVS strain may be used.
Test dose of virus. The test dose of virus is such that each
mouse receives by intracerebralinjection between 20 and 100()
LD so preferably about 100 LD so ' To determine the number of
LD so ofvirus used, mix the virus dilution used in the test with
an equal quantity of a 2 per cent v/v solution of heatinactivated normal horse serum in water and maintain the
mixture at 37 for 1 hour. Prepare 10-fold dilutions in a 2 per
cent v/v solution of heat-inactivated normal horse serum
and inject them into mice. Carry out this test at the same time
as the test for determining the potency ofthe rabies antiserum.
The test is not valid unless the quantity of virus used lies
between 20 and 1000 LDso'
Determination ofpotency ofthe rabies antiserum. Prepare a
series of six 2-fold dilutions of the Standard Preparation and
ofthe preparation under examination with water containing 2
per cent v/v of heat inactivated normal horse serum. To each
dilution add a quantity of a suspension of the test virus
containing the test dose and keep the mixtures at 37 for 1
hour. Inject intracerebrally 0.03 ml ofeach mixture into 10 mice.
Observe the mice for 14 days after the injection. Mice dying
before the fifth day after inoculation with the virus are
eliminated from the test; all the mice dying between the fifth
and fourteenth days after showing signs of rabies are
considered to have died of rabies; mice living upto the
fourteenth day but showing signs of rabies are also counted
as having died from rabies. Calculate the result ofthe test by
standard statistical methods. The preparation under
examination passes the test if, in a single comparative assay,
His f()lllld t() hav<l~O or lllore lJnits p<lrllll~ Ifitis fOlilld tohaye
less than 80 Units per ml, two more similar assays may be
carried out; the preparation passes the test if, in hoth these
additional assays, it is found to have 80 or more Units per ml.
Labelling. The label states (1) the number of Units per ml in

case ofliquid preparation; (2) the total volume in the container;
(3) the nameand volume of the diluent to be used for
reconstituting the freeze-dried preparation; (4) the
recommended dose; (5) the name and concentration of any
added preservative; (6) the animal species in which the
antiserum has been raised; (7) that the liquid preparation
should not be allowed to freeze.
Rabies Vaccine, Human

Rabies Vaccine for Human use is a freeze-dried or liquid
(adsorbed) preparation ofa suitable approved, strain of fixed
rabies virus grown in an approved cell culture/ embryos of
duck/chicken and inactivated by a validated method.
The freeze-dried vaccine is reconstituted immediately before
use as stated on the label to give a clear or slightly opalescent
solution/suspension. It may be coloured owing tothe presence
of a pH indicator.
The vaccine complies with the General Requirements of
Production
General provisions
The vaccine is produced on the basis of virus seed lot system
and if a cell line is used for virus propagation, a cell-bank
system shall be followed. The production method shall have
been shown to yield consistently vaccines that comply with
the requirements for immunogenicity, safety and stability.
Unless otherwise justified and authorized, the virus in the
the master seed lot than was used to prepare the vaccine
efficacy.
The virus is propagated in any suitable approved cell substrate
like a human diploid cell line (2.7.2), a continuous cell line, or
in duck embryos or in cultures of chicken embryos derived
from a flock certified as free from specified pathogens(2.7.7).
SEED LOT
'[he Straill of r~~iesvirtl~ll~eds~all bei<ientifie4 ~y ~is~ori~~
records that include information on the origin of the strain
and its subsequent manipulation.
Working seed lots are prepared by not more than five passages
from the master seed lot.
2436
IP 2010
RABIES VACCINE, HUMAN

test for identification and with the requirements for extraneous

agents (2.7.3).
Identification
Control eggs. Control eggs shall be tested for freedom from

haemagglutinating agents, and other extraneous agents.
Each working seed lot is identified as rabies virus using specific

antibodies by an approved method.
Virus concentration. The virus concentration ofeach working
seed lot is determined by a cell culture meth9d using
immunofluoresence or any other approved method.
the requirements for the virus seed lots.
All processing of the cell banle and subsequent cell cultures
are done under aseptic conditions in an area where no other
serum; the media may contain human albumin. Serum proteins,
if present are reduced to an acceptable level by a suitable
method of purification. Serum and trypsin used in the
preparation ofcell suspension and media are shown to be free
from infectious extraneous agents. The cell culture media may
contain a pH indicator such as phenol red and approved
antibiotics at the lowest effective concentration. Not less than
500 ml of the cell cultures employed for vaccine production
are set aside as uninfected cell cultures (control cells). The
virus suspension is harvested on one or more occasions
during incubation. Multiple harvests from the same production
cell culture may be pooled and considered as a single harvest.
When vaccine is prepared in embryonated eggs, the egg
proteins are minimized by an appropriate method ofpurification.
The eggs are inoculated with virus seed by the yolle sac route.
The infected sterile living embryos are harvested, minced and
emulsified in suitable diluent, and stabilizer with aseptic
precautions. Emulsions are centrifuged, supernatants are
collected and stored as raw virus harvest at a suitable
temperature.
Viral harvests that comply with the following requirements are
pooled in the preparation of the inactivated viral harvest.
PURIFICATION AND INACTIVATION

The virus harvests may be concentrated and/or purified by
suitable methods; the virus harvest is inactivated by a validated
method at a fixed, well defined stage ofthe process which may
be before, during or after any concentration or purification.
The method shall have been shown to be capable of
inactivating rabies virus without destruction of the
immunogenic activity. If betapropiolactone is used, the
concentration shall at no time exceed 1:3500.
Cell culture vaccines
Only an inactivated viral suspension that complies with the
following requirements may be used in the preparation of the
Inactivation. Inactivation is confirmed by carrying out an
amplification test for residual infectious rabies virus, not more
than 4 days after inactivation or on the sample frozen after
inactivation and stored at -70. Inoculate a quantity of
inactivated viral suspension equivalent to. not less than 25
vaccine doses into cell cultures of the same type as those
used for production of the vaccine. Make a passage after 7
days. Maintain the cultures for a further period of14 days and
then examine the cell cultures for rabies virus using an immune
fluorescence test. No rabies virus is detected. Alternatively, 5
m1 ofeach culture fluid is pooled on days 14and21 and 0.03 m1
is inoculated intracerebrally into each ofthe. I0 mice weighing
12 to 15 g. The mice are observed for 14 days for symptoms
caused by rabies virus, and mice showing symptoms ofrabies
are sacrificed and virus presence is confirmed by
immunofluorescence test or tested for live virus in cell culture
by immunofluorescence test or method of equal sensitivity.
No rabies virus should be detected.
Residual host-cell DNA (2.2.15). If a continuous cell line is
used for virus propagation, the content of residual host-cell
DNA, detennined using a suitable method, should not be
greater than lOng per single human dose.
Identification
Embryonated egg vaccine
The single harvest contains virus that is identified as rabies
virus using specific antibodies by an approved method.
Virus concentration. Titrate for infective virus in cell cultures
or by any other approved method. The titre is used to monitor
consistency of production.
from which the single harvest is derived should comply with a
Only concentrated viral suspensions that comply with the

test for sterility, antigen content and endotoxin requirements
may be used for preparation of bulle for inactivation.
Inactivation is confirmed by carrying out Mice Inoculation
Test for residual infectious rabies virus, not more than four
days after inactivation or on the sample frozen4 days after
inactivation are stored at-70. Inoculate intracerebrally 0.03
2437
RABIES VACCINE, HUMAN
IP 2010
ml into each of 20 mice weighing between 12 and 15 g. The

mice are observed for 14 days for symptoms caused by rabies
virus and mice showing symptoms ofrabies are sacrificed and
virus presence is confirmed by an immunofluorescence test
or tested for live virus in cell culture by immunofluorescence
test or method of equal sensitivity. No rabies virus should be
detected.
FINAL BULK VACCINE
viral suspensions. An approved stabilizer may be added to
maintain the activity of the product during and after freezedrying. Thiomersal can be used as preservative.
Antigen content. Determine the antigen content by a suitable
approved in vitro or in vivo method. The content should be
FINAL LOT
containers and can be freeze-dried in case of lyophilized
products. The containers are then sealed so as to prevent
contamination and the introduction of moisture.
released for use. Provided that the test for inactivation has
been carried out with satisfactory results on the inactivated
virus suspension and the test for bovine serum albumin has
been carried out with satisfactory results on the final bulk
vaccine, these tests may be omitted on the final lot.
Identification
The vaccine is shown to contain rabies virus antigen by a
suitable immunochemical method using specific antibodies,
alternatively, the Assay also serves to identify the vaccine.
Tests
test is already performed on inactivated virus used for final

lot, it may be omitted from the test on final lot.
Bacterial endotoxins (2.2.3). Less than 25 IU per single human
dose.
Pyrogens (2.2.8). Complies with the test for pyrogens. Unless
otherwise justified and authorized, inject into each rabbit a
single human dose of the vaccine diluted to ten times its
volume.
Water (2.3.43). Not more than 3.0 per cent determined by an
approved method.
toxicity.
Accelerated degradation. The potency determined by method
described under "Assay" ofa sample ofthe preparation under
examination after storage at 37 for 4 weeks is not less than 2.5
units per single human dose. This may not be mandatory for
lot release, once the consistency of the product is approved
by National Regulatory Authority.
Bovine serum albumin (for cell culture vaccine). Not more
than 50 ng per single human dose, determined by a suitable
immunochemical method (2.2.14).
Aluminium content (for gel absorbed vaccine) (2.3.9). Not
more than 1.25 mg per single human dose.
Ovalbumin (for egg based vaccines). Not more than Illg of
ovalbumin per human dose, determined by a suitable technique
using a suitable reference preparation of ovalbumin.
Residual host-cell DNA (for continuous cell line vaccines)
(2.2.15). Should not be greater than 10 ng per single human
dose.
Assay
The Standard preparation is the international standard or
another suitable preparation, the potency of which has been
determined in relation to the International standard. The
potency of rabies vaccine is determined by comparing the
dose necessary to protect mice against a lethal intracerebral
dose of rabies vaccine necessary to provide the same
protection. For this comparison, a reference preparation of
rabies vaccine, calibrated in international units, and a suitable
Inactivation. Inoculate a quantity equivalent to not less than

25 human doses of vaccine into cell cultures ofthe same type
as those used for production of the vaccine. Make a passage
after 7 days. Maintain the culture for a further 14 days and
then examine the cell culture for rabies virus using an
. immunofluorescence test. No rabies virus is detected.
Alternatively, injectO.03mlofthe vaccine intracerebrallyintoprep~aratioIiofrablesvrrus~foJ:useastlieclial1engepreparatlon
each of the 10 mice weighing between 12 and 15g. Neither are necessary.
symptoms of disease in the central nervous system nor death The international unit is the activity contained in a stated
occurs in any ofthe animal within 14 days. Ifthe inactivation quantity of the international standard. The equivalence in
2438
IP 2010
RUBELLA VACCINE (LIVE)
international units of the international standard is stated by

the World Health Organization.
The test described below uses a parallel-line model with at
least three points for the vaccine under examination and the
reference preparation.
Test animals. Use mice ofa suitable strain, drawn from a uniform
stock three to four weeks old, weighing between 11 and 15 g.
Distribute the mice into six groups ofat least 16 mice each and
four groups of 10 mice each and must be ofthe same sex or the
sexes must be equally distributed among the groups.
Throughout the test all mice that die before the fifth day after
challenge are excluded from the test and all mice that die with
signs of rabies between the fifth and fourteenth day after
challenge are counted as failing to resist the challenge.
The strain of mice suitable for the test is such that when
0.03 ml containing 5 to 50 LD so of the challenge virus
suspension is injected intracerebrally per mouse there is 100
per cent mortality.
Standard challenge virus suspension. A working pool ofthe
challenge virus strain is prepared by injecting intracerebrally
0.03ml ofa 10 fold dilution ofthe CVS strain ofrabies virus in
2 per cent v/v sterile inactivated normal horse serum in water
for injection or another suitable diluent approved by the
competent authority into a suitable number of test animals.
The animals when moribund after showing characteristic signs
of rabies are sacrificed and their brains harvested aseptically.
They are then washed in chilled saline solution to remove
blood clots. A 10 per cent suspension ofthe brains is prepared
in a suitable diluent approved by the competent authority and
thoroughly homogenised. After centrifuging lightly, the
supernatant liquid is distributed into sterile vials and freeze
dried. The sealed and freeze-dried supernatant liquid containing
vials are stored at -20. When stored under prescribed
conditions the virus titre of the freeze-dried preparation may
be expected to be maintained for not less than 3 years.
Alternatively, the washed brains are homogenised in a suitable
diluent approved by the competent authority to give 10 per
cent suspension. It is then centrifuged lightly, distributed into
sterile ampoules or sterile plastic vials and sealed. The sealed
ampoules or plastic vials can be stored at -60 or below. When
stored under prescribed conditions, the virus titre may be
expected to be maintained for not less than one year. Storage
time needs to be validated by the manufacturer.
Virus titre of the challenge virus. Prepare ten fold serial
dilutions of the standard challenge virus suspension. Using
the four groups of 10 mice each, inject 0.03 ml of the virus
suspension intracerebrally into each mouse, using a different
group for each suspension. Observe the mice for 14 days.
Calculate the virus titre of the standard challenge virus
suspension in LD so per dose of 0.03 ml by standard statistical
methods.
Determination of potency of the vaccine. Reconstitute the

standard preparation with a suitable diluent. Prepare at least
three 5-fold serial dilutions of the solution of the standard
preparation lJ,nd three 5-fold serial dilutions of the vaccine
under examination. For both, the standard preparation and
the preparation under examination, the serial dilutions should
be prepared in such a way that the lowest dilution protects
more than 50 per cent ofthe injected mice. Allocate one dilution
to each of the six groups of 16 mice each. Inject
intraperitoneally each mouse in each group with dilutions of
the vaccine and reference preparation and repeat the injections.
After 7 days, prepare identical dilutions of the vaccine and
reference preparation and repeat the injections.
After a further 7 days, inject each vaccinated mouse
intracerebrally with 0.03 ml of the standard challenge virus
suspension such that on the basis of preliminary titration,
0.03 rnl contains between 5 to 50 LD so ' Observe the mice for 14
days and record the number of mice surviving the challenge
in each group. Calculate the potency ofthe preparation under
examination by standard statistical methods.
The vaccine complies with the test ifthe estimated potency is
not less than 2.5 ill per single human dose.
The test is not valid unless (a) for both the preparation under
examination and the standard preparation, the 50 per cent
protective dose lies between the largest and smallest doses
given to the mice; (b) there is not deviation from linearity or
parallelism of the dose response lines, the confidence limit
(P = 0.95) are not less than 25.0 per cent and not more than 400
per cent ofthe estimated potency; (c) the titre ofthe challenge
virus suspension lies between 5 to 50 LD so '
Labelling. The label states the biological origin of the cells
used for the preparation of the vaccine.
Rubella Vaccine (Live)

Rubella Vaccine (Live) is a freeze-dried preparation ofa suitable
attenuated strain ofrubella virus. The vaccine is reconstituted
immediately before use to give a clear liquid or may be coloured
owing to the presence of a pH indicator.
Production
General provisions
been shown to yield consistently live rubella vaccines of
adequate immunogenicity and safety in man. Unless otherwise
justified and authorised, the virus in the final vaccine shall
have undergone no more passages from the master seed lot
than were used to prepare the vaccine shown in clinical studies
2439
IP 2010
RUBELLA VACCINE (LIVE)
to be satisfactory with respect to safety and efficacy.

efficacy.
production cell culture may be pooled and considered as a

single harvest.
requirements may be used in the preparation ofthe final bulk
vaccine.
Identification
The virus is propagated in human diploid cells (2.7.2).

SEED LOT
The strain of rubella virus used in the production of rubella
vaccine shall be identified by historical records that include
manipulation.
To avoid unnecessary use of monkeys in the test for
neurovirulence virus seed lots are prepared in large quantities
and stored at temperatures below -20 iffreeze-dried, or below
-60 ifnot freeze-dried.
Identification
The master and working seed lots are identified as rubella
antibodies.
and working seed lots is determined to ensure consistency of
production.
and Cercopithecus monkeys are suitable for the test.
All processing of the cell bank and subsequent cell cultures is
used in the growth medium, but the final medium for maintaining
suspensions and culture media are shown to be free from
indicator such as phenol red and suitable antibiotics at the
than5_QQ_ml._oLthe_produ.ctionc_ell_culture is set aside as
_uninfected cell culture (control celIs). The temperatur.e of
incubation is controlled during the growth of the virus. The
virus suspension is harvested, on one or more occasions,
within 28 days ofinoculation. Multiple harvests from the same
The single harvest contains virus that is identified as rubella

antibodies.
harvest is detennined as prescribed under Assay to monitor
used for the final bulle vaccine.
Extraneous agents (2.7.3). The single harvest complies with
the tests for extraneous agents.
Control cells. The control cells comply with a test for
identification and with the tests for extraneous agents (2.7.3).
FINAL BULK VACCINE
Single harvests that comply with the above tests are pooled
and clarified to remove cells. A suitable stabilizer may be added
FINAL LOT
established such as to ensure, in the light of stability data,
that the minimum concentration stated on the label will be
present at the end of the period of validity.
under Identification and Tests may be released for use.
Provided that the test for bovine serum albumin has been
carried out with satisfactory results on the final bulk vaccine,
more than 1.0 10glO lower than that of the unheated vaccine.
Identification- -------------- ------------- ------------When the vaccine reconstituted as stated. 011 tIle label is mixed
with specific rubella antibodies, it is no longer able to infect
susceptible cell cultures.
2440
IP 2010
TETANUS ANTITOXIN
Tests
Identification

test for sterility.
Specifically renders the corresponding venom or venoms

harmless to susceptible animals. Itmay also be identified by
any other alternate suitable in vitro method.
Water (2.3.43). Not more than 3.0 per cent, detennined by Karl
Fischer, semi-micro detennination ofwater or by any suitable
validated method.
Tests

(2.2.14).
Potency. In view of the numerous toxic fractions in venoms

and varying requirements of different localities, no standard
for potency is prescribed.

toxicity.
The potency of the snake venom antiserum is detennined by

estimating the ability ofthe antiserum to protect mice or other
suitable animals against the lethal effect of a fixed dose of a
reference preparation of snake venom ofthe relevant species
or by comparing its ability to do so with that of a reference
preparation of antiserum of established potency at two or
more dose levels ofthe venom.
Assay
using at least five cell cultures for each 0.5 lag lo dilution step
less than 1 x 103 CCID so per human dose. The assay is not
valid ifthe confidence limits (P=0.95) of the logarithm ofthe
Rubella vaccine (Live) RS is suitable for use as a reference

preparation.
Labelling. The label states (1) the strain ofvirus used for the
concentration; (4) the time within which thevaccine must be
used after reconstitution; (5) that the vaccine must not be
given to a pregnant woman and that a woman must not become
pregnant within two months.

Storage. Store the freeze-dried preparation in a cool, dark place
and avoid exposure to excessive heat. Store the liquid
preparation at a temperature between 2 and 8. It should not
Labelling. The label states (1) the volume ofthe contents and
in case of freeze-dried preparation, the directions for
reconstitution; (2) the species of snake against whose venom
the antiserum is effective; (3) the animal species from which
the antiserum has been obtained; (4) the name and proportion
of any preservative added; (5) that in case of a liquid
preparation, it should not be allowed to freeze.
Tetanus Antitoxin
Snake Venom Antiserum
Snake Antivenin; Snake Antivenom Serum; Snake
Venom Antitoxin
Snake Venom Antiserum is a sterile preparation containing
antitoxic globulins or their derivatives that have the power of
specifically neutralising the venom of one or more species of
snakes. It may be obtained from the serum or plasma ofhealthy
equines or other suitable animals immunised against venoms
of specific species usually elapine and viperine. It may be a
purified liquid preparation or a freeze-dried product. It may
contain a suitable preservative.
Description. A clear to slightly opalescent, colourless or pale
yellow liquid, free from suspended particles or cream-coloured
powder or pellet which when reconstituted with the diluent
supplied by the manufacturer with the freeze-dried product
yields a clear, colourless or pale yellow liquid.
Tetanus Antitoxin is a preparation containing the specific

antitoxic globulins or their derivatives obtained by purification
of hyperimmune serum of horses or other suitable animals
and have the specific activity ofneutralising the toxin fonned
by Clostridium tetani. The liquid preparation may contain a
suitable antimicrobial preservative.
Tetanus Antitoxin has a potency of not less than 1000 Units
per ml when intended for prophylactic use and not less than
3000 Units per ml when intended for therapeutic use.
Description. A clear, colourless or pale yellow liquid or a freezedried, cream-coloured powder or pellet.
Identification
Specifically neutralises and renders the toxin formed by
C. tetani hannless to susceptible animals or by any other
suitable in vitro test.
2441
TETANUS ANTITOXIN
IF 2010
Tests
Potency. Cany out the biological assay of tetanus antitoxin
described below.
Biological Assay of Tetanus Antitoxin

The potency of tetanus antitoxin is determined by comparing
the dose necessary to protect mice against the paralytic effects
ofa fixed dose of tetanus toxin with the dose ofthe Standard
Preparation of tetanus antitoxin necessary to give the same
protection. For this purpose the Standard Preparation of
tetanus antitoxin and a suitable preparation of toxin, for use
as a test toxin, are required. The test dose of the toxin is
determined in relation to the Standard Preparation and the
potency of the preparation under examination is then
determined in relation to the Standard Preparation using the
test toxin.
The Standard Preparatioil is the 2nd International Standard
for Tetanus antitoxin, equine, established in 1969, consisting
of freeze-dried hyperimmune horse serum (supplied in
ampoules containing 1400 Units), or another suitable
preparation the potency of which has been determined in
relation to the International Standard.
Suggested Method
NOTE - The severity ofthe tetanic paralysis to be regarded

as the end-point is such that the paralysis is readily
recognised but not sL@ciently extensive to cause significant
suffering. For humane reasons the animals should be
examined at least Mice a day and should be killed as soon
as the end-point is reached.
Test animals. Use healthy mice, weighing between 17 to 22 g,
from the same stock.
Preparation oftest toxin. Prepare tetanus toxin from a sterile
filtrate ofan 8 to 10 day culture of C. tetani. Test toxin may be
prepared by adding this filtrate to glycerin in the propOltion
of 1 volume ofthe filtrate to 1 or 2 volumes of glycerin. This
solution oftest toxin is stored below 0. Test toxin may also be
prepared in stable form by saturating the filtrate with
ammonium sulphate, collecting the resulting precipitate,
drying it over phosphorus pentoxide at a pressure of 1.5 to 2.5
kPa and reducing it to a fine powder. The powder so obtained
is preserved in the dry condition at a low temperature either in
sealed ampoules or over phosphorus pentoxirjf!~~ ~J)!~~~~!~
of 1.5 to 2.5 kPa.
Determination of test dose of toxin (Lp/l0 dose). First
determine the Limes paralyticum/l 0 (Lp/10) dose of the test
toxin. This is the smallest quantity of the toxin which when
mixed with 0.1 Unit of the Standard Preparation and injected

subcutaneously into mice causes tetanic paralysis within 4
days. Prepare a solution of the Standard Preparation in a
suitable liquid such that 1 ml contains 0.5 Unit. Accurately
measure or weigh a quantity ofthe test toxin and dilute it with,
or dissolve it in, a suitable liquid. Prepare mixtures such that
each contains 2.0 ml ofthe solution ofthe Standard Preparation
(1 Unit); one of a series of graded volumes ofthe solution of
the toxin, and sufficient of a suitable liquid to give a final
volume of 5.0 ml. Allow the mixtures to stand at room
temperature, protected from light, for 60 minutes and then
inject 0.5 ml of each mixture subcutaneously into mice, six
mice being used for each mixture, and observe the mice for 4
days.
The test (Lp/lO) dose ofthe toxin is the amount present in 0.5
ml ofthatmixture that contains the smallest amount of toxin
sufficient to cause tetanic paralysis in all six mice injected
with it within 4 days.
Determination ofpotency ofthe antitoxin. Prepare a solution
bfthe StahdardPtepll.ratiCll1 in a suitableliquidsllch thatit
contains 0.5 Unit per ml. Accurately measure or weigh a
quantity of the test toxin and dilute it with, or dissolve it in, a
suitable liquid so that 1.0 ml contains five times the Lp/lO
dose as previously determined. Prepare mixtures such that
each contains 2.0 ml of the solution of the test toxin and one
of a series of graded volumes of the preparation under
examination, and sufficient of a suitable liquid to give a final
volume of5.0 ml. Prepare similar mixtures containing 2.0 ml of
the test toxin and one of a series of graded volumes of the
solution of the Standard Preparation centred on that volume
(2.0 ml) that contains 1 Unit. Allow the mixtures to stand at
room temperature, protected from light, for 60 minutes and
then inoculate 0.5 ml of each mixture subcutaneously into
each mouse, six mice being used for each mixture, and observe
the mice for 4 days. The mixture that contains the largest
volume of the preparation under examination that fails to
protect the mice from paralysis contains 1 Unit. The test is not
valid unless all the mice injected with mixtures containing 2.0
ml or less of the solution of the Standard Preparation show
paralysis and all those injected with more do not. Calculate
the potency of the preparation under examination in Units
perml.
Labelling. The label states (1) the number ofUnits per ml; (2)
species of animal from which the preparation has been made;
(3) whether the preparation is for prophylactic or for therapeutic
use; (4)Jhe_xecommendeddose;_(5)_the name and proportion
of any added preservative; (6) thl:!t the preparation, if liquid,
should not be allowed to freeze; (7) that the preparation, if
dried, should be used immediately after reconstitution in the
stated quantity of the diluent supplied by the manufacturer.
2442
IP 2010
TETANUS VACCINE (ADSORBED)

Tetanus Vaccine (Adsorbed) is a preparation oftetanus formol
toxoid adsorbed on mineral carrier. The formol toxoid is
prepared ii-om the toxin produced by the growth of Clostridium
tetani.
Production
sensitive assay for active tetanus toxin, such as inoculation

into mice or guinea-pigs. The toxoid complies with the test if
neither sample produces any sign of a toxic reaction attributable to tetanus toxin.
Antigenic purity. Not less than 1,000 Lf per mg of protein
nitrogen.
FINAL BULK VACCINE
General provisions
The maximum number ofLfper single human dose oftetanus
vaccine is 25. The production method is validated to
demonstrate that the product, if tested, would comply with
the following test.
BULK PURIFIED TOXOID
For the production of tetanus toxin, from which toxoid is

prepared, seed cultures are managed in a defined seed-lot
system in which toxinogenicity is conserved and, where
necessary, restored by deliberate reselection. A highly
toxinogenic strain of C. tetani with lmown origin and history
is grown in a suitable liquid medium. At the end ofcultivation,
the purity of each culture is tested and contaminated cultures
are discarded. Toxin-containing culture medium is collected
aseptically. The toxin content (Lfper ml) is checked to monitor
consistency of production. Single harvests may be pooled to
prepare the bulle purified toxoid. The toxin is purified to remove
components likely to cause adverse reactions in humans. The
purified toxin is detoxified with formaldehyde by a method
that avoids destruction of the immunogenic potency of the
toxoid and reversion oftoxoid to toxin, particularly on exposure
to heat. Alternatively, purification may be carried out after
detoxification.
Only bulk purified toxoid that complies with the following
vaccine.
Sterility (2.2.11). Cany out test for sterility using 10 ml of
The final bulk vaccine is prepared by adsorption of a suitable

quantity ofbulk purified toxoid onto a mineral carrier such as
hydrated aluminium phosphate or aluminium hydroxide; the
resulting mixture is approximately isotonic with blood. Suitable
antimicrobial preservatives may be added. Certain antimicrobial
preservatives, particularly those of the phenolic type,
adversely affect the antigenic activity and must not be used.
Specific toxicity. Use five nonnal, healthy guinea pigs weighing between 250 g and 350 g which have been maintained for
at least one week on a uniform, unrestricted diet, have not lost
weight during this period and have not been previously treated
with any material that will interfere with the test. Weigh the
animals separately and record their weights. Inject subcutaneously into each animal five times the dose stated on the
label. Weigh all the animals at weeldy intervals for 3 weeks.
None of the animals shows any symptoms of tetanus toxaemia or dies from tetanus within 21 days or loses weight at
the end of the test. If more than one animal dies from nonspecific causes or loses weight repeat the test. If an animal
dies or loses weight in the second test, the vaccine fails the
test.
Absence of tetanus toxin. Inject subcutaneously at least 500

Lf of purified toxoid in a volume of 1 ml into each of five
healthy guinea pigs, each weighing 250 to 350 g, that have not
previously been treated with any material that will interfere
with the test. None of the animals shows any symptoms of
tetanus toxaemia or dies from tetanus within 21 days or loses
weight at the end ofthe test. Ifmore than one animal dies from
non-specific causes or loses weight repeat the test. Ifan animal
dies or loses weight in the second test the toxoid fails the test.
Irreversibility oftoxoid. Using the buffer for the final vaccine,
prepare a dilution of the bulle purified toxoid containing the
same toxoid concentration as the final vaccine. Divide the
dilution into two equal parts. Keep one ofthem at 2 to 8 and
the other at 37 for 6 weeks. Test both dilutions by a suitable
pH (2.4.24).6.0 to 7.0.
Sterility (2.2.11). Cany out test for sterility using 10 ml ofbulle
Abnormal toxicity (2.2.1). Complies with the test for
abnormal toxicity for antisera and vaccine.
FINAL LOT
tamper-proof containers. The containers are closed. so as to
prevent cont~mination.
Assay may be released for use. Provided the tests for
2443
IP 2010
antimicrobial preservative, free formaldehyde and the assay

have been carried out with satisfactory results on the final
bulle vaccine, they may be omitted on the final lot.
Identification
Tetanus toxoid is identified by a suitable immunochemical
solution. Maintain at 37 for about 16 hours and centrifuge
until a clear supernatant liquid is obtained. The clear
supernatant liquid reacts with a suitable tetanus antitoxin,
giving a precipitate.
Tests
Biological assay of adsorbed tetanus vaccine

The potency of adsorbed tetanus vaccine is detennined by
comparing the dose ofthe vaccine required to protect guineapigs or mice from the paralytic effects of a subcutaneous
injection of tetanus toxin with the dose of the Standard
preparation needed to give the same protection. For this
comparison, the Standard preparation of adsorbed tetanus
toxoid and a suitable preparation oftetanus toxin, for use as a
challenge toxin, are necessary.
The Standard preparation is International standard for Tetanus
toxoid, adsorbed or another suitable preparation, the potency
of which has been determined in relation to the International
standard.
Suggested method
The paralysis method is not obligatory and the lethal method
may be used. For this method the number of animals and the
procedure are identical with those describedfci:dhepariilysis
method but the end-point is the death of the animal rather
than the onset of paralysis and the LD IO dose is used instead
ofthe LD 50 dose.
(a) Test on guinea pigs

toxicity for antisera and vaccine.
Test animals. Use healthy guinea-pigs from the same stock

and weighing between 250 and 350 g. Distribute them into six
groups of sixteen each. The guinea-pigs should all be of the
same sex or the males and females should be distributed equally
between the groups. If the challenge toxin to be used has not
been shown to be stable or has not been adequately
standardised, include four further groups of five guinea pigs
to serve as unvaccinated controls.
Potency of tetanus component

Assay. Detennine by either ofthe following methods.
(1) Inject subcutaneously on each of two occasions separated
by an interval of not more than 4 weeks, one-tenth of the
stated human dose diluted to 1 ml with saline solution into
each of9 normal, healthy guinea pigs weighing between 250
and 350 g. Not more than 2 weeks after the second injection,
collect the serum from each animal and carry out the biological
test for tetanus antitoxin, described under Tetanus antitoxin
or any other method approved by National Regulatory
. Authority.
Challenge toxin. Select a preparation of tetanus toxin

containing not less than 50 times the 50 per cent paralytic
dose per ml. Ifthe challenge toxin preparation has been shown
to be stable, it is not necessary to verify the paralytic dose for
every assay.
(2) Carry out the biological assay ofadsorbed Tetanus Vaccine

(Adsorbed) described below.
Preparation ofthe challenge toxin solution. Immediately prior

to use, prepare from the challenge toxin by dilution with
phosphate buffered saline pH 7.4 a challenge toxin solution
containing fifty times the 50 per cent paralytic dose per ml. If
necessary, dilute portions of this challenge toxin solution 16,
50 and 160 fold with the same buffer solution.
If the lower limit of the 95.0 per cent confidence interval of

estimated potency is less than 40 IU per single human dose
then the limitsofthe 95;0 percent confidence interval ofthe
estimaterofpotency shall be within 50 to 200 per cent of the
estimated potency unless the lower limit of the 95.0 per cent
confidence interval of the estimated potency is greater than
40 IU per single human dose.
Determination of potency. Prepare in saline solution three

dilutions ofthe vaccine under examination and three dilutions
ofasolution-ofthe-Standard-preparation such -that,.for-eaGh,
the dilutions forma series differing by-not more than 2.5 fold
when injected subcutaneously in 1 ml volumes into guineapigs, protect approximately 50 per cent ofthe animals from the
Sera of at least 6 guinea pigs out of 9 should contain not less

than 0.5 Unit oftetanus antitoxin per ml.
2444
IP 2010
paralytic effects ofthe subcutaneous injection of the quantity

of tetanus toxin prescribed for this test. Allocate the six
dilutions one to each of the six groups of sixteen guinea pigs
and inject subcutaneously 1.0 ml of each dilution into each
guinea pig in the group to which that dilution is allocated.
After 28 days inject each animal subcutaneously with 1.0 ml
of the challenge toxin solution containing fifty times the
50 per cent paralytic dose. Ifnecessary, allocate the challenge
toxin solution and the three dilutions made from it one to each
of the four groups of five guinea pigs and inject
subcutaneously 1.0 ml ofeach toxin solution into each guineapig in the group to which that toxin solution is allocated.
Examine the guinea pigs twice daily, remove and kill all animals
showing definite signs oftetanus paralysis. Count the number
of guinea pigs without paralysis 5 days after injection of the
challenge toxin and calculate the potency ofthe vaccine under
examination relative to the potency ofthe Standard preparation
on the basis of the number of animals without paralysis in
each of the six groups of sixteen, using standard statistical
methods.
The test is not valid unless (a) for both the vaccine under
examination and the Standard preparation, the 50 per cent
protective doses lie between the largest and smallest doses of
the preparations given to the guinea pigs; (b) if applicable,
the number of paralysed animals among the four groups of
five injected with the challenge toxin solution and its dilutions
indicate that the challenge was approximately 50 times the 50
per cent paralytic dose; (c) the fiducial limits of assay lie
between.50.0 per cent and 200.0 per cent of the estimated
potency; (d) the statistical analysis shows no deviations from
linearity or parallelism. The test may be repeated any number
o~times but when more than one test is performed the results
of all valid tests must be combined in the estimate ofpotency.
(b) Test on mice

Test animals. Use healthy mice from the same stock, weighing
between 14 and 20 g. Distribute them into six groups ofsixteen
each. If the challenge toxin to be used has not been shown to
be stable or has not been adequately standardised, include
four further groups of six. mice to serve as unvaccinated
controls. The mice should all be of the same sex or the males
and females should be distributed equally among the groups.
Challenge toxin. Select a preparation of tetanus toxin
containing not less than 100 times the 50 per cent paralytic
dose perml.
to use prepare from the challenge toxin by dilution with
phosphate buffered saline pH 7.4 a challenge toxin solution
containing fifty times the 50 per cent paralytic dose in each 0.5
ml. Ifnecessary, dilute portions ofthis challenge toxin solution
16, 50 and 160 fold with the same buffer solution.
Determination of potency. Prepare in saline solution three

dilutions ofthe vaccine under examination and three dilutions
of a solution of the Standard preparation such that, for each,
the dilutions form a series differing by not more than 2.5 fold
when injected subcutaneously in 0.5 ml volumes into mice,
protect approximately 50 per cent. of the animals from the
paralytic effects of the subcutaneous injection ofthe quantity
of tetanus toxin prescribed for this test. Allocate the six
dilutions one to each of the six groups of sixteen mice and
inject subcutaneously 0.5 ml ofeach dilution into each mouse
in the group to which the dilution is allocated. After 28 days
inject each animal subcutaneously with 0.5 ml ofthe challenge
toxin solution containing fifty times the 50 per cent paralytic
dose. If necessary, allocate the challenge toxin solution and
the three dilutions made from it one to each ofthe four groups
of six mice and inject subcutaneously 0.5 ml of each toxin
solution into each mouse in the group to which that toxin
solution is allocated. Count the number of mice without
paralysis 4 days after injection of the challenge toxin and
calculate the potency ofthe vaccine under examination relative
to the potency ofthe Standard preparation on the basis of the
numbers ofanimals without paralysis in each ofthe six groups
of sixteen, using standard statistical methods.
examination and the Standard preparation, the 50 per cent
protective doses lie between the largest and smallest doses of
the preparations given to the mice; (b) ifapplicable, the number
of paralysed animals among the four groups of six injected
with the challenge toxin solution and its dilutions indicate
that the challenge was approximately 50 times the 50 per cent
paralytic dose; (c) the fiducial limits ofthe assay lie between
50.0 per cent and 200.0 per cent of the estimated potency;
(d) the statistical analysis shows no deviation from linearity
or parallelism. The test may be repeated any number oftimes
but when more than one test is performed the results of all
valid tests must be combined in the estimate of potency.
(c) Determination ofantibodies in guinea pigs

Preparation of serum samples. For preparation of serum
samples, the following technique has been found suitable.
Invert the tubes containing blood samples 6 times and allow
to stand at 37 0 for 2 h, then at 4 0 for 2 hours, centrifuge at room
temperature at 800 g for 20 min. transfer the serum to sterile
tubes and store at a temperature below -20 0 At least 40.0 per
cent yield of serum is obtained by this procedure.
Determination of antibody titre. The ELISA and ToBI tests
shown below are given as examples ofimmunochemical
methods that have been found suitable for the determination
of antibody tire.
Determination of antibody titre in guinea pig serum by
enzyme-linked immunosorbent assay (ELISA). Dilutions of
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IP 2010
TETANUS VACClNE (ADSORBED)

test and reference sera are made on ELISA plates coated with
tetanus toxoid. A positive guinea"pig serum control and a
negative guinea-pig serum control are included on each plate
to monitor the assay performance. Peroxidase-conjugated
rabbit or goat antibody directed against guinea-pig-IgG is
added followed by a peroxidase substrate. Optical density is
measured and the relative antibody titre is calculated using
the usual statistical methods;
Reagents and equipment

ELISAplates: 96 wells, columns 1-12, rowsA-H.
Clostridium tetani guinea-pig antiserum (jor vaccines-human

use) reference preparation (positive control serum).
Peroxidase conjugate. Peroxidase-conjugated rabbit or goat
antibody directed against guinea-pig IgG.
Tetanus toxoid.
Carbonate coating buffer pH 9.6. Dissolve 1.59 g of anhyd,;ous
sodium carbonate and 2.93 g of sodium hydrogen carbonate
in 1000 ml of wate/: Distribute into 150 m1 bottles and sterilise
by autoclaving at 121 for 15 min.
Phosphate buffered saline pH 7.4 (PBS). Dissolve with stirring
80.0 g of sodium chloride, 2.0 g of potassium dihydrogen
phosphate, 14.3 g of disodium hydrogen phosphate dihydrate
and 2.0 g ofpotassium chloride in 1000 ml ofwate/: Store at
room temperature to prevent crystallisation. Dilute to 10 times
its volume with water before use.
Citric acid solution. Dissolve 10.51 g of citric acid in 1000 m1
of water and adjust the solution to pH 4.0 with a 400 gil solution
of sodium hydroxide.
Washing buffer. PBS containing 0.5 gil ofpolysorbate 20.
Diluent block buffer. PBS containing 0.5 gil ofpolysorbate 20
and 25 gil ofdried skimmed milk.
Peroxidase substrate. ShOltly before use, dissolve 10 mg of
diammonium 2, 2'-azinobis(3-ethylbenzothiazoline-6sulphonate) (ABTS) in 20 ml of citric acid solution.
Immediately before use add 5 III of strong hydrogen peroxide
solution.
thoroughly with washing buffer. Block the plates by addition

of 100 III of diluent block buffer to each well. Incubate in a
humid atmosphere at 379 for I h. Wash the plates thoroughly
with washing buffer. Place 100 III of diluent block buffer in
each well ofthe plates, except those ofrow A. Prepare suitable
dilutions of negative control serum; positive control serum
(from about 0.01 IU/ml) and test sera. Allocate the negative
control sentm to column l,positive control serum to columns
2 and 3 and test sera to columns 4-12 and add 100 III of each
serum to the fIrst 2 wells ofthe column to which it is allocated.
Using a multi channel micropipette, make twofold serial
dilutions from row B down the plate to row H by transferring
100 III to the following well. Discard 100 III from the last row so
that all wells contain 100 Ill. Incubate at 37 for 2 h. Wash
thoroughly with washing buffer. Prepare a suitable dilution (a
1 in 2000 dilution has been found suitable) of peroxidase
conjugate in diluent block buffer and add 100 III to each well.
Incubate at 37 in a humid atmosphere for 1 h. Wash the plates
thoroughly with washing bzif.[er. Add 100 III of peroxidase
substrate to each well. Allow to stand at room temperature,
protected from light, for 30 min. Read the plates at 405 nm in
the same order as addition of substrate was made.
Determination ofantibody titre in guinea-pig serum by toxinor toxoid-binding inhibition(ToBI). Tetanus toxin or toxoid is
added to serial dilutions oftest and reference sera; the seruml
antigen mixtures are incubated overnight. To determine
unbound toxin or toxoid, the mixtures are transferred to an
ELISA plate coated with tetanus antitoxin. Peroxidaseconjugated equine anti-tetanus IgG is added followed by a
peroxidase substrate. Optical density is measured and the
antibody titre is calculated using the usual statistical methods.
A positive control serum and a negative control serum are
included on each plate to monitor assay performance.
Reagents and equipment

Round-bottomed, rigid polystyrene microtitre plates.
Flat-bottomed ELISA plates.
Tetanus toxin or tetanus toxoid.
Clostridium tetani guinea-pig antiserum (jor vaccines-human

use) reference preparation.
Method
The description below is given as an example of a suitable
plate lay-out but others may be used. Wells 1A-H are for
negative control serum and wells 2A-H and 3A-H are for
positive control serum for assay monitoring. Wells 4-12A-H
are for test samples.
Equine anti-tetanus IgG.

Peroxidase-conjugated equine anti-tetanus IgG.
Carbonate buffer pH 9.6. Dissolve 1.5 g of anhydrous sodium
carbonate, 2.39 g of sodium hydrogen carbonate and 0.2 g of
sodium azide in 1000 m1 of wateJ; adjustto pH 9.6 and autoclave
<::;Qlt(ll<:11 W(lll QfJhe.E1ISAplate~wi!11 100 Ill. ()f tetanus

toxoid solution (0.5 Lflml in carbonate coating buffer).
aTi2T'5Tor20J:rilii.~-~-
Allow to stand overnight at 4 in a humid atmosphere. To

avoid interference from temperature gradient, do not stack
more than 4 plates high. On the following day, wash the plates
Sodium acetate buffer pH 5.5. Dissolve 90.2 g of anhydrous

sodium acetate in 900 ml ofwateJ; adjust to pH 5.5 using a
saturated solution of citric acid monohydrate and dilute to
2446
.---- -.. --.--- -- --
._~--~-
IP 2010
TICK-BORNE ENCEPHALITIS VACCINE (INACTIVATED)
1000 ml with wate!:

Phosphate buffered saline pH 7.2 (PBS). Dissolve 135.0 g of
sodium chloride, 20.55 g of disodium hydrogen phosphate
dihydrate and 4.80 g of sodium dihydrogen phosphate
monohydrate in water and dilute to 15 litres with the same
solvent. Autoclave at 100 for 60 min.
Diluent buffer. PBS containing 5 gil of bovine albumin and
0.5 gil of polysorbate 80.
Block buffer. PBS containing 5 gil of bovine albumin.
Tetramethylbenzidine solution. 6 gil solution of tetramethylbenzidine in alcohol. The substance dissolves within 30-40
min at room temperature.
Peroxidase substrate. Mix 90 ml of wate!; 10 ml of sodium
acetate buffer pH 5.5, 1.67 ml of tetramethylbenzidine solution
and 20 !J.I of strong hydrogen peroxide solution.
Washing solution. Tap water containing 0.5 gil ofpolysorbate
80.
Method
Block the round-bottomed polystyrene microtitre plates by
placing in each well 150 !J.I of block buffer. Cover the plates
with a lid or sealer. Incubate in a humid atmosphere at 37 for
1 h. Wash the plates thoroughly with washing solution. Place
100 !J.I ofPBS in each well. Place 100 !J.I of reference guineapig
tetanus antitoxin in the first well of a row. Place 100 !J.I of
undiluted test sera in the first well of the required number of
rows. Using a multichannel micropipette, make it two fold serial
dilutions across the plate (up to column 10), by transfer of 100
!J.I to the following well. Discard 100 !J.l from the last column so
that all wells contain 100 !J.l. Prepare 0.1 Lfiml solution oftetanus
toxin or toxoid using PBS a diluent. Add 40 !J.I ofthis solution
to all wells except those column 12. The wells ofrow 11 are a
positive control. Add 40 !J.I of PBS to the wells of column 12
(negative control). Shake the plates gently and cover them
with lids. Coat the ELISA plates: immediately before use make
a suitable dilution of equine anti-tetanus IgG in carbonate
bufferpH 9. 6 and add 100 !J.I to all wells. Incubate the 2 series
of plates overnight in a humid atmosphere at 37. To avoid
temperature gradient effects, do not stack more than 4 plate
high. Cover the plates with lids. On the following day, wash
the ELISA plates thoroughly with washing solution. Block
the plates by placing in each well 125 !J.I of block bzlffir. Incubate
at 37 in a humid atmosphere for 1 h. Wash _the plates
thoroughly with washing solution. Transfer 100 !J.I ofthe preincubation mixture from the polystyrene plates to the
corresponding wells ofthe ELISA plates, starting with column
12 and then from 1 to 11. Cover the plates with a lid. Incubate
at 37 in a humid atmosphere for 2 h. Wash the ELISA plates
thoroughly with washing solution. Make a suitable dilution
(a 1 in 4000 dilution has been found suitable) ofthe peroxidase-
conjugated equine anti-tetanus IgG in diluent buffer. Add

100 !J.I of the dilution to each well and cover the plates with a
lid. Incubate at 37 in a humid atmosphere for 1.5 h. Wash the
ELISA plates thoroughly with washing solution. Add 100 !J.I
ofperoxidase substrate to each well. A blue colour develops.
Incubate the plates at room temperature. Stop the reaction at
a given time (within 10 min) by the addition of 100 !J.l of2 M
sulphuric acid to each well in the same order as the addition
ofsubstrate. The colour changes from blue to yellow. Measure
the absorbance (2.4.7) at 450 nm immediately after addition of
the sulphuric acid or maintain the plates in the dark until
reading.
(d) Any other validated serological assay in guinea-pigs/mice
approved by National RegulatoryAuthority.
Labelling. The label states (1) the human dose (ml); (2) the
minimum units per single human dose or the minimum
International Units per single human dose ifpotency test done
by challenge method; (3) the name and the amount of the
adsorbent and preservative; (4) that the vaccine must be
shaken before use; (5) that the vaccine is not to be frozen.
Tick-borne Encephalitis Vaccine

(Inactivated)
Tick-borne Encephalitis Vaccine (Inactivated) is a liquid
preparation ofa suitable strain ofticle-borne encephalitis virus
grown in cultures of chick-embryo cells or other suitable cell
cultW"es and inactivated by a suitable, validated method.
Production
General provisions
The vaccine complies with the General Requirements of
Production of the vaccine is based on a virus seed lot system.
clinical efficacy and safety in man. The virus in the final vaccine
shall not have undergone more passages from the master seed
lot than the virus in the vaccine used in clinical trials.
toxicity and immunogenicity.
The virus is propagated in chick embryo cells prepared from
eggs derived from a chicken flock free from specified pathogens
(2.7.7) or in other suitable cell cultures.
2447
IP 2010
TICK-BORNE ENCEPHALITIS VACCINE (INACTIVATED)
Inactivation
SEED LOT
To avoid interference, viral aggregates are removed by :filtration

immediately before the inactivation process. The virus
suspension is inactivated by a validated method; the method
shall have been shown to be consistently capable of
Only a seed lot that complies with the following requirements inactivating tick-borne encephalitis virus without destroying
the antigenic and immunogenic activity; as part of the
validation studies, an inactivation curve is plotted representing
Identification
residual live virus concentration measured on not fewer than
three
occasions. If fonnaldehyde is used for inactivation, the
Each seed lot is identified as containing the vaccine strain of
presence
of an excess offree fonnaldehyde is verified at the
tick-borne encephalitis virus by a suitable immunochemical
end
of
the
inactivation process.
method, preferably using monoclonal antibodies.
The strain ofvirus used is identified by historical records that
include information on the origin of the strain and its
subsequent manipulation. Virus seed lots are stored at or below
-60.
Virus concentration. The virus concentration of each seed

lot is determined by titration in suitable cell cultures to monitor
consistency of production.
Only an inactivated harvest that complies with the following

vaccine.
Extraneous agents (2.7.3). Each seed lot complies with the

requirements for extraneous agents in viral vaccines for human
use; the tests in cell cultures are carried out in human and
simian cells only.
Residual infective virus. Inoculate a quantity ofthe inactivated

harvest equivalent to not less than ten human doses ofvaccine
in the final lot into primary chicken fibroblast cell cultures, or
other cells shown to be at least as sensitive to tick-borne
encephalitis virus with not less than 3 cmz of cell sheet per ml
of inoculum. Incubate at 37 1 for 14 days. No cytopathic
effect is detected at the end ofthe incubation period. Collect
the culture fluid and inoculate 0.03 ml intracerebrally into each
of not fewer than ten mice about 4 weeks old. Observe the
mice for 14 days. They show no evidence of tick-borne
encephalitis virus infection.

All processing of the cell cultures ifperfonned under aseptic
conditions in an area where no other cells are being handled.
Serum and trypsin used in the preparation of cell suspensions
and media used must be shown to be free from extraneous
agents. The cell culture media may contain a pH indicator
such as phenol red and approved antibiotics at the lowest
effective concentration. At least 500 ml of the cell cultures
employed for vaccine production is set aside as uninfected
cell cultures (control cells).
requirements may be used in the preparation ofthe inactivated
harvest.
Identification
The single harvest is shown to contain tick-borne encephalitis
virus by a suitable immunochemical method, preferably using
monoclonal antibodies, or by virus neutralization in cell
cultures.
Sterility (2.2.11). Complies with the test for sterility carried
out using 10 ml for each medium.
Mycoplasma (2.7.4). Complies with the test for mycoplasmas
carried out using I ml for each medium.
Control cells. The control cells comply with the tests for
extraneous agents (2.7.3). If the vaccine is produced using a
for
identification.
Virus concentration. Determine the virus concentration by
titration in suitable cell culture to monitor consistency of
production.
Purification
Several inactivated single harvests may be pooled before
concentration and purification by suitable methods, preferably
by continuous-flow, sucrose density-gradient centrifugation.
Only a purified, inactivated harvest that complies with the
following requirements may be used in the preparation offinal
bulle vaccine.
Sterility ( 2.2.11). Complies with the test for sterility, carried
out using 10 ml for each medium.
Specific activity. Detennine the antigen content ofthe purified,
inactivated harvest by a suitable immunochemical method
(2.2.14). Detennine the total protein content by a suitable
method. The specific activity, calculated as the antigen content
per unit mass of protein, is within the limits approved for the
specific product.
FINAL BULK VACCINE
The final bulle vaccine is prepared from one or more purified,
inactivated harvests.
Qnly a final bulle vaccin.e that cOlnplies. with the following
requirement may be used in the preparation ofthe final lot.
2448
TUBERCULIN PURIFIED PROTEIN DERIVATIVE
IP 2010
FINAL LOT
Assay may be released for use. Provided that the tests for :free
formaldehyde, bovine serum albumin (where applicable) and
pyrogens and the assay have been carried out satisfactory
final lot.
Identification
The vaccine is shown to contain tick-borne encephalitis virus
antigen by a suitable immunochemical method using specific
antibodies or by the mouse immunogenicity test described
under Assay.
Tests
validity criteria four to five dilutions will usually be necessary.

Prepare dilutions such that the most concentrated suspension
is expected to protect more than 50 per cent ofthe animals and
the least concentrated suspension less than 50.0 per cent.
Allocate each dilution to a different group of mice and inject
subcutaneously into each mouse 0.2 ml ofthe dilution allocated
to its group. Seven days later make a second injection using
the same dilution scale. 14 days after the second injection
prepare a suspension of the challenge virus containing not
less than 100 LD so in 0.2 m!. Inject 0.2 ml of this virus
suspension intraperitoneally into each vaccinated mouse. To
verify the challenge dose, prepare a series of not fewer than
three dilutions ofthe challenge virus suspension at not greater
than one-hundred fold intervals. Allocate the challenge
suspension and the four dilutions, one to each of the five
groups often mice, and inject intraperitoneally into each mouse
0.2 ml ofthe challenge suspension or the dilution allocated to
its group. Observe the animals for 21 days after the challenge
and record the number of mice that die in the period between
7 and 21 days after the challenge.
Calculations. Calculate the results by the usual statistical

Bovine serum albumin. If bovine serum albumin has been methods for an assay with quantal responses (5.7).
used during production, the vaccine contains not more than Validity criteria. The test is not valid unless (l) the
50 ng per single human dose, determined by a suitable . concentration ofthe challenge virus is not less than 100 LD 50;
(2) for both the vaccine under examination and the reference
preparation'the 50.0 per cent protective dose (PD so) lies
between the largest and smallest doses given to the mice; (3)
the statistical analysis shows a significant slope and no
into each rabbit, per kg of body mass, one dose of vaccine.
significant deviation from linearity and parallelism ofthe doseresponse lines; (4) the fiducial limits (P = 0.95) are not less
Assay
The potency is determined by comparing the dose necessary
to protect a given proportion of mice against the effects of a estimated potency.
lethal dose of tick-borne encephalitis virus, administered
intraperitoneally, with the quantity of a reference preparation
of tick-borne encephalitis vaccine necessary to provide the
same protection. For this comparison an approved reference
preparation and a suitable preparation of tick-borne
encephalitis virus from an approved strain for use as the
challenge preparation are necessary.
Potency requirement. Include all valid tests to estimate the

mean potency and the fiducial limits (P = 0.95) for the mean
potency; compute weighed means with the inverse of the
squared standard error as weights. The vaccine complies with
the test ifthe estimated potency is not less than that approved
by the competent a)lthority, based on data from clinical efficacy
trials.
The following is cited as an example ofa method that has been

found suitable for a given vaccine.
Labelling. The label states (1) the strain of virus used in

preparation; (2) the type of cells used for production of the
vaccine.
Selection and distribution oftest animals. Use healthy mice

weighing between 11 and 17 g derived from the same stock.
Distribute the mice into not less than six groups of a suitable
size to meet the requirements for validity ofthe test; for titration
of the challenge suspension, use not fewer than four groups
often mice. Use mice of the same sex or distribute males and
females equally between groups.
Determination ofpotency of the vaccine. Prepare not less
than three suitable dilutions ofthe vaccine under examination
and of the reference preparation; in order to comply with
Thberculin Purified Protein Derivative

Tuberculin PPD; Tuberculin Purified Protein Derivative for
Human Use
Tuberculin Purified Protein Derivative is a preparation made
from the heat-treated products of growth and lysis of one or
more strains of Mycobacterium tuberculosis that reveal
2449
IP 2010
TUBERCULIN PURIFIED PROTEIN DERIVATIVE
delayed hypersensitivity in animals sensitised by a

organism of the same species.
micro~
Production
It is prepared from the water-soluble fraction obtained by
heating in free-flowing steam or in an autoclave and
subsequently filtering cultures of the mycobacteda grown in
a suitable liquid medium. The active fraction in the filtrate,
which is predominantly protein, is separated by precipitation,
washed and redissolved. The preparation is free from
mycobacteria. An antimicrobial preservative that does not give
rise to false positive reactions, such as 0.5 per cent w/v of
phenol, and a suitable stabiliser may be added. Phenol is not
added to preparations that are to be freeze-dTied. The final
sterile product is distributed into sterile glass containers which
are then sealed so as to prevent microbial contamination or
alternatively it is freeze-dTied and the containers subsequently
sealed.*
* To ensure availability of a preparalion of uniform pOlency, Tuberculin

Purified Prolein Derivative is produced and issued by Ihe Sialens Serum
Inslilule, Denmark as a powder 10 be reconslilllied as slaled on Ihe
label.
The preparation may be issued either as a sterile liquid or as a
freeze-dTied product. If issued as a liquid, it is in a ready-touse form and 0.1 ml constitutes one intradermal dose
containing appropriate number ofUnits. If issued as a freezedTied product, it should yield a ready-to-use preparation when
reconstituted as per manufacturer's instructions.
Description. A colourless or pale, straw-coloured liquid, or

dry cream-coloured powder, or pellet.
The preparation, reconstituted if necessary as stated on the
label, complies wi(h the follOWing requirements.
Identification
A. When progressively increasing doses are injected
intradellllally into specifically sensitised guinea-pigs, reactions
occur at the points of injection, varying from erythema to
necrosis. When similar injections are .administered to nonsensitised guinea pigs no such reactions occur.
B. The potency test described below serves as a test for
identity ifit is perfoll11ed on material from the final containers.
Tests
pH (2.4.24). 6.5 to 7.5.
Live mycobacteria
A. Inject 5.0 ml intraperitoneally or subcutaneously into each

oftwo guinea-pigs weighing between 300 and 400 g. Observe
the animals for not less than 42. days. Kill the animals and
carry out an autopsy. No guinea-pig shows signs of infection
with mycobacteria.
B. Carry out tests for live mycobacteria in the preparation
under examination using suitable culture media. No growth of
mycobacteria should occur.
Sensitising effect. Inject intradell11ally into each of three

guinea-pigs three times, at intervals of 5 days, a dose of the
preparation under examination containing about 500 Units in
a volume of0.1 m!. Two to three weeks after the third injection
inoculate the same dose intradellllally into these animals and
into a control group of three guinea-pigs of the same weight
but that have not had any previous injections of tuberculin.
The reactions ofthe two groups are not significantly different
after 48 to 72 hours.
Toxicity. Inject subcutaneously into 2 healthy guinea-pigs,
weighing not less than 250 g and which have not previously
been treated with any material which will interfere with the
test, 0.5 ml ofa solution containing 1,00,000 Units per m!. No
hall11ful effects are produced within 7 days.
Biological assay
The potency of tuberculin purified protein derivative is
detell11ined by comparing the dose necessary to reveal delayed
hypersensitivity in guinea-pigs or other animals
hypersensitised with mycobacteria of the same type as that
used in the preparation of the tuberculin purified protein
derivative with the dose of the appropriate Standard
Preparation necessary to give the same effect.
The estimated potency is not less 80 per cent and not more
than 125 per cent ofthe stated potency. The fiducial limits of
error are not less than 64 per cent and not more than 156 per
cent of the stated potency.
The Standard Preparation is the 1st International Standard for
Tuberculin, purified protein derivative (PPD), mammalian,
established in 1951, consisting ofPPD derived from cultures
ofM tuberculosis (supplied in ampoules containing 5,00,000
Units) or another suitable preparation the potency of which
has been detellllined in relation to. the International Standard.
Phenol (ifpresent) (2.3.36). Not more than 0.5 per cent w/v.
Method

Potency. Carry out the biological assay oftuberculin purified
protein derivative described below.
Tuberculin Purified Protein Derivative in fi'eeze-dried form
complies with the following additional requirements.
Sensitise 12 guinea-pigs eachweighingnotlessthan400g by

the intramuscular injection of a total of about 0.5 ml of a
suspension in a suitable mineral oil with or without emulsifier
and containing 0.1 mg per ml of heat-inactivated, dried
mycobacteria ofthe same type as that used in the preparation
2450
IP 2010
TYPHOID (STRAIN Ty 21a) VACCINE, LIVE (ORAL)
of tuberculin. Use phosphate-buffered saline pH 7.4

containing 0.005 per cent w/v of polysorbate 80 to prepare
the dilutions of the Standard preparation and of the
preparation under examination and to reconstitute freeze~dried
preparations containing no stabiliser. Not less than 1 month
and not more than 6 months later carry out the following
test:
Shave the flanks to provide space for at least three reactions
on each side, but not more than a total of 12 injection sites per
animal. Use at least three doses of the Standard preparation
and at least three doses of the preparation under examination,
the highest dose being about 10 times as strong as the lowest.
Dilute the preparations so that the lesions produced are 8 to
25 mm in diameter. Allocate the doses to the available sites in
a random manner, in a Latin square design. Inject each dose
intradermally in the same volume (0.1 or 0.2 ml) at the sites to
which it has been allocated. Measure the diameters of the
lesions after 24 to 48 hours and calculate the result ofthe test
using standard statistical methods on the basis that the lesion
diameters are directly proportional to the logarithm of the
concentration of the tuberculin.
Storage. Store in light-resistant containers at a temperature
between 2 and 8. It should not be allowed to freeze.
Labelling. The label states (1) the number ofUnits per dose of
0.1 ml or per ml or per mg; (2) the total volume in the container
(for liquid preparation); (3) the nature and quantity of the
reconstituting liquid (for the freeze-dried preparation); (4) the
name and proportion of any added substances; (5) the species
or strain used; (6) the storage conditions; (7) the date after
which the contents are not intended to be used; (8) that care
should be taken to avoid inhaling the powder (for the freezedried preparation).
flagellar H:d antigen and does not produce hydrogen sulphide

on Kligler iron agar. The strain is nonvirulent for mice. Cells of
strain Ty 21 a lyse if grown in the presence of 1.0 per cent of
galactose.
SEED LOT
The vaccine is prepared using a seed-lot system. The working
seed lots represent not more than one subculture from the
master seed lot. The final vaccine represents not more than
four subcultures i:om the Oliginal vaccine on which were made;
the laboratory and clinical tests showing the strain to be
suitable.
Only a master seed lot that complies with the following
requirements may be used in the preparation of working seed
lots.
Galactose metabolism
In a spectrophotometric assay, no activity of the enzyme
uridine diphosphate-galactose-4-epimerase is found in the
cytoplasm of strain Ty 21a compared to strain Ty 2.
Biosynthesis of lipopolysaccharide
Lipopolysaccharides are extracted by the hot-phenol method
and examined by size-exclusion chromatography (2.4.16). Strain
Ty 21a grown in medium free of galactose shows only the
rough (R) type of lipopolysaccharide.
Serological characteristics
Strain Ty 21a grown in a synthetic medium without galactose
does not agglutinate to specific anti-O:9 antiserum. Whatever
the growth conditions, strain Ty 21 a does not agglutinate to
Vi antiserum. Strain Ty 21a agglutinates to H:d flagellar
antiserum.
Biochemical markers
Typhoid (Strain Ty 21a) Vaccine, Live

(Oral)
StrainTy 21a does not produce hydrogen sulphide on Kligler

iron agar. This property serves to distinguish Ty 21 a from
other galactose-epimerase-negative S. typhi strains.
Typhoid Vaccine (Live, Oral, strain Ty 21a) is a freeze-dried

preparation of the live Salmonella typhi strain Ty 21a grown
in a suitable medium. When presented in capsules, the vaccine
complies with the tests stated under Capsules.
Strain Ty 21a cells lyse when grown in the presence of 1.0 per
cent of galactose.
Production
The bacteria from the working seed lot are multiplied in a

preculture, subcultured once and are then grown in a suitable
medium containing 0.001 per cent ofgalactose at 30 for 13 to
15 hours. The bacteria are harvested. The harvest must be
free from contaminating micro-organisms.
The main characteristic of the strains is the defect of the

enzyme uridine diphosphate-galactose-4 epimerase. The
activities of galactopermease, galactokinase and galactose-lphosphate uridyl-transferase are reduced by 50 to 90 per cent.
Whatever the growth conditions, the strain does not contain
Vi antigen. The strain agglutinates to anti-O : 9 antiserum only
if grown in medium containing galactose. It contains the
CeUgrowth

requirements may be used for the preparation of the freezedtied harvest.
2451
TYPHOID (STRAIN Ty 21a) VACCINE, LIVE (ORAL)
IP 2010
each dosage unit must contain not less than 4 x 109 live
bacteria.
pH (2.4.24).6.8 to 7.5.
Optical density
The optical density of the culture, measured at 546 nm, is 6.5
to 11.0. Before carrying out the measurement,dilute the culture
so that a reading in the range 0.1 to 0.5 is obtained and correct
the reading to take account of the dilution.
Identification
Culture bacteria on an agar medium containing 1.0 per cent of
galactose and bromothymol blue. Light blue, concave
colonies, transparent due to lysis of cells, should be found.
No yellow colonies (galactose-fermenting) should be found.
FREEZE-DRIED HARVEST
The harvest is mixed with a suitable stabilizer and freeze-dried
by a process that ensures the survival of at least 10.0 per cent
of the bacteria and to a water content shown to be favourable
to the stability ofthe vaccine. No antimicrobial preservative is
added to the vaccine.
Only a freeze-dried harvest that complies with the following
tests may be used for the preparation of the final bulle.
Identification
Culture bacteria are examined on an agar medium containing
1.0 per cent of galactose and bromoethymol blue. Light blue,
concave colonies, transparent due to lysis of cells, should be
found. No yellow colonies (galactose-fermenting) should be
found.
Number oflive bacteria

Not less than 1 x 1011 live S. lJphi strain Ty 21a per gram.
Water (2.4.43). 1.5 to 4.0 per cent, determined by the semimicro detennination of water or any other validated method.
Identification ,
Culture bacteria from the vaccine under examination on an
agar medium containing 1.0 per cent of galactose and
bromothymol blue. Light blue, concave colonies transparent
due to lysis of cells, should be found. No yellow colonies
(galactose-fermenting) should be found.
Tests
Microbial contamination (2.2.9). Carry out the test using
suitable selective media. Determine the total viable count using
the plate-count method. The number of contaminating microorganisms per dosage unit is not greater than 102 bacteria and
20 fungi. No pathogenic bacterium, particularly Escherichia
coli, Staphylococcus aureus, Pseudomonas aeruginosa, and
Salmonella other than strain Ty 21 a are found.
Water (2.4.43). 1.5 to 4.0 per cent.
Number of live bacteria
Carry out the test using not less than five dosage units.
Homogenise the contents ofthe dosage units in a 0.9 per cent
w/v solution of sodium chloride at 4 0 using a mixer in a cold
room with sufficient glass beads to emerge from the liquid.
Immediately after homogenization prepare a suitable dilution
of the suspension using cooled diluent and inoculate brain
heart infUsion agar, incubate at 36 10 for 20 to 36 hours. The
vaccine contains not less than 2 x 109 live S. typhi Ty 2la
bacteria per dosage unit.
Labelling. The label states (1) the minimum number oflive

bacteria per dosage unit; (2) that the vaccine is for oral use
only.
FINAL BULK VACCINE
Typhoid Polysaccharide Vaccine
The final bulle vaccine is prepared by aseptically mixing one or

more freeze-dried harvests with a suitable sterile excipient.
Typhoid Polysaccharide Vaccine is a preparation of purified

Vi capsular polysaccharide obtained from Salmonella typhi
Ty2 strain or some other suitable strain of known origin and
history that has the capacity to produce Vi polysaccharide,
and which have been characterized by suitable biochemical,
physicochemical, serological or molecular methods.
Only a final bulle that complies with the following requirement

may be used in the preparation of the final lot.
Number of live bacteria. Not less than 40

strain Ty 21 a per gram.
109 live S. typhi
FINAL LOT
The final bulle vaccine is distributed under aseptic conditions
c:aJ~~lllles with a
shell or into suitable
containers.
Only a final lot that is satisfactory with respect to Identification,
Tests and Number of live bacteria will be released for use,
except that in the determination of number of live bacteria
Capsular polysaccharide is a partly 3-0-acetylated repeated

units of 2-acetylamino-2-deoxy-D-galactopyranuronic acid
with a-(1-74)-linkages.
Ginerarpiovisi()-lis'
The production of Vi polysaccharide is based on a defined
seed-lot system. The method of production shall have been
2452
IP 2010
TYPHOID POLYSACCHARIDE VACCINE
shown to yield consistently Viapolysaccharide typhoid

vaccines of adequate immunogenicity and safety in man. The
production method is validated to demonstrate that the product,
if tested, would comply with the tests for abnormal toxicity.
SEED LOT
The strain of S. typhi used for the master seed lot shall be
identified by historical records that include information on its
origin and by its biochemical and serological characteristics.
Cultures from the working seed lot shall have the same
characteristics as the strain that was used to prepare the master
seed lot, shall be demonstrated along with adequate
documentation.
Only a strain that has the following characteristics may be
used in the preparation ofthe vaccine: (a) stained smears from
a culture are typical of S. typhi; (b) the culture utilises glucose
without production of gas; (c) colonies on agar are oxidasenegative; (d) a suspension of the culture agglutinates
specifically with an appropriate Vi antiserum or colonies form
haloes on an agar plate containing a suitable Vi antiserum.
The worldng seed lot is cultured on a solid medium, which
may contain blood-group substances, or a liquid medium; the
inoculum obtained is transferred to a liquid medium, which is
used to inoculate the fmal medium. The liquid medium used
and the final medium are semi-synthetic, fi'ee from substances
that are precipitated by cetrimonium bromide and do not
contain blood-group substances or high-molecular-mass
polysaccharides, unless it has been demonstrated that they
are removed by the purification process. The bacterial purity
ofthe culture is verified by microscopic examination ofGramstained smears and by inoculation into appropriate media.
The culture is then inactivated at the beginning of the
stationary phase by the addition ofJormaldehyde. Bacterial
cells are eliminated by centrifugation; the polysaccharide is
precipitated from the culture medium by addition of
hexadecyltrimethylammonium bromide (cetrimonium
bromide). The precipitate is harvested and may be stored at
or below -20 0 before purification.
Purified Vi Polysaccharide
The polysaccharide is purified, after dissociation of the
polysaccharide/cetrimonium bromide complex, using suitable
procedures to eliminate successively nucleic acids, proteins
and lipopolysaccharides. The polysaccharide is precipitated
in its salt form and dried at 53; the powder obtained
constitutes the purified Vi polysaccharide. The loss on drying
is determined by thermogravimetry, Karl Fischer or any other
suitable method and is used to calculate the results of the
chemical tests shown below with reference to the dried
substance.
Only those pools of purified Vi-polysaccharide that comply

with the following requirements may be used in the preparation
ofthe final bulle:
Protein (2.7.1). Not more than 10 mg per gram of
polysaccharide, calculated with reference to the dried
substance.
Nucleic acids (2.7.1). Not more than 20 mg per gram of
substance.
O-Acetyl groups (2.7.1). Not less than 2 mmol per gram of
substance.
Molecular size. Examine by gel filtration or size-exclusion
chromatography (2.4.16) using cross-linked agarose for
chromatography. Use a column 0.9 m long and 16 mm in intemal
diameter equilibrated with a solvent having an ionic strength
of 0.2 mol per kg and a pH of7.0 to 7.5. Apply about 5 mg of
polysaccharide in a volume of 1 ml to the column and elute at
about 20 ml/h. Collect fractions ofabout 2.5 ml. Determine the
point corresponding to K o = 0.25 and make two pools
consisting of fractions eluted before and after this point.
Determine O-acetyl groups on the two pools. Not less than 50
per cent ofthe polysaccharide is found in the pool containing
fractions eluted before K o = 0.25.
Identification
Carry out an identification test using a suitable
Bacterial endotoxins (2.2.3). Determine by a suitable method,
the content should not be more than 150 IU per mg of
polysaccharide.
FINAL BULK VACCINE
One or more batches of purified Vi polysaccharide are
dissolved in a suitable solvent, which may contain an
antimicrobial preservative, so that the volume corresponding
to one dose contains 25 J.tg ofpolysaccharide and the solution
is isotonic with blood (250 mosrn/kg to 350 mosrn/kg).
requirements may be used in the preparation of the final lot;
Sterility (2.2.11). Cany out test for sterility using 10 ml of
Antimicrobial preservative. Where applicable, deterniine the
greater than 115.0 per cent of the intended amount. Ifphenol
has been used in the preparation, the content is not more than
2.5 gil.
2453
TYPHOID POLYSACCHARIDE VACCINE
IF 2010
FINAL LOT
The final bulle vaccine is distributed and filled aseptically into
sterile containers. The containers are closed so as to prevellt
contamination.
requirements prescribed below under Identification, Tests and
Assay and with the requirements for Bacterial endotoxins may
be released for use. Provided the tests for free fonnaldehyde
and antimicrobial preservative have been carried out on the
final bulle vaccine, they may be omitted on the final lot.
The vaccine contains minimum of 25 J.Lg purified ViPolysaccharide per dose of 0.5 ml.
absorbance for the five reference solutions and the

corresponding content ofacetylcholine chloride and read from
the curve the content of acetylcholine chloride in the test
solution for each vo1mne tested. Calculate the mean of the
two values.
1 mol ofacetylcholine chloride (181.7 g) is equivalent to 1 mol
ofO-acetyl(43.05 g).
Free formaldehyde(2.3.20). Maximum 0.2 gil.
Identification
If phenol has been used in the preparation, the content is not

more than 2.5 gil.
Carry out an identification test using a suitable

Assay
Tests
Sterility (2.2.11). Complies with the tests forsterility;
toxicity.
pH (2.4.24). 6.5 to 7.5.
O-Acetyl groups (2.7.1). 0.085 ( 25 per cent) J.Lmol per dose
(25 J.Lg ofpolysaccharide).
Test solution. Place 3 ml of the vaccine in each ofthree tubes

(two reaction solutions and one correction solution).
Reference solutions. Dissolve 0.150 g of acetylcholine
chloride in 10 ml of water (stock solution containing 15 gil of
acetylcholine chloride). Immediately before use, dilute 0.5 ml
of the stock solution to 50 ml with water (working dilution
containing 150 J.Lg/ml of acetylcholine chloride). In ten tubes,
place in duplicate (reaction and correction solutions) 0.1 ml,
0.2 ml, 0.5 ml, 1.0 ml and 1.5 ml ofthe working dilution.
Prepare a blank using 3 ml ofpurified water.
Make up the volume in each tube to 3 ml with water. Add
0.5 ml of a mixture of 1 volume of water and 2 volumes of
dilute hydrochloric acid to each of the correction tubes and
to the blank. Add 1.0 m1 of alkaline hydroxylamine solution
to each tube. Allow the reaction to proceed for exactly 2 min
and add 0.5 ml ofa mixture ofl volume of water and 2 volumes
of dilute hydrochloric acid to each of the reaction tubes.
Add 0.5 ml of a 20 per cent wlv solution offerric chloride in
a.2M hydrochloric acid to each tube, stopper the tubes and
shCl!~~_"igQr()ll.sly torelll()"e. bll1:>1:>l~s~
Measure the absorbance of each solution at 540 TIm using the
blank as the compensation liquid. For each reaction solution,
subtract the absorbance of the corresponding correction
solution. Draw a calibration curve from the corrected
Determine Vi polysaccharide content by a suitable

immunochemical method (2.2.14) using a reference purified
polysaccharide. The estimated amount of polysaccharide per
dose is 80.0 per cent to 120.0 per cent ofthe content stated on
the label. The fiducial limits oferror (P = 0.95) ofthe estimated
amount of polysaccharide are not less than 80.0 per cent and
Labelling. The label states (1) the number of micrograms of
polysaccharide per human dose (25 J.Lg); (2) the total quantity
of polysaccharide in the container.
Typhoid Paratyphoid A Vaccine

Typhoid Paratyphoid A Vaccine is a sterile suspension or a
freeze-dried solid prepared from one or more strains of
Salmonella typhi and S. paratyphi A that are smooth and
have the full complement of 0, H and Vi antigens in S. typhi
strain, and 0 and H antigens in S. paratyphi A strain. The
bacteria are killed by heat or by a bactericide such as phenol
or fonnaldehyde or by a chemical such as acetone. The liquid
vaccine and any diluent supplied by the manufacturer with
the freeze-dried vaccine contains a suitable preservative. The
dried vaccine contains no preservative.
Typhoid Paratyphoid A Vaccine for adults contains not less
than 1000 million S. typhi and 500 million S. paratyphi A
organisms per ml. The vaccine meant for children contains
333 million S. typhi and 167 million ofS. paratyphi A organisms
pennl.
;Q~~~r:ipti()!1~ I~eliqlli(t()l:t.l!l:l.~l:l<:()l1sti!Ute<ly~<:cineis~Vv'1:>ite
or pale yellow turbid liquid free from clumps.
Identification
Identify by specific agglutination.
2454
IP 2010
TYPHUS VACCINE
Tests
pH (2.2.24). 6.0 to 7 A.
Freeze Dried Typhoid Vaccine is a freeze-dried preparation of

inactivated Salmonella typhi. The vaccine is reconstituted as
stated on the label to give a uniform suspension containing
not less than 5 x 108 and not more than 1 x 109 bacteria per
human dose. The human dose does not exceed 1.0 ml of the
reconstituted vaccine.
Other tests. Complies with the tests stated under Vaccines

with the following modifications.
Abnormal toxicity (2.2.1 ). Complies with the test for abnormal
toxicity injectirig subcutaneously, intramuscularly or
intraperitoneally.
Storage. Store at a temperature 2 to 8. The liquid vaccine
must not be allowed to freeze.
Labelling. The label states (1) the number of organisms per
ml; (2) whether the vaccine is meant for use in adults or for use
in children; (3) the recommended dose; (4) the name and
proportion ofany added preservative; (5) that in case ofliquid
preparation, the vaccine should not be allowed to freeze.
Production
The vaccine is prepared from a seed-lot system from a suitable
strain of S. typhi, such as Ty 2. The final vaccine represents
not more than 3 subcultures from the strain on which were
made the laboratory and clinical tests that showed it to be
suitable. The bacteria are inactivated either by acetone or by
formaldehyde or by heat. Phenol is not used in the preparation.
The vaccine is distributed into sterile containers and freezedried to a moisture content favourable to the stability of the
vaccine.
Typhoid Vaccine is a sterile suspension of inactivated

Salmonella typhi containing not less than 5 x 108 and not
more than 1 x 109 bacteria per human dose. The human dose
does not exceed 1.0 ml.

product, iftested, would comply with test for abnormal toxicity
(2.2.1), modified to the extent that 0.5 ml of the vaccine is
injected subcutaneously or intramuscularly or
intraperitoneaUy into each mouse and 1.0 ml into each guineapig.
Production
Identification
The vaccine is prepared using a seed-lot system from a

suitable strain of S. typhi such as, Ty 2. The final vaccine
represents not more than 3 subcultures from the strain on
which were made the laboratory and clinical tests that showed
it to be suitable. The bacteria are inactivated by acetone, by
formaldehyde, by phenol or by heating or by a combination
of the last two methods.
The vaccine reconstituted as stated on the label is identified

by specific agglutination.
Typhoid Vaccine

toxicity (2.2.1) modified to the extent that 0.5 rnl ofthe vaccine
is injected subcutaneously or intramuscularly or
intraperitoneally into each mouse and 1.0 ml into each guineapig.
Antigenic power. When injected into susceptible laboratory

animals, the reconstituted vaccine elicits anti-O, anti-H and,
to a lesser extent, anti-Vi agglutinins.
tests for sterility.
Water. Not more than 5.0 percent.
Labelling. The label states (1) the method used to inactivate
the bacteria; (2) the number of bacteria per human dose; (3)
that the vaccine should be used within 8 hours of
reconstitution.
Identification
It is identified by specific agglutination.
Typhus Vaccine
Phenol (2.3.36). If phenol has been used in the preparation,

the concentration is not more than 0.5 per cent w/v.
Antigenic power. When injected into susceptible laboratory
animals, it elicits anti-O, anti-H and, to a lesser extent, anti-Vi
agglutinins.
Labelling. The label states (1) the method used to inactivate
the bacteria; (2) the number of bacteria per human dose.
Typhus Vaccine is a sterile suspension ofkiUed rickettsiae of

a strain or strains of epidemic typhus rickettsiae (Rickettsia
prowazeki) selected for antigenic efficiency.
The vaccine is prepared by injecting virulent rickettsiae into
the yolk sacs of fertile eggs which have been incubated for 7
days. Within 9 to 13 days after the injection, the yolk sacs are
collected under aseptic conditions and subjected to suitable
treatment to liberate the maximum number ofrickettsiae. The
2455
TYPHUS VACCINE
IP 2010
material is suspended in saline solution or other suitable

solution isotonic with blood to which formaldehyde solution
has been added so that the concentration of formaldehyde is
0.2 per cent to 0.5 per cent. The suspension so formed contains
10 per cent to 15 per cent w/w ofyolk sac tissue. It is purified
by treatment with ether. The aqueous middle layer of the
resultant mixture is collected and distributed under aseptic
conditions into sterile containers which are then sealed so as
to exclude micro-organisms.
The vaccine may also be prepared from the lungs of small
rodents in which rickettsial pneumonias have been caused by
inhalation ofmassive doses ofvirulent rickettsiae, or from the
peritoneal cavities of gerbils which have received
intraperitoneal injections of rickettsiae.
Description. A slightly turbid liquid; a white deposit of the

rickettsiae may separate on standing which can be readily
redistributed on shaking.
Identification
The vaccine specifically protects laboratory animals against
epidemic typhus.
Tests
toxicity injecting subcutaneously, intramuscularly or
intraperitoneally.
Potency. Carry out the biological assay of typhus vaccine
described below. The vaccine passes the test if the serum of
immunised guinea-pigs, when diluted 32-fold, protects mice
against the toxin of epidemic typhus rickettsiae.
ampoules for freezing. Dilute a small amount ofthis suspension

8-, 16-, 32- and 64-fold with saline solution. Inject
intravenously doses of 0.5 ml of each dilution into 8 white
mice, each weighing between 11 and 15 g. 18 hours later,
estimate the toxicity of the preparation as the LD so per g of
yollc sac, calculated from the numbers of mice killed by each
dilution. Those toxic suspensions showing 160 or more LDso
per g ofyollc sac are satisfactory for use in the neutralisation
test. The remainder of the suspension may be sealed in
ampoules and preserved for 18 months in liquid nitrogen.
Prepare mixtures of the toxic substance and the sera under
test for the neutralisation test in mice and allow to stand for 2
hours before injection. Inject intravenously into mice, each
weighing between 11 and 15 g, 0.5 ml containing 2 LDso ofthe
toxic substance together with serial 1 to 2 dilutions of serum.
Keep the mice in an incubator at a temperature between 28
and 30 until the results of the test are recorded. Record the
deaths at the end of 24 hours and express the results in terms
of the highest dilution giving complete protection.
Storage. Store at a temperature 2 to 8 ; When stored under

the prescribed conditions, the vaccine may be expected to
retain its potency for at least 1 year.
Labelling. The label states (1) the nature of the preparation,
i.e., whether it has been prepared in eggs, rodent's lungs or
otherwise and whether it has been purified; (2) the name and
proportion of any added preservative.
Varicella Vaccine, Live

Varicella Vaccine (Live) is a freeze-dried preparation ofa suitable
attenuated strain of Helpesvirus varicellae.
Production
Biological Assay of Typhus Vaccine

Inject subcutaneously 0.5 ml ofthe vaccine under examination
into each of 10 or more healthy guinea-pigs, each weighing
between 400 and 500 g. After 7 days inject once again 0.5 ml of
the vaccine into the animals. Fourteen days after the second
injection bleed the animals and pool aliquots of the sera. Test
the pools against a toxic substance prepared from injected
yolle sacs of living fertile eggs as described below.
Incubate for 4 to 10 days after inoculation with epidemic typhus
rickettsiae. Harvest the yolk sac and stain a smear by the
Macchiavello technique. Use only yolle sac of living embryos
showing 4+ or more rickettsiae. Determine the wet weight of
the yolk_sac, grind the yolk sac with a suitable grade of
aluminilllnoxide -anrlsuspend-il1 sterile-mi1lC'(pHadjusted-to
7.6) or in a mixture ofone part offresh eggyollc and 99 parts of
sodium chloride injection using, for each g ofthe yolk sac, 10
ml of the vehicle. Centrifuge this suspension for 5 minutes at
low speed, decant the supernatant liquid and distribute in
General provisions
been shown to yield consistently live varicella vaccines of
adequate immunogenicity and safety in man. The virus in the
final vaccine shall not have been passaged in cell cultures
beyond the 38th passage from the original isolated virus.
efficacy.

The virus is propagated in human diploid .cells (2.7.2).
SEED LOT
The strain ofvaricella virus shall be identified as being suitable
by historical records which shall include information on the
2456
VARICELLAVACCINE, LIVE
IP 2010
origin ofthe strain and its subsequent manipulation. The virus

shall at no time have been passaged in continuous cell lines.
Seed lots are prepared in the same kind of cells as those used
for the production of the final vaccine. To avoid the
unnecessary use of monkeys in the test for neurovirulence,
virus seed lots are prepared in large quantities and stored at
temperatures below -20, iffreeze-dried, or below -60, ifnot
freeze-dried.
Only a virus seed lot that complies with the following
monitor consistency of production and to determine the

dilution to be used for the final bulle vaccine.
Extraneous agents (2.7.3). Use 50 ml for the test in cell cultures.
from which the single harvest is derived comply with a test for
identity and with the requirements for extraneous agents
(2.7.3).
FINAL BULK VACCINE
Identification
Virus harvests that comply with the above tests are pooled
and clarified to remove cells. A suitable stabiliser may be added
The master and working seed lots are identified as varicella

antibodies.


and working seed lots is detelmined as prescribed underAssay
to monitor consistency of production.
Extraneous agents (2.7.3). Complies with the requirements for
seed lots for live virus vaccines; a sample of 50 ml is taken for
the test in cell cultures.
Neurovirulence (2.7.5). Complies with the test for
neurovirulence of live virus vaccines.

are handled. Approved animal (but not human) serum may be
used in the media. Serum and trypsin used in the preparation
of cell suspensions and media are shown to be free from
indicator such as phenol red and approved antibiotics at the
substrate free from antibiotics during production. 5.0 per cent,
but not less than 50.0 ml, of the cell cultures employed for
vaccine production is set aside as uninfected cell cultures
(control cells). The infected cells constituting a single harvest
are washed, released from the support surface and pooled.
The cell suspension is disrupted by sonication.
Only a virus harvest that complies with the following
requirements may be used in the preparation of the final bulk
vaccine.
Identification
The virus harvest contains virus that is identified as varicella
antibodies.
Virus concentration. The concentration of infective virus in
virus harvests is determined as prescribed under assay to
Sterility (2.2.11). Cany out test for sterility using 10 ml ofbulle

FINAL LOT
tamper-proofcontainers and freeze-dried to a moisture content
shown to be favourable to the stability of the vaccine. The
containers are then closed so as to prevent contamination
and the introduction of moisture.
Only a final lOt that is satisfactory with respect to each of the
Assay may be released for use. Provided that the test for
bovine serum albumin has been carried out with satisfactory
results on the final bulle vaccine, it may be omitted on the final
lot.
Identification
with specific Herpesvinls varicellae antibodies, it is no longer
able to infect susceptible cell cuIhIres.
Tests
Sterility (2.2.11) Complies with the test for sterility.
toxicity.
Bovine serum albumin. Not more than 0.5 J.Lg per human dose,
detelmined by a suitable immunochemical method (2.2.14).
Water (2.3.43). Not more than 3.0 per cent, detelmined by the
.
Assay
Tih'ate for infective virus, using at least ten cell culhu'es for
each fourfold dilution or by a technique of equal precision.
Use a suitable virus reference preparation to validate each
assay. The virus concentration is not less than the minimum
2457
IP 2010
VIPER VENOM
used for the preparation of the vaccine; (3) that contact with
disinfectants is to be avoided; (4) the minimum virus
concentration; (5) that the vaccine is not to be administered
to pregnant women; (6) the time within which the vaccine
must be used after reconstitution.
intravenously into each of 10 mice weighing between 18 and

20 g and observe the animals for 24 hours. Not less than 3 and
not more than 8 ofthe mice die in 2 to 24 hours. Ifthe number
of deaths is not within this range, change the dilution of the
venom suitably.
.
Express the result in terms ofnumber ofMouse Units per mg.
NOTE - The quantity in mg ofthe venom which will/dll in

2 to 24 hours not less than 3 and not more than 8 mice
represents one Mouse Unit.
Viper Venom
Storage. Store in single dose, light-resistant containers.
Daboia Venom
Viper Venom is the dried secretion obtained from the poison
glands of Viperae russelli and other species of Viperae (Fam.
Viperidae).
Labelling. The label states (1) the number ofMouse Units per
container; (2) the volume of waterfor injection to be used for
reconstitution.
Viper Venom contains not less than 50 Mouse Units per mg.
Yellow Fever Vaccine (Live)
Production
Immediately afterextraction;thepoisonoussecretion is dried
from the frozen state. The dried venom is pooled, mixed,
dissolved in ice-cold water for injection and then filtered
through a bacteria-prooffilter to give a stock solution. Further
dilutions of the stock solution are made with water for
injection under aseptic conditions to give solutions with the
required number of Mouse Units per ml. These solutions are
then distributed in single dose sterile glass containers, dried
from the frozen state and sealed at a pressure not exceeding
2.75kPa.
Yellow Fever Vaccine (Live) is a freeze-dried preparation of

the l7D stiairiofyellow fever virus gfoWif iIi fertilised hert
eggs.
Production
General provisions
The production ofvaccine is based on a virus seed-lot system.
consistently the yellow fever vaccine (live) of acceptable
immunogenicity and safety for man.
Description. An almost white or very light yellow, dry powder

which when mixed with water yields a clear solution with
some insoluble residue.

efficacy.
Identification
Referencepreparation. In the test for neurotropism, a suitable

batch of vaccine known to have satisfactory properties in
man is used as the reference preparation.
A. Produces almost immediate coagulation of blood and

citrated human plasma.
B. Mix the soluble fraction from at least 0.6 mg with 1 mlof
polyvalent antisnake venom serum and incubate the mixture
at 370 for 30 minutes. Inject 0.5 ml ofthe mixture intravenously
into a group of mice weighing between 18 and 20 g. Observe
the animals for 24 hours; no animal dies.

Virus for the preparation ofmaster and working seed lots and
for all vaccine batches is grown in the tissues ofchick embryos
from a flock free from specified pathogens (2.7.7).
SEED LOT
Tests
The 17D strain shall be identified by historical records that

include information on the origin of the strain and its
Assay. Carry out the biological assay of snake venom subsequent manipulation. Virus seed lots are prepared in large
. quantities and stored at a temperature below _60 0 Master and
described below:
Sterility (2.2.11). Complies with tests for sterility.
worklrtg seed Ioisshitu- nofcoiifain-any-humanprotein or
Biologicalassay of snake venom
added serum.
Dissolve a quantity of the freeze-dried venom equivalent to

50 Mouse Units in 25 ml of saline solution. Inject 0.5 ml

[mal vaccine shall be between passage levels 204 and 239
2458
IP 2010
YELLOW FEVER VACCINE (LIVE)
from the original isolate ofstrain l7D. A working seed lot shall
be only one passage from a master seed lot. A working seed
lot shall be used without intervening passage as the inoculum
for infecting the tissues used in the production of a vaccine
lot, so that no vaccine vims is more than one passage from a
seed lot that has passed all the safety tests.
Only a vims seed lot that complies with the following
Identification
The master and working seed lots are identified as containing
yellow fever virus by semm neutralisation in cell culture, using
specific antibodies.
Extraneous agents (2.7.3). Each worldng seed lot complies
with the test for extraneous agents.
All processing of the fertilised eggs is done under aseptic
conditions in an area where no other infectious agents or cells
are handled at the same time. Two per cent but not less than
twenty and not more than fifty eggs are set aside as uninfected
control eggs. After inoculation and incubation at a controlled
temperature, only living and typical chick embryos are
harvested. The age of the embryo at the time of vims harvest
is reckoned from the initial introduction of the egg into the
incubator and shall not be more than 12 days. After
homogenisation and clarification by centrifugation, the extract
of embryonic pulp is tested as described below and kept at 70 or colder until further processing. Vims harvests that
comply with the prescribed tests may be pooled. No human
protein is added to the vims suspension at any stage during
production. Ifstabilisers are added, they shall have been shown
to have no antigenic or sensitising properties for man.
Only a single harvest that complies with the following tests
may be used in the preparation of the final bulle vaccine.
Identification
The single harvest contains vims that is identified as yellow
fever virus by semm neutralisation in cell culture, using
specific antibodies.
Extraneous agents (2.7.3). Complies with the tests for
extraneous agents.
Control eggs. Complies with the tests for extraneous agents
(2.7.3).
Virus concentration. In order to calculate the dilution for
formulation ofthe final bulle, each single harvest is titrated as
described under Assay.
content is carried out. A suitable stabiliser may be added and

the pooled harvests diluted as appropriate.
tests may be used in the preparation of the final lot.
Protein nitrogen content (2.3.30). The protein nitrogen
content, before the addition of any stabiliser, is not more than
0.25 mg per human dose.
FINAL LOT
tamper-proofcontainers and freeze-dried to a moishrre content
shown to be favourable to the stability of the vaccine. The
containers are then closed so as to prevent contamination
and the introduction of moishrre.
Only a final lot that is satisfactory with respect to thermal
stability and each ofthe tests given under Identification, Tests
and Assay may be released for use. Provided that the test for
ovalbumin has been performed with satisfactory results on
the final bulk vaccine, it may be omitted on the final lot.
vims concentration as described under Assay in parallel for
the heated vaccine and for unheated vaccine. The difference
in the virus concentration between unheated and heated
vaccine does not exceed 1.0 loglo' and the vims concentration
ofthe heated vaccine is not less than the number of TCID so or
plaque-forming units (PFU) equivalent to 1 x 103 mouse LDso
per human dose.
Identification
with specific yellow fever virus antibodies, there is a significant
reduction in its ability to infect susceptible cell culhlres.
Tests
.Ovalbumin. Not more than 5 Ilgofovalbumin per hrrman dose,
determined by a suitable imrnunochemical method (2.2.14).
,

toxicity.
Bacterial endotoxins (2.2:3). Not more than 5 ill ofbacterial
endotoxin per human dose.
Water (2:3.43). Not more than 3.0 per cent, detelmined by the
FINAL BULK VACCINE
Assay
Single harvests that comply with the tests prescribed above

are pooled and clarified again. A test for protein nitrogen
Titrate for infective vims in cell cultures. Use an appropriate

vims reference preparation to validate each assay.
2459
IP 2010
The virus concentration is not less than the equivalent in

TCID or PFU of 1 x 103 mouse LD 50 per human dose. The
m
relationship
between mouse LD 50 and TCID 50 or PFU'IS
established by each laboratory and approved by the competent
authority.
The method shown below, or another suitable technique, may
be used to detennine the mouse LD 50
Mouse LD . The statistically calculated quantity of virus
suspensio~ that is expected to produce fatal specific
encephalitis in 50 per cent of mice of a highly susceptible
strain, 4 to 6 weeks of age, after intracerebral inoculation.
Appropriate serial dilutions of the reconstituted vaccine are
made in diluent for yellow fever virus (0.75 per cent solution
of bovine albumin in phosphate-buffered saline pH 7.4, or
any other diluent that has been shown to be equivalent for
maintaining the infectivity ofthevirus).
inoculation and prepare serum from each sample. Prepare 1: 10,

1:100 and 1: 1000 dilutions from each serum and inoculate each
dilution into a group ofat least six cell culture vessels used for
the detennination of the virus concentration. The seed lot
complies with the test if none of the sera contains more than
the equivalent of 500 mouse LD50 in 0.03 ml and at most one
serum contains more than the equivalent of 100 mouse LD 50 in
O.03ml.
Immunogenicity. Take blood from each monkey 30 days after
the injection of the test dose and prepare serum from each
sample. The seed lot complies with the test if at least 90.0 per
cent of the test monkeys are shown to be immune, as
detennined by examining their sera in the test for neutralisation
of yellow fever virus described below.
Each master and working seed lot complies with the following
tests in monkeys for viraemia (viscerotropism),
immunogenicity and neurotropism.
It has been shown that a low dilution of serum (for example,

1: 10) may contain non-specific inhibitors that influence this
test; such serum shall be treated to remove inhibitors. Mix
dilutions of at least 1: 10, 1:40 and 1: 160 ofserum from each
monkey with an equal volume of l7D vaccine virus at a dilution
that will yield an optimum number ofplaques with the titration
method used. Incubate the serum-virus mixtures in a waterbath at 37 for 1 h and then cool in iced water; add 0.2 ml of
each serum-virus mixture to each of four cell-culture plates
and proceed as for the detennination of virus concentration.
Inoculate similarly ten plates with the same amount of virus
plus an equal volume ofa 1: 10 dilution ofmonkey serum Imown
to contain no neutralising antibodies to yellow fever virus. At
the end of the observation period, compare the mean number
ofplaques in the plates receiving virus plus non-immune serum
with the mean number ofplaques in the plates receiving virus
plus dilutions of each monkey serum. Not more than 10 per
cent of the test monkeys have serum that fails to reduce the
number ofplaques by 50.0 per cent at the 1: 10 dilution.
The monkeys shall be Macaca spp. susceptible to yellow

fever virus and shall have been shown to be non-immune to
yellow fever at the time ofinjecting the seed virus. They shall
be healthy and shall not have received previously intracerebral
or intraspinal inoculation. Furthennore, they shall not have
been inoculated by other routes with neurotropic viruses or
with antigens related to yellow fever virus. Not fewer than ten
monkeys are used for each test.
Neurotropism. Neurotropism is assessed from clinical

evidence of encephalitis, from incidence of clinical
manifestations and by evaluation of histological lesions, in
comparison with ten monkeys injected with the reference
preparation. The seed lot is not acceptable if either the onset
and duration of the febrile reaction or the clinical signs of
encephalitis and pathological findings are such as to indicate
a change in the propeIties of the -virus.
Use a test dose of 0.25 ml containing the equivalent of not

less than 5000 mouse LD 50 and not more than 50,000 mouse
LD 50' determined by a titration for infectious virus and using
the established equivalence between virus concentration and
mouse LD 50 (see under Assay). Inject the test dose into one
lobe of each
under anaesthesia and observe
the monkeys for not less than 30 days.
Clinical evaluation. The monkeys are examined daily for 30

days by personnel familiar with clinical signs of encephalitis
in primates (ifnecessary, the monkeys are removed from their
cage and examined for signs ofmotor wealmess or spasticity).
The seed lot is not acceptable ifin the monkeys injected with
l11(;J(1lenc:e of severe signs of-encephalitis,such.-as
paralysis or inability to stand when stimulated, or mortality is
greater than for the reference vaccine. These and other signs
of encephalitis, such as paresis, in-coordination, lethargy,
tremors or spasticity are assigned numerical values for the
Mice of a highly susceptible strain, 4 to 6 weeks of age, are

injected intracerebrally under anaesthesia with 0.03 ml of the
vaccine dilution. Groups of not less than eight mice are used
for each dilution; the series of dilutions is chosen so as to
cover the range 0 to 100.0 per cent mortality of the mice.
Injection of the mice is performed immediately after the
dilutions have been made. The mice are observed for 21 days
and all deaths are recorded. Only survivors and deaths caused
by typical yellow fever infections are counted in the
computations. Mice paralysed on the twenty-first day of
observation are counted as survivors.
Tests in monkeys for Yellow Fever Vaccine
Viraemia (Viscerotropism). Viscerotropism is indicated by the

amount ofvirus present in serum. Take blood from each ofthe
test monkeys on the second, fourth and sixth days after
2460
IP 2010
severity of symptoms by a grading method. Each day each

monkey in the test is given a score based on the scale:
Grade 1-
rough coat, not eating,
Grade 2-
high-pitched voice, inactive, slow moving,
Grade 3-
shaky, tremors, unco-ordinated, limb weakness,
Grade 4-
inability to stand, limb paralysis or death (a dead

monkey receives a daily score of4 from the day
of death until day 30).
A clinical score for a particular monkey is the average of its

daily scores; the clinical score for the seed lot is the mean of
the individual monkey scores. The seed lot is not acceptable
if the mean of the clinical severity scores for the group of
monkeys inoculated with it is significantly greater (P = 0.95)
than the mean for the group of monkeys injected with the
reference preparation. In addition, special consideration is
given to any animal showing unusually severe signs when
deciding on the acceptability of the seed lot.
Histological evaluation. Five levels ofthe brain are examined

including:
Block 1
the corpus striatum at the level of the optic

chiasma,
Block II
the thalamus at the level ofthe mamillary bodies,
Block III -
the mesencephalon at the level of the superior

colliculi,
BlockIV -
the pons and cerebellum at the level of the

superior olives,
Block V
the medulla oblongata and cerebellum at the

level ofthe mid-inferior olivary nuclei.
anatomical fonnations of the central nervous system with

varying frequency and severity (I. S. Levenbook et al., Journal
o/Biological Standardization, 1987,15,305-313). Based on
these two indicators, the anatomical structures can be divided
into target, spared and discriminator areas. Target areas are
those which show more severe specific lesions in a majority
of monkeys ilTespective of the degree of neurovirulence of
the seed lot. Spared areas are those which show only minimal
specific lesions and in a minority of monkeys. Discriminator
areas are those where there is a significant increase in the
frequency ofmore severe specific lesions with seed lots having
a higher degree of neurovirulence. Discriminator and target
areas for Macaca cynomolgus and Macaca rhesus monkeys
are shown in the table below:
Table 1 - The discriminator and target areas for monkey.
Type ofmonkey
Macaca cynomolgus globus pallidus

putamen
median thalamic
nucleus lateral
thalamic nucleus
Macaca rhesus
Cervical and lumbar enlargements of the spinal cord are each

divided equally into six blocks; 15 /lm sections are cut from
the tissue blocks embedded in paraffin wax and stained with
gallocyanin. Numerical scores are given to each hemisection
of the cord and to structures in each hemisection of the brain
as listed below. Lesions are scored as follows:
Grade 1. Minimal: 1 to 3 small focal inflammatory infiltrates.
Degeneration or loss of a few neurons.
Grade 2. Moderate: 4 or more focal inflammatory infiltrates.
Degeneration or loss of neurons affecting not more than one
third of cells.
Grade 3. Severe: moderate focal or diffuse inflammatory
infiltration. Degeneration or loss of up to two third of the
neurons.
Grade 4. Overwhelming: vmiable but often severe inflammatory
reaction. Degeneration or loss of more than 90.0 per cent of
neurons.
It has been found that inoculation of yellow fever vaccine
into the monkey brain causes histological lesions in different
Discriminator areas Target areas

substantia nigra
anterior/
caudate nucleus
substantia nigra
globus pallidus
cervical
putamen anterior/ lumbm'
enlargement
median thalamic
nucleus lateral
thalamic nucleus
cervical enlargement
lumbar enlargement
enlargement
Scores for discriminator and target areas are used for the final
evaluation of the seed lot. The individual monkey score is
calculated from the sum of individual target area scores in
each hemisection divided by the number ofareas examined. A
separate score is calculated similarly for the discriminator areas.
Mean scores for the test group are calculated in two ways: (1)
by dividing the sum of the individual monkey discriminator
scores by the number ofmonkeys and (2) by dividing the sum
of the individual monkey target and discriminator scores by
the number of monkeys. These two mean scores are taken
into account when deciding on the acceptability of the seed
lot. The seed lot is not acceptable if either ofthe mean lesion
scores is significantly greater (P = 0.95) than for the reference
preparation.

preparation; (2) that the vaccine has been prepared in chick
embryos; (3) the minimum virus concentration; (4) that contact
with disinfectants is to be avoided; (5) the period of time
within which the vaccine is to be used after reconstitution.
2461
MONOGRAPHS
HERBS AND HERBAL PRODUCTS

....
2467
Monographs
Acacia
2469
Ajwain
Amalaki
2470
2471
Amla Juice Powder
2472
Amra
2473
Anantmula
2474
Arachis Oil
2475
AIjuna
2476
Aljuna Dry Extract
2477
Artemisia
2478
Ashwagandha
Ashwagandha Dry Extract
2479
2480
BelladonnaLeaf
2481
Belladonna Dry Extract
2483
Belladonna Tincture
2483
Bhibhitaki
2484
BhibhitakiAqueous Extract
2485
Bhringraj
2486
Bhuiarnla
2488
Brahmi
2489
Brahmi Extract
2490
Castor Oil
2490
2491
Clove Oil
Coleus
2492
2492
Coleus Dry Extract
2493
Coriander Oil
2494
Daruharidra Roots
2495
Coconut Oil
2463
MONOGRAPHS
2496
2498
2499
2500
Daruharidra Stems
Eucalyptus Oil
Garcinia
GarciniaAqueous Extract
2500
2501
Gokhru
GuarGum
2502
2503
2504
2505
2506
2507
2508
2508
2510
2510
2511
2512
Gudmar
Guduchi
GuggulResin
Gugulipid
Gugulipid Tablets
Haridra
Haridra Dry Extract
Haritaki
Haritaki Extract
Haritaki Aqueous Extract
Hydrogenated Castor Oil
Ipecac Tincture
2513
2513
2514
2515
2516
2517
2518
2519
2520
2521
2522
2523
Ispaghula Husk
Kalmegh
Kalmegh Dry Extract
Kunduru
Kutki
Lasuna
Lavang
Malt Extract
Mandukaparni
Manjistha
Maricha
Mentha Oil
2524
2525
Neem
Opium
2464
MONOGRAPHS
2527
2528
2529
2530
2531
Opium Powder
Papain
Peppennint Oil
Pippali Large
Pippali Small
2532
2533
2535
Punarnava
Sarpagandha
Sarpagandha Powder
2535
2536
2537
2538
2539
2540
2540
2542
Sarpagandha Tablets
Saunf
Senna Leaf
Senna Pods
Senna DIY Extract
Senna Tablets
Shatavari
Shati
2543
2543
2544
2545
2546
2547
2548
2549
Shellac
Starch
Sunthi
Sunthi Extract
ToluBalsarn
Tragacanth
Tulasi
Tulasi DIY Extract
Yasti
2550
2550
2551
Yasti DIY Extract
2552
Vasaka
Vasaka Extract
2465
IP 2010
Herbs and Herbal Products

Herbs and products containing herb(s) have been in trade
and commerce and are currently used for a variety ofpurposes.
As a country, India has a rich history ofuse ofherbs, processed
herbs and formulations containing herbs both from traditional
wisdom as well as cultural usage. Herbs and herbal products
are also regulated by various laws. For the purposes of
pharmacopoeial standards various considerations have been
given. This monograph provides a general outline and policies
towards the same.
Crude Herbs
This term means, unless specified otherwise, mainly whole,
fragmented or cut, plants, parts of plants, algae, fungi, and
lichen in a form which is not processed. Herbs are usually in
dried form, but sometimes, when specified, may also be in a
fresh form. In specific cases exudates which have not been
processed further also are covered under the term herbs.
Processing, does not include, normally expected value
addition steps like grading, sizing, removal of weeds or parts
of plants other than those specified herb and removal of
adulterants. The term herbs, though botanically generally refer
to plants of specified height and nature, for the purposes of
pharmacopoeial reference, shall mean and include plants and
parts of plants not necessarily from herbs and shrubs, but
cover the entire range namely creepers, climbers, trees etc.
Each monograph ofa herb in the pharmacopoeia shali specify
the botanical scientific name according the binomial system
specifying the genus, species, variety and author. In cases
where there are controversial botanical identity, as is seen
with mainly herbs known in the Indian traditional system, the
monograph shall specify the official name of the herb along
with its botanical scientific name and guidance is taken from
Ayurvedic Pharmacopoiea of India' to decide the same. In
cases where, the same herb is available in different grades or
sizes, if found appropriate and necessary, separate
monographs may be introduced in the pharmacopoeia to cover
each ofthem with appropriate standards. For example- Pippali
(large) and Pippali (small).
While deciding to introduce a monograph for a herb in the
pharmacopoeia, the criteria that would be kept in mind, but
not limited to are - herbs with specific name and a definitive
botanical identity up to species, availability and usage in trade
and commerce, regulatory interest, knowledge of and
availability of a specific chemical compound of well
characterised structure [either responsible for the biological
activity of the herb (bio-marker) or a chemical compound
known to be present in the herb even if not responsible for
biological activity(chemical/analytical marker)], availability of
a quantitative method for estimation of such a compound,
lmowledge of safety of the herb, and its sustainability. Herbs
which may figure in a regulated list under appropriate forest

and other laws, may still be taken up for a monograph for
inclusion in pharmacopoeia, if there is knowledge of efforts
to cultivate or take care ofsustainability issues and lor specific
permission is available under law for use ofthe herb. As already
specified under "General Notices" in the pharmacopoeia,
appearance of a monograph does not mean its approval as a
drug under the law. Monograph ofa herb in the pharmacopoeia
is to provide qualitative and quantitative standards of quality
for the herb for its use either as a food item or food ingredient
or food supplement! nutraceuticals, as a drug, and/or as an
ingredient in cosmetics. Each such use would need to comply
with applicable regulations. Each herb is regarded as one active
substance, irrespective of the lmowledge about the active
constituents of the herb is available of not.
* Ayurvedic Pharmacopoeia of India, Vol. 1-6, Ministry of Health and

Family Welfare, Govt. of India.
Herbs may be exposed to low dose gamma radiation for
purposes of reducing their microbial contamination. Herbs
treated with low dose gamma radiation shall meet national
laws related to such treatment and shall be .labeled as per law.
Processed Herbs
Processed herbs means preparations obtained by subjecting
herbs to treatment such as extraction, distillation, expression,
fractionation, purification, concentration and partial or full
fermentation. Processing may also be done by way of
powdering herbs, preparing tincture, preparing extract,
distilling to get essential oils, fatty oils (either expressed or
solvent extracted or a blend of both) expressedjuices, extracted
exudates, gums and oleo resins, liquid extract where the
solvent is evaporated to yield concentrated semi solid mass
or dried mass. Extraction may be performed by means of
appropriate technology such as infusion, maceration,
soxbleting, boiling under ambient or higher pressure, with or
without specified enzymes, with or without agitation and
combination thereof. Drying ofliquid extracts for removal of
the solvent may be done by using various appropriate
technologies like air drying, sun drying, drying under vacuum
or with forced air circulation, drying at low temperature with
air circulation, by way of lyophilization or freeze drying.
Extracts of herbs may also be prepared by using carbon dioxide as a SOlvent-super critical fluid extraction.
Extracts may be liquid extracts and tinctures, semi solid (soft
extracts) or solid dry extracts of known consistency obtained
from herbs. Standardized extract, a term commonly employed,
would for pharmacopoeial purposes, mean an extract adjusted
with in an acceptable tolerance to a given content of biomarker or chemical/ analytical marker. Standardization may be
achieved by adjusting the extracts with approved inert material
or by blending one or more batches of extracts. Wherever
2467
IP 2010
possible, extracts shall specify the defined range of the

constituents (bio-marker or chemical! analytical marker).
Extracts not covered in the above description would be defined
by the process ofproduction ofthe herb to the extract, solvent
used and technology applied. The difference between extracts
and tinctures would be, in the type of solvent used for
extracting a herb, and tincture would normally mean an extract
where aqueous-ethanol is used as a solvent for extraction.
Dry extracts usually have a loss on drying or water content
not greater than 5 per cent w/w, unless specified otherwise in
any monograph. It is normal to extrapolate safety aspects and
history ofuse information for extracts as long as the process,
solvents, extraction ratios are comparable to the processes
used in documented traditional Imowledge. Additionally in
cases of standardized extracts the inert excipients(s), if any
used for standardization or adjustment of the content of
constituents should also be declared on the label of such
extracts. Extracts shall be free from solvent used for extraction
and shall comply with the respective limts as given in
Appendix 5.4. Harmful and carcinogenic solvents shall not be
used fof extraction purposes. Solvents and solvent systems
may include use of propylene glycol, glycerin, sorbitol and
such other polyhydroxy alcohols, as long as the content of
such polyhydroxy alcohol are within safe limit in the final
product.
In cases where extraction and fractionation process leads to

preparation of an extract, which consists of a single chemical
compound of more than at least 70 per cent purity, such
extracts shall be treated as an active pharmaceutical ingredient
or a food additive or a cosmetic ingredient and would be
required to meet relevant laws.
Extracts may also be offered as purified or enriched extracts.
Such extract of!l herb is processed in such a way to provide
higher than normal proportion ofthe active constituent (s) of
the herb as long as the active constituent (s) is/are Imown.
Such purified or enriched extracts may contain additiona~
valuable components which may provide specific properties
like enhanced efficacy or stability or solubility and availability
of the active constituent (s). Purified and enriched extracts
may also be prepared to reduce or remove other specific
compound or group of compounds that is scientifically
considered undesirable in the herb extracts. Pharmacological,
toxicological, pharmaceutical considerations need to be
applied while preparing such purified or enriched extracts.
Mixed extracts may also be offered which would cover
combination of more than one herb extract for purposes of
providing simplification or economical way to manufacture
Approved preservative~ or preservatives system may be used

during preparation of extracts. The names of such
preservatives used which would remain in the final extract
shall be listed on the label of such extract, and the proportion
ofpreservatives used shall not exceed normally accepted safe
limits of their usage as per relevant laws or pharmacopoeial
standards. No artificial colours may be used in extracts of
herbs unless and otherwise specified in the specific
monograph.Only approved colours shall be used.
Extracts may be exposed to ethylene oxide fumigation or low
dose gamma radiation for purposes ofreducing their microbial
contamination. In cases where they are fumigated, the final
extracts exposed shall meet residual levels of ethylene oxide
limits as applicable. Herbs treated with low dose gamma
radiation shall meet national laws related to such treatment
and shall be labelled as per law.
Appearance ofa monograph ofan extract in the pharmacopoeia
does not mean its approval as a drug under the law. Monograph
of an extract in the pharmacopoeia is to provide qualitative
and quantitative standards ofquality for the extract for its use
either as a food item or food ingredient or food supplement!
nutraceuticals, as a drug and / or as an ingredient in cosmetics.
Each such use would need to comply with applicable
regulations. Each extract is regarded as one active substance
irrespective of the knowledge about the active constituents
ofthe herb is available or not.
Herbal Formulations
Herbal formulation shall mean a dosage form consisting of
one or more herbs or processed herb(s) in specified quantities
to provide specific nutritional, cosmetic benefits, and/or other
benefits meant for use to diagnose treat, mitigate diseases of
human beings or animals and/or to alter the structure or
physiology of human beings or animals. Dosage forms
commonly employed for food or cosmetic or pharmaceuticals
may be employed to formulate one or more herb or processed
herbs. Dosage forms Imown in traditional medicines may also
be adopted for preparing herbal formulations, either for external
use or for internal administration. Adequate consideration for
uniform distribution of herb or processed herbs as well as
stability of the same in the dosage form shall be provided
during formulation development.
Herbal formulation shall be labelled to comply with relevant

labelling requirements under food or drug or cosmetics laws
as applicable. Additionally, adequate information shall be
provided on label ofsuch formulations to include the name of
the herb, parts used, nature and type of extract or processed
herb used, extraction ratios, quantity per unit dose or ,per
~nerbal-formulationS:-------~------~~--'--'---~~
-serving:name (s) ofinert-excq;ients used and any preserVatives
.
Herbs may also be extracted using vegetable oils (approved added shall be provided on the label.
by Food Law) for extraction purposes and such extracts shall Appearance of a monograph of a herbal formulation in the
specify the oil used for processing.
pharmacopoeia does not mean its approval as a drug under
2468
ACACIA
IP 2010
the law. Monograph of a herbal formulation in the

pharmacopoeia is to provide qualitative and quantitative
standards of quality for the formulation for its use either as a
food item or food ingredient or food supplement/
nutraceuticals, as a drug and / or as a cosmetic. Each such use
would need to comply with applicable regulations.
C. Al 0 per cent w/v solution is either dextro-rotatory or slightly

laevo-rotatory.
Tests
Sterculia gum and agar. To 50 mg ofthe powdered substance
under examination add 0.2 ml of freshly prepared ruthenium
red solution and examine microscopically; the particles do
not acquire a red colour after irrigation with water.
Agar and tragacanth. To 10 ml of a 10 per cent w/v solution
add 0.2 ml of lead acetate solution; no precipitate is produced.
Acacia
Gum Acacia; Indian Gum
Starch and dextrin. Boil 10 ml of a 10 per cent w/v solution

and cool, add 0.1 ml of 0.05 M iodine; no blue or brown colour
is produced.
Tannins. To 10 ml of a 10 per cent w/v solution add 0.1 mlof
ferric chloride test solution; a gelatinous precipitate is formed,
but neither the precipitate nor the liquid shows. a dark blue
colour.
Sucrose and fructose. To 1 ml of a 10 per cent w/v solution
add 4 ml of water, 0.1 g of resorcinol and 2 ml of hydrochloric
acid and heat on a water-bath; no yellow or piDk colour
develops.
Acacia is the air-hardened, gummy exudation from the stem

and branches of Acacia nilotica (Linn.) Del. subsp. indica
(Benth.) Brenan (syn. A. arabica Willd. var. indica Benth.)
(Fam. Leguminosae), or other species ofAcacia. It is available
as pieces (tears) or in the form of a powder.
Description
Tears - Irregular and broken pieces ofvarying size, yellowishwhite, yellow or amber in colour, with numerous minute
fissures; brittle fractured surface, glassy and occasionally
iridescent; odourless.
Powder - A white or yellowish-white powder; odourless; on
treatment with water it dissolves to give a mucilaginous liquid
which is colourless or yellowish, dense, viscous, adhesive
and translucent.
Water-insoluble matter. Dissolve 5 g, in fine powder, in 100

ml of water in a 250-ml flask, add 10 ml of dilute hydrochloric
acid and boil gently for 15 minutes. Filter by suction while hot
through a sintered-glass crucible, previously tared, wash
thoroughly with hot water, dry at 105 and weigh; the residue
does not exceed 50 mg.
Microbial contamination (2.2.9). 1.0 g is free from Escherichia
coli.
Acid-insoluble ash (2.3.19). Not more than 1.0 per cent,
determined on 1.0 g by Method C.
. Storage. Store protected from heat, moisture and against
attack by insects and rodents.
Identification
A. An aqueous solution is gelatinised by the addition of lead
subacetate solution.
B. To 5 ml of a 10 per cent w/v solution add gradually, while

shaking, 10 ml of ethanol (95 per cent). The cloudy liquid, on
addition of 0.5 ml of acetic acid, gives a white precipitate.
Filter and add to the clear filtrate 50 ml of ammonium oxalate
solution; the filtrate becomes cloudy.
2469
AJWAlN
IP 2010
Test solution. To 1 g of the coarsely powdered substance

under examination, add 25 ml ofdichloromethane, reflux for
15 minutes, cool and filter. Reflux the residue further for two
times with 25 m1 ofdichloromethane, cool and filter. Combine
all the filtrates and concentrate under vacuum to 25 m!.
Ajwain
Bishop's weed
Reference solution. To 1 g of the qjwain RS, add 25 ml of

dichloromethane, reflux for 15 minutes, cool and filter. Reflux
the residue further for two times with 25 ml of
dichloromethane, cool and filter. Combine all the filtrates and
concentrate under vacuum to 25 m!.
9
8
7
Apply to the plate 10 III ofeach solution as bands 10 mm by 2

mm. Allow the mobile phase to rise 8 cm. Dry the plate in air
and examine in ultraviolet light at 254 nm and 365 nm, spray
with dnisaldehyde sulphuric acid reagent. Heat at 11 0 for 10
minutes arid examine the plate in daylight. The
chromatographic profile of the test solution is similar to that
of the 'reference solution.
'. .
Tests
Ajwain consists of the dried fruits of Trachyspermum ammi
Mill. (Fam.Apiaceae).
Ajwain contains not less than 1.0 per cent w/w of thymol,
Description. The fruits are oval-oblong, greenish brown to
yellowish brown in colour. They have an aromatic
characteristic odour and the taste is sweet aromatic.
Foreign organic matter{2.6.1). Not more than 2.0 per cent.

Ethanol-soluble extractive (2.6.2). Not less than 2.0 per cent.
Water-soluble extractive(2:6.3); Not less than 15.0 per cent
by method!.
'
'
,
Total Ash (2.3.19). Not more than 15.0 percent.
Acid-insoluble ash (2.3 .19). Not more than 7.0 per cent.
Identification
A. Macroscopic - The fruit consists of two portions each
called mericarp and connected by central stalk (carpophore).
A single seed is seen in each mericarp. Fruit surface is glabrous
and the five primary ridges of each mericarp are prominent,
straight and pale straw coloured.
B. Microscopic - Epicarp is composed ofpolygonal cells. In
the mesocarpic region, reticulate and lignified parenchymas
are seen at vascular strands. Tracheids show helical
thickening. Endocarp consists of narrow elongated cells
having a parquetry arrangement. Polyhedral, thick walled
endospenn contains aleurone grains and oil globules. Vittae
are six in number, four on the dorsal surface at the mesocarpic
region below the secondary ridges and two on the commissural
surface ofthe mericarp. Vittae long, slender composed ofthin
walled polygonal cells and is lined by an epithelium of small
polygonal tubular cells; 10-15 separate, septum transverse or
curved.
C. Detennine by thin-'layer chromatography (2.4.17), coating

the plate with silica gel GF 254..
Mobile phase. A mixture of 93 volumes of toluene and 7
Loss on drying (2.4.19). Not more than 10 per cent, detennined

contamination tests.
Test solution. Weigh 2.0 g ofthe coarsely powdered substance
under examination, add 50 ml of methanol, and reflux on a
water-bath for 15 minutes, cool and filter. Reflux the residue
further with methanol till the extract turns colourless, cool
and filter. Combine all the filtrates and concentrate to a volume
slightly less than 100 m!. Dilute with methanol to 100.0 m!.
Reference solution. A 0.01 per cent w/v solution of thymol RS
in methanol.
- a capillary column 30 m x 0.25 m x 0.25 mm, packed with
_..... methylpolysiloxane,._ ..... _ ....__ .. - ._- ....
temperature:
oven 90 to 260 @1 0 per minute (initially and finally
hold for 5 minutes respectively),
injector 240,
2470
IP 2010
AMALAKI
detector 280,
split flow 20 ml per minute.


of ethyl acetate, 20 volumes of glacial acetic acid and 5
volumes offormic acid.

Calculate the content of thymol.
Storage. Store protected from light in well-filled containers, at
a temperature not exceeding 30.
Amalaki
Test solution. Reflux 2 g of the coarsely powdered substance

under examination with 50-75 ml of methanol for 15 minutes,
cool and filter. Reflux the residue further for two times with
75 mlofmethanol, cool and filter. Combine all the filtrates and
concentrate under vacuum to 50 ml.
Reference solution. Reflux 0.4 g of the coarsely powdered
amalaki RS with 50-75 ml of methanol for 15 minutes, cool
and filter. Reflux the residue further for two times with 75 ml of
methanol, cool and filter. Combine all the filtrates and
Emblic Myrobalan; Indian Gooseberry
Apply to the plate 10 /ll ofeach solutioh as bands 10 mm by 2

with anisaldehyde sulphuric acid reagent. Heat the place at
100 for 5-10 minutes and examine in day light. The
of the reference solution.
Tests
Foreign organic matter (2.6.1). Not more than 3 per cent.
Ethanol-soluble extractive (2.6.2). Not less than 30 per cent.
Amalaki consists of 'the dried fruit pericarp of Emblica

offiCinalis Gaertn. (Phyllanthus emblica Linn.) (Fam.
Euphorbiaceae).
Amalaki contains not less than 1.0 per cent w/w gallic acid
Water-soluble extractive (2.6.3). Not less than 40 per cent by

Method I.
Total Ash (2.3.19). Not more than 5.0 per cent.
Acid-insoluble ash (2.3.19). Not more than 2.0 per cent.
Description. The dried fruit has a highly shriveled and wrinlded

external surface. The taste is sour and astringent followed by
delicately sweet tinge.

determined on 5 g by drying in an oven at 105.
Identification

A. Macroscopic - The dried fruit shows a broad, highly

shriveled and wrinkled external convex surface, lateral surface
transversely wrinlded, external surface exhibits few whitish
specks, occasionally some pieces show a portion of stony
testa.
E. Microscopic - The epicarpic cells are rectangular in shape
and their walls are highly cuticularized. Anomocytic type of
stomata is found rarely. Collateral fibrovascular bundles are
scattered throughout the inner mesocarp. Pitted and helical
tracheids with tapering ends are seen. At places in the phloem,
large cavities filled with crystal mass are present.
Test solution. Weigh accurately about 0.5 g of coarsely

powdered substance under examination, add 50 ml of water,
sonicate for 3 minutes and heat on a boiling water-bath for
15 minutes, cool and dilute to 100.0 ml with water and filter.
Reference solution. A 0.01 per cent w/v solution of gallic
acid RS in water.
2471
IP 2010
AMLA JUICE POWDER
mobile phase: A. a solution prepared by dissolving 0.136

g of potassium di-hydrogen orthophosphate in 500 ml
of water, add 0.5 ml of orthophosphoric acid and dilute
to 1000 ml with water,
B. acetonitrile
below,
Time
(in min)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
o
18
25
100
55
20
45
30
. 100
and examine in ultraviolet light at 254 urn. Spray the plate with
anisaldehyde-sulphuric acid reagent. Heat the plate at 110
for 10 minutes and examine the plate under 365 nm and in day
light. The chromatographic profile ofthe test solution is similar
to that of the reference solution.
Tests
~
Water - soluble extractive (2.6.3). Not less than 85.0 per cent
by method I.
Total ash (2.3.19). Not more than 15.0 percent.
80

contamination tests:
than2;0 per cent.
Gallic acid. Not less than 0.1 per cent and not more than 2.0
per cent.
Test solution. Dissolve about 50 mg of the extract under

examination or quantity equivalent to 10 mg of gallic acid in
water, by frequent shaking and dilute to 50.0 ml with water
and filter.
Calculate the content of gallic acid.

Storage. Store protected from light, heat, moisture and against

acid RS in water.
Amla Juice Powder

Amla Juice Powder is obtained by spray drying cold pressed
juice of fresh fruits of Phyllanthus emblica Linn. (Emblica
officinalis Gaertn, Fam. Euphorbiaceae)
Amla Juice Powder contains not less than 90.0 per cent w/w
and not more than 110 per cent w/w of the stated amount of
polyphenols, calculated on the anhydrous basis. It may contain
suitable added substances.
Description. A beige to off-white powder.
octadecylsilane bonded porous silica (5 11m),
mobile phase: A. 0.1 per centformic acid in water,
B. methanol,
below,
Identification
Mobile phase. A mixture of 15 volumes of ethyl acetate, 1.5

volumes of methanol and 1 volume of water.
Test solution. Dissolve 0.5 g of the extract under examination
with 10 ml of water and filter.
acid RS in water.
Time
(in min)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
95
25
95
30
40
42
95
100
100
5
ieTiitivestanaara-creviii1ion-for therep1icateiiijections isiiof
more than 2.0 percent.
Calculate the content ofthe gallic acid in extract.
2472
AMRA
IP 2010
Assay. Weigh accurately about 2 g of the extract, add 50 ml

boiling water and heat it on a water-bath for 30 minutes with
frequent stirring. Allow the solution to settle and carefully
decant it through a piece ofcotton wool to a 500-ml volumetric
flask. Repeat the extraction for 5 times with 50 ml of boiling
water. To confirm that all tannins have been extracted, add 34 drops offerric ammonium sulphate solution to 5 ml of the
extract. Blue colour will not be produced, if all tannins have
been extracted. If blue colour develops extract again with 2
times with 50 ml of boiling water and check with ferric
ammonium sulphate solution again. Cool the extracts and
make up to the mark with water. Take 50 ml into a 250-ml
conical flask and add 50 ml of indigo suiphonic acidsolution,
prepared by dissolving 1 g of indigo carmin in 50 ml of
sulphuric acid, add 500 ml of water and dilute this solution to
1000.0 m!. Titrate, with 0.1 M potassium permanganate using
indigo sulphonic acid as indicator until a golden yellow colour
is produced. Carry out a blank titration.
1 ml of 0.1 M potassium permanganate is equivalent to
0.004157 g ofpolyphenoIs calculated as tannic acid.
Storage. Store protected from heat and moisture.
Identification
A. Macroscopic - The surface is rough due to longitudinal
cracks, fissures and scattered, raised lenticels, gr~yish to dark
brown externally and yellowish~white to reddish internally. .
B. Microscopic -
The mature bark shows a wide cork which

has tangentially elongated cells a few outer layers are brown
and inner lighter in colour, resin canals and yellow coloured
tannin sacs are found in the phloem region, stone cells are
thick walled and lignified, prismatic calcium oxalate crystals
are present.
C. Determine by thin-layer ohromatography (2.4.17), coating

11 volumes of formic acid, 11 volumes of acetic acid and
25 volumes of water.
Test solution. To 5.0 g of the coarsely powdered substance
under examination, add 50 ml of methanol and reflux for
15 minutes, cool and filter. Reflux the residue further for three
times with 50 ml of methanol, cool and filter. Combine all the
filtrates and evaporate under vacuum to 10m!.
Reference solution. Weigh about 2.0 g of amra RS, add 50 ml
of methanol and reflux for 15 minutes, cool and filter. Reflux
the residue further three times with 50 ml of methanol, cool
and filter. Combine all the filtrates and concentrate under
vacuum to 4 m!.
Amra
Mango; Mangifera indica
Apply to the plate 10 III ofeach solution as bands 10 rnm by 2

rnm. Allow the mobile phase to rise 8 cm. Dry the plate in air
with vanillin glacial acetic acid reagent. Heat the plate at
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent.
Ethanol-soluble extractive (2.6.2). Not less than 10.0 percent.
Water-soluble extractive (2.6.3). Not less than 10.0 per cent
by method I.
Total ash (2.3.19). Not more than 16.0 per cent.
Amra consists ofdried stem bark of Mangifera indica L.(Fam.
Anacardiaceae).
Amra contains not less than 1.5 per cent of mangiferin

Loss on drying (2.4.19). Not rllorethan 12.0 per cent,

Description. The dried stem bark occurs in pieces ofvariable

size and thickness, surface rough. Odour pleasant and taste
astringent.

2473
AMRA
IP 2010
Test solution. Weigh 2 g of coarsely powdered substance

under examination, add 10 ml ofdimethylformamide, sonicate
for 5 minutes and add 50 ml of methanol and boil on a waterbath for 10 minutes, cool and dilute to 100.0 ml with methanol
and filter. Dilute 5.0 rnl ofthis solution to 50.0 ml with methanol.
Reference solution. Dissolve 10 mg ofmangiferin RS in 10 ml
ofdimethylformamide and dilute to 100.0 ml with methanol.
- mobile phase: filtered and degassed mixture of 15
volumes of acetonitrile and 85 volumes of buffer pH
2.8 prepared by dissolving 1.36 g of potassium dihydrogen orthophosphate in 950 ml of water, adjust
the pH 2.8 with orthophosphoric acid and make upto
1000rnl,
than 2.0 per cent.
Calculate the content of mangiferin.
Storage. Store protected from moisture and against attack by
insects and rodents.
Anantmula
Sariva; Indian Sarsaparilla
A. Macroscopic - Root is 30 cm or more long and from 3 to

6 rom thick, rigid, tortuous, cylindrical, and little branched,
consisting of a ligneous center, and a brownish, corky bark,
marked with longitudinal furrows and transverse fissures. The
odour is aromatic, recalling that of tonqua bean, the taste is
aromatic and sweetish. On one side of the root the cork is
frequently separated from and raised above the cortex, and is
transversely fissured.
B. Microscopic - Transverse section of root shows 2-3
layered cork cambium having compressed cells filled with
brown contents. Xylem traversed by narrow medullary rays.
Pith absent and central region occupied by woody tissues.
Vessel shows pitted thickening. The transverse section
exhibits numerous laticiferous cells in the secondary cortex.
Powder in chloral hydrate solution shows unicellular and
branched latex ducts; lignified elements and vessels with pitted
thickenings.
the plate with silica gel GF 254.
of ethyl acetate, 5 volumes of glacial acetic acid and 5
under examination, add 40 rnl ofmethanol, reflux for 15 minutes,
cool and filter. Reflux the residue further for two times with 25
ml of methanol, cool and filter. Combine all the filtrates and
Reference solution. To 2 g of the anantmula RS, add 40 ml of
methanol, reflux for 15 minutes, cool and filter. Reflux the
residue further for two times with 25 ml ofmethanol cool and
filter. Combine all the filtrates and concentrate unde~ vacuum
t025rnl.
8
7
Apply to the plate 20 /ll of each soluti~n as bands 10 rom by

2 rom.Allow the mobile phaseto rise 8 cm. Dry the plate in air
with anisaldehyde sulphuric acid reagent. Heat at 105" for 510 minutes and examine the plate in day light. The
5
4
3
2
1
Identification
Tests
1
Anantrnula consists ofthe dried roots ofHemidesmus indicus
(Linn) R.Br. (Fam.Asc1epiadaceae).
Allani:ffiulacontalnsnotfessthano.02ifper-centoflso=vanmill,
Water,;;soluble extractive (2:6.3): Not less than 12:0 percent

by method I.
Description. The roots are cylindrical, yellowish brown in

colour. They have vanillin like odour and the taste is acrid.
2474
ARACHIS OIL
IP 2010
.Heavy metals (2.3.13). 1.0 g complies with the limit test for
Description. A clear, colourless or pale yellow oily liquid;

odour, faint and nutlike.

Identification

Determine by the thin-layer chromatography (2.4.17), coating

the plate with Ideselguhr G.
Mobile phase. Glacial acetic acid.
Test solution. Dissolve 20 mg (one drop) of the substance

under examination in 4 ml of chloroform.
Test solution. Weigh 2 g ofthe coarsely powdered substance

under examination, add 30 ml of water, and reflux on a waterbath for 15 minutes, cool and filter. Reflux the residue further
with water till the extract turns colourless, cool and filter.
Combine all the filtrates and concentrate to a volume slightly
less than 50 ml. Dilute to 50.0 ml with methanol.
Reference solution (a). Dissolve 20 mg (one drop) of arachis

oil in 4 ml of chloroform.
Reference solution. A 0.002 per cent w/v solution of isovanillin RS in methanol.
Reference solution (c). Dissolve 20 mg (one drop) of sesame

oil in 4 ml of chloroform.
mobile phase: A. 0.1 per cent w/v solution of formiC
acid in water
B. acetonitrile,
below,
Impregnate the plate by placing it to a depth of about 5 mm in

a tank containing a shallow layer ofa mixture of95 volumes of
light petroleum (60 to 80) and 5 volumes of liquidparqlfin,
allowing the solvent to rise at least 14 cm, removing the plate
and allowing it to dry in air for 5 minutes; use with the flow of
the mobile phase in the direction in which impregnation was
carried out.
Time
Mobile phase A
(in min.) (per cent v/v)
o
90
10
70
35
10
36
90
40
90
Mobile phase B
(per cent v/v)
10
30
90
10
10
than 2.0 per cent.
Calculate the content ofiso-vanillin.
Storage. Store protected from heat, moisture and against attack
of insects and rodents.
Arachis Oil
Groundnut Oil; Peanut Oil
Arachis Oil is the refined fixed oil obtained from the seed
kernels of one Of more of the cultivated varieties of Arachis
hypogaea Linn. (Fam. Leguminosae) and may contain suitable
antioxidants as stabilisers.
Reference solution (b). Dissolve 20 mg (one drop) of

cottonseed oil in 4 ml of chloroform.

phase to rise 12 cm. Dry the plate at 110 for 10 minutes, allow
to cool and expose it to iodine vapour in a saturated chamber.
Remove the plate and allow it to stand for a few minutes until
the brown background colour has disappeared. Spray the plate
with starch solution; blue spots appear which become brown
on drying and become blue again on spraying with water. The
spots in the chromatogram obtained with the test solution
correspond to those in the chromatogram obtained with
Tests
Alkaline impurities. Mix 10 ml of recently distilled acetone
and 0.3 ml of water in a test-tube, add 0.05 m1 ofa 0.04 per cent
w/v solution of bromophenol blue in ethanol (95 per cent)
and neutralise the solution, if necessary, with 0.01 M
hydrochloric acid or 0.01 M sodium hydroxide. Add 10 m1 of
the substance under examination, shaIce and allow'to stand.
Not more than 0.1 ml of 0.01 M hydrochloric acid is required
to change the colour of the upper layer to yellow. .
Semi-drying oils. Boil 1 g in a flask under a reflux condenser
for 5 minutes with 5 ml of a mixture of 3 volumes of 2 M
ethanolic potassium hydroxide and 1 volume of ethanol
(95 per cent), add 1.5 m1 of 6 M acetic acid and 50 ml of
ethanol (70 per cent), warm until the solution is clear. Cool
2475
ARACHIS OIL
IP 2010
slowly with a thennometer in the liquid; the temperature at

which the solution becomes turbid is not lower than 36.
AIjuna consists ofdried stem bark ofTerminalia arjuna (Roxb)

Wight & Am (Fam. Combretaceae)
Arjuna contains not less than 0.02 per cent of arjungenin

.calculated on the dried basis.
Description. A flat or minutely curved thick pieces of bark

with reddish gray colour and astringent taste.
Iodine value (2.3.28). 85 to 105.

Saponification value (2.3.37). 185 to 196.
Rancidity. Shake 1 ml of a 10 per cent v/v solution in ether
with 1 ml of hydrochloric acid, add 1 ml of a 0.1 per cent w/v
solution of phloroglucinol in ether; no red or pink colour is
produced.
Unsaponifiable matter (2.3.39). Not more than 1.0 per cent.
Cottonseed oil. In the Identification test, the chromatogram
obtained with the test solution does not correspond to that
Sesame oil. In the Identification test, the chromatogram
obtained with the test solution does not correspond to that
obtained with reference solution (c).
Arachis Oil intendedforuse in the manufacture ofparenteral

preparations complies with the follOWing additional
requirement.
Storage. Store protected from light in a well-filled container.
Labelling. The label states (1) whether the contents are
suitable for use in the manufacture ofparenteral preparations;
(2) when the addition of antioxidants is authorised, the name
and quantity of the added antioxidants.
Arjuna
Terminalia arjuna Bark
Identification
A. Macroscopic - Stem bark pieces, flat or minutely curved,

with reddish gray external surface and darker inner surface.
Internal surface has longitudinal minute ridges. Fractures
longitudinal.
B. Microscopic - Cork consisting of6-10 layers ofelongated
cells, phloem broad, medullary rays uruseriate. Calcium oxalate
clusters abundant. Few of the parenchyma cells contain
colouring matter.
C. Detennine by thin-layer chromatography (2.4.17), coating

Mobile phase. A mixture of 5 volumes of toluene,S volumes

of ethyl acetate and 0.5 volume of acetic acid.
Test solution. Reflux 2 g ofcoarsely powdered substance under
examination with 50 ml of chloroform for 15 minutes, cool and
filter. Reflux the residue further with 50 rnl of chloroform.
Combine the filtrate and concentrate under vacuum to dryness.
Dissolve the residue in 10 ml of ethanol at 50 for 10 minutes
and filter.
Reference solution. Reflux 1 g of arjuna RS with 50 ml of
chloroform for 15 minutes, cool and filter. Reflux the residue
further with 50 ml of chloroform. Combine the filtrate and
concentrate under vacuum to dryness. Dissolve the residue
in 5 ml of ethanol at 50 for 10 minutes and filter.
Apply to the plate 10 ~l ofeach solution as bands 10 mID by 2
mID. Allow the mobile phase to rise 8 cm. Dry the plate in air
and examine in ultraviolet light at 254 urn and 365 urn, spray
with 10 per cent w/v solution of sulphuric acid in methanol.
Heat the plate at 110 for 5 minutes and examine in day light.
The chromatographic profile of the test solution is similar to
that of the reference solution.
Tests
Water",solubleextractive(2.6.3).Notlessthan20per~centby
method!.
2476
ARJUNA DRY EXTRACT
IP 2010
.
arjunolic acid on the dried basis. It may contain suitable added

substances.

Description. A light brown to beige powder with or without

red tinge.

Identification


examination with 50 ml of chloroform for 15 minutes, cool and
filter. Reflux the residue further with 50 ml of chloroform, cool
and filter. Combine the filtrates and concentrate under vacuum
to dryness, then extract dried residue with 10 ml of ethanol at
50 for 10 minutes and filter.
Reference solution. A 0.1 per cent w/v solution of aljungenin
RS in ethanol.
mobile phase: A. acetonitrile (70 per cent) in water,
B. acetonitrile (30 per cent) in water,
injection volume.20 III
Time
(in min)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
30
70
10
50
50
30
70
30
50
30
70
Mobile phase. A mixture of 92 volumes of chloroform and 8

Test solution. Dissolve 50 mg ofthe extract under examination
with 50.0 ml of methanol and filter.
Reference solution. A 0.1 per cent w/w solution of arjunolic
acid RS in the methanol.
Apply to the plate 5 /ll of each solution as bands 10 mm by 2
and spray with anisaldehyde sulphuric acid reagent. Heat
the plate at 110 for 10 minutes and examine the plate in
ultraviolet light at 365 nm and day light. The chromatographic
profile of the test solution is similar to that of the reference
solution.
Mobile phase. A mixture of 35 volumes of chlorofom, 10

volumes of methanol and 2 volumes of water.
with 10.0 ml of methanol and filter.
relative standard deviation for the replicate injections for the
analyte peak corresponding to atjungenin is not more than 2. 0
per cent.
Calculate the contents of arjungenin.
Reference solution. A 0.05 per cent w/w solution of aljugenin

RS in the methanol.
Apply to the plate 5 III of each solution as bands 10 mm by 2
and spray with vanilline sulphuric acid reagent. Heat the
plate at 110 for 10 minutes and examine the plate in ultraviolet
light at 365 urn and day light. The chromatographic profile of
the test solution is similar to that of the reference solution.
Tests
Arjuna Dry Extract
Arjuna Dry Extract is obtained by extracting Arjuna

(Terminalia aljuna Wight and Arn,Fam. Combretaceae) bark
with methanol or any other suitable solvent and evaporation
of solvent.
Atjuna Dry Extract contains not less than 90.0 per cent w/w
and not more than 110.0 percent w/w of stated amount of the

2477
ARTEMISIA
IP 2010
Test solution. Shake a quantity ofthe extract under examination

containing about 50 mg of mjunolic acid in 50.0 ml of the
methanol, filter.
Reference solution. A 0.1 per cent w/v solution of aljunolic
a stainless steel column 25 cm x 4.6 mm packedwith
mobile phase: a mixture of 35 volumes of 5 mM
a-cyclodextrin and 65 volumes of methanol,
than 2.0 per cent.
Calculate the content ofthe arjunolic acid in the extract.
3-pinnatipartite, petiolate and much variable in size (2.510.0 cm long). Leaf lobes narrow, oblong to elliptical with
acuminate tip, about 1.0 mm wide. Petioles up to 1.0 cm long,
base amplexicaul. Inflorescence panicled raceme. Dry capitula
yellowish brown, pedicellate, heterogamous globose to subglobose or disc shaped, 2.0 mm in diameter, flower heads
arranged in lax or drooping, involucral bracts 3- seriate,
greenish yellow, glabrous and oblong in shape, measure
1.0-1.2 x 0.5 mm in size. Inner involucral bracts elliptic having
a median greenish streak on its outer surface. Ray florets
pistillate, 6-8 in number per capitulum and 1.0-1.2 mm long.
Disc florets hermaphrodite, 20-36 florets per capitulum and
0.8-1.0 mm long. All florets possess capitate oil glands on the
middle of the outer surface that are 54-83 ~m in diameter.
Stamens 0.7 mm long attached to the corolla base, anther
appendages lanceolate to triangular with acuminate tip. Anthers
oblong and introrse. Pollen grains tricolpate, rounded 21-33
~m in diameter. In dry condition, capitula become empty
because florets/achenes come out of it. Receptacle globular
to oblong. Achenes minute; possess striated surface,
yellowish brown in colour, 0.7-1.0 mm long, and oval to elliptic
in shape. Stomatal index: 8-10-12.
B. The powder ofthe herb is grayish green to greenish yellow.
Examined under a microscope using chloral hydrate solution.
The powder shows the following diagnostic characteristics:
T-shaped and globular trichomes, epidermal cells with wavy
walls, anomocytic to anisocytic stomata, minute druse crystals,
tricolpate rounded pollen grains, stone cells, annular vessels,
rod shaped palisade cells and stigma with small and club
shaped papillae.
Artemisia
Artemisia annua

Mobilephase. Amixtureofl volume of hexane and 1 volume

of diethyl ether.
Test solution. Boil 0.1 g of the coarsely powdered substance
under examination with 10 ml of hexane for 10 minutes and
filter. Evaporate the filterate to 1 ml.
artemisia RS in hexane.
Artemisia consists of dried leaves or the dried leaves and
flowering tops of Artemisia annua L. (Fam. Asteraceae),
Imown as Qinghao.
Artemisia contains not less than 0.8 per cent of artemisinin,
odour and bitter in taste.
Apply to the plate 5 ~l of each solution as bands 10 mm by 2

with anisaldehyde sulphuric acid reagent. Heat the plate at
100 for 15 minutes and examine in day light. The
Identification
Tests
A. The leaves grayish green, slightly darker upper surface,

glabrous to sparsely hairy, break easily into small fragments,
2478
ASHWAGANDHA
IP 2010
Identification
Assay. Determine by high performance thin-layer

GF254.
A. Macroscopic - Primary roots are straight, conical or finger

like in shape, variable in thickness with the age. Secondary
roots are thin and fibrous. Surf~ce buff to greyish-yellow
with longitudinal wrinkles.
Mobile phase. A mixture of 1 volume of hexane and 1 volume

of diethyl ether.
Test solution. To 0.1 g of the coarsely powdered substance
under examination, add 10 ml of hexane and keep for 12 hours,
filter. Repeat the process of extraction 3 times. Combine the
extracts, evaporate and dissolve in 1.0 ml of hexane.
B. Microscopic - Vessels with bordered pits and horizontal

perforations. Fibres aseptate with pointed ends. Wood
elements lignified. Starch grains abundant, simple, mostly
spherical, reniform - oval with central hilum. Microcrystals in
parenchyma cells.
Reference solution. A 0.1 per cent w/v solution of artemisinin

RS in hexane.


2 rom. Allow the mobile phase to rise 8 cm. Dry the plate in air
and spray with a mixture of50 volumes ofglacial acetic acid,
1 volume of sulphuric acid and 0.5 volume of anisaldehyde.
Heat the plate at 100 for 15 minutes, scan the plate in
absorbance mode at 540 nm. Record the chromatograms and
measure the responses for the analyte peak.

1 volume of methanol.
Calculate the content of artemisinin.

examination with 50 ml methanol for 15 minutes, cool and
filter.
Reference solution. Reflux 0.6 g of coarsely powdered
ashwagandha RS with 10 ml methanol for 15 minutes, cool
and filter.
Storage. Store protected from light and moisture and against

Ashwagandba
Indian Ginseng; Withania somnifera
Apply to the plate 10 III of each solution as bands 10 rom by 2

rom. Allow the mobile phase to rise 8 cm. Dry the plate in air
100 for 5-10 minutes and examine the plate in day light. The
Tests
9
8

Method I.
6
5
4

Ashwagandha consists of the dried mature roots of Withania

somnifera (L.) Dunal (Fam. Solanaceae).
Ashwagandha contains not less than 0.02 per cent w/w of
total withanolide (sum of withanolide glycosides and
withanolide aglycones), calculated on the dried basis.
Description. Buff to greyish-yellow roots. Taste, slightly
mucilaginous, bitter and acrid.

Test solution. Reflux about 5 g of the coarsely powdered

substance under examination with 50 ml of acetonitrile on a
water-bath for 15 minutes, cool and filter. Reflux the residue
2479
ASHWAGANDHA
IP 2010
further with acetonitrile till the last extract turns colourless,

cool and filter. Combine all the filtrates and concentrate to 50.0
ml.
Reference solution. A solution containing 0.01 per cent
w/v each of withanolide A RS and withanoside IV RS in
acetonitrile, prepared by heating gently on a water-bath.
- mobile phase: A. a buffer solution prepared by
dissolving 1.36 g of potassium dihydrogen phosphate
in 900 ml water, adjust pH to 2.8 with dilute phosphoric
acid and diluting it to 1000 ml with water,
B. acetonitrile,
- a linear gradient programme using the conditons given
below,
- spectrophotometer set at 227nm,
- injection volume.20 Ill.
Time
(in min)
Mobile phase A
(per cent v/v)
Mobile phase B
( per cent v/v)
95
18
55
45
25
20
80
27
95
37
95
Withanoside V and VI
0.89

Ashwagandha Dry Extract is obtained by extracting
Ashwagandha (Withania somnifera (L.) Dunal, Fam.
Solanaceae) roots with methanol or any other suitable solvent
and evaporation of solvent.
Ashwagandha Dry Extract contains not less than 90.0 per
withaferin A, not less than 85.0 per cent and not more than
110.0 per cent w/w ofthe total withanolides calculated as the
sum of free withanolides and glycowithanolides calculated
on the dried basis. The extract also contains not less than 1.0
per cent of alkaloids, calculated on the dried basis.
Identification
Determine by thin layer chromatography (2.4.17), coating the
Mobile phase. A mixture of 5 volumes of toluene, 5 volumes

of ethyl acetate and 1 volume offormic acid.
Withanolide glycoside
0.7

by insects and rodents.
Description. A pale brown to light brown powder.
Inject the reference solution.The relative retention time of

withanolide A is 1.0 and withanoside IV is about 0.7.The test
is not valid unless the relative standard deviation for the
replicate injections for both the analyte peaks is not more
than 2.0 per cent and resolution is not less than 2.0. The relative
retention time of withanolide glycosides and withanolide
aglycones are as follows.
Withanoside IV
withanone. Sum the content of withanolide glycosides and

withanolide aglycones to get total withanolides.
Withanolide aglycones
Test solution. Dissolve about 0.2 g of the extract under

examination with 10 ml of methanol and filter.
withaferin A RS in methanol.
2 mm. Allow the mobile phase to rise 8 em. Dry the plate in air
and examine in ultraviolet light at 254 nm. Spray the plate with
anisaldehyde-sulphuric acid reagent. Heat the plate at 110 0
for 10 minutes and examine the plate at 365 nm and under day
12, deoxywithastramonolide 0.96
Tests
Withanolide A
1.0
Withanolide B
1.14

by method!.
Withanone
1.01

Ca,lc1.!~t~!h~cont~!!t1i. Qfl1!tfhC!n~lc.1e.glyc:gLrki!!th~
1ia,IIlPLe
as withanoside IV by summing the peak areas of withanoside

IV,ValJ.d Vi. Calulate the content ofwithanolide aglycones in
the sample as withanolide A by summing the peak areas of 12,
deoxywithastramonolide, withanolide A, withanolide Band
Loss on drymg(2.4: 19):Nofmorethiiii 5.Operceiit, deteiiTImed

bn 1 g by drying in an ovenatl05.

2480
BELLADONNA LEAF
IP 2010
Assay
Calculate the content oftotal withanolides as the sum of free

withanolides and glycowithanolides.
A. Total aUmloids
Test solution. Shake about 20 g ofthe extract under examination

with 400 ml ofa solution containing 4 volumes of ether and 1
volume of ethanol. Add 20 ml of 5 per cent v/v ammonia
solution into 1000-ml conical flask and shake for one hour.
Decant and filter through cotton.
Transfer the filtrate into a separator. Wash the residue with a
mixture containing 80 volumes of ether and 20 volumes of
ethanol. To the filtrate add 100 ml of 0.5 M sulphuric acid.
Collect the lower layer into another separator. To the ether
layer; add 100 ml ofa mixture containing 80 volumes of 0.25 M
sulphuric acid and 20 volumes of ethanol and extract.
Continue the extraction 3 times with 80 ml of the 0.25 M
sulphuric acid and a mixture containing 80 volumes of ether
and 20 volumes of ethanol until aqueous layer is colourless.
Combine the acid solution and wash with 40 ml of chloroform
followed by 2 times with 20 ml of chloroform. Wash, the
combined chloroform layer, with acid alcohol mixture. Discard
the chloroform layer. Combine the acid alcohol solutions and
make it alkaline with 5 per cent v/v ammonia solution and add
10 ml excess. Extract 3 times with 100 ml of chloroform. Add 2
mlof 0.1 M hydrochloric acid to 0.5 ml ofthe extract, remove
the chloroform by evaporation, transfer the aqueous residue
to a test tube and add 0.05 ml of potassium mercury-iodide
solution; not more than a very faint opalescence is produced.
If the opalescence is more, the extraction is not complete.
Extract further 2 times with 100 ml of chloroform.
Combine the chloroform extract and wash with 20 ml of
distilled water. Filter the chloroform layer through cotton plug
into a tared beaker. Wash the residue with a little chloroform,
transferring the chloroform layer to the same tared beaker.
Evaporate on a water-bath. Add 5 ml of alcohol to the residue
and dry to constant weight at 105. Finally weigh the residue
and calculate the content of total alkaloids.
D.Withaferin A
Test solution. Dissolve a quantity of the extract under

examination containing about 25 mg ofwithaferin A in 25.0 ml
of water, filter.
Reference solution. A 0.1 per cent w/v solution of withaferin
A RS in methanol.
octadecylsilane bonded to porous silica (10 J.l.m),
mobile phase: a mixture of65 volumes ofO.lper cent
of acetonitrile,
injection volume. 20 J.l.l.
than 2.0 per cent.
Calculate the content of the withaferin A in the extract.
Usual strength. 2.5 per cent withaferin A and 7.5 to 10 per cent
w/w of total withanolides and glycowithanolides.
Belladonna Leaf
9
B. Total withanolides. Shake about 5 g ofdry extract with 25

ml of methanol and 25 ml of water in a separating funnel. It is
defatted by extraction 4 times with 50 m1 ofhexane. This fraction
is discarded. The remaining aqueous methanolic solution is
extracted with hexane and then with 5 times with 25 ml of
ether. The ether extracts are combined, washed twice with
water and dried under vacuum to give the free withanolides
content. Finally weigh the residue and calculate the content
offree withanolides.
C. Glycowithanolides. The above remaining aqueous

methanolic solution is extracted 3 times with 2 volumes of
chloroform and 1 volumes of methanol. The chloroformmethanol extracts are combined and evaporated on waterbath and dried under vacuum at 80 to constant weight. Finally
weigh the residue and calculate the content of
glycowithanolides.
7
6
5
4
3
2
o
Belladonna Leaf consists of the dried leaf and flowering tops
of Atropa belladonna Linn. or of A. acuminata Royle ex
Lindley (Fam. Solanaceae) or a mixture ofboth species.
2481
BELLADONNA LEAF
IP 2010
Belladonna Herb contains not less than 0.30 per cent of total
alkaloids, calculated as hyoscyamine with reference to the
material dried at 100 to 105.
Description. Green to greenish-brown leaves, slightly darker
on the upper surface, often crumpled and rolled and partly
matted together in the drug. When whole, the lamina is 5 to
25 cm long and 3 to 12 cm wide, elliptical to ovate; acuminate
at the apex, narrowing at the base; margin entire. Petiole 0.5 to
4 cm in length. The young leaves are highly pubescent, the
older leaves are slightly pubescent along the veins. In the
flowering tops, the stems are hollow and flattened, with leaves
in pairs ofunequal size, in the axils ofwhich are single flowers
with campanulate corolla, about 2 cm long and 1.5 cm wide,
purple or yellow-brown in colour, with five short, reflexed lobes,
five epipetalous stamens and one bilocular ovary with
numerous ovules.
Identification
A. When examined under a microscope it shows epidermal
cells with sinuate anticlinal walls and cuticle which is often
striated and furrowed. Covering and glandular hairs infrequent,
though more frequent in the young leaves and around the
veins; covering hairs, multicellular, uniseriate, with thin smooth
walls; glandular hairs; short clavate glands with multicellular
heads and glands with a long uniseriate static and ovoid
unicelluar head. Stomata, anisocytic, more frequent on the
lower epidermis. The midrib is characterised by an open arc of
vascular bundles with isolated groups ofperimedullary phloem.
Mesophyll dorsiventral with a single palisade layer.
Throughout the parenchyma and particularly just below the
palisade layer are cells containing microsphenoidal crystals
of calcium oxalate or, very rarely, cluster crystals. The stems
show pericyclic fibres and perimedullary bundles ofphloem,
few trichomes; the cortical parenchymatous cells and the pith
cells contain microsphenoidal crystals of calcium oxalate.
B. Powder 1 g and shake for 2 minutes with 10 ml of O. 05 M
sulphuric acid. Filter and add to the filtrate 1 ml of strong
ammonia solution and 5 ml of water. Extract with 15 ml of
ether, taking care to prevent the formation ofan emulsion. Dry
the ether extract over anhydrous sodium. sulphate and filter.
Evaporate the filtrate to dryness, add 0.5 ml ofjUming nitric
acid and evaporate to dryness. Add 10 ml of acetone and,
dropwise, a 3 per cent w/v solution ofpotassium hydroxide in
ethanol (95 per cent); a. deep violet colour develops.
MOEiilephase.Airiixfiiie 6f90 volumes ofcicelone, 7v6liimes

of water and 3 volumes of strong ammonia solution.
Test solution. Add 15 ml of 0.05 Msulphuric acid to 0.6 g of
the material under examination, in fine powder, shake for
15 minutes, filter and wash the filter with 0.05 M sulphuric

acid until 20 m1 of filtrate is obtained; add 1 ml of strong
ammonia solution to the filtrate, extract with two quantities,
each of 10 ml, ofperoxide-free ether, separate the ether layer,
by centrifugation ifnecessary, dry the combined ether extracts
over anhydrous sodium sulphate, filter, evaporate to dryness
on a water-bath and dissolve the residue in 0.5 ml of methanol.
Reference solution. Add 15 ml of O. 05 M sulphuric acid to 0.6

g of the belladonna leaf RS, shake for 15 minutes, filter and
wash the filter with 0.05 M sulphuric acid until 20 ml offiltrate
is obtained, add 1 m1 ofstrong ammonia solution to the filtrate,
extract with two quantities, each of 10 ml, of peroxide-free
ether, separate the ether layer, by centrifuging if necessary,
dry the combined ether extracts over anhydrous sodium
sulphate, filter, evaporate to dryness on a water-bath and
dissolve the residue in 0.5 ml of methanol. or
Dissolve 50 mg of hyoscyamine sulphate in 9 ml of methanol
(solution A) and dissolve 15 mg of hyoscine hydrobromide
in 10 mlofmethanol (solution B), mix 8 mlofsolutionAwith
1.8 ml ofsolution B.
Apply to the plate 10 III and 20 III ofeach solution as bands 10
mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate
at 105 for 15 minutes, allow to cool and examine in ultraviolet
light at 254 nm and 365 nm, spray with 10 ml of modified
potassium iodobismuthate solution until the bands become
visible as orange or brown on a yellow background. The bands
in the chromatogram obtained with test solution have similar
Rfvalues to those in the chromatogram obtained with reference
solution (hyoscyamine in the lower third ofthe chromatogram;
hyoscine in the upper third) and are similar in colour and
atleast equal in size. Faint secondary bands may appear,
particularly in the middle ofthe chromatogram obtained with
20 III of the test solution or near the line of application in the
chromatogram obtained with 10 III oftest solution. Spray the
plate with a freshly prepared 10 per cent w/v solution of
sodium nitrite until transparent and examine after 15 minutes.
The colours due to hyoscyamine in the chromatogram change
from brown to reddish-brown but not to greyish-blue
(atropine); any secondary bands are no longer visible.
Tests
Acid-insoluble ash (2.3.19). Not more than 3 per cent.
Assay. Powder 50 g and determine the loss on drying (2.4.19),
by drying 2 g, accurately weighed, in an oven at 105. From
the remaining sample, weigh accurately about 109, moisten
withamixtureofSmlofdiluteammoniasolution,lO.mLof
ethanol (95 per cent) and 30 m1 of ether and mix thoroughly.
Transfer the mixture to a percolator with the aid ofan extracting
solvent mixture consisting on volumes of ether and 1 volume
of chloroform. Allow to macerate for 4 hours and percolate
2482
BELLADONNA TINCTURE
IP 2010
with the solvent mixture until complete extraction of the

alkaloids is effected, (2.6.4).
.
Concentrate the percolate to about 50 ml by distilling off the
solvent mixture on a water-bath, and transfer to a separator,
previously rinsed with ether. Add a quantity of ether at least
equal to 2.1 times the volume ofthe percolate and extract with
three quantities, each of 20 ml, of 0.5 M sulphuric acid.
Transfer each acid extract to another separating funnel.
Combine the acid extracts, make the solution alkaline with
dilute ammonia solution and extract with chloroform until
complete extraction of the alkaloids has been effected. Wash
the combined chloroform extracts with 10 ml water, discard
the watel; evaporate the chloroform layer to dryness and heat
the residue for 15 minutes on a water-bath. Redissolve the
residue in successive small quantities of chloroform,
evaporating to dryness on a water-bath each time before
adding the solvent. Heat for 15 minutes on a water-bath and
dissolve the residue in 5 ml of chloroform. Add 20.0 ml of
0.01 M sulphuric acid, remove the chloroform by evaporation
on a water-bath and titrate the excess of acid with 0.02 M
sodium hydroxide using methyl red solution as indicator.
total alkaloids calculated as hyoscyamine. Calculate the
content oftotal alkaloids with reference to the dried material.
solutions with successive quantities ofa mixture on volumes

of 0.1 M sulphuric acid and 1 volume of ethanol (95 per
cent) until complete extraction of the alkaloids is effected,
filtering each extract through a plug of absorbent cotton
previously moistened with water. Wash the mixed acid
solutions with 10,5 and 5 ml of chloroform, extracting each
chloroform solution with the same 20 ml of O. 05 M sulphuric
acid and discard the chloroform. Combine the acid solutions,
neutralise with dilute ammonia solution, add 5 ml in excess
and shake with successive quantities, each of 25 ml, of
chloroform until complete extraction ofthe alkaloids is effected,
washing each chloroform solution with the same 10 ml of water
and filtering into a flask through a plug of cotton wool
previously moistened with chloroform. Distil most of the
chloroform from the combined extracts and transfer the
remainder ofthe chloroform to a shallow open dish. Evaporate
the remainder ofthe chloroform without the aid ofa current of
air, heat the residue in an oven at 1000 for 15 minutes, dissolve
in a little chloroform, evaporate to dryness without the aid of
a current ofair and again heat in an oven at 1000 for 15 minutes.
Dissolve the residue in 2 ml of chloroform, add 5.0 ml of
0.025 M sulphuric acid, warm to remove the chloroform, cool
and titrate the excess of acid with 0.05 M sodium hydroxide
using methyl red solution as indicator.
I ml of 0.025 M sulphuric acid is equivalent to 0.01447 g of
allcaloids, calculated as hyoscyamine.
Storage. Store in small, wide-mouthed, tightly-closed
containers in a cool place.

Belladonna Dry Extract is obtained from the dried leaf and
flowering top of Atropa belladonna Linn or ofA. acuminata
Royle ex Lindley (Fam. Solanaceae) by extraction with ethanol
or any other suitable solvent.
Belladonna DIy Extract contains not less than 0.95 per cent
and not more than 1.05 per cent w/w ofallcaloids, calculated as
hyoscyamine.
Tests
Assay. Weigh accurately about 3 g and wash into a separating
funnel with 12 ml of a mixture of equal 'volumes of ethanol
(95 per cent) and water, shake-well and frequently for
30 minutes, add 2 ml of dilute ammonia solution and 25 mlof
chloroform. Shake well, allow to separate and filter the
chloroform layer into a second separating funnel through a
plug ofabsorbent cotton moistened with chloroform. Continue
the extraction with further quantities, each of 25 ml, of
chloroform until complete extraction ofthe alkaloids is effected
(2.6.4), running each chloroform solution through the same
plug of absorbent cotton. Extract the combined chlorofonn
Belladonna Tincture
Belladonna Tincture obtained from Belladonna leaf or roots
ofone or more ofthe cultivated varieties ofAtropa belladonna
Linn. or A. acuminata Royle ex Lindley (Fam. Solanaceae) or
a mixture of both species.
Belladonna Tincture contains not less than 90 per cent w/w
and not more than 110 per cent w/w oftotal alkaloids, calculated
as hyoscyamine, C 17H23N0 3
Description. A clear green or brownish green liquid.
Identification
Mobile phase. A mixture of 10 volumes of anhydrousformic

acid, 10 volumes of water, 30 volumes of methyl ethyl ketone
Test solution. Evaporate 10 ml ofthe tincture under examination
in a water-bath at 40 under reduced pressure. Dissolve the
residue in 1.0 ml ofthe methanol.
2483
BELLADONNA TINCTURE
IP 2010
Reference solution. Dissolve 1 mg of chlorogenic acid RS

and 2.5 mg of rutin RSin 10 ml ofthe methanol.
free ether. Add a quantity of peroxide-free ether equal to at

least 2.1 times the volume of the solution to produce a layer
having
a density well below that ofwater. Extract the resulting
solution
with minimum of three quantities, each of20 ml of
mm. Allow the mobile phase to rise 15 cm. Dry the plate in air,
0.25
M
sulphuric
acid until the alkaloids are completely
heat the plate at 110 for 10 minutes. Spray the warm plate with
extracted.
Separate
the layers by centrifugation if necessary
1 per cent v/v solution of diphenylboric acid aminoethyl
and
transfer
the
layers
to a separating funnel. Make the
ester in methanol. Allow to cool and spray with 5 per cent
combined
layers
alkaline
with strong ammonia solution and
v/v solution of macrogol 400 in methanol, allow the plate to
extract
with
minimum
of
three quantities, each of 30 ml of
dry in air for 30 minutes and examine in ultraviolet light at 365
dichloromethane
until
the
alkaloids are completely extracted.
nm. The chromatographic profile ofthe test solution is similar
Combine
the
dichloromrthane
and ether extracts, add 4 g of
anhydrous sodium sulphate and allow to stand for 30 minutes
B. Atropine. Detennine by thin-layer chromatography (2.4.17), with occasional shaking. Decant the dichloromethane and
filter. Wash the sodium sulphate with three quantities, each
Mobile phase. A mixture of 3 volumes of strong ammonia of 10 ml of dichloromethane. Combine the dichloromethane
and ether extracts, evaporate to dryness on a water-bath.
solution, 7 volumes of water and 90 volumes of acetone.
Heat the residue in an oven at 105 for 15 minutes. Dissolve
Test solution. To 15.0 ml of the tincture under examination,
the residue in a few ml of dichloromethane, evaporate to
add 15 ml of 0.05 M sulphuric acid and filter. Add 1 mlof
dryness on a water-bath and heat the residue in an oven at
strong ammonia solution to the filtrate, with two quantities,
105 for 15 minutes again. Dissolve the residue in a few ml of
each of 10 ml, of peroxidefree ether, separate the ether layer
dichloromethane. Add 20.0 ml of 0.01 Msulphuricacid and
by centrifugation ifnecessary, dry the combined ether extracts
remove the dichloromethane by evaporation on a waterover anhydrous sodium sulphate, filter and evaporate to
bath. Titrate the excess ofacid with 0.02 M sodium hydroxide
dryness on a water-bath. Dissolve the residue in 0.5 ml of
using methyl red mixed solution as indicator.
methanol.
Reference solution. Dissolve 50 mg of hyoscyamine sulphate
total alkaloids calculated as hyoscyamine. Calculate the
in 9 ml of methanol and 15 mg of hyoscine hydrobromide in
content of total alkaloids with reference to the dried material.
10 ml of methanol, separately. Mix 1.8 ml of the hyoscine
hydrobromide solution and 8 ml ofthe hyoscyamine sulphate Usual strength. 0.03 per cent w/w.
solution.
Apply to the plate 20 III and 40 III ofeach solution as bands 10
mm by 2 mm. Allow the mobile phase to rise 10 cm. Dry the
plate at 105 for 15 minutes, spray with potassium
iodobismuthate solution, dry the plate and spray with sodium
nitrite solution until the plate is transparent. Examine the plate
after 15 minutes in day light. The chromatogram obtained with
the test solution corresponds to the chromatogram obtained
Bhibhitaki
Belliric Myrobalan; Terminalia bellirica
Tests
Ethanol (2.3.45). 64 to 69 per cent v/v by method Ill.
Assay. Evaporate 50.0 g ofthe tincture under examination to a

volume ofabout 10 ml. Transfer quantitatively to a separating
funnel, with the minimum volume of ethanol (70 per cent
v/v). Add 5 ml ofstrong ammonia solution and 15 ml of water.
Extract with three quantities, each of 40 ml of a mixture of 1
volume of dichloromethane and 3 volumes of peroxide-fi-ee
ether, 9aIiefully toaYQig iemulsiOIl, llIlliUhe_a.ll~alQLds_are
completely extracted. Combinethe dichloromethane and ether
extracts, concentrate the solution to a volume of about 50 ml
by heating on a water-bath. Transfer the resulting solution
quantitatively to a separating funnel, rinsing with peroxide-
5
4
3
2
Bhibhitald consists of the dried fruit pericarp of Terminalia

bellirica (Gaertn.) Roxb. (Fam. Combretaceae).
2484
BHIBHITAKI AQUEOUS EXTRACT
IP 2010
Bhibhitaki contains not less than 0.3 per cent w/w of ellagic
acid and 0.75 per cent w/w of gallic acid, calculated on the
dried basis.
Description. The dried pericarp appears as curved pieces of
irregular shapes, the external surface is velvety, wrinkled grey
to brown in colour and has astringent taste
Identification
A. Macroscopic - The dried pericarp of the ripe fruit occurs

as curved pieces of irregular shapes with convex external
surface. The external surface appears velvety, slightly wrinlded
grey to brown in colour. Internal surface is pale yellow. The
cut surface is with occasional projecting threads, representing
the vascular bundles.
Test solution. Weigh 0.5 g of coarsely powdered substance

under examination, add 50 ml of water, sonicate for 3 minutes
and heat on a boiling water bath for 15 minutes, cool and
dilute to 100.0 ml with water and filter.
B. Microscopic- The cells ofthe epidermis has a characteristic

and a slightly bulged based with a hair like prolongation.
S~veral vascular strands traverse the mesocarp in various
directions. Peripheral layers of mesocarp have tangentially
elongated cells, devoid of starch grains, containing rosettes
of calcium oxalate crystals and few small stone cells.
Mobile phase. A mixture of20 volumes of toulene, 45 volumes

of ethyl acetate and 20 volumes of glacial acetic acid and
5 volumes offormic acid.
Test solution. Reflux 2 g ofthe coarsely powdered substance
75 ml of methanol, cool and filteE Combine allthe filtrates and
concentrate under vacuum to 50 m1.
bhibhitald RS with 50-75 ml of methanol for 15 minutes, cool
and filter. Reflux the residue further for two times with 75 ml of
concentrate under vacuum to 10 m1.
Apply to the plate 10 /-ll ofeach solution as bands 10 mm by 2
Tests

Method 1.
Acid-insoluble ash (2.3 .19). Not more than 2 per cent.
NOTE - Use freshly prepared solution and protectedfrom

light.
mobile phase: A. a gradient mixtures of acetonitrile and
a buffer solution prepared by dissolving 0.136 g of
potassium di-hydrogen orthophosphate in 500 ml of
water, add 0.5 ml of orthophosphoric acid and make
upto 1000 ml with wate];
B. acetonitrile
mobile phase B
Time
mobile phase A
(per cent v/v)
(in min)
(per cent v/v)
5
o
95
35
18
65
55
25
45
5
30
95
for the replicate injections is not more than 2.0 per cent.
Calculate the content of gallic acid and ellagic acid.
Bhibhitaki Aqueous Extract
Total ash (2.3.19). Not more than 8 percent.

each of gallic acid RS and ellagic RS in wate]:
Bhibhitaki Aqueous Extract is obtained by extracting Bhibhitaki

(Terminalia bellirica (Gaeltn.) Roxb., Fam. Combretaceae) fruit
with water.
Bhibhitaki Aqueous Extract contains not less than 90 per cent
w/w and not more than 120.0 per cent w/w ofthe stated amount
of gallic acid and ellagic acid.
2485
BHIBHITAKl AQUEOUS EXTRACT
IP 2010
Description. A light brown to dark brown powder.
Time
(in min)
Identification
plate with silica gel G.F 254.
Mobile phase. A mixture of35 volumes of benzene, 10 volumes

of methanol, 4 volumes of acetone and 1 volume of water.
Test solution. Dissolve 0.5 g ofthe extract under examination
in 20 ml of methanol under reflux at 80 0 on a water bath and
filter.
Mobile phase A
(per cent v/v)
0-8
95
8-25
95~0
25-30
30-32
0~95
Mobile phase B
(per cent v/v)
5
5
~100
100
5
~5
Inject reference solution (a) and (b). The test is not valid
unless the relative standard deviation for the replicate
Inject the test solution, reference solution (a) and (b).

Calculate the content of gallic acid and ellagic acid in the

extract

and examine in ultraviolet light at 254 run. The chromatographic
solution.

Bhringraj
Tests
Eclipta alba
Acid insoluble ash (2.3.19). Not more than 4.0 per cent.
Loss ofdrying (2.4.19). Not more than 5.0 per cent, determined
7
6

5
4

3

examination or a quantity containing 10 mg ofgallic acid and
1 mg ofellagic acid in 80 ml of methanol and dilute to 100.0 ml
with water and filter.
Reference solution (a). A 0.01 per cent w/v solution of gallic
acid RS in 80 per cent v/v methanol in water.
o
Bhringraj consists of the dried whole plant of Eclipta alba
(L.) Hassle. (Fam. Asteraceae).
Reference solution (b). A 0.001 per cent wIv solution of ellagic

acid RS in 80 per cent v/v methanol in water.
Bhringraj contains not less than 0.1 per cent ofwedelolactone,

. - mobile phase: A mixture of acetonitrile and a 0.1 per
cent v/v orthophosphoric acid in water,
Description. A green to greenish brown colour when

completely dry.

spectrophotometer set at 252 nm for ellagic aCid a.nd a.t
271 urn for gallic acid,
injection volume 20 Ill.
Identification
A. Macroscopic - Root. Well developed, a number of
secondary branches arise from main root up to about 7 mm in
Stem. Herbaceous, branched occasionally rooting at nodes,
cylindrical or flat, rough due to oppressed white hairs, node
distinct, greenish, occasionally brownish.
2486
BHRINGRAJ
IP 2010
Leaf. Opposite, sessile to sub sessile, usually oblong,

lanceolate, sub-entire, sub-acute or acute, strigose with
appressed hairs on both surfaces.
further with 3 x 25 ml of methanol, cool and filter. Combine all

the filtrates and concentrate under vacuum to 5 ml.
Flower. Solitary or 2, together on unequal axillary peduncles,

involucral bracts about 8, ovate, obtuse or acute, herbaceous,
strigose with oppressed hairs; ray flowers ligulate, ligule small,
spreading, scarcely as long as bracts, not toothed; white disc
flowers tubular, corolla often 4 toothed; pappus absent, except
occasionally very minute teeth on the top of achene; stamen
5, filaments epipetalous, free, anthers united into a tube with
base obtuse; pistill bicarpellary; ovary inferior; unilocular with
one basal ovule.
Fruit. Achenial cypsella, one seeded, cuneate, with a nan-ow

wing, covered with warty excrescences, brown.
Seed. Dark brown, hairy and non endospennic.
B. Microscopic - Powder. Dark green; shows vessels in
large groups or single broken pieces with pitted walls,
numerous fibres entire or in pieces, trichomes entire or in
pieces, warty, a few attached with epidennal and subsidiary
cells, anomocytic and anisocytic stomata.
Root. The cells of outer one or two rows of secondary cortex,
elongated or rounded with air cavities, while cells of inner
secondary cortex, elongated to irregular in shape. Stone cells
scattered in secondary cortex. Phloem rays broader towards
the periphery, cells rounded. Xylem rays distinct, run straight
in tangential section, rarely uniseriate and biseriate, cells pitted.
Stem. A few epidennal cells elongate to fonn characteristic
non-glandular trichomes. Secondary cortex composed oflarge,
rounded parenchymatous cells having wide air space. Vascular
bundle in a ring, collateral, endarch, of varying size. Vessels
barrel-shaped, some elongated with simple perforations, pitted
with spiral thickening. A few xylem fibres bifurcate. Xylem
rays uniseriate or biseriate.
Leaf. Anomocytic and anisocytic stomata and non-glandular
hairs are present on both surface, more abundant on lower
side. Vascular bundle, fine in mid rib, central one largest while
four other small flanking either side of central bundle.

of acetone and 1 volume offormic acid.

100 for 5-10 minutes and examine the plate in day light. The
Tests
Water- soluble extractive (2.6.3). Not less than 15.0 per cent
by Method 1.
Total ash (2.3.19). Not more than 22 per cent.
Acid-insoluble ash (2.3 .19). Not more than 11 per cent.
detennined on 5 g by drying in an oven at 105.

under examination with 30 ml of methanol on a water- bath for
30 minutes, cool and filter. Reflux the residue further with
methanol till the last extract turns colourless, cool and filter.
Combine all the filtrates and concentrate to 100 ml.
Reference solution. 0.01 per cent w/v solution of
wedelolactone RS in methanol.
- mobile phase: 35 volumes of acetonitrile and 60 volumes
of0.1 per cent v/v phosphoric acid prepared by diluting
1ml ofphosphoric acid to I000 ml with water,
relative standard deviation for the replicate injections is not
Test solution. Reflux Ig of the coarsely powdered substance

under examination with 25 ml of methanol for 30 minutes, cool
and filter. Reflux the residue further with 3 x 25 ml of methanol,
cool and filter. Combine all the filtrates and concentrate under
vacuum to 10 ml.
Calculate the content of wedelolactone.
Reference solution. Reflux 0.5 g bhringrqj RS with 25 ml of

methanol for 30 minutes, cool and filter. Reflux the residue

2487
BHUIAMLA
IP 2010

mm. Allow the mobile phase to rise 8 em. Dry the plate in air
Bhuiamla
Phyllanthus amarus
with methanolic sulphuric acid (10 per cent, v/v) Heat the
plate at 1200 for 5-10 minutes and examine in day light. The
Tests
by Method 1.
Acid-insoluble ash (~.3 .19). Not more than 5.0 per cent.
Bhuiamla consists of the dried aerial parts of Phyllanthus
amarus Schum. & Thorn. (Fam. Euphorbiaceae).
Bhuiamla contains not less than 0.25 per cent w/w of total
phyllanthin and hypophyllanthin, calculated on the dried
basis.
Description. A green to greenish yellow in colour, taste,
slightly bitter.
Identification
A. Macroscopic - Stem teret, 1-4 mm in diameter. Leaves
oblong 5 x 3 mm, short stalked, greenish brown in colour.
B. Microscopic - Stem, inner cortex chlorenchymatous; xylem
rays 1-2-seriate. Leafstomata mostly paracytic; epidermal cell
wall markedly sinuous; rosette and prismatic crystals ofcalcium
oxalate along the veins and midrib.


of ethyl acetate, 1 volume offormic acid and 0.2 volume of
methanol.
examination with 50 ml methanol on a boiling water-bath for
30 minutes, cool and filter. Reflux the residue further with 2 x
50 m1 of methanol, cool and filter. Combine all the filtrates and
Reference solution;--Reflux: -l-gof-bhuiamla-RS-with- 50-m1
methanol on a boiling water-bath for 30 minutes, cool and
filter. Reflux the residue further with 2 x 50 ml of methanol,
vacuum to 5 mL

detennined on 5 g by drying in an oven at 105.

substance under examination with 50 ml of methanol on a
water bath for 15 minutes, cool and filter. Reflux the residue
further with methanol till the last extract turns colorless, cool
and filter. Combine all the filtr~tes and concentrate to 10.0 ml.
Reference solution (a). 'A 0.020 per cent w/v solution of
phyllanthin RS in methanol.
hypophyllanthin RS in methanol.
octadecylsilane bonded to porous silica (5Ilm),
Inject the reference solution (a) and (b). The test is not valid
injections for both the analytepeaks corresponding to
phyllanthin andhypophyllanthin--is not-more than-2.0-per
cent.
Inject the test solution, reference solutions (a) and (b).
Calculate the contents of phyllanthin and hypophyllanthin.
2488
BRAHMI
IP 2010

vacuum to 25 ml.
Reference solution. Reflux 0.4 g ofcoarsely powdered brahmi
RS with 5 ml methanol for 15 minutes, cool and filter.
Brahmi
Apply to the plate 10 fll of each solution as bands 10 mm by 2

with methanolic sulphuric acid (20 per cent v/v). Heat the
Bacopa monnieri
D. In the Assay, the chromatogram obtained with test solution

corresponds to the chromatogram obtained with reference
solution.
Tests
Method l.
Brahmi consists of the dried whole plant, preferably leaves
and stem of Bacopa monnieri (Linn.) Pennell (Fam.
Scrophulariaceae).
Brahmi contains not less than 2.5 per cent w/w ofbacoside A,
Description. A brown to reddish brown colour when
completely dried or green colour when partially dried with
slightly bitter taste.

Identification
A. Macroscopic
Herbaceous comprising ofstems, runner
stems and leaves. Stems glabrous, leafless towards the base;
internodes long. Leaves spatulate-obovate, sessile, and
glabrous.
B. Microscopic - Cortex in stem composed ofparenchyma
cells enclosing large air spaces; xylem vessels radially arranged
xylem rays uniseriate; pith parenchyma collapsed. In leaf,
midrib indistinct, mesophyll isobilateral ofspongy cells, a few
prismatic crystals of calcium oxalate in mesophyll; stomata
anomocytic on both the surfaces of leaf.

substance under examination with 50 ml of methanol on.a
and filter. Combine all the filtrates and concentrate to 100.0 ml.
Reference solution. A 0.2 per cent w/v solution of bacoside A
RS in methanol, prepared by heating gently on a water bath.
a stainless steel column 25 cm x 4.6 rum packed with
mobile phase: A.a gradient mixtures ofacetonitrile and
a buffer solution prepared by dissolving 0.5 g
phosphoric acid in 800 ml of water, adjust pH to 2.8
with dilute phosphoric acid and dilute to 1000 ml with
water,
B. acetonitrile,
2489
BRAHMI EXTRACT
Time
(min)
o
25
35
36
45
mobile phase A
( per cent v/v)
70
60
40
70
70
IP 2010
Tests
mobile phase B
( per cent v/v)
30
40
60
30
30
heavy metals, (Method B) 20 ppm.
Inject the reference solution. The relative retention times are

1.00 for bacoside A3, about 1.04 for bacopaside II, 1.13 for
jujubogenin isomer of bacopasaponine C and 1.19 for
bacopasaponine C as bacoside A. The test is not valid unless
the relative standard deviation for the replicate injections is
Calculate the content of bacoside A.
Brahmi Extract
Brahmi Extract is obtained by extracting Brahmi (Bacopa
monnieri, Fam. Plantaginaceae) from the dried leaves and stems
of with aqueous ethanol or any other suitable solvent.

contamination test.
Test solution. Dissolve about 0.5 g of the extract in methanol

by gentle heating, dilute to 100 ml and filter.
Reference solution. A 0.1 per cent w/v solution of bacosideA RS in methanol.
- mobile phase: A. 0.5 g ofphosphoric acid in water,
B. Acetonitrile
- a Hnear gradient programme using the conditions given
below,
- flow rate 1.5 ml per minute,
- spectrophotometer set at 205 llill,
- injection volume.20 /ll.
Time
(in min)
Brahmi Extract contains not less than 90.0 per cent w/w and
not more than 120.0 percent w/w of bacoside-A (sum of
bacoside-AJ , bacopaside-II, bacopasaponin-C, jujubogenin
isomer ofbacopasaponin-C).
o
25
35
36
45
Description. Light green to dark green powder with

characteristic odour and bitter taste.
Identification
Mobile phase. A mixture of 7 volumes of Ethyl acetate, 2
volumes of methanol and 1 volume of water.
mobile phase A
(per cent v/v)
70
mobile phase B
(per cent v/v)
30
60
40
40
60
70
70
30
30
than 2.0 per cent.The relative retention time with refemce to
bacoside-A J for Bacopaside II is about 1.04, for jujubogenin
isomer of bacopasaponin C is about 1.13 and for
bacopasaponin C is about 1.19.
Test solution. Shakewell 0.5 g ofthe extract under examination

with 50 ml methanol and filter.
Reference solution. A 0.1 per cent w/v solution of bacosideA RS in methanol.
Usual strengths. 10 per cent w/w; 20 per cent w/w.
Apply to the plate 10 /ll of each solution as bands 10 mm by 2

andexaminein_ultravioletlight_at_25Anm.Spraythe_plate with
vanillin sulphuric acid sQJJJt!QI.l..l:Ie!3JJlle plate at 60 t070
for 10 minutes and examine the plate at 365 nm and in day
Calculate the content of bacoside-A.

Castor
en
Castor Oil is the fixed oil obtained by cold expression from the
seeds of Ricinus communis Linn. (Fam. Euphorbiaceae). It
may contain suitable antioxidants.
2490
CLOVE OIL
IP 2010
Description. A pale yellowish or almost colourless,

transparent, viscid liquid; odour, slight and characteristic.
Tests
solution in ethanol (95 per cent) at the maximum at about 269
nm, not more than 1.0.
Optical rotation (2.4.22). +3.5to +6.00
Acetyl value (2.3.22). Not less than 143.
Hydroxyl value (2.3.27). Not less than 150.
Saponification value (2.3.37).176 to 187.
Foreign fatty substances. A mixture of2 ml ofthe substance
under examination and 8 ml of ethanol (95 per cent) is clear.
B. Shake 10.0 ml with 20.0 ml of light petroleum (60 0 to 800)

and allow to separate; the volume ofthe lower layer is not less
than 16.0 ml.
temperature not exceeding 15 0
Labelling. The label states (1) the name and quantity of any
added antioxidant; (2) whether the contents are suitable for
use in the manufacture of parenteral preparations.
Apply to the plate 20 III of the test solution and 10 III of the
reference solution as bands 20 mm by 3 mm. Use an unlined
tanle, develop the chromatogram immediately after pouring
the mobile phase into the tanle and allow the mobile phase to
rise 10 cm. Dry the plate, allow to stand for 5 minutes and
again allow the mobile phase to rise 10 cm under the same
conditions. Following the second development, dry the plate
in air, examine in ultraviolet light at 254 nm and mark the
quenching bands. In the chromatogram obtained with the test
solution there is a quenching band in the middle of the plate
corresponding to the quenching band due to eugenol in the
chromatogram obtained with the reference solution. A weak
quenching band may also be seen in the chromatogram
obtained with the test solution with an Rfvalue slightly lower
than that of the band corresponding to eugenol
(acetyleugenol). Spray the plate with about 10 ml of
anisaldehyde solution, heat at 1000 to 105 0 for 10 minutes and
examine in daylight. In the chromatogram obtained with the
test and reference solutions the bands corresponding to
eugenol are strongly coloured brownish-violet and any band
corresponding to acetyleugenol in the chromatogram obtained
with the test solution is faintly violet-blue. Other coloured
bands may be visible in the chromatogram obtained with the
test solution, in particular a faint red band in the lower part of
the chromatogram and a reddish-violet band in the upper part
(caryophyllene).
Tests
Optical rotation (2.4.22). 00 to -1.50.
Weightperml(2.4.29).1.038gto 1.060g.
Refractive index (2.4.27). 1.527 to 1.535, determined at 200
Clove Oil
Clove oil is the oil distilled from the dried flower buds of
Syzygium aromaticum (Linn.) Merrill and Perry Eugenia
caryophyllus (Spreng.) Bull. and Harr.(Fam. Myrtaceae).
Description. A clear, colourless or pale yellow liquid when
freshly distilled, becoming darker and thicker by ageing or
exposure to air; odour as of clove.
Clove Oil contains not less than 85.0 per cent w/w and not
more than 95.0 per cent w/w of phenolic substances, chiefly
eugenol, C IOH I20 2
Identification
Mobile phase. Toluene
Test solution. Dissolve 20 III of the substance under
examination in 2 ml oftoluene.
Reference solution. Dissolve 20 III of eugenol RS in2 ml of
toluene.
Phenol. Shake 1 ml with 20 ml ofhot water; the mixture shows
not more than a scarcely perceptible acid reaction with blue
litmus paper. Cool the mixture, pass the aqueous layer through
a wetted filter and treat the clear filtrate with 1 drop offerric
chloride test solution. The mixture has only a transient greyishgreen colour but not a blue or violet colour.
Alkali-soluble matter. Place 80 ml ofa 5 per cent w/v solution
of potassium hydroxide in a 150-ml flask with a long neck
which is graduated in tenths of a ml and is ofsuch a diameter
that not less than 15 em in length has a capacity of 10 ml.
Clean the flask with sulphuric acid and rinse well with water
before use. Add 10 ml of the oil and shake thoroughly at
5 minute intervals for 30 minutes at ambient temperature. Raise
the undissolved portion of the oil into the graduated part of
the neck of the flask by the gradual addition of more of the
potassium hydroxide solution; allow to stand for not less
than 24 hours and read off the volume of the undissolved
portion ofthe oil which measures between 1.0 and 1.5 ml.
2491
CLOVE OIL
IP 2010
Identification
Test solution (aj: A 0.2 per cent w/v solutiOlj ofthe oil under
examination in ethanol (95 per cent).
A. It complies with the test for melting range (2.4.21).
Test solution (b). A 0.2 per cent w/v solution of the oil under
examination and 0.15 w/v of I-decanol (internal standard) in
B. It complies with the test for composition offatty acids.

Tests
Melting range (2.4.21). 23 to 26.

solution of eugenol RS and 0.15 per cent w/v of the internal
standard in ethanol (95 per cent).
Refractive index (2.4.27). About 1.449, at 400.

a glass column 1.5 m x 4 mm, packed with 3 per cent w/w
ofdimethyl silicone fluid on acid-washed diatomaceous
.support (120 mesh),
- temperature:
column. 11 0 for 18 minutes, then increased to 170 at a
rate of 12 per minute and maintained at this temperature
for 2 minutes,
inlet port at 220 and
detector at 300,
- flow rate 40 ml per minute ofthe carrier gas.
Unsaponifiable matter (2.3.39). Not more than 1.0 per cent,

determined on 5.0 g.
Calculate the eugenol content in the oil under examination

using the ratios of the area of the peak corresponding to
eugenol to the area ofthe peak due to the internal standard in
the chromatogram obtained with test solutions (b) and the
reference solution.
Storage. Store protected from light in well-filled containers at
Acid value (2.3.23). Not more than 0.5, determined on 20.0 g
Composition offatty acids
Test Solution. Refined coconut oil is melted under gentle

heating to a homogeneous liquid.
Reference solution. Dissolve 15 mg of tricaproin RS, 80 mg of
tristearin RS, 0.15 g of tricaprin RS, 0.2 g of tricaprylin RS,
0.45 g of trimyristin RS and 1.25 g of trilaurin RS in a mixture
of2 volumes of dichloromethane and 8 volumes of heptane,
then dilute to 50 ml with the same mixture of solvents heat at
45to 500. Transfer 2 ml ofthis mixture to a 10 ml centrifuge
tube with a screw cap and evaporate the solvent in a current
of nitrogen. Dissolve with 1 ml of heptane and 1 ml of dimethyl
carbonate and mix vigorously under gentle heating (500 to
60 0). Add, while still warm, I ml ofa 1.2 per cent w/v solution
. of sodium in anhydrous methanol, prepared with the
necessary precautions, and mix vigorously for about 5 minutes.
Add 3 ml of distilled water and mix vigorously for about 30
seconds. Centrifuge for 15 minutes at 1500 rpm. Inject 1 III of
the organic phase.
Coconut Oil
Storage. Store protectedfrom light in well-filled containers.
Coconut Oil is the refined fixed oil obtained from the dried,
solid part of the endosperm of Cocos nucifera L.
(Fam.Arecaceae).
Coleus
Coconut Oil contains not less than 1.5 per cent w/w ofcaproic
acid, not less than 5.0 percent and not more than 1l.0 per cent
w/w of caprylic acid, not less than 4.0 per cent and not more
than 9.0 per cent w/w ofcapric acid, not less than 40.0 per cent
and not more than 50.0 per cent w/w of lauric acid, not less
than 15.0 per cent and not more than 20.0 per cent w/w of
myristic acid, not less than 7.0 percent and not more than 12.0
per cent w/w of palmitic acid, not less than 1.5 per cent and
not more than 5.0 per cent w/w ofstearic acid, not less than 4.0
per cent and not more than 10.0 w/w per cent ofoleic acid, not
less than l.0 per cent and not more than 3.0 per cent w/w of
lin:(j1eicacid;~n:(jrlessthan0:2p ercent w/Woflin51enicacid;
not less than 0.2 per cent w/wof arachidic acid and not less
than 0.2 per cent w/w of eicosenoic acid.
Coleus forskohlii
9
8
7
6
5
4
3
2
Description. A white or almost white, unctuous mass.

2492
COLEUS DRY EXTRACT
IP 2010
Coleus consists of the whole or cut dried roots of Coleus
forskohlii Briq. (Fam. Lamiaceae).

Coleus contains not less than 0.4 per cent w/w of forskolin,

Description. The roots are light brown in color, generally long

and radially spread. They have an aromatic characteristic odor
and the taste is slightly pungent.
Identification
A. Macroscopic - Roots are brown, longitudinally wrinlded,
fracture short, cut surface yellowish white.
B. Microscopic - The outermost layer consists ofrectangular
cork cells, cork cambium, rectangular parenchymatous region
containing sclereids and calcium oxalate crystals. Vascular
cambium is present in the form of a continuous ring. The
tracheids and tracheidal fibres have bordered pits.
Mobile phase. A mixture of 75 volumes of benzene, and

under examination, add 50 ml of acetonitrile and reflux for
times with 50 ml of acetonitrile, cool and filter. Combine all the
filtrates and concentrate under vacuum to 100 ml.
Reference solution. To 1 g of coleus RS add 50 ml of
acetonitrile and reflux for 15 minutes, cool and filter. Reflux
the residue further for two times with 50 ml of acetonitrile,
vacuum to 20 mi.

under examination, add 50 ml of acetonitrile and reflux on a
two times with 75 ml of acetonitrile, cool and filter, Concentrate
the filterate to 100.0 ml.
Reference solution. A 0.1 per cent w/v solution offorskolin
RS in acetonitrile.
mobile phase: filtered and degassed mixture of 45
volumes of acetonitrile and 55 volumes of water,
Calculate the content offorskolin.
Coleus Dry Extract
Apply to the plate 20 /ll ofeach solution as bands 10 rom by 2

with vanillin glaCial acetic acid reagent. Heat the plate at
Coleus Dry Extract is obtained by extracting Coleus (Coleus

Forskohlii Willd. Briq.,Fam. Lamiaceae) roots with methanol
or any other suitable solvent and evaporation of solvent.
Tests
Description. A light brown to brown colour powder.
Coleus Dry Extract contains not less than 90 per cent w/w to
not more than 120 per cent w/w of the stated amount of
forskolin, calculated on the dried basis. It may contain suitable
added substances.
Identification
by method I.

Mobile phase. A mixture of40 volumes of ethyl acetate and 60

volumes of hexane.

examination with 10 ml methanol, filter.

RS in methanol.
2493
COLEUS DRY EXTRACT
IP 2010
Apply to the plate 10 J!l of each solution as bands 10 rom by

2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air
and spray the plate with a anisaldehyde-sulphuric acid
reagent. Heat the plate at 110 for 10 minutes and examine in
ultraviolet light at 365 urn and in day light. The chromatographic
solution.
not less than 0.5 per cent and not more than 4.0 per cent ofpcymene, not less than 3.0 per cent and not more than 6.0 per
cent of camphor, not less than 0.1 per cent and not more than
1.5 per cent of a-terpineol, not less than 0.5 per cent and not
more than 4.0 per cent ofgeranyl acetate and not less than 0.5
per cent and not more than 3.0 per cent of geraniol.
Tests
Description. A clear, colourless or pale yellow liquid;

characteristic and spicy odour .
Totalash (2.3.19). Not more than 10.0 per cent.
Identification



Test solution. Dissolve 10 J!l of the substance under

examination in 1 mloftoluene.
Reference solution. Dissolve 10 J!l of linalol and 2 J!l of

geranyl acetate in Iml of toluene.
Test solution. Dissolve a quantity of the extract under

examination containing about 50 mg offorskolin in 50.0 ml of
methanol and filter.

RS in the methanol.
- a stainless steel column, 25 cm x 0.46 mm packed with
octadecylsilane bonded to porous silica (5J!m),
- mobile phase: a mixture of50 volumes of water and 50
i~ection volume. 20 J!l.
than 2.0 per cent.
Calculate the content of the forskolin in the extract.
Apply to the plate 10 J!l ofeach solution as bands of 10 rom by

2 rom. Allow the mobile phase to rise 10 cm. Dry the plate in air,
spray with anisaldehyde sulphuric acid reagent. Heat the
plate at 100 to 105 for 10 to 15 minutes and examine the plate
in day light. The chromatographic profile of the test solution
is similar to that ofthe reference solution.
Tests
Relative density (2.4.29.).0.860 to 0.880.
Optical rotation (2.4.22). + 7 to + 13.
Enantiomeric purity. Not more than 14 per cent w/w of-{R) linalol.

examination in 10 ml ofpentane.
Reference solution. Dissolve 10 J!l of linalol and 5 mg of
borneol in 10 ml ofpentane.
- a fused silica column 25 m x 0.25 mm, packed with modified
a-cyclodextrin (0.25 /lm),
Coriander Oil
- temperature:
Coriander Oil is obtained by steam distillation from the fruits
column. 50 for 5 minutes, then increased to 180 at a
of Coriandrum sativum L.(Fam.Apiaceae).
rate of 12 per minute and maintained at this temperature
for 65 minutes,
CorianderDiLcontainsnotJess_than..65.0_peLcenLandnoL
'iiiletport andCletectof at 1J(}0~--'~"'" .~._-~--~_....
more.tl111n 78.0 perc~llt.9Jljna1o],llQU~.~~tl111n 3.Qp~I cellt
- flowraten-mlpefmiiiliteoftlfecamergas: ....
and not more than 7.0 per cent of ,*pinene, not less than 1.5
per cent and not more than 5.0 per cent of limonene, not less Inject I /ll of the reference solution. The test is not valid
than 1.5 per cent and not more than 8.0 per cent of 'Y -terpinene, unless the resolution between the peaks due to (R)-linalol
2494
DARUHARIDRA ROOTS
IP 2010
and (S) linalol is not less than 5.5 and the resolution between
the peaks due to (S)-linalol and borneol is not less than 2.9.
Inject 1 fll of the reference solution and the test solution.
Description. The roots are cylindrical, yellowish brown in

colour. They have a slightly characteristic odour and the taste
is bitter.
Identification
Test solution. The substance under examination dissolve in I

ml ofhexane.
Reference solution (a). Dissolve 10 fll of a-pinene, 10 fll of
limonene, 10 fll of a-terpinene, 10 fll ofp-cymene, 10 mg of
camphor, 20 fll oflinalol, 10 fll ofa-terpineol, 10 fll ofgeranyl
acetate and 10 fll ofgeraniol in 1 ml of hexane.
Reference solution (b). Dissolve 5 fll of geraniol in hexane
- a fused silica column 60 m x 0.25 mm, packed with
macrogol20000 (0.25 flm),
- temperature:
column. 60 0 for 10 minutes, then increased to 1900 at a
rate of 12 0 per minute and maintained at this temperature
for two minutes,
- inlet port at 220 0 and detector at 240 0 ,
- flow rate 1ml per minute ofthe carrier gas.
Inject 1 fll of the reference solution: The test is not valid
unless the resolution between the peaks due to linalol and
camphor is not less than 1.5.
Inject 1 fll of the refernce solution and the test solution.
exceeding 300
Daruharidra Roots
A. Macroscopic - Root cylindrical, more or less knotty,

strongly branched, usually cut into pieces of varying length
and up to 45 mm in diameter; externally light yellowish-brown,
longitudinally wrinlded and short scaly; fracture hard and
tough; bark 1 mm. in thickness, easily separable into layers;
wood yellow.
B. Microscopic - The powder is yellowish-brown; composed
chiefly of fragments of wood fibers associated with a few
tracheae and medullary rays; wood fibers yellowish, scarcely
giving any reaction with phloroglucinol and hydrochloric
acid, and with large, simple, transverse pores; trachea chiefly
scalariform with bordered pits, occasionally reticulate;
medullary rays one to twelve cells wide, and in very .long
rows; starch grains simple or two- to three-compound, the
individual grains being irregularly spherical.

volumes offormic acid, 10 volumes of glacial acetic acid
and 20 volumes of water.
under examination, add 40 mI ofmethanol, reflux for 15 minutes,
ml of methanol, cool and filter. Combine all the filtrates and
Reference solution. To 1 g of the daruharidra roots RS, add
40 mI ofmethanol, reflux for 15 minutes, cool and filter. Reflux
the residue further for two times with 25 ml ofmethanol, cool
vacuum to 25 mI.
Berberis, Berberis arisata

9
Apply to the plate 10 fll of each solution as bands 10 mm by 2

and examine in ultraviolet light at 254 urn and 365 nm and also
under day light The chromatographic profile of the test
solution is similar to that ofthe reference solution.
6
5
4
Tests
Water-soluble extractive (2.6.3). Not less than 6.0 per cent by
methodL
.
o
Daruharidra Roots consist of the dried roots of Berberis
aristata DC (Fam. Berberidaceae).
Daruharidra Roots contain not less than 0.70 per cent w/w of
berberine, calculated on the dried basis.

2495
DARUHARIDRA ROOTS
IP 2010
Loss on drying (2.4.19). Not more than 10.0 per centdetennined

.
Assay. Perform the Assay by following method I or by
method I!.
MethodI
Test solution. Weigh 2 g ofthe coarsely powdered substance

under examination, add about 20 ml 2 M hydrochloric acid
and 30 ml of methanol, reflux on a water-bath for 15 minutes,
cool and filter. Reflux the residue further with 2 Mhydrochloric
acid and methanol, till the extract turns colourless, cool and
filter. Combine all the filtrates and concentrate to a volume
slightly less than 50 ml. Dilute to 50.0 mI with methanol.
Reference solution. A 0.01 per cent w/v solution of berberine
mobile phase: A. a buffer solution pH 2.5 prepared by
dissolving 0.136 g of potassium dihydrogen
orthophosphate in 500 ml of water, add 0.5 ml of
orthophosphoric acid and make upto 1000 ml with
water,
B. acetonitrile,
below,
Time
(in min.)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
80
50
80
80
20
50
20
20
25
26
30
solution of strong ammonia solution in methanol for 30

minutes, cool and filter. Boil under reflux the residue with
further 2 quantities, each of30 mI, of 0.1 per cent v/v strong
ammonia solution in methanol, cool and filter. Combine all
the filtrates and dilute to 100 m1 with 0.1 per cent v/v solution
ofstrong ammonia solution in methanol. Dilute 5.0 ml ofthis
solution to 50 ml with a 0.1 per cent v/v strong ammonia
solution in methanol.

hydrochloride RS in 0.1 per cent v/v strong ammonia solution
in methanol.
(5/lm),
w/v solution of potassium dihydrogen orthophosphate
in water and 27 volumes of acetonitrile,
than 2.0 per cent.
Calculate the content of berberine.
1 mg ofberberine hydrochloride is equivalent to 0.9045 mg of
berberine.
Darubaridra Stems
Berberis; Berberis aristata
8
7
1 mg of berberine hydrochloride is equivalent to 0.9045 mg of

berberine.
MethodU
. ----------.-.
3
1=::::J
2
DeteriiiilieoyliqUid cbf6mat6jifapliY(2:4.T4:). ... Test solution. Boil under reflux 1.0 g ofthe coarsely powdered
substance under examination with 40 m1 of a 0.1 per cent v/v
2496
"'=C7--,
,-'----,~J"':,:'c.=:~
DARUHARIDRA STEMS
IP 2010
Daruharidra Stems consist of cut dried stems of Berberis

aristata, DC (Fam. Berberidaceae).
Daruharidra Stems contain not less than 0.5 per cent w/w of
berberine, calculated on the dried basis.
Description. Yellowish-brown, cut chips of varying sizes,
fracture fibrous, and taste bitter.
Identification
A. Macroscopic - Cut tortuous pieces of varying length and
thickness. Bark thick, yellowish to brownish grey, wrinkled
irregularly, deeply furrowed; surface hard, rough, fracture short
and fibrous.
B. Microscopic - Cork consists of rectangular to squarish
yellow coloured thin walled cells, radially arranged; sieve
elements irregularly shaped, few cells containing yellowish
brown to orange brown masses; phloem fibers thick walled
arranged tangentially; phloem ray cells have calcium oxalate
crystals, scattered stone cells; secondary phloem broad, sieve
elements arranged as tangential bands, phloem fibers short,
thick walled; secondary xylem broad, consisting of small to
medium sized xylem vessels, tracheids, xylem fibers.
Powder is greenish yellow and shows the following diagnostic
characters when observed under a microscope using chloral
hydrate solution. Abundant thin walled parenchyma,
fragments ofyellowish-brown cork; ovoid or spherical orange
brown granular masses. Examine under a microscope using
50 per cent v/v solution ofglycerol. The powder shows starch
granules, simple, small, spherical to ovoid.
C. Determine by thin-layer chromatography (2.4.17) coating
Mobile phase. A mixture of 70 volumes of n-butanol, 10

volumes of glacial acetic acid and 20 volumes of water.
Test solution. Shake 1 g of powdered substance under
examination with 10 ml methanol for 10 to 15 minutes and
filter. Wash the residue with 5 ml of methanol and add washing
to the filtrate.
Reference solution. Shake 1 g ofpowdered daruharidra stem
RS with 10 ml of methanol for 10-15 minutes and filter. Wash
the residue with 5 ml of methanol and add washing to the
filtrate.
Apply to the plate, 10 III of each solution as bands of 10 mm
by 2 mm. Allow the mobile phase to rise 8 em. Dry the plate in
air and observe in ultraviolet light at 254 urn and 365 urn and
also under day light. The chromatographic profile of the test
solution is similar to that of reference solution.
Tests

Method 1.
Loss on drying (2.4.19). Not more than 12.0 per cent deterrnined
Assay. Perform the Assay by following method I or by method
ll.
Method I
Test solution. Boil under reflux 1.0 g ofthe coarsely powdered

substance under examination with 40 ml of a 0.1 per cent v/v
solution of strong ammonia solution in methanol for 30
minutes, cool and filter. Boil under reflux the residue with
further 2 quantities, each 000 ml, of 0.1 per cent v/v strong
ammonia solution in methanol, cool and filter. Combine all
the filtrates and dilute to 100 ml with 0.1 per cent v/v solution
of strong ammonia solution in methanol. Dilute 5.0 m1 ofthis
solution to 50 ml with a 0.1 per cent v/v strong ammonia
solution in methanol.
hydrochloride RS in 0.1 per cent v/v strong ammonia solution
in methanol.
(5Ilm),
w/v solution of potassium dihydrogen orthophosphate
in water and 27 volumes of acetonitrile,
Methodll
Determine by liquid chromatography (2.4.14.).
:Test solution. Weigh 2 g ofthe coarsely powdered substance

under examination, add about 20 ml 2 M hydrochloric acid
and 30 ml of methanol, reflux on a water-bath for 15 minutes,
cool and filter. Reflux the residue further with 2 M hydrochloric
acid and methanol, till the extract turns colourless, cool and
filter. Combine all the filtrates and concentrate to a volume
slightly less than 50 ml. Dilute to 50.0 m1 with methanol.
2497
DARUHARIDRA STEMS
IP 2010

Eucalyptus Oil contains not less than 60 per cent w/w of

cineole, C IOH 180.
water,
B. acetonitrile,
below,
- spectrophotometer set at 346 Dill,
Description. A colourless or pale yellow liquid; odour, aromatic

and camphoraceous; taste, pungent and camphoraceous,
followed by a sensation of cold.
Time
Mobile phase A
o
25
26
30
80
50
80
80
Mobile phase B
(per cent v/v)
20
50
20
20
1 mg of berberine hydrochloride is equivalent to 0.9045 mg of
berberine.
than 2.0 per cent.
Identification

in 100 ml of toluene.
Reference solution. A 1 per cent w/v solution of cineole RS in
toluene.
dry the plate in air, spray with anisaldehyde solution, using
about 10 ml for a 200 mm x 200 mm plate, heat at 105 for
10 minutes and examine in daylight and in ultraviolet light at
365 nm. In the chromatogram obtained with the reference
solution a dark brown spot due to cineole is visible in daylight
in the middle part; when examined in ultraviolet light at
365 nm, the spot shows a brown fluorescence. The principal
corresponds to that ofcineole; no carmine-brown spot appears
in daylight in the upper third of the chromatogram and when
examined in ultraviolet light at 365 nm no spot showing a
greenish brown fluorescence appears in the upper third
(citronellal). Other spots may be visible in the upper and lower
thirds of the chromatogram.
Tests
Optical rotation (2.4.22). 0 to +10.
1 mg ofberberine hydrochloride is equivalent to 0.9045 mg of

berberine.

Eucalyptus Oil
NilgiriOil
~!1:(;alyptlIsQil is tl!(l(ls(ll1til!IQ~l()t>:tajl1(l<it>ys!(ll!Il1<1jtiIMi()l1
and rectification from the fresh leaves or the fresh terminal

branches of varIous speCies of ellcalyptlls
globulus Labill., E. fruticetorum F. von Muell., and E. smithii
(R. T. Balcer)(Fam. Myrtaceae).
iikeEucalyptus
Aldehydes. Place 10 ml in a glass-stoppered tube (150 mm x

25 mm) add 5 ml of toluene and 4 ml of ethanolic
hydroxylamine solution, shake vigorously and titrate
immediately with 0.5 M potassium hydroxide in ethanol
(60 per cent) until the red colour changes to yellow. Continue
the shaking and neutralising until the pure yellow colour of
the indicator is permanent in the lower layer after shaking
vigorously for 2 minutes and allowing separation to take place;
the reaction is__(;~Il1J>lelej!!_~t>_()1!t 1~_Il1il1ute_~~~l!Lt1J:~
operation using a further 10 ml of the substance under
ex.amination, and as the standard for the end-point, the titrated
liquid ofthe first determination with the addition of 0.5 mlof
0.5 M potassium hydroxide in ethanol (60 per cent). Not
2498
GARCINIA
IP 2010
more than 2.0 ml of 0.5 M potassium hydroxide in ethanol

(60 per cent) is required in the second determination.
Tests
Citric Acid. Not more than 2.0 per cent.
Phellandrene. Mix 1 ml with 2 ml of glacial acetic acid and

5 ml of light petroleum (40 to 60), add 2 ml of a saturated
solution of sodium nitrite and shake gently; no crystalline
precipitate is produced in the upper layer within 1 hour.
Test solution, reference solutions (a), (b) and chromatographic

system as described under Assay.
Assay (2.3.24) Determine the content of cineole in the oil.
Inject test solution and reference solution (b).
Storage. Store in well-filled, tightly-closed containers at a

Calculate the content of citric acid.
by Method I.
Garcinia
Vilayati Imli; Garcinia cambogia
Acid-insoluble ash (2.3.19). Not more than 1.5 percent.

Test solution. Weigh about 5 g of the coarsely powdered

substance under examination and transfer to a 250-ml beaker.
Add 5 ml of dilute phosphoric acid and 100 ml of water,
concentrate to half the volume by heating, cool and filter.
Extract the residue further with water till the last extract turns
colorless, cool and filter. Combine all the filtrates and
concentrate under vacuum to 250.0 ml.
Garcinia is the dried deseeded fruit of Garcinia cambogia
Desr. {Garciniagummi-gutia (L.) N: Robson}(Fam. Guttiferae).

hydroxycitric acid calcium salt RS in dilute phosphoric acid.
Garcinia contains not less than 12.0 per cent of total

hydroxycitric acid and hydroxycitric acid lactone, calculated
on the dried basis.
Reference solution (b). A 0.1 per cent w/v solution of citric

acid in dilute phosphoric acid.
Description. Dark brown to blackish brown fruits. Taste, acidic.
Identification
A. Macroscopic -
Dark brown to blackish brown fruits,

ovoid, longitudinally grooved.
B. Microscopic - Mesocarp very wide composed of

parenchymatous cells ofvarious sizes and shapes. Compound
starch grains and prismatic crystals ofcalcium oxalate traverse
throughout the parenchymatous cells of the mesocarp.
obtained with test solution has a retention time similar to that
of the peak due to hydroxycitric acid in the chromatogram
- a stainless steel column 25 em x 4.6 rom packed with
mobile phase: a buffer solution prepared by dissolving
1.36 g ofpotassium dihydrogen phosphate in 900 ml of
water, adjusting the pH to 2.5 with dilute phosphoric
acid and diluting to 1000.0 ml with water,
the relative retention times are about 0.8 for hydroxycitric
acid lactone, 1.0 for hydroxycitric acid and 2.0 for citric
acid, the resolution factor between hydroxycitric acid lactone
and hydroxycitric acid is not less than 1.8, the tailing factor is
2499
GARCINIA AQUEOUS EXTRACT
IP 2010
Inject the referene solution and the test solution.
not more than 1.5 and the relative standard deviation for the
Calculate the content ofthe hydroxy citric acid in the extract.
Calcium -
Calculate the sum of the contents of hydroxycitric acid and

hydroxycitric acid lactone.
Test solution. Weigh accurately about 0.2 g ofthe extract and

transfer into a 500 ml conical flask. Dissolve in 2 ml of 3 M
hydrochloric acid and add 200 ml of water. Add 15 ml of 1 M
Sodium hydroxide and titrate with 0.05 M EDTA using hydroxy
naphthol blue as indicator until deep blue colour persist.

Each ml of 0.05 M EDTA solution is equivalent to 0.002004 g

ofcalcium.
Garcinia Aqueous Extract

Garcinia Aqueous Extract is obtained by extracting Garcinia
(Garcinia cambogia Desr., Garcinia gummi-gutta (L.) N:
Robson, Fam. Guttiferoe) fruit rinds with water as calcium saIts.
Garcinia Aqueous Extract contains not less than 90.0 per cent
w/w to not more than 120.0 per cent w/w ofthe stated amount
ofhydroxy citric acid, and calcium not less than 15.0 per cent
w/w calculated on the dried basis. It may contain suitable
added substances.
Usual strengths. 50 per cent; 60 per cent.

Storage. Store in air-tight containers, protected from light.
Gokhru
Tribulus terrestris
Description. A pale brown to brown powder.
Tests
on I g by drying in an oven at 105.
Assay. hydroxy citric acid chromatography (2.4.14).
Determine by liquid
Test Solution. Shake well 0.2 g ofthe extract under examination

add 2 ml of 3 M hydrochloric acid and add 10 ml of water,
.sonicate it to dissolve, make up to volume 100.0 ml with water
and mix.
Gokhru consists of dried fruits of Tribulus terrestris

L.(Fam.zygophyllaceae).
Reference solution. A 0.1 per cent w/v solution of hydroxy

citric acid RS with 2 ml of 3 M hydrochloric acid in water.
Description. Pedicellate and globose fruits, having wedgeshaped cocci, covered with short and stiff spines. Possesses
faintly aromatic smell and acrid taste.
octadecylsilane bonded to porous silica (5J.1m),
mobile phase: o. I per cent orthophosphoric acid in
water, filter and degas,
- flow rote. 0.6 ml per minute,
___ ~ectr~~~()met~~~~tat_~~nm,,
_
- injection volume. 20 J.11.
Gokhru contains not less than 0.5 per cent of diosgenin,

Identification
A. Macroscopic - Fruit is pedicellate, having wedge-shaped
cocci, covered with spines. Surface of schizocarp is rough.
B. Microscopic -
Pericarp is differentiated into epicarp,
mes6'carp and eiidocail): Epicarp issuif6uiidedoyiion'::'

. glandular trichomes;
2500
GUAR GUM
IP 2010
Mobile phase. A mixture of8 volumes oftoluene and 2 volumes

of ethyl acetate.
examination with 50 ml of methanol for 15 minutes, cool and
filter. Reflux the residue further with 50 ml ofmethanol, cool
and filter. Combine both the filtrates and concentrate under
vacuum to dryness. Extract the dried residue with 10 ml of
methanol at 50 for 10 minutes, filter the solution and use
filtrates for analysis.
Reference solution. Reflux 2.5 g ofgokhnl RS with 50 ml of
further with 50 ml ofmethanol, cool and filter. Combine both
the filtrates and concentrate under vacuum to dryness. Extract
the dried residue with 5 ml ofmethanol at 50 for 10 minutes,
filter the solution and use the filtrates for analysis.
and examine in ultraviolet light at 254 nm and 365 llill, spray
105 for 5 minutes and examine in day light. The chromatographic
solution.
mobile phase: 80 volumes ofacetonitrile and 20 volumes

of methanol,
Inject the test solution.
Calculate the content of diosgenin.
Guar Gum
Guar Gum is a gum obtained from the ground endosperms of
the seeds of Cyamopsis tetragonolobus (Linn.) Taub or other
species of Cyamopsis (Fam. Leguminosae). It consists mainly
of a high molecular weight hydrocolloidal polysaccharide,
composed of galactan and mannan units combined through
glycosidic linkages.
Tests
Description. An almost white to pale yellowish white powder;

odour, characteristic.
Identification
A. When mounted in lactophenol and examined under a

microscope, irregular, angular particles of various sizes and
shapes are seen.

by method I.
B. To 0.1 g add 1 ml of 0.2 M iodine; the mixture does not

acquire an olive-green colour.
Total ash (2.3 .19). Not more than 11.0 per cent.
Test solution. Reflux 5.0 g ofthe substance under examination
with 50 m1 of sulphuric acid (10 per cent) for 4 hours. Cool
and transfer to separating funnel. Extract with 50 ml of ethyl
acetate. Repeat the extraction 3 times. Pass the ethyl acetate
layer through sodium sulphate and evaporate. Dissolve the
residue with 50 rn1 of methanol.
Reference solution. A 0.1 per cent w/v solution of diosgenin
RS in methanol.
C. Dissolve 0.1 gin 20 ml ofwater by shaking and add 0.5 ml

of hydrogen peroxide solution (20 vol) and 0.5 ml ofa 1 per
cent w/v solution of benzidine in ethanol (90 per cent), shake
and allow to stand; no blue colour is produced (distinction
from acacia).
D. Mount a small quantity in ruthenium red solution and
examine under a microscope; the particles do not acquire a
pink colour (distinction from sterculia gum and agar).
E. To 2 ml of a 0.5 per cent w/v solution add 2 ml of a 20 per
cent w/v solution of lead acetate; a flocculent precipitate is
produced (distinction from acacia, ghatti gum and sterculia).
Tests
Acidity or alkalinity. A 0.5 per cent w/v solution is neutral to
litmus paper.
Tannin. To 5 ml of a 0.5 per cent w/v solution add 0.1 ml of
ferric chloride test solution; no bluish black colour is produced.
carbonate, add 10 ml ofbromine solution and mix thoroughly.
2501
GUAR GUM
IP 2010

dissolve the cooled residue in a mixture of 16 ml of brominated
(3 ppm).
Gudmar contains not less than 1.0 per cent w/w of gymnemic
acids (calculated as gymnemagenin), calculated on dried basis.
Identification
Protein. Not more than 5.0 per cent, determined by the

following method. Carry out the determination of nitrogen
(2.3.30), using about 3.5 g, accurately weighed, and multiplying
the percentage of nitrogen determined by 6.25 to obtain the
percentage of protein.
Acid-insoluble matter. Not more than 3.0 per cent, determined
by the following method. Weigh accurately about 1.5 g and
disperse in 150 ml of water and 1.5 ml ofsulphuric acid. Warm
on a water-bath for 6 hours, replacing the water lost by
evaporation. Add about 0.5 g ofa suitable filter-aid, accurately
weighed, and filter through a suitable ashless filter paper. Wash
the residue several times with hot water, dry the filter and its
contents at 105 for 3 hours. Cool in a desiccator, weigh and
subtract the weight of the filter aid.
Microbial contamination (2.2.9). Total bacterial count: Not
more than 5000 per g. 1 g is free from Escherichia coli and 10
g is fi'ee from salmonellae.
Total ash (2.3.19). Not more than 2.0 per cent, determined on
l.Og.
Gudmar
Gymnema sylvestre
Description. Greenish-yellow in colour, surface pubescent

on both sides and characteristic odour with extremely bitter
and acrid taste.
A. Macroscopic - Leaves, simple, petiolate about 2 to 6 em

long and 1 to 4 em broad, yellowish brown on adaxial and dark
green on abaxial side.
B. Microscopic - Upper and lower epidermis covered with
cuticle having uni to tri cellular covering trichomes which are
slightly curved at the bulbous base. Below the epidermis is
single layer ofpalisade cells followed by 2-3 layered spongy
parenchyma. Starch gains are simple and present in spongy
parenchyma. Midrib region shows 2-7 layers of
collenchymatous cells. Stomata are ofparacytic type, mostly
on lower the surface. There is a fan shaped vascular bundle in
the centre. Each vascular bundle is collateral, closed and
surrounded by parenchymatous sheath. Rosette crystal of
calcium oxalate present in the spongy parenchyma.
Mobile phase. A mixture of5 volumes of chloroforms, 1 volume

of methanol and 1 volume of ethyl acetate.
under examination with 50 ml of ethanol (50 per cent v/v) for
50 ml of ethanol (50 per cent v/v), cool and filter. Combine all
the filtrates and concentrate under vacuum to 25 ml. Take 5 ml
of resulting solution, add 5 ml ethanol and 2 ml ofpotassium
hydroxide and reflux for 1 hour. Cool and add 1.8 ml of 12 M
hydrochloric acid and heat on water bath. Cool and adjust
the pH to 7.5-8.5 with 11 per cent potassium hydroxide. Dilute
the solution with ethanol (50 per cent v/v) to 100 ml and filter.
Reference Solution. Reflux 1 g of gudmar RS with 50 ml of

ethanol (50 per cent v/v) for 15 minutes, cool and filter. Reflux
the residue further with 2 x 50 ml of ethanol (50 per cent v/v) ,
vacuum to 10 ml. Take 5 ml of resulting solution, add 5 ml
ethanol and 2 ml ofpotassium hydroxide and reflux for 1 hour.
Cool and add 1.8 ml of 12 M hydrochloric acid and heat on
water bath. Cool and adjust the pH to 7.5-8.5 with 11 per cent
potassium hydroxide. Dilute the solution with ethanol
(50 per cent v/v) to 50 mi and filter.
8
7
6
o
Gudmar consists of the dried mature leaves of Gymnema
sylvestre R.Br. (Fam. Asclepiadaceae).
Apply to the piatelO III ofeach solution as ~ands 10 mm

2
rom. Allow the mobile phase to rise 8 em. Dry the plate in air
and examine in ultraviolet light at 254 urn and 365 nm, spray
2502
GUDUCHI
IP 2010

Tests
Calculate the content of gymnemagenin.


by method 1.
Guduchi
Giloe; Amrita; Tinospora cordifolia

8
7


under examination with 50 ml of ethanol (50 per cent v/v) for
50 ml of ethanol (50 per cent v/v) , cool and filter. Combine all
the filtrates and concentrate under vacuum to 25 ml. Take 5 ml
of resulting solution, add 5 ml ethanol and 2 ml ofpotassium
hydroxide and reflux for 1 hour. Cool and add 1.8 ml of 12 M
hydrochloric acid and heat on water bath. Cool and adjust
the pH to 7.5-8.5 with 11 per cent potassium hydroxide. Dilute
the solution with ethanol (50 per cent v/v) to 100 ml and filter.
gymnemagenin RS in methanol.
- mobile phase (A). 80 per cent v/v acetonitrile,
(B). 0.1 percentw/v dihydogenpotassium
phosphate,
- a linear gradient prograriune using the conditions given
below,
- flow rate. 1.5 mlperminute,
Time
(min)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
25
75
20
50
50
30
25
75
o
Guduchi consists of the dried, mature pieces of stem of
Tinospora cordifolia (W~lld.) Miers (Fam. Menispermaceae).
Guduchi contains not less than 0.02 per cent w/w of
cordifolioside A, calculated on the dried basis.
Description. A greyish-black in colour, fibrous fracture and
no distinct odour with bitter taste.
Identification
A. Macroscopic - Stem-pieces glabrous, cylindrical, solid,
lenticillate, 5-15 rom in diameter having light brown surface
marked with warty protuberances due to circular lenticels.
Transversely smoothened surface shows a radial structure
with conspicuous medullary rays traversing porous tissues.
B. Microscopic - Transverse section of stem shows
outermost layer ofcork which is differentiated in to outer zone
ofthick walled, compressed cells and inner zone ofthin walled,
tangential cells. Cork broken at some places due to lenticels.
Cortex consists of 3-5 rows ofirtegularly arranged tangential,
chlorenchymatous cells with numerous intercellular spaces.
Inner cortex filled with plenty of starch gains. Vascular zone
consists of 10-12 wedge-shaped strips of xylem externally
surrounded by semi-circular strips of phloem. Cambium
2503
GUDUCHI
IP 2010
composed ofone to two layers oftangentially elongated cells.

Primary phloem appears crushed and obliterated; secondary
phloem groups are massive. Pith composed oflarge, thin walled
cells mostly containing starch gains.

vacuum to 5 ml.
Reference solution. Reflux 2 g ofthe guduchi RS with 25 mlof
Apply to the plate 10 /-ll ofeach solution as bands 10 mm by 2
and examine in ultraviolet light at 254 nm and 365 urn, spray
- mobile phase: A. a gradient mixtures of water
B. .acetonitrile,
below,
Time
Mobile phase A Mobile phase B
(per cent v/v)
(per cent v/v)
(in min)
80
20
25
20
80
30
80
20
Calculate the cont~nt of cordifolioside A.
Tests
Guggul Resin
Guggul; Commiphora wightii

method!.



under examination with 50 ml of methanol on a water-bath for
methanol till the extract turns colourless, cool and filter.

. -Comoinealnhe~filtratesandcoifcentrateto~avoluinesliglit1y
less than25 ml. Dilute with methanol to 25;0 ml.

cordifolioside A RS in methanol.
Guggul Resin is the oleoresin exudation from Commiphora

wightii (Arnott) Bhandari (Commiphora mukul (Am.)
Bfian(Jan~73atsiiiiioaenaroii-miilCiil
H ool(~
ex StoCks) (Fam.
Btitsetaceae). .
Guggul Resin contains not less than 1.0 per cent w/w and not
more than 1.5 per cent w/w of gugulsterones (Z and E).
2504
GUGULIPID
IP 2010
Description. Light to dark-brown conglomerates of tears,

rounded or irregular, slightly sticky to touch; odour, faintly
balsamic.
allowing to stand for 18 hours. Filter rapidly taking care to

avoid loss ofethanol, evaporate 25 ml ofthe filtrate to dryness,
dry at 105 and weigh.
Identification
onO.5 g.
A. Prepare a 0.005 per cent w/v solution in ethanol (95 per

cent) of the residue obtained in the test for Ethanol-soluble
extractive.
resulting solution shows absorption maxima at about 245 nm
and 327 nm.
Mobile phase. A mixture on volumes of light petroleum (60
to 80) and 1 volume of ethyl acetate.
Test solution. Dissolve 0.5 g of the residue obtained in the
test for Ethanol-soluble extractive in 100 ml of ethanol (95 per
cent).
Reference solution. A 0.5 per cent w/v solution ofthe residue
obtained similarly from guggul resin RS in ethanol (95 per
cent).
Apply to the plate 20 /Ll of each solution as bands 10 mm by 2

until the odour of the solvent is no longer detectable and
examine in ultraviolet light at 254 om and 365 om, spray with a
10 per cent v/v solution of sulphuric acid in methanol. Heat
the plate at 100 for 10 minutes and examine in day light. The

Test solution. Weigh accUrately about 3.0 g of the substance
under examination, add 50 ml of acetonitrile, reflux on a waterbath for 30 minutes, cool and filter. Reflux the residue further
with three portions, each of 30 ml, of acetonitrile, cool and
filter. Combine the filtrates and concentrate to 100.0 ml.
gugulsterones (Z and E) RS in acetonitrile.
octadecylsilane silica gel (5 /Lm),
mobile phase: a filtered and degassed mixture of 45
volumes of acetonitrile and 55 volumes of water,
injection volume. 20 /Ll.
relative retention times are about 0.69 for gugulsterone E and
1.0 for gugulsterone Z and the relative standard deviation for
the contents of gugulsterones (Z and E).
exceeding 30.
Tests
Ethyl acetate-soluble extractive. Not less than 25.0 per cent,
determined by the following method. Crush the substance
under examination to a coarse powder. Shake 5.0 g of the
powder with 25 ml of light petroleum (60 to 80) for 1 hour
and separate the liquid by filtration. Repeat the extraction
twice and dry the defatted material over phosphonLS pentoxide
at room temperature at a pressure not exceeding 2.75 kPa for
8 hours. Crush the dried material and extract with four quantities,
each of25 ml, of ethyl acetate by shaking each time for 1 hour
followed by filtration through a sintered-glass funnel (porosity
No.3) and combining the filtrates. Evaporate the combined
filtrates, dry over phosphorus pentoxide at room temperature
at a pressure not exceeding 2.75 kPa for 12 hours and weigh.
Ethanol-soluble extractive (2.3.46). Not less than 35.0 percent,
determined by the following method. Crush the substance
under examination to a coarse powder. Macerate 5.0 g of the
powder with 100 ml of ethanol (95 per cent) in a closed flask
for 24 hours, shaldng frequently during the first 6 hours and
Gugulipid
Gugulipid is the ethyl acetate extractive ofGuggul Resin.
Gugulipid contains not less than 4.0 per cent w/w and not
more than 6.0 per cent w/w of gugulsterones (Z and E).
Description. A brown, viscous liquid.
Identification
A. When examined in the range 230 om to 360 om (2.4.7), a
0.005 per cent w/v solution in chloroform shows absorption
maxima at about 245 om and 327 om; absorbance at about
Mobile phase. A mixture of 3 volumes of light petroleum
(60 to 80) and 1 volume of ethyl acetate.
2505
GUGULIPID
IP 2010

examination in 100 ml of ethanol (95 per. cent).
gugulipid RS in ethanol (95 per cent).
Apply to the plate 5 f.ll of each solution as bands 10 mm by 2
until the odour of the solvent is no longer detectable and
examine in ultraviolet light at 254 om and 365 om, spray with a
10 per cent v/v solution of sulphuric acid in methanol. Heat
15 m1, of ethyl acetate, combine the extracts, filter and

evaporate to dryness. The residue complies with the following
tests.
om
A. When examined in the range 230 om to 360

(2.4.7),
a 0.005 per cent w/v solution in chloroform shows absorption
maxima at about 245 nm and 327 om; absorbance at about
245 nm, about 0.87 and at about 327 om, about 0.52.


(60 to 80) and 1 volume of ethyl acetate.
under examination in ethanol (95 per cent).
Tests

under examination, add 50 ml of acetonitrile and warm on a
boiling water-bath for 10 minutes. Cool and add sufficient
acetonitrile to produce 100.0 m1.
gugulsterones (Z and E) in acetonitrile.
octadecylsilane silica gel (5 f.lm),
injection volume. 20 f.l1.
Calculate the contents of gugulsterones (Z and E).
exceeding 30.
Gugulipid Tablets
Gugulipid Tablets contain not less than 90.0 per cent and not
more than 110.0 per cent ofthe stated amount ofgugulsterones
(Z and E). The tablets may be coated.
Identification
20 mg of gugulsterones (Z and E) with two quantities; each of
Reference solution. A 0.25 per cent w/v solution of gugulipid

Apply to the plate 5 f.ll of each solution. After development,
detectable and spray with 50 per cent w/v solution of sulphuric
acid. The principal spots in the chromatogram obtained with
the test solution correspond to those in the chromatogram
Tests
Disintegration (2.5.1).60 minutes.

gugulsterones (Z and E) and extract with five quantities, each
of 20 ml, of acetonitrile, with the aid of heat. Combine the
extracts and concentrate to 50.0 ml. Filter the solution through
than 1.0 f.lm and use the filtrate.
gugulsterones (Z and E) in acetonitrile.
a stainless steel colwI1ll 25 cm x 4.6 mm, packed with
octadecylsilyl silica gel (5 f.lm),
the reference solution. The test is not valid unless the
2506
IP 2010
HARlDRA

the contents of gugulsterones (Z and E) in the tablets.
Reference solution. Reflux 1 g of coarsely powdered haridra

RS with 5 ml methanol for 15 minutes, cool and filter.

exceeding 30.
Apply to the plate 10 f..ll of each solution as bands 10 rom by 2

and examine in ultraviolet light 254 run, 365 run and also under
day light. The chromatographic profile of the test solution is
similar to that ofthe reference solution.

equivalent amount of gugulsterones (Z and E).
Tests
Haridra
Haldi; Turmeric; Curcuma longa

by Method 1.


0.2g.

Haridra consists ofthe dried rhizomes of Curcuma longa Linn.

(Fam. Zingiberaceae).
.
Haridra contains not less than 1.5 per cent w/w of curcumin,
Description. Externally yellowish to yellowish brown with
root scars and annulations. Odour, aromatic; taste, warmly
aromatic and bitter.
Identification
A. Macroscopic - Rhizome oblong, conical or cylindrical to
elongate, finger-like; internally orange yellow. Texture hard
and heavy; fracture short.
B. Microscopic - Ground tissue of parenchyma cells; cells

filled with gelatinized starch grains and yellow pigment.
Fibrovascular bundles and oil cells scattered through out
ground tissue.

5 volumes of ethanol and 1 volume glacial acetic acid.
Test solution. Extract 1 g of the coarsely powdered substance
under examination with 5 ml methanol for 10 minutes with
slight warming. Filter and use the filtrate.

water bath for 15 minutes cool and filter. Reflux the residue
the filtrates and concentrate to 100.Omi.
Reference solution. A 0.01 per cent w/v solution of curcumin
RS in methanol.
silicagel consisting of porous spherical particles with
chemically bonded nitrile group,
- mobile phase: a mixture of35 volumes of tetrahydrofuran
109 of citric acid in 1000 ml of water, adjusting the pH
to 3.0 with dilute ammonia solution,
injection volume. 20 f..lI.
Calculate the content of curcumin.
2507
HAiuDRA DRY EXTRACT
IP 2010
Haridra Dry Extract

Haridra Dry Extract is a partially purified natural complex of
diaryl heptanoid derivatives isolated by extracting Haridra
(Curcuma longa L., Fam. Zingiberaceae), rhizomes with
acetone or ethanol or any other suitable solvent and
evaporation of solvent under vacuum..
Haridra Dry Extract contains not less than 95.0 per cent w/w
and not more than 102.0 per cent w/w ofthe stated amount of
total curcuminoids calculated on the dried basis, as the sum
ofcurcumin, demethoxycurcumin and bisdemethoxycurcumin.
It contains not less than 70.0 per cent w/w to not more than
80.0 per cent w/w ofcurcumin, not less than 15.0 per cent w/w
to not more than 25.0 per cent w/w ofdemethoxycurcumin and
not less than 2.5 per cent w/w to not more than 6.5 per cent
w/w ofbisdemethoxycurcumin.
Description. An orange yellow crystalline powder.
Identification
A. Determine by thin-layer chromatography (2.4.17) coating
Mobile phase. A mixture of 10 volumes of chloroform, 1.0

volume of methanol, 0.5 volume of glacial acetic acid and 0.5
volume offormic acid.
examination with 100.0 ml of methanol and filter.
Test Solution. Dissolve a quantity of the extract under

examination containing about 50 mg curcumin in 100.0 ml of
tetrahydrofuran. Dilute 5.0 ml ofthis solution to 50.0 ml with
the mobile phase.
Reference solution. A solution 'containing 0.01 per cent w/v
each of bisdemethoxycurcumin RS, demethoxycurcumin RS,
curcumin RSin tetrahydrojitran. Dilute 5ml ofthis solution to
a stainless steel column, 25 em x 0.46 mm packed with
octadecylsilane bonded to porous silica (5f..lm),
w/v solution of citric acid and 60 volumes of
tetrahydrofuran,
injection volume. 20 f..ll.
curcumin for bisdemethoxycurcumin is about 1.3 and for
demethoxycurcumin is about 1.14.
Calculate the content of the bisdemethoxycurcumin,
demethoxycurcumin and curcumin in extract.

each of bisdemethoxycurcumin RS, demethoxycurcumin RS,
curcumin RS in the methanol.
Apply to the plate 10 f..ll ofeach ofsolution as bands 10 mm by
2 mm. Allow the mobile phase to rise 8 em. Dry the plate in air
a anisaldehyde- sulphuric acid reagent. Heat the plate at
110 for 10 minutes and examine the plate at 365 nm and day
Haritaki
Harad; Chebulic myrobalan; Terminalia chebula
Tests
Microbialcontarnination(2:2:9): Complies withthemicrobial
contamination 'tests;- -,
Assay. Total curcuminoids chromatography (2.4.14).
Determine by liquid
Haritaki consists of pericarp of the dried fruit of Terminalia

chebula Retz. (Fam. Combretaceae).
2508
HARITAKI
IP 2010
Haritaki contains not less than 5 per cent w/w of chebulagic

acid and not less than 12.5 per cent w/w of chebulinic acid,
Description. It has a shine on its external part and has
longitudinal ridges. The colour varies from yellowish brown
to light black. It has a astringent taste and is also slightly
bitter
Identification
Test C may be omitted if tests A, BandD are carried out and
test D may be omitted if tests A, Band C are carried out.
A. Macroscopic - The fruit is 2 to 3 cm in length and 1 to
2 cm in diameter with hard stony appearance. Externally it is
shining and is adorned with longitudinal ridges. Color of the
fruit rind varies from yellowish brown, uniform brown to light
black. Internally the fruit is light yellow.
B. Microscopic - Epicarp has thick walls covered with cuticle.
The mesocarp has many stone cells of various sizes and
shapes which forms a reticulum. Large quantity of tannin is
present in the mesocarp..Simple starch granules are present in
plenty.

Mobile phase. Amixture of 35 volumes of toulene, 50 volumes

of acetone, 15 volumes of glacial acetic acid and 5 volumes
offormic acid.
being examined, add 50-75 ml of methanol and reflux for
15 minutes, cooland filter. Reflux the residue further for two
times with 75 ml of methanol, cool and filter. Combined all the
Reference solution. To 0.1 g ofthe haritald RS, add 50-75 ml
of methanol and reflux for 15 minutes, cool and filter. Refluf(
the residue further for two times with 75 ml of methanol, cool
vacuum to 10 ml.
Apply to the plate 10 III or'each solution as bands 10 mm by 2
with 10 per cent w/v ferric chloride solution in water. Heat

Method I.
heavy metals, Method B (20 ppm)
Test solution. Weigh 0.5 g of coarsely powdered sample, add

50 ml of water, sonicate for 3 minutes and heat on a boiling
water bath for 15 minutes, cool and dilute to 100.0 ml with
water andfilter. Dilute 10.0 ml ofthe solution to 25.0 ml with
water.
Reference solution (a). Weigh 0.5 g of haritaki RS, add 50 ml
of water, sonicate for 3 minutes and heat on a boiling water
bath for 15 minutes, cool and dilute to 100.0 ml with water and
filter. Dilute 10.0 ml ofthe solution to 25.0ml with water.
chebulagic acid RS in water.
chebulinic acid RS in wate]:
octadecylsilane bonded to porous silica (5 11m).
- mobile phase: A.a buffer solution pH 2.5 prepared by
dissolving 0.136 g of potassium di~hydrogen
water,
B. acetonitrile,
flow rate. 1.5 ml per minute
Time
(in min.)
a
18
25
D. In the Assay, the chromatogram obtained with the test

solution corresponds to the chromatogram obtained with
Tests
28
35
Mobile phase A
(per cent v/v)
95
65
45
45
95
Mobile phase B
(per cent v/v)
5
35
55
55
5
Inject the reference solution (b) and (c). The relative standard
deviation for the replicate injections is not more than 2.0 per
cent:
2509
HARITAKI EXTRACT
IP 2010
Calculate the content of Chebulagic acid and Chebulinic

acid.
Test solution. Dissolve about 0.5 g ofthe extract or quantity

equivalent to 100mg ofpolyphenoIs in water, make up to 100
ml and filter.
Storage. Store protected from heat, moishrre and against attack

Reference solution (a). 0.01 per cent w/v solution of

Reference solution (b). 0.01 per cent w/v solution of
chebulinic acid RS in watel:
Haritaki Extract
Haritaki extract is obtained by extracting Haritaki (Terminalia
chebula Retz., Fam. Combretaceae) from the dried fruit pericarp
with ethanol or any other suitable solvent.
Haritaki extract contains not less than 90 per cent w/w and not
more than 120 percent w/w ofthe stated amount ofchebulinic
acid and chebulagic acid.
Description. Light yellowish to yellowish brown powder with
odour, characteristic; taste, bitter.
Identification
mobile phase:A. a buffer solution prepared by dissolving
0.136 g of potassium di-hydrogen orthophosphate in
500 ml of watel; add 0.5 mI of orthophosphoric acid and
dilute to 1000 ml with water,
B. acetonitrile,
below,

volumes offormic acid, 2 volumes of toluene and 1 volume of
methanol.
Test solution. Dissolve 0.5 g ofextract under examination with
100 ml methanol and filter.
Reference solution. A. 0.1 per cent w/v solution of chebulagic
acid RS and 0.1 per cent w/v solution of chebulinic acid RS
in methanol.
and examine in ultra-violet light at 254 urn. Spray the plate with
ferric chloride reagent. Heat the plate at I 10 for 10 minutes
and examine the plates at 365 nm and in day light. The
B. In the assay, the peaks due to c hebulinic acid and chebulagic
acid in the chromatogram obtained with test solution
corresponds to the peak obtained with reference solutions.
Tests
Time
(in min)
mobile phase A
(per cent v/v)
mobile phase B
(per cent v/v)
95
18
75
25
25
65
35
28
65
35
35
95
Inject reference solution (a) and (b). The test is not valid rrnless
Calculate the content of chebulinic acid and chebuliagic
acid.
Heavy metals (2~3 .13). 1.0 gcomplies with the limit fest for
heavy metals, (Method B) 20 ppm;
Haritaki Aqueous Extract is obtained from the dried fruit

pericarp of Haritaki (Terminalia chebula Retz., Fam.
Combretaceae) by extraction with water.
--_._-", .. _-.,'------"'--_._----
Haritaki Aqueous Extract contains not less than 90.0 per cent
w!waIlcl [lot more than 120.0 percen1:w/w ()filie stated am()rrnt
of total polyphenols (sum of chebulagic acid, chebulic acid,
gallic acid, coriagin, 1,3,6 trigalloyl glucose and ellagic acid).
2510
HYDROGENATED CASTOR OIL
IP 2010
B. acetonitrile
Description. Brown to brown powder; with characteristic odour

and bitter taste.

below,
Identification

volumes offormic acid, 2 volumes of toluene and 1 volume of
methanol.
100 ml methanol and filter.
Reference solution. A 0.1 per cent w/v solution of chebulagic
acid RS and 0.1 per cent w/v solution of gallic acid in
methanol.
and examine in ultra-violet light at 254 nm: Spray the plate with
ferriC chloride reagent. Heat the plate at 1100 for 10 minutes
and examine the plates under 365nm and under day light. The
B. In the assay the peak in the chromatogram obtained with

the test solution corresponds to the peak in the chromatogram
obtained with the chebulinic acid and gallic acid.
Time
(in min)
mobile phase A
(per cent v/v)
mobile phase B
(per cent v/v)
95
18
75
25
25
28
65
65
35
35
95
35
Inject the reference solutions (a) and (b). The test is not valid
Calculate the content of Total polyphenols by summing the
peak areas of chebuliagic acid, el/agic acid, 1,3,6 tri gal/oyl
glucose, corilagin, gallic acid and chebulic acid using
chebulagic acid. The relative retention times of various
polyphenols with reference to gallic acid and chebulagic acid
are as follows.
Chebulic acid
0.80
025
Tests
Gallic acid
1.00
0.32
Corlagin
2.57
0.81
1,3,6 Trigalloyl glucose
2.81
0.89
Chebulagic acid
3.17
1.00
Ellagic acid
3.49
Chebulinic acid
3.64
1.10
1.15

contamination test.

Test solution. Dissolve about 0.5 g of the extract containing

100 mg ofpolyphenols in 100 m1 of water,and filter.
Reference solution (a). 0.01 per cent w/v solution of

Reference solution (b). 0.01 per cent w/v solution of gallic
acid RS in water.
- mobile phase:A.a buffer solution prepared by mixtures
dissolving 0.136 g of Potassium di-hydrogen
orthophosphoric acid and dilute to 1000 ml with water.

Castor Wax; Opalwax
Hydrogenated Castor Oil is refined, bleached, hydrogenated
and deodorised castor oil. It consists mainly ofthe triglyceride
ofhydroxystearic acid.
Description. A white to yellow powder ofuniform consistency
and texture. It may have a hard, waxy consistency.
Tests
Melting range (2.4.21). 85 to 88, determined by Method II.
2511
IPECAC TINCTURE
IP 2010
Free fatty acids. Weigh accurately about 20 g, melt on a waterbath, add 75 ml of hot ethanol (95 per cent), previously
neutralised to phenolphthalein solution with 0.1 M sodium
hydroxide, swirl, add 1 ml of phenolphthalein solution and
titrate with 0.1 M sodium hydroxide, swirling vigorously until
the solution remains faintly pink after being shaken for
60 seconds; not more than 11.0 mlofO.I Msodiumhydroxide
is required.
in day light. An intense yellow, fluorescent zone of emetine is

observed in chromatogram of test solution corresponding to
intense yellow fluorescent zone of emetine in chromatogram
of reference solution.
Tests
Ethanol. Not less than 24.0 per cent and not more than 28.0 per
cent.
Test solution. A 0.8 per cent v/v solution ofthe sample under
examination and 02 per cent v/v of acteonitrile as internal
standard.
Acetyl value (2.3.22). Not less than 143.

Reference solution. A solution in water containing 0.2 per

cent v/v of ethanol and 2 per cent v/v of acetonitrile (internal
standard).
Iodine value (2.3.28). Not more than 5.0.

Saponification value (2.3.37). 176 to 182.
Storage. Store at a temperature not exceeding 30. Avoid
exposure to excessive heat.
Ipecac Tincture
Ipecac Tincture obtained from roots and rhizomes ofCephaelis
ipecacuanha A. Rich (Fam. Rubiaceae).
Ipecac Tincture contains not less than 90 per cent w/w and
not more than 110 per cent w/w ofalkaloids ofIpecac calculated
as emetine.
Description. A yellowish brown liquid.
Identification
Mobilephase. A mixture of 2 volumes of ammonia, 15 volumes
of methanol, 18 volumes of ethyl acetate and 65 volumes of
toluene.
Test solution. Shake 2.0 ml oftincture under examination with
2 ml ofwater and 0.1 ml ofammonia. Add 10 ml ofether and
shake. Separate the ether layer, dry it over about 2 g of
anhydrous sodium sulphate & filter.
Reference solution. Dissolve 2.5 mg ofemetine hydrochloride
RS in methanol and dilute to 10 ml with the same solvent.
Applytotheplate~IO_!-LlQfeach.sQlution~as_bandsj)LLQmmby
2111l]1.l\.llQW the mQl:>ilepl1l1~e t()ris~toJOCIl1' .J)ryt.l1~plat.e

in air. Spray the plate with 0.5 per cent w/v solution of iodine
in ethanol (95 per cent v/v) solution. Heat the plate at 60 0 for
10 min and examine the plate in ultraviolet light at 365 urn and
a stainless steel column 1.8 m x 3 mm, packed with
copolymer of ethylvinylbenzene and divinylbenzene
(100 to 150 mesh),
temperature:
column. 1200 ,
inlet port. 21 00 ,
- a flame ionization detector at 210 0 ,
- flow rate. Adjust the carrier flow so that acetonitrile,
internal standard, elutes in 5 to 10 minutes.
Calculate the percentage v/v of ethanol.
Test solution. Transfer 5 ml ofsample into a 50 ml volumetric
flask. Dilute to volume with O.OlM hydrochloric acid and
mix.
Reference solution. A 0.010 per cent emetine hydrochloride
heptahydrate RS and 0.010 per cent of cephaeline in O.OlM
hydrochloric acid.
octadecyl silane chemically bonded to porous silica
(5!-Lm).
- mobile phase: a mixture of50 volumes ofmethanol and
50 volumes of buffer prepared by dissolving 2.0 g of
I-heptane sulfonate in 500 volumes water,
- column temperature: 50 0
- flow rate. Adjust the flow rate so that the retention time
of emetine is about 14 minutes,
inje~ctiatfvolurn~:tOf.l.t
Injecti:he referencesoiution. fhe test IS not valid unless the

than 2.0 per cent.
2512
KALMEGH
IP 2010

Calculate the content of cephaeline and emetine as total
alkaloids in the sample.
1 mg of emetine hydrochloride contains 0.868 mg of emetine
and correction factor for cephaeline is 0.971.
Usual strengths. 0.1 per cent w/w; 0.2 per cent w/w.
layers ofmucilage are more resistant and swell to form rounded

papillae. When mounted in 0.005 M iodine, occasional simple
and two- to four-compound starch granules, about 2 mm to
10 mm, can be seen in some ofthe cells. Occasional fragments
of thick-walled, reddish brown endosperm, cells with pitted
walls and elongated fragments ofgrey embryo may be present.
Swelling power. Transfer 1 g to a 100-ml stoppered cylinder
containing 90 ml of water, shake well for 30 seconds and allow
to stand 24 hours, shaking gently on three occasions during
this period. Add sufficient water to produce 100 mI, mix gently
for 30 seconds, avoiding the entrapment of air, allow to stand
for 5 hours and measure the volume ofn:mcilage. Repeat the
determination three times. The average offour detenninations
is not less than 40 m!.
Ispaghula Husk
Isapgol Husk; Plantago ovata
1 g.

Storage. Store protected from moisture and from attack by

insects and. rodents.
Kalmegh
o
Andrographis paniculata
Ispaghula Husk consists of the epidermis and collapsed

adjacent layers removed from the dried ripe seeds ofPlantago
ovata Forssk.(Fam. Plantaginaceae).
Description. Pale buff, brittle flakes, more or less lanceolate,
up to 2 mm long and 1 mm wide at the centre, much broken into
smaller fragments; many ofthe flakes having a small, brownish,
oval spot, about 0.8 to 1.0 mm long, in the centre; the material
swells rapidly in water, forming a stiffmucilage.
Identification
When mounted in cresol and examined under a microscope,
the particles are found to be transparent and angular, the edges
straight or curved and sometimes rolled. They are composed
ofpolygonal prismatic cells with four to six straight or slightly
curved walls; the cells vary in size in different parts of the
seed coat, from about 25 mm to 60 mm at the summit of the
seed, that is, near and over the brown spot, to 25 mm to
100 mm for the remainder ofthe epidermis except at the edges
ofthe seed, where the cells are smaller, about 45 mm to 70 mm.
When mounted in ethanol (95 per cent) and irrigated with
water, t4e mucilage in the outer part of the epidermal cells
swells rapidly and goes into solution, while the two inner
Kalmegh consists of the dried aerial parts, mainly stems and

leaves, of Andrographis paniculata Nees. (Fam,
Acanthaceae).
Kalmegh contains not less than 1.0 per cent w/w of
andrographo1ide, calculated on the dried basis.
Description.Taste, intensely bitter.
2513
KALMEGH
IP 2010
Identification
A. Macroscopic -
Mixture of crisp, dark green-coloured

broken leaves and quadrangular stems; leaves brittle. Stem
fracture short, fibrous.
B. Microscopic - Stems quadrangular with collenchyma

strands at angles and on side; small acicular crystals ofcalcium
oxalate present in pith and cortex. Trichomes 1-3 celled,
glandular hair disc-shaped and multicellular.

cool and filter. Combine all the filtrates and concentrate to
lOml.
Reference solution. Reflux 0.5 g of kalmegh RS with 50 ml
the filtrates and concentrate to 5 ml.
and examine in ultraviolet light at 254 Dill and 365 Dill, spray
with methanolic sulphuric acid (20 per cent, v/v). Heat the
ofthe reference solution.
Tests
by Method 1.

andrographolide RS in methanol.
- mobile phase: 65 volumes of methanol and 35 volumes
of water,
Calculate the content of andrographolide.
Kalmegh Dry Extract

Kalmegh Dry Extract is obtained by extracting Kalmegh with
aqueous ethanol or ethanol or any other suitable solvent.
Kalmegh Dry Extract contains not less than 90.0 per cent w/w
and not more than 120.0 per cent w/w of the labelled amount
of andrographolides (sum of andrographolide, neoandrographolide and andrograpanin) The content of 14-deoxy11,12-didehydroandrogapholide content shall not be more than
one sixth of the amount of andrographolide.
Description. A light green to dark green powder.
Identification
Total ash (2.3.19), Not more than 15 per cent.

Assay: DefeItrline by liqtlidchrbmatography(2:4:14): ....

Test solution. Reflux about 2.5 g of the coarsely powdered


with 50 ml methanol and filter.
andrographo1ldeJISinliieth7inol.
Apply to the plate 10 III of each solution as
10 mm
2 mll. Allow the mobile phase to rise 8 cm. Dry the plate in air
and examine in ultraviolet light at 254 Dill and 365 Dill and also
2514
KUNDURU
IP 2010
under day light. Spray the plate with methanolic sulphuric

acid (20 per cent) and heat at 120 for 10 minutes. The
chromatogram obtained with test solution shows a band
corresponding to the band obtained using reference solution
indicating the presence of andrographolide.
Calculate the content of l4-deoxy-ll, l2-didehydroandrographolide and andrographolides in the extract.

Calculate andrographolide by summing the peak areas of
andrographolide, neo-andrographolide, and andrograpanin.
Tests
Acid insoluble ash (2.3.19). Not more than 3.0 percent.
Kunduru
Loss of drying (2.4.19). Not more than 5.0 percent.
Sallaki Gum; Gum of Boswellia serrata

contamination test.

examination or quantity equivalent to 20 mg of
andrographolide in methano~ by gently heating, make it up to
100.0 ml and filter.
andrographolide RS in methanol.
14-deoxy-ll,12-didehydroandrographolide RS in methanol.
orthophosphate in 500 ml of water and 0.5 ml of
orthophosphoric acid, dilute to 1000 ml with water,
B. acetonitrile,
below,
Time
Mobile phase
Mobile phase
(in min.)
(per cent v/v)
(per cent v/v)
95
55
45
25
20
80
30
95
Kunduru contains not less than 1.0 per cent w/w of total 11keto-~-boswellic acid and acetyl-ll-keto-~-boswellicacid,
Description. Translucent, brittle, whitish yellow substance,
in roundish, club-shaped, pear-shaped, or irregular tears.
Identification
A. Macroscopic - Fracture dull. Slightly sticky to touch;
odour, balsamic; taste slightly mucilaginous, bitter and
aromatic.
Mobile phase. A mixture of7 volumes of hexane and 3 volumes

of ethyl acetate.
more than 2.0 per cent. The relative retention time with respect
to andrographolide, for neo-andrographolide is about 1.2 and
for andrograpanin is about 1.6.
Kunduru is the gum-resin from Boswellia serrata Roxb. (Fam.

Burseraceae).
Test solution. Reflux I g ofcoarsely powdered substance under

examination with 50 ml methc;mol on a boiling water-bath for
30 minutes, cool and filter. Evaporate the filtrate to dryness
and dissolve the residue in 10 ml of methanol.
Reference solution. Reflux 1 g ofcoarsely powdered lamduru
RS with 50 ml methanol on a boiling water-bath for 30 minutes,
2515
KUNDURU
IP 2010
cool and filter. Evaporate the filtrate to dryness and dissolve

the residue in 10 ml of methanol.
Calculate the sum ofthe contents of ll-keto-p-boswellic acid

and acetyl-ll-keto-p-boswellic acid.
Apply to the plate 10 Jll ofeach solution as bands 10 rom by 2

and examine in ultraviolet light 254 urn and 365 urn, spray with
methanolic sulphuric acid (1 0 per cent, v/v). Heat the plate at

Kutki
Picrorhiza kurroa
Tests
9

0.2g.

further with methanol till the last extract tums colorless, cool
Kutlci consists of dried roots of Picrorhiza lalrroa Royle ex

Benth.(Fam. Scrophulariaceae).
Kutki contains not less than 5 per cent w/w ofkutkin, calculated
on the dried basis.

ll-keto-p-boswellic acid RS in methanol.
Description. Rhizomes are sub cylindIical, straight or slightly

curved, externally greyish with wrinkled surfaces, circular scars
of roots and bud scales, with cork exposed at places.

acetyl-ll-keto-p-boswellic acid RS in methanol.
Identification
octadecylsilane bonded to porous silica (5 l..un),
mobile phase: 90 volumes of methanol and 10 volumes
ofa mixture containing 5 ml of acetonitrile and 95 ml of
water, adjusting the pH to 2.8 with dilute
Inject the reference solution (a) and (b). The test is not valid
unless the relative retention times are about 0.65 for ll-keto-
and 1.0 foracetyr.:n~kao,:;p~boswelIic acid
P~oo~swellicacid
arid thefelativestandard deviation for theteplicateihjectioIiS

Inject the test solution.
A. Macroscopic - 3-6 cm long and about 1 cm thick, subcylindrical, straight, grayish brown, wrinkled roots. Odour is
pleasant and tastes bitter.
B. Microscopic - There is well-developed periderm. About
7-10 layers of cork cells are seen. Cells ofphelloderm loosely
arranged.

Mobile phase. A mixture of 7.5 volumes of ethyl acetate,

2.2 volumes of methanol and 0.1 volume glacial acetic acid.
Testsolution.Refluxl.. g.ofcoarselypowdered.substance~unde r
examiIJatiQI), withSQml ofm?thm1Q! forI Smj!1lJt~~,c.gQLl!n~d
filter. Reflux the residue fmther with 50 ml of methanol, cool
and filter. Combine both the filtrates and concentrate under
vacuum to dryness. Extract the dried residue with 50 ml of
2516
LASUNA
IP 2010
methanol at 50 for 10 minutes, filter the solution and use the

filtrate for analysis.
Apply to the plate 20 )..ll ofeach solution as bands 10 rom by 2

100 for 10 minutes and examin in day light. The
Garlic; Allium sativum bulb
Calculate the content ofkutkin.

Reference solution. Reflux 5 g of leutlei RS with 50 ml of
. by insects and rodents.
further with 50 ml of methanol, cool and filter. Combine both
the filtrates and concentrate under vacuum to dryness. Extract
the dried residue with 10 ml of methanol at 50 for 10 minutes,
filter the solution and use the filtrate for analysis.
Lasuna
Tests
by method I.
Total ash (2.3 .19). Not more than 6.0 per cent.
Lasuna consists of the fresh or dried compound bulbs of

Allium sativum Linn. (Fam. Liliaceae).
Lasuna contains not less than 0.2 per cent w/w of alliin,
Description. Bulbs made up of cloves and is wrapped in a
white papery sheath with pungent taste and odour
Identification
A. Macroscopic - Each bulb has several cloves which are

arranged in concentric rings and enclosed in a shining white
or pinkish papery envelope. The cloves are attached to a flat,
Reference solution. A 0.1 per cent wN solution of leut/an RS circular hard disc with numerous thin wiry roots from its
in methanol.
underside and short, cylindrical out growth from the upper
surface. Each clove is ovoid and further covered by papery
sheath with a tail like structure at one and opposite to its
attachment. The cloves in the outer ring are loose and white in
octadecylsilane bonded to porous silica (5 )..lm),
colour where as cloves in the inner ring are adherent and pale
mobile phase: 83 volumes of 1 per cent v/v.
pinkish in colour.' Each clove covered with a white scale leaf
orthophosphoric acid in water and 17 volumes of
and a pinkish white epidermis, easily separated from the solid
acetonitrile,
portion. In the middle of the bulb a hollow, cylindrical, linear
remnant of the scape is seen.
B. Microscopic
The protective leaf contains an epiderniis
injection volume. 20 )..l1.
enclosing a mesophyll free from chlorophyll. The outer
Inject the reference solution. The relative standard deviation epidermis consists of lignified sclereid cells of thick, pitted
walls, elongated, covered with thin cuticle. The cortical cells

examination in 25.0 ml of methanol, filter.
2517
IP2010
LASUNA
are thick walled, non lignified, tending to collapse on maturity,

isodiametric and contain purple pigments. The vascular
bundles consist of lignified spiral and annular vessels. The
storage leaves show an outer epidermis of thin, delicate cells
ofvariable shape. Stomata are present on the outer epidermis
only at extreme tip near the base of the foliage leaves.The
mesophyll consists ofswollen storage parenchyma cells filled
with fme granular reserve material.
Mobile phase. A mixture of 3 volumes of butyl alcohol,

1 volume of n-propyl alcohol, 1 volume ofglacial acetic acid
and 1 volume of water.
Test solution. Reflux 1 g ofsubstance under examination with
20 ml of methanol (50 per cent) for 10 minutes, cool and filter.
Reflux the residue with another 20 ml of methanol (50 per
cent), cool and filter. Combine all the filtrates and concentrate
under vacuum to 5 ml.
Reference solution. Reflux 1 g of lasuna RS with 20 ml of
methanol (50 per cent) for 10 minutes, cool and filter. Reflux
the residue with another 20 ml of methanol (50 per cent), cool
vacuum to 5 m!.
with 0.2 per cent w/v solution of ninhydrin in mixture of 95
volumes of butyl alcohol and 5 volumes of 2 M acetic acid.
Heat the plate at 100 to 105 for about 10 minutes and examine
in day light. The chromatographic profile ofthe test solution
is similar to that ofthe reference solution.

Test solution. Weigh accurately about 2 gofthe freshly peeled

substance under examination. Add 15 ml of hot water and
grind in a porcelain mortor and filter. Grindthe residue further
with 2 x 15 ml ofhot water and filter. Combine all the filtrates
and make up to volume 50 ml with distilled water.
Reference solution. A 0.004 per cent w/v solution of alliin RS
in water.
- mobile phase: 0.1 per cent v/vphosphoric acid prepared
by diluting 1ml ofphosphoric acid to 1000 ml with water,
Inject the test and reference solution.
Calculate the content of alliin.
Lavang
Syzigium aromaticum
D. Transfer about 10 g of garlic bulbs that have been cut into

small pieces to a beaker, add 10 ml of 1 M sodium hydroxide
and 10 ml of water, heat the beaker in a boiling water for
10 minutes, cool and filter. To 2 ml ofthis filtrate add few drops
of freshly prepared solution of sodium nitroprusside.
Appearance ofa red or orange red colour indicates the presence
of sulphur containing compounds in the sample.
9
8
7
6
Tests

Heavymefals (2.3.n): LO gcompliesWithtnelimit test for
heavy metals; Method B (20 ppm).
Liiviiiig consistsofdriea-floweroudsofSyzigiulrfCi;'OmaticuiiI
(L.) Merr. & L.M. Perry. (Fam.Myrtaceae}-
Loss on drying (2.4.19). Not more than 65.0 per cent for fresh
bulbs, determined on 5 g by drying in an oven at 105.
Lavang contains not less than 7.0 per cent w/w of eugenol,
calculated on the dry basis.
2518
MALT EXTRACT
IP 2010
Test solution. A 0.5 per cent w/v of substance under

examination in methanol.
Description. Aromatic odour and spicy taste.
Identification
A. Macroscopic-The flower bud is reddish brown in colour
and consist of quadrangular stalked portion, the hypanthium,
surrounded by four lobes of sepals.
B.Microscopic-Epidermis
followed
by
thick
cuticle,characteristic zone of bilateral vascular bundles,
numerous schylogenous oil gl!mds present beneath the
epidermis. Central collumella and air spaces or lacuna in
parenchyma regeion.

volumes of ethylacetate.
Test solution. Reflux 2.0 g ofthe coarsely powdered substance
under examination with 50 ml dichloromethane for 15 minutes,
50ml dichloromethane, coo1and filter. Combine all the filtrates
and concentrate under vacuum to 25 ml.
Lavang RS with 25 ml dichloromethane for 15 minutes, cool
and filter. Reflux the residue further for two times with 50 ml
dichloromethane, cool and filter. Combine all the filtrates and
and examine in ultraviolet light at 254 mn. Spray the plate with
a vanillin sulphuric acid reagent. Heat the plate at 105 for 5
minutes and examine the plate at 365 mn and in day light. The
Reference solution. A 0.04 per cent w/v of eugenol RS in

methanol.
a stainless steel 30m xO.53mm capillary column
temperature:
column.70 for 1minute, then increased to 100 at a rate
of 10 per minute, then increased to 200 at a rate of20
per minute and maintained at this temperature for 2
minutes,
inlet port. 220,
detector. 250,
flow rate 1.5 ml per minute ofthe carrier gas.
Inject reference solution and the test solution.
Calculate the eugenol content in the substance under
examination using the ratios ofthe area ofpeak corresponding
to eugenol to the area of the peak obtained with test solution
and the reference solution.
Storage. Store protected from light, moisture and against
attack by rodents and insects.
Malt Extract
Malt Extract is a product obtained by extracting malted grains
of cereals (barley, cholam or wheat) with water at a suitable
temperature and evaporation of the strained liquid until a
viscous product is obtained. Itmay be mixed with 10 per cent
by weight ofGlycerin.
Malt Extract contains nitrogen equivalent to not less than
4.0 per cent w/w ofprotein.
Tests
Description. A sweet, viscous, light brown liquid; odour,

pleasant and characteristic.
Foreign organic matter (2.6.1 ).Not more than 2.0 per cent.
Tests
Ethanol soluble extractive (2.6.2). Not less than 7.0 per cent.
Water soluble extractive (2.6.3). NotJess than 20.0 per cent
by Method 1.
Heavy metals (2.3.13). LOg complies with the limit test for
heavy metals, Method B (20ppm):
Microbial contamination (2,2.9). Complies with the microbial

Arsenic (2.3.10). Dissolve 10.0 g in 10 ml of water, add 10ml
of brominated hydrochloric acid, allow to stand for 5 minutes
and remove the excess ofbromine with a few drops ofstannous
chloride solution AsT. The resulting solution complies with
'
Lipase. To 95 ml of water add 6.5 ml of triacetin and 0.2 mlof
a 0.1 per cent w/v solution of bromocresol purple, neutralise
with 0.5 M sodium hydroxide and add sufficient water to
produce 110 ml. Place 50 ml of this solution in each of two
large test-tubes (20 cm X 3 cm)AandB keptina water-bath at
30 1. Insert in each tube a rubber stopper having two
holes, one for the tip of a burette and the other for a short
2519
MANDUKAPARNI
IP 2010
glass tube through which passes a thread operating a glass

stirring coil. Stir the contents of the tube untilthey attain the
temperature of the water-bath. Prepare a solution of 5.0 g of
the substance under examination in 10 ml of water. To tube A
add 1 ml of this solution; to tube B add 1 ml of this solution
after previous boiling. Adjust and maintain the pH ofthe two
tubes to between 6.2 and 6.4 by the dropwise addition of 0.05
M sodium hydroxide, stirring frequently. After 6 hours, the
difference between the amounts of 0.05 M sodium hydroxide
added to the tubes is not more than 1.0 ml.
Assay. Weigh accurately about 5.0 g into a 200-rnllong-necked
flask and carry out the determination of nitrogen, Method A
(2.3.30), using 0.05 M sulphuric acid instead of 0.1 M
sulphuric acid.
1 ml of 0.05 Msulphuricacidis equivalentto 0.001401 g ofN
and multiply the result by 6.25 to obtain the protein content.
B. Microscopic - Animocytic stomata on both surfaces,

more on lower surface; petiole epidermis has calcium oxalate
prisms; vascular bundles seven arranged in 'U' shape,
parenchyma cells contain simple and compound starch
granules.

32 volumes of glacial acetic acid, 12 volumes of methanol
and 8 volumes of water.
10 ml under reduced pressure.
Reference solution. Reflux 1 g of mandukaparni RS with

50 rnl methanol for 15 minutes, cool and filter. Reflux the residue
Mandukaparni
Apply to the plate 10 )ll ofeach solution as bands 10 rom by 2

Gotu Kola; Centella asiatica
Tests
by Method I.
Mandukaparni consists of the dried aerial parts of Centella

asiatica (Linn.) Urban. (Fam. Umbelliferae).
Mandukaparni contains not less than 0.5 per cent w/w of
asiaticoside, calculated on the dried basis.
Description. A green to greenish yellow in colour, taste,
slightly bitter and sweet.
Identification
Assay. Detelmine by liquid chromatography (2.4.14).
Test sdutiQIJ. RdhlJI. alwut 19_Qf the.-J<9arseJY_P-QW<;l~.l'ed

and filter. Combine all the filtrates and concentrate to 100.0 rnl.
_ .. __ ...
A. Macroscopic - A slender trailing herb with rooted nodes

and internodes. Leaves with elongated petioles and sheathing
leafbases; lamina reniform with crenate margin.
2520
. __
_.____
__
_ ... _ _ _ . _
__
_ _ _ ....
MANJISTHA
IP 2010

asiaticoside RS in methanol.

octylsilane bonded to porous.silica (10 J..1m),
of water,
injection volume. 20 J..1l.

of ethyl acetate and 0.5 volumes glacial acetic acid.
Calculate the content of asiaticoside.
Test solution. To 4 g of coarsely powdered substance under

examination, add 100 ml of methanol, reflux for 15 minutes,
ml methanol, cool and filter. Combine all the filtrates and
Reference solution. To I g of coarsely powdered manjistha,
add 100 ml of methanol, reflux for 15 minutes, cool and filter.
Reflux the residue further for two times with 50 ml methanol,
vacuum to 25 ml.
Manjistha
Apply to the plate 20 J..11 of each solution as bands 10 mm by 2

the plate with anisaldehyde sulphuric acid reagent. Heat the
plate at 100 for 5 minutes and examine in day light. The
Indian Madder; Rubia cordifolia
Tests

9

by method I.
7
6

Loss on drying (2.4.19). Not more than 5 per cent, detennined
o
Manjistha consists of dried stem of Rubia cordifolia
Linn.sensu Hook. (Fam. Rubiaceae).
Manjistha contains not less than 0.02 per centw/w ofrubiadin,
Description. A cylindrical, slightly flattened, wiry pieces of
brown to purple coloured root with mild bitter taste.

under examination, add 100 ml of methanol, reflux on a waterbath 15 minutes, cool and filter. Reflux the residue 2 x 50 ml
concentrate under vacuum to 100.0 m!.
Identification
Reference solution. A 0.002 per cent w/v solution of rubiadin

RS in methanol.
A. Microscopic - Exfoliating cork, consisting of4-12 or more

layered radially arranged, thin walled cells.
a stainless steel column 25 em x 4.6 rom packed with
octadecylsilane bonded to porous silica (5 J..1m),
dissolving 0.136 g of potassium di-hydrogen
B. Macroscopic ~ Stem, slender, cylindrical, wiry and about

0.5 cm thiclc. It is brown to purple coloured and with
longitudinal cracks.
2521
MANJISTHA
IP 2010

B. acetonitrile,
below,
- injection volume. 20 J..tl.
Time
Mobile phase A
(per cent v/v)
(in min.)
65
5
50
15
20
30
20
35
50
40
65
Mobile phase B
(per cent v/v)
35
50
80
80
50
35
Identification
A. Macroscopic - The fruits are globular or oblong, 4-6 mm
in diameter. The outer cover is blackish brown, with raised
reticulated wrinkles. One seeded, seeds are white and hollow.
B. Microscopic - The fruit has a well differentiated pericarp,
testa and perisperm. Isolated, tangentially elongated oil cells
are in the outer region of the mesocarp. Endocarp has beaker
shaped stone cells and numerous polyhedral masses of starch
grains. Testa has a single layer of yellow colored cells.


of ethyl acetate and 10 volumes of diethyl ether.

Calculate the content of rubiadin.

under examination, add 50 ml of methanol and reflux for
times with 75 ml of methanol, cool and filter. Combine all the
Reference solution. To 2 g of the maricha RS, add 50 ml of
methanol and reflux for 15 minutes, cool and filter. Reflux the
residue further for two times with 75 ml of methanol, cool and
filter. Combine all the filtrates and concentrate under vacuum
t050ml.
Apply to the plate 10 J.!l ofeach solution as bands 10 mm by 2
and examine in ultraviolet light at 254 nm and 365 om, spray
the plate with vanillin sulphuric acid reagent. Heat the plate
at 100 for 5-10 minutes and examine in day light. The
Maricha
Black pepper; pepper; Piper nigrum
9
Tests

method I.
Maricha consists of the unripe fruits of Piper nigrum Linn.

(Fam. Piperaceae).

Maricha contains not less than 2.5 per cent w/w of piperine,

ciilCl.ihited on the drieu basis.
Assay.
Description. Fruits are globular or oblong. They have a blackish

brown cover, with raised reticulated wrinkles. The odour is
aromatic and the taste is strong and pungent.

under examination, add 50 ml of methanol, sonicate for
3 minutes and heat on a boiling water bath for 15 minutes, cool
2522
I>eterrnme by Hqui(lclrr()niatognlphy (2.4. 14): ..
METHI
IP 2010
and dilute to 100.0 ml with methanol and filter. Dilute further if

necessary.

piperine RS in methanol.
- mobile phase.A. a buffer solution pH 2.5 prepared by
B.acetonitrile,
below,
- spectrophotometer set at 345 Dill,
Time
(per centv/v)
o
95
5
18
55
45
25
20
80

Inject the test solution and reference solution. The relative
retention time ofpiperine is 1.
~alculate
a reflux condenser and boil for 2 hours. Cool, add 30 ml of

water and warm on a water-bath for 15 minutes with occasional
shaking. Transfer the contents of the flask to a separating
funnel, reject the water layer and wash the remaining oil with
water until the last washing no longer shows acid reaction.
Dry the resulting oil by shaking with 2 g of anhydrous sodium
sulphate, allow to stand for 30 minutes and filter through a
dry filter paper. Weigh accurately about 1.5 g of the dry
acetylated oil, add 3 ml of ethanol (95 per cent) and 0.1 mlof
phenolphthalein solution and dropwise, 0.5 M ethanolic
potassium hydroxide until the solution acquires a faint pink
colour. Add a further 20.0 ml of the alkali, attach a reflux
condenser and boil for 1 hour on a water-bath. Cool, add 1 ml
of phenolphthalein solution and titrate the excess of alkali
. with 0.5 M hydrochloric acid: Repeat the operation with the
same quantities of the same reagents in the same manner
without the oil and calculate the amount oftotal menthol from
the following expression.
.
(a-b)x7.813
Total menthol (per cent) = ----''-----'----S -(a-b)xO.021
where, S is the amount, in g, ofthe acetylated sample taken, a
is the amount, in ml, of 0.5 M hydrochloric acid consumed in
the blank test, and b is the amount, in ml, of 0.5 M
hydrochloric acid consumed in saponification of the
acetylated oil.
the content ofpiperine.

Methi
Trigonella foenum-graecum
Mentha Oil
9
Mentha
Mentha Oil is the volatile oil distilled with steam from various
species of Mentha (Fam. Labiatae) and rectified ifnecessary.
7
6
Mentha Oil contains not less than 50.0 per cent w/w of total
menthol, CIOHzoO.
Description. A colourless or yellowish, clear liquid; odour,
characteristic and pleasant.
Tests
Acidity or alkalinity. A solution of 1 ml in 3.5 ml of ethanol
(70 per cent) is neutral to litmus.
Weight per ml (2.4.29). 0.892 gtoO.910 g.
Specific optical rotation (2.4.22). -18.00 to-33.0.
Methi is dried, ripe seeds of Trigonella foenum-graecum L.

(Fam.Fabaceae).
Assay. Place about 10.0 gin an acetylation flask, add 10 mlof

acetic anhydride and 1 g of anhydrous sodium acetate, attach
Methi contains not less than 0.1 per cent w/w of trigonelline
on dry basis.
2523
METHI
IP 2010
Description. The seed is hard, flattened, brown or reddishbrown and more or less rhomboidal with rounded edges. 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. They have a strong
aromatic characteristic odour.
Identification
A. Macroscopic - Seed is rhomboidal with rounded edges.
It is 3 mm to 5 rom long, 2 rom to 3 rom wide and 1.5 rom to 2 rom
thick. The widest surfaces are marked by a groove that divides
the seed into two unequal parts. The smaller part contains the
radicle; the larger part contains the cotyledons.
B. Reduce to a powder. The powder is yellowish-brown.

Examine under a microscope using chloral hydrate solution.
The powder shows fragments of the testa in sectional view
with thick cuticle covering lageniform epidermal cells, with an
underlying hypodermis of large cells, narrower at the upper
end and constricted in the middle, with bar-like thickenings of
the radial walls; yellowish-brown fragments of the epidermis
in surface view, composed of small, polygonal cells with
thickened and pitted walls, frequently associated with the
hypodermal cells, circular in outline with thickened and closely
beaded walls; fragments ofthe hypodennis viewed from below,
composed ofpolygonal cells whose bar-like thickenings extend
to the upper and lower walls; parenchyma of the testa with
elongated, rectangular cells with slightly thickened and beaded
walls; fragments of endosperm with irregularly thickened,
sometimes elongated cells, containing mucilage.
Mobile phase. A mixture of 30 volumes of water and 70

Test solution. To 1.0 g of the powdered substance under
examination, add 5.0 ml ofthe methanol. Heat on a water-bath
at 65 for 5 minutes, cool and filter.
Reference solution. Dissolve 3 mg of trigonelline
hydrochloride in 1.0 ml of methanol.
with potassium iodobismuthate solution. Heat the plate at
of the reference solution. It also shows in its upper half, a
broad light brownish-yellow zone (triglycerides).
Tests
Transfer 1g to a 100 ml stoppered cylinder containing 90 ml of

water, shake well for 30 seconds and allow to stand 24 hours,
shaking gently on three occasions during this period. Add
sufficient water to produce I00 ml, mix gently for 30 seconds,
avoiding the entrapment of air, allow to stand for 5 hours and
measure the volume of mucilage. Repeat the determination
three times. The average of four determinations is not less
than40ml.
Ash (2.3.19). Not more than 5.0 per cent.
Neem
Azadirachta indica
9
8
7
6
5
4
3
2
1
o
Neem consist of dried leaves of Azadirachta indica A.Juss.
(Fam. Meliaceae).
Neem leaves contain not less than 1.0 per cent w/w of the
stated amount of rutin, calculated on the dry basis.
Description. Characteristic odour and bitter taste.
Identification
A. Macroscopic-The leaves dark green, the petioles are
short.The shape of mature leaflets is more assymetrical and
their margins are dentate with the exception of the base of
their basiscopal half, which is very strongly reduced and
cuneate or wedge-shaped leaves.
B. Microscopic-Upper and lower epidermis, exhibiting two

layers ofpalisade cells below the upper epidermis. The spongy
parenchyma with intercellular spaces abundant on the border
line of palisade cells. Midrib shows numerous
ConeiiCliym~atouscen1n)elowi.Ipperand~loweTepidermis:A
Swelling Power. Not less than 6.0, determined on the powdered

substance under examination, determined by the following
method.
characteristic Zone of vascUlar bundles is present.

C. Determine by thin-layer chromatography (2.4.17) coating
2524
OPIUM
IP 2010

volumes of n-butanol ,5 volumes of formic acid and 5
volumes of water.
Inject the reference solution .The test is not valid unless the
more than 2 per cent.

under examination with 100 ml of n-hexane for 2 hours, cool
and filter. Reflux the residue further for 1hour with 100 ml of
methanol, cool and filter. Concentrate under vacuum to 25ml.
Reference solution. Reflux 2.5g ofthe coarsely powdered drug

with 100 ml n-hexane for 2 hours, cool and filter. Reflux the
residue further for Ihour with 100 ml of methanol, cool and
filter. Concentrate under vacuum to 10 ml.
Apply to the plate 1Of.ll of each solution as bands 10mm by 2
Allow the mobile phase to rise 8 cm. Dry the plate in air
and examine in ultraviolet light at 254. Spray the plate with
anisaldehyde sulphuric acid reagent. Heat the plate at 105
for 5 minutes and examine the plate at 365 urn and in day light.
The chromatographic profile oftest solution is similar to that
Calculate the content of rutin.

by rodents and insects.
Opium
Raw Opium; Papaver somniferum
mill.
9
8
7
Tests
Ethanol soluble extractive (2.6.2). Not less than 7.0 per cent.
Water soluble extractive (2.6.3). Not less than 19.0 per cent
by Method I.

heavy metals, Method B (20ppm).
Loss on drying (2.4.19).Not more than 12.0 per cent, determined

under examination with 100 ml of n-hexane for 2 hours, cool
and filter. Reflux the residue further for 1 hour with 100 ml of
methanol, cool and filter. Concentrate under vacuum to 25ml.
Reference solution. A 0.05 percent w/v of rutin RS in
methanol.
stainless steel column 25cm x4.6mm packed with
octadecylsilane bonded to porous silica(5f.lm),
mobile phase:30 volumes of acetonitrile and 70 volumes
of 0.5 per cent formic acid prepared by diluting 5ml of
formic acid to 1000ml with water.
flow rate, 1ml per minute,
injectiion volume.20 f.ll.
Opium is the air-dried latex obtained by incision from the unripe

capsules of Papaver somniferum Linn (Fam. Papaveraceae).
Opium contains not less than 10.0 per cent w/w of morphine,
C 17H I9N03, and not less than 2.0 per cent w/w of codeine,
ClsH2IN03, both calculated on the dried basis.
Description. Masses of various sizes which tend to be soft
and shiny and, after drying, hard and brittle; usually in
somewhat irregularly shaped masses (natural opium) or
moulded into masses of more uniform size and shape
(manipulated opium); colour, blacldsh brown; odour, strong
and characteristic.
Identification
Strip off any covering, cut the substance under examination
into thin slices, ifnecessary, dry at about 60 for 48 hours and
reduce to a powder.
A. When examined under a microscope, a suspension in a
2 per cent w/v solution of potassium hydroxide appears as
granules of latex agglomerated in irregular masses and light
brown elongated filaments. Some fragments of vessels and
rather elongated, refringent crystals are also visible, as well as
2525
OPIUM
IP 2010
a smaller number ofround pollen grains and fragments of

elongated fibres. Hairs of various lengths with sharp points
and a few grains of starch introduced during the handling of
the latex may be present. Fragments of epicarp consisting of
polygonal cells with thick walls defining a stellate lumen may
also be present.

Mobile phase. A freshly prepared mixture of 20 volUmes of

acetone, 20 volumes of toluene, 3 volumes of ethanol (95 per
cent) and 1 volume of strong ammonia solution.
Test solution. Triturate OJ g of the powdered substance with
5 ml of ethanol (70 per cent), add 3 ml of ethanol (70 per
cent), transfer to a 25-ml conical flask and heat in a water-bath
at 50 to 60 for 30 minutes, with stirring. Cool, filter, wash the
filter with ethanol (70 per cent) and dilute the filtrate to 10 ml
Reference solution. Dissolve 2 mg of papaverine

hydrochloride RS, 12 mg of codeine phosphate RS, 12 mg of
noscapine hydrochloride RS, and 25 mg of morphine
hydrochloride RSin ethanol (70 per cent) and dilute to 25 ml
Apply to the plate 20 ).tl of each solution as 20 mm bands.
After development, dry the plate at 100 to 105 for 15 minutes,
allow to cool and spray with potassium iodobismuthate
solution and then with a 0.4 per cent w/v solution ofsulphuric
acid. The chromatogram obtained with the reference solution
shows in the lower part an orange-red or red band (morphine),
above it a similarly coloured band (codeine) and in the upper
part an orange-red or red band (papaverine) and above it a
similarly coloured band (noscapine). The chromatogram
obtained with the test solution shows orange-red or red bands
corresponding to those in the chromatogram obtained with
the reference solution. The chromatogram obtained with the
test solution may also show a dark red band (thebaine) situated
between those due to codeine and to papaverine.
C. To 1 g ofthe powdered substance add 5 ml of water, shake
for 5 minutes, filter and add to the filtrate 0.25 ml offerric
chloride solution; a red colour develops which does not
disappear on the addition of 0.5 ml of 2 M hydrochloric acid.
Chromatographic system as described in the Assay.

The test is not valid unless the capacity factor for thebaine is
at least 3.0 and the number of theoretical plates is at least
3000. Calculate the percentage content of thebaine from the
expression given in the assay.
Total ash (2.3.19). Not more than 6.0 percent, determined on

0.5g.
determined on 0.25 g cut into thin slices, by drying in an oven
at 105 for 4 hours.
Test solution. Suspend about 1.0 g, accurately weighed

substance under examination, cut into thin slices, in 50 ml of
ethanol (50 per cent), mix with the aid of ultrasound for
1 hour, allow to cool, dilute to 100.0 ml with the same solvent
and allow to stand. To 10.0 ml of the supernatant liquid add
5 ml of ammonia buffer pH 9.5, dilute to 25.0 ml with water,
mix, transfer 20.0 ml ofthe solution to a column (about 15 cmX
30 mm) containing 15 g of kieselguhr for column
chromatography and allow to stand for 15 minutes. Elute with
two quantities, each of 40 ml, of a mixture of 85 volumes of
dichloromethane and 15 volumes of 2-propanol, evaporate
the eluate to dryness at 40 at a pressure of2 kPa, transfer the
residue to a 25-ml volumetric flask with the aid ofthe mobile
phase and dilute to volume with the same solvent.
Reference solution. Weigh accurately 0.1 g of morphine
hydrochloride and 25 mg of codeine, dissolve in sufficient of
the mobile phase to produce 25.0 ml and dilute 10.0 ml ofthis
octylsilane bonded to porous silica (5 ).tm), fitted with a
guard column 4 cm x 4.6 mm packed with octylsilane
bonded to porous silica (5 ).tm),
mobile phase: 1.0 g of sodium heptanesulphonate in
420 ml of water, adjusting to pH 3.2 with phosphoric
acid that has been diluted to contain 0.49 per cent w/v
solution of H3P04 (about 5 ml) and adding 180 ml of
acetonitrile,
spectrophotometer set at 280 nID.
Tests
Thebaine. Not more than 3 per cent, calculated on the dried
basis.
Teslsoluiion:UsefheTesfsoliifioiipreparediiitheassay.
Reference solution. Dissolve 25 mg of thebaine in sufficient

mobile phase to produce 25.0 ml and dilute 10.0 ml of this
solution to 100.0 mlwith the mobile phase.
Inject suitable volumes of each solution. The assay is not

valid unless the resolution between the peaks corresponding
to morphine and codeine is at least 2.5. If necessary, adjust
the volume of acetonitrile in the mobile phase. Iiiject the
reference solution six times; The assayisnotvalid unless the
relative standard deviation ofthe peak area for morphine is
2526
OPIUM POWDER
IP 2010
Calculate the percentage content of each alkaloid from the

expression
WI xA z x625xl00
W z xA I x5(lOO-h)
where, WI
weight, in g, ofthe alkaloid used to prepare the

reference solution,
Wz
weight, in g, of the substance under
examination used to prepare the test solution,
AI
area ofthe peak corresponding to the alkaloid
in the chromatogram obtained with the
reference solution,
A z = area ofthe peak corresponding to the alkaloid
in the chromatogram obtained with the test
solution,
h = percentage loss on drying.
For calculation, I mg Of morphine hydrochloride may be taken

as equivalent to 0.759 mg of morphine and I mg of codeine
phosphate may be taken as equivalent to 0.943 mg ofcodeine.
wide, in groups of from one to six, traversing a spongy

parenchyma of thin-walled cells about 40 to 60 rom in either
direction and united by arm-like projections enclosing almost
circular intercellular spaces; fragments of the
sclerenchymatous layer, consisting of thick-walled lignified
brown, rectangular to polyhedral cells in a single layer,
individual cells about 5 to 10 J!m wide and 10 to 30 J!m long;
fragments of mucilage staining in ruthenium red solution.
the plate with silica gel OF 254.

of toluene, 3 .volumes of ethanol (95 per cent) and I volume
Test solution. Triturate 0.10 g of the powdered substance with
5 ml of ethanol (70 per cent), add 3 ml of ethanol (70 per
cent), transfer to a 25-ml conical flask and heat in a water-bath
at 50 to 60 for 30 minutes, with stirring. Cool, filter, wash the
filter with ethanol (70 per cent) and dilute the filtrate to 10 ml
Reference solution. Dissolve 2 mg of papaverine
hydrochloride RS, 12 mg of codeine phosphate RS, 12 mg of
noscapine hydrochloride RS, and 25 mg of morphine
hydrochloride RS in ethanol (70 per cent) and dilute to 25 ml
Opium Powder
Opium Powder is Opium dried at a temperature not exceeding
70, reduced to a fine or moderately fine powder and adjusted
by the addition ofLactose, suitably coloured with Caramel, or
other suitable diluent to contaIn about 10 per cent ofmorphine
and 2 per cent of codeine.
Opium Powder contains not less than 9.5 per cent w/w and
not more than 10.5 per cent w/w ofmorphine, C 17H I9N03, and
not less than 1.9 per cent w/w and not more than 2.1 per cent
w/w ofcodeine, ClsHzIN03, both calculated on the dried basis.
Description. A light brown powder consisting of yellowish
brown or brownish red particles; odour, characteristic.
Identification
A. When examined under a microscope, the residue obtained
after extraction with water, appears as granules of latex
agglomerated in irregular masses and light brown elongated
filaments. Some fragments of vessels and rather elongated,
refringent crystals are also visible, as well as a smaller number
ofround pollen grains and fragments ofelongated fibres. Hairs
ofvarious lengths with sharp points and a few grains ofstarch
introduced during the handling of the latex may be present.
Fragments ofepicarp consisting ofpolygonal cells with thick
walls defining a stellate lumen may also be present and if
powdered cocoa husk is present, the following: brown colour
of the fragments; narrow spiral vessels about 10 to 20 rom
Apply to the plate 20 J!l of each solution as 20 mm bands.

After development, dry the plate at 100 to 105 for 15 minutes,
allow to cool al1d spray with potassium iodobismuthate
solution and then with a 0.4 per cent w/v solution of sulphuric
acid. The chromatogram obtained with the reference solution
shows in the lower part an orange-red orred band (morphine),
above it a similarly coloured band (codeine) and in the upper
part an orange-red or red band (papaverine) and above it a
similarly coloured band (noscapine). The chromatogram
obtained with the test solution shows orange-red or red bands
corresponding to those in the chromatogram obtained with
the reference solution. The chromatogram obtained with the
test solution may also show a dark red band (thebaine) situated
between those due to codeine and to papaverine.
C. To I g ofthe powdered substance add 5 ml of water, shake
for 5 minutes, filter and add to the filtrate 0.25 ml offerric
chloride solution; a red colour develops which does not
disappear on the addition of 0.5 ml of2 M hydrochloric acid.
Tests
Thebaine. Not more than 3.0 per cent, calculated on the dried
basis.
Test solution. Use the test solution prepared in the Assay.
2527
OPIUM POWDER
IP 2010
Reference solution. Dissolve 25 mg of thebaine in sufficient

mobile phase to produce 25.0 ml and dilute 10.0 ml of this

Calculate the percentage content of each alkaloid from the
expression
Chromatographic condition as described in the Assay.

The test is not valid unless the capacity factor for thebaine is
at least 3.0 and the number of theoretical plates is at least
3000. Calculate the percentage content of thebaine from the
expression given in the Assay.
WI
W
where,
0.5g.
z xA I x5(100-h)
WI
Wz =

AI =
Test solution. Suspend about 1.0 g, accurately weighed, of

the substance under examination, cut into thin slices, in 50 ml
of ethanol (50 per cent), mix with the aid of ultrasound for
1 hour, allow to cool, dilute to 100.0 ml with the same solvent
and allow to stand. To 10.0 ml of the supernatant liquid add
5 ml of ammonia buffer pH 9.5, dilute to 25.0 ml with water,
mix, transfer20.0ml ofthe solution to a column (about 15 cmX
30 mm) containing 15 g of kieselguhr for column
chromatography and allow to stand for 15 minutes. Elute with
two quantities, each of 40 ml, of a mixture of 85 volumes of
dichloromethane and 15 volumes of 2-propanol, evaporate
the eluate to dryness at 40 at a pressure of2 kPa, transfer the
residue to a 25-ml volumetric flask with the aid ofthe mobile
phase and dilute to volume with the same solvent.
Reference solution. Weigh accurately 0.1 g of morphine
hydrochloride RS and 25 mg of codeine phosphate RS,
dissolve in sufficient of the mobile phase to produce 25.0 ml
and dilute 10.0 ml of this solution to 100.0 ml with the same
solvent.
xA z x625x100
Az
weight, in g, ofthe alkaloid used to prepare the

reference solution,
weight, in g, of the substance under
examination used to prepare the test solution,
in the chromatogram obtained with the
reference solution,
in the chromatogram obtained with the test
solution,h
percentage loss on drying.
For calculation, 1 mg of morphine hydrochloride may be taken

as equivalent to 0.759 mg of morphine and 1 mg of codeine
phosphate may be taken as equivalent to 0.943 mg ofcodeine.
Papain
Carica papaya
Papain is an enzyme or a mixture ofenzymes obtained from the
juice of the unripe fruit of Carica papaya Linn. (Fam.
Caricaceae). It may contain a suitable diluent such as Lactose.
Papain contains not less than the minimum protease activity
determined under the conditions of the Assay.
Description. A white to light brown, amorphous or slightly
a stainless steel column 25 cm x 4.6 rom, packed with granular powder; odour, characteristic.
octylsilane bonded to porous silica (5 /lm), fitted with a
guard column 4 cm x 4.6 rom packed with octylsilane Tests
bonded to porous silica (5 /lm),
_ mobile phase: 1.0 g of sodium heptanesulphonate in Microbial contamination (2.2.9). 1.0 g is free from Escherichia
420 m1 of water, adjusting to pH 3.2 with phosphoric coli and 10.0 g is free from salmonellae.
acid that has been diluted to contain 0.49 per cent w/v Loss on drying (2.4.19). Not more than 7.0 per cent, determined
solution of H3P0 4 (about 5 ml) and adding 180 ml of on 1 g by drying in an oven at 60 at a pressure not exceeding
acetonitrile,
Assay. Weigh accurately about 0.5 g, triturate with 10 ml of
- spectrophotometer set at 280 urn.
cysteine hydrochloride solution and dilute to 100.0 ml with
Inject suitable volume of reference solution. The test is not water. To 30 ml of water in each oftwo flasks add 15.0 m1 of
valid unless the resolution between the peaks corresponding . . casein solution andmaintain at 60 by heating on a water~~~to~m(;rp1ilnean(rcodeIne~is aTieast-2:S. If nec-essary, ~adjust ~ bath.~ To~thetlfstllasl(liaa:~5~0 n:ironllesolution~ortlie~
thevohimeofacetoniirlle iii themooilepnase: ThefeIative sli5stiiiice u.11def exaiiiiiiiitiori;and .fOthesecond flask-add
standard deviation of the peak area for morphine is not more 5.0 ml of the same solution, previously boiled for 2 minutes
than 1.0 per cent.
and cooled. Maintain the solutions at 60 for 30 minutes, cool
2528
PEPPERMINT OIL
IP 2010
rapidly to room temperature and add to each flask 0.75 ml of

phenolphthalein solution and 10 ml offormaldehyde solution,
previously neutralised to phenolphthalein solution. Titrate
both solutions with 0.1 M sodium hydroxide to the same
definite pink colour; the difference between the two titrations
is not less than 4.5 ml.
Labelling. The label states the name of any added substance.
Peppermint Oil
Peppermint Oil is obtained by steam distillation from the aerial
parts of the flowering plant of Mentha piperita L. and M.
arvensis var. piperascens (Fam. Labiatae).
. Peppermint Oil contains not less than 4.5 per cent w/w and
not more than 10.0 per cent w/w ofesters, calculated as menthyl
acetate, C 12H 22 0 2, not less than 44.0 per cent w/w of free
alcohols, calculated as menthol, C IOH 200, and not less than
15.0 per cent w/w and not more than 32.0 per cent w/w of
ketones, calculated as menthone, CIOH1SO.
Description. A colourless, pale yellow or pale greenish yellow
liquid; odour, characteristic; taste, characteristic followed by
sensation of cold.
Identification
plate with silica gel G F254.

Test solution. Dissolve 1 g of the oil under examination in
100 ml of toluene.
Reference solution. Dissolve 50 mg of(-)menthol RS, 20 III of
cineole RS, 10 mg of thymol RS and 10 III of menthyl acetate
RS in sufficient toluene to produce 10 ml.
Apply to the plate 20 III oftest solution and 10 III ofreference
solution as bands 20 mrn by 3 mrn. After development, dry the
plate in air until the odour of solvent is no longer detectable
and examine in ultraviolet light at 254 urn. In the chromatogram
obtained with test solution there are no quenching bands at
Rf values slightly lower than that of the faint band due to
thymol in the chromatogram obtained with reference solution
(carvone and pulegone). Spray the plate with anisaldehyde
solution and examine in daylight after heating at 105 for
5 minutes. The chromatogram obtained with reference solution
shows, in order of increasing Rf value, an intense blue to
violet band (menthol) in the lower third, a violet-blue to brown
band (cineole), a pink band (thymol) and a violet-blue band
(menthyl acetate). The chromatogram obtained with test

solution shows an intense band corresponding to menthol, a
band corresponding to cineole, a violet-blue band
corresponding to menthyl acetate in the middle of the
chromatogram and at a slightly lower Rfvalue a greenish band
(menthone). The chromatogram obtained with test solution
does not show intense greyish green or faint bluish grey bands
at Rf values between those of cineole and thymol in the
chromatogram obtained with reference solution (carvone,
pulegone, isomenthone). The chromatogram obtained with
test solution shows an intense reddish violet band near the
solvent front (hydrocarbons) and a brownish yellow band
(menthofuran) at a slightly lower Rfvalue; other less intensely
coloured bands may also be seen.
Tests
Acidity. To 2 g add 0.25 ml of phenolphthalein solution; not
more than 0.1 ml of 0.5 M ethanolic potassium hydroxide is
Optical rotation (2.4.22). -10 to -30.
Dimethyl sulphide. Distil 25 ml, collect the first 1 ml of the
distillate and carefully superimpose it on 5 ml ofa 6.5 per cent
w/v solution of mercuric chloride; no white film is produced
within 1 minute at the interface of the two liquids.
Fixed oils and resinified volatile oils. Allow 0.05 m1 to fall on
a filter paper. The oil evaporates completely within 24 hours
without leaving a translucent or greasy mark.
Assay. For esters - To 2 g in a borosilicate glass flask add
2 ml of ethanol (90 per cent) and 0.25 ml ofphenolphthalein
solution, neutralise with 0.5M ethanolic potassium
hydroxide, add an additional 25.0 m1 and a little pumice powder
or a few pieces ofporous pot and heat under a reflux condenser
on a water-bath for 30 minutes. Add 1 ml ofphenolphthalein
solution and immediately titrate with 0.5 M hydrochloric acid.
Repeat the operation without the substance under examination.
The difference between the titrations represents the volume
of alkali required to saponifY the esters.
1 ml of 0.5 M ethanolic potassium hydroxide is equivalent to
0.09915 g ofesters, calculated as menthyl acetate, CI2H2202
Forfree alcohols - To 1 g in a dry, 150-ml acetylation flask,

add 3 ml ofa mixture oD volumes ofpyridine and I volume of
acetic anhydride. Determine the weight of the acetylation
mixture to the nearest mg, keeping the flask closed while
weighing. Boil under a reflux condenser in a water-bath for
3 hours, maintaining the water level 2 to 3 cm above the level
of the liquid in the flask throughout. Remove the flask from
the water-bath and add 50 ml of water through the condenser,
2529
PEPPERMINT OIL
IP 2010
remove the condenser and wash the walls of the flask with
10 ml of water. Allow to stand for 15 minutes and titrate with
0.5 M sodium hydroxide using 1 ml of phenolphthalein
solution as indicator. Repeat the operation without the
titrations represents the volume ofsodimn hydroxide required.
free alcohols, calculated as menthol, C IOH2o O. Ifthe quantities
ofacetic anhydride in pyridine used in the two determinations
differ by more than 5 mg, adjust the volume of alkali used in
the second titration by multiplying with alb where a is the
weight, in g, of acetic anhydride in pyridine used in the fIrst
determination and b is the weight, in g, of acetic anhydride in
pyridine used in the second test.
Pippali, Large contains not less than 1.0 per cent w/w of
piperine, calculated on the dried basis.
Description. The spikes are blacking green to green in colour,
cylindrical, erect and blunt. It has pungent taste and the odour
is aromatic and characteristic. The spikes are 2 to 4 cm long
and 0.4-0.7 cm in diameter.
Identification
A. Macroscopic - The spikes are blackish green to green in
colour, surface rough. The spikes bear bracts and numerous
small fruits sunle in solid spike.
Forketones- To 2 g add 25 ml ofa 5.0 per centw/v solution

of hydroxylamine hydrochloride in ethanol (95 per cent),
heat on a water-bath for 1 hour, allow to cool, add about 1 mg
of methyl orange and titrate with 0.5 M ethanolic potassium
hydroxide until an orange-yellow colour is obtained. Repeat
the heating for further periods of 1 hour until, after cooling,
not more than 0.1 ml of 0.5 M ethanolic potassium hydroxide
is required to neutralise the solution.
B. Microscopic - The penduncle is circular in outline,

trichomes are absent, epidermis is a single layer oftangentially
elongated cells. Vascular traces are in fIve to six bundles. Pith
is star shaped. Endosperm cells are larger, cells are packed
with abundant starch grains and oil droplets. Crystals are
present in some of the cells, they appear unequal in size. The
epicarp is a single layer ofthick walled cells having greenish
content. Endocarp is wavy in outline. Mostly, endocarp and
seed coat are fused together to form a deep zone with hyaline
content in outer layer and orange red (brown) inner region.
1 ml of 0.5 M ethanolic potassium hydroxide is equivalent to

0.07710 g ofketones, calculated as menthone, CIOH1SO.

Storage. Store protected from light and moisture, in well-fIlled

containers.

Pippali, Large

cool and fIlter. Reflux the residue further for two times with
75 ml of methanol, cool and fIlter. Combine all the fIltrates and
Long Pepper; Catkins (Big); Piper retrofractum

pippali, Big RSwith 50-75 rnl of methanol for 15 minutes, cool
and fIlter. Reflux the residue further for two times with 75 ml of
methanol, cool and fIlter. Combine all the filtrates and
9
8
7
6
5
4
3
2
1
I
Apply to the plate 10 I-LI ofeach solution as bands 10 mm by 2

the plate with vanillin sulphuric acid reagent.. Heat the plate
chromatographic profIle of the test solution is similar to that
11~1
Pippali, Large consists of the fruiting

of Piper
retrofractum Vahl.(Syn. P. latifolium Hunter, (Fam.
Piperaceae).
2530
PIPPALI, SMALL
IP 2010

by Method I.
Pippali, Small
Small Pepper; Catkins (small); Piper longum

I.'

Heavy metals (2.3.13). 1.0 g complies'with the limit test for
heavy metals, Method B (20 ppm). ,
Assay. Determine by liquid chromatography (2.4.14)..

and dilute to 100.0 ml with methanol and filter. Dilute further if
necessary.
Reference solution. A ,0.01 per cent w/v solution of
octadecylsilane bonded to porous silica (5 /-un),
mobile phase: A.a buffer solution prepared by dissolving
0.136 g of potassium di-hydrogen orthophosphate in
900 ml of water, adjust the pH to 2.5 with
B. acetonitrile,
below,
injection volume. 20 iiI.
Time
(in min)
(per cent v/v)
(per cent v/v)
95
18
55
45
25
20
80
30
95
Pippali, small consists of the fruiting spike of Piper longllln

Linn. (Fam. Piperaceae).
Pippali, small contains not less than 0.4 per cent w/w ofpiperine,
Description. The spikes are greenish black to black in colour,
cylindrical, erect and blunt. It has pungent taste and the odour
is aromatic and characteristic. The spikes are 1.0 to 1.9 cm
long and 0.2 to 0.3 cm in diameter.
Identification
A. Macroscopic - The spikes are greenish black to black in
colour, surface rough. The spikes bear bracts and numerous
small fruits sunk in solid spike.
B. Microscopic - The peduncle has uniseriate septate
trichomes, epidermis is a single layer oftangentially elongated
cells. The penduncle vascular traces are in 2-3 bundles, pith is
dumbbell shaped. Cells are small, packed with abundant strach
grains and oil droplets. Crystal are present in some of the
cells, they appear unequal in size. The epicarp is a single layer
of thick walled cells having greenish content. Endocarp is
wavy in outline. Mostly, endocarp and seed coat are fused
together to form a deep zone with hyaline content in outer
layer and orange red (brown) inner region.
C. Determine by thin-layer c:hromatography (2.4.17), coating



75 ml of methanol, cool and filter. Combine all the :filtrates !;lnd
concentrate under vacuum to 50 mI.
Calculate the content of piperine.

Storage. Store protected from heat, moisture and against attach
by insects and rodents. '
2531
PIPPALI, SMALL
IP 2010

pippali, small RS with 50-75 ml of methanol for 15 minutes,
75 m1 of methanol, cool and filter. Combine all the filtrates and
Time
(min)
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
o
18
25
95
55
20
5
45

the plate with vanillin sulphuric acid reagent.. Heat the plate
30
95
Tests
80
5

retention time ofpiperine is 1.
Calculateihe content of piperine.
Storage. Store protected from heat, moisture and against attach
by Method I.
Punarnava
Ragweed; Boerhaavia diffusa
Acid-insoluble ash (2.3 .19). Not more than 3.0 per cent.
9
8

7
6

5
4

and dilute to 100.0 m1 with methanol and filter. Dilute further if
necessary.
Reference solution. A 0.01 per cent wlv solution of Punarnava consists of the dried root of Boerhaavia diffilsa
Linn. (syn. B. reperis Linn) (Fam. Nyctaginaceae).
Punarnava contains not less than 0.005 per cent wlw of
a stainless steel column 25 cm x 4.6 mm packed with boeravinone B, calculated on the dried basis.
octadecylsi1ane bonded to porous silica (5 Ilm),
orthophosphate in 900 ml of water, adjust the pH to 2.5
with orthophosphoric acid and dilute to 1000 ml with
water,
B. acetonitrile,
__
Description. A greyish-brown in colour, short and fibrous

fracture and no distinct odour with bitter taste.
Identification
A. Macroscopic - Stout, tapering, somewhat lmotty and
twisted roots upto 30 cm or more long and 0.5-1.5 cm thick
.=-~alineargradientprogramme.usingJhe.conditions.given ;~~h ~~e;~~i~~~~:iul~~~~ffi~;~~~~:~~i:su~~c;o~~

B. Microscopic -
Transverse section of root shows

outermost layer of cork which consists of thin walled
2532
.-
SARPAGANDHA
IP 2010
tangentially elongated cells with brownish walls followed by

thin walled 1-2 layered cork cambium. Cortex many layered
and composed of thin walled cells. Secondary cortex 2-4
layered and parenchymtous. Concentric bands ofxylem tissues
alternating with parenchymatous tissues. Vessels are radial
with reticulate thickening. Simple and compound starch grains
with centric hilum and raphide crystals of calcium oxalate in
cortex region. Fibres are aseptate and spindle shaped with
pointed ends.

6 volumes of methanol and 1 volume of glacial acetic acid.
vacuum to 5 ml.
Reference solution. Reflux 2 g ofthe punarnava RS with 25 ml
of methanol for 15 minutes, cool and filter. Reflux the residue
Apply to the plate 10 /ll ofeach solution as bands 10 mm by 2
and examine in ultraviolet light at 254 TIm and 365 nm, spray
plate at 110 for 10 minutes and examine in day light. The

boeravinone B RS in methanol.
- mobile phase: A.a gradient mixtures of water;
B. acetonitrile,
below,
Time
(min)
(per cent v/v)
( per cent v/v)
90
25
10
35
90
10
90
10
Calculate the content of boeravinone B.
Tests
Sarpagandha
Rauwolfia serpentina Root

methodl.
\
9
8


Assay. Determine by liquid chromatography (2'.4.14).

Sarpagandha consists of the dried roots of Rauwolfia

serpentina Bentham ex Kurz (Fam Apocynaceae).
2533
SARPAGANDHA
IP 2010
Sarpagandha contains not less than 0.15 per cent of reserpine

ajmalicine, calculated on the dried basis.
an~
Description. Taste bitter, odour indistinct.
band may also appear at the line of application in the

chromatogram obtained with test solution.
Tests
Identification
Foreign organic matter (2.6.1). Not more than 2.0 per cent
A. Macroscopic - Roots are sub cylindrical to tapering,

tortuous or curved, rarely branched. Occurs as segments
usually from 5 to 15 cm in length and 3 to 20 mID in diameter.
Externally grayish yellow to brown, wood pale yellow. Roots
tough with longitudinal marking and slightly wrinkled surface.
When scraped, bark separates readily from the wood. Fracture
is short and irregular.
B. Microscopic - In transverse section, cork cells in 2 to 8

alternating bands of radically narrow and broader cells, thin,
lignified up to 75 /lm in tangential width, broader cells up to
about 90 /lm in radial length, phelloderm, tangentially elongated
to isodimetric parenchyma cells containing starch and short
latex cells with brown resinous matter; secondary cortex
consists of parenchyma cells, heavily packed with starch
grains secondary phloem contains phloem parenchyma and
sieve elements, parenchyma contains starch and angular
crystals of calcium oxalate 3 to 20 /lm in length. Xylem is
aboiIt 4/5 ofthe diameter of the root, wood is transversed by
medullay rays 1 to 5 cells in width. Xylem consists ofvessels,
tracheids, wood parenchyma and wood fibers. Xylem vessels
are elongated up to 350 /lm in length and 50 /lm in width and
contains simple or bordered pits; tracheids lignified, pitted;
wood parenchyma with moderately thick, lignified and pitted
walls containing starch; wood fibres highly thickened with
pointed ends, stone cells absent.
C. Determine by thin-layer chromatography (2.4. I7), coating
Test solution. To 250 mg ofthe coarsely powdered substance
under examination, add 5 ml methanol, shake for 10 minutes,
and filter. Wash the residue with 5 ml of methanol and add the
washing to the filtrate.
Reference solution. To 250 mg of sGipagandha RS, add 5 ml
methanol, shake for 10 minutes, and filter. Wash the residue
with 5 ml of methanol and add the washing to the filtrate.
Apply to the plate 10 /ll of each solution as bands 10 mID by 2

the plate_with anisaldehYdesltlphuricaCidxeagent.Heatthe
plllte at 100 for 5 minutes and examine the plate in day light.
The chromatogram obtained with test solution shows two
pinkish-violet bands corresponding to the bands in the
chromatogram obtained with reference solution. A dark brown
Water-soluble extractive (2.6.3). Not less than 5.0 per cent.

Total ash (2.3.19). Not more than 8.0 perceut.
Acid-insoluble ash (2.3.19): Not more than 2.0 per cent.
Microbial contamination limits (2.2.9). Complies with the
microbial contamination tests.
substance under examination with 50 ml of
ethanol (95 per cent), on a water bath for 15 minutes, cool
and filter. Reflux the residue further with ethanol (95 per
cent), till the last extract turns colorless, cool and filter. Combine
all the filterates and concentrate to 100.0 ml.
Reference solution (a). A solution containing a 0.002 per cent
w/v reserpine RS and 0.002 percent w/v ajmalicine RS in
Reference solution (b). A 0.002 per cent w/v reserpine RS in
ethanol (95 per cent) for peak identification.
a stainless steel column 25 cm x 4.6 mID packed with
in 1000 ml water, adjust the pH to 3.0 with dilute
.
not more than 2.0 per cent. Inject reference solution (b) for
peak identification.
Inject the test solutionandTeference solution(a}
CalcUlate the content of reserpine and ajrnalicine.
2534
SARPAGANDHA TABLETS
IP 2010
Sarpagandha Powder
Reference solution (a). A solution containing a 0.002 per cent

w/v reseJpine RS and 0.002 per cent w/v ajmalicine RS in
Rauwolfia serpentina Powder
Sarpagandha powder is obtained by Rauwolfia serpentina

Bentham ex Kurz roots (Fam. Apocynaceae) reduced to a fine
powder.
It contains not less than 0.15 per cent w/w and not more than
0.2 per cent w/w ofreserpine-ajmalicine alkaloid calculated on
dried basis.
Description. Taste bitter, odour indistinct.
Identification
plate with silica gel OF 254.
Mobile phase. A mixture ono volumes of chloroform and 30
volumes of acetone.
Test solution. To 0.25 g of the substance under examination,
add 5 ml methanol, shake for 10 minutes, and filter. Wash the
residue with 5 ml of methanol and add the washing to the
filtrate.
.Reference solution. To 0.25 g ofRauwolfia serpentina powder
RS, add 5 ml methanol, shake for 10 minutes, and filter. Wash
the residue with 5 ml of methanol and add the washing to the
filtrate.
Apply to the plate 20 f..ll ofeach solution as bands 10 rom by 2

anisaldehyde sulphuric acid reagent. Heat the plate at 100
for 5 minutes and examine the plate at 365 nm. The
Tests
Reference solution (b). A 0.002 per cent w/v reserpine RS in

ethanol (95 per cent) for peak identification.
octadecylsilane bonded to porous silica (5 f..lm),
mobile phase: a mixture of35 volumes of acetonitrile
in 1000 ml water, adjust the pH to 3.0 with dilute
not more than 2.0 per cent. Inject reference solution (b) for
peak identification.
Calculate the content ofreserpine and ajmalicine.
Sarpagandha Tablets
Rauwolfia serpentina Tablets
Sarpagandha Tablets contain not less than 85.0 per cent w/w
and not more than 115.0 per cent w/w of stated amount of the
alkaloids contents mainly ofreserpine together with ajmalicine.
Identification

Heavy metals (2.3.13). 1.0 gm complies with the limittest for
heavy metals, method B (20 ppm).
In the assay the principal peak in chromatogram obtained

on 1 gm by drying in an oven at 105.
Tests
Microbial contamination limits (2.2.9). Complies with the

microbial contamination tests.
Test solution. Reflux about 2.0 g of the substance under
examination with 50 ml of ethanol (95 per cent), on a water
bath for 15 minutes, cool and filter. Reflux the residue further
with ethanol (95 per cent), till the last extract turns colourless,
cool and filter. Combine all the filterates and concentrate to
100.Ornl.

.
in 1.0 gm by drying in an oven at 105.
a quantity of the powder containing about 1 g of rauwolfia
serpentina into a 200 ml flat bottom flask. Add 50 ml of
ethanol (95 per cent), reflux on a water bath for 30 minutes,
cool and filter. Reflux the residue further with ethanol
2535
SARPAGANDHA TABLETS
IP 2010
(95 per cent) till the last extract turns colorless, cool and filter.
Combine all the filtrates and concentrate to 100.0 ml.
Reference solution. A 0.001 per cent w/v Reserpine RS and
0.001 % w/v Ajmalicin RS in ethanol(95 per cent).
octadecyl silane chemically bonded to porous silica (5/l),
and 65 volumes of a buffer (pH 3.0), prepared by
in 1000 ml water, pH of which is adjusted to 3.0 with
dilute orthophosphoric acid,
- flow rate. Iml per minute,
than 2.0 per cent.
Inject the refernce solution and the test solution.
Description. The fruits are oval-oblong, greenish brown to

yellowish brown in colour. They have an aromatic
characteristic odour and the taste is sweet aromatic.
Identification
A. Macroscopic - Fruit cylindrical to oval with pedicel
attached, consists of mericarps. Each mericap is 10 mm long
and 4 mm broad, five sided with a wider commissural surface,
tapered at apex and base, crowned with a conical stylepod,
glabrous, greenish or yellowish brown with five ridges.
B. Microscopic - Transverse section of fruit shows pericarp
with outer epidermis of quadrangular to polygonal cells with
smooth cuticle. Vittae 4 dorsal and 2 commissural extending
with length of each mericarp, having brown cells and volatile
oil in cavity. Mesocarp with reticulate lignified parenchyma.
Endocarp cells thin walled arranged parallel to one another in
groups of5-7. Endosperm consists of thick walled, cellulosic
parenchyma containing fixed oil cells and rosette crystals of
calcium oxalate.

Calculate the content of ajmalicin and reserpine.

Usualstrengths. 0.1 mg; 0.25 mg.
exceeding 30.
Labelling. The quantity ofactive ingredient is stated in terms
ofreserpine-ajmalicine content in labelled amount.

under examination, add 25 ml of dichloromethane, reflux for
times with 25 ml ofdichloromethane, cool and filter. Combine
all the filtrates and evaporate under vacuo to 25 ml.
Reference solution. To 2 g of the saunf RS, add 40 ml of
methanol, reflux for 15 minutes, cool and filter. Reflux the
residue further with 25 ml of methanol twice, cool and filter.
Combine all the filtrates and concentrate under vacuum to
25ml.
Saunf
Fennel; Foeniculum vulgare
Apply to the plate 10 /ll of each solution as bands 10 mm by

with a anisaldehyde sulphuric acid reagent. Heat at 110 for
10 minutes and examine the plate in day light. The
8
7
6
5
4
Tests
3
2
.~~l.l~f(;Q~~!~t Qfthe.. d.l'ie.4fr.t1it~Qf EQ?rz.ifltlYf!I y.y Igg!:?J'4!11-

by Method 1.
Totalash (2-:-:r.T9rNOtmore tlianlKO per cent.-------
(Fam. Apiaceae).
Acld=insolubie-ashC2.iT9YNot-morethanCOper'cent.
Saunf contains not less than 0.60 per cent of anethole,

2536
SENNA LEAF
IP 2010

Senna leaf contains not less than 1.0 percent w/w of

sennosides A and B, calculated on the dried basis.

Description. Pale yellowish green coloured leaflets with

mucilaginous and faint odour.
Identification
Test solution. Weigh 2.0 g ofthe coarsely powdered substance

under examination, add 50 ml of methanol, reflux on a waterbath for 15 minutes, cool and filter. Reflux the residue further
with methanol till the extract turns colourless, cool and filter.
Reference solution. A 0.01 per cent w/v solution of anethole
RS in methanol.
- a capillary column 30 m x 0.25 m, packed with DB 1,
temperature:
oven 90 to 260 @10 per minute (initially and finally
hold for 5 minutes respectively),
injector 240,
detector 280,
split flow 20 ml per minute.
Inject 1 fll of the reference solution. The test is not valid
Inject 1 fll of the test solution and the reference solution.
A. Macroscopic -Leaflets, 2.5 to 8 cm long and 5-15 mm

wide at centre, pale yellowish green, elongated lanceolate,
slightly asymmetric at base; margins entire, flat, apex acute
with a sharp spine; both surface smooth with sparce trichomes;
odour, faint but distinctive; taste, mucilaginous and
disagreeable but not distinctly bitter.
B. Microscopic
Transverse section shows outer single
layered mucilaginous epidermal cells. Unicellular hairs
presents. Stomata paracytic, numerous on both surface.
Mesophyll consists of upper and lower palisade layers with
spongy layer in between, primatic crystals ofcalcium oxalate
present on larger veins.
C. In the Assay, the chromatogram obtained with test solution
solution.
D. Determine by thin-layer chromatography (2.4.17), coating
Mobile phase. A mixture of 40 volumes of n- propyl alcohol,

40 volumes of ethyl acetate, 29 volumes of water and 1 volume
Calculate the content of anethole.

Storage. Store protected from light in well-filled containers, at
Senna Leaf
Test solution. Take 1 g of the dried leaves powder substance

under examination. Add 25 ml of methanol, reflux for 10 minutes,
cool and filter. Reflux the residue with another 20 ml of
concentrate to 10 ml.
Reference solution. Boil 0.5 g ofdried senna leaves RS powder
with 25 ml of methanol under reflux for 10 minutes, cool and
filter. Boil under reflux the residue with another 20 ml of
methanol, cool and filter. Combine all the filtrates and evaporate
to 5 ml.
Cassia leaf; Cassia angustifolia
Apply to the plate 10 fll ofeach solution as bands 10 mm by 2

and examine in ultraviolet light at 254 nm and 365 urn, spray
the plate with 20 per cent v/v of nitric acid solution. Heat the
plate at 100 to 105 for about 10 minutes and immediately
examine the plate in day light. The chromatographic profile of
the test solution is similar to that of the reference solution.
5
4
Tests
o
Senna leaf consists of the dried compound leaves of Cassia
angustifolia or Cassia senna Vahal. (Fam.Leguminosae).

2537
SENNA LEAF
IP 2010

determined on 5 g by drying in an oven at 105 .
Description. Pale yellowish green coloured pods with slight

odour. Leaflets with mucilaginous and faint odour.

contamillation tests.
Identification

Test solution. Weigh accurately 1.0 g ofthe coarsely powdered
substance in a round bottom flask, add about 10 ml of 1 per
cent v/v acetic acid and 25 ml of methanol and reflux on a
water bath for about 30 minutes. Cool to room temperature;
make up the volume up to 50.0 ml with methanol and filter.
sennasides RS in methanol.
.
mobile phase: a mixtures of 82 volumes of 1 per cent
v/v acetic acid in water and 18 volumes of acetonitrile,
Calculate the content of sennoside A and B.
A. Macroscopic - Flattened reniform pods, brownish yellow

at the edges, dark brown in the central area about 40 to 50 mm
long and about at least 20 mm wide. At one end is a stylar
point and at the other a short stalk. The pods contains 5 to 7
flattened and obovate seeds, green to pale brown, with a
continuous network of prominent ridges on the tests andin
complate wavy transverse ridges on the testa. Leaflets, 2.5 to
8 em long and 5-15 mm wide at centre, pale yellowish green,
elongated lanceolate, slightly asymmetric at base; margins
entire, flat, apex acute with a sharp spine; both surface smooth
with sparce trichomes; odour, faint but distinctive; taste,
mucilaginous and disagreeable but not distinctly bitter.
B. Microscopic - The pods present an epicarp with strongly
cuticularised isodiametric cells, occasional anomocytic or
paracytic stomata, and very few conical, unicellular and warty
trichomes. Hypodermis with collenchymatous cells, mesocarp
with parenchymatous tissue, a layer of prism s of calcium
oxalate and containing vascular bundles incompletely
surrounded by fibres with a crystals sheth of calcium oxalate
prisms, endocarp consisting of thick-walled and inter lacing
fibres. The seeds present a sub epidermal layers of palisade
cells with thick outer walls, endosperm composed of
polyhedral cells with mucilaginous wall.
Reduce to moderately fine powder examine microscopically
using chloral hydrate solution. The powder consists ofepicarp
with polygonal cells and a small number of warty trichomes
and occasional anomocytic stomata, fibres in two crossed
layers, accomparied by a crystal sheath of calcium oxalate
prism, characteristic palisade cells in the seeds and stratified
cells in the endosperm, cluster and prisms of calcium oxalate
Senna Pods
. Senna Fruit; Pods of cassia; Cassia angustifolia
9
C. In the Assay, the chromatogram obtained with test solution

solution.
D. Determine by thin-layer chromatography (2.4.17), coating

Mobile phase. A mixture of40 volumes of n- propyl alcohol,

40 volumes of ethyl acetate, 29 volumes of water and 1volume
4
3
o
Se.I1Ila. PQcl~cQl1sist()f.tlle cb:teg g()J.:I1lJ()l,l!!d..:fl()As.()f(:;'qss.iCl
angustifolia or Cassia senna Vahal. (Fam.Leguminosae).
Senna Pods contains not less than 1.0 per cent w/w of
sennosides A and B, claculated on the dried basis.
Test solution. Take 1 g of the dried pods powder substance

under examination. Add 25 ml ofmethanol, reflux for 10 minutes,
cool and filter. Reflux the residue with another 20 ml of
11'l~!h'1..nc~~co~o~lJ;a~n~(d filter. Combine all the filtrates and
concentrate to
Reference solution. Boil 0.5 g ofdried senna'jJods RS powder
with 25 ml of methanol under reflux for 10 minutes, cool and
filter. Boil under reflux the residue with another 20 ml of
2538
SENNA DRY EXTRACT
IP 2010
methan~l, cool and filter. Combine all the filtrates and evaporate
t05m!.
min. Allow the mobile phase to rise 8 cm. Dry the plate in air
' and examine in ultraviolet light at 254 nm and 365 urn, spray
.. the plate with 20 per cent v/v of nitric acidsolution. Heat the
ptatt,l at r00 to 105 for about 10 minutes and immediately
examine the plate in day light. The chromatographic profile of
the ,test solution is similar to that of the reference solution.
Tests
determined on 5 g by drying in an oven at 105 .
Test solution. Weigh accurately 1.0 g ofthe coarsely powdered

substance in a round bottom flask, add about 10 ml of 1 per
cent v/v acetic acid and 25 ml of methanol and reflux on a
water bath for about 30 minutes. Cool to room temperature;
make up the volume up to 50 ml with methanol and filter.
Reference solution. A 0.004 percent w/v solution of
sennosides RS in methanol.
Chromatographic system:
mobile phase. a mixtures of 82 volumes of 1 per cent
v/v acetic acid in water and 18 volumes of acetonitrile.
Senna Dry Extract contains not less than 90.0 per cent w/w
and not more than 110.0 per cent w/w of stated amount of
sennoside A and sennoside B, calculated on the dried basis.
Description. A light brown to dark brown powder.
Identification
Mobile phase. A mixture of40 volumes of n-propyl alcohol,

40 volumes of ethylacetate, 30 volumes of water and 1 volume
Test solution. Shake well 0.1 g ofthe extract under examination
with 5 ml ofa mixture ofequal volumes of methanol and water
for 5 minutes, heat to 60, cool and allow to settle. Use the
supernatant liquid.
'Reference solution. Dissolve 10 rng of senna dry extract RS
in 1 ml of a mixture of equal volumes of methanol and water.
Apply to the plate, 10 III of each solution as bands of 10 mm
by 2 mm. Allow the mobile phase to rise 10 cm. Dry the plate
in air and examine in ultraviolet light at 254 urn and 365 nm,
spray the plate with 20 per cent v/v of nitric acid solution.
Heat the plate at 110 for 10 minutes and examine the plate in
day light. Allow to cool and spray with a 5 per cent w/v
solution of potassium hydroxide in ethanol (50 per cent v/v)
until the zones appear. The chromatographic profile of the
test solution is similar to that of the reference solution.
B. To about 25 mg ofthe extract add 50 ml of water and 2 ml

of hydrochloric acid. Heat in a water-bath for 15 minutes.
Cool and shake with 40 ml of ether. Separate ether layer, dry
over anhydrous sodium sulphate and evaporate 5 ml to
dryness. To the cooled residue add 5 ml of dilute ammonia. A
yellow or orange colour develops. Heat on a water-bath for 2
minutes. A reddish-violet colour develops.
Tests
pH (2.4.24).5.5 to 7.5, determined in a 10 per cent solution in
water.
on 1.0 gm by drying in an oven at 105.
Microbial contamination (2.2.9). Total viable aerobic count
not more than 104 micro organisms per gm, fungi not more
than 102 micro organisms per g and Escherichia coli and
salmonellae absent.
Calculate the content of sennoside A and B.

Senna Dry Extract
Solvent mixture. A 0.3 per cent v/v solution of acetic acid

with the pH adjusted to 5.9 with 1 M sodium hydroxide.
Senna Dry Extract is produced from Senna leaves or pods of

Cassia angustifolia (Tinnevelly Senna) or Cassia acutifilia
(Cassia Senna) as calcium salts.
Test solution. Dissolve an accurately weighed quantity of

substance under examination containing about 10 mg of
sennosides in 50.0 ml of the solvent mixture.
2539
SENNA TABLETS
IP 2010

of calcium sennosides RS containing about 10 mg of
sennosides in 50.0 ml ofthe solvent mixture.
(5J.lm),
mobile phase: a mixture of 83 volumes of a 1 per cent
v/v solution of glacial acetic acid and 17 volumes of
acetonitrile,
injection volume. 10 J.ll.
a stainless steel column 15 cm x 4.6 mID, packed with
(5J.lm),
mobile phase: a mixture of 83 volumes of a 1 per cent
v/v solution of glacial acetic acid and 17 volumes of
acetonitrile,
than 2.0 per cent.

exceeding 30.
Calculate the content of sennosides A and B, as sennoside B

in the extract.
Calculate the content of sennosides A and B, as sennoside B

in the tablets.
Storage. Store protected from light, in air-tight containers.
Labelling. The quantity ofactive ingredient is stated in terms

of total sennoside A and B, expressed as the equivalent
content of sennoside B.
Senna Tablets
Shatavari
SennaTablets contain not less than 85.0 per cent w/w and not
more than 115.0 per cent w/w of the stated amount of
sennosides A and B, calculated as sennoside B.
Asparagus racemosus root
Identification
In the assay, the principal peak in the chromatogram obtained

7
6
Tests
Solvent mixture. A 0.3 per cent v/v solution of acetic acid,

with the pH adjusted to 5.9 with 1 M sodium hydroxide.
Test solution. Weigh and powder 20 tablets. Weigh accurately Shatavari consists of the tuberous roots of Asparagus
a quantity of the powder containing about 10 mg of racemosus Willd. (Fam. Liliaceae).
sennosides, dissolve in about 40 ml ofsolvent mixture and mix Shatavari contains not less than 0.1 per cent of shatavarin IV,
.._ - . - - - - -.....
withtheai&ofultrasoundfor15-millutes:-Diluteto-50mlwitb ca1culatedoni:hedriedbasis:the solventmixture and filter.
DescrIptloil:fhetuberous roofbIts are-dirtY whIte lri. color,
Reference solution. A 0.02 per cent w/v solution of calcium

sennaside RS in the solvent mixture.
longitudinally wrinkled with yellow hard central core. It is

starchy and slightly bitter followed by sweet taste.
2540
SHATAVARl
IP 2010
Identification
A. Macroscopic - The tuberous roots are borne in a compact
bunch and are fleshy, and spindle shaped. They are marketed
in pieces 5-15 cm in length and 2 cm in thiclmess. They are
silvery white or ash-colored externally and white internally,
more or less smooth when fresh, developing longitudinal
wrinldes when dry.
B. Microscopic - The inner parenchymatous zone of cortex
is composed of 18-24 layers in upper and 42-47 layers in the
middle tuberous portion ofthe roots. The cells are thin - walled
cellulosic, with circular to oral outlines and distinct inter cellular
spaces. In some roots 3-4 layers ofcortex immediately adjacent
to the endodermis are modified into a sheath of stone cells
round the endodermis. The number ofvascular bundles is 3035 in the upper levels and 35-45 in the middle tuberous portions
of the roots.

and filter. Reflux the residue further with 2 x 30 mI of methanol,
vacuum to 10.0 mI.
Reference solution. Reflux 1 g of shatavari RS with 30 ml of
the filtrates and concentrate under vacuum to 10.0mI.
Apply to the plate 5 /-l1 of each solution as bands 10 mm by 2

at 120 for 10 minutes and examine the plate in day light. The

Assay. Perform the Assay by following method I or by method
II. The result ofthe method II will be official in case ofdispute.
Method I
Mobile phase. A mixture of 6.5 volumes of chloroform and
3.5 volumes of methanol.
Combine all the filtrates and concentrate to 50 mi.
Reference solution. A 0.008 per cent w/v solution ofshatavarin
IV RS in methanol.
Apply to the plate 5 /-ll of each solution as bands 10 mm by

2 mm.Allowthe mobile phase to rise 8 cm.Dry the plate in air
and spray with a 10 per cent v/v solution of sulphuric acid in
methanol. Heat the plate at 100 for 5 minutes, scan the plate
in absorbance mode at 500 urn. Record the chromatograms
and measure the responses for the analyte peak.
Calculate the content of shatavarin Iv.
Methodll
Combine all the filtrates and concentrate to 50.0 mi.
shatavarin IV RS in methanol. Dilute suitably to prepare
0.0075- 0.075 percentw/v solution.
use evaporative light scattering detector,
temperature
evaporator 110,
nebulizer 90,
nebulizer gas nitrogen and gas flow 1 SLM,

regression coefficient is not more than 0.9.
Tests
Water- soluble extractive (2.6.3). Not less than 20.0 per cent
by Method I.
2541
SHATI
IP 2010
Calculate the content of shatavarin lV.
grains tissue are simple, circular or oval in shape, found in

ground cells.


Mobile phase. A mixture of 80 volumes of n-hexane and

20 volumes ofacetone.
vacuum to 25 mi.
Shati
Hedychium spicatum
Reference solution. Reflux 0.5 g ofthe shati RSwith 25 mlof

the filtrates and concentrate under vacuum to 12.5 mi.
9
8
'7
Apply to the plate 10 J!l of each solution as bands 10 mm by 2

and examine in ultraviolet light at 254 om and 365 om, spray
plate at 110 0 for 10 minutes and examine the plate in day light.
The chromatographic profile of the test solution is similar to
that ofthe reference solution.
5
4
3
2
Tests
Shati consists of the dried rhizomes of Hedychium spicatum
Buch.-Ham.ex Smith (Fam. Zingiberaceae).
Shati contains not less than 0.80 per cent w/w ofp-methoxy
cinnamic acid ethyl ester, calculated on the dried basis.
Description. A reddish-brown outer surface and white in side,
short and uneven fracture and camphoraceous odour with
aromatic and pungent taste.
Identification
A. Macroscopic - Tuberous rhizome having reddish brown,
rough outer surface with round root scars or rootlets which
remain attached at some places. Transversely sliced pieces of
dried rhizome are spherical, flat, 1 cm in thickness and 2-3 cm
in diameter having white and starchy surface.

by method!.
less than 100 mi. Dilute to 100.0 ml with methanol.
B. Microscopic - Transverse section of rhizome shows

outermost layer of cork having 2-6 layers of isodiametic,
nonlignified and suberised cells which are radially arranged.
In old rhizome, the cork cells are exfoliated or crushed. Cortex
is a broad zone with 20-25 layers of thin walled parenchymatous
cells. Cortex region is filled with abundant starch grains and Reference solution. A 0.01 per cent w/v solution of
~nlliiieroi.iSo1eo-resincells. Vascular bundles are closed
p;methoxy~cinnamic~acid~ethyl-esterRS~inmethanol~--
collateral arid .scattered fhrougl1ourtne-grouiidlissues. Chromat6graphicsystem ..... -.~ .....~-~ ..... _....
Cambium has 4-5 rows oftangentially elongated cells. It forms
a complete ring between the xylem and phloem groups. Starch
octadecylsilane bonded to porous silica (5 J!m),
and
2542
STARCH
IP 2010
mobile phase: 40 volumes of water and 60 volumes of

acetonitrile,
Calculate the content ofp-methoxy cinnamic acid ethyl ester.
Shellac
Lac
Shellac consists of a resinous substance prepared from a
secretion that encrusts the bodies of a scale insect, Laccifer
lacca Kerr (Fam. Ciccidae).
Description. Lemon-yellow to brownish orange thin scales or
hard, brittle masses; odourless or with a faint odour.
wash with two successive portions, each of 50 ml, of water,

filter the washed light petroleum solution, and evaporate to
dryness; to the residue add 2 ml of a mixture of 1 volume of
liquified phenol and 2 volumes of carbon tetrachloride and
transfer to a cavity of a colour-reaction porcelain tile; fill an
adjacent cavity with a mixture of 1 volume of bromine and
4 volumes of carbon tetrachloride, and cover both cavities
with an inverted watch-glass; no purple or deep indigo-blue
colour is produced in the liquid containing the residue.
Arsenic (2.3 .10). Heat gently 5.0 g with 2 ml of nitric acid and
0.5 ml of sulphuric acid in a long-necked flask, until the first
reaction has subsided, cool, add carefully and in small portions,
15 ml of nitric acid and 6 ml of sulphuric acid taking care to
avoid excessive foaming, and continue heating, adding further
small portions of nitric acid, if necessary, until white fumes
are evolved and the solution becomes colourless or almost
colourless. Cool, add carefully 10 ml of water, evaporate until
white fumes are evolved, and repeat the addition of water and
evaporation until all the nitric acid has been removed, cool,
dilute to 50 ml with water, and add 10 ml of stannated
hydrochloric acid AsT. The resulting solution complies with
the limittest for arsenic (2 ppm). Use 0.5 ml ofarsenic standard
solution (10 ppm As) for the standard stain.
Identification
To 50 mg add a few drops of a mixture of 1 g of ammonium

molybdate and 3 ml of sulphuric acid; a green colour is
produced and it becomes lilac on standing for 5 minutes.
on 0.5 g.
Tests
Acid value (2.3.23). 50 to 70, determined by the following
method. Weigh accurately about 2.0 g and dissolve with the
aid ofgentle heat, in 50 ml of ethanol (95 per cent) previously
neutralised to ethanolic thymol blue solution. Titrate with
0.1 M ethanolic potassium hydroxide using ethanolic thymol
blue solution as an external indicator. Calculate the acid value
from the expression
5.61 x alw
where, a
number of ml of 0.1 M ethanolic potassium

hydroxide and
w = weight, in g, ofthe sample.
Ethanol-insoluble matter. Not more than 2.0 per cent,

determined by the following method. Weigh accurately about
5.0 g in an extraction thimble, cover with ethanol (95 per
cent) and allow to stand for 16 hours. Place in an apparatus
for the continuous extraction of drugs, extract with ethanol
(95 per cent) for 4 hours, dry the residue at 1000 for 3 hours
and weigh.
Colophony. Dissolve 2.0 g by shaking with 10 ml ofethanol,
add slowly, with shaking 50 ml of lightpetroleum (40 0 to 60"),
Starch
Starch consists ofpolysaccharide granules obtained from the
caryopsis ofmaize or com, Zea mays Linn.(Fam. Poceae), or of
rice, Oryza sativa Linn., or of wheat, Triticum aestivum
Linn.(Fam. Graminae) , or from the tuber ofpotato, Solanum
tuberosum Linn (Fam. Solanaceae), or from the rhizomes of
tapioca, Manihot utilissima PoW.(Fam. Euphorbiaceae).
Description. A very fme, white or slightly yellowish powder
or irregular white masses which are readily reducible to powder,
creaks when pressed between the fmgers; odourless and
tasteless. The presence of granules showing cracks or edge
irregularities is exceptional in starches other than wheat starch;
wheat starch may contain granules with cracks on the edges.
Identification
A. Corn or maize starch - Polyhedral granules, 2 to 23 Ilm in
size, or rounded granules, 25 to 32 Ilm in diameter. The central
hilum consists of a distinct cavity or two- to five-rayed cleft;
no concentric striations. Viewed between crossed nicol prisms,
a distinct black cross is seen intersecting at the hilum.
2543
STARCH
IP 2010
Potato starch - Single granules, either irregular, ovoid or

pear-shaped, 30 to 100 11m in size, or rounded, 10 to 35 11m in
size; oompound granules consisting of groups of two to four
elements are rare. Eccentric hilum; clearly visible concentric
striations. Viewed between crossed nicol prisms, a distinct
black cross is seen intersecting at the hilum.
Rice starch - Polyh<;ldral granules, 2 to 5 11m in size, either
isolated or aggregated in ovoid masses, 10 to 20 11m in size.
Central hilum poorly visible; no concentric striations. Viewed
between crossed nicol prisms, a distinct black cross is seen
intersecting at the hilum.
Tapioca starch - Principally simple granules, sub-spherical,
muller-shaped or rounded polyhedral; smaller granules 5 to
10 11m, larger granules 20 to 35 11m in diameter; hilum, central,
punctate, linear or triradiate; striations, faint, concentric;
compound granules, few, oftwo to three unequal components.
Wheat starch - Large discoid or, more rarely, reniform
granules, 10 to 45 11m in size; profile, elliptical and fusiform, slit
along the main axis. Small rounded or polyhedral granules,
2 to 10 11m in size. Granules of intermediate size very rarely
occur. Hilum and striations invisible or barely visible. Viewed
between crossed mcol prisms, a distinct black cross is seen
intersecting at the hilum.
Microbial Contamination (2.2.9). 1 g is free from Escherichia

coli and salmonellae.
Sulphated ash (2.3.18). Not more than 0.6 per cent (for all
starches except rice starch) and not more than 0.8 per cent (for
rice starch), determined on 2.0 g.
Loss on drying (2.4.19). Not more than 15.0 per cent (for all
starches except potato starch) and not more than 20.0 per cent
(for potato starch), determined on 0.2 g by drying in an oven
at 105.
Labelling. The label states the type of starch.
Sunthi
Saunth; Ginger; Zingiber officinale
9
8
7
B. Heat to boiling for 1 minute a suspension of 1 g in 50 ml of

water and cool; a thin and cloudy mucilage is produced with
all starches except potato starch which gives a thick and more
transparent mucilage.
5
4
C. To 10 ml ofthe mucilage obtained in test B add 0.05 mlof
0.01 M iodine; a dark blue colour is produced, which
disappears on heating and reappears on cooling.
Tests
Acidity. Add 10.0 g to 100 ml of ethanol (70 per cent)
previously neutralised to phenolphthalein solution, shake
for 1 hour, filter and titrate 50 ml of the filtrate with 0.1 M
sodium hydroxide. Not more than 2.0 ml is required to change
.
Iron (2.3.14). Dissolve the residue obtained in the test for
sulphated ash in 4 ml of hydrochloric acid with the aid of
gentle heat, dilute with water to 50 ml and mix; 25 ml of the
resulting solution complies with the limit test for iron (40 ppm).
Fluorescence. No fluorescence should be visible under
screened ultra-violet light.
Sunthi is the whole or cut scraped or unscraped, dried rhizomes

of Zingiber officinale Roscoe. (Fam. Zingiberaceae).
Sunthi contains not less than 0.8 per cent w/w of total
gipgerols, calculated on the dried basis.
Description. Odour, agreeable and aromatic; taste, agreeable
and pungent.
Identification
A. Macroscopic - Rhizome laterally compressed, bearing
short, flattened, oblique branches; outer surface buff-coloured,
longitudinally striate; inner surface pale yellow, starchy and
fibrous. Fracture short with projecting fibers.
Oxidising substances. To 5.0 gadd 10mlofwaterand 1 mlof

--acetic aci~ranastiruni;iiaI;omogeneoussu-spensionTs B.
obfamea:Aaa-03 illJ. Of:.i freshly prepared safuiated solution

ofpotassium iOdide, mix and allow to stand for 5 minutes; no
distinct brown or blue colour is observed.
MicroscopiC=- Fibro-vascular bundles"-tilld-6Ieore-sin cells---
witli-yellowpigmenfsc:.itteredingroUiiatissue~Sfarcligraiiis
abundant in parenchyma cells, mostly simple, sack shaped,

spherical; hilum eccentric.
2544
-".,,-,-,,~ . _,---
SUNTHI EXTRACT
IP 2010


70 volumes of diethyl ether.

under examination with 25 rnl of methanol for 15 minutes, cool
and filter. Wash the residue with 10 ml of methanol. Combine
all the filtrates and concentrate to 10 ml.
Calculate the contents oftotal gingerols by summing the peale

areas of 6-gingerol with all other peaks, which elute after 6gingerol and have a peak area ofat least 5 per cent ofthe peak
area of6-gingerol.
Reference solution. Reflux 0.5 g of coarsely powdered

sunthi RS with 5 ml methanol for 15 minutes, cool and filter.

Apply to the plate 10 III of each solution as bands 10 rom by 2

at 100 for 5-10 minutes and examine the plate in day light. The
Suothi Extract
Tests
by Method I.
Sunthi extract is obtained by extracting Sunthi (Zingiber

officinale Roscoe, Fam. Zingiberaceae) dried rhizome ofwith
ethanol or any other suitable solvent. The powdered extract
may contain suitable excipients.
Sunthi extract contains not less than 90.0 per cent w/w and
not more than 120 percent w/w of the stated amount of total
gingerols and shogaols (sum of 6 gingerol, 8 gingerol, 10
gingerol and 6 shogaol). The content of6 shogaol shall not be
more than the content of 6 gingerol.
Description. Very light yellow to light brown powder or thick
liquid with characteristic odour and pungent taste.

Acid-:-insoluble ash (2.3 .19). Not more than 1.5 per cent.
O.2g.

substance under examination with 100 rnl of methanol on a
water-bath for 15 minutes cool and filter. Reflux the residue
and filter. Combine all the filtrates and concentrate to 50.0 rnl
6-gingerol RS in methanol.
of water,
- flow rate. 1.3 rnl per minute,
Identification
Mobile phase. A mixture of 30 volumes of hexane and 70

volumes of diethyl ether.
50 ml of methanol and filter.
Reference solution. 0.1 per cent w/v solution of 6 gingerol RS
in methanol.
Apply to the plate 10 III ofeach solution as bands 10 rom by 2
and examine in ultraviolet light at 254 urn. Spray the plate with
vanillin sulphuric acid Heat the plate at 100 for 10 minutes
and examine the plate at 365 nm and in day light. The
ofthe reference solution.
Tests
Water (2.3.43).Notmorethan 5.0 percent, determined on2.0 g
when dried at 105 for 3 hours.
2545
SUNTHI EXTRACT
IP 2010

contamination test
Test solution. Dissolve about Ig ofthe extract containing 100

mg ofgingerols in methanol by heating, make up to 100 ml and
filter.
Identification
A. To a solution in ethanol (90 per cent) add ferric chloride

test solution; a green colour is produced.
B. To 1 g add to 5 ml ofwater, heat to boiling, filter, add 30 mg
of potassium permanganate and continue heating; the odour .
of benzaldehyde is produced.
Tests
Reference solution. A 0.1 per cent wlv solution of 6 gingerol

RS in methanol.
Acidity. A solution in ethanol (90 per cent) is acidic to litmus

solution.
- mobile phase: A mixture of 55 volumes of Acetonitrile
and 45 volumes of water
- flow rate 1.3 ml per minute,
- injection volume.20 Ill.
Acid value (2.3.23).97 to 160, calclliated on the dry, ethanolsoluble matter, determined by the following method. Dissolve
5.0 g in 50 ml of boiling ethanol (90 per cent), add 3 mlof
phenolphthalein solution and titrate the hot solution with
1 M ethanolic potassium hydroxide. When the colour
becomes dark brown, attach to a reflux condenser, boil for a
few minutes to break up the precipitate and complete the
titration.
Ethanol-insoluble matter. Not more than 5 per cent, determined

by the following method. Digest 2.5 g with 50 ml of ethanol
(90 per cent), filter through a sintered glass crucible, transfer
the residue to the filter crucible with the aid of more ethanol
(90 per cent), wash with hot ethanol (90 per cent) until all
soluble matter is removed and dry to constant weight at 100.

Calculate the content oftotal gingerols by summing the peak
areas of 6 gingerol, 8 gingerol, 10 gingerol and 6 shogaol. The
relative retention time of 6 gingerol is 1.0, 8 gingerol is about
2.1, 10 gingerol is about 5.0 and 6 shogaol is about 2.6. The
area ofthe peak of 6 gingerol in the sample chromatogram is
more than the area of 6 shogaol indicating the content of 6
gingerol is more than the content of 6 shogaol.
Colophony. Add 5 g to 25 ml ofcarbon disulphide, warm gently

on a water-bath under a reflux condenser, filter, evaporate the
solution to dryness, dissolve the residue in 6 ml of light
petroleum (40 to 60) and shake with 10 ml of a 0.5 per cent
wlv solution of cupric acetate; the light petroleum layer is
not coloured green.

Ester value (2.3.26).47 to 95, calculated on the dry, ethanolsoluble basis.
Saponification value (2.3.37). 170 to 230, calculated on the

dry, ethanol-soluble basis.
Tolu Balsam
than 4 per cent, determined

Loss on drying (2.4.19). Not more
a
on 2.0 g by drying in a thin layer in an oven at 60 over
phosphorus pentoxide at a pressure not exceeding 2.7 kPa.
Balsam ofTolu
Tolu Balsam is a solid or semi-solid, balsam obtained by incision
from the trunk of Myroxylon balsamum (Linn.) Harms (Fam.
Leguminosae).
Tolu Balsam contains not less than 35.0 per cent and not more
than 50.0 per cent of total balsamic acids, calculated as
cinnamic acid, C9H gOz, on the dry, ethanol-soluble matter.
.... ~_._ ....Qes_cription.AsQft,.teml&i~:ms,b[Qwnis.hy_ellQ\YOTb.ro}'\T!:!
Assay. Weigh accurately about 2.0 g and boil with 25 ml of

dilute ethanolic potassium hydroxide solution under a reflux
condenser for 1 hour. Remove the ethanol and digest the
residue with 50 ml ofhot water until diffused. Cool the liquid,
add 150 ml of water and 1.5 g of magnesium sulphate
dissolved in 50 mlofwater. Mix thoroughly and set aside for
10 minu!es.Filt~r,\\,ashthe residue on the filter with 20 ml of
.!ll.a..~s,'Y_l!~1!fuLc.<:ln~.<:.t~Q;~tlQ~~qt!~I!!ly,1?-~C:<:>Il1!l1K~.~cl~~-water:a:cTCliiY the combined filtrate- an-Cl washings with--~-
hydroClilorlcaczdaiidextracfwiilisuccessivequanntiesoC
and finally brittle. Transparent in thin films; odour, aromatic .

and vanilla-like. Warmed and pressed between pieces ofglass 50,40,30,30 and 30 ml ofether. Combine the ether extracts and
and examined with a lens, it exhibits crystals ofcinnamic acid. discard the aqueous portion. Extract with successive
2546
TRAGACANTH
IP 2010
quantities of 20, 20, 10, 10 and 10 ml of sodium bicarbonate

solution, washing each aqueous extract with the same 20 ml
of ether. Discard the ether layers, acidify the combined
aqueous extracts with hydrochloric acid and extract with
successive quantities of 30, 20, 20 and 10 ml of chloroform,
filtering each chlorofOlm extract through a plug ofcotton wool
on which a layer of anhydrous sodium sulphate is placed.
Evaporate the chlorofonn on a water-bath until about 10 ml
remains and remove the remainder in a current ofair stopping
immediately when the last trace ofsolvent is removed. Dissolve
the residue by wanning with 10 ml of ethanol (95 per cent),
previously neutralised to phenol red solution, cool and titrate
with 0.1 M sodium hydroxide using phenol red solution as
indicator.
total balsamic acids, calculated as cinnamic acid, C9H sOz.
Storage. Store protected from light and moisture. Avoid
exposure to excessive heat.
Tragacanth
Tragacanth is the air-hardened gummy exudate, flowing
naturally or obtained by incision, from the trunk and branches
of Astragalus gummifer Labill. and certain other species of
Astragalus (Fam. Fabaceae).
Description. Pale yellow, thin, flattened ribbons or brittle
pieces; odourless and almost tasteless. On the addition of
about 10 times its weight of water, it forms a mucilaginous gel.
It has the macroscopic and microscopic characteristics
described under Identification tests A and B.
Identification .
A. Macroscopic - Occurs as thin, flattened pieces, 30 mm
long, 10 mm wide and up to I mm in thic1mess, more or less
curved, marked on the surface by fine longitudinal striae and
concentric transverse ridges; white, translucent, horny;
fracture, short. May also be in the fonn ofthicker, less brittle
pieces, white to pale yellow and more opaque.
B. Microscopic - Reduce to powder. Examine under a
microscope; the powder shows in the gummy mass numerous
stratified cellular membranes which turn violet on the addition
of iodinated zinc chloride solution. The mass includes starch
granules, isolated or in small clusters, rounded or occasionally
defonned, diameter 4 to 10 /lm, and up to 20 /lm, with a central
hilum, visible in polarised light.
C. Moisten 0.5 g ofthe powdered material with 1 ml of ethanol
(95 per cent) and add gradually, while shaking, 50 ml of water
until a homogeneous mucilage is obtained. To 5 ml of the
mucilage add 5 m1 of water and 2 ml of barium hydroxide
solution. A slightly flocculent precipitate is fonned which,

when heated for 10 minutes on a water-bath, gives an intense
yellow colour.
D. Add 4 ml ofa 0.5 per cent w/v dispersion in water to 0.5 m1
of hydrochloric acid and heat on a water-bath for 30 minutes.
To one half of the resulting liquid add 1.5 ml of sodium
hydroxide solution and 3 ml of alkaline cupric-tartrate
solution and heat on a water-bath; a reddish brown precipitate
is fonned. To the other half of the liquid, add a few drops of
barium chloride solution; no precipitate is fonned (freedom
from agar).
Tests
Acacia and other soluble gums. To 20 ml ofa 2.5 per cent w/v
suspension of the powdered material prepared with freshly
boiled water add 10 ml of lead acetate solution; a flocculent
precipitate is fonned. Filter and add to the filtrate 10 ml of lead
subacetate solution; a slight cloudiness may appear, but there
is no precipitate.
Karaya gum. Boil 1 g with 20 ml of water until a mucilage is
fonned, add 5 ml of hydrochloric acid and again boil for
5 minutes; no pink or red colour develops.
Sterculia. A. Shake 0.2 g ofthe powdered material with 10 ml
of ethanol (60 per cent) in a 10-ml stoppered cylinder; any
gel fOlmed occupies not more than 1.5 ml.
B. Shake 1 g ofthe powdered material with 100 m1 of water and

titrate with 0.01 M sodium hydroxide, using methyl red
solution as indicator. Not more than 5.0 ml is required to change
Foreign matter. Not more than 1.0 per cent, detennined by the
following method. To 2.0 g ofthe powdered material in a 250ml round-bottomed flask add 95 ml of methanol, swirl to
moisten the powder and add 60 m1 of 7 M hydrochloric acid.
Add a few glass beads and heat under a reflux condenser in a
water-bath for 3 hours, shaking occasionally. Remove the glass
beads and filter the hot suspension under reduced pressure
through a sintered-glass crucible (porosity No.1). Rinse the
flask with a small quantity of water, passing the rinsings
through the filter. Wash the residue on the filter with about
40 ml of methanol and dry to constant weight at 110.
Evaporate to dryness on a water-bath, gently ignite, and
hydrochloric acidAsT and 45 ml of water. Remove the excess
ofbromine with 2 ml of stannous chloride AsT. The resulting
solution complies with the limit test for arsenic (3 ppm).
2547
TULASI
IP 2010
1.0g.
Microbial contamination (2.2.9). 1 g is free from Escherichia
coli and 10 g is free from salmonellae.
and thin smooth cuticle. Trichomes bent, consisting of 2-6

cells; glandular trichomes short, lamiaceae type, consisting of
one stock cell and 2-4 cells with rounded heads. Palisade
parenchyma consists of layer of long cylindrical cells
containing chlorophyll; spongy parenchyma consists of
polygonal cells with thin, straight or slightly wavy side walls.
Vascular bundles collateral type. Stomata diacytic.
Tulasi

Basil; Ocimum sanctum

vacuum to 10 ml.
Reference solution. Reflux 1 g of tulasi RS with 25 ml of
Apply to the plate 10 fJ.I of each solution as bands 10 mm by 2
with anisaldehyde sulphuric acid reagent. Heat the plate
110 for 10 minutes and examine the plate in day light. The
Tulasi consists of leaves of Ocimum sanctum Linn (Fam.
Lamiaceae).
Tulasi contains not less than 0.40 per cent w/w of eugenol,
Description. Greyish-black in colour having characteristic
odour with slightly pungent and aromatic taste.
.... _._.
Tests
by method!.
Identification
A. Macroscopic - Leaves simple, elliptic, 2.7-7.5 cm long,

1-3 cm wide, with acute top, cuneate, obtuse to rounded base,
margin entire, undulate or serrate, both surfaces thinly
pubescent and dotted; petiole 0.2-3.0 cm long. Flowers are
5-7 mm in length. It has both male and female parts. Calyx:
There are 5 sepals and it is greenish in colour. Corolla: There
are 5 petals, bilabiate in shape and covered with scattered
h' P t 1 h't' h
1
aIrs. e a s w 1 IS -purp e.

Heavy metals (2.3.13). 1.0 g complies with the limit testfor
heavy metals, Method B(20 ppm).
~hap~::{i~ppe~:~~~::~s~e~~~~:s~~i:~~;:~::s~~~:
__ Ass~~:.~~t~~i~~~!'~~~~~~~~:~~a~~~ (2:~:13)..
. .... _..
... Jlll!!clral1glJJ~rtr!!!l~12!!1:{ll1t9_e..U~.witJ;1Jmll_W!!11~!!!lgJ.bi!t~.rllQQt1L
. r?...s:Qltl!j()I1,R~j'lIIX_Q~~g.Q.f1b.e <::QaI~IYP()w..Q~r<:l.g1.!1:>~!IlJ1'<::~ .....

cuticle. On tangential view, these cells are polygonal with under examination with 50 ml of methanol on a water-bath for
straight or wavy walls. Lower epidermis consists ofa layer of

small, quadrangular transparent cells with thin walls and thin

methanol till the extract turns colourless, cooi and filter.
2548
.__
TULASI DRY EXTRACT
IP 2010

at 365 nm and in day light. The chromatographic profile ofthe

test solution is similar to that of the reference solution.

eugenol RS in methanol.
Tests
- a capillary column 30 m x 0.25 x 0.25 mm coated with100
per cent dimethylpolysiloxane
temperature:
oven 60 to 260 @l 0 per minute, (Initially and finally
hold for 5 minutes respectively)
Injector 240,
detector 280,
- split flow 20 ml per minute.
Inject 1 III of the reference solution. The test is not valid

by method I.
Test solution. Shakewell about 0.5 g of the extract under

examination in 100 ml ofthe methanol.
Inject the refemce solution and the test solution.

Calculate the content of eugenol.
Thlasi Dry Extract

Tulasi Dry Extract is obtained from the Tulasi leaves ( Ocimum
sanctum Linn, Fam. Laminaceae) by extraction with methanol
or other any suitable solvent and evaporation of solvent.
Tulasi Dry Extract contains not less than 90.0 per cent w/w
and not more than 120.0 per cent w/w ofthe stated amount of
the ursolic acid, calculated on the dried basis. It may contain
suitable added substances.
Reference solution. A 0.03 per cent w/v solution of ursolic

- a stainless steel column, 25 cm x 0.46 mm packed with
octadescylsilane bonded to porous (0.5 Ilm),
mobile phase: a mixture of30 volumes of mefhanol and
- flow rate. 0.6mlperminute,
than 2.0 per cent.
Description. A pale green powder.
Calculate the content of the ursolic acid in extract.

Identification
A. Determine by thin layer chromatography (2.4.17) coating

Storage. Store in a container purged with Nitrogen under the

refrigerated condition, protected from heat and moisture.
Mobile phase. A mixture of 95 volumes of chloroform and 5

volume of methanol.
Test solution. Shakewell 0.2 g ofthe extract under examination
with 10.0 ml methanol, filter.
Reference solution. A 0.02 per cent w/v solution of ursolic
and spray with anisaldehyde-sulphuric acid reagent. Heat
the plate at 110 for 10 minutes and examine in ultraviolet light
2549
VASAKA
IP 2010
Vasaka
Tests
Adulasa; Adhatoda vasica
Method I.

Vasaka consists ofthe dried mature leaves ofAdhatoda vasica
,substance under examination with 50 ml of methanol on a
Neese (Fam. Acanthaceae).
Vasaka contains not less than 0.6 per cent w/w of vasicine, further with methanol till the last extract turns colorless, cool
Reference solution. Dissolve 25 mg ofvasicine hydrochloride
RS in 50 ml of methanol. Dilute 5.0 ml ofthis solution to 50.0 ml
with methanol.
Description. Taste, bitter.
Identification
A. Macroscopic - Leafpieces membranous, brittle, greyishbrown, a few pieces green coloured. Floral bracts leaf-lilee.
B. Microscopic - Stomata diacytic, more on the lower
epidermis; glandular and non-glandular hair on both surfaces
ofleaf; elongated cystoliths present in palisade cells.

2 volumes of methanol and 0.2 volume of strong ammonia
solution.
IOml.
Reference solution. Reflux 0.5 g of vasaka RS with 50 ml
.~~._.~
- mobile phase: a mixture of 3 volumes of the solution
prepared by dissolving 1 g of sodium hexanesulphonate in 1000 ml of water, 1 volume of acetonitrile
and 20 volumes of glacial acetic acid,
injection volume. 20 f.!l.
Calculate the content of vasicine.
Apply to the plate 10 f.!l ofeach solution as bands 10 rom by 2 Vasaka Extract
mm.Allo.wthemobilephasetorise.8cm.Dry the.plateinaiL.~__ ._
~......................_
_~ __
llIld tl:x:alllille iIlllltrayioM ligl1tllt254IllllaIl<l36.5Illll,spraYYll~alelle:x:trll<::tj.s()1J!lliJJ~41Jye:x:trll<::til1gya,sllka(4d,hgtQclg
with dragendOiff's reagent. Heat the plate at 100 for 5-10 vasica Nees, Fam. Acanthaceae) dried matured leaves with
minutes and examine in day light. The chromatographic profile ethanol or any other suitable solvent. The powdered extract
ofthe test solution is similar to that ofthe reference solution. may contain suitable excipients.
2550
.~_._
YASTI .
IP 2010
Vasaka extract contains not less than 90.0 per cent w/w and
not more than 120.0 percent w/w of the stated amount of
vasicine.
Description. Light green to greenish brown powder;
characteristic odour and bitter taste.

Inject the refernce solution and the test solution.
Identification
Calculate the content of vasicine.

Usual strengths. 1 per cent w/w; 1.5 per cent w/w.


volumes of methanol and 1 volume of strong ammonia.
Test solution. Dissolve 1.0 g ofeJ:(.tract under examination with
25 ml of methanol and filter.
Reference solution. A 0.1 per cent w/v solution of vasicine
Yasti
Liquorice root; Mulethi; Glycyrrhiza glabra

2 mm. Allow the mobile phase to rise 8 cm.Spray the plate with
Dragendorff reagent.Dry the plate in air and examine in
ultraviolet light at 254nm and 365 urn.The chromatographic
solution.
7
6
Tests
Loss ofdrying (2.4.19). Not more than 6.0 per cent, detennined
on 2.0 g when dried at 105 for 3 hours.
. Microbial contamination (2.2.9). Complies with the microbial
contamination test.
Test solution. Dissolve about 1.5 g of the extract containing

20 mg ofvasicine in 100 ml of methanol by gently heating, and
filter.
Reference solution. A 0.025 per cent w/v solution ofvasicine
silica bonded with cyanopropyl groups (5 Ilm),
mobilephase: A mixture of 92 volumes ofbuffer solution
ortho phosphate in 500 ml of water and 2 ml of
orthophosphoric acid, dilute to 1000 ml with water, 5
volumes of acetonitrile and 3 volumes of
tetrahydrofitran,
Yasti consists of the dried, unpeeled roots and stolons of

Glycyrrhiza glabra Linn. (Fam. Leguminosae).
Yasti contains not less than 3.0 per cent w/w of glycyrrhizinic
acid.
Description. Odour, characteristic and slightly aromatic; taste,
very sweet and faintly astringent; the bark is not bitter.
Identification
A. Macroscopic - Root with few branches, up to 1 m long
and 0.5 to 3 cm in diameter. Bark, brownish-grey to brown with
longitudinal striations, bearing traces oflateral roots. Stolons,
cylindrical, 1 to 2 cm in diameter and up to several meters long,
but may be cut into lengths of 10 to 15 cm; similar in external
appearance to the root but with occasional small buds. Fracture
of the root and stolon, granular and fibrous. Cork layer, thin;
secondary phloem region, wide, light yellow with radial
striations; xylem, compact, yellow, with radiate structure. The
stolon has a central pith which is absent from the root.
E. Microscopic - Cork and phelloderm are narrow. Phloem

consisting ofbundles ofthick-walled, yellow fibres with narrow
2551
YASTI
IP 2010
lumina surrounded by cells each containing a calciumoxalate

prism, alternatingin the external layers with areas of strongly
hyaline keratenchyma; functional sieve tissue near the
cambium. Medullary rays parenchymatous, widening towards
the exterior, 3 to 12 cells wide. Xylem composed ofradial rows
oftracheids and vessels alternating with bundles of lignified
fibres with crystal sheaths similar to those of the secondary
phloem; vessels 30 J-lm to 150 J-lm in diameter with thick walls
(5 J-lm to 10 J-lm) having reticulate thickenings or numerous
bordered pits with slit-shaped openings associated with
lignified xylem parenchyma. Medullary rays, 2 to 5 cells wide.
Parenchymatous cells throughout containing simple, round,
oval or fusiform starch granules 2 J-lm to 20 J-lm, mostly 5 J-lm to
12 J-lm, in diameter; parenchymatous pith present solely in the
stolon.
Mobile phase. A mixture of 70 volumes of butyl alcohol, 20
volumes of water and 10 volumes of acetic acid.
Test solution. Add 10 ml of 70 per cent v/v methanol to 1 g of
dried yasti powder, heat by shaking on a water bath for
5 minutes, cool and filter.
Reference solution. Add 10 ml ono per cent v/v methanol to
1 g of dried yasti RS powder, heat by shaking on a. water bath
for 5 minutes, cool and filter

Test solution. Weigh accurately about 1.0 g of the coarsely
powdered substance under examination in a 250-ml conical
flask, add 100 ml of 0.1 M ammonia and mix with the aid of
ultrasound for 30 minutes. Centrifuge a part ofthe supernatant
liquid and dilute 1.0 m1 to 5;0 rn1 with 0.1 M ammonia. Filter the
solution through a membrane filter disc with an average pore
diameter not greater than 1.0 J-lm and use the filtrate.
glycyrrhizinic acid RS in 0.1 M ammonia.
octadecylsilane bonded to porous silica (5 J-lm),
mobile phase: a mixture of 6 volumes of glacial acetic
injection volume. 20 J-ll.
than 2.0 per cent.
Inject the test solution and the reference solution arid measure
the responses for the analyte peak.
Calculate the content of glycyrrhizinic acid.
Apply to the plate 10 J-ll of each solution as bands 10 mm by 2

105 for 10 minutes and examine the plate in day light. The
Yasti Dry Extract
D. Mix a small quantity, in powder, with 0.05 ml of sulphuric

acid; the powder particles become orange-yellow and some
fragments change, more slowly, to pinkish red.
Yasti Dry Extract contains not less than 90.0 per cent w/w and
not more than 120.0 per cent w/w of the stated amount of
glycyrrhizinic acid.
Yasti Dry Extract is obtained by extracting Yasti with water or

aqueous ethanol or any other suitable solvent.
Description. A yellowish-brown to dark brown powder.

Tests
Curcuma. When examined under a microscope, none of the
fragments ofthe powder in sulphuric acid (see Identification
test D) should immediately take on a carmine-red colour.
Identification

Water-soluble extractive (2.6.3). Not less than 20 per cent, Mobile phase. A mixture of70 volumes of butanol, 10 volumes
determined by the following method. Mix 2.5 g of the finely of acetic acid and 20 volumes of water.
powdered drug with 50 ml of water and allow to stand for Test solution. Dissolve 200 mg ofthe extract under examination
2 hours, shaking frequently. Filter, evaporate 10.0 g of the with 50 ml of methanol (70 per cent) and filter.
-----filtrateto-dryness-on-a-water-bath,drytheresidueatl05()and -Rejerencesolflti{fii.AO.1percent-w/vsolijlionofglycytthizin-weigh._ --- --------.---.-.---.-.-.--...-----.-.---.- .--ammonical hydrateRS in methanol 00 percent);
Apply to the plate 10 J-ll of each solution as bands 10 mm by
2552
YASTI DRY EXTRACT
IP 2010
and examine in ultraviolet light at 254 nm and 365 nm. Spray

the plate with anisaldehyde sulphuric acid reagent and heat
the plate at 105 for 10 minutes and examine the plate in day
light. The chromatogram obtained with test solution shows a
band corresponding to the band obtained with the reference
solution indicating the presence of glycyrrhizin.
Tests
Acid insoluble ash (2.3.19). Not more than 2.0 per cent.
mobile phase by mixing for 30 minutes with the aid ofultrasound

and dilute to 1OO.Oml with the mobile phase and filter.

glycyrrhizin ammonical hydrate RS in the mobile phase.
a stainless steel column 25 cm x 4.6 llllll, packed with
acid, 67 volumes of methanol and 33 volumes of 0.2 M
ammonium acetate in water,
flow rate. Iml per minute,
Test solution. Dissolve about 500 mg ofthe extract or a quantity

containing 7 mg of glycyrrhizinic acid and dissolve in the
Calculate the content of glycyrrhizinic acid in the extract.

2553
MONOGRAPHS
BLOOD AND BLOOD-RELATED PRODUCTS

Anti-A Blood Grouping Serum
Anti-B Blood Grouping Serum
Anti-D (Rho) ImrnlUloglobulin
Anti-D ImrnlUloglobulin for Intravenous Use
Anti-Hmnan Globulin Serum
Blood Grouping Serums Anti-D, Anti-C, Anti-E, Anti-c, Anti-e
Concentrated Hmnan Red Blood Corpuscles
CryoprecipitatedAntihaemophilic Factor
Dried HmnanAntihaemophilic Fraction
Fibrin Sealant Kit
HumanAlbumin
Hmnan Coagulation Factor IX
Hmnan Coagulation Factor vn
Hmnan Coagulation Factor VIII (rDNA)
HumanNonnal ImmlUloglobulin
Human Plasma Protein Fraction
Hmnan Prothrombin Complex
HmnanNonnallmmlUloglobulin for Intravenous Use
Plasma for Fractionation
Platelet Concentrate
Whole Human Blood
2555
2557
2557
2558
2559
2560
2560
2562
2562
2563
2566
2568
2570
2571
2572
2574
2576
2578
2579
2586
2588
2589
ANTI-B BLOOD GROUPING SERUM
IP 2010

Anti-A Blood Grouping Serum is a sterile, liquid or dried
preparation containing the particular blood group antibodies
derived from high-titered blood plasma or serum of human
subjects, with or without stimulation by the injection ofBlood
Group Specific Substance A (or AB). It contains a suitable
antimicrobial preservative. It is of two types polyclonal &
monoclonal.
Production
The monoclonal antibody technique devised by Kohler and
Milsten has proved useful in producing high titre and specific
antibodies. Laboratory animals, usually mice are immunized
for the production of monoclonal antibodies. After suitable
immune response, mouse spleen cells containing antibody
secreting lymphocytes are fused to neoplastic plasma cells of
infinite reproductive capacity from a mouse (that is myeloma
cells). The resulting hybridomas are screened for antibody
with the required specificity and affinity. The antibody
secreting clones may then be propagated in tissue culture or
by inoculation into mice in which case the antibodies are
collected as ascites. The clonal line produces a single antibody,
there is no need for absorption or to remove heterospecific
antibodies. All antibody molecules produced by a clone of
hybridoma cells are identical in terms of antibody structure
and antigen specificity. Once one antibody secreting clone of
cells has been established, antibody with same specificity
and reaction characteristics will be available indefmitely.
Identification
It agglutinates human red cells containing A-antigens that is
blood groups A and AB (including subgroups A lo A2, AlB,
and A2B but not necessarily weaker subgroups).
Tests
after the date of issue from manufacturer's cold storage (5,1

year; or 0, 2 years), provided that the expiration date for dried
serum is not later than 1 year after constitution.
Storage. Store at a temperature between 2 and go.
Labelling. The label states that the source material was not
reactive for hepatitis B surface antigen, but that no known
test method offers assurance that products derived from human
blood will not transmit hepatitis in case of polyclonal. Label
also states that it is for in vitro diagnostic use.
NOTE-The labeling is in black lettering imprinted on paper

that is white or is coloured completely or in part to match
the specified blue colour standard.

Anti-B Blood Grouping Serum is a sterile, liquid or dried
preparation containing the particular blood group antibodies
derived from high-titered blood plasma or serum of human
subjects, with or without stimulation by the injection ofBlood
Group Specific Substance B (or AB). It contains a suitable
antimicrobial preservative.
Production
The monoclonal antibody technique devised by Kohler and
Milsten has proved useful in producing high titre and specific
antibodies. Laboratory animals, usuallymice are immunized
for the production of monoclonal antibodies. After suitable
immune response, mouse spleen cells containing antibody
secreting lymphocytes are fused to neoplastic plasma cells of
infinite reproductive capacity from a mouse (that is myeloma
cells). The resulting hybridomas are screened for antibody
with the required specificity and affinity. The antibody
secreting clones may then be propagated in tissue culture or
by inoculation into mice in which case the antibodies are
collected as ascites. The clonal line produces a single antibody,
there is no need for absorption or to remove heterospecific
antibodies. All antibody molecules produced by a clone of
hybridoma cells are identical in terms of antibody structure
and antigen specificity. Once one antibody secreting clone of
cells has been established, antibody with same specificity
and reaction characteristics will be available indefinitely.
It complies with the test for potency, in parallel with, and not
less than equivalent to, the Reference Blood Grouping Serum
Anti-A, in agglutinating red blood cells from Group Al and
Group A2B donors. It complies with the tests for specificity
with Group AI, A2B, B, and 0 cells and confirms the absence
of contaminating antibodies reactive with Mg, Wra antigens
as well as other antigens having an incidence of I per cent or
greater in the general population (see under Blood Grouping
Serums Anti-D, Anti-C, Anti-E, Anti-c, rAnti-e). It complies Identification
with the tests for avidity with Group Al and A2B cells. All
fresh or frozen red blood cell suspensions used for these It agglutinates human red cells containing B-antigens, that is
tests are prepared under specified conditions and meet blood groups Band AB (including subgroups AlB and A2B).
specified criteria. Anti-A Blood Grouping Serum may be
"Tests
artificially coloured blue.
Expiration date. The expiration date for liquid serum is not

later than I year, and for dried serum not later than 5 years
It complies with the test for potency, in parallel with, and not
less than equivalent to, the Reference Blood Grouping Serum
2557
ANTI-B BLOOD GROUPING SERUM
IP 2010
Anti-B, in agglutinating red blood cells from Group B donors.

It complies with the tests for specificity with Group AI> B, and
o cells and confirms the absence of contaminating antibodies
reactive with Mg, WI" antigens as well as other antigens having
an incidence of 1.0 per cent or greater in the general population
(see under Blood Grouping Serums Anti-D, Anti-C, Anti-E,
Anti-c, Anti-e). It complies with the test for avidity with Group
B cells. All fresh or frozen red blood cell suspensions used for
these tests are prepared under specified conditions and meet
specified criteria. Anti-B Blood Grouping Serum may be
artificially coloured yellow.
Expiration date. The expiration date for liquid serum is not
later than 1 year and for dried serum not later than 5 years after
the date of issue from manufacturer's cold storage (5, 1 year;
or 0, 2 years), provided that the expiration date for dried serum
is not later than 1 year after constitution.
Storage. Store at a temperature between 2 and 8.
Labelling. The label states that the source material was not
reactive for hepatitis B smface antigen, but that no known
blood will not transmit hepatitis in case of polyclonal. Label
also states that it is for in vitro diagnostic use.
NOTE-The labelling is in black lettering imprinted on paper

that is white or is coloured completely or in part to match
the specified yellow colour standard.
Anti-D (Rho) Immunoglobulin

Human Anti-D Immunoglobulin
Human anti-D immunoglobulin is a liquid or freeze-dried
preparation containing immunoglobulins, mainly
immunoglobulin G. The preparation is intended for
intramuscular administration. It contains specific antibodies
against erythrocyte D-antigen and may also contain small
quantities of other blood-group antibodies. Human normal
immunoglobulin may be added.
It complies with the monograph on Human Normal
Immunoglobulin, except for the minimum number of donors
and the minimum total protein content. For products prepared
by a method that eliminates hmnunoglobulins with specificities
other than anti-D, where authorised, the test for antibodies to
hepatitis B surface antigen is not required.
Production
with D-positive erythrocytes that are compatible in relevant

blood group systems in order to avoid fonnation ofundesirable
antibodies.
Erythrocyte donors
Erythrocyte donors comply with the requirements for donors

prescribed in the monograph on Human Plasma for
Fractionation.
Immunisation
Immunisation ofthe plasma donor is carried out under proper

medical supervision. Recommendations concerning donor
immunisation, including testing of erythrocyte donors, have
been formulated by the World Health Organisation
(Requirements for the collection, processing and quality
control ofblood, blood components and plasma derivatives,
WHO Technical Report Series, No. 840, 1994 or subsequent
revision).
Pooled plasma
To limit the potential B19 virus burden in plasma pools used

for the manufacture ofanti-D immunoglobulin, the plasma pool
is tested for B 19 virus using validated nucleic acid
amplification techniques (2.8.1).
B19 virus DNA. Maximum 104 ill per ml.
A positive control with 104 ill ofB 19 virus DNA per ml and, to
test for inhibitors, an internal control prepared by addition of
a suitable marker to a sample ofthe plasma pool are included
in the test. The test is invalid if the positive control is nonreactive or if the result obtained with the internal control
indicates the presence of inhibitors.
BI9 virus DNA for NAT testing reference preparation is

suitable for use as a positive control.
IfHuman Normal Immunoglobulin is added to the preparation,
the plasma pool from which it is derived complies with the
above requirement for B 19 virus DNA.
Tests
Potency. Determine the assay of human anti-D
immunoglobulin by Method A (2.8.2). The estimated potency
is not less than 90.0 per cent of the stated potency. The
confidence limits (P = 0.95) are not less than 80.0 per cent and
not more than 120.0 per cent ofthe estimated potency.
Method B or C (2.8.3) may be used for potency determination

if a satisfactory correlation with the results obtained by
Method A has been established for the particular product.
Storage. For the liquid preparation, store protected from light,

..... _H!1!I!!ll1!ll1ti:I::> i!I!!I!1ll1og1()1:>ulil1i~.J:lr:t'lfeI'l1lJ1Y()1:>tl1i.l1t'ltlft()fl1 .ina-plasticcontainer;Forthefreeze-driedpreparation,store
the plasma of donors with a sufficient titre of previously
acqulre(ranti.~D
-protectedfromlight,inaplastic container;
.
anti.bbClIes. Where necessary; In order to
ensure an adequate supply ofhuman anti-Dimmunoglobulin,

it is obtained from plasma derived from donors immunised
Labelling. The label states (1) for liquid preparations, the

volume of the preparation in the container and the protein
2558
_.
IP 2010
ANTI-D IMMUNOGLOBULIN FOR INTRAVENOUS USE
content expressed in grams per litre; (2) for freeze-dried

preparations, the quantity of protein in the container; (3) the
route of administration; (4) for freeze-dried preparations, the
name or composition and the volume of the reconstituting
liquid to be added; (5) where applicable, that the preparation
is suitable for use in the prophylaxis of hepatitis A infection;
(6) where applicable, the anti-hepatitis A virus activity in
International Units per ml; (7) where applicable, the name and
amount of antimicrobial preservative in the preparation; (8)
the number ofInternational Units per container.
3.
Antibodies against hepatitis C virus (anti-HCV).
4.
Hepatitis B surface antigen (HBsAg),
Pending complete harmonisation of the laboratory tests to be

carried out, the competent authority may require that a test for
alanine aminotransferase (ALT) also be carried out.
The test methods used are of suitable sensitivity and
specificity and comply with the regulations in force. Ifa repeatreactive result is found in any of these tests, the donation is
not accepted.
Immunisation
Anti-D Immu.noglobulin for

Intravenous Use
Anti-D Immunoglobulin Human for Intravenous Use
Anti-D Immunoglobulin for intravenous administration is a
liquid or freeze-dried preparation containing immunoglobulins,
mainly immunoglobulin G. It contains specific antibodies
against erythrocyte D-antigen and may also contain small
quantities of other blood-group antibodies. Human normal
immunoglobulin for intravenous use may be added.
It complies with the monograph on Human Normal
Immunoglobulin for Intravenous Administration, except for
the minimum number of donors, the minimum total protein
content, the limit for osmolality and the limit for prekallikrein
activator. For products prepared by a method that eliminates
immunoglobulins with specificities other than anti-D where
authorised, the test for antibodies to hepatitis B surface antigen
is not required; a suitable test for Fc function is carried out.
Production
Polyclonal (human) anti Rh (D) serum
Immunisation ofthe plasma donor is carried out under proper

medical supervision. Recommendations concerning donor
immunisation, including testing of erythrocyte donors, have
been formulated by the World Health Organisation
(Requirements for the collection, processing and quality
control ofblood, blood components and plasma derivatives,
WHO Technical Report Series, No. 840, 1994 or subsequent
revision).
Pooled plasma
To limit the potential B 19 virus burden in plasma pools used
for the manufacture of anti-D immunoglobulin, the plasma
pool is tested for B 19 virus using validated nucleic acid
amplification techniques (2.8.1).
Identification
Examine by a suitable immunoelectrophoresis technique. Using
antiserum to normal human serum, compare normal human
serum and sample under examination in a dilution of 109 per
litre ofprotein. The main component ofthe sample corresponds
to the IgG component ofnormal human serum.
Tests
Human anti-D immunoglobulin is preferably obtained from

the plasma of donors with a sufficient titre of previously
acquired anti-D antibodies. Where necessary, in order to
ensure an adequate supply ofhuman anti-D immunoglobulin,
it is obtained from plasma derived from donors immunised
with D-positive erythrocytes that are compatible in relevant
blood group systems in order to avoid formation ofundesirable
antibodies.
B19 virus DNA. Maximum 104 IU per ml.

A positive control with 104 IU ofB 19 virus DNA per ml and,
to test for inhibitors, an internal control prepared by addition
ofa suitable marker to a sample ofthe plasma pool are included
in the test. The test is invalid if the positive control is nonreactive or if the result obtained with the internal control
indicates the presence of inhibitors.
Bi9 virus DNA for NAT testing RP is suitable for use as a
positive control.
Erythrocyte donors
Laboratory tests are carried out for each donation to detect
the following viral markers:
1.
Antibodies against human immunodeficiency virus 1 (antiHlV-I),
If Human normal immunoglobulin for intravenous

administration is added to the preparation, the plasma pool
from which it is derived complies with the above test for B19
virus DNA.
2.
Antibodies against human immunodeficiency virus 2 (antiHlV-2),
Assay. Determine by Method A of human anti-D

immunoglobulin (2.8.2). The estimated potency is not less
2559
IP 2010
ANTI- HUMAN GLOBULIN SERUM
than 90.0 per cent ofthe stated potency. The confidence limits
(P = 0.95) are not less than. 80.0 per cent and not more than
120.0 per cent of the estimated potency.
MethodB or C (2.8.3) may be used for potency detennination
ifa satisfactory correlation with the results obtained by Method
A has been established for the particular product.
Storage. For the liquid preparation, store protected from light,
in a colourless glass container, at the temperature stated on
the label. For the freeze-dried preparation, store protected from
light, in colourless glass container, at a temperature not
exceeding 25 0
content expressed in gram per litre; (2) for freeze-dried
amount of immunoglobulin in the container; (4) the route of
administration, (5) for freeze-dried preparations, the name or
composition and the volume of the reconstituting liquid to be
added; (6) the distribution of subclasses of immunoglobulin
G present in the preparation; (7) where applicable, the amount
of albumin added as a stabilizer; (8) the maximum content of
immunoglobulin A; (9) the number ofIntemational Units per
container.
suspended in isotonic saline sensitized with decreasing

amounts of nonagglutinating Anti-D (Anti-Rho) serum, and
with cells sensitized in the same manner with an
immunoglobulin IgG Anti- Fya serum ofsimilar potency. AntiHuman Globulin Serum containing one or more Anticomplement components complies with the tests for potency
in giving a 2+ agglutination reaction (Le., agglutinated cells
dislodged into many small clumps of equal size) by the lowionic sucrose or sucrose-trypsin procedures when tested as
recommended in the labelling. Anti-Human Globulin Serum
containing Anti-3Cd activity meets the requirements for
stability, by potency testing of representative lots every 3
months during the dating period.
Expiration date. Its expiration date is not later than 1year after
the date of issue from manufacturer's cold storage (5, I year;
or 0, 2 years).
Labelling. The label states the animal source of the product,
the specific antibody activities present; the application for
which the reagent is intended; a cautionary statement that it
does not contain antibodies to immunoglobulins or that it
does not contain antibodies to complement components,
wherever and whichever is applicable; and states that it is. for
in vitro diagnostic use.
NOTE-The lettering on the label of the general-purpose

polyspecijic reagent is black on a white background. The
Anti-Human Globulin Serum is a sterile, liquid preparation of label ofall other Anti-Human Globulin Serum containers is
serum produced by immunizing lower animals such as rabbits in white lettering on a black background.
or goats with human serum or plasma, or with selected human
plasma proteins. It is free from agglutinins and from hemolysins
to nonsensitized human red cells of all blood groups. It
contains a suitable antimicrobial preservative.
Blood Grouping Serums Anti-D, Anti-
Anti-Human Globulin Serum
Three varieties ofAnti-Human Globulin Selum are recognized:

(1) a general-purpose polyspecific reagent which, as a
minimum, contains antibodies specific for immunoglobulin IgG,
and at least the C3d component of human complement (for
use in the direct antiglobulin test, it contains this Anti-C3d
and Anti-IgG activity) and which may be artificially coloured
green; (2) a reagent containing antibodies only against
immunoglobulin IgG (not heavy chain specific) intended for
use in the indirect antiglobulin test, and which may be
artificially coloured green; and (3) reagents containing
antibodies specific for individual or selected components of
human complement, such as Anti-C3, and Anti-C3b-C3d-C4,
or a single class ofimmunoglobulins, such as Anti-IgG (heavy
chainspecific),used onlyt03dentify plasma components
C, Anti-E, Anti-c, Anti-e

Anti-Rh Blood Grouping Serums
Blood Grouping Serums Anti-D, Anti-C, Anti-E, Anti-c, Antie (Anti-Rh Group) are sterile, liquid or dried preparations derived
from the blood plasma or serum of human subjects who have
developed specific Rh antibodies. They are free from
agglutinins for the A or B antigens and from alloantibodies
other than those for which claims are made in the labelling.
They contain a suitable antimicrobial preservative. Liquid
serums are not artificially coloured. Two varieties ofAnti-Rh
Blood Grouping Serums are recognized, i.e., (1) saline
agglutinating "complete" antiserums, which specifically
agglutinatehumalLIed blood. cells suspended in saline and
cQat~c::l Qll t1;l~sllrfac:eofI't:c::l1::>looc::lc:c:lli>.Anti~II1ll11a!1.<::i:19_1::>1lIil1 (2) ''1::>locking or inc:ol11pl<::t~"a.t;ltii>~I'lll11i>,wllicl1C:Ql1ta.inPI9t e:!11
Serum containing Anti-IgG complies with the test for potency, or other macromolecular substances, usually require the cells
in parallel with the Reference Anti-Human Globulin (Anti- to be suspended in serum or plasma,and generally are for
IgG) Serum (at a 1:4 dilution) when tested with red cells slide or rapid tube tests. The most commonly used of these
2560
BLOOD GROUPING SERUMS ANTI-D, ANTI-C, ANTI-E, ANTI-c, ANTI-e
IP 2010
blood grouping serums are listed in Table 1 each reacting with

the antigen(s) designated by the corresponding letter(s) with
the alternative nomenclature indicated parenthetically.
Table - 1
antigens having an incidence of 1.0 per cent or greater in the

general population, except where some ofthese confirmatory
tests can not be done by the manufacturer, in which event
such omissions are noted.
Table-3
Serum
Antigen(s) Reacting
Anti-D (Anti-Rho)
D(Rho)
Serum
Cells
Anti-C (Anti-rh')
C(rh')
Anti-D
Anti-E (Anti-rh")
E(rh")
Anti-CD (Anti-Rho')
D (Rho) and C (rh')
CcDe, cDe, Ccde, cdEe, Al cde, B cde, 0 cde,

and where recommended for use by indirect
antiglobulin technique, cde Bg(a+) cells from 3
different donors
Anti-DE (Anti-Rh o")
D (Rho) and E (rh")
Anti-C
Anti-CDE (Anti-Rho
Anti-c (Anti-hr')
D (Rho), C (rh'), and E (rh')

c (hr')
cDe, Ccde, cdEe, C + rh i neg. cells, Al cde, B

cde, 0 cde
Anti-E
cDe, Ccde, cdEe, Al cde, B cde, 0 cde
Anti-e (Anti-hr")
e (hr")
Anti-CD
cDe, Ccde, cdEe, Al cde, B cde, 0 cde, and

where recommended for detection ofthe G
antigen, rGr
Anti-DE
cDe, Ccde, cdEe, Al cde, B cde, 0 cde
Anti-CDE
cDe, Ccde, cdEe, Al cde, B cde, 0 cde, and

where recommended for detection ofthe G
antigen, rOr
Anti-c
Ccde, Al CDe, B CDe, 0 CDe, and CDEe or

CDEorCdE
Anti-e
cdEe, Al cDE, B cDE, 0 cDE, and CcDE or

CDEorCdE
lll
)
Each serum complies with the test for potency in the case of
serums for saline tube test in parallel with, and not less than
equivalent to, the Reference Blood Grouping Serum for AntiD, Anti-C, or Anti-E, whichever is applicable, or, in the case of
Anti-c and Anti-e for saline tube test which have no reference
preparations, the test for minimum agglutination reactivity at
a specified dilution; and in the case of serums for slide or
rapid tube test in parallel with, and not less than equivalent to,
the Reference Blood Grouping Serum for Anti-D, Anti-C, AntiE, Anti-c, or Anti-e, whichever is applicable, in agglutinating
as a minimum red blood cells from the donors indicated in
Table 2 (which may be from Group A, B, AB, or 0).
Each serum for slide or rapid tube test complies with the tests
for avidity with the cells as indicated under tests for potency
above.
Monoclonal Rh (D) antibodies. Monoclonal antibodies are

derived from hybridoma cell lines produced by fusing mouse
antibody produced by B lymphocyte with mouse myeloma
cells or are derived from a human B cell line through EpsteinBarr Virus (EBV) transformation. Each hybridoma cell line
produces homogenous antibodies ofonly one immunoglobulin
class, which are identical in their chemical structure and
immunological activity.
Table - 2
Serum
Phenotype of Cells
Anti-D
cDe
Anti-C
Ccde
Anti-E
cdEe
Anti-CD
cDe,Ccde
Anti-DE
cDe,cdEe
Anti-CDE
cdEe, cDe, Ccde
Anti-c
CcDEe
Anti-e
cdEe
All fresh or frozen red blood cell suspensions used for these
tests are prepared under specified conditions and meet
specified criteria.
The type of monoclonal anti-D reagents are:
Each serum complies with the tests for specificity by the most
sensitive method recommended by the manufacturer, in which
not less than 4 positive and 4 negative phenotypes are included
(see Table 3), and confirms the absence of contaminating
antibodies reactive with Mg, WI:" antigens as well as other
1.
IgM anti-D monoclonal reagent,
2.
Blend ofIgM and IgG monoclonal antibodies reagent,
3.
Blend of monoclonal IgM and polyclonal (human)

IgGanti-D.
IgM anti-D monoclonal antibodies are highly specific and

saline reacting equally well at room temperature and at 37.
They are good for slide test or immediate spin tube tests as
well as routine Rh(D) typing in tube. IgM anti D are unreliable
for detection ofweak D (Du) by AHG test. Blend ofIgM and
2561
IP 2010
CONCENTRATED HUMAN RED BLOOD CORPUSCLES
IgG (monoclonal) anti-D or blend ofIgM (monoclonal) and

polyclonal (human) IgG anti-D can be used for testing weak D
(Du) antigen by AHG test. Mostly blended IgM and IgG
(monoclonal) anti- Rh(D) or blended monoclonal IgM and
polyclonal (human) IgG anti Rh (D) antibodies are used now
in routine.
Expiration date. The expiration date for liquid serums is not
later than 1 year and for dried serums not later than 5 years
after date of issue from manufacturer's cold storage (5, I
year; or 0, 2 years), provided that the expiration date for dried
serums is not later than 1 year after constitution.
Labelling. The label each to state that the source material was
not reactive for hepatitis B surface antigen, but that no known
blood will not transmit hepatitis. Label each to state that it is
for in vitro diagnostic use.
Concentrated Human Red Blood Corpuscles should be

administered only with suitable equipment meant for the
transfusion of blood and blood components.
Concentrated Human Red Blood

Corpuscles
Concentrated Human Red Blood Corpuscles contains not less'

than 15.5 per cent w/v of haemoglobin.
Concentrate ofHuman Red Blood Cells; Packed Red Cells

Concentrated Red Blood Cells (RBCs) are units ofwhole blood
with most of the plasma removed. Additive red cell
preservative systems consist of a primary collection bag
containing an anticoagulant preservative with at least two
satellite bags integrally attached, one is empty and one
contains an additive solution (AS).
Each 100 ml citrate phosphate dextrose (CPD) contains
Citric acid (monohydrate)
0.327 g
Sodium citrate (dihydrate)
2.63 g
Sodium acid phosphate (dihydrate)
0.251 g
Dextrose (monohydate)
2.55 g
Water for injection
100 ml
solution equivalent to not less than 40.0 per cent of the total
volume of Whole Human Blood. A portion of the plasma,
sufficient to ensure optimal cell preservation, shall be left. All
surfaces that come in contact with the red cells and plasma
shall be sterile 'and pyrogen-free and the entire processing of
the blood shall be conducted in a sterile system or in a closed
system by use of satellite bags. The final containers used for
the Concentrated Human Red Blood Corpuscles shall be the
original blood containers unless the method of processing
requires a different container. Immediately after processing,
the containers are stored at a temperature between 2 and 8
and not opened until immediately before transfusion. Human
Red Blood Corpuscles may be stored for a period not longer
than that for which the Whole Human Blood from which it is
prepared. However, if the hermetic seal is broken during
processing, the product must be used within 24 hours. From
the tube of the blood bag sample is taken for compatibility
and infections markers testing.
Description. A dark red fluid when prepared; after standing,

the red corpuscles may form a sediment, leaving a small
supernatant layer of yellowish plasma.
Tests
Sterility (2.2.11 ). Complies with the tests for sterility,
determined by Method B.
Assay. Determine the haemoglobin content by photometric
haemoglobinometly (2.8.12).
Storage. Store in containers which are made ofcolourless and
transparent glass, or of a suitable plastic material, are sterile
and sealed so as to exclude micro-organisms. Store at a
temperature between 2 and 8.
AS contains sodium chloride, dextrose, adenine and other

substances that support red cell survival and function up to
42 days. The volume of AS in 350 ml is 49 ml and in 450 ml is
63 m!. AS is added to the red cells remaining in the primary bag
after most ofthe plasma has been removed. This allows blood
centres to use or recover a maximum amount of plasma, yet
still prepare a red cell component with a final haematocrit
between 55.0 per cent and 65.0 per cent, a level that facilitates
excellentflowratesand allows_easy administration.
.__
It may be prepared by centrifugation or undisturbed

sedimentation for the separation ofplasma and anticoagulant
Labelling. The label states (1) the reference number of the

Whole Human Blood from which the preparation was made;
(2) the ABO and Rh groups of the Whole Human Blood; (3)
the date ofcollection ofthe Whole Human Blood from which
the preparation was made; (4) the storage conditions; (5) the
date after which the preparation is not suitable for transfusion;
(6) that the preparation should not be used if there is any
visible evidence ofhaemolysis or other deterioration.
Cryoprecipitated,Antihaemophilic
Factor
Cryoprecipitated Antihaemophilic Factor is a sterile, frozen
concentrate of human antihaemophilic factor prepared from
2562
DRIED HUMAN ANTIHAEMOPHILIC FRACTION
IP 2010
the Factor VIII-rich cryoprotein fraction of human venous

plasma obtained from suitable whole-blood donors from a
single unit of plasma derived from whole blood or by
plasmapheresis, collected and processed in a closed system.
It contains no preservative. It complies with the test for
potency by comparison with the Standard Antihaemophilic
Factor (Factor VIII) or with a working reference that has been
calibrated with it, in having an average potency of not less
than 80 Antihaemophilic Factor Units per container, made at
intervals of not more than 1 month during the dating period.
human donors who are, as far as can be ascertained after

clinical examination, laboratory tests on their blood and
consideration of their medical history, free from detectable
agents of infection transmissible by blood transfusion. The
examinations and tests to be carried out are decided by the
National Regulatory Authority. In particular, the blood must
be tested with negative results for (a) evidence of syphilitic
infection; (b) hepatitis B surface antigen and (c) HIV antibodies
by suitably sensitive methods. The haemoglobin value of the
donor's blood is not less than 12.5 per cent w/v.
Expiration date. The expiration date is not later than I year

from the date of collection of source material.
The blood is withdrawn aseptically through a closed system

of sterile tubing into a sterile container in which a suitable
anticoagulant solution has been placed before sterilisation.
During the withdrawal there is.no interruption in the flow from
the donor, and the container is gently agitated. Immediately
after the withdrawal is completed, the blood is cooled t04; if
the plasma is to be stored frozen it is separated from the cellular
components by centrifugation and frozen to -30 0 or below,
preferably within 12 hours ofcollection; ifthe plasma is not to
be frozen it is separated from the cellular components by
centrifugation as so.on as possible and not later than 18 hours
after collection, and fractionation begun without delay.
Storage. Store in hennetic containers at a temperature of - 18

or lower.
Labelling. Label it to indicate (1) the ABO blood group
designation and the identification number of the donor from
whom the source material was obtained; (2) with the type and
result of a serologic test for syphilis, or to indicate that it was
non-reactive in such test; with the type and result of a test for
hepatitis B surface antigen, or to indicate that it was nonreactive in such test; with a warning not to use it if there is
evidence of breakage or thawing; with instructions to thaw it
before use to a temperature between 20 and 37, after which
it is to be stored at room temperature and used as soon as
possible but within 6 hours after thawing; (3) to state that it is
to be used within 4 hours after the container is entered; (4) to
state that it is for intravenous administration; (5) that a filter is
to be used in the administration equipment.
Dried Human Antihaemophilic

Fraction
Dried Factor VIII Fraction; Freeze-dried Human Coagulation
FactorVII1
Dried Human Antihaemophilic Fraction is a preparation of
antihaemophilic factor which is obtained from human plasma.
It is rich in clotting factor VIII.
When the contents of a sealed container of Dried Human
Antihaemophilic Fraction are dissolved in a volume ofwater
equal to the volume of waterfor injection stated on the label,
the resulting solution contains not less than 3.0 Units per ml,
not less than 0.1 Unit per mg ofprotein, not more than 80.0 per
cent ofwhich is fibrinogen, and not more than 200 millimoles
of sodium ions per litre.
Dried Human Antihaemophilic Fraction may be prepared :Ii-om

human plasma so obtained by precipitation under controlled
conditions ofpH, ionic strength and temperature with organic
solvents, or by freezing and thawing. The precipitate may be
washed by extraction with suitable solvents, dissolved in a
solution ofsodium citrate adjusted to a pH of6.8 to 7.2, which
may also contain sodium chloride. The solution is sterilised
by filtration through a membrane filter, distributed in sterile
containers and dried from the frozen state. The air is removed
or replaced by oxygen-free nitrogen and the containers are
sealed so as to exclude micro-organisms. No antimicrobial
preservative is added but an antiviral agent may be added
provided that it can be demonstrated to have no deleterious
effect on the final product in the amount present and to cause
no adverse reaction in man. Heparin may be used.
For the following tests, where it is directed that a solution is to
be used, dissolve the contents ofa sealed container in a volume
of the appropriate solvent equal to the volume of water for
injection stated on the label.
Description. A white or pale yellow powder or friable solid.
Identification
Production
A. Precipitation tests with a suitable range of species specific

antisera give positive results for the presence of plasma
proteins of human origin and negative results with antisera
specific to plasma proteins of the other species.
The plasma to be used for preparing Dried Human

Antihaemophilic Fraction is 'obtained from blood of healthy
B. A fi:eshly prepared solution in water causes a reduction in

clotting time when treated as directed under Assay.
2563
IP 2010
Tests
pH (2.4.24).6.8 to 7.4, determined on the reconstituted solution.
Loss on drying (2.4.19). Not morethan 2.0 per cent, determined
by drying 0.5 g over phosphorus pentoxide at a pressure not
exceeding 3 kPa for 24 hours.
Haemagglutinins, anti-A and anti-B. Dissolve in water. Dilute
the solution with saline solution to produce a solution
containing 3 Units per ml. Carry out the test for
haemagglutinins anti-A and anti-B using a suitable indirect
method such as that described below.
Prepare in duplicate serial dilutions of the preparation under
examination in saline solution. To each dilution of one series
add an equal volume ofa 5.0 per cent v/v suspension ofgroup
AI red blood cells previously washed three times with saline
solution. To each dilution of the other series add an equal
volume of a 5.0 per cent v/v suspension of group B red blood
cells previously washed three times with saline solution.
Incubate the suspensions at 37 for 30 minutes and then wash
the cells three times with saline solution. Leave the cells in
contact with a polyvalent anti-human globulin reagent for 30
minutes. Without centrifuging, examine each suspension for
agglutination under a microscope. The 1 in 64 dilutions do not
show agglutination.
Hepatitis B surface antigen. Dissolve in water. Examine the
solution by a suitably sensitive method such as
radioimmunoassay. Hepatitis B surface antigen is not detected.
Abnormal toxicity (2.2.1). When dissolved in water for
injection, complies with the test for abnormal toxicity, Method
B, injecting into each mouse a volume containing 1.5 Units
and into each guinea-pig a volume containing 15 Units.
Pyrogens (2.2.8). When dissolved in water for injection,
complies with the test for pyrogens, using a volume containing
10 Units per kg of the rabbit's weight in rabbits that have not
previously received blood products.
Assay
For potency - Carry out the biological assay of human
antihaemophilic fraction described below. The estimated
potency is not less than 80.0 per cent and not more than 125.0
per cent of the stated potency. The fiducial limits of error are
not less than 64.0 per cent and not more than 156.0 per cent of
the stated potency.
Biological assay
to produce the same effect under the conditions of the

following method ofassay.
The Standard preparation is the 4th International Standard for
Blood coagulation factor VIII:C,concentrate, human,
established in 1989, consisting of an intermediate purity
concentrate of human blood clotting factor VIII (supplied in
ampoules containing 6.3 Units of clotting factor VIII), or
another suitable preparation the potency of which has been
determined in relation to the International Standard
The Unit is the specific antihaemophilic factor contained in
such an amount of the Standard preparation as the Ministry
ofHealth & Family Welfare, Govt. ofIndia may from time to
time indicate as the quantity exactly equivalent to the Unit
accepted for international use.
Special reagents
Normal serum reagent. Collect normal human blood in a dry,
sterile, glass bottle, shake continuously until coagulation is
complete, incubate at 37 for 3 hours, maintain at 4 overnight,
remove the serum, store at -20, dry from the frozen state and
keep in a vacuum desiccator over phosphorus pentoxide.
Dissolve a quantity of the dried serum calculated to have
been obtained from 1 ml of the serum in sufficient imidazole
bufferpH 7.4 to produce 10 ml and allow to stand at 4 for 16
to 24 hours.
Phospholipid. Wash a quantity of normal human or bovine
brain freed from meninges and blood vessels and macerate in
a suitable blender. Weigh 1,000 to 1,300 g ofthe macerate and
measure its volume (V). Extract with three quantities, each of
4 V ml, of acetone, filter by suction and dry the precipitate at
37 for 18 hours. Extract the dried precipitate with two
quantities, each of2 V ml, ofa mixture oftwo volumes of light
petroleum (boiling range 30 to 40) and 3 volumes of light
petroleum (boiling range 40 to 60), filtering each extract
through a filter paper previously washed with the light
petroleum mixture. Combine the extracts' and evaporate to
dryness at 45 at a pressure not exceeding 0.7 kPa. Dissolve
the residue in 0.2 V ml of ether and allow to stand at 4 until a
deposit forms. Centrifuge and evaporate the clear supernatant
liquid under reduced pressure until the volume is about 100 ml
per kg of the original macerate. Allow to stand at 4 until a
precipitate forms (12 to 24 hours) and centrifuge. To the clear
supernatant liquid add 5 times its volume of acetone, centrifuge,
discard the supernatant liquid, dry the precipitate and store
protected from light in a vacuum desiccator.
Thep()tel1cy.()fl111111al1al1ti1Jfl~p:1()Pl1ili()fracti()l1js4~!~l1J1iJ.1~(1
. . . I'ltosPIt()lipilil"eagellt.. ~llspen4 0,125 g ()fphospho.Pp.(cijl1
by comparing the amount necessary to reduce the clotting

time ofa test mixture containing substances that cause clotting
ofblood with the amount ofthe Standard preparation necessary
S.
ml of water, shake and stir until a uniform suspension is

obtained. Prepare a dilution with saline solution that will give
minimum clotting time consistent with the largest clotting time
2564
IP 2010
differences between consecutive dilutions of the Standard should have a clotting time, when tested by the assay method
preparation and the preparation under examination. The . given under clottingfactor V solution, of 70 to 100 seconds;
concentration usually lies between 50 and 250 Ilg per ml. The ifthe clotting time is less than 70 seconds, incubate the plasma
for a further 12 to 24 hours.
diluted suspension may be kept at -20 for 6 weeks.
Clotting factor V solution. Prepare from fresh oxalated bovine
plasma by fractionation at 4 with a saturated solution of
ammonium sulphate prepared at 4. Use the fraction
precipitating between 38 per cent and 50 per cent saturation
(which contains clotting factor V not significantly contaminated
with clotting factor VIII), dialysed to remove ammonium
sulphate and diluted with saline solution to give a solution
containing between 10 per cent and 20 per cent ofthe amount
of clotting factor V present in fresh normal human plasma.
Detelmine the clotting factor V content of the solution as
follows. Prepare two dilutions in imidazole blif.fer pH 7.4 to
contain 1 volume of the solution under examination in 10
volumes and 20 volumes respectively. Test each dilution as
follows. Mix 0.1 ml each of substrate plasma deficient in
clotting factor V, the dilution under test, thrombokinase
extract and O.025M calcium chloride. Record as the clotting
time the interval between the addition ofthe calcium chloride
solution and the first indication of fibrin formation, which
may be observed visually or by mechanical means.
Similarly determine the clotting times, in duplicate, for four
dilutions ofpooled normal human plasma in imidazole blif.fer
pH 7.4 containing 1 volume in 10 volumes (equivalent to 100
per cent of clotting factor V), in 50 volumes (20 per cent), in
100 volumes (10 per cent), and in 1,000 volumes (1 per cent),
respectively.
To calculate the result, plot the mean of the clotting times for
each dilution of human plasma on double cycle log/log paper
against the equivalent percentage of clotting factor V and
read the percentage of clotting factor V for the two dilutions
of clottingfactor V solution by interpolation from the curve.
The mean of the two results is taken as the percentage of
clotting factor V in the solution.
Substrate plasma. Separate the plasma from 9 volumes of
human or bovine blood collected in 1 volume ofa 3.8 per cent
w/v solution of sodium citrate or from 3.5 volumes ofhuman
or bovine blood collected in 1 volume ofa solution containing
2.0 per cent w/v of sodium acid citrate and 2.5 per cent w/v of
dextrose. In the former case, prepare the substrate plasma on
the day of collection of the blood. In the latter case, the
substrate plasma may be prepared up to 2 days after collection
ofthe blood. Store at-20.
Substrate plasma deficient in clotting factor V. Preferably
use congenitally deficient plasma or, alternatively, prepare as
follows. Separate the plasma from human blood collected in
one-tenth its volume ofa 1.34 per cent w/v solution of sodium
oxalate and incubate at 37 for 24 to 36 hours. This plasma
Storage. Store in small amounts, at -20 or below.

Suggested method
Dissolve the contents of the sealed container of the substance
under examination in the volume of the liquid stated on the
label and use immediately. Reconstitute the entire contents of
one ampoule of the Standard preparation as stated on the
label and use immediately.
To the reconstituted Standard preparation and the preparation
under examination, add sufficient imidazole blif.fer pH 7.4 to
produce solutions containing between 0.5 and 2 Units per ml;
these solutions are stable for 15 minutes at200. Using a mixture
of 1 volume of a 3.8 per cent w/v solution of sodium citrate
and 5 volumes of saline solution as the diluent, make from the
solutions three successive 2-fold dilutions in the range 1 in16
to 1 in 256 so that all the clotting times are between 17 and 35
seconds; the dilutions must be accurately made and used
immediately.
Introduce into each of six glass incubation tubes (75 rom to
100 min) 0.1 ml each of clottingfactor V solution,phospholipid
reagent and normal serum reagent. To the first tube add 0.1
ml of the highest dilution of the Standard preparation, place
the tube in a water-bath at 37, add 0.1 ml ofO.05M calcium
chloride and start a. stop-watch. During the next minute add
0.1 ml of the second highest dilution of the standard to a
second tube, place it in the water-bath, and add 0.1 ml of
O.05M calcium chloride at exactly 1 minute by the stop-watch.
Repeat the procedure with the lowest dilution of the standard
and the highest to lowest dilutions of the preparation under
examination so that the calcium chloride solution is added at
2,3,4 and 5 minutes by the stop-watch, respectively.
Place in a water-bath at 37, twelve glass tubes each containing
0.2 ml of O.025M calcium chloride and a further tube
containing about 3 ml of substrate plasma. At 14 minutes, 40
seconds by the stop-watch, transfer 0.1 ml ofthe mixture from
the first incubation tube to one ofthe tubes containing 0.2 ml
of O.025M calcium chloride solution and mix. At 15 minutes
add 0.2 ml ofthe warmed substrate plasma and, using a second
stop-watch, record as the clotting time the interval between
the addition ofthe substrate plasma and the first indication of
fibrin formation, which may be observed visually or by
mechanical means. Repeat the procedure with the other
incubation tubes at I-minute intervals and carry out a second
series of determinations at 21 to 26 minutes. The period of
incubation should, ifnecessary, be adjusted so that the clotting
times recorded in the corresponding tests in the two series of
determinations do not differ by more than 5.0 per cent, showing
2565
DRIED HUMAN ANTlHAEMOPHILIC FRACTION
IP 2010
that a stable plateau of prothrombin activator formation has

been reached.
Carry out a blank determination using in place of the
preparation under examination, an equal quantity ofa mixture
of 1 volume of a 3.8 per cent w/v solution of sodium citrate
and 5 volumes of saline solution. The result of the assay is
not valid unless the dotting time in the blank determination is
more than 40 seconds. Calculate the result of the assay by
standard statistical methods.
For totalprotein. Dilute 1.0 ml to 10.0 ml with saline solution.

Determine the assay described under Human Plasma using
5.0 ml ofthe dilution and beginning atthe words "add 0.2 ml of
a 7.5 per cent w/v solution....".
Forfibrinogen. Dilute 1.0 ml ofthe solution to 10.0 ml with a
phosphate-saline buffer pH 6.5 and ionic strength 0.15. Clot
5.0 ml ofthe dilution with the minimum amount of thrombin,
collect the clot and add three drops ofa 30 per cent w/v solution
of copper sulphate and 1 ml of nitrogen-fi-ee sulphuric acid
and boil gently for 10 minutes; cool, add 1 g of anhydrous
sodium sulphate and 10 mg of selenium, boil gently for 1 hour
and cool. Transfer to an ammonia distillation apparatus, add
6 ml of a saturated solution of sodium hydroxide and pass
steam through the flask; distil for seven minutes, collecting
the distillate in a mixture of 5 ml of a saturated solution of
boric acid, 5 ml of water; and 1 drop of a saturated solution of
methyl red in alcohol containing 0.1 per cent of methylene
blue, and titrate with 0.02 M hydrochloric acid
the solution is not complete or if a gel forms on constitution,

the preparation should not be used; (11) that the solution
should be used as soon as possible and in any case within 3
hours of constitution and any unused solution' should be
discarded; (12) that the solution sho,!ldbe administered only
with equipment that includes a filter; (13) the storage
conditions.
Fibrin Sealant Kit

Fibrin Sealant Kit is essentially composed oftwo components,
namely fibrinogen concentrate (component 1), a protein
fraction containing human fibrinogen and a preparation
containing human thrombin (component 2). A fibrin clot is
rapidly formed when the two thawed or reconstituted
components are mixed. Other ingredients (for example, human
coagulation factor XlII, a fibrinolysis inhibitor or calcium ions)
and stabilisers (for example, Human albumin solution may be
added). No antimicrobial preservative is added.
Human constituents are obtained from plasma that complies
with the requirements ofthe monograph on Human Plasma for
Fractionation. No antibiotic is added to the plasma used.
When thawed or reconstituted as stated on the label,
component 1 contains not less than 4.0 per cent of clottable
protein; the thrombin activity of component 2 varies over a
wide range (approximately 4-1 000 IV per ml).
1 ml ofO.02Mhydrochloric acid is equivalent to 0.00175 g of

fibrinogen.
Production
For sodium ions. To 10.0 ml of the solution add sufficient
The method of preparation includes a step or steps that have

been shown to remove or to inactivate known agents of
infection; if substances are used for inactivation of viruses
water to produce 100 ml, dilute 10.0 ml to 500 ml with water

and detennine the content of sodium ions by Method B tior
flame photomeny (2.4.4), measuring at about 589 urn and using
sodium solution FP suitably diluted with water as the standard
solution.
Storage. Store protected from light, in an atmosphere of

nitrogen at a temperature below 8. The containers are sterile
and sealed so as to exclude micro-organisms.
Labelling. The label states (1) the ABO blood group
during production, the subsequent purification procedure

must be validated to demonstrate that the concentration of
these substances is reduced to a suitable level and any
residues are such as not to compromise the safety of the
preparation for patients.
Constituents or mixtures of constituents are passed through
a bacteria-retentive filter and distributed aseptically into sterile
containers. Containers of freeze-dried constituents are closed
under vacuum or filled with oxygen-free nitrogen or other
suitable inert gas before being closed. In either case, they are
closed so as to exclude micro-organisms.
designation of the source ofblood; (2) the number ofUnits in

the container; (3) that 1 Unit is approximately equivalent to
the antihaemophilic activity of 1 ml ofaverage normal plasma;
(4) the concentration of protein in g per litre and of sodium
Ifthe human coagulation factor XIII content in component 1
ions in millimoles per litre of the solution constituted as
is greater than 10 Units per ml, the assay ofcoagulation factor
directed; (5) the maximum fibrinogen content; (6) where XIII is carried out.
.applicahle,Jhe.numhecoLUnits_ofheparin.inJhe_c.ontaineG.(1) .
._ ...
.__.
.
._
Qt)~(:r!pfu>!1-"Ere~:Z;\'l.-4ri.e:<igQ!1sti1:ll.e:I!!~!lrejl)'gr2sc()pic,,,,1J.itel
t1J.e .l1a111~. al1(:l.a11101l.n1.()fl:ll1y()t1J.era<i<:l~<:lsll1:Jstal1<:~_c0l1tajJ:l~cl
in it; (8) the volume of Water for Injections necessary to or pale yellow powders or friable solids. Frozen constituents
constitute the solution; (9) the instructions for constitution are colourless or pale yellow, opaque solids. Liquid
and that reconstitution may take upto 30 minutes; (10) that if constituents are colourless or pale yellow.
2566
IP 2010
FIBRIN SEALANT KIT
For the freeze-dried or frozen constituents, reconstitute or

thaw as stated on the label immediately before carrying out
the Identijication and the Tests, except those for solubility
and water.
Component 1 (Fibrinogen Concentrate)
Identification
plasma or another suitable diluent. Add to each dilution

suitable amounts of the following reagents:
a. activator reagent, containing bovine or human thrombin,
a suitable buffel; calcium chloride and a suitable inhibitor
such as Gly-Pro-Arg-Pro-Ala-NH2 which inhibits clotting of
the sample but does not prevent coagulation factor XIII
activation by thrombin,
b. detection reagent, containing a suitable factor XIIIa-specijic
peptide substrate, such as Leu-Gly-Pro-Gly-Glu-Ser-Lys-ValIle-Gly-NH2 andglycine ethyl ester as 2nd substrate in a suitable
buffer solution,
A. Complies with the limits of the assay of fibrinogen.

B. Complies with the limits ofthe assay offactor XIII
(where applicable).
c. NADH reagent, containing glutamate dehydrogenase, aketoglutarate and NADH in a suitable buffer solution.
Tests
pH (2.4.24). 6.5 to 8.0.
Stability of solution. No gel formation appears at room
temperature during 120 minutes following thawing or
reconstitution.
Water. Determine by semi-microdetermination (2.3.43), loss
on drying (2.4.19) or near infrared spectrophotometry (2.4.6),
the water content is within the limits approved by the
competent authority.
After mixing, the absorbance changes (M per min) are

measured at a wavelength of 340 nm, after the linear phase of
the reaction is reached.
I Unit of factor XIII is equal to the activity of 1 mlofhuman
normal plasma.
Calculate the activity of the test preparation by the usual

statistical methods. The confidence limits (P = 0.95) are not
estimated activity.
Assay
Component 2 (Thrombin Preparation)
Fibrinogen (Clottable Protein)

The estimated content in mg of clottable protein is not less
content stated on the label.
Mix 0.2 ml of the reconstituted preparation with 2 ml of a
suitable buffer solution pH 6.6 to 7.4 containing sufficient
human thrombin (approximately 3 IU per ml) and calcium
(0.05mol per I). Maintain at 37 for 20 minutes, separate the
precipitate by centrifugation (5000 g, 20 minutes), wash
thoroughly with a 0.9 per cent solution of sodium chloride
and determine the protein as nitrogen by sulphuric acid
digestion (2.3.30)..Calculate the protein content by multiplying
the result by 6.0. If for a particular preparation this method
cannot be applied, use another validated method for
determination of fibrinogen.
Identification
Complies with the limits ofthe assay ofthrombin.
Tests
pH (2.4.24). 5.0 to 8.0.
Water. Determine by semi-microdetermination of water
(2.3.43), loss on. drying (2.4.19) or near infrared
spectrophotometry (2.4.6), the water content is within the
limits approved by the competent authority.
Assay
Thrombin
The estimated activity is not less than 80.0 per cent and not
more than 125.0 per cent of the activity stated on the label.
Factor XIII
Where the label indicates that the human coagulation factor
XIII activity is greater than 10 Units per ml, the estimated
activity is not less than 80.0 per cent and not more than 120.0
per cent of the activity stated on the label.
Make at least 3 suitable dilutions of thawed or reconstituted
component 1 and of human normal plasma (reference
preparation) using as diluent coagulation factor XIII deficient
If necessary, dilute the reconstituted preparation under

examination to approximately 2-20 IV ofthrombin per ml using
as diluent a suitable buffer pH 7.3 to 7.5, such as imidazole
buffer solution pH 7.3 containing 1.0 per cent of human
albumin or bovine albumin. To a suitable volume of the
dilution, add a suitable volume offibrinogen solution (0.1 per
cent ofclottable protein) warmed to 37 and start measurement
2567
IF 2010
. HUMAN ALBUMIN
of the clotting time immediately. Repeat the procedure with

each of at least 3 dilutions, in the range stated above, of a
reference preparation ofthrombin, calibrated in International
Units. Calculate the activity of the test preparation by the
usual s.tatistical methods. The confidence limits (P = 0.95) are
the estimated activity.
then heated to and maintained at 60 0;5 for 10 hours so as

to prevent the transmission ofagents ofinfection transmissible
by transfusion of blood or blood derivatives. Finally, the
containers are stored for not less than 14 days at 30 to 32 or
for not less than 4 weeks at 20 and examined visually. Those
showing abnormalities such as abnormal colour, turbidity,
microbial contamination, or presence ofatypical particles must
be discarded.
Labelling. The label states (I) the amount of fibrinogen (mg

of clottable protein), thrombin (International Units) per
container, and coagulation factor XIII, ifthis is greater than 10
Units per ml; (2) where applicable, the name and volume of
solvent to be used to reconstitute the components.
Albumin Solution should be tested in accordance with the

requirements decided by the National Regulatory Authority;
in particular, tests for the absence ofhepatitis B surface antigen
and HIV antibodies are carried out by suitably sensitive
methods.
Human Albumin
Human Albumin contains not less than 95.0 per cent and not
more than 105.0 per cent of the stated amount of protein. It
contains not less than 95.0 per cent and not more than 105.0
per cent ofthe contents ofNa and K stated on the label which
are, in any case, not more than 160 millimoles ofNa per litre
and 2 millimoles ofK per litre.
Human Normal Albumin; Human Albumin Solution
Human Albumin is a sterile non-pyrogenic solution of the

Description. A clear, slightly viscous liquid, ranging in colour
albumin component obtained from pooled human blood or
from almost colourless to greenish-yellow or amber depending
from normal placentae frozen immediately after collection. It is
on protein concentration and the method offractionation used.
obtained by fractionating source material such as blood,
plasma, serum or placentae from healthy human donors and
tested individually for the absence of hepatitis B surface Identification
antigen, HCV antibodies and HIV antibodies and complies A. It contains plasma proteins of human origin only as
with other tests and requirements prescribed by the appropriate determined by precipitation tests with specific antisera.
national control authority. Source material obtained from
donors who do not meet all the requirements stated may be B. Determine the cellulose acetate by electrophoresis (2.4.12),
used provided that it has been demonstrated to the national using barbitone buffer pH 8.6, ionic strength 0.1 and human
control authority that the process offractionation will remove albumin for electrophoresis RS; 96.0 per cent of the protein
any known agent capable of adversely affecting the health of has the mobility of human albumin.
subjects treated with the preparation. It may be prepared from
pooled source materials by precipitation with organic solvents Tests
under controlled conditions of pH, ionic strength and
Acidity or alkalinity (2.4.24). Dilute with sufficient saline
temperature or by chromatography or by any other method
solution to produce a solution containing 1.0 per cent w/v of
which does not affect the integrity of the product and has
protein; pH of the resulting solution, 6.7 to 7.3.
been shown to yield consistently a product containing not
less than 95.0 per cent w/v of the total protein as albumin Alkaline phosphatase. Not more than 0.1 Unit per g ofprotein,
which is safe for intravenous injection. Residual organic determined by the following method. Transfer a mixture of
solvent, if present, is removed by freeze-drying or other
0.5 ml of the substance under examination and 0.5 ml of
suitable treatment. The product is dissolved in sufficient water diethanolamine buffer pH 10.0 to a spectrophotometer cell
to obtain a suitable concentration, and a suitable stabilising maintained at a temperature of37 0.2 0 and add 0.1 mlof
agent is added to stabilise it to heat. It is prepared as a solution nitrophenyl phosphate solution. Using a continuously
containing 15.0 to 25.0 per cent w/v of total protein or as an recording spectrophotometer record the absorbance of the
isotonic solution containing 4.0 to 5.0 per cent w/v of total solution at about 405 nm (2.4.7), over a period of at least 30
protein. No antimicrobial agent is added at any stage during seconds from the time of addition of the nitrophenyl
preparation.and.allprocessing stepsareconducted.in.. a.rnanner .phosphate solutian .__CakulaJe_Jb.e_alkaJifle_pb_Q.sp.h.atas.s<.
to.miI:liIllist;:ri:;lc.ofcolltamiI:latiOil frOm t;:itl1er micI:O:QrgllIlisIl1s .11ctiyity .at37 iIllJIlitsper g ofPI:ot<eillfrOIl1!I1t) t))(pr(;:siQI1
or other deleterious matter. The solution is sterilised by 118.3xJP, where x is the rate of increase of absorbance per
filtration and distributed aseptically into containers which are minute and P is the content of total protein in g per litre, as
then sealed so as to exclude micro-organisms. The solution is determined in the Assay:
2568
LP 2010
HUMAN ALBUMIN
Haem content. Dilute with sufficient saline solution to produce

a solution containing 1.0 per cent w/v of protein; absorbance
of the resulting solution at about 403 nm, not more than 0.15
(2.4.7).
Denatured protein. Equilibrate a column (60 to 75 cm x 2.5 to
3.0 cm) of a gel of a cross-linked dextran suitable for
fractionation ofproteins in the range ofmolecular weight from
5,000 to 3,50,000 with a lower molecular weight for complete
exclusion of globulin proteins of molecular weight between
4,00,000 and 5,00,000 (Such as Sephadex G 150) at 20 to 25
with a saline-phosphate solution prepared by mixing 2
volumes of saline solution and I volume of mixedphosphate
bufferpH 7.0 with azide. Apply to the column 2.5 ml ofnormal
human serum, previously clarified by centrifuging, and elute
with the saline-phosphate solution ata rate of20 ml per hour.
Prepare a chromatogram by recording the absorbance (2.4.7),
of the eluate at about 280 nm in relation to its volume. The
chromatogram exhibits three well-defined peaks. Determine
the volume, V, of the eluate from the entry of the sample into
the column to the apex of the first peak. ,
Dilute the substance under examination with the salinephosphate solution to contain about 5.0 per cent w Iv ofprotein,
apply 2.5 ml to the column and elute under the above
conditions, collecting the eluate in 5-ml portions. Three peaks
may appear in similar positions to those in the chromatogram
obtained from the normal human serum but the relative peak
heights may be different. To the fraction eluted between
volume 0.85 V and 1.15 V, add for each 10 ml, 0.4 rnl ofa 7.5 per
cent w/v solution of sodium molybdate and 0.4 ml ofa mixture
of 1 part of nitrogen-fi-ee sulphuric acid and 30 parts of water,
shake, centrifuge for 5 minutes and complete the Assay
described under Human Plasma beginning at the words
"decant the supernatant...". The weight of protein in the
fraction of the eluate is not more than 3.5 per cent of the
weight of protein in the volume of the substance under
examination applied to the column.
Heat the substance under examination for 50 hours at 56.5 0 to
57.5 and repeat the chromatographic separation and the
detennination of the weight of protein in the fraction eluted
between 0.85 V and 1.15 V. When expressed as a percentage of
the weight of protein in the volume of the substance under
examination applied to the column, it exceeds the percentage
obtained before heating by not more than 1.5 per cent w/v.
0
Protein composition. Not less than 95.0 per cent w/v of the
total protein as albumin, when determined by the following
method. Carry out Method II for cellulose acetate
electrophoresis (2.4.12), using one strip of cellulose for each
solution.
Test solution. Dilute the substance under examination with
saline solution to contain 2.0 per cent w/v of total protein.
Reference solution. Dilute human albuminfor electrophoresis

RS with saline solution to obtain a solution containing 2.0 per
cent w/v of total protein.
Not more than 5.0 per cent of total protein is contained in
bands other than the principal band in the strip obtained with
test solution. The test is not valid if the proportion of the
protein in the principal band is not within the limits stated in
the leaflet supplied with human albumin for electrophoresis
RS.
Stability. The contents ofthe final containerremain unchanged,

as determined by visual inspection, after heating at 57 for 50
hours, when compared to its control consisting of a sample
from the same lot which has not undergone this heating.
Pyrogens (2.2.8). Complies with the test for pyrogens, using
3 ml per kg of the rabbit's weight, inespective of the protein
content, in rabbits that have not previously received blood
products.
toxicity, using Method Band 0.5 ml of the solution for each
mouse and 5 ml for each guinea-pig irrespective ofthe protein
content.
Assay
For protein. Dilute to about 0.75 per cent w/v of total protein
with saline solution. Take 2 ml of this solution in a roundbottomed centrifuge tube, add 2 ml of a 7.5 per cent w/v
solution of sodium molybdate and 2 ml of a mixture of 30
volumes of water and I volume of nitrogen-free sulphuric
acid. Shake, centrifuge for 5 minutes, decant the supernatant
liquid and let the inverted tube stand on a filter paper to drain
the fluid. Carry out Method E for determination of nitrogen
(2.3.30), on the residue thus obtained and multiply the result
by 6.25 to obtain the protein content.
For sodium. Dilute toO.Ol per cent w/v ofprotein with water
and determine by Method A for atomic absorption
spectrophotometry (2.4.2), or by Method B for flame
photometry (2.4.4), measuring at about 589 urn and using
sodiwn solution FP suitably diluted with water as the standard
solution.
For potassium. Dilute to 0.25 per cent w/v of protein with
water and determine by Method A for atomic absorption
spectrophotometry (2.4.2), or by Method B for flame
photometry (2.4.4), measuring at about 767 nm and using
potassium solution FP suitably diluted with water as the
standard solution.
Human Albumin intended for administration to patients
undergoing dialysis or to premature infants complies with
the following additional test.
2569
IP 2010
HUMAN ALBUMIN
Aluminium (2.3.8). Not more than 200 Ilg of Al per litre.

Determine by atomic absorption spectrophotometry (2.4.2),
with a furnace as atomic generator and measuring at 309.3 nm
and using as standard solutions a suitable range of dilutions
in water of aluminium standard solution (10 ppm AI) further
diluted, as necessary, with a solution containing 0.17 per cent
w/v of magnesium nitrate and 0.05 per cent w/v of octoxinol
lOin a solution of nitric acid containing 1 per cent w/v of
nitric acid. Prepare suitable dilutions ofthe preparation under
examination and human albumin for aluminium validation
RS with water. Dilute the solutions, as necessary, with the
magnesium nitrate-octoxinol 10-nitric acid solution used
for dilution of the standard solution. The test is valid only if
the aluminium content determined for human albumin for
aluminium validation RS is within 20 per cent of the stated
value.
NOTE - Wash all equipments with a solution containing

20.0 per cent w/v of nitric acid before use and use plastic
containers only to prepare all solutions.
2 and 25. Human Albumin stored at 2 to 8 may be expected
to continue to meet the requirements of the monograph for 5
years from the date on which it was heated at 60 for 10 hours.
Human Albumin stored at a temperature not exceeding 25
may be expected to continue to meet the requirements of the
monograph for 3 years from the date on which it was heated at
60 for 10 hours.
Labelling. The label states (1) the volume in the container; (2)
the total amount ofprotein in the container expressed in g per
litre or as percentage; (3) the concentration of sodium and
potassium ions expressed in millimoles per litre; (4) the names
and concentrations of any stabilising agents and any other
additives in the final solution; (5) the type of source material
used to manufacture the product; (6) the words "Do not use if
turbid"; (7) that the contents must not be used more than 4
hours after the container has been penetrated and any remnant
portion must be discarded; (8) the storage conditions; (9) the
date after which the solution is not intended to be used; (10)
either that the preparation is suitable for administration to
patients undergoing dialysis and to premature infants or that
it is not intended for such purpose.
The potency of the preparation, reconstituted as stated on

the label, is not less than 20 IV of factor IX per ml.
Production
The method ofpreparation is designed to maintain functional
integrity offactor IX, to minimise activation ofany coagulation
factor (to minimise potential thrombogenicity) and includes a
step or steps that have been shown to remove or to inactivate
known agents of infection; if substances are used for
inactivation of viruses during production, the subsequent
purification procedure must be validated to demonstrate that
the concentration of these substances is reduced to a suitable
level and that any residues are such as not to compromise the
safety of the preparation for patients.
The specific activity is not less than 50 IV offactor IX per mg
of total protein, before the addition of any protein stabiliser.
The factor IX fraction is dissolved in a suitable liquid. Heparin,
antithrombin and other auxiliary substances such as a
stabiliser may be included. No antimicrobial preservative is
added. The solution is passed through a bacteria-retentive
filter, distributed aseptically into the final containers and
immediately frozen. It is subsequently freeze-dried and the
containers are closed under vacuum or under an inert gas.
Consistency ofthe method
The consistency of the method of production is evaluated by
suitable analytical procedures that are determined during
process development and which normally include (1) assay
offactor IX; (2) determination ofactivated coagulation factors;
(3) determination of activities offactors II, VII and X which
shall be shown to be not more than 5.0 per cent ofthe activity
offaetor IX.
Description. A white or pale yellow, hygroscopic powder or
friable solid.
Reconstitute the preparation under examination as stated

on the label, immediately before carrying out the
Identification, Tests (except those for solubility and water)
and Assay.
Identification
It complies with the limits ofthe Assay.
Tests
pH (2.4.24). 6.5 to 7.5.
Human Coagulation Factor IX
Osmolality (2.4.23). Minimum 240 mosmol per kg.
Human Coagulation Factor IX is a plasma protein fraction

containing coagulation .factor. IX,preparedby_a.methodjhat
effectively seParates factor lXJrom other .. prothrombin
complex factors (factors II, VII and X). It is obtained from
human plasma that complies with the monograph on Human
Plasma for Fractionation.
Total protein. If necessary, dilute an accurately measured

:v:olume_ofthe_pr,eparation_undecexamination_w.ith_a_Q.2..p.eI
, c:ellLw/v .SQ1lltiQIl ofs.o.dJz1l11
fh]oTic/e, tQ()lJtaiI1a,~Qlll,ti.QI1.
which may be expected to contain about 15 mg ofprotein in 2

ml. To 2.0 ml ofthat solution, in a round-bottomed centrifuge
tube, add 2 rnl ofa 7.5 per cent w/v solution ofsodium molybdate
2570
HUMAN COAGULATION FACTOR VII
IP 2010
and 2 ml of a mixture of 1 volume of nitrogen-:fi-ee sulphuric

acid and 30 volumes of water. Shake, centrifuge for 5 minutes
decant the supernatant liquid and allow the inverted tube to
drain on filter paper. Determine the nitrogen in the residue by
the method of sulphuric acid digestion (2.3.30) and calculate
the amount of protein by multiplying the result by 6.25.
For some products, especially those without a protein

stabiliseI' such as albumin, this methodmay not be applicable.
Another validated method for protein determination must
therefore be pe/jormed.
Activated coagulation factors (2.8.4). Ifnecessary, dilute the
preparation under examination to contain 20 IU of factor IX
per ml. For each of the dilutions the coagulation time is not
less than 150 seconds.
Heparin. If heparin has been added during preparation,
determine the amount by the assay of heparin in coagulation
factor concentrates (2.8.10). The preparation under
examination contains not more than the amount of heparin
stated on the label and in any case not more than 0.5 ill of
heparin per International Unit of factor IX.
Water. Detennine by semi-micro determination of water
(2.3.43), loss on drying (2.4.19) or near infrared
spectrophotometry (2.4.6), the water content is withinthe limits
approved by the competent authority.'
per kg of the rabbit's mass a volume equivalent to not less
than 50 ill offactor IX.
Assay. Determine the assay ofhuman blood coagulation factor
IX (2.8.8).
more than 125.0 per cent ofthe stated potency. The confidence
limits (P = 0.95) are not less than 80.0 per cent and not more
than 125.0 per cent ofthe estimated potency.
Labelling. The label states (1) the number ofInternational
Units offactor IX per container; (2) the amount ofprotein per
container; (3) the name and quantity of any added substances
including, where applicable, heparin; (4) the name and volume
of the liquid to be used for reconstitution; (5) that the
transmission of infectious agents cannot be totally excluded
when medicinal products prepared from human blood or
plasma are administered.
also contain small amounts of the activated fonn, the twochain derivative factor VIla. It may also contain coagulation
factors II, IX and X and protein C and protein seconds. It is
obtained from human plasma that complies with the
monograph on Human Plasma for Fractionation.
the label, is not less than 15 ill of factor VII per ml.
Production
The method ofpreparation is designed to minimise activation
of any coagulation factor (to minimise potential
thrombogenicity) and includes a step or steps that have been
shown to remove or to inactivate known agents of infection;
if substances are used for inactivation of viruses during
production, the subsequent purification procedure must be
validated to demonstrate that the concentration of these
substances is reduced to a suitable level and that any residues
are such as not to compromise the safety of the preparation
for patients.
The specific activity is not less than 2 IU offactor VII per mg
The factor VII fraction is dissolved in a suitable liquid. Heparin,
antithrombin and other auxiliary substances such as a
stabi1iser may be added. No antimicrobial preservative is added.
The solution is passed through a bacteria-retentive filter,
distributed aseptically into the final containers and
immediately frozen. It is subsequently freeze-dried and the
containers are closed under vacuum or under an inert gas.
Consistency ofthe method
The consistency of the method of production with respect to
the activities of factors II, IX and X of the preparation,
expressed in International Units relative to the activity of~actor
VII, shall be demonstrated.
The consistency of the method of production with respect to
the activity of factor VIla of the preparation shall be
demonstrated. The activity offaCtor VIla may be detennined,
for example, using a recombinant soluble tissue factor that
does not activate factor VII but possesses a cofactor function
specific for factor VIla; after incubation of a mixture of the
recombinant soluble tissue factor with phospholipids reagent
and the dilution of the test sample in factor VII-deficient
plasma, calcium chloride is added and the clotting time
determined; the clotting time is inversely related to the factor
VIla activity of the test sample.
Description. A hygroscopic powder or friable solid that may
be white, pale yellow, green or blue.
Human Coagulation Factor VII

Human Coagulation Factor VII is a plasma protein fraction
that contains the single-chain glycoprotein factor VII and may
Reconstitute the preparation under examination as stated

on the label immediately before carrying out the
Identification, Tests (except those for solubility and water)
and Assay.
2571
IP 2010
HUMAN COAGULATION FACTOR VII
Identification
The estimated content is not more than 125.0 per cent of the
stated content. The confidence limits (P = 0.95) are not less
estimated potency.
It complies with the limits ofthe Assay.
Tests
pH (2.4.24). 6.5 to 7.5.
Factor X
Determine the assay ofhuman coagulation factor X (2.8.9).

volume of the reconstituted preparation with a 0.9 per cent
solution w/v of sodium chloride to obtain a solution expected
to contain about 15 mg of protein in 2 ml. To 2.0 ml of the
solution in a round-bottomed centrifuge tube add 2 ml ofa 7.5
per cent w/v solution of sodium molybdate and 2 ml of a
mixture of 1 volume of nitrogen-free sulphuric acid and 30
volumes of water. Shake, centrifuge for 5 minutes, decant the
supernatant liquid and allow the inverted tube to drain on
filter paper. Determine the nitrogen in the residue by the
method of sulphuric acid digestion (2.3.30) and calculate the
amount ofprotein by multiplying the result by 6.25.
estimated potency.
Activated coagulation factors (2.8.4). For each ofthe dilutions,

the coagulation time is not less than 150 seconds.
determine the amount present by the assay of heparin in
coagulation factor concentrates (2.8.10). The preparation under
heparin per International Unit offactor VII.
Thrombin. If the preparation under examination contains
heparin, determine the amount present as described in the
test for heparin and neutralise the heparin by addition of
protamine sulphate (10 /lg ofprotamine sulphate neutralises
1 ill ofheparin). In each of2 test-tubes, mix equal volumes of
the reconstituted preparation and a 0.3 per cent solution of
fibrinogen. Keep one of the tubes at 37 for 6hours and the
other at room temperature for 24 hours. In a third tube, mix a
volume of the fibrinogen solution with an equal volume of a
solution of human thrombin (l IU per ml) and place the tube
in a water-bath at 37. No coagulation occurs in the tubes
containing the preparation under examination. Coagulation
occurs within 30 seconds in the tube containing thrombin.
Water. Determine by semi-micro determination ofwater (2.3 .43),

loss on drying (2.4.19) or near-infrared spectrophotometry
(2.4.6), the water content is within the limits approved by the
per kg of the rabbit's mass a volume equivalent to not less
than 30 ill offactor VII.
A~say
Determine the assay ofhuman coagulation factor VII (2.8.6).

than 125.0 per cent of the estimated potency.

Labelling. The label states (1) the number of International
Units of factor VII per container; (2) the maximum content of
International Units of factor II, factor IX and factor X per
container; (3) the amount of protein per container; (4) the
name and quantity of any added substances, including where
applicable, heparin; (5) the name and volume of the liquid to
be used for reconstitution; (6) that the transmission of
infectious agents cannot be totally excluded when medicinal
products prepared from human blood or plasma are
administered.
Factorll
Determine the assay of human coagulation factor II (2.8.5).
t~aIl90.0 per cf:llt and n()t l11()rethall 111 ~ 0 per (;~nt of the
estimated potency.
Factor IX
Determine the assay of human coagulation factor IX (2.8.8).
Human Coagulation Factor VIII

(rDNA)
Human Coagulation Factor VIII (rDNA) is a freeze-dried
preparationofglycoproteinshaving the same activity as
coagulation fa.ctor VUI in.humanplasma.Jtactsas_a.c_Qfactor
of the activation of factor X in the presence of factor IXa,
phospholipids and calcium ions. It circulates in plasma
mainly as a two-chain glycosylated protein with 1 heavy
2572
IP 2010
HUMAN COAGULATION FACTOR VIII (rDNA)
(relative molecular mass ofabout 2,00,000) and 1 light (relative

molecular mass 80,000) chain held together by divalent metal
ions. Human coagulation factor VIII (rDNA) is prepared as
full-length factor VIII (octocog alfa), or as a shortened twochain structure (relative molecular mass 90,000 and 80,000), in
which the B-domain has been deleted from the heavy chain
(moroctocog alfa).
Full-length human rDNA coagulation factor VIII contains 25
potential N-glycosylation sites, 19 in the B domain of the
heavy chain, 3 in the remaining part ofthe heavy chain (relative
molecular mass 90,000) and 3 in the light chain (relative
molecular maSS 80,000). The different products are
characterised by their molecular size and post-translational
modification and/or other modifications.
Production
Human coagulation factor VIII (rDNA) is produced by
recombinant DNA technology in mammalian cell culture. It is
produced under conditions designed to minimise microbial
contamination.
Purified bulle factor VIII (rDNA) may contain added human
albumin and/or other stabilising agents, as well as other
auxiliary substances to provide, for example, correct pH and
osmolality.
The specific activity is not less than 2,000 ill offactor VIII:C
per mg of total protein before the addition of any protein
stabiliser, and varies depending on purity and the type of
modification ofmolecular structure offactor VIII.
The quality of the bulk preparation is controlled using
reference preparations.
Reference preparations
During development, reference preparations are established
for subsequent verification of batch consistency during
production, and for control ofbull( and final preparation. They
are derived from representative batches ofpurified bulk factor
VIII (rDNA) that are extensively characterised by tests
including those described below and whose procoagulant
and other relevant functional properties have been
ascertained and compared, wherever possible, with the
International Standard for factor VIII concentrate. The
reference preparations are suitably characterised for their
intended purpose and are stored in suitably sized aliquots
under conditions ensuring their stability.
applicable, the characterisation tests may alternatively be

carried out on the finished product.
Specific biological activity or ratio offactor VITI activity to
factor VITI antigen
Determine the assay ofhuman coagulation factor VIII (2.8.7).
The protein content, or where a protein stabiliser is present,
the factor VIII antigen content, is determined by a suitable
method and the specific biological activity or the ratio offactor
VIII activity to factor VIII antigen is calculated.
Protein composition
The protein composition is determined by a selection of
appropriate characterisation techniques which may include
peptide mapping, Western blots, HPLC, gel electrophoresis,
capillary electrophoresis, mass spectrometry or other
techniques to monitor integrity and purity. The protein
composition is comparable to that ofthe reference preparation.
Molecular size. Using size-exclusion chromatography
(2.4.16), the molecular size distribution is comparable to that
of the reference preparation.
Peptide mapping (2.3.47). There is no significant difference
between the test protein and the reference preparation.
Carbohydrates/sialic acid. To monitor batch-to-batch
consistency, the monosaccharide content and the degree of
sialylation or the oligosaccharide profile are monitored and
correspond to those of the reference preparation.
Final lot
It complies with the tests under Identification, Tests and
Assay.
Excipients. 80.0 per cent to 120.0 per cent ofthe stated content,
determined by a suitable method, where applicable.
Description. A white or slightly yellow powder or friable mass.
Identification
A. It complies with the limits of the Assay.
B. The distribution ofcharacteristic peptide bands corresponds .
with that ofthe reference preparation (SDS-PAGE or Western
blot).
Tests
Purified bulk factor VITI (rDNA)
Reconstitute the preparation as stated on the label

The purified bulk complies with a suitable combination of immediately before canying out the Tests (except those for
the following tests for characterisation of integrity of the solubility and water) and Assay.
factor VIII (rDNA). Where any substance added during
pR(2.4.24).6.5t07.5.
preparation of the purified bulk interferes with a test, the
test is carried out before addition ofthat substance. Where Osmolality (2.4.23). Minimum 240 mosmol per kg.
2573
IP 2010
HUMAN NORMAL IMMUNOGLOBULIN
Water. Determine by serrli-rrlicro determination ofwater (2.3.43),

loss on drying (2.4.19) or near infrared spectrophotometry
Bacterial endotoxins (2.2.3). Less than 3 ill in the volume
that contains 100 ill offactor VIII activity.
Assay
Determine the assay ofhuman coagulation factor VIII (2.8.7).
than 120.0 per cent of the estimated potency.
Labelling. The label states (1) the factor VIII content in
International Units; (2) the name and amount ofany excipient;
(3) the composition and volume of the liquid to be used for
reconstitution.
Human Normal Immunoglobulin

Normal Immunoglobulin; Immune Human Serum Globulin;
Human Gamma Globulin
Human Normal Immunoglobulin is a sterile solution or freezedried preparation containing immunoglobulins, mainly
immunoglobulin G (IgG), together with smaller amounts of
other plasma proteins.
Production
It is obtained from source materials such as the blood, plasma,
serum or placentae frozen immediately after collection from
healthy donors who must as far as can be ascertained after
clinical examination, laboratory tests on their blood and a study
of their medical history, be free from disease transmissible by
transfusion of blood or blood products. The examinations
and tests to be carried out are decided by the National
Regulatory Authority; in particular, tests for hepatitis B surface
antigen, HCV antibodies and for HIV antibodies must be
can-ied out by suitable sensitive methods and must show
negative results in both cases. Plasma, serum or placentae
obtained from donors who do not meet all the requirements
stated above may be used as source material provided that it
It is prepared from pooled material ofa minimum volume of25

litres by a method which has been shown (a) to be capable of
concentrating tenfold from source material at least two different
antibodies, one viral and one bacterial", for which an
International Standard or Reference Preparation is available;
(b) not to affect the integrity of the globulins; (c) to
consistently yield a product which is safe for intramuscular
injection and; (d) to yield a product .that does not transmit
viral hepatitis or any other infection.
The liquid preparation is prepared as a stabilised solution in

saline solution or a 2.25 per cent w/v solution of glycine or
other suitable agent and is sterilised by filtration and
distributed into previously sterilised containers which are then
sealed so as to exclude micro-organisms. An antimicrobial
preservative may be added except when the preparation is to
be freeze-dried. Any antimicrobial preservative or stabilising
agent added must be such that neither deleterious effect on
the final product in the amounts present nor capability to
cause untoward reactions in human beings is demonstrated.
An accelerated degradation test is can-ied out on the final
liquid or freeze-dried preparation by heating at 37 for 4 weeks.
The difference between the percentages of protein eluted in
the fractions following the main peak as determined by sizeexclusion chromatography (2.4.16), before and after exposurt?
at 37 does not exceed 5.0 per cent.
Human Normal Immunoglobulin contains not less than 90.0
per cent and not more than 110.0 per cent of the quantity of
protein stated on the label and in any case, not less than 10.0
per cent w/v and not more than 18.0 per cent w/v of protein.
Description. The liquid preparation is clear pale yellow or
brownish in colour; on storage it may show turbidity or a
small amount ofparticulate matter. The freeze-dried preparation
is a white to slightly yellow powder or solid, friable mass.
Identification
A. Precipitation tests with a suitable range ofspecies-specific
antisera which give positive results for the presence ofproteins
of human origin and negative results with antisera specific to
plasma proteins of the other species.
B. Examine by electrophoresis (2.4.12), using the moving
boundary technique and a 1.0 per cent w/v solution in
barbitone buffer solution pH 8.6 of ionic strength 0.1. At
least 90.0 per cent w/v ofthe protein has a mobility not greater
than -2.8 x 10-5 cm2V- 1S-I .
has_bee~demonstratedjo_the-llationaLauthority.thaLprocess'l'ests--------~----- ---------.------._-.-..-. -----------
Qfft~<::tiQnllJjQl11111<:l_.prQ<:ll1<::.tiQl1.rf)mQ~fill.l1Y
.mQ\:Y.l111gfJ.l1t
capable of adversely affecting the health of subjects treated

with the preparation. No antibiotic is added to the source
materials used for the preparation of immunoglobulin.
pH
(2.LCi4Y.-6.4 to 7.2, deterrnlnedTn a solution prepared by

dilution of a quantity with saline solution so as to contain 1.0
per cent w/v of protein.
2574
IP 2010
Protein composition. Determine by cellulose acetate

electrophoresis (2.4.12), but applying an electric field such
that the albumin band of normal human serum applied in a
control strip migrates at least 30 mm and using one strip of
cellulose acetate for each of the following solutions:

saline solution to produce a solution containing 5 per cent
w/v ofprotein.
Reference solution. Reconstitute human immunoglobulinfor
electrophoresis RS with saline solution to produce a solution
containing 5 per cent w/v of protein.
Calculate the result as the mean ofthree measurements of the
absorbance of each strip. In the electrophoretogram obtained
with test solution not more than 10.0 per cent ofthe protein is
contained in bands other than the principal band. The test is
not valid unless the proportion of protein in the principal
band in the electrophoretogram obtained with reference
solution is within the limits stated in the leaflet supplied with
human immunoglobulin for electrophoresis RS.
Molecular size. Determine by size-exclusion chromatography
(2.4.16), applying 2 ml of the preparation under examination
diluted with mixed phosphate buffer pH 7.0 with azide to
produce a solution containing 4.0 to 5.0 per cent wIv ofprotein.
a column 1 m x 25 mm packed with agarose trapped
within a cross-linked polyacrylamide network and
having a linear fraction range suitable for fractionation
of globular proteins in the range of molecular weights
1i-om 20,000 to 350,000,
- mobile phase: mixedphosphate buffer pH 7.0 with
azide,
flow rate. 20 ml per hour (4 ml per cm2 ofcolumn
cross-sectional area),
spectrophotometer set at 280 nm.
Collect the eluate in fractions of about 4 ml. Examine the
chromatogram in comparison with that in Fig.l. The sum of
f\
the areas ofthe peaks containing IgG monomer, dimer, albumin

and other proteins ofsimilar molecular size (area B) is not less
than 85.0 per cent ofthe total area of the chromatogram. Not
more than 10.0 per cent of the total area ofthe chromatogram
represents proteins eluted ahead ofigG dimer (area A); ifarea
A can be subdivided into two distinct areas, the area
corresponding to larger proteins does not exceed 5.0 per cent
of the total area ofthe chromatogram. Not more than 5.0 per
cent ofthe total area ofthe chromatogram represents proteins
eluted after IgG monomer and albumin (area C).
Stability. Heat approximately 2 ml at 57 for 4 hours in a
stoppered glass tube (75 mm x 12 mm); no gelation or
flocculation occurs.
1 ml ofthe preparation under examination per kg ofthe rabbit's
weight.
toxicity, Method A, injecting 0.5 ml into each mouse and 5 ml
into each guinea-pig.
Assay. Dilute a suitable volume with water to produce a
solution containing 1.0 per cent w/v ofprotein. Take 1.5 ml of
the dilution in a round-bottomed centrifuge tube. Add 5 ml of
wate/; mix, add 0.2 ml on.5 per cent w/v solution of sodium
molybdate and 2 ml of a mixture consisting of 1 volume
nitrogen free sulphuric acid and 30 volumes of water. Shake,
centrifuge for five minutes, decant the supernatant liquid and
allow the inverted tube to drain on filter paper. To the residue
in the tube add three drops of a 30 per cent w/v solution of
copper sulphate and 1 ml of nitrogen-free sulphuric acid
and boil gently for 10 minutes; cool, add 1 g of anhydrous
sodium sulphate and 10 mg of selenium, boil gently for 1 hour
and cool. Transfer to an ammonia distillation apparatus, add
6 ml of a saturated solution of sodium hydroxide and pass
steam through the flask; distil for seven minutes, collecting
the distillate in a mixture of 5 ml of a saturated solution of
boric acid, 5 ml of water, and 1 drop saturated solution of
methyl red in alcohol containing 0.1 per cent of methylene
blue, and titrate with 0.02 M hydrochloric acid.
1 ml of 0.02 Mhydrochloric acid is equivalentto 0.00175 g of
protein.
/'~!
Freeze-dried Human Normal Immunoglobulin complies with

the following additional requirements.
llicOO:::i'1:-'-~~==::-iiB-::=~~~~C~:;;;a,~5~OOIll1
ELUATE VOLUME
Fig.l Typical Chromatogram for Human Normal Immunoglobulin
Solubility rate. Add the volume of the liquid stated on the

label and allow it to stand for 15 minutes at a temperature of
20 to 25; it dissolves completely.
2575
IP 2010
Loss on drying (2.4:1 9). Not more than 2.0 per cent, determined
on 0.5 g, by drying over phosphorus pentoxide at a pressure
not exceeding 3 Pa for 24 hours.
Human Normal Immunoglobulin intended for use in the
prevention of infective hepatitis (hepatitis A) complies with
the follOWing additional requirement.
Anti-hepatitis Aactivity. Determine the anti-hepatitis A activity

by comparison with the activity of the Standard preparation,
using an immunoassay of suitable sensitivity and specificity.
The stated potency is not less than 100 Units per m!. The
estimated potency is not less than the stated potency. The
fiducial limits of error are not less than 80.0 per cent and not
Standard Preparation. The Standard Preparation is the 1st
International Reference Preparation for Hepatitis A
immunoglobulin, established in 1981, consisting offreeze-dried
material derived from fractionated plasma (supplied in
ampoules containing 100 Units), or another suitable
preparation the antigen binding ofwhich has been determined
in relation to the International Reference Preparation.
Storage. Store protected from light, the liquid preparation in
sealed, colourless, glass containers, at a temperature between
2 and 8. Store the freeze-dried preparation under vacuum or
under an inert gas.
Labelling. The label states (1) the volume and the protein
concentration expressed in g per litre or, for freeze-dried
preparations, the total amount of protein in the container; (2)
the type of source material; (3) the name and quantity of any
added preservative or stabilising agent; (4) the recommended
human dose; (5) that it is meant for intramuscular injection
only; (6) the storage conditions.

Plasma Protein Solution; Human Albumin Fraction (Saline);
Plasma Protein Fraction; PPF
Human Plasma Protein Fraction is a sterile isotonic aqueous
solution of proteins of plasma or serum containing albumin
and globulins. It is prepared as an isotonic solution containing
4.0 to 5.0 per cent w/v oftotal protein. It contains no fibrinogen
or antibodies.
in particular tests for hepatitis B surface antigen and for HIV

antibodies are carried outby suitable sensitive methods and
must give negative results in .both cases. Other diseasecausative agents that are not destroyed or removed by the
processing method must not be present. Plasma or serum
obtained from donors who do not meet all the stated
requirements may be used as source material provided that it
has been demonstrated to the national authority that the
process offractionation will remove any lmown agent capable
of adversely affecting the health of recipients of the Human
Plasma Protein Fraction.
The separation of the protein may be done by precipitation
with suitable organic solvents under controlled conditions,
particularly of pH, ionic strength and temperature, so that in
the final product not less than 85.0 per cent ofthe total protein
is albumin. Residual solvent, if present, may be removed by
freeze-drying or other suitable treatment. Alternative methods
ofpreparation which shall not affectthe integrity ofthe product
and shall have been shown to yield consistently a product
which is safe for intravenous injection may be adopted.
The product is dissolved in water and sufficient quantities of
a suitable stabiliser against the effect of heat, like sodium
caprylate in a suitable concentration, and sufficient sodium
chloride to adjust the sodium ions to between 130 and 160
millimoles per litre may be added but no antibiotic or
antimicrobial preservative is added at any stage during
preparation. The solution is sterilised by filtration through a
bacteria-retentive filter and distributed aseptically into sterile
containers which are then closed so as to prevent microbial
contamination. The solution in its final container is heated at
60 0.5 and maintained at this temperature for 10 hours. The
containers are then incubated at 30 to 32 for not less than 14
days or at 20 to 25 for not less than 4 weeks and examined
visually for evidence of microbial contamination. Those
showing abnormalities such as abnormal colour, turbidity,
presence of atypical particles or microbial contamination are
discarded.
Human Plasma Protein Fraction contains not less than 95.0
of total protein.
Description. A clear, almost colourless or pale yellow liquid;

almost odourless. On storage a dust-like precipitate may
develop but it disappears on shaking.
Identification
Production
The plasma or serum is obtained froin healthy human donors
who.must, .after cJinicaLexarninaJiQl1,la.b.otat.oryJ:esJs_QDJ:heir
bl(){)cLiil1c1 a. .~tllcly()f tl1()ir .l11ecli()all1i~t()ry ,J:>()ft-()()ft-()l11
detectable agents of infection transmissible by transfusion of
blood or blood derivatives. The examinations and tests to be
carried out are decided by the National Regulatory Authority;
A. Precipitation tests with specific antisera show that the

preparation consists only ofplasma proteins of human origin
onl)lano gives negativefesiiltsWitli-antiseraspeeifictoplasma

proteins of"other species: "
B. Examine by a suitable immunoelectrophoresis technique.
Using antiserum to normal human serum, compare nOlmal
2576
IP 2010
HUMAN PLASMA PROTEIN FRACTION
human serum and the preparation under examination, both

diluted to contain 1.0 per cent w/v of protein. The main
component ofthe preparation under examination corresponds
to the main component of the normal human serum. The
solution may show the presence of small quantities of other
plasma proteins.
Sodium. Not less than 95.0 per cent and not more than 105.0
per cent of the stated amount and, in any case not more than
160 millimoles ofNa per litre, determined by Method A for
589 nm and using sodium solution AAS suitably diluted with
water to prepare the standard solutions.
Tests
Potassium. Not more than 50 !!mol of K per g of protein,

determined by atomic absorption spectrophotometry (2.4.2),
measuring at 766 nm and using potassium solution AAS,
suitably diluted with water to prepare the standard solutions.
pH (2.4.24).6.7 to 7.3, determined in a solution prepared by

diluting with sufficient of saline solution to produce a solution
containing 1.0 per cent w/v of protein.
- a column 1mx25 mmpacked with a cross-linked dextran
suitable for fractionation ofglobular proteins in the range
of molecular weights from 5,000 to 3,50,000 (such as
Sephadex 0-150),
mobile phase: mixedphosphate bufferpH 7.0 with azide,
- flow rate. 20 ml per hour (4 ml per square centimeter of
column cross-sectional area),
spectrophotometer set at 280 nm.
Alkaline phosphatase. Not more than 0.1 Unit per g ofprotein,

detennined by the following method. Transfer a mixture of0.5
ml of the substance under examination and 0.5 ml of
diethanolamine buffer pH 10.0 to a spectrophotometer cell
maintained at a temperature of 37 0.2 and add 0.1 ml of
nitrophenyl phosphate solution. Record the absorbance of
the solution at about 405 nm (2.4.7), over a period ofat least 30
seconds from the time of addition of the nitrophenyl
phosphate solution. Calculate the alkaline phosphatase
activity at 37 in Units per g of protein from the expression
118.3x1P, where x is the rate of increase of absorbance per
minute and P is the content of total protein in g per litre, as
Collect the eluate in fractions of about 4 ml and combine the

fractions corresponding to each peak. For each combined
fraction, determine by Method E for determination ofnitrogen
(2.3.30).
Haem content. Dilute with sufficient saline solution to produce

a solution containing 1.0 per cent w/v of protein; absorbance
of the resulting solution at about 403 nm, not more than 0.15
(2.4.7).
1 ml of 0.02Mhydrochloric acid is equivalent to 0.00028 g of

nitrogen. Not more than 10.0 per cent of the total nitrogen is
present in the combined fraction associated with non-retained
proteins.
Polymers and aggregates. Determine by size-exclusion

chromatography (2.4.16), applying 2 ml ofthe preparation under
examination.
Protein composition. Carry out by cellulose acetate

electrophoresis (2.4.12), Method II using one strip for each
solution.

saline solution to obtain a solution containing 2.0 per cent
w/v of protein.
Reference solution. Dilute human plasma protein fraction
for electrophoresis RS with saline solution to obtain a solution
containing 2.0 per cent w/v of protein.
Calculate the result as the mean ofthree measurements ofthe
absorbance ofeach ofthe 10 strips. In the electrophoretogram
obtained with test solution not more than 15.0 per cent of the
protein is contained in bands other than the principal band.
The test is not valid unless the proportion of protein in the
principal band in the electrophoretogram obtained with
reference solution is within the limits stated in the leaflet
supplied with human plasma protein fraction for
electrophoresis RS.

toxicity, using Method Band 0.5 ml of the solution for each
mouse and 5 ml for each guinea-pig irrespective ofthe protein
content.
3 ml per kg of the rabbit's weight in rabbits that have not
previously received blood products.
Assay
For protein. Dilute to about 0.75 percent w/v oftotalprotein

with saline solution. Take 2 ml of this solution in a roundbottomed centrifuge tube, add 2 mlof a 7.5 per cent w/v
solution of sodium molybdate and 2 ml of a mixture of 30
volumes of water and 1 volume of nitrogen-free sulphuric
acid. Shake, centrifuge for 5 minutes, decant the supernatant
liquid and let the inverted tube stand on a filter paper to drain
the fluid. Carry out Method E for determination of nitrogen
(2.3.30), on the residue thus obtained and multiply the result
by 6.25 to obtain the protein content.
2 and 25.
2577
HUMAN PROTHROMBIN COMPLEX
IP 2010
Labelling. The label states (1) the volume in the container; (2)
the total amount of protein in the container expressed as a
percentage or in grams per litre; (3) the concentration ofsodium
ions in millimoles per litre; (4) the names and concentrations
of stabilising agents and of any other added substances
present in the final solution; (5) that the solution should not
be used if the solution is cloudy or shows a deposit which
does not disappear on shaking; (6) that, once the container
has been penetrated, the contents must be used within 4 hours
and any unused solution discarded; (7) the storage conditions.
Human Prothrombin Complex

Human Prothrombin Complex is a plasma protein fraction
containing blood coagulation factor IX together with variable
amounts of coagulation factors II, VII and X; the presence
and proportion of these additional factors depends on the
method of fractionation. It is obtained from human plasma
that complies with the monograph on Human Plasma for
Fractionation.
the label, is not less than 20 ill offactor IX per ml.
Production
The method ofpreparation is designed to minimise activation
of any coagulation factor (to minimise potential
thrombogenicity) and includes a step or steps that have been
shown to remove or to inactivate known agents of infection;
if substances are used for inactivation of viruses during
production, the subsequent purification procedure must be
validated to demonstrate that the concentration of these
substances is reduced to a suitable level and that any residues
are such as not to compromise the safety of the preparation
for patients.
Identification
It complies with the limits of the assay for coagulation factor
IX activity and, where applicable, those for factors II, VII and
X
Tests
pH (2.4.24). 6.5 to 7.5.
volume of the reconstituted preparation with a 0.9 per cent
w/v solution of sodium chloride to obtain a solution expected
to contain about 15 mg of protein in 2 ml. To 2.0 ml of the
solution in a round-bottomed centrifuge tube add 2 ml ofa 7.5
per cent w/v solution of sodium molybdate and 2 ml of
mixture of 1 volume of nitrogen-free sulphuric acid and 30
volumes of water. Shake, centrifuge for 5 minutes, decant the
supernatant liquid and allow the inverted tube to drain on
filter paper. Determine the nitrogen in the residue by the
amount of protein by multiplying the result by 6.25.
Activated coagulation factors (2.8.4). Ifnecessary, dilute the

preparation under examination to contain 20 ill of factor IX
per ml. For each of the dilutions, the coagulation time is not
less than 150 seconds.
determine the amount present by the assay of heparin in
coagulation factor concentrates (2.8.10). The preparation under
heparin per International Unit of factor IX.
Thrombin. If the preparation under examination contains
heparin, determine the amount present as described in the
test for heparin and neutralise it by addition of protamine
sulphate (10 Jlg of protamine sulphate neutralises 1 ill of
heparin). In each of 2 test-tubes, mix equal volumes of the
reconstituted preparation and a 0.3 per cent w/v solution of
fibrinogen. Keep one of the tubes at 37 for 6 hours and the
other at room temperature for 24 hours. In a third tube, mix a
volume of the fibrinogen solution with an equal volume of a
solution of human thrombin (1 IU per ml) and place the tube
in a water-bath at 37. No coagulation occurs in the tubes
containing the preparation under examination. Coagulation
occurs within 30 seconds in the tube containing thrombin.
The specific activity is not less than 0.6 ill offactor IX per mg
The prothrombin complex fraction is dissolved in a
suitable liquid. Heparin, antithrombin and other auxiliary
substances such as a stabiliser may be added. No antimicrobial
preservative is added. The solution is passed through a
bacteria-retentive filter, distributed aseptically into the final
containers and immediately frozen. It is subsequently freezedried and the containers are closed under vacuum or under
an inert gas.
.
Description. A white or slightly coloured powder or friable
s~lid, ~e_l}'~~~'<:>~~?l'i.c-.:_ ... ... ._~ ._.~.__ . . __._..
Water. Determine by semi-micro determination of water
.... R~GQ/Js.titljt~ th~ . p,.epq[(JljQ!J..ljnr1er.~~Clmill.qtion as stated (23.43)~loss-on-drying(2:4.-19)ornear~infraredspectrometry
..... _.__.
on the label immediately before carryi~g~~~
(2;4;6); the watercontentiswithinthelimitsapprovedbythe
Identification, Tests (except those for solubility and water) competent authority.
and Assay.
-;iz-;
2578
HUMAN NORMAL IMMUNOGLOBULIN FOR INTRAVENOUS USE
IP 2010
Q

per kg of the rabbit's mass a volume of the reconstituted
preparation equivalent to not less than 30 IU of factor IX.
Human Normal Immunoglobulin for

Intravenous Use
Assay
Human Normal Immunoglobulin for Intravenous

Administration
Factor IX
Determine the assay ofhuman coagulation factor IX (2.8.8).
interval (P = 0.95) ofthe estimated potency is not greater than
80.0 per cent to 125.0 per cent.
Factor II
Determine the assay of human coagulation factor II (2.8.5).
Factor VII
If the label states that the preparation contains factor VII,
Determine the assay of human coagulation factor VII
(2.8.6).
80.0 per centto 125.0 per cent.
Factor X
Determine the assay of human coagulation factor X (2.8.9).
Units of factor IX, factor II and factor X per container;
(2) where applicable, the number of International Units of
factor VII per container; (3) where applicable, that the
preparation contains protein C and/or protein S; (4) the amount
of protein per container; (5) the name and quantity of any
added substances, including where applicable, heparin;
(6) the name and quantity of the liquid to be used for
reconstitution; (7) that the transmission of infectious agents
cannot be totally excluded when medicinal products prepared
from human blood or plasma are administered.

Administration is a liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G (IgG). Other
proteins may be present. Human normal immunoglobulin for
intravenous administration contains the IgG antibodies of
normal subjects. This monograph does not apply to products
intentionally prepared to contain fragments or chemically
modified IgG.
Human normal immunoglobulin for intravenous administration
is obtained ii-om plasma that complies with the requirements
of the monograph on Human plasma for fractionation. No
antibiotic is added to the plasma used.
Production
The method of preparation includes a step or steps that have
been shown to remove or to inactivate known agents of
infection; if substances are used for inactivation of viruses, it
shall have been shown that any residues present in the final
product have no adverse effects on the patients treated with
the immunoglobulin.
The product shall have been shown, by suitable tests in
animals and evaluation during clinical trials, to be well tolerated
when administered intravenously.
is prepared from pooled material from not fewer than 1,000
doriors by a method that has been shown to yield a product
that (a) does not transmit infection; (b) at an immunoglobulin
concentration of 50 g per litre, contains antibodies for at least
2 of which (one viral and one bacterial) an International
Standard or Reference Preparation is available, the
concentration of such antibodies being at least 3 times that in
the initial pooled material; (c) has a defined distribution of
immunoglobulin G subclasses; (d) complies with the test for
Fc function of immunoglobulin.
Test for Fc function ofimmunoglobulin
Stabilised human blood. Collect group 0 human red blood
into ACD anticoagulant solution. Store the stabilised blood
at 4 for not more than 3 weeks.
Phosphate buffered saline pH 7.2. Dissolve 1.022 g of
anhydrous disodium hydrogen phosphate, 0.336 g of

anhydrous sodium dihydrogen phosphate and 8.766 g of
sodium chloride in 800 ml of water and dilute to 1,000 ml with
the same solvent.
2579
IP 2010
Magnesium and calcium stock solution. Dissolve 1.103 g of

calcium chloride and 5.083 g of magnesium chloride in water
barbital buffer solution, thereby obtaining the final adjusted

volume Vf = Vr x A of sensitised human red blood cells and
adjusting A to 1.0 0.1 for a tenfold dilution.
Barbital buffer stock solution. Dissolve 207.5 g of sodium

chloride and 25.48 g of barbital sodium in 4,000 ml of water
and adjust to pH 7.3 using 1 M hydrochloric acid. Add 12.5
ml of magnesium and calcium stock solution and dilute to
5,000 m1 with water. Filter through a membrane filter (pore size
0.22 gm). Store at 4 in glass containers.
Antibody binding ofantigen-coated tanned human red blood

cells. Prepare the following solutions in succession and in
duplicate, using for each solution a separate half-micro cuvette
(for example, disposable type) or test-tube:
Albumin barbital buffer solution. Dissolve 0.150 g of bovine

albumin in 20 ml of barbital buffer stock solution and dilute
Tannic acid solution. Dissolve 10 mg of tannic acid in 100 ml
of phosphate-buffered saline pH 7.2. Prepare immediately
before use.
Guinea-pig complement. Prepare a pool of serum from the
blood of not fewer than 10 guinea-pigs. Separate the serum
from the clotted blood by centrifugation at about 4. Store the
serum in small amounts below _70. Immediately before starting
complement-initiated haemolysis, dilute to 125 to 200 CHso
per ml with albumin barbital buffer solution and store in an
ice-bath during the test.
Rubella antigen. Suitable rubella antigen for
haemagglutination-inhibition titre (HIT). Titre> 256 HA units.
Preparation oftanned human red blood cells. Separate human
red blood cells by centrifuging an appropriate volume of
stabilised human blood and wash the cells at least 3 times
with phosphate-buffered saline pH 7.2 and suspend at 2 per
cent v/v in phosphate-bufferedsaline pH 7.2. Dilute 0.1 mlof
tannic acid solution to 7.5 ml with phosphate-bufferedsaline
pH 7.2 (final concentration 1.3 mg perlitre). Mix 1 volume of
the freshly prepared dilution with 1 volume of human red
blood cell suspension and incubate at 37 for 10 minutes.
Collect the cells by centrifugation (400 to 800 g for 10 minutes),
discard the supernatant and wash the cells once with
phosphate-buffered saline pH 7.2. Resuspend the tanned
cells at 1.0 per cent v/v in phosphate-buffered saline pH 7.2.
Antigen coating of tanned human red blood cells. Take a
suitable volume (Vs) of tanned cells, add 0.2 ml of rubella
antigen per 1.0 ml of tanned cells and incubate at 37 for 30
minutes Collect the cells by centrifugation (400 to 800 g for 10
minutes) and discard the supernatant, leaving a volume of
200 gl. Add a volume of albumin barbital buffer solution
equivalent to the discarded supernatant, resuspend and collect
the cells as described and repeat the washing procedure.
Make.upJheremaining200g1Jothree::quartersofV".thereby
ob4tiD4Ig tl1<;l ipitialYQIl1fQ<;l.( Vi),.MJx._9QQ.fllQfqlbUl11il1.QqrbjtClI
buffer solution with 100 gl of Vi, which is thereby reduced to
the residual volume (V,), and determine the initial absorbance
. at 541 nm (A). Dilute Vr by a factor equal to A using albumin
Test solutions. Ifnecessary, adjust the immunoglobulin under

examination to pH 7, for example by addition of 1 M sodium
hydroxide. Dilute volumes of the preparation under
examination containing 30 mg and 40 mg ofimmunoglobulin
with albumin barbital buffer solution and adjust the volume
to 900 Ill.
Reference solutions. Prepare as for the test solutions using
human immunoglobulin RS.
Complement control. 900 III of albumin barbital buffer
solution.
Add to each cuvette/test-tube 100 III of sensitised human red

blood cells and mix well.
Incubate at room temperature for 15 minutes, add 1,000 III of
albumin barbital buffer solution, collect the cells by
centrifugation (1,000 g for 10 min) of the cuvette/test-tube
and remove 1,900 III ofthe supernatant. Replace the 1,900 III
with albumin barbital buffer solution and repeat the whole
ofthe washing procedure, finally leaving a volume of200 Ill.
Test samples may be stored in sealed cuvette/test-tubes at 4
for 24 hours.
Complement-initiated haemolysis. To measure haemolysis,
add 600 gl of albumin barbital buffer solution wanned to 37
to the test sample, re-suspend the cells carefully by repeated
pipetting (not fewer than 5 times) and place the cuvette in the
thermostatted cuvette holder of a spectrophotometer. After 2
minutes, add 200 III of diluted guinea-pig complement (125
to 200 CHso/ml), mix thoroughly by pipetting twice and start
immediately after the second pipetting the time-dependent
recording of absorbance at 541 nm, using albumin barbital
buffer solution as the compensation liquid. Stop the
measurement if absorbance as a function of time has clearly
passed the inflexion point.
Evaluation. Determine the slope (8) of the haemolysis curve
at the approximate inflexion point by segmenting the steepest
section in suitable time intervals At (for example, At = 1minute)
and calculate S between adjacent intersection points,
<::xp!_e~~~d_~~_.&tE~~_Il1!l111!eJ:T~eJ.!~~~eJ~t."-ll}ll_e. .t'.o~!! seJrveJs.ll:~

($cxp). IIJ,aA~ition, .~(lt(ll1:l1il1t::th(lll~~()!~lll1c:elltthe:~t~!!()f
measurement (As) by extrapolating the curve, which is almost
linear and parallel to the time axis within the first few minutes.
Correct (Scxp) using the expression:
2580
IP 2010
Tests
pH (2.4.24). 4.0 to 7.4.
Calculate the arithmetic mean of the values of S' for each
preparation. Calculate the index ofFc function (he) from the
expression:
S' -S'
s

Total protein. Minimum 3.0 per cent w/v and between 90.0 to
110.0 per cent of the quantity of protein stated on the label.
(Si -
100 x
S' e )
- -=--'---,=---'Fe -
Dilute the preparation under examination with a 0.9 per cent

w/v solution of sodium chloride to obtain a solution
containing 1.0 per cent of protein.
S' = arithmetic mean ofthe corrected slope for the

preparation under examination,
S's = arithmetic mean of the corrected slope for the
reference preparation,
S'c = arithmetic mean of the corrected slope for the
complement control.
Calculate the index ofFc function for the preparation under
examination; the value is not less than that stated in the leaflet
accompanying the reference preparation.
is prepared as a stabilised solution or as a freeze-dried
preparation. A stabiliser may be added. In both cases the
preparation is passed through a bacteria-retentive filter. The
preparation may subsequently be freeze-dried and the
containers closed under vacuum or under an inert gas. No
antimicrobial preservative is added either during fractionation
or at the stage of the final bulk solution.
The stability of the preparation is demonstrated by suitable
tests carried out during development studies.
Description. The liquid preparation is clear or slightly
opalescent and colourless or pale yellow. The freeze-dried
preparation is a hygroscopic, white or slightly yellow powder
or solid friable mass.
For the freeze-dried preparation, reconstitute as stated on
the label immediately before carrying out the identification
and the tests, except those for solubility and water.
Identification
Examine by a suitable immunoelectrophoresis technique. Using
antiserum to normal human serum, compare normal human
serum and the preparation under examination, both diluted to
contain 1.0 per cent w/v of protein. The main component of
the preparation under examination corresponds to the IgG
component of normal human serum. The preparation under
examination may show the presence of small quantities of
other plasma proteins; ifhuman albumin has been added as a
stabiliser, it may be seen as a major component.
Dilute the preparation under examination with a 0.9 per cent

solution of sodium chloride to obtain a solution containing
about 15 mg ofprotein in 2 ml. To 2.0 ml ofthis solution in a
round-bottomed centrifuge tube add 2 ml of a 7.5 per cent
w/v solution of sodium molybdate and 2 ml of a mixture of 1
volume of nitrogen-free sulphuric acid and 30 volumes of
water. Shake, centrifuge for 5 minutes, decant the supernatant
liquid and allow the inverted tube to drain on filter paper.
Determine the nitrogen in the centrifugation residue. by the
content of protein by multiplying the result by 6.25.
Protein composition. Determine by zone electrophoresis
(2.4.12).
Use strips of suitable cellulose acetate gel as the supporting
medium and barbital buffer solution pH 8.6 as the electrolyte
solution.
Test solution. Dilute the preparation under examination with a
0.9 per cent w/v solution of sodium chloride to an
immunoglobulin concentration of3.0 per cent w/v.
Reference solution. Reconstitute human immunoglobulin for electrophoresis reference preparation and dilute with a 0.9
per cent w/v solution of sodium chloride to- a protein
concentration of 3.0 per cent w/v.
To a strip apply 4 III of the test solution as a 10 rom band or
apply 0.4 III per mm ifa narrower strip is used. To another strip
apply in the same manner the same volume of the reference
solution. Apply a suitable electric field such that the albumin
band ofnormal human serum applied on a control strip migrates
at least 30 mm. Stain the strips with amido black lOB solution
for 5 minutes. Decolourise with a mixture of 10 volumes of
glacial acetic acid and 90 volumes of methanol so that the
background is just free of colour. Develop the transparency
of the strips with a mixture of 19 volumes of glacial acetic
acid and 81 volumes of methanol. Measure the absorbance
ofthe bands at 600 nm in an instrument having a linear response
over the range of measurement. Calculate the result as the
mean of3 measurements of each strip.
System suitability. In the electropherogram obtained with the
reference preparation, the proportion ofprotein in the principal
band is within the limits stated in the leaflet accompanying
the reference preparation.
2581
Results. In the electropherogram obtained with the test

solution, not more than 5.0 per cent ofprotein has a mobility
different from that of the principal band. This limit is not
applicable if albumin has been added to the preparation as a
stabiliser; for such preparations, a test for protein composition
is carried out during manufacture before addition of the
stabiliser.
Molecular size. Determine by liquid chromatography (2.4.14).
Test solution. Dilute the preparation under examination with a
0.9 per cent w/v solution of sodium chloride to obtain a
concentration in the range of 0.4 to1.2 per cent w/v and
injection of 50 to 600 Jlg ofprotein are usually suitable.
Reference solution. Dilute human immunoglobulin RS with a
0.9 per cent w/v solution of sodium chloride to the same
protein concentration as the test solution.
- a stainless steel column 60 cmx 7.5 mmor30 cmx 7.8 mm
packed with hydrophilic silica,
mobile phase. dissolve 4.873 g ofdisodium hydrogen
phosphate dihydrate, 1.741 g of sodium dihydrogen
phosphate monohydrate, 11.688 g of sodium chloride
and 50 mg ofsodium azide in 1000 ml ofwater.
- spectrophotometer set at 280 urn.
In the chromatogram obtained with the reference solution, the
principal peak corresponds to IgG monomer and there is a
peak corresponding to dimer with a relative retention to the
principal peak of about 0.85. Identify the peaks in the
chromatogram obtained with the test solution by comparison
with the chromatogram obtained with the reference solution;
any peak with a retention time shorter than that of dimer
corresponds to polymers and aggregates.
Results. In the chromatogram obtained with the test solution:
a. relative retention: for monomer and dimer, the relative
retention to the corresponding peak in the chromatogram
obtained with the reference solution is 1 02;
b. peak area: the sum ofthe peak areas ofmonomer and dimer
represent not less than 90.0 per cent of the total area of the
chromatogram and the sum of the peak area of polymers and
aggregates represents not more than 3.0 per cent of the total
area ofthe chromatogram. This requirement does not apply to
products where albumin has been added as a stabiliser; for
products stabilised with albumin, a test for distribution of
molecular size is carried out during manufacture before
addition ofthe stabiliser.
Anticomplementary activity. The consumption ofcomplement
isnof greafeffhan 5U:OperceriI(rCH;07mgofiIDiliilii6g1615U1ill):
TesHorantlcomplementary'actlViijofii:i:lmiinoglobullii
For the measurement ofanticomplementary activity (ACA) of
immunoglobulin, a defined amount oftest material (10 mg of
IP 2010
immunoglobulin) is incubated with a defined amount of

guinea-pig complement (20 CH so) and the remaining
complement is titrated; the anticomplementary activity is
expressed as the percentage consumption of complement
relative to the complement control considered as 100.0 per
cent.
The haemolytic unit of complement activity (CHso) is the
amount of complement that, in the given reaction conditions,
will produce the lysis of 2.5 x 108 out of a total of 5 x 108
optimally sensitised red blood cells.
Magnesium and calcium stock solution. Prepare as stated
earlier.
Barbital buffer stock solution. Prepare as stated earlier.
Gelatin solution. Dissolve 12.5 g ofgelatin in about 800 ml of
water and heat to boiling in a water-bath. Cool to 20 and
dilute to 10 litres with water. Filter through a membrane filter
(pore size: 0.22 Jlm). Store at4. Use clear solutions only.
Citrate solution. Dissolve 8.0 g of sodium citrate, 4.2 g of
sodium chloride and 20.5 g of glucose in 750 ml of water.
Adjust to pH 6.1 using a 10.0 per cent w/v solution of citric
acid and dilute to 1,000 ml with water.
Gelatin barbital buffer solution. Add 4 volumes ofgelatin
solution to 1 volume of barbital buffer stock solution and
mix. Adjust to pH 7.3, ifnecessary, using 1 M sodium hydroxide
or 1 M hydrochloric acid. Maintain at 4. Prepare fresh
solutions daily.
Stabilised sheep blood. Collect one volume of sheep blood
into one volume ofcitrate solution and mix. Store at 4 for not
less than 7 days and not more than 28 days. (Stabilised sheep
blood and sheep red blood cells are available from a number
ofcommercial sources.)
Haemolysin. Antiserum against sheep red blood cells prepared
in rabbits.
Guinea-pig complement. Prepare a pool of serum from the
blood of not fewer than ten guinea-pigs. Separate the serum
from the clotted blood by centrifugation at about 4 0 Store the
serum in small amounts below -70.
Method
Preparation ofstandardised 5 per cent sheep red blood cell
suspension. Separate sheep red blood cells by centrifuging
an appropriate volume of stabilised sheep blood and wash
the cells at least three times with gelatin barbital buffer
sQlutiQnand_prepare..as_a_5.0_pJ~LC_enLyLv_s.usp_ensiolljnths<
s.a.mesgMi<:m,.Ml'laslll"l'lJlle. c:t)n<il'll1sjty()fJI1f:Sll~Pf:l1si()l1a~
follows: add 0.2 to 2.8 ml of water and centrifuge the lysed
solution for 5 minutes at 1,000 g; the cell density is suitable if
the absorbance (2.4.7) ofthe supernatant liquid at 541 urn is
2582
IP 2010
0.62 0.01. Correct the cell density by adding gelatin barbital

buffer solution according to the fonnula:
v _
f -
maximum degree ofhaemolysis is not in this range, repeat the

titration with more or less diluted complement solution.
. Table-l
xA
0.62
The adjusted suspension contains about 1 x 109 cells per m!.
Required
Prepared using
dilution of
haemolysin
Gelatin barbital buffer solution
Volume
Dilution
ml
(l : ... )
Haemolysin titration
7.5
Prepare haemolysin dilutions as shown in Table 1.
10
0.65
0.90
75
100
1.80
1.80
7.5
10
150
200
300
1.00
1.00
75
100
150
Vr = final adjusted volume,

V; = initial volume,
A = absorbance of the original suspension at 541 nm.
Add 1.0 ml of 5.0 per cent sheep red blood cell suspension to
each tube of the haemolysin dilution series, starting at the
1:75 dilution, and mix. Incubate at 37 for 30 minutes.
Transfer 0.2 ml of each of these incubated mixtures to new
tubes and add 1.10 ml of gelatin barbital buffer solution and
0.2 ml ofdiluted guinea-pig complement (for example, 1: 150).
Perform this in duplicate.
As the unhaemolysed cell control, prepare three tubes with
1.4 ml of gelatin barbital buffer solution and 0.1 ml of 5.0 per
cent sheep red blood cell suspension.
As the fully haemolysed control, prepare three tubes with 1.4
ml of water and 0.1 ml of 5.0 per cent sheep red cell
suspension.
Incubate all tubes at 37 for 60 minutes and centrifuge at 1,000
g for 5 minutes. Measure the absorbance (2.4.7) of the
supernatants at 541 urn and calculate the percentage degree
of haemolysis in each tube using the expression:
Aa
Ab
AI
absorbance oftubes with haemolysin dilution,

mean absorbance of the three tubes with full
haemolysis,
mean absorbance of the three tubes with no
haemolysis.
Plot the percentage degree of haemolysis as the ordinate

against the corresponding reciprocal value ofthe haemolysin
dilution as the abscissa on linear graph paper. Determine the
optimal dilution of the haemolysin from the graph by
inspection. Select a dilution such that further increase in the
amount of haemolysin does not cause appreciable change in
the degree ofhaemolysis. This dilution is defined as one minimal
haemolytic unit (1 MHU) in 1.0 m!. The optimal haemolytic
haemolysin dilution for preparation of sensitised sheep red
blood cells contains 2 MHU per m!.
400
600
800
1200
1600
2400
3200*
4800*
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
undiluted
undiluted
200
300
400
600
800
1200
1600
2400
Haemolysin
Volume
ml
0.1
0.1
0.2
0.2
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
* discard 1.0 ml ofthe mixture
Preparation of optimised sensitised sheep red blood cells

(haemolytic system)
Prepare an appropriate volume of diluted haemolysin

containing 2 MHU/ml and an equal volume of standardised
5.0 per cent sheep red blood cell suspension. Add the
haemolysin dilution to the standardised cell suspension and
mix. Incubate at 37 for 15 minutes, store at 2 to 8 and use
within 6 hours.
Titration ofcomplement. Prepare an appropriate dilution of
complement (for example, 1:250) with gelatin barbital buffer
solution and perform the titration in duplicate as shown in
Table 2.
Add 0.2 ml of sensitised sheep red blood cells to each tube,

mix well and incubate at 37 for 60 minutes. Cool the tubes in
an ice-bath and centrifuge at 1,000 g for 5 minutes. Measure
the absorbance of the supernatant liquid at 541 nm and
calculate the degree ofhaemolysis (Y) using the expression:
The haemolysin titration is not valid unless the maximum

degree ofhaemolysis is 50.0 per cent to 70.0 per cent. If the
2583
Ac
Ab
AI
absorbance of tubes 1 to 12,

mean absorbance of tubes with 100 per cent
haemolysis,
mean. absorbance of cell controls with 0 per
cent haemolysis.
Plot Y/(l - Y) as the abscissa against the amount of diluted

complement in ml as the ordinate on log-log graph paper. Fit
the best line to the points and determine the ordinate for the
50.0 per cent haemolytic complement dose where Y/(l - Y) =
1.0. Calculate the activity in haemolytic units (CHso/ml) from
the expression:
Cd
Co
5 =
reciprocal value ofthe complement dilution,

volume of diluted complement in ml resulting
in 50.0 per cent haemolysis,
scaling factor to take account ofthe number of
red blood cells.
The test is not valid unless the plot is a straight line between
15.0 per cent and 85.0 per cent haemolysis and the slope is
0.15 to 0040, and preferably 0.18 to 0.30.
Table - 2
Tube Number
Volume ofdiluted
complement in ml
(for example 1:250)
Volume ofgelatin
barbital buffer
solution in ml
0.1
1.2
0.2
1.1
0.3
1.0
0.4
0.9
0.5
0.8
0.6
0.7
0.7
0.6
0.8
0.5
0.9
004
10
1.0
0.3
11
1.1
0.2
12
1.2
0.1
Three tube as cell

c0 l11:r01 11t Qperc;ent
haemolysis
......... .,. .. __ _--_
.
__
--_
1.3
.....
Three tube at 100

per cent haemolysis
IP 2010
Test for anticomplementary activity

Prepare a complement dilution having 100 CHso per ml by
diluting titrated guinea-pig complement with gelatin barbital
buffer solution. Ifnecessary, adjust the immunoglobulin under
examination to pH 7. Prepare incubation mixtures as follows
for an immunoglobulin containing 50 mg per ml:
Table- 3
Immunoglobulin
under examination
Complement
control
(in duplicate)
Immunoglobulin (50 mg per ml)
O.2ml
Gelatin barbital buffer
0.6ml
0.8ml
Complement
O.2ml
O..2ml
Carry out the test on the immunoglobulin under examination

and prepare ACA negative and positive controls using human
immunoglobulin RS, as indicated in the leaflet accompanying
the reference preparation. Higher or lower volumes of sample
and of gelatin barbital buffer solution are added if the
immunoglobulin concentration varies from 50 mg per ml; for
example, 0047 ml of gelatin barbital buffer solution is added
to 0.33 ml ofimmunoglobulin containing 30 mg per ml to give
0.8 ml. Close the tubes and incubate at 37 for 60 minutes.
Add 0.2 ml of each incubation mixture to 9.8 ml of gelatin
barbital buffer solution to dilute the complement. Perform
complement titrations as described above on each tube to
determine the remaining complement activity (Table 2).
Calculate the anticomplementary activity of the preparation
under examination relative to the complement control
considered as 100.0 per cent, from the expression:
a - b xlOO
a
a
b
mean complement activity (CHso/ml) of

complement control,
complement activity (CHso/ml) oftested sample.
The test is not valid unless:

a. the anticomplementary activities found for ACA negative
control and ACA positive control are within the limits stated
in the leaflet accompanying the reference preparation,
b. the complement activity ofthe complement control (a) is in
the range 80 to 120 CH so perml.
Prekallikrein activator. Maximum 35 IV per ml, calculated
with reference to a dilution ofthe preparation under examination
containing.3.O_per centw/v ofimmunoglobulin.
Testforprekllllilrreifillctivator
1.3 ml of water
Prekallikrein activator (PKA) activates prekallikrein to kallikrein

and may be assayed by its ability to cleave a chromophore
2584
IP 2010
from a synthetic peptide substrate so that the rate of cleavage

. can be measured spectrophotometrically and the
concentration of PKA calculated by comparison with a
reference preparation calibrated in International Units.
The International Unit is the activity ofa stated amount ofthe
International Standard which consists of freeze-dried
prekallikrein activator. The equivalence in International Units
of the International Standard is stated by the World Health
Organisation.
Reagents. Prekallikrein activator in albumin RS is calibrated
in International Units by comparison with the International
Standard.
Buffer A. Dissolve 6.055 g of tris(hydroxymethyl)aminomethane, 1.17 g of sodium chloride, 50 mg of
hexadimethrine bromide and 0.100 g ofsodium azide in water.
Adjust to pH 8.0 with 2 M hydrochloric acid and dilute to
1000 ml with water.
Buffer B. Dissolve 6.055 g of tris(hydroxymethyl)aminomethane and 8.77 g of sodium chloride in water. Adjust
to pH 8.0 with 2 M hydrochloric acid and dilute to 1,000 ml
with water.
Preparation of prekallikrein substrate
To avoid coagulation activation, blood or plasma used for

the preparation of prekallikrein must come into contact
only with plastics or silicone-treated glass sUifaces.
Draw 9 volumes ofhuman blood into 1volume ofanticoagulant
solution (ACD, CPD or 3.8 per cent w/v solution of sodium
citrate) to which 1 mg per ml of hexadimethrine bromide has
been added. Centrifuge the mixture at 3,600 g for 5 minutes.
Separate the plasma and centrifuge again at 6,000 g for 20
minutes to sediment platelets. Separate the platelet-poor plasma
and dialyse against 10 volumes ofbuffer A for 20 hours. Apply
the dialysed plasma to a chromatography column containing
agarose-DEAE for ion exchange chromatography which has
been equilibrated in buffer A and is equal to twice the volume
ofthe plasma. Elute from the column with bufferA at20 ml per
cm 2 per hours. Collect the eluate in fractions and record the
absorbance (2.4.7) at 280 nm. Pool the fractions containing
the first protein peak so that the volume of the pool is about
1.2 times the volume ofthe platelet-poor plasma.
Test the substrate pool for absence of kallikrein activity by
mixing I part with 20 parts of the pre-warmed chromogenic
substrate solution to be used in the assay and incubate at 37
for 2 minutes. The substrate is suitable if the increase in
absorbance is less than 0.001 per minute. Add to the pooled
solution 0.7 per cent w/v ofsodium chloride and filter using a
membrane filter (porosity 0.45 /lm). Freeze the filtrate in portions
and store at _25; the substrate may be freeze-dried before
storage.
Carry out all procedures from the beginning of the

chromatography to freezing in portions during a single working
day.
Method. The assay may be carried out using an automated
enzyme analyser or a suitable microtitre plate system allowing
kinetic measurements, with appropriate software for calculation
ofresults. Standards, samples and prekallikrein substrate may
be diluted as necessary using buffer B.
Incubate diluted standards or samples with prekallikrein

substrate for 10 minutes such that the volume ofthe undiluted
sample does not exceed 1/10 of the total volume of the
incubation mixture to avoid errors caused by variation in ionic
strength and pH in the incubation mixture. Incubate the
mixture or a part thereof with at least an equal volume of a
solution ofa suitable synthetic chromogenic substrate, known
to be specific for kallikrein (for example, N-benzoyl-L-prolylL-phenylalanyl~L-arginine 4-nitroanilide acetate or Dpro ly 1- L-p h eny Ia Iany 1- L-argi n in e- 4 -n itroan iiidedihydrochloride), dissolved in buffer B. Record the rate of
change in absorbance per minute for 2 to 10 minutes at the
wavelength specific for the substrate used. Prepare a blank
for each mixture ofsample or standard using buffer B instead
ofprekallikrein substrate.
Depending on the method used, ~ A per minutes has to be
corrected by subtracting the value obtained for the
corresponding blank without the prekallikrein substrate. The
results may be calculated using a standard curve, a parallelline or a slope ratio assay or any other suitable statistical
method. Plot a calibration curve using the values thus obtained
for the reference preparation and the respective
concentrations; use the curve to determine the PKA activity
ofthe preparation under examiantion.
Anti-A and anti-B haemagglutinins. Carry out the tests for
anti-A and anti-B haemagglutinins as stated under Dried
human haemophilic fraction. If the preparation under
examination contains more than 3.0 per cent of
immunoglobulin, dilute to this concentration before preparing
the dilutions to be used in the test. The 1 to 64 dilutions do
not show agglutination.
Anti-D antibodies. It complies with the test for anti7D
antibodies in human immunoglobulin for intravenous
\
administration.
.
Test for anti-D antibodies in human immunoglobulin for
intravenous administration
'
Materials
Phosphate-buffered saline (PBS). Dissolve 8.0 g ofsodium
chloride, 0.76 g of anhydrous disodium hydrogen phosphate,
0.2 g ofpotassium chloride, 0.2 g of potassium dihydrogen
2585
IP 2010
phosphate and 0.2 g of sodium azide in water and dilute to

1,000 ml with the same solvent.
of the well indicates a positive result. A stream of cells

represents a negative result.
Papain solution. Use serological grade papain from a

commercial source, the activity of which has been validated.
Record the endpoint titre as the reciprocal of the highest

dilution that gives rise to a positive result.
Red blood cells. Use pooled red blood cells from not fewer
than 3 donors of group ORzRz and 3 donors of group Orr
respectively. Wash the cells 4 times with PBS or until the
supematant is clear. Centrifuge the cells at 1, 800 g for 5 minutes
to pack. Treat the packed red cells with papain solution
according to the manufacturer's instructions. Store in
Alsever's solution for not more than 1 week.
The titre of the preparation under examination is not greater

than the titre of the reference preparation.
Microtitre plates. Use V-bottomed rigid micro-titre plates.
Reference standards. Immunoglobulin (anti-D antibodies

test) reference preparation and Immunoglobulin (anti-D
antibodies test negative control) reference preparation are
suitable for use as the reference preparation and negative
control, respectively.
Method
Reference preparation and negative control solutions.
Reconstitute the reference preparation and the negative
control according to instructions. Dilute the reconstituted
preparations with an equal volume of PBS containing 0.2 per
cent w/v of bovine albumin and then prepare a further 7 serial
two-fold dilutions using PBS containing 0.2 per cent w/v of
bovine albumin to give a total dilution range from 1/2 to
1/256. Make 2 independent sets of dilutions for each
preparation. Add 20 III ofeach dilution to the microtitre plate.
Test solutions. Initially dilute the test samples to give a starting

immunoglobulin G (IgG) concentration of 2.5 per cent w/v
using PBS containing 0.2 per cent w/v of bovine albumin and
then prepare a further 7 serial two-fold dilutions using PBS
containing 0.2 per cent w/v of bovine albumin to give a total
dilution range from 1/2 to 1/256. Make 2 independent sets of
dilutions for each test sample. Add 20 III of each dilution to
the microtitre plate.
Prepare 3.0 per cent v/v suspensions of papain-treated Dpositive (ORzR z) and D-negative (Orr) red cells in PBS
containing 0.2 per cent w/v of bovine albumin. Add 20 III of
D-positive cells to one dilution series ofeach ofthe test sample,
the reference preparation and the negative control, and 20 III
of D-negative cells to the other dilution series of each of the
test samples, the reference preparation and the negative
control. Mix by shaking the plate on a shaker for 10 seconds.
Water. Determine by semi-micro determination ofwater (2.3.43),

loss on drying (2.4.19) or near infrared spectrophotometry
per kg of the rabbit's mass a volume equivalent to 0.5 g of
immunoglobulin but not more than 10 ml per kg of body mass.
Antibody to hepatitis B surface antigen
Minimum 0.5 ill per g of immunoglobulin, determined by a
suitable immunochemical method.
Storage. For the liquid preparation, store in a colourless glass
container, protected from light, at the temperature stated on
the label. For the freeze-dried preparation, store in an airtight
colourless glass container, protected from light, at a
content expressed in grams per litre; (2) for freeze-dried
amount of immunoglobulin in the container; (4) the route of
administration; (5) for freeze-dried preparations, the name or
composition and the volume ofthe reconstituting liquid to be
added; (6) the distribution of subclasses of immunoglobulin
G present in the preparation; (7) where applicable, the amount
of albumin added as a stabilizer; (8) the maximum content of
immunoglobulin A.

Human Plasma for Fractionation
Human Plasma for Fractionation is the liquid part of human
blood remaining after separation ofthe cellular elements from
blood collected in a receptacle containing an anticoagulant,
or separated by continuous filtration or centrifugation of
anticoagulated blood in an apheresis procedure; it is intended
for the manufacture of plasma-derived products.
Centrifuge the plate at 80 g for 1 minutes to pellet the cells.

Place the plate at an angle ofapproximately 70. Read after 4 to
mInutes {or.untli.tl1eceIIs-Iiavestrealnedlntile weIls
containing the negative control and the wells where the

D-negative cells have been added). A cell button at the bottom
Donors
Only a carefully selected, healthy donor who, as far as can be
ascertained after medical examination, laboratory blood tests
2586
PLASMA FOR FRACTIONATION
IP 2010
and a study of the donor's medical history, is free from

detectable agents ofinfection transmissible by plasma-derived
products may be used.
Immunisation ofdonors
Immunisation of donors to obtain immunoglobulins with
specific activities may be carried out when sufficient supplies
of material of suitable quality can not be obtained from
naturally immunised donors. Recommendations for such
immunisations are formulated by the World Health
Organisation (Requirements for the collection, processing
and quality control ofblood, blood components and plasma
derivatives, WHO Technical Report Series, No. 840, 1994 or
subsequent revision).
Records
Records ofdonors and donations made are kept in such a way
that, while maintaining the required degree of confidentiality
concerning the donor's identity, the origin of each donation
in a plasma pool and the results of the corresponding
acceptance procedures and laboratory tests can be traced.
Laboratory tests
Laboratory tests are carried out for each donation to detect
the following viral markers:
1.
Antibodies against human immunodeficiency virus 1(antiHIV-l),
2.
Antibodies against human immunodeficiency virus 2 (antiHIV-2),
3.
Antibodies against hepatitis C virus (anti-HCV),
4.
Hepatitis B surface antigen (HBsAg).
Pending complete harmonisation of the laboratory tests to be

carried out, the competent authority may require that a test for
alanine aminotransferase (ALT) also be carried out.
The test methods used are of suitable sensitivity and
specificity and comply with the regulations in force. Ifa repeatreactive result is found in any of these tests, the donation is
not accepted.
Individual plasma units
The plasma is prepared by a method that removes cells and
cell debris as completely as possible. Whether prepared from
whole blood or by plasmapheresis, the plasma is separated
from the cells by a method designed to prevent the introduction
of micro-organisms. No antibacterial or antifungal agent is
added to the plasma. The containers comply with the
requirements for plastic containers for blood and blood
components (6.2). The containers are closed so as to prevent
any possibility of contamination.
If 2 or more units are pooled prior to freezing, the operations

are carried out using sterile connecting devices or under
aseptic conditions and using containers that have not
previously been used.
When obtained by plasmapheresis, plasma intended for the
recovery of proteins that are labile in plasma is frozen by
cooling rapidly in a chamber at _30 0 or below as soon as
possible and at the latest within 24 hours of collection.
When obtained from whole blood, plasma intended for the
recovery of proteins that are labile in plasma is separated
from cellular elements and is frozen by cooling rapidly in a
chamber at -30 0 or below as soon as possible and at the latest
within 24 hours of collection.
When obtained from whole blood, plasma intended solely for
the recovery of proteins that are not labile in plasma is
separated from cellular elements and frozen in a chamber at 20 0 or below as soon as possible and at the latest within 72
hours of collection.
It is not intended that the determination oftotal protein and

factor VIII shown below be carried out on each unit ofplasma.
They are rather given as guidelines for good manufacturing
practice, the test for factor VIII being relevant for plasma
intendedfor use in the preparation ofconcentrates oflabile
proteins.
The total protein content ofa unit ofplasma depends on the
serum protein content ofthe donor and the degree ofdilution
inherent in the donation procedure. When plasma is obtained
fi-om a suitable donor and using the intended proportion of
anticoagulant solution, a total protein content complying
with the limit of5 per cent is obtained. Ifa volume ofblood or
plasma smaller than intended is collected into the
anticoagulant solution, the resulting plasma is not
necessarily unsuitable for poolingfor fractionation. The aim
of good manufacturing practice must be to achieve the
prescribed limit for all normal donations.
Preservation offactor VIII in the donatio,n depends on the
collection procedure and the subsequent handling of the
blood andplasma. With goodpractice, O. 71U1ml can usually
be achieved, but units ofplasma with a lower activity may
still be suitable for use in the production of coagulation
factor concentrates. The aim ofgood manufacturingpractice
is to conserve labile proteins as much as possible.
Total protein. Carry out the test using a pool ofnot less than
10 units. Dilute the pool with a 0.9 per cent solution of sodium
chloride to obtain a solution containing about 15 mg ofprotein
in 2 ml. To 2.0 ml ofthis solution in a round-bottomed centrifuge
tube add 2 ml of a 7.5 per cent solution of sodium molybdate
and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric
acid and 30 volumes of water. Shake, centrifuge for 5 minutes,
decant the supernatant liquid and allow the inverted tube to
2587
PLASMA FOR FRACTIONATION
IP 2010
drain on filterpaper. Determine the nitrogen in the residue by

the method of sulphuric acid digestion (2.3.30) and calculate
the protein.content by multiplying the quantity ofnitrogen by
6.25. The total protein content is not less than 5.0 per cent.
FactorVID
Carry out the test using a pool ofnot less than 10 units. Thaw
the samples under examination, ifnecessary, at 37. Determine
the assay of factor VIII (2.8.7), using a reference plasma
calibrated against the International Standard for human
coagulation factor VIII in plasma. The activity is not less than
0.7IUperml.
Pooled plasma
Production
Platelet Concentrate is prepared from units. of whole blood
that have not been allowed to cool below 20. Platelet rich
plasma (PRP) is separated within 4-6 hours after completion
of the phlebotomy. The final component should contain
resuspended platelets in an amount of plasma adequate to
maintain an acceptable pH - generally 40 to 70 ml is used.
Tests
Four PC per month should be assayed for pH and for platelet,
RBC and leucocyte counts.
PRP
During the manufacture of plasma products, the first

homogeneous pool ofplasma (for example, after removal of
cryoprecipitate) is tested for HBsAg, HCV antibodies and for
HIV antibodies using test methods of suitable sensitivity and
specificity; the pool must give negative results in these tests.
The plasma pool is also testedfor hepatitis C virus RNA using
a validated nucleic acid amplification technique (2.8.1). A
positive control with 100 IU per ml of hepatitis C virus RNA
and, to test for inhibitors, an internal control prepared by
addition of a suitable marker toa sample of the plasma pool
are included in the test. The test is invalid if the positive
control is non-reactive or ifthe result obtained with the internal
control indicates the presence of inhibitors. The plasma pool
complies with the test ifit is found non-reactive for hepatitis C
virus RNA.
Description. Before freezing, a clear to slightly turbid liquid
without visible signs of haemolysis; it may vary in colour
from light yellow to green.
Storage. Frozen plasma is stored in conditions designed to
maintain the temperature at or below -20; for accidental
reasons, the storage temperature may rise above -20 on one
or more occasions during storage but the plasma is
nevertheless considered suitable for fractionation if all the
following conditions are fulfilled (1) the total period of time
during which the temperature exceeds -20 does not exceed 72
hours; (2) the temperature does not exceed -15 on more than
one occasion; (3) the temperature at no time exceeds _5.
Labelling. The label enables each individual unit to be traced
to a specific donor.
Counts
Volume
Total Count
3.0 - 4.5 x 105per JlI (approximately)

170 - 250 ml (approximately)
9 x 10 10 per bag (approximately)
PRC
Counts
8 -10 x 105per Jll350 ml bag

(approximately)
10 -12 x 105per Jll450ml bag
(approximately)
Volume
40 - 50 ml per bag
10
Platelet counts> 5.5 x 10 per bag in 75 per cent ofbags

(ifprepared fi:om 450 ml bags)
> 4.2 X 10 10 per bag in 75 per cent of bags
(ifprepared from 350 ml bags)
Leucocyte count < 0.12 x 109 per bag.

RBC counts < 1.2 x 109 per bag.
pH < 6.3 at the end of 5 days storage.
Expiration time. The expiration time is not more than 72 hours
from the time of collection ofthe source material.
Storage. Store at 20 to 22 in polyvinylchloride plastic bags.
Preserve at the temperature relevant to the volume of
resuspension plasma, either between 20 and 22 or between
I and 6, the latter except during shipment, when the
temperature may be between 1 and 10.
Labelling. In addition to the labelling requirements ofWhole
Blood applicable to this product, label it to state the volume of
original plasma present, the kind and volume ofanticoagulant
solution present in the original plasma, the blood group
designation ofthe source blood, and the hour ofexpiration on
th~~till~(L~pjIl!tiQ!1gl:lt~,Wb:r:.ll:lJ?~~U~MQrtQ@g~.l:ltlQo
ill
PlafeIetsseparated from wholehlood withiri4 to 6 hollis of
collection and suspended in 40 to 50 ml of plasma are
designated as platelet concentrate.
~~o, .11l~~1 ital~()to state t~llta c:ontinu()lls gentleagitlltioll
shall be maintained, or where labelled for storage at I to 6, to

state that such agitation is optional. Label it also with the type
and result of a serologic test for syphilis, or to indicate that it
2588
WHOLE HUMAN BLOOD
IP 2010
was nonreactive in such test; with the type and result of a test
for hepatitis B. surface antigen, or to indicate that it was
nonreactive in such test; with a warning that it is to be used as
soon as possible but not more than 6 hours after entering the
container; to state that a filter is to be used in the administration
equipment; and to state that the instruction circular provided
is to be consulted for directions for use.
Whole Human Blood

Whole Blood (Human)
Whole Human Blood is blood drawn aseptically from selected
human donors and mixed with a suitable anticoagulant.
Whole Human Blood is the final mixture of blood and
anticoagulant solution contains not less than 9.7 per cent
w/v ofhaemoglobin, calculated from the haemoglobin content
ofthe donor's blood and the dilution due to the anticoagulant
solution. It is obtained from healthy donors who must:
(a) be in the age range of 18 to 60 years and be in good health
as indicated in part by normal temperature and blood
pressure within normal limits;
(b) not be pregnant, if females;
(c) not have undergone major surgery within 6 months of
donation;
(d) as far as can be ascertained after clinical and laboratory
examination and the study ofmedical history ofthe donor
be free from disease transmissible by blood transfusion;
(e) have blood containing not less than 12.5 per cent w/v of
haemoglobin;
(f) be free from acute respiratory diseases;

(g) be free from any infectious skin disease at the site of
phlebotomy;
(h) have no history of malarial fever within 12 months of
donation;
(i)
G)
have no history of viral hepatitis or of close contact with

an individual having viral hepatitis within 12 months of
donation and have blood that has given negative results
in tests for the presence of hepatitis-B antigen;
have blood that has been tested with negative results
for evidence ofsyphilitic infection, HCV antibodies, HIV
antibodies and malarial parasites.
The examinations and tests to be carried out are decided by

the National Regulatory Authority.
The frequency of donations of whole blood shall not exceed
once every 3 months with a maximum volume of 1.5 litres in
any consecutive 12 month period.
The blood is drawn aseptically through a closed system into

a suitable sterile container containing a specific amount of
Anticoagulant Citrate Dextrose Solution (ACD Solution) or
Anticoagulant Citrate Phosphate Dextrose Solution (CPD
Solution) which is placed before the container is sterilised.
The quantity of anticoagulant solution should not exceed
22.0 per cent v/v of the final volume of the mixture. No
antimicrobial preservative is added.
During the withdrawal ofblood the container is gently agitated
to ensure thorough mixing. When withdrawal is complete the
container is immediately sealed and cooled to 2 to 8. It is not
opened until immediately before transfusion. With every
container of blood, a separate sample mixed with the
appropriate quantity of anticoagulant solution, is collected
for compatibility and other tests; this small container is firmly
attached to the main container.
Whole Human Blood in containers from which samples have
been removed for tests should not be used for transfusion.
Consequently, it is not intended that the Tests and the Assay
should be carried out on the contents of the container. The
Blood Banle or the service collecting the blood is responsible
for ensuring that the conditions in which the blood is collected
and stored are such that, if and when tested, the blood will
comply with the requirements ofthe monograph.
Blood group. Determine the blood group (in the sample

accompanying each donation) under the ABO system (2.8.11),
by examination of both corpuscles and serum, and under the
Rh system (2.8.11), by examination ofthe corpuscles.
Description. Deep red fluid which, on standing separates into

a lower layer of sedimented-red cells and a yellowish, almost
clear upper layer of plasma, free from visible signs of
haemolysis, with a greyish layer between the two consisting
ofleucocytes and thrombocytes. A layer containing emulsified
fat may form on the surface.
Tests
Sterility (2.2.11). Complies with the tests for sterility,
determined by Method B.
Assay. Detennine the haemoglobin content by photometric
haemoglobinometry (2.8.12).
Storage. Store in colourless, transparent and sterile containers

into which it was originally drawn. The containers should be
provided with a hermetic, contamination-proofclosure. Store
at a temperature between 2to 8.
Labelling. The label states (1) the distinctive code number by

reference to which the details of the donor are available; (2)
the ABO group with the approved colour scheme for different
groups as specified by the National Regulatory Authority; (3)
the Rh group; (4) the total volume of fluid, the proportion of
2589
IP 2010
WHOLE HUMAN BLOOD
blood, and the nature and volume of anticoagulant solution;

(5) the date on which the blood was withdrawn; (6) the expiry ,
date which should not exceed 21 days from the date of
withdrawal of blood; (7) the storage conditions; (8) that the
blood must not be used for transfusion if there is any visible
evidence ofhaemolysis or other deterioration; (9) for blood of
group 0 whether haemolysins are present or not and if they

are, that the blood must be administered only to recipients of
blood group 0; (10) that the blood has given negative results
in the tests for the presence of malarial parasites, hepatitis-B
antigen, syphilis and HIV antibodies and any other tests
prescribed by the National Regulatory Authority.
2590
MONOGRAPHS
BIOTECHNOLOGY PRODUCTS
General Monograph
Biotechnology Derived Therapeutic Products
....
2593
Monographs
Erythropoietin Concentrated Solution
2602
Filgrastim Concentrated Solution
2610
Interferon Alfa-2 Concentrated Solution
2613
Streptokinase Bulle Solution
2617
2591
IP 2010
BIOTECHNOLOGY DERIvED THERAPEUTIC PRODUCTS
Biotechnology Derived Therapeutic

Products
the current edition of the Indian Pharmacopoeia does not

contain products produced by this process and hence no
further discussion on this aspect is included in this chapter
Biotechnology has brought about the discovery and

development of a new class ofhuman therapeutics. Advances
in genetics, cellular and molecular biology have allowed
scientists to identitY and develop a number of new products.
These products provide significant clinical benefits, and in
many cases, provide therapies where no effective treatment
existed.
MonoclonalAntibodies
Proteins are the worldlOrse molecules ofthe cell. Due to their

biological significance, proteins can make excellent drug
candidates. In the living cell, genes, in the form of DNA,
provide the basic instructions in creating biologically
functional proteins. Biotechnology is the process that
produces protein molecules in sufficient quantities for
therapeutic use, which have very specific physiological roles
that ah'eady exist in the human body albeit in limited quantities.
Techniques of genetic engineering using bacteria, yeast and
eukaryotic cells in culture or cultured animal cells are employed
to incorporate in these cells the information needed to produce
a human protein with therapeutic potential. Once engineered,
these cells can be grown in large quantities by fermentation or
large-scale cultivation of animal or human cells. The addition
ofgenetic material to cells gives the engineered proteins their
name, recOl;nbinant proteins
Gene Cloning and Protein Expression
The basic principle involved in gene cloning includes the
insertion of a select fragment of DNA representing the gene
for a peptide/protein into an episomal circular DNA, called the
vector, which transports the gene into a host cell. The vehicle
containing the inserted gene then replicates along with the
host cell. The cellular machinery of the host cell transcribes
and translates the genes including the inserted gene into
proteins. The vector with an inserted foreign gene is called a
recombinant DNA molecule and the protein produced as a
consequence of the inserted gene by the host cell is called
recombinant Protein. The commonly used vectors for
bacterial transformations are Plasmids, which are small circular
DNAs found in bacteria and other microorganisms. Other
vectors include viruses such as bacteriophages (for bacterial
hosts) or animal viruses for eukaryotic host cells (e.g., EpsteinBarr virus, Simian Virus etc). Major advances have been made
in the past decade for efficient and stable expression offoreign
proteins in mammalian cells in culture.
In addition, transgenic animals and plants have been produced
which express foreign genes inserted into their genome for
tissue/organ specific expression. Although large-scale
production of therapeutic proteins has been accomplished,
Antibodies are proteins produced by differentiated blymphocytes. Kohler and Milstein first fused a normally shortlived antibody producing b-cell obtained from the spleen of
mice immunized with an antigen to a rapidly dividing cancer
cell (mouse myeloma) to produce a cell line (hybridoma), which
was both immortal and a producer of antibodies that
recognized a single epitope on an antigen, hence, the name
monoclonal antibodies (mAbs). Monoclonal antibodies can
be produced in cell culture or in live animals. When the
hybridoma cells are injected into the peritoneal cavity ofmice,
they produce tumors containing an antibody-rich fluid called
ascites fluid. Production in cell culture is usually preferred
since cell culture in fermentation chambers can be used to
produce antibodies on a larger scale.
Humanized monoclonal antibodies
The standard procedure of producing monoclonal antibodies
yields mouse antibodies. For therapeutic applications in
humans these proteins are unsuitable since the human immune
system recognizes mouse antibodies as foreign, causing
systemic inflammatory effects and are removed rapidly from
circulation. Application ofrecombinant DNA technology has
overcome this problem. The DNA that encodes the binding
portion of monoclonal mouse antibodies is ligated to the
human antibody producing DNA. Such a construct is then
clo~ed suitably in a vector and introduced into a mammalian
expression system to produce these half-mouse and halfhuman antibodies. Depending on how big a part ofthe mouse
antibody is used,such proteins are eitherreferred to as Chimeric
or humanized monoclonal antibodies. Monoclonal antibodies
hav:e been generated and approved to treat cancer,
cardiovascular disease, inflamatory diseases, muscular
degeneration, transplant rejection, viral infection and others.
Therapeutic recombinant monoclonal antibodies
Recombinant monoclonal antibodies for therapeutic
applications belong to the IgG class of immunoglobulins.
Immunoglobulins are made up of four polypeptide chains,
two light (A. and K) and two heavy chains (a, ~, 'Y, e, !J.). The
type of heavy chain determines the immunoglobulin isotype
(IgA, IgD, IgG, IgE and IgM respectively). The fourpolypeptide chains are cross-linked by disulfide bridges (-SS- bonds). Light chains comprise 220 amino acid residues
whereas heavy chains are made of440-550 amino acids. Each
chain has two distinct regions namely, "constant" and
"variable", the former being proximal to the C-terminal region
(111-220 or 440-550 residues) whereas the latteris towards the
N- terminal sequence of the polypeptide (1-110). The amino
2593
IP 2010
BIOTECHNOLOGY DERIVED THERAPEUTIC PRODUCTS
acids of the constant region of immunoglobulin are uniform

from one antibody to another within the same isotype (e.g.,
IgG), while the variable region found in both. the light and
heavy chains consist of different amino acids. These regions
are referred to as hypervariable region or "Complementary
Determining Regions (CDRs)"representing antibody diversity,
each specific for the recognition of the antigen. An antibody
molecule is generally represented as a "Y" shaped structure.
The.arms of the Y structure are connected to the stem region
by a flexible hinge. IgG can be proteolytically cleaved by
partial digestion with papain, which results in three distinct
~50 KD fragments: two identical fragments designated Fab
fragments and one Fc fragment. The Fab fragments are the
arms ofthe antibody containing the entire light (L) chain and
the N-terminal half ofthe Heavy (H) chain. These fragments
contain the IgG's antigen binding domains. The Fc fragment
derives from the stem portion of the Y structure and contains
the c-terminal halves of the two H chains. The Fc region is
responsible for the activation of phagocytosis, complement
dependent cellular toxicity and antibody dependent cellular
cytotoxicity (ADCC).
The IgG-Fc region is glycosylated atAsn 297, which is essential
for the conformation of the antibody molecule that allows
interaction with effector ligands. The IgG-Fc glycoform pattern
of monoclonal chimeric and "humanized" recombinant
therapeutic antibodies produced in CHO or NSO cells show
wide variation from clone to clone, which is dependent on the
process and cell culture conditions. Under certain conditions
a number of abnormally glycosylated products that lack
potency and potentially immunogenic products are produced
(e.g., galactose-a (1,3)-galactose and N-glycolylneuraminic
acid) which are not therapeutically acceptable. However,
successive rounds of selection of cell lines overcome these
problems. Another important variable in efficacy due to
glycosylation is the ability of the IgG molecule to mediate
ADCC. This property is particularly important in antibody
therapeutics for oncology indications. The ability to elicit
ADCC greatly varies with different glycoforms. Therefore,
the cell lines have been engineered by the introduction of a
glycosyl transferase (GnTIII) and/or inactivation/deletion of
fucosyl transferase in the CHO cells, which has resulted in
products with 20 to 100-fold increase in ADCC.
Processes for the production of recombinant therapeutic
proteins including mAbs
Prokaryotes
The qualitative and quantitative demand for recombinant

proteins is. steadi!y'jl!fr_~l!.i!!g. MQ1~ulllX.1:>!ologjst~_~~
constalltly challenged by. the. need to iIIlproveaIldoptiIIlize
the existing expression systems to meet the steadily increasing
qualitative and quantitative demand for recombinant proteins.
The continuous evolution of novel expression systems is
paralleled by growing concerns about the safety ofthese novel

pharmaceuticals, which has resulted in the regulatory
authorities setting high standards for certification. One ofthe
strategies used by researchers in this field involves sourcing
new genetic elements for incorporation into expression
systems by systematically analyzing the rich natural diversity
ofmicroorganisms. There are, in addition, numerous tool~ for
modifying microorganisms and for re-engineering existing
biological pathways or processes to meet the needs of the
pharmaceutical industry. Although many prokaryotic hosts
are described in literature for the production of recombinant
proteins, E.coli is the most preferred host because its genetics
are well understood. The popular cloning host has been E. coli
BL21 and its derivative BL 21A.DE3. However there are some
disadvantages such as the recombinant protein produced may
not have a proper secondary structure due to the non-formation
of intra-molecular disulfide bonds. Many recombinant
proteins tend to aggregate when over expressed in E.coli.
E.coli proteins begin their sequence with an N-formyl
methionine, which may not be removed by its enzyme systems.
Proteolytic enzymes of E.coli may partially degrade the
recombinant protein.
The principal element in recombinant protein expression
technology is the recombinant plasmid, which contains the
gene that codes for the protein ofinterest. The gl:!ne expression
regulatory elements such as the promoter sequence in the
plasmid play a key role in the production of mRNA of the
recombinant gene. The other important regulatory process
employed in gene expression is the application of gene
induction. Induction is the phenomenon wherein the
transcription of the gene is turned on by the addition of a
chemical to the growth medium. The most popular system
used in academic research is the lac promoter, which is induced
by Isopropyl thiogalactopyranoside (IPTG). However, in the
industrial production of recombinant therapeutic proteins,
addition of chemical inducers is not desired, hence autoinducible promoters which are up-regulated upon changes in
the culture medium or the growth phase of the bacteria or the
regulation of cell cycle, are employed for improvement in
production efficiency. Overproduction ofproteins most often
results in misfolded structures, which are insoluble and
accumulate in the cytoplasm as "inclusion bodies". Cloning
and expressing a group of proteins collectively called
"Chaperones" overcome this problem. Some ofthe chaperones
whose mode ofaction is partially understood include, Gro ELI
Gro ES, DnaK, Dnal and GrpE. Fusion ofthe gene of interest
with a partner gene results in a recombinant protein, which
produces both gene products linked in tandem. Such fusions
also increase.expression levels as. well as provide ameansfor
1l.CQllVj:: l1 if)IJ,t . proces~ . oJ pll:rificatioll pyaffinity
chromatography. A number offusion partner genes are reported
in literature but the most common partner proteins are maltose
binding protein, glutathione-S-transferase and thioredoxin.
2594
IP 2010
For ease of recombinant protein purification, short sequence

tags at N- or C-tenninal are genetically attached which result
in recombinant protein products with additional amino acid
sequences. These additional sequences have to be cleaved
from the final therapeutic product. Several methods for precise
excision of tag sequences have been described which include
enzymatic and chemical processes. Another strategy employed
for enhancing protein expression is the use of "bicistronic"
vectors. In this strategy, a short sequence of a gene efficiently
expressed is linked as a non-translatable transcriptional fusion
to the gene of interest. Secretion of recombinant proteins or
their translocation to periplasmic space of bacteria is a
procedure employed in the expression of foreign proteins in
prokaryotes. This procedure requires the addition of signal
sequences from secretory pathways or leader sequences for
transport to periplamic space. OmpA and PelB signal peptides
have been shown to efficiently transport recombinant proteins
to bacterial periplasm. A good example is the production of
correctly folded full-length antibodies within the periplasm of
E. coli.
Kukaryotes
Yeasts
Similar to prokaryotes in growth kinetics achieving high
density, yeasts secrete large amounts ofrecombinant proteins.
Saccharomyces sp, Pichia pastoris, Hansenula polymorpha,
and Kluyveromyces lactis are some of the yeasts, which are
currently in use for the production oftherapeutic recombinant
proteins. Yeasts are the preferred eukaryotes for the
production of recombinant therapeutic proteins that cannot
be produced from prokaryotes because ofincorrect folding or
the requirement for glycosylation. Sacharomyces cerevisiae
is genetically well characterized, its growth physiolQgy well
understood and hence, it is the prevalent yeast species in
phannaceutical production process. However, Pichia pastoris
is also gaining ground as a preferred host for heterologous
protein expression because of its superior secretion efficiency
and high yields. Pichia pastoris and Hansenula polymorpha
are methanol metabolizing yeasts (methylotrophic yeasts),
which contain strong promoters for the genes of methanol
assimilating enzymes such as methanol oxidase and alcohol
oxidase. As the enzymes ofthese pathways account for more
than 30% of the total protein content of the organism, the
metabolic efficiency with respect to secreted protein is
significantly higher. Although attempts are being made to
humanize N-linked glycosylation in the yeastPichiapastoris,
the employability of yeasts for the expression of human
therapeutic proteins might reach a limit if the product has a
highly specific post-translational modification for its
phannacological activity/properties. In such cases, mammalian
cell expression systems are employed for the production of
therapeutic proteins.
Mammalian Cell Lines

Animal Cell expression systems produce proteins, which have
high degree ofsimilarity to human proteins with respect to the
pattern and capacity of post translational modifications. The
most commonly used cel1lines for the expression oftherapeutic
proteins are as follows:
Chinese Hamster Ovary (CHO) Cell Line
Baby Hamster Kidney (BHK) Cell Line
HEK Human Embryo Kidney (HEK) Cell Line
Human Retinal Embryonic Cell Line (pER C.6)
Mouse Myeloma Cel1 Line (NSO)
Mammalian cel1lines produce therapeutic proteins with proper
folding, assembly and post-translational modifications, which
results in phannacological1y active products in tenns ofquality
and reproducibility. However, there remains a possibility of
transmission of viral or pyrogenic substances carried over in
the final product. There is a trend now to use cell lines, which
are of human origin since they possess all mechanisms to
produce products most similar if not identical to naturally
occurring proteins in the human body.
Immortalized cel1 lines transfected with foreign therapeutic
protein genes are used for the production of the respective
proteins. There are basical1y two processes of immortalizing
mammalian cells; (1) Viral transformation: Induction of
immortalization by inactivating the tumor suppressor genes
ofmammalian cel1s. Viral genes including Epstein - Barr virus
(EBV), Simian Virus 40 (SV 40) T antigen, adenovirus and
human papillomavirus (HPV) can induce immortalization.
Although the method is quite reliable, there are reports of
aneuploidy and loss of some properties of the primary cells.
(2) Modification of telomere of somatic cells. Telomerase
reverse transcriptase protein (TERT), which is inactive in most
somatic cells, when exogenously expressed, the cells are able
to maintain telomere lengths sufficient to avoid replicative
senescence thus, rendering the cells immortal. The latter
method is preferred for therapeutic protein expression.
Analysis ofseveral telomere-immortalized cel1lines has verified
that the cells maintain a stable genotype and retain critical
phenotype markers.
Development ofa cell line stably expressing a desired protein
gene starts with the construction of expression vectors.
Different expression vectors both viral and non-viral have
been designed to transfer foreign genes into mammalian cells.
The choice of the vector is dependent on the host cell, the
level of the desired product fonned and safety. Typically an
expression vector consists of (i) a constitutive or inducible
promoter (ii) a transcription tenninator and (iii) a cassette with
an origin of replication and a selection marker for vector
production in bacteria. Strong constitutive promoters are of
2595
viral genomes (e.g., human cytomegalovirus (HCMV), simian

virus 40 and Rous sarcoma virus). Amongst the non-viral
promoters the promoter of human elongation factor-I-a (EFIa) is similar in strength to the HCMV promoter. The desired
therapeutic protein gene is isolated as a cDNA, with one intron
sequence, which is preferably located between the promoter
and the coding sequence of the gene of interest and inserted
into the vector at a suitable site. The vector containing the
gene insert is then propogated in the bacterial host. The vector
is then recovered from the bacteria, linearized and introduced
into the mammalian cell by transfection. There are many
reagents/methods to effectively mediate gene transfer. Calcium
phosphate facilitated transfection, electroporation, lipofection,
micro iiDection, biolistic and polymer mediated transfer are
routinely used. For selection of recombinant cell, a second
gene is transferred that confers to the recipient cells a selective
advantage. The most common selector genes are dihydrofolate
reductase (DHFR) and glutamine synthetase (OS). In both
cases, selection occurs in the absence of appropriate
metabolite (hypoxanthine and thymidine in the case ofDHFR
and glutamine in the case of OS), thus preventing the growth
ofnon-transfonned cells. When the transfected foreign gene
is integrated into a "good site" on the chromosome of the
host by recombination, the resultant cell line will stably express
the desired gene. Selection pressure results in several fold
amplification of genes including the integrated genes when
high concentrations ofrespective inhibitors are used (for. e.g.,
Methotrexate inhibition of DHFR and Methionine
sulphoximine inhibition ofOS).
IP 2010
General Techniques used in down-stream processing of

recombina.nt proteins
The design ofthe purification process ofa recombinant protein
and its scale-up capabilities assumes significant importance
since the regulatory authorities would approve the "product
by process". Thus, the approved process must be followed
even ifnew developments in separation teclmologies provide
significant advantages.
Liquid chromatography is the core of preparative protein
purification and all supplementary procedures like extraction,
centrifugation, ultrafiltration, and dialysis serve to prepare
the protein solution for chromatography. A series of
chromatographic steps usually tenned as capture, intennediate
purification and polishing, make use of different intrinsic
features of proteins, is usually required to achieve sufficient
separation of the target from contaminants. Common modes
of chromatography include ion exchange chromatography,
affinity chromatography, hydrophobic interaction
chromatography, gel filtration and reverse phase
chromatography. The selection of chromatographic
procedures and their arrangements in a suitable order are based
on the properties of the recombinant protein particularly its
tolerance to organic solvents, its susceptibility to denaturation
due to changes in pH, temperature, degradation and oxidation.
In the case of secreted recombinant proteins, downstream

processing starts with the collection of the fermentation
supernatant after separating the cell mass while if the
recombinant protein is intracellular, the process begins with
There are two main ways of culturing animal cells for the the extraction of soluble proteins from the cell mass. In the
production of recombInant proteins, viz., (a) Adherent cell fonner case the target protein may be highly dilute with large
culture and (b) Suspension culture. In adherent cell culture amounts of inorganic salts and media proteins such as fetal
technology the mammalian cells (e.g., CHO cells) are seeded calf serum, yeast extract and other growth promoting
into roller bottles that are filled to about 25 per cent capacity substances, which may limit the choice ofthe capture step. In
with the medium and slowly rotated allowing cells to adhere. the case of the target protein expressed as an intracellular
The rotation provides the continuous contact of the cells protein, the process of extraction from the cell mass is
with the medium and oxygen is supplied by the "head space" employed, which leads to a highly complex mixture of
in the bottle. Following attainment of confluency, the product recombinant and host biomolecules including host proteins,
is harvested from the decanted supernatant. The suspension nucleic acids, lipids, carbohydrates and other cell components.
culture method is more widely used for the mass production In addition to commonly employed technologies such as
ofrecombinant proteins, where the capacity ofthe mammalian continuous centrifugation, tangential flow filtration and fast
cells for single cell suspension growth is exploited for flow chromatography two novel techniques viz.; affinity tags
scalability to very large volumes. The transition of adherent and expanded bed adsorption techniques are gaining
cells to suspension cultivation requires a selection of media importance in the purification protocols for recombinant
fonnulations. The seeding inoculum begins with a small volume proteins. Affinity tags are short peptide sequences genetically
of cell suspension, which is gradually expanded so that fused to a target protein. A peptide ofsix consecutive histidine
sufficient cell numbers are generated for the final production residues (His-6-tag) is the most widely used tag peptide, which
~~ase. J~~~~p~nsi()~'<::l:llt.1:!!:~p~~~~~~!~"~~~~ll~g!gg is fused either at the N- or C-tenninal ofthe protein ofinterest.
facllitateSthe punflcation ofthe proteIn by irnrrlObilized
composition of the cell medium during the production phase This
metii!ioii{generiillYNiClcel)iiffiiiiiYcolmnii(lMAC).Forthis
iiie~product
enzymes released by the cells as well as the molecular technique, a metal chelating ligand, e.g., iminodiacetic acid or
nitrilotriacetic acid is immobilized on a chromatographic matrix
composition of the product due to limitations of nutrients.
can "iiffectiiie"quaHijo:i:'
.dueiodegradatfve
2596
tag
IP 2010
and charged with transition metal ions leaving one or more

coordinating sites free for interaction with the analyte. Proteins
with the His-6-tag will bind to the ligand tightly to allow
quantitative capture of the target molecule from a complex
feed stream. The bound target protein is recovered by stepgradient elution using imidazole or by acidification. A potential
problem is the presence of leached metal ions in the eluate,
which must be removed in a subsequent step of purification.
His tag facilitated purification process has yielded therapeutic
recombinant proteins without affecting the folding, bioactivity
and biodistribution profiles.
Expanded bed adsorption (EBA) is another novel technique,
which is employed for preparative protein purification when
large volumes ofparticulate raw materials are involved in the
initial purification step. EBA combines the advantages ofbatchor fluidized bed methods with superior adsorption
characteristics of packed bed columns by a novel desi,gn of
matrix particles. With the exception ofgel filtration, all modes
ofbiochromatography are compatible for EBA. However, ionexchange media are most extensively used.
Post-Translational Modifications ofProteins
xiI) Formation ofgamma carboxyglutamic acid

xiii) Methylation
xiv) Succinimide forms

xv) Aspartate isomerization
xvi) Disulfide linkage
xvii) Oxidation
Amongst these only a few are relevant from the perspective
oftherapeutic recombinant proteins. Glycosylation represents
the most important PTM since a great diversity exists among
different expression systems. For example, prokaryotic
expression systems result in aglycosylated proteins often
resulting in insoluble inclusion bodies. Yeasts on the other
hand, add carbohydrate side chains of high mannose content,
CHO and murine cells can also add sugars not normally found
in human proteins some ofthem are known to be immunogenic.
Posttranslational modifications of recombinant therapeutic
proteins are significant in terms of potency, stability and
toxicity including immunogenecity of the biopharmaceutical
product/so This emphasizes the need for inclusion in the
pharmacopoeial monographs, meticulous description of
specifications and rigorous analytical data to characterize each
biopharmaceutical product.
The primary structure ofproteins, which is the linear sequence

of amino acids, is encoded by the gene sequence of the cell.
The mere demonstration that variations in the post translational
However, proteins undergo a variety of modifications
modifications are found in recombinant glycoproteins
subsequent to their synthesis. Enzymes within the cell mediate
produced
in different expression systems, does not
these modifications and such modifications are attributed to
automatically
negate the biological efficacy or the safety of
certain properties, such as solubility, transportability,
the product. In many instances it has been clearly demonstrated
resistance to proteolysis, folding, receptor binding, signal
transduction etc., in the native milieu of the cell. However, ' through clinical trials that the products are safe and efficacious
(e.g. recombinant factor VIII produced by BHK cells and CHO
with the advent of production. of proteins for therapeutic
cells have different glycosylation profiles but hoth are safe
applications in heterologous expression systems, postand efficacious). Therefore, comparability ofthe same protein
translational modifications (PTMs) have assumed special
significance from the point of view of setting specifications produced by different processes has to be governed by a
pragmatic approach. However, the secretory pathway ofPichia
and quality assurance. Proteins undergo a broad range of
pastoris
has been genetically re-engineered to perform
PTMs; the most common ofthem are listed below.
sequential glycosylation reactions that mimic early processing
Post-Translational Modifications of Proteins
ofN-glycans in humans'
Acylation
Glycosylation ofProteins
ii). Phosphorylation
iii) Sulphonation
iv) Proteolysis (terminal or domain deletion)
v)
Glycosylation (N-linked, O-linked)
vi) Aggregation
vii) High order structural change (conformational change or
denaturation)
viii) Arnidation or deamidation

ix) Carbamylation
x)
Carboxylation
Xl)
Formylation
More than one third of the biopharmaceticals currently in

clinical use have an absolute requirement for glycosylation
and hence they are produced in eukaryote expression systems.
Oligosaccharides are attached co-translationally through
specific aspargine (N-linked) or serine or threonine residues (
O-linked). While the consensus sequence for N-glycosylation
has been shown to be Asn-x-ser/Thr as an essential but not
sufficient sequence, there is no such consensus sequence
Imown for O-glycosylation sites. In some cases ofrecombinant
proteins, glycosylation may not be essential for therapeutic
application, but as pharmaceutical products, glycosylated
preparations have been found to have lowered protein
2597
IP 2010
aggregation and better thermal stability which are desired

properties.
Immunogenicity is yet another parameter, which is a matter of
concern with regard to recombinant therapeutic proteins,
produced in different expression systems.
'Y-Carboxylation and j3-hydroxylation ofProteins

These are particularly found in proteins involved in the
process of Blood Coagulation. An enzyme, carboxylase,
converts target glutamic acid residues in the protein to 'Ycarboxyglutamic acid residues, while hydroxylase enzyme
catalyses the conversion of either the target aspartic acid or
the target aspargine residues to their corresponding~-hydroxy
derivatives (~-hydroxyaspartate and ~-hydroxyaspargine
respectively
O-sulfonation
Tyrosine O-sulfonation is a post-translational modification
limited to a few proteins. Tyrosine O-sulfonation is catalyzed
by two enzymes, viz., tyrosylprotein sulfotransferases (TPSTI and TPST-2) that transfer the sulfuryl (-S03') group from
phosphoadenosylphosphosulphate to certain tyrosyl residues
in the protein. Tyrosine sulfation occurs in all mammalian cell
lines but not in yeasts and prokaryotes. The functional
importance oftyrosine sulfation is presumed to be in proteinprotein interactions.
Amidation
Modification ofthe carboxy terminal ofproteins and peptides
with an amide group is widely found in vertebrate and
invertebrate proteins and peptides but not in yeasts and
prokaryotes. Bioactive peptides such as vasopressin,
oxytocin, gastrin, calcitonin, substance P and Neuropeptide
Yare amidated at their C-terminal. Although amidation is of
widespread occurrence in mammalian bioactive peptides and
polypeptides, the biological significance of amidation is less
understood. It is presumed that this PTM may contribute to
stability and in increasing the hydrophobicity of the peptide
to facilitate binding to its receptor. Most bioactive peptides
are chemically synthesized and subsequently amidated
enzymatically using a-amidating enzyme.
Covalent modification of Therapeutic Proteins
to proteolytic degradation, less immunogenic due to masking

of epitopes and have reduced renal clearance rates. Early
procedures of PEgylation resulted in mixtures of PEG
substituted positional isomers. But later developments in the
chemistry of ligation which includes different coupling
techniques using different activated polyethylene glycols
and introduction of linkers between PEG and the protein
targets, have resulted in site-specific PEgylation. Several
PEGylated biopharmaceuticals have received regulatory
approvals, which include PEG-IFN, PEG-human growth
hormone and PEG-granulocyte macrophage colonystimulating factor. In all cases PEGylation increased product
plasma half-life which resulted in reduced frequency of
administration.
Proteins by nature are heterogeneous but in the context of
recombinant DNA derived protein products produced in
various systems add to more complexities. In the chain of
processes starting from cloning of the gene to the production
of therapeutic protein, there could be many products formed
such as truncated polypeptides, differently folded proteins,
scrambled disulfide linkages, varying extents and different
pattern of glycosylation. In addition, there could be process
related contaminants resulting in impurities in the fmal product.
Proteins have pleomorphic physiological effects. Thus,
although the recombinant therapeutic protein is optimized for
one biological activity, it may have a deleterious effect on
another activity, which may result in adverse effects. Therefore,
pharmacopoeial monographs include precise specifications
for each formulation of therapeutic proteins, and analytical
characterization.
Specifications
They comprise ICH recommendations of characterization
parameters that include appearance, identity, quantity, and
purity including impurity profile, stability, and biological
potency. These parameters that are numerical limits are
established by a list of tests, analytical procedures, and other
criteria, to which a drug substance or drug product should
conform. Conformity of each batch of production to
"specification test accept-criteria" is an important part of the
batch quality control release (certificate of analysis, COA).
The specifications are developed in the early part of process
and product development, scale up, non-GMP manufacture,
and cGMP manufacture for clinical trials wherein the active
drug is fully characterized for its physiochemical properties,
biological activity, immunochemical properties, purity, and
quantity. The acceptance criteria are related to the analysis
method chosen and established during the development
process
----- ------------ -----------------
Many therapeutic recombinant proteins are characterized by

low protease stability, quick renal excretion and high
immunogenicity. Covalent modification ofproteins is a process,
which to some extent, ifnot all overcomes problems. Covalent
conjugation_oLpolyethyleneglycoL(EEG)totherapeutic
pJ:Qteinl'; is one of the: most important tegbniql.!es (Mole:cule: _
Altering Structural Chemistry (MASC)), employed for the IdentitY development of second-generation biopharmaceuticals. An important part ofthe overall characterization program for a
PEgylated proteins have been shown to be less susceptible recombinant protein is the identity tests confirming molecular
2598
IP 2010
weight, isoelectric point, primary structure, higher order

structures (secondary, tertiary and quaternary) and possible
posttranslational modifications. Protein structure and function
are closely related. Even minor deviations in the threedimensional conformation or in the posttranslational
modifications (e.g., glycosylation, phosphorylation, or
acylation pattern) may result in altered biological activity or in
an adverse imunogenic/allergic response. During the drug
development phase extensive characterizations are carried out
in order to confirm the chemical and physical properties ofthe
recombinant product with its natural counterpart. Only a
minority of the identity tests will be used for batch releases
that are specified in the relevant monographs.
The standard methods for determination of molecular weight
(MW) are Electrospray Mass Spectra (EMS), Matrix Assisted
Laser Desorption/lonization (MALDI)-Time ofFlight (TOF),
High Performance Size Exclusion Chromatography (HP-SEC),
and Analytical Ultracentrifugation (AVC). Various
sedimentation velocity measurements (sedimentation velocity,
difference sedimentation, and sedimentation equilibrium) are
used to gain information on shape and conformation. This is
a useful check of homogeneity.
The isoelectric point (PI) is the pH level where the protein has
no net charge. The standard methods for determination of pI
are Isoelectric focusing in polyacrylamide gels (IEF) and
Capillary Electrophoresis (CE)-IEF. The separation principle
is based on charge heterogeneity caused by differences in
amino acid residue charges. The pI may also be determined by
means of2D-electrophoresis, where molecules are separated
according to pI and MW.
The primary structure provides information on the amino acid
sequence ofthe protein. For most proteins a primary structure
analysis comprises N-terminal sequencing by Edman
degradation, C-terminal analysis, and peptide mapping
followed by High Performance Reversed Phase
Chromatography (HP-RPC) purification and subsequent
determination of the MW of the fragments by mass
spectrometry. The amino acid sequence is often supported by
total amino acid analysis, comparison of the cloned gene
sequence, and the molecular weight determination.
Comparative fingerprints between the natural and the
recombinant protein are also used to confrrm primary structure
identity.
The secondary structure provides information on disulfide
bond arrangement, a-helix, and ~-sheet content. The standard
methods for determination of disulfide arrangements are
peptide mapping by HP-RPC or Sodium dodecyl sulphate
(SDS)-Polyacrylamide Gel Electrophoresis (pAGE). Structural
information is obtained by means of fluorescence, far UV
circular dichroism, Raman scattering, and infrared absorption
using Fourier Transformed Infra Red Spectroscopy (FTIR).
The spectroscopic methods are very powerful when used for

comparison analysis with the natural counterpart. The
confrrmation ofcorrect disulfide linkage is usually carried out
on appropriate enzymatic or chemical digests of the target
protein followed by HP-RPC purification ofthe fragments and
subsequent mass analysis using ESI-MS or MALDI-TOF.
The tertiary structure is the three~dimensionalstructure ofthe
molecule. The standard methods for tertiary structure
determination are NMR (in solution), X-ray diffraction using
crystals at high atomic resolution 3A), and near UV circular
dicroism. The latter method is very powerful for comparison
analysis with the natural counterpart.
The quaternary structure ofa protein represents the interaction
between individual polypeptide chains (Subunits) resulting
in larger assemblies. The standard methods for determination
of quaternary structure are HP-SEC, Raman scattering, and
light scattering, useful for determination of large
macromolecular assemblies.
Posttranslational modifications include chemical modifications
of side groups (e.g., oxidation of
Met, de-amidation of Asn of Gin); phosphorylation,
glycosylation, fatty acid acylation, farnesylation, sialic acid
capping, N-methylation, and acetylation. Typical standard
methods for the detection of posttranslational modifications
are HP-RPC, HP-IEC, or mass spectrometry. In mass
spectrometry, the MW of the molecule can be determined
with high accuracy and precision (better than 0.01 %). This
performance is normally sufficient to identify modifications
such as missing residues or additional groups. Peptide
mapping, using specific enzymes and subsequent HP-RPC
purification of the fragments prior to MW detection is also
used, for example, for determination ofglycosylation patterns.
Heterologous glycosylated products are often identified by
their isoelectric focusing slab gel pattern or more rarely by
2D-electrophoresis. A thorough carbohydrate structural
analysis may include glycosylation site(s), carbohydrate chain
structure, the oligosaccharide pattern, and the content of
neutral sugars, amino sugars, and sialic acids.
The extinction coefficient can be determined from a known
protein quantity (protein concentration) and the absorbance
at 277 to 280 urn. In chromatographic evaluation, thetarget
protein retention time using HP-IEC or HP-RPC can be used
as an identity marker. For biomolecular interaction analysis,
the method uses surface plasmon resonance to detect
biomolecular interactions.
Biological Activity
The biological activity (Potency) describes the ability of the
drug substance to achieve a defined biological effect. Examples
ofprocedures used to measure the biological activity include
2599
IP 2010
animal based biological assays; cell culture-based biological

assays, biochemical assays, and ligand binding assays. A
biological assay may be replaced by physicochemical tests
provided sufficient information and correlation between the
bioassay and the said tests can be demonstrated and there
exists a well-established manufacturing history (ICH
Harinonized Tripartite Guideline. Specifications: Test
Procedures and Acceptance Criteria for Biotechnological
Products (Q6B).
Structural variants of therapeutic proteins in general and
recombinant proteins in particular which include isoforms (e.g.,
variations in post translational modifications, varying degrees
of processing of C- and N- terminal substitutions etc) often
show variations in biological activity. Thus, the batch release
of the product should be shown to possess the expected
degree ofbiological activity using relevant bioassays. In order
to use the same dosing protocol as a reference medicinal
product, the content and specific activity of the clinically
comparable product should be compared to that of one or
more reference medicinal products, or to an international
reference standard to which the reference product is calibrated.
(WHO Guideline for abbreviated licensing pathways for certain
therapeutic proteins)
Purity
Protein purity has been historically linked to the specific
biological activity in terms of units of biological activity per
mass unit of the product. The purest product is that of the
highest specific biological activity. In contrast to drugs based
on small molecules, which could be controlled on the drug
product level, protein-based pharmaceuticals are closely linked
to the process itself due to the complexity of the active
pharmaceutical ingredient and the lack of proper
characterization ofthe final product. With the introduction of
recombinant technology and modem analytical methods, a
much better drug substance/product characterization has
become possible resulting in the well-characterized protein
concept and the widespread use of comparability studies.
The importance of a: stronger focus on presence of
adventitious agents and specific impurities are also
recognized, as the presence of even minor amounts oftoxic,
immunogenic, or adventitious compounds proved to have
severe side effects. The acceptable level ofimpurities depends
on the nature of the drug product and the dose.
forms are detected by analytical HP-lEC, HP-RPC, native PAGE,

lEF, MS, or CEo The content accepted depends on the nature
of the drug product and the dose.
Oxidized Forms. Oxidized forms are target protein derivatives
in which one or several Met, Cys, His, Trp, or Tyr residues
have been oxidized. The oxidation ofcystinyl residues results
in formation of a disulfide bond (cystinyl residue). Oxidized
forms are detected by analytical HP-lEC, HP-RPC, native PAGE,
lEF, MS, or CEo The content accepted depends on the nature
of the drug produet and the dose.
Scrambled Forms. Scrambled forms are target protein
molecules with a disulfide bond pattern different from that of
the native molecule. Scrambled forms are typically formed
during in vitro folding ofproteins, but disulfide bond shuffling
at neutral pH or above also occurs. This requires studies on
control of protein stability during downstream processing.
The formation of scrambled forms is closely linked to the
folding procedure and is protein specific, which are detected
by analytical HP-lEC, HP-RPC, CE, or peptide mapping. The
content accepted depends on the nature of the drug product
and the dose.
Cleaved Forms. Cleaved forms are those where a peptide bond
is cleaved, resulting in loss ofa N- or C-terminal site or where
an internal peptide bond is cleaved while at the same time the
resulting fragments are kept together by means of disulfide
bonds. These forms are detected by N- and C- terminal amino
acid analysis.
Aggregates. Aggregates are target protein derivatives in which

two or more molecules are linked together either by covalent
inter-disulfide bonds or by hydrophobic interaction. Target
protein aggregates are formed as a result of hydrophobic
intermolecular reactions or because ofintermolecular disulfide
bond formation under oxidizing conditions. Aggregates are
very often antigenic, resulting in formation of antibodies
against the target protein. Proteins exposed to even mildly
denaturing conditions may partially unfold, resulting in the
exposure of hydrophobic residues to the aqueous-solvent,
favoring aggregation. It is generally believed that the
aggregation process is controlled by the initial dimerization
step in a second order reaction. Consequently, high protein
concentrations will increase the aggregation rate.
Intermolecular disulfide bond formation between cystinyl
residues takes place at alkaline pH under oxidizing conditions.
Proteins with reactive free thiol groups ~hould be purified
Impurity ProfIle
under reducing conditions (typically 1 to 10 mM reducing
agent) in the presence of EDTA. Even proteins with disulfide
Product related impurities
.bonds-may-participate--in-intermolecular-disulfide-bond
DeS:3midoForms. Des-amido forms are target protein reactions due todisulfide bond shuffling at neutraland alkaline
derivatives in which one or several of the glutaminyl or pH. The aggregation reaction based on intermolecular disulfide
asparagyl amino acid residues are converted to. the bond formation is prevented at pH < 6 and under reducing
corresponding acids (glutamyl and asparagyl). Des-amido conditions. The hydrophobic aggregation compounds,
2600
lP 2010
enzymes, detergents, and stabilizers must be accounted for

and their removal validated. Large amounts of hydrophobic
reagents may affect hydrophobic interaction chromatography.
Analytical HP-IEC, HP-RPC, native PAGE, IEF, MS, or CE
detects oxidized forms. The content accepted depends on the
nature of the drug product and the dose. Non-reducing IDSDS, HP-SEC, MS, or CE can detect disulfide-based
aggregates. Hydrophobic aggregates may be detected by HPSEC. The content accepted depends on the nature of the drug
product and the dose as specified in the product monographs.
Process Related Impurities
Pyrogens and Endotoxins
Pyrogens are a group of chemically diverse substances,
including endotoxins ofbacteria and debris of dead bacterial
cells that cause fever and shock in severe cases. The most
important pyrogenic substances in pharmaceutical industry
are bacterial endotoxins. Endotoxins come from Gram-negative
bacteria (e.g., E. coli), if it is used as the expression system.
Presence of endotoxins indicates bacterial contamination in
raw materials, columns, water, and buffers. There are two
methods of detection: the pyrogen test, which is based upon
the measurement of body temperature of rabbits before and
after injection of the specimen, and the Limulus Amebocyte
lysate (LAL) test, which is based upon the clotting reaction of
an enzyme complex of cells of the horseshoe crab together
with bacterial endotoxins (in vitro test).
Nucleic acids
Nucleic acid contamination comes from host cell DNA/RNA
or retroviral RNA. Nucleic acids are detected by monitoring
absorption of light at 260 om. The residual content in drug
substance (DS) or drug product (DP) is usually measured by
PCR or amplification teclmiques. The maximum allowable
content of nucleic acid per dose remains under continuous
evaluation by regulatory agencies. "Lot-to-lot" testing for
DNA content in biological products produced in cell lines
should be performed and lot release limits established which
reflects a level of purity that can be achieved reasonably and
consistently.
Host cell proteins
Host cell proteins (HCP) come from the host organism and
constitute a major purification problem due to variability
structure and surface properties. The amount released into
the culture medium depends on the expression system used,
the culture conditions and the process related cell lysis.
Extraneous proteins may be introduced in the downstream
process also. Several analytical methods have been used to
monitor HCP including SDS-PAGE, 2D-electrophoresis (IEF
in combination with SDS-PAGE), Western blot (WB), and
immunoassays. However, Generic HCP assays are preferred
for lot release. If the assays are based on competently

produced antibodies raised against cell lysates, the quality
may be adequate
Viruses
Virus contamination comes from the host cell, the culture
medium, and infections during manufacture. The host cell may
contain a genomic virus or virus vectors used to transform
the cell line. The type of viral genome or vector depends on
the cell line history. Chronic or latent viruses may be present
in continuous cell lines, and the retroviruses associated with
continuous cell lines are non-infectious, but oncogenic.
Epstein-Barr virus or Sendai virus is often used for cell
transformations. Contaminants such as BVDV, IBR, reovirus,
PI-3, bovine leukemia virus, and bovine polyoma virus should
be expected from serum-supplemented media. The cell line
history reveals all information on the origin and identity ofthe
cell line and the host genome vectors used to establish the
cell line
Prions
Prions come from transmissible spongiform encephalopathies
(TSE), The major source of contamination of a recombinant
product is the use ofanimal-derived raw materials, which could
harbor bovine prions (BSE agent). Currently, there are no assays
that are sensitive or specific' enough to test raw materials or
sources, and the only reliable prevention is to include barriers,
such as avoidance of animal or human raw materials (e.g.,
trypsin, serum, transferin, bovine/human serum albumin,
protein supplements, peptones).
Microbial agents including Mycoplasma
Microbial agents and fungi come from infection of the
bioreactor during cell culture. Other sources are contaminated
water, buffers, raw materials, chromatographic columns, and
equipment. Fermentation and cell culture bioreactors are prone
to microbial infections. Viable cells can be identified by spread
out of the cell suspension or sample solution on agar plates.
Mycoplasma
Mycoplasmas have for long been recognized as a contaminant
of continuous cell cultures caused by an infection of the cell
line or bioreactor. Working in closed systems under GMP will
reduce the risk ofinfection. The end ofproduction test includes
screening for mycoplasmas. Mycoplasmas are difficult to
detect, the only reliable way of demonstrating infection is by
agar plating, fluorescent dying of DNA, or by PCR
Quantity
Protein content
Quantitative estimation of protein is measured by several
methods (Kjeldahl analysis, Lowry assay, Biuret assay,
2601
ERYTHROPOIETIN CONCENTRATED SOLUTION
IP 2010
Bicinchoninic acid (BCA) assay, Bradford assay, UV

absorbance andamino acidanalysis). It is desirable to confIrm
the quantity by more than one method.
Host cell-derived proteins. This must be routinely monitored

using a scientifically accepted method that demonstrates:
1.
The assay is sensitive (a useful target for the limit of

detection is 1 to 100 ppm)
2.
The assay is specifIc for the HCP (defined by proprietary

detection reagents such as antibodies elicited against
process-specifIc contaminating HCP) consistently found
in the product from a specifIc manufacturing / purifIcation
process.
Stability
Formulation development of biopharmaceutical therapeutic
protein will provide a fInal dosage form that offers ex vivo
stability during processing, handling, and long-term storage
under specifIed conditions (e.g., temperature, humidity etc). It
is also expected to provide adequate in vivo bioavailability
that meets the desired pharmacokinetic/pharmacodynamic (pK/
PD) properties. Therapeutic Proteins do not survive terminal
sterilization procedures commonly employed for small
therapeutic molecule. Therefore, formulation components and
excipients should also undergo microbiological tests including
analysis for endotoxins and pyrogens in addition to biological,
chemical and physical functions.
Limits are approved by the competent authority.

Host cell and vector-derived DNA
The limit is as prescribed by the competent authority.
Description. A clear and colourless solution.
Identification
A. It gives the appropriate response when examined using the
conditions described under Assay.
B. Determine by isoelectric focusing (2.4.33).
EHCSLNENIT
VPDTKVNFYA
WKRMEVGQQA
Test solution. Dilute, the preparation in water, if necessary

and desalt the preparation, using a membrane fIltration system
suitable for desalting proteins. Make up the volume to the
original volume with water.
VEVWQGLALL
SEAVLRGQAL
LVNSSQPWEP
Reference solution. Dissolve erythropoietin RS in water to

produce a solution containing 1mg per ml and desalt as above.
LQLHVDKAVS
GLRSLTTLLR
ALGAQKEAIS
PPDAASAAPL
RTITADTFRK
LFRVYSNFLR
GKLKLYTGEA
CRTGD
APPRLlCDSR
I
VLERYLLEAK
EAENITTGCA
I
Structure of Erythropoietin
The isoelectric focusing procedure may be carried out using a

0.5 mm thick polyacrylamide slab gel containing ampholytes
covering the pH of range 3 to 10, prepared as follows.
Mol. Wt. 30,600 (approx)
Erythropoietin Concentrated Solution contains a family of

closely related glycoproteins which are not different from the
naturally occurring human erythropoietin (urinary
erythropoietin) in terms ofthe amino acid sequence (165 amino
acids) and average glycosylation pattern, at a concentration
of 0.5 mg per ml to 10 mg per ml. It has a potency of not less
than 100000 ill per mg of active substance.
Production
Erythropoietin is produced in rodent cells in vitro by a method
based on recombinant DNA technology. All batches are tested
as described below, prior to release (unless exemption has
granied'bythecompeteriiautIi'orlty)'.
Host cell-derived proteins (Hep)

The limit is as prescribed by WHO.
_.,--_.-
Mix 9 g of urea, 6.0 ml of 30 per cent acrylamide/bisaclylamide solution, 1.05 ml of pH 3 to 5 ampholyte, 0.45 ml of
pH 3 to 10 ampholyte and 13.5 ml of water. Degas the mixture
. Add 15 ).11 of tetramethylethylene diamine and 0.3 ml ofa 100
g per litre freshly prepared solution of ammonium persulphate.
Pour into a suitable gel cassette, with approximate dimensions
ofl5 cmx 15 em x 0.05 em. Insert a suitable sample well comb
and allow to polymerize.
Use the anode solution the anolyte for isoelectric focusing
at pH 3 to 5 and the cathode solution the catholyte for
isoelectric focusing pH 3 to 5. Allow prefocusing to take
place for 1 hour at a constant power of 10 W, with maximum
voltage and current settings of 2000 V and 100 rnA,
respectively.
Dilute the test solution to 0.5 mg per ml with water. Apply to
the-geHOfJ;lofeach solution;-GarryoutfoGusing-for-a-further
30 minutes at-thesamepowersupply settings. Take the gel
out ofthe focusing chamber, and transfer the proteins onto a
membrane suitable for immobilization of proteins (such as
Polyvinylidene Fluoride), using commercially available
2602
IP 2010
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 per litre ofdried mille), for 1 hour, followed by
incubation in the same blocking solution with a suitable dilution
of a polyclonal anti-erythropoietin antibody. Detect the
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
optimized using the principles set out in Immunochemical
methods.
The test is not valid unless the distribution of bands in the
electrophoretogram obtained with the reference solution
contains at least 6 well separated bands. If necessary, the
voltage settings and duration may be altered to optimize the
separation of the isofonns.
In the electrophoretogram obtained with the test solution, the
pH range of the bands observed corresponds to that of the
electrophoretogram obtained with the reference solution. The
predominant bands correspond to isoforms 4, 5, 6 and 7.
Additional, fainter bands corresponding to isoforms 1, 2, 3
and 8 may also be present. Other bands are present in no more
than trace amounts.
C. Determine by capillary electrophoresis (capillary zone
electrophoresis) (2.4.32).
.
All the solutions should be filtered through a 0.45 /lm membrane

filter before use.
Test solution. Dilute the substance under examination with
water or concentrate it to obtain a concentration on mg per
ml. Desalt 0.25 ml ofthe solution by passage through a microconcentrator cartridge provided with a membrane with a
molecular mass cut-off not more than 10000. Add 0.2 ml of
water to the sample and desalt again. Repeat the desalting
procedure once more. Dilute the sample with watel; determine
its protein concentration as described under Tests and adjust
to a concentration ofapproximately 1 mg per ml with water.
Reference solution. Dissolve elythropoietin RS in water to
produce a solution containing 1 mg per ml. Desalt the sample
as described for the test solution.
Capillary system
material. uncoated fused silica,
size. effective length = about 100 cm, internal diameter
=50/lm,
temperature. 35,
spectrophotometer set at 214 llID,
- injection. under pressure or vacuum.
CZE buffer concentrate (0.1 M sodium chloride, 0.1 M tricine,
0.1 M sodium acetate). Dissolve 0.584 g of sodium chloride,
1".792 g oftricine and 0.820 g of anhydrous sodium acetate in
water and dilute to 100.0 ml with the same solvent.
1 M putrescine solution. Dissolve 0.882 g ofputrescine in 10
ml of water. Distribute in 0.5 ml aliquots.
on
'"
<!
"'-
'"
'"~-
'"E
oS
"
_ "'Vl
5.....
e.EC1
8
--'-----_..J>.._.,J-.
..,
g
~HI._ _
00
~ .... _ . .:r-'I IA!~;:
I.--,-:-r--...,..--.,.---,.....----,.--,....-.,..._,--.I=:I,_-,._...,....._.,.-_...-_,...---
g'"
Reference electropherogram ofErythropoietin
2603
IP 2010
CZE buffer. (0.01 M tricine, 0.01 M sodium chloride, 0.01 M

sodium acetate, 7 M urea, 2.5 mMputrescine). Dissolve 21.0
g of urea in 25 ml of water by warming in a water-bath at 30.
Add 5.0 ml of CZE buffer concentrate and 125 ml of 1 M
putrescine solution. Dilute to 50.0 ml with water. Using dilute
acetic acid, adjust the pH to 5.55 at room temperature and
filter through a 0.45 /-lm membrane filter.
Set the auto sampler to store the samples at 4 during
analysis.
D. Immunoblotting. Determine by electrophoresis (sodium

dodecyl sulphate polyacrylamide gel electrophoresis) (SDSPAGE) (2.4.12).
Use a 1-mm thick spacer with six-well comb, a well capacity of
451 and suitable plate size e.g. 100 x 120 mm
Assemble the gel casting as recommended by the
manufacturer.
Use the composition of gel given below:
Preconditioning ofthe capillQ1Y. Rinse the capillary for 60

minutes with 0.1 M sodium hydroxide filtered through a 0.45
/-lm membrane filter and for 60 minutes with CZE buffer. Apply
voltage for 12 hours (20 kV).
Separating gel 12 per cent

Add the reagents into a clean glass/plastic container in
the same sequence as given in the table below:
Between-run rinsing. Rinse the capillary for 10 minutes with

water, for 5 minutes with 0.1 M sodium hydroxide filtered
through a 0.45 /-lm membrane filter and for 10 minutes with
CZEbuffer.
Migration. Apply a field strength of 143 V/cm (15.4 kV for
capillaries of 107 cm total length) for 80 min, using CZE bliffer
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 reference electropherogram
ofelythropoietin RS is seen, and the largest peak is atleast 50
times greater than the baseline noise. If necessary, adjust the
sample load to give peaks of sufficient height. Identify the
peaks corresponding to isoforms 1 to 8. The peak
corresponding to isoform 8 is detected; the resolution between
the peaks corresponding 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 ofpeaks is qualitatively
and quantitatively similar to the distribution of peaks in the
reference electropherogram ofelythropoietin RS. The relative
standard deviation of the migration time of the peak
corresponding to isoform 2 is less than 2 per cent.
Identify the peaks corresponding to isoforms 1 to 8 in the
electropherogram obtained with the test solution by
comparison with the electropherogranl 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 Number
Content (per cent)
1
2
3
0-15
0-15
1-20
10'-:'35
7
8
Solutions
For7rnl
(for 1gel)
For 14ml
(for 2 gels)
Water
2.16rnl
4.32rnl
AClylamide (30 per cent)
2.8ml
5.6rnl
1.5 MTris-CI pH8.8
1.75rnl
3.4rnl
20 per cent SDS
35rnl
70rnl
Ammonium persulphate
(iO per cent w/v)
70rnl
140ml
TEMED
3rnl
6ml
Mix the above contents by swirling gently (do not mix

vigorously) in the container and pour into the casting unit
using a 1-ml pipette to 1 to 1.5 cm from the top edge of the
plate. After addition of ammonium persulphate and TEMED,
mix the solution and pour quickly (in less than 1minute) or it
will begin to polymerize. Pour 200 to 1000 /-ll of water-saturated
butanol on the top of separation gel. Keep aside for at least
45 minutes for polymerization at room temperature.
Casting the Stadang gel: 4 per cent

Decant the water-saturated butanol and rinse the separating
gel with water. (If the gel has not polymerized and flows out,
discard and prepare fresh). Place the comb in position above
the separating gel. Prepare the stacking mix by adding the
reagents in the same sequence as given in the table below:
1<)"-35
5-25
0-15
2604
Solutions
For5rnl
(1 gel)
For lOrnl
(2 gels)
Water
1.8ml
3.6rnl
Acrylamide (30 per cent)
0.6rnl
1.2rnl
0.5 MTris CI pH 6.8
2.5rnl
5.0rnl
Ammonium pel'sulphate
(10 per cent w/v) (APS)
50/-ll
100/-ll
TEMED
5/-ll
10/-ll
IP 2010
Mix the above ingredients by swirling gently and pour over

the separating gel. Avoid bubbles. Keep aside for at least 45
minutes for polymerization at room temperature.
3.
Equilibrate the membrane in chilled transfer buffer for

15 - 20 minutes.
4.
Equilibrate the gel in chilled transfer buffer for 15 - 20

minutes.
Soak the nylon pads in transfer buffer and place on the
pad on the cathode.
Preparation ofsamples and loading

5.
Samples include:
1.
Test sample
1.0 Ilg
2.
Reference standard RS
1.0 Ilg
3.
Prestained Marker
20 III
6.
Test solution. Take X III of the test sample and add an equal
volume of 2X non-reducing sample bU.ffer where,
7.
Take 5 sheets of a suitable filter paper, cut to the size of

the gel and soak in chilled transfer buffer. Place them on
the nylon pad placed on cathode plate.
Carefully place the equilibrated gel on the filter papers.
8.
Place the equilibrated membrane onto the gel.
X (Ill) = lllg per (concentration ofthe test sample in mg per ml)
9.
Roll a clean glass rod dipped in transfer buffer on top of

the membrane to get rid of any air bubbles trapped
between the gel and the membrane.
Reference solution. Dilute a part of Img per ml reference

standard RS stock 10 times to get a final concentration of
0.1 mg per ml with water as follows:
To 10 III of 1 mg per ml reference standardRS solution add 90
mlwater.
10. Place 5 sheets of a suitable filter paper soaked in chilled

transfer buffer over the membrane. Once again roll the
glass rod to remove any air bubbles.
To 10 III of 0.1 mg per ml reference standardRS solution add

10 ml of 2X non-reducing sample buffel:
11. Close the cassette.
Prestained marker. Take 20 ml of prestained marker,

reconstituted as recommended by the manufacturer.
13. Connect the electrodes with the Power pack and set
following parameters for transfer:
12. Place the cassette in the running unit.
Loading ofsamples.
Current: 200 rnA
Boil the samples for two minutes, centrifuge, bring to room

temperature and load the entire volume on the gel.
Sample
No.
Samples
Test Sample
Reference
Marker
Amount of protein Total Vol. to be

loaded/well (Ilg)
loaded.
1
.1
20
20
Running the gel.

1.
Fix the gel apparatus in the running unit according to the

manufacturer's instructions.
2.
Chill the IX running buffer to 4 for at least 1 hour.
3.
Pour IX running buffer in the upper as well as lower

chambers of the running unit.
4.
5.
Set parameters on the Power pack as follows:

-
Constant Voltage: 130 V
Max Current: 200 rnA.
Run until dye front just goes out at the gel bottom (usually
about 1.5 hrs)
Transfer, blotting and development of membrane:

1.
Disassemble the running unit according to the

2.
Cut Nitrocellulose Membrane (NCM) having the same

dimensions as of the gel.
Voltage: 100 V
Time: 1 hour.
14. After the transfer is over, disassemble the cassette.

15. Transfer the membrane to the blocldng solution. Incubate
the membrane in the blocldng buffer for 1 hour with gentle
shaking at room temperature.
16. Discard the blocldng buffer. Add 25 ml of rabbit anti
r-Hu EPO antibody (Primary antibody) solution.
17. Incubate for Ihour with gentle agitation at room
temperature.
18. Wash the membrane with 1 X transfer btifJer saline with
change of buffer in-between at room temperature with
gentle agitation (3 times X 5 minutes).
19. Discard the transfer btifJer saline. Add 25 ml ofgoat antirabbit ALP conjugated antibody (secondary antibody)
solution. Incubate for 1 hour with gentle shaking at room
temperature.
20. Wash the membrane with 1 X transfer buffer saline (3
times X 5 minutes)
21. Develop the color by adding 5 ml ofthe substrate solution
(Usuallyittakes l5-20minutes).
22. Stop the reaction by pouring off the substrate solution
and washing it with water before the color of the
background gets dark.
23. Scan the blot while it is wet.
24. Air dry the blot and preserve it.
2605
IP 2010
The molecular mass markers are resolved on the membrane

into discrete bands with a linear relationship between distance
migrated and logarithm lO ofthe molecular mass.
Equilibrate at initial conditions for at least 15 minutes. Carry

out a blank run using the above-mentioned gradient.
To evaluate the linear relationship between the distance

migrated and 10garithmIO ofthe molecular mass, calculate
The chromatogram obtained with each solution is qualitatively

similar to the reference chromatogram oferythropoietin RS
digest.
a)
b)
c)
The profile of the chromatogram obtained with the test

solution corresponds to that of the chromatogram obtained
log molecular weights corresponding to marker bands

migration distance of protein band
plot (a) vs (b) and perform linear regression analysis
F. Determine by N-terminal sequence analysis
The graph should be linear

The single broad band of the test solution and of reference
standard RS match in position and intensity.
Peptide mapping (2.3.47).
Test solution. Dilute the substance under examination in trisacetate buffer solution pH 8.5 to a concentration of 1.0 mg
per ml. Equilibrate the solution in tris-acetate buffer solution
pH 8.5 using a suitable procedure (such as dialysis against
tris-acetate buffer solution pH 8.5, or membrane filtration using
the procedure described under Identification C, but
reconstituting the desalted sample with tris-acetate buffer
solution pH 8.5). Transfer the dialyzed solution to a
polypropylene centrifuge tube. Freshly prepare a solution of
trypsin for peptide mapping at a concentration of 1 mg per ml
in water, and add 5 ml to 0.25 ml ofthe dialysed solution. Cap
the tube and place in a water-bath at 37 for 18 hours. Remove
the sample from the water-bath and stop the reaction
immediately by freezing.
erythropoietin RS in 0.25 ml of water. Prepare as for the test
solution, ensuring that all procedures are carried out
simultaneously, and under identical conditions.
butylsilyl silica gel (5-10 /-lm),
- mobile phase: A. 0.06 per cent v/v solution of
trifluoroacetic acid,
B. to 100 m1 of water add 0.6 ml of
trijluoroacetic acid and dilute to 1000 ml with
acetonitrile,
below,
Time
Flow rate Mobile phase A Mobile phase B
(in min)
(ml/min)
(per cent v/v)
(per cent v/v)
0-10
m=125'
125"': 135
135 -145
145-150
0.75
0:75
1.25
1.25
1.25
100
100=+39
39 ~T7
17 --70
100
o
, 0=+'61
61~83'
83 --7100
The first 15 amino acids are: Alanine - Proline - Proline Arginine - Leucine - Isoleucine - (no recovered peak) - Aspartic
acid - Serine - Arginine - Valine - Leucine - Glutamic acid Arginine - Tyrosine.
Perform the Edman degradation using an automated solidphase sequencer, operated in accordance with the
Desalt the equivalent of50 /-lg oferythropoietin. For example,
dilute a volume ofthe substance under examination containing
50 /-lg of the active substance in 1 ml of a 0.1 per cent v/v
solution of trifluoroacetic acid. Pre-wash C 18 reverse-phase
sample preparation cartridge according to the instructions
supplied by the manufacturer and equilibrate the cartridge in
a 0.1 per cent v/v solution of trifluoroacetic acid. Apply the
sample to the cartridge, and wash successively with a 0.1 per
cent v/v solution of trifluoroacetic acid containing 0 per cent,
10 per cent and 50 per cent v/v of acetonitrile according to
the manufacturer's instructions. Lyophilise the 50 per
cent v/v acetonitrile eluate.
Redissolve the desalted sample in 50 /-ll of a 0.1 per cent v/v
solution of trifluoroacetic acid 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 second and third 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 ofPTH-aminoacids.
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 blanksequencing cycle obtained
. asrecommended'by the equipment-manufactureE --
Tests
Protein. 80 per cent to 120 per cent of the stated amount.
2606
IP 2010
Test solution. Dilute the substance under examination in a 0.4

per cent w/v solution of ammonium hydrogen carbonate or
the appropriate blank solution.
For20ml
(1 gel)
For40ml
(2 gels)
Water
Acrylamide (30 per cent)
6.l7ml
1234ml
8.0ml
16.0ml
1.5 MTris Cl pH 8.8

20 per cent SDS
5.0ml
1O.Oml
100 III
200 III
200 III
400J.lI
TEMED
8 III
16 J.lI
Solutions

solution shows an absorption maximum between 276 nin and
282 nm. After correction of any light scattering measured up
to 400 nm, calculate the content of erythropoietin taking the
specific absorbance to be 7.43.
Dimers and related substances of higher molecular mass
A. Detennine by size-exclusion chromatography (2.4.16).
Mix the above ingredients by swirling gently and pour.
Test solution. Dilute the substance under examination in the

mobile phase to obtain a concentration of 0.2 mg per ml.
Reference solution. To 0.02 ml ofthe test solution add 0.98 ml
of the mobile phase (2 per cent).
hydrophilic silica gel, ofa grade suitable for fractionation
of globular proteins in the molecular mass range of
20000 to 200 000,
- mobile phase: Dissolve 1.15 g of anhydrous disodium
hydrogen phosphate, 0.2 g of potassium dihydrogen
phosphate and 23.4 g of sodium chloride in 1 litre of
water (1.5 mM potassium dihydrogen phosphate,
8.1 mM disodium hydrogen phosphate, 0.4 M sodium
chloride,pH 7.4); adjusted to pH to 7.4, if necessary,
- spectrophotometer at 214 nm,
Mix the above contents by swirling gently (do not mix

vigorously) in the container and pour into the casting unit
using a I-ml pipette till 1 - 1.5 cm from the top edge ofthe plate.
After addition of ammonium persulphate and TEMED, the
solution should be mixed and poured quickly (less than
1 minute) or it will begin to polymerize.
Pour 200 - 1000 III of water-saturated butanol on the top of
the separation gel.
Keep aside for at least 45 minutes for polymerization at room
temperature (RT).
b) Casting the Stacking gel: 4 per cent
Decant the water-saturated butanol and rinse the separating
gel with water. (If the gel has not polymerized and flows out,
discard and prepare fresh)
Place the comb in position above the separating gel.
Prepare the stacking mix by adding the reagents in the same
sequence as given in the table below:
Inject the test solution and the reference solution. Continue

the chromatography for 1 hour. The area ofthe principal peak
1.5 to 2.5 per cent of the area of the principal peak in the
chromatogram obtained with the test solution. The total area
ofany peaks eluted before the principal peak is not more than
with the reference solution (2 per cent).
B. Detennine by electrophoresis (sodium dodecyl sulphate

polyacrylamide gel electrophoresis (SDS-PAGE) (2.4.12).
Solutions
For5ml
ForlOml
Water
1.8ml
3.6ml
AClylamide (30 per cent)
0.6ml
1.2ml
0.5 MTris Cl pH 6.8
2.5ml
5.0ml
20 per cent SDS
25J.lI
50J.lI
50 III
100 III
TEMED
5 III
lOJ.ll
Casting the gels
Mix the above contents by swirling gently and pour over the
separating gel. Avoid bubbles.
Use a I-mm thick spacer with eight-well comb.

The well capacity is 65 Ill.
Assemble the gel-casting unit according to the
manufacturer's insttuctions.
Use the assembly ofsuitable plate size e.g. 160 x 160 mm.
Keep aside for at least 45 minutes for polymerization at room

temperature.
Preparation ofsamples
Samples include:
a) Casting the separating gel: 12 per cent

Add the reagents into a clean glass/plastic container in the
same sequence as given in the table below:
2607
1.
2.
3.
Test sample
Reference standard RS
Marker for non-reducing gel
IP 2010
Test solution. To a volume containing 10 Ilg ofprotein add an

equal volume of 2X non-reducing sample buffel:
Reference solution. Take 10 III of the reference standard RS
from Imgperml stock in a micro-centrifuge tube and add 10 III
of 2X non-reducing sample btiffer.
Molecular weight marker. Take 20 III of low molecular weight
markers for SDS-PAGE, which is reconstituted according to
the manufacturer's instruction:
The dye front is run for at least 80 per cent of the total gel
length. Molecular weight markers are resolved on the gel into
discrete bands, with a linear relationship between distance
migrated and 10garithmlO ofthe molecular mass.
To evaluate the linear relationship between distance migrated
and 10garithmlO of the molecular mass, calculate
a)
log molecular weights corresponding to marker bands
b)
migration distance of protein bands
Sample loading
c)
plot (a) vs (b) and perform linear regression analysis.
Keep all the samples in a boiling water-bath for 2 minutes.

Centrifuge, bring to room temperature and load the entire
volume on to the gel.
The graph should be linear.
Sample
No.
Samples
Amount of protein Total Vol. to be

loaded/well (Ilg)
loaded. (Ill)
Test Sample
Reference
20
Marker
20
The single diffuse band ofthe test solution and ofthe reference
solution match in position and intensity.
Sialic acids. Not less than 10 mol ofSialic acids (calculated as
N-acetylneuraminic acid) per mole of erythropoietin,
determined in the following manner.
Test solution (a). Dilute the preparation under examination in

the mobile phase used in the test for dimers and related
substances ofhigher molecular mass to obtain a concentration
of0.3 mg per ml.
Running the gel

Fix the gel apparatus in the running unit as given in the
instruction manual ofthe manufacturer.
Chill the IX running buffer to 40 for at least 1 hour.
Pour IXrunning btiffer in the upper as well as lower chambers
of the running unit.
Test solution (b). To 0.5 ml oftest solution (a) add 0.5 mlofthe
mobile phase used in the test for dimers and related substances
ofhigher molecular mass.
Reference solution (a). Dissolve a suitable amount of Nacetylneuraminic acid in water to produce a solution
containing 0.1 mg per ml.
Reference solution (b). To 0.8 ml ofreference solution (a) add
0.2 ml ofwater.
Set parameters on the Power pack as follows:

Constant Voltage: 130 V
Reference solution (c). To 0.6 ml ofreference solution (a)add

0.4 rnl ofwater.
Max Current: 200 rnA.

Run until the dye front reaches the bottom ofthe gel (usually
about 5.0 hrs. The dye front should have run at least 80 per
cent of the gel).
Staining of Gel
Stop the run. Disassemble the casting unit and transfer the
gel carefully into a staining tray. Do not touch the gel with
naked hands; wear gloves while handling the gel.
Detect proteins in the gel by commassie stain.
Scanning
Scan and save the image of the stained gel.
Gel drying
The stained gel is placed in between two cellophane sheets
and clamped with the gel dryerframes(taking-careihatnoair
bubbles are-present in between the two cellophane sheets);
The set up is placed in the gel dryer apparatus and left at least
2 hrs for drying.
Reference solution (d). To 0.4 ml ofreference solution (a) add

0.6mlofwater.
Reference solution (e). To 0.:2 ml ofreference solution (a) add
0.8 ml ofwater.
Reference solution (f). Use water
Carry out the test in triplicate. Transfer 100 ml each ofthe test
and reference solutions to 10-ml glass test tubes. To each
tube add 1.0 ml of resorcinol reagent. Stopper the tubes and
incubate at 100 for 30 minutes. Coolon ice. To each tube, add
2.0 ml ofa mixture of 12 volumes ofbutanol and 48 volumes of
butyl acetate. 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 phases. Measure the absorbance of all
samplesaiS8(rnJJ.i(2:LL7T---_.
-_.~._
. ,-..-. __..
~.
_ -
_.
- .- _---._-.-'_"_-+"'_'_-----".,.- _ _-...,
Using the calibration curve generated by the reference

solutions, determine the content of sialic acids in each of the
two test solutions and calculate the mean. Calculate the
2608
IP 2010
number of moles of sialic acids per mole of erythropoietin

assuming that the relative molecular mass oferythropoietin is
30,600 and that the relative molecular mass of
N-acetylneuraminic acid is 309.
System suitability. The individual replicates agree to within
10 per cent of each other; the value obtained from reference
solution (a) is between 1.5 and 2.5 times that obtained with
test solution (a).

in the volume that contains 100000 IV of erythropoietin.
Assay. The activity of the preparation is compared with that
of elythropoietin RS and expressed in International Units
(lU).
more than 125 per cent of the stated potency. The fiducial
limits of error ofthe estimated potency (P = 0.95) are not less
than 64 per cent and not more than 156 per cent of the stated
potency.
Determine by Method A or Method 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 g to 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 to 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 hour at about 11 :00 a.m., in order to clean the
cages and feed the animals). At the end ofthe 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 under examination in
phosphate-albumin buffered saline pH 7.2 to obtain a
concentration of 0.2 IV perml.
Test solution (b). Mix equal volumes of test solution (a) and
phosphate-albumin buffered saline pH 7.2.
Test solution (c). Mix equal volumes of test solution (b) and
Reference solution (a). Dissolve erythropoietin RS in
concentration of 0.2 IV per ml.
Reference solution (b). Mix equal volumes of reference

solution (a) and phosphate-albumin buffered saline pH 7.2.
solution (b) and phosphate-albumin buffered saline pH 7.2.
Radiolabelledferric [59Fe] chloride solution, concentrated.
Use a commercially available solution of[59Fe] ferric chloride
(approximate specific activity: 100-1000 MBq per mg ofFe).
Radiolabelled [59Fe] ferric chloride solution. Dilute the
concentrated radiolabelled [59Fe] ferric chloride solution in
sodium citrate buffer solution pH 7.8 to obtain a solution
with an activity of 3.7 x 104 Bq per 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
recommen4ed. 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 ofthe
erythropoietin and the radiolabelled ferric chloride solution
must be the same for each animal. After a further 48 hours,
anaesthetise each animal by injection ofa suitable anaesthetic,
record body weights and withdraw blood samples (0.65 ml)
into haematocrit capillaries from the bifurcation ofthe 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:
A s xMx7.5
AtxVs
Where, 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.
2609
IP 2010
Test solution (a). Dilute the substance under examination in

concentration of 80 IU per ml.
Granulocyte Colony Stimulating Factor Solution
Test solution (b). Mix equal volumes of test solution (a) and
Test solution (c). Mix equal volumes oftest solution (b) and
Reference solution (a). Dissolve erythropoietin RS in
phosphate-albumin buffered saline pH 7.2 to produce a
solution containing 80 IU per ml.
Reference solution (b). Mix equal volumes of reference
solution (a) and phosphate-albumin buffered saline pH 7.2.
solution (b) and phosphate-albumin bziffered saline pH 7.2.
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 ofthe assay procedure, randomly distribute
mice ofa suitable age and strain (8-week old B6D2Fl mice are
suitable. Other strains like Swiss Albino, Balb/C of suitable
age can also be used) into 6 cages. A minimum of 8 mice per
cage is recommended. Inject each animal subcutaneously with
0.5 ml ofthe 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 ofthe 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 the following procedure.
The volume of blood, dilution procedure and fluorescent

reagent may need to be modified to ensure maximum
development and stability offluorescence.
Colorant solution, concentrated. Use a solution of thiazole
orange suitable for the determination ofreticulocytes. 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
ofreticulocytes is determined using a biparametric histogram:
number ofcells/red fluorescence (620 nm).
Calculate the potency by the usual statistical methods for a
parallel line assay.
,---'I
MTPLGPASSL PQSFLLKCLE QVRKIQGDGA ALQEKLCATY KLCHPEELVL LGHSLGIPWA
PLSStpSQAL QLAGtLSQLH SGLFLYQGLL QALEGISPEL GPTLDTLQLD VADFATIlWQ
QMEELGMAPA LQPTQGAMPA FASAFQRRAG GVLVASHLQS FLEVSYRVLR HLAQP
Mol. Wt. 18799

Filgrastim Concentrated Solution is a solution of a protein
having the structure of the granulocyte colony-stimulating
factor (G-CSF) produced and secreted by various human blood
cell types. The protein stimulates the differentiation and
proliferation ofleucocyte stem cells into mature granulocytes.
It is produced by a method based on rDNA technology, using
bacteria as host cells.
Filgrastim Concentrated Solution contains not less than
0.9 mg per ml, and not less than 1.0 x 108 IU offilgrastim per mg
of protein.
Prior to release, the following tests are carried out on each
batch ofthe final bulle product, unless the regulatory authority
has granted exemption.
Description. A clear, colourless to slightly yellowish liquid.
Identification
A. It shows the biological activity as decribed under Assay.
B. Determine by isoelectric focusing (2.4.33).
In the test for impurities with charges different from that of
filgrastim the principal band in the electropherogram obtained
with the test solution is similar in position to the principal
band in the electropherogram obtained with the reference
solution.
C. In the test for impurities of molecular masses higher than
that of filgrastim, the retention time, ofthe principal peak
obtained with the test solution is similar to that ofthe principal
peak obtained with the reference solution.
D. In the test for impurities with molecular masses differing
from that of rG-CSF under both reducing and non-reducing
conditions, the principal band in the electropherogram obtained
with test solution (a) is similar in position to the principle
band in the electropherogram obtained with reference solution
(a).
E. Determine by peptide mapping (2.3.47).
!t:!l'~g~.~!()~<: il1~l1nl1i~tig~1contai~e!:.~t a ~~p~!ature bel.2_~

-20.
Avoid
repeated freezing
and thawing..
- ..
.
. . ,
Test solution .Introduce~50'/-1lof a 0.05 Msodiumphosphate

bufjerpH 8. ointo. a polypropylene tube. Add a volume of the
Labelling. The label states (l) the erythropoietin content in

mg per ml; (2) the protein content in mg per ml; (3) the name
and the concentration of any other excipients.
substance under examination corresponding to 25 /-1g of

protein, add 25 /-11 ofa 0.1 mg perml solution ofGlu-C2 protease;
dilute to 1 ml with water; stopper the tube and incubate at
2610
IP 2010
FILGRASTIM CONCENTRATED SOLUTION
about 37 for 18 hours. Add 125 /!1 of a 7.64 per cent w/v
solution of guanidine hydrochloride and mix well. Add 10 /!l
ofa 1.542 per cent w/v solution of dithiothreitol and mix well.
Place the capped tube in boiling water for 1 minute. Allow to
cool to room temperature.
Rejerence solution. Prepare at the same time and in the same

manner as for the test solution but use G-CSF RS instead of
the test preparation under examination.
octadecylsily1 silica gel (5 /!m) with a pore size of20 nm,
mobile phase: A. Dilute 0.5 ml oftrijluoroacetic acid to
950 ml with water add 50 ml of acetonitrile and mix,
E. Dilute 0.5 ml oftrijluoroacetic acidto
50 ml with water; add 950 ml of acetonitrile,
flow t:ate. 0.2 ml per minute,
A linear gradient programme using the conditions given
below,
injection volume. 10 /!l.
Time
Mobile phase A
Mobile phase B
(in min.)
(per cent v/v)
(per cent v/v)
0-8
8-25
97-94
94-66
25~
66-10
40-45
45-4Q
10
10-97
45-65
97
3-6
6-34
34-90
90
90-3
3
Test solution (b). To 0.20 ml oftest solution (a) add 0.20 ml of

sample buffer A.
Test solution (c). Dilute 0.20 ml of test solution (b) to 1.0 ml
with sample buffer A.
Test solution (d). Dilute 0.20 ml of test solution (c) to 1.0 ml
with sample buffer A.
Test solution (e). To 0.20 ml oftest solution (d) add 0.20 ml of
sample buffer A.
Reference solution. Solution of molecular mass markers
suitable for calibrating SDS-polyacrylamide gels in the range
ofl4.4-94 kDa.
Sample treatment: boil for 5 minutes.
Apply 20 /!l of each reduced and non-reduced solutions to
separate gels.
Detection: Silver staining as described below.
Immerse the gel for 30 minutes in a mixture of 10 volumes of
acetic acid, 40 volumes of water and 50 volumes of methanol.
Transfer the gel to 5 per cent methanol and shake for 5 minutes.
Repeat this washing step thrice. Replace the 5 per cent v/v
solution of methanol with 0.2 g per litre sodium thiosulphate.
Wash the gel thrice in water for 30 seconds each. Transfer the
gel to 0.2 per cent w/v solution of silver nitrate.
Equilibrate the column at the initial conditions for at least

30 minutes.
The chromatograms obtained with the reference solution and
the test solutions are qualitatively similar.
The profile of chromatogram obtained with the reference
solution and the test solution corresponds to that of the
Tests
Impurities with molecular masses differing from that of
Filgrastim. Determine by electrophoresis (sodium dodecyl
sulphate polyacrylamide gel electrophoresis) (2.4.12) under
both reducing and non-reducing conditions.
This solution is prepared immediately before use. Place the

gel on shaker for 25 minutes. Wash the gel for 1 minute. Repeat
this washing step thrice. Transfer the gel into a mixture
containing 30 g per litre solution of sodium carbonate,
0.05 per cent v/v solution offormaldehyde and 0.2 g per litre
solution of sodium thiosulphate in water. Protein bands
become visible during this step. Keep the gel in the solution
until sufficiently stained and then stop the staining by soaking
the gel in a 14 g per litre solution of disodium edetate.
The test is not valid unless the proteins ofthe molecular weight
marker are distributed along 80 per cent ofthe gel and over the
required separation range (the range covering the product
and its dimer or the product and its related impurities); a band
is seen in the electropherogram obtained with test solution
(e), and a gradation of intensity is seen in the
electropherograms obtained with solutions (a) to (e).
In the electropherogram obtained with test solution (a) no
band is more intense that the principal band in the
electropherogram obtained with reference solution (t).
Impurities with charges differing from that of Filgrastim.
Detennine by isoeloelectric focusing (2.4.33).
Resolving gel. 13 per cent Acrylamide

Sample buffer A.
Sample buffer B (reducing conditions).
Test solution (a). Dilute the preparation under examination

with sample buffer A to obtain a protein concentration of
100 /!g per ml.
Test solution. Dilute the preparation under examination to

produce a solution containing 0.3 mg per ml.
Reference solution (a). A solution ofjUgrastim RScontaining
0.3 mg per ml.
2611
IP 2010
FILGRASTIM CONCENTRATED SOLUTION
Reference solution (b). A solution ofjilgrastim RS containing

0.03 mg per ml.
Detection. Proceed as described in Isoelecric Focusing

(2.4.33).
Detect the product and its dimer or the product and its related
impurities.
In the electropherogram obtained with reference solution (c),
relevant isoelectric point markers are distributed along the
entire length of the gel.
In the electropherogram obtained with reference solution (a),
the pI ofthe principal band is 5.7-6.3.
No band is more intense that the principal band in the
electropherogram obtained with reference solution (b).
Related Proteins. Determine by liquid chromatography
(2.4.14).

mobile phase A to obtain a concentration of 0.3 mg per ml.
Reference solution (a). Dilute G-CSFRSwith the same mobile
phase to obtain a concentration of 0.3 mg per ml.
Reference solution (b). To 570 III of reference solution (a),
add 6.8 III ofa 0.45 per cent v/v solution of hydrogen peroxide;
mix and incubate at 25 for Ihour, then add 2.5 mg of
methionine RS.
octadecylsilane bonded to porous sili<;a (3 Ilm) and a
12.5 em x 4.6 mm, packed with octadecylsilane bonded
to porous silica (20 Ilm),
mobile phase: A. dilute 1 ml of trifluoroacetic acid to
500 ml with water and add 499 ml of acetonitrile,
B. dilute 1 ml of trifluoroacetic acid to
950 ml with acetonitrile and add 49 ml of water,
ilQ~~l~, 1!!!lp~~min1l1e, ....
a linear gra~iellt progrfll11lIle usiIlgthe c()Il~itions given
below,
Mobile phase A
(per cent v/v)
Mobile phase B
(per cent v/v)
0-4
92
4-19
92~72
28
~O
28
100
Time
Reference solution (c). Use an isoelectric point (PI) calibration

solution, in the pI range of 2.5-6.5, prepared according to
Focusing:
pH gradient. 4.5 - 8.0,
- catholyte. 1 M sodium hydroxide,
- anolyte: 0.04 M glutamic acid in a 0.0025 per cent v/v
- Application 20 Ill.
(in min)
19-19.1
72
19.1-21
100
21-21.1
0~92
100~8
21.1-25
92
Inject the test solution and reference solutions (a) and (b).
Relative retention with reference to filgrastim (retention
time = about 12 minutes.): oxidized filgrastirn 2 = about 0.95.
The profile of the chromatogram obtained with reference
solution (b) is similar to that ofthe chromatogram ofoxidized
filgrastim supplied with jilgrastim RS. The chromatogram
shows two peaks corresponding to oxidized filgrastim 1 and
oxidized filgrastim 2 that elute before the principal peak, the
second peak not being completely separated from the principal
peak.
of any peak other than the principal peak is not greater than
2.0 per cent ofthe total area of all the peaks. The sum of the
areas of any peaks other than the principal peak is not greater
than 3.5 per cent ofthe total area of all of the peaks.
Dimers and Related Substance of Higher Molecular Mass.
Determine by size-exclusion chromatography (2.4.16).
. Solution A. Dissolve 4.1 g of sodium acetate in 400 ml of

water, adjust to pH 4.0 with acetic acid and dilute to 500 ml
with water.
solution A to obtain a concentration of 0.4 mg per ml.
Reference solution. Dilute jilgrastim RS with solution A to
obtain: a concentration of 0.4 mg per ml.
Resolution solution. Mix a sample of the reference solution
for about 30 seconds using a vortex mixer.
hydrophilic silica gel, ofa grade suitable for fractionation
ofglobular proteins in the relative molecular mass range
of I 0,000 to 5,00,000,
mobile phase: dissolve 7.9 g of ammonium hydrogen
CCl.rlJ()llaJ?iIlI0QQ!!!J.Qi1'1llf.~:and~dj!lst tg-.plL7,Q':Y!!!!.
phosphoric acid; dilute to 2000 ml with water,
2612
flowra.te.6.5 IIl}perIIl1nl.lte, ..

IP 2010
INTERFERON ALFA-2 CONCENTRATED SOLUTION
Relative retention times with reference to filgrastim monomer

(retention time=about 19 minutes): aggregates = about 0.60;
filgrastim oligomer 1= about 0.75; filgrastim oligomer 2= about
0.80; filgrastim dimer=about 0.85.
Inject the resolution solution. The retention time offilgrastim
monomer is 17 minutes to 20 minutes. The resolution between
the peaks due to filgrastim dimer and filgrastim monomer is
not less than 4.0..
Calculate the content ofdimer, oligomers and aggregates. The
sum of the peaks with retention times less that that of the
principal peak is not more than 2.0 per cent.
Bacterial endotoxins (2.2.3). Not more than 20 Endotoxin
Units per mg ofprotein.
Assay. A. Protein - Determine by liquid chromatography (2.4.14)
as described under the test for Related proteins.
Calculate the content offilgrastim.
B. Potency- Determination of the biological activity of
rG-CSF concentrated solution is based on the stimulation of
NFS-60 cells (murine myeloblastic cell line) or any other suitable
myeloblastic cell line by rG-CSF.
The following method uses the conversion of tetnlzolium
bromide (MTT or any other suitable dye) as a staining method.
Alternative methods of quantifying cell proliferation, such as
measurement of intracellular ATP by luciferase
bioluminescence have also been found suitable, and may be
used as the assay readout, subject to appropriate validation.
NFS-60 cells (murine myeloblastic cell line) or any other suitable
myeloblastic cell line are incubated with varying dilution of
test and reference preparations of rG-CSF. They are then
incubated with a solution of MTS, MTT, XTT or any other
suitable dye. This cytochemical stain is converted by cellular
dehydrogenases to a purple formazan product. The formazan
is then measured spectrophotometrically. The potency of the
test preparation is determined by comparison of the dilutions
of the test preparation with the dilutions of the appropriate
International Standard of rG-CSF or with a reference
preparation calibrated in International Units, which yield the
same response (50 per cent maximal stimulation).
The International Unit is the activity contained in a stated
amount of the appropriate International Standard. The
equivalence in International Units ofthe International standard
is stated by the World Health Organization.
Add 50 III of the dilution medium to all wells ofa 96 -well
microtitre plate. Add an additional 50 III ofthis solution to the
wells designed for blanks. Add 50 III of each solution to be
tested in triplicate (test preparation and reference preparation
at a concentration of about 800 ill per ml, plus a series of 10
twofold dilutions to obtain a standard curve). Prepare a
suspension ofNFS-60 cells (murine myeloblastic cell line) or

any other suitable myeloblastic cell line containing 7xl 0 5cells
per ml immediately before use, add 2-mercaptoethanol to a
final concentration of 0.1 mM, and add 50 III of the prepared
cell suspension to each well, maintaining the cells in a uniform
suspension during addition.
Incubate the plate at 36 to 38for a minimum of24 hours in a
humidified incubator using 6 1per centCO 2, 20 III ofa 5.0 g
per litre sterile solution of tetrazolium bromide to each well
and re-incubate for 4 hours. Estimate the quantity offormazan
produced using a microtitre well plate reader at 490 urn.
Analyze the data by fitting a sigmoidal dose-response curve
to the data obtained and by using a suitable statistical method,
for example the 4-parameter or parallel line models.
more than 125 per cent of the stated potency. The confidence
limits (P=0.95) of the estimated potency are not less than 74
per cent and not more than 136 per cent of the stated potency.
Labeling. The label states the content, in mg ofprotein per ml;
the potency, in ill per mg of protein.
Interferon Alfa-2 Concentrated

Solution
SRRTLMLLAQ
L-
MRXjISLFSCL
KDRHDFGFPQ
QQIFNLFSTK
DSSAAWDETL
QGVGVTETPL
MKEDSILAVR
LDKFYTELYQ
QLNDLEACVI
KYFQRITLYL
KEKKYSPCAW EVVRAEIMRS
FSLSTNLQES
LRSKE
alfa-2a: CS60Hj353N2270255S9
Mol. Wt.19,241
alfa-2b: CS6oHI353N2290255S9
Mol. Wt.19,269
Interferon alfa-2 concentrated solution is a solution ofan rDNA

derived therapeutic protein which exhibits non-specific
antiviral activity, at least in homologous cells. Interferon
alfa-2 concentrated solution also exerts antiproliferative and
immunomodulator activity. Two different types of alfa-2
interferon, varying in the amino acid residue at position 23,
are found and are named as alfa-2a and alfa-2b.
Designation
Residue at position 23 (Xl)
alfa-2a
Lys
alfa-2b
This monograph applies to interferon alfa-2a and 2b
concentrated solutions.
2613
IP 2010
Interferon alfa-2 concentrated solution contains not less than

1.4 x 108 ill per mg ofprotein and not less than 2 x 108 ill of
Interferon alfa-2 per ml.
Prepare a mixture of 32 volumes of glacial acetic acid;

100 volumes of ethanol and 268 volumes of water. Transfer
the gel to the mixture and soak for 5 minutes.
Production
Immerse the gel for 10 minutes in a staining solution prewanned

to 60.The staining solution is prepared by adding 1.2 g per
litre of acid blue 83 to the mixture of glacial acetic acid,
ethanol and water.
Interferon alfa-2 concentrated solution is produced by a

method based on recombinant DNA technology using bacteria
as host cells. It is produced under controlled conditions
designed to minimise microbial contamination ofthe product.
Interferon alfa-2 concentrated solution complies with the
following additional requirements.
Host-cell-derived proteins
The limit is approved by WHO guidelines.
Host-cell- or vector-derived DNA
The limit is approved by WHO guidelines.
Description. A clear,colourless or slightly yellowish liquid.
Identification
A. It shows the expected biological activity as described under
Assay for potency.
B. Detennine by isoelectric focusing (2.4.33).

water to obtain a solution containing 0.5 mg protein per ml.
Reference solution. A 0.5 mg per ml solution of intelferon
alfa-2 RS in water.
Isoelectric poii1t calibration solution pI range 3.0 to 10.0.
Prepare and use according to the manufacturer's instructions.
Isoelectric focusing is carried out using either horizontal
electrophoresis system or by vertical electrophoresis system
as per the procedure described below or by any appropriate
validated method.
Horizontal Electrophoresis
Select and use a suitable horizontal isoelectric focusing
apparatus with facility for connecting a circulating bath chiller
capable ofmaintaining 10. Select gels for isoelectric focusing
with a pH gradient from 3.5 to 9.5.
Use phosphoric acid as anode solution (98 g per litre
phosphoric acid) and 1 M sodium hydroxide as the cathode
solution. Using filter paper apply 15 f!l ofthe test solution and
the reference solution to the gel close to the cathode.
Start the isoelectric focusing at 1500 V and 50 rnA. Turn off
the power after 30 minutes. Remove the application filters and
reconnect the power supply for 1 hour. Keep the power
cc)'ns6irifdilllng--the '''''tocusmg'' pr"6c't~"ss.~""~"~'-'" "
Wash the gel several times to destain with the mixture ofglaeial
acetic acid, ethanol and water and keep the gel in this mixture
for about 12-24 hours until the background is clear.
Add glycerol, 10 per cent v/v to the mixture of glacial acetic
acid, ethanol and water. Soak the gel for 1 hour in the solution.
The principal bands of the electropherogram obtained with
the test solution correspond in position to the principal bands
of the electropherogram obtained with the reference solution.
Plot the migration distances of the isoelectric point markers
versus their isoelectric points and detennine the isoelectric
points of the principal components of the test solution and
the reference solution. They do not differ by more than 0.2 pI
unit. The test is not valid unless the isoelectric point markers
are distributed along the entire length of the gel and the
isoelectric points ofthe principal bands in the electropherogram
obtained with the reference solution are between 5.8 and 6.3.
C. Examine the electropherograms obtained under reducing
conditions in the test for impurities ofmolecular masses differing
from that of interferon alfa-2. The principal band in the
electropherogram obtained with test solution (a) corresponds
in position to the principal band in the electropherogram
D. Detennine by peptide mapping (2.3 ~4 7).

water to produce a solution containing 0.5 mg protein per ml.
Transfer 25 f!l to a microfuge tube of 1.5 ml capacity. Add 1.6 f!l
of 1 M phosphate buffer solution pH 8.0, 2.8 f!l of a freshly
prepared 1.0 mg per ml solution of tlypsin in water (suitable
for peptide mapping) and 3.6 III of water and mix vigorously.
Cap the tube and place it in a water-bath at 37 for 18 hours.
Add 100 III of a 573 g per litre solution of guanidine
hydrochloride and mix well. Add 7 III of 154.2 g per litre solution
of dithiothreitol and mix well. Place the capped tube in boiling
water for 1 minute and cool to room temperature.
Reference solution. A 0.5 mg per ml solution ofthe appropriate
inteiferon alfa-2 RS prepared at the same time and in the same
manner as the test solution.
Immerse the gel in a solution containing 115 g per litre of

trichloroacetic acid and 34.5 g per litre of sulphosalicylic
acid in water and agitate the container gently for 60 minutes.
2614
a~sJainlesf:Lstedc~QlllmnlQQcm..x 4,.6~mm,~p.Jlc1~(lwi1b
a
pore size oDO nm,
mobile phase: A.l ml of trifluoroacetic acid dilute to
1000 ml with water,
IP 2010
B. Add 1 ml of trifluroacetic acid to

100 ml ofwater and dilute to 1000 ml with acetonitrile,
below,
Time
Mobile
Mobile
Comment
phase A
phase B
(in min) (per cent v/v) (per cent v/v)
0.8
100
0
isocratic
8.68
100 -7 04
0 -7 60
linear gradient
68.72
40
60
isocratic
72.75
40 -7 100
60 -70
linear gradien
75.08
100
0
re-equilibration
Equilibrate the column with inobile phase A for at least 15
minutes maintaining tlle temperature ofthe column at 30.
Inject the test solution and the reference solution. The
chromatogram obtained with each solution should be
qualitatively similar to the chromatogram ofinterferon alfa-2.
The profile of the chromatogram obtained with the test
solution should also correspond to that of the chromatogram
Tests
Impurities of molecular masses differing from that of
interferon alfa-2. Determine by electrophoresis (sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) (2.4.12). The test is pelformed under both reducing
and non-reducing conditions, using resolving gels of
14 per cent aClylamide and silver staining as the detection
method.
Sample buffer (non-reducing conditions). Mix equal volumes
of water and concentrated SDS-PAGE sample buffel~
Sample buffer (reducing conditions). Mix equal volumes of
water and concentrated SDS -PAGE sample bufferfor reducing
conditions containing 2-mercaptoethanol as the reducing
agent.
Test solution (a).Dilute the preparation under examination in
the sample buffer to obtain a solution containing a
concentration of 0.5 mg protein per ml.
Test solution (b). Dilute 0.2 ml oftest solution (a) to 1ml with
the sample buffer.
Reference solution (a). Prepare a 0.625 mg per ml solution of
the appropriate interferon alfa-2 RS in the sample buffer.
to 1 ml with the sample buffer.
Reference solution (c). Dilute 0.2 ml of reference solution (b)
to I ml with the sample buffer.
Reference solution (d). Dilute 0.2 ml of reference solution (c)

to I ml with the sample buffer.
Reference solution (e). Dilute 0.2 ml of reference solution (d)
to 1 ml with the sample buffer.
Reference solution (I). Use a solution of molecular mass
standards suitable for calibrating SDS-PAGE gels in the range
141illa to 99 lilla.
Place the test and reference solutions, contained in covered
test-tubes, on a water-bath for 2 minutes.
Apply 10 III of reference solution (f) and 50 III of each ofthe
other solutions to the stacking gel wells. Perform the
electrophoresis under the conditions recommended by the
manufacturer of the equipment. Detect proteins in the gel by
silver staining.
The test is not valid unless (I) the validation criteria are met;
(2) a band is seen in the electropherogram obtained with
reference solution (e); (3) a gradation of intensity of staining
is seen in the electropherograms obtained, respectively, with
test solution (a) and test solution (b) and with reference
solutions (a) to (e).
The electropherogram obtained with test solution (a) under
reducing conditions may show, additional bands but no such
band should be more intense than the band obtained with
reference solution (d). Further, not more than 3 such bands
should be more intense than the principal band obtained with
reference solution (e).
The electropherogram obtained with test solution (a) under
non-reducing conditions may show, in addition to the principal
band, less intense bands with molecular masses higher than
the principal band. No such band is more intense than the
principal band in the electropherogram obtained with reference
solution (d) and not more than three such bands are more
intense than the principal band in the electropherogram
obtained with reference solution (e).
Related proteins. Determine by liquid chromatography (2.4.14).

0.25 per cent w/w hydrogen peroxide solution. Dilute
hydrogen peroxide solution with water to obtain 0.25 per
cent w/w solution.
water to obtain a solution containing 0.5 mg protein per ml.
Reference solution. To a volume of the test solution, add a
suitable volume of the 0.25 per cent hydrogen peroxide
solzition to give a final hydrogen peroxide concentration of
0.005 per cent, and allow to stand at room temperature for 1
hour, to generate about 5 per cent oxidised interferon. Add
12.5 mg ofL-methionine per ml ofthe solution. Allow to stand
at room temperature for I hour. Store the solutions for not
longer than 24 hours at a temperature of 2 to 8.
2615
IP 2010
a stainless steel column 25 cmx 4.6 mm, packed with
octadecylsilane bonded to porous silica (5 f.lm) with a
pore size oBO nm,
- mobile phase: A. To 700 ml of water add 2 ml of
trifluoroacetic acid and 300 ml of acetonitrile,
B. To 200 ml of water add 2 ml of
trifluoroacetic acid and 800 ml of acetonitrile,
below,
Time
Mobile
Mobile
phase A
phase B
(in min) (per cent v/v) (per cent v/v)
0.1
72
28
1.5
72 -+ 67
28 -+ 33
Comment
linear gradient
5 - 20
67 -+ 63
33 -+ 37
linear gradient
63-+57
37 -+ 43
linear gradient
30-40
57-+40
43 -+ 60
linear gradient
40-42
40
60
42-50
40-+ 72
60 -+ 28
linear gradient
50-60
72
28
re-equilibration
Prepare 30-fold and 50-fold dilutions of the test solution.

Prepare a mixture of 2.0 ml of a 2.0 per cent w/v solution of
copper sulphate in water, 2.0 ml ofa 4.0 per cent w/v solution
of sodium tartrate in water and 96.0 ml of a 4.0 per cent w/v
solution of sodium carbonate in 0.2 M sodium hydroxide.
Add 1.25 ml ofthe above mixture to the test-tube containing
1.5 ml of water to prepare the blank, 1.25 ml to the test tube
containing different dilutions ofthe sample and 1.25 ml to the
test tube with the reference solution.
Mix after each addition and after approximately 10 minutes,
add to each test-tube 0.25 ml ofa mixture ofequal volumes of
water and phosphomolybdotungstic reagent. Mix after each
addition. After 30 minutes, measure the absorbance of each
solution at 750 nm (2.4.7) using the blank as the compensation
liquid.
isocratic
20-30
Reference solutions. Prepare a stock solution of0.5 mg per ml

of bovine albumin. Prepare eight dilutions of the stock
solution containing between 3 f.lg per ml and 30 f.lg per ml of
bovine albumin.
Draw a calibration curve from the absorbances of the eight

reference solutions and the corresponding protein contents
and read from the curve the content of protein in the test
solution.
isocratic
B.Potency
Equilibrate the column with the mobile phases in the initial

gradient ratio for at least 15 minutes.
Interferon alfa-2 elutes at a retention time ofabout 20 minutes
in the chromatogram. With the reference solution a peak related
to oxidized interferon appears at a retention time of about 0.9
relative to the principal peak.
corresponding to oxidised interferon and interferon is at least
1.0. Consider only the peaks whose retention time is 0.7 to 1.4
relative to that of the principal peak.
of any peak, apart from the principal peak, is not greater than
3.0 per cent ofthe total area ofall ofthe peaks. The sum ofthe
areas of any peaks other than the principal peak is not greater
than 5.0 per cent of the total area of all ofthe peaks.
Bacterial endotoxins (2.2.3). Not more than 100 Endotoxin

Units per mg ofprotein.
Assay

water to obtain a concentration of about 0.5 mg of interferon
alfa-2 per ml.
The potency of interferon alfa-2 is estimated based on its

ability to protect cells against a viral cytopathic effect compared
to the protection accorded by an appropriate International
Standard of human recombinant interferon alfa-2 or of a
reference preparation calibrated in International Units.
The International Unit is the activity contained in a stated
amount of the appropriate International Standard. The
equivalence in International Units ofthe International Standard
is stated by the World Health Organisation.
Carry out the assay by a suitable method, based on the
following design.
Use, an established cell line sensitive to the cytopathic effect
of a suitable virus, responsive to interferon.
The following cell cultures and virus have shown to be suitable:
MDBK cells (ATCC No. CCL22), or Mouse L cells (NCTC
clone 929; ATCC No.CCL 1) as the cell culture and vesicular
stomatitis virus VSV; Indiana strain (ATCCNo. VR-158) as the
infective agent; or human diploid fibroblast FS-71 cells
responsive to interferon as the cell culture, and
encephalomyocarditis virus (ATCC No.VR-129B) as the
infective agent.
Incubate in at least three groups, cells with three or more
different concentrations oftbe preparat)onlin<:lerexaml:nat)()Il
and one with the reference preparation in a microtitre plate.
Include appropriate controls ofuntreated cells in each group.
2616
IP 2010
STREPTOKINASE BULK SOLUTION
Choose the concentrations of the preparations such that the

lowest ,concentration produces some protection and the
largest concentration produces less than maximal protection
against the viral cytopathic effect.
per ml in phosphate buffer pH 7.2. Mix 'immediately. A clot

cUrated bovine plasma. The clot does not lyse within 60
minutes.
Add the cytopathic virus after the cells have established to all
wells except the control wells.
B. Dissolve 0.6 g ofagar in 50.0 ml of barbitone bufferpH 8.6,
Determine the cytopathic effect of virus quantitatively and

calculate the potency of the preparation to be examined by
the usual statistical methods for a parallel line assay.
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.
Storage. Store protected from light, at or below -20.
Labelling. The label states (1) The type of interferon (alfa-2a
or alfa-2b); (2) the type ofproduction.
Streptokinase Bulk Solution
heating until a clear solution is obtained. Use glass plates

50 mm square (transparency mounts) free from traces ofgrease.
Using a pipette, apply to each plate 4 ml ofthe agar solution.
Maintain the plates horizontal. Allow to cool. Pierce a cavity
6 mm in diameter in the centre ofthe agar and an appropriate
number of cavities (not exceeding 6) at distances of II mm
from the central cavity. Remove the residual agar from the
cavities using a cannula connected to a vacuum pump. Using
pipettes graduated in microlitres, place in the central cavity
about 80 III ofgoat or rabbit antistreptokinase serum containing
10,000 units ofantistreptokinase activity per ml; place in 'each
of the surrounding cavities about 80 III of a dilution of the
preparation under examination containing 125,000 IU of
streptokinase activity per ml. Allow the plates to stand in a
humidified tank for 24 hours. Only one precipitation arc appears
and it is well defined and localised between the application
point of the serum and each cavity containing the solution of
the preparation under examination.
Streptokinase Bulk Solution is a fibrinolytic enzyme present

in certain strains ofhaemolytic Streptococcus group C. It has
the property of combining' with human plasminogen to form
plasminogen activator. Streptokinase is also produced by a
method based on recombinant DNA technology using bacteria
or suitable genetically engineered host cells.

diluting the preparation under examination in carbon dioxidefree water to produce a solution containing 5000 IU of
streptokinase activity per ml.
Streptokinase Bulle Solution has a potency of not less than

96,000 ill per mg ofprotein.
Streptodornase. Not more than 10 ill ofstreptodornase activity

per 100,000 ill ofstreptokinase activity.
Production
Test solution. Dilute the preparation under examination in

imidazole buffer pH 6.5 to obtain a solution containing
150,000 ill ofstreptokinase activity per ml.
If intended for use in the manufacture of parenteral

preparations, the method of manufacture is validated to
demonstrate that the. product, if tested, would comply with
the following test.
Abnormal toxicity
Inject into each mouse a quantity of the preparation under
examination (diluted, if necessary, with water for injections)
containing 50,000 ill of streptoldnase activity in 0.5 ml, the
injection lasting 15 to 20 seconds.
Description. A clear, colourless liquid.
Identification
A. Place 0.5 ml of citrated human plasma in a haemolysis
tube maintained in a water-bath at 37. Add 0.1 ml ofa dilution
ofthe preparation under examination containing 10,000 ill of
streptokinase activity per ml in phosphate buffer pH 7.2 and
0.1 ml of a solution of human thrombin RS containing 20 ill
Tests
Reference solution. Dissolve in imidazole buffer pH 6.5 a

reference preparation of streptodornase, calibrated in
International Units against the International Standard of
streptodornase, to obtain a solution containing 20 IU of
streptodornase activity per m!. The equivalence in International
Units of the International Standard IS stated by the regulatory
authority.
To each of 8 numbered centrifuge tubes, add 0.5 ml ofa 0.1 per
cent solution of sodium deoxyribonucleate in imidazole buffer
pH 6.5. To tube number 1 and tube number 2 add 0.25 ml of
imidazole buffer pH 6.5, 0.25 ml of the test solution and,
immediately, 3.0 ml of2.5 per cent w/v ofperchloric acid. Mix,
centrifuge at about 3000 rpm for 5 minutes and measure the
absorbances ofthe supernatant liquids at 260 nm (2.4.7), using
as the compensation liquid a mixture of 1.0 ml of imidazole
buffer pH 6.5 and 3.0 ml of 2.5 per cent w/v solution of
perchloric acid (absorbances Al and A2). To the other 6
2617
STREPTOKINASE BULK SOLUTION
IP 2010
tubes (numbers 3 to 8) add 0.25 ml, 0.25 ml, 0.125 mI, 0.125 ml,
oml and 0 ml respectively of imidazole buffer solution pH 6.5;
add to each tube 0.25 mI of the test solution and 0 ml, 0 mI,
0.125 ml, 0.125 ml, 0.25 ml and 0.25 ml respectively of the
reference solution. Mix the contents of each tube and heat at
37 for 15 minutes. To each tube add 3.0 ml of2.5 percentw/v
of perchloric acid, mix and centrifuge. Measure the
absorbances ofthe supernatant liquids at 260 nm (204.7), using
the compensation liquid described above (absorbances A3 to
A8). The absorbances comply with the following test.
(A3+A4)-(Al+A2)< (A5+A6+A7+A8)
2
(A3+A4)
Streptolysin
In a haemolysis tube, use a quantity of the preparation under
examination containing 500,000 ill of streptokinase activity
and dilute to 0.5 ml with a mixture of 1 volume ofphosphate
bziffer pH 7.2 and 9 volumes of a 0.9 per cent w/v solution of
sodium chloride. Add 004 ml ofa 2.3 per cent w/v solution of
sodium thioglycollate. Heat in a water-bath at 37 for
10 minutes. Add 0.1 ml ofa solution ofa reference preparation
of human antistreptolysin 0 containing 5 ill per ml. Heat at
37 for 5 minutes. Add 1 ml ofrabbit erythrocyte suspension.
Heat at 37 for 30 minutes. Centrifuge at about 1000 rpm. In the
same manner, prepare a haemolysis tube in which the solution
of the preparation under examination has been replaced by
0.5 ml ofa mixture of 1 volume ofphosphate bz!fferpH 7.2 and
9 volumes of a 0.9 per cent w/v solution of sodium chloride.
Measure the absorbances of the supernatant liquids at
550 nm (204.7). The absorbance ofthe test solution is not more
than 50 per cent than that of the reference solution.
Protein. Determine the nitrogen content (2.3 .30).
1 mg ofN is equivalent to 6.25 mg ofprotein.
Potency
The potency of streptokinase is determined by comparing its
capacity to activate plasminogen to form plasmin with the
same capacity of a reference preparation of streptokinase
calibrated in International Units; the formation of plasmin is
determined using a suitable chromogenic substrate.
Substrate solution. Mix 1.0 ml of tris (hydroxymethyl)

aminomethanebuffer pH 7.4 with 1.0 ml of chromophore
substrate. Add 5 III ofa 10 per cent w Iv solution of polysorbate
20. Keep at 37 in a water-bath. Immediately before
commencing the activation assay, add 45 III of a 1 mg per ml
solution of human plasminogen.
Analyse each streptokinase dilution, maintained at 37, in
duplicate. Initiate the activation reaction by adding 60 III of
each dilution to 40 III of substrate solution. For blank wells,
use 60 III of tris (hydroxymethyl) aminomethane sodium
chloride bziffer solution pH 7.4 instead of the reference and
test solutions. Allow the reaction to proceed at 37 for
20 minutes and read the absorbance at 405 nm (204.7). If a
suitable thermostated plate reader is available, this may be
used to monitor the reaction. Alternatively, it may be necessary
to stop the reaction after 20 minutes using 50 III of a 50 per
cent v/v solution of glacial acetic acid. Best results are
obtained when the absorbance for the highest streptokinase
concentration is between 0.1 and 0.2 (after blank subtraction).
If necessary, adjust the time of incubation in order to reach
this range of absorbances.
Calculate the regression of the absorbance on log
concentrations of the solutions of the substance under
examination and ofthe reference preparation of streptokinase
and calculate the potency ofthe substance under examination
using the usual statistical methods for parallel-line assays.
limits (P = 0.95) of the estimated potency are not less than
80 per cent and not more than 125 per cent of the stated
potency.
Streptokinase Bulk Solution intended for use in the

appropriate procedure for the removal of bacterial
endotoxins complies with the following additional
requirement.
Bacterial endotoxins (2.2.3). Less than 0.02 Endotoxin Unit
per 100 ill of streptokinase activity.
The International Unit is the activity of a stated amount of the

International Standard for streptokinase. The equivalence in
International Units of the International Standard is stated by
the regulatory authority.
Storage. Store protected from light and at a temperature of

about - 20. If it is intended for the manufacture of parenteral
preparations, the container should be sterile and sealed so as
to exclude micro-organisms.
Reference and test solutions. Prepare 2 independent series of
4 dilutipmLQLeac]LoDhe sUh~anc~!1I1dt<I~examination anggi Units~of streptokina~~cJiyi!Ym~!:mg,..9!ll.9_ul~eg.QIlthe~:ieg

the .referellce pr~Pllrllti()Il. ofstr~pt()lcinase ill. tris.
(hydroxymethyl) aminomethane sodium chloride bziffer pH

7.4, in the range of 0.5-4.0 ill per ml. Prepare and maintain all
solutions at 37.
~llsi:>; (2) thelllllJJellng qllillltity()[a.l1y agg<;:g sll~stance; (3)

where applicable, that the substance is free from bacterial
endotoxins; (4) where applicable, thatthe substance is suitable
for use in the manufacture of parenteral preparations.
2618
GENERAL MONOGRAPHS
VETERINARY PRODUCTS
c;eneral~onographs
Veterinary Tablets
2621
2621
2622
2622
2622
2623
2623
2623
2623
Veterinary Vaccines
2623
Dip Concentrates
IntramammaryInfusions
Premixes
VeterinmyAerosols
Veterinary Diagnostics
Veterinary Oral Liquids
Veterinary Oral Powders
Veterinary Parenteral Preparations
~onographs
2627
2705
2747
Non Biological
Biological
.Diagnostics
2619
IP 2010
GENERAL MONOGRAPHS
Veterinary Preparations
The general requirements relating to a specific type of dosage
form of an active pharmaceutical ingredient or ingredients,
that have been given in the chapter on General Monographs
on Dosage Forms ofActive Pharmaceutical Ingredients apply
to all veterinary dosage forms or preparations of the type
defined. However, a valid interpretation ofthe appropriateness
of a test or requirement should be done in the context of the
monograph as a whole and of the relevant General Notices.
The requirement for compliance with the tests given under
each dosage form or preparation is indicated in each
monograph ofa drug product or preparation under the heading
'Other tests'. These tests are mandatory and are additional to
the tests given in the individual monograph.
Dip Con.centrates
Dip concentrates are preparations for the prevention and
treatment ofectoparasitic infestations ofanimals. They contain
one or more medicaments, usually in the form of wettable
powders,pastes or solutions from which diluted suspensions
or emulsions are prepared by appropriate dilution with the
recommended liquid. The diluted preparations are applied by
complete immersion of the animal or by spraying, as
appropriate. They contain suitable antimicrobial preservatives.
Labelling. The label states (1) the name(s) and proportion(s)
ofmedicament(s); (2) the name and proportion of any added
antimicrobial preservative; (3) the name and quantity of the
diluent and the manner of preparing the diluted dip solution
or spray; (4) any special precautions to be taken for use ofthe
preparation; (6) the storage conditions; (6) the date after which
the preparation is not intended to be used.
If the preparation contains an organophosphorus compound
the label also states (I) that the preparation contains an
organophosphorus compound; (2) and special precautions
on the use of the preparation.
Intramammary In.fusions
Intramammary Infusions for Veterinary Use;
Intramammary Injections.
Intramammary Infusions are sterile products intended for
injection into the mammary gland through the teat canal. They
are solutions, emulsions or suspensions or semi-solid
preparations containing one or more active ingredients in a
suitable vehicle. They may contain stabilizing, emulsifying,

suspending and thickening agents. If a sediment is formed in
a suspension, it is readily dispersible on shaking. In emulsions,
phase separation may occur but this is readily miscible on
shaking.
There are two main types ofIntramammary Infusions. One is
intended for administration to lactating animals as qualified
by the term Lactating Cow/Buffalo and the other, qualified as
Non-lactating or Dry Cow/Buffalo, is intended for
administration to animals at the end oflactation or during the
non-lactating period for the prevention or treatment ofinfection
during the dry period.
Intramammary Infusions are prepared by dissolving or
suspending the sterile medicaments in the sterilized vehicle
using aseptic precautions, unless a process of terminal
sterilisation is employed.
Containers. Intramammary Infusions are usually supplied in
single dose containers for administration into a single teat
canal of an animal. If supplied in multiple dose containers,
aqueous preparations contain an antimicrobial preservative
in adequate concentration except when the preparation itself
has antimicrobial properties. The containers are made as far
as possible from materials that meet the requirements for
Parenteral Preparations intended for use in human beings.
The containers are sealed so as to exclude micro-organisms
and each container is fitted with a smooth, tapered nozzle to
facilitate the introduction of the infusion into the teat canal.
The containers are sterilised and filled aseptically unless the
preparation is subjected to a process ofterminal sterilisation.
Tests
Sterility. Intramammary Infusions comply with the test for
sterility (2.2.11), using Method A or B, as appropriate, using
the contents of 10 containers mixed thoroughly before use in
the test. Use for each medium 0.5 to 1.0 g or 0.5 to 1.0 ml, as
appropriate, ofthe mixed sample.
Storage. Store in sterile, single dose or multiple dose, tamperevident containers.
weight or the number of Units of activity of the active
ingredient(s) or that may be expressed from the container using
normal techniques; (2) whether the preparation is intended
for use in lactating cow/buffalo or in dry or non-lactating
cow/buffalo; (3) for Intramammary Infusions (Non-lactating
or Dry Cow/Buffalo), that the preparation is not intended for
use in lactating animals; (4) in the case ofinfusions in multiple
dose containers, the name of any added antimicrobial
preservative.
2621
IP 2010
GENERAL MONOGRAPHS
Premixes
Premixes are mixtures of one or more active ingredients with
suitable bases intended for mixing with feedstuffs before
administration to the animals. They are used to dilute
medicament(s) with the feed and are usually issued as pellets,
granules or powders. If issued as granules, these are freeflowing and free from aggregates. Suitable precautions are
taken during manufacture for ensuring that the premix is
homogeneous.
Labelling. The label states (1) that the aerosol is intended for
external use only; (2) the instructions for use; (3) any special
precautions in the use of the preparation.
Unless otherwise stated in the individual monograph, the

concentration ofthe premix in medicated feedstuffs is not less
than 0.5 per cent.
Veterinary Diagnostics are antigenic materials of bacterial or

viral origin employed for various tests. These will also include
polyclonal or monoclonal antibodies. The preparations are
examined for their purity at various critical stages of
production. The diagnostic kits may be prepared using
bacterial or viral antigens and antisera.
Tests
Tests

determined on 3 g by drying in an oven at 105" for 2 hours.
Veterinary Diagnostics, reconstituted where necessary,

comply with the following tests unless otherwise stated in
the individual monograph.
Labelling. The label states (1) the strength in tenns of the

amount ofactive ingredient(s) as a percentage; (2) the category
of animal for which the premix is intended to be used; (3) the
directions for the preparation ofthe medicated feed; (4) where
applicable, the minimum interval between the stoppage of
feeding ofthe diluted premix and the slaughter of the animal
for human consumption; (5) any special precautions to be
taken for use ofthe premix; (6) the storage conditions; (7) the
date after which the preparation is not intended to be used.
Identification
Unless otherwise stated in the individual monograph,
Veterinary Diagnostics give specific reaction when injected
into the skin of a healthy white guinea-pig or rabbit that has
not been previously treated with any material that will interfere
with the test but fails to produce this reaction when mixed
with a sufficient quantity ofthe specific antitoxin or antiserum.
Sterility. Unless otherwise stated, Veterinary Diagnostics
comply with the test for sterility (2.2.11), except that in the
case of preparations containing living bacteria there may be
growth of the organism from which the diagnostic was
prepared.
Veterinary Aerosols
Veterinary Sprays
Veterinary Aerosols are solutions, suspensions or emulsions
of one or more active ingredients intended for use by external
application. They may contain auxiliary substances such as
solvents, solubilising agents, emulsifying agents and
suspending agents. They are delivered in the form ofan aerosol
by the actuation of an appropriate valve or by means of a
suitable atomizing device that is either an integral part of the
container or is supplied separately.
Use suitable solid media for streaking the preparation under

examination and incubate at 32" to 37" for 72 hours for detecting
bacteria and at 20" to 25" for 72 hours for detecting fungi: The
media selected will depend upon the nature of the product to
be tested. The contents of each randomly selected sealed
container of the preparation under examination or portions or
dilutions thereof, as appropriate, are used for the test.
They may be presented in special containers under pressure

of a gas and contain propellants or mixtures of propellants.
The medicaments are released from the container in the form
of an aerosol upon actuation of an appropriate valve.
Other tests to determine the nature and identity of

contaminating microorganisms, if any, detected during the
test include examination for mobility of the organisms,
fermentation reactions, thermo-agglutination tests and dye
inhibitor tests (in the case of Brucella cultures).
Veterinary Aerosols supplied in special pressurized containers

comply with the appropriate requirements for Inhalation
Preparations. The following requirements also apply for any
. veteiTrlaryaerosoTthaf is The--subjeCtO:fan indiVIdual
Containers. A suitable atomizing device may form part ofthe
container or is supplied separately.
Unless otherwise stated in the monograph, the preparation

passes the test if no growth of microorganisms, other than
those from which the Yeterinarydiagnostic.wasprepared,.is
o!JseJ:YedinliPY .Qf th.e Jrre<iili d\lripKtl1e .i l1<:\llJa.ti()l1. P~ri()cl
Repeat the tests if growth oforganisms, other than those from
which the veterinary diagnostic was prepared, is observed.
The vaccine passes the test if no growth of microorganisms,
2622
IP 2010
GENERAL MONOGRAPHS
other than those from which the diagnostic was prepared, is

observed in any ofthe media. The preparation fails the test if
growth of a microorganism that was seen after the first test,
other than those from which the veterinary diagnostic was
prepared, is observed. Ifgrowth ofa different microorganism
is observed, the test may be repeated a second time. The
preparation passes the test if no growth of a microorganism,
other than those from which the veterinary diagnostic was
prepared, is observed in any of the media.
The number of containers recommended to be drawn by the
manufacturer for performing the test for sterility depends on
the environmental conditions of manufacture, the volume of
preparation per container and any other special considerations
applicable to the preparation concerned. For preparations
intended for veterinary use, 1 per cent of the containers in a
batch, with a minimum of three and a maximum of ten, is
considered a suitable number assuming that the preparation
has been manufactured under appropriately validated
conditions designed to exclude contamination.
Storage. Store protected from light in a refrigerator (2" to 8")
unless otherwise stated in the individual monograph.
Labelling. The label states (1) the name and quantity of any
antibacterial substance added; (2) for a dried preparation, the
nature and quantity ofthe liquid to be used for reconstitution.
given in the chapter on General Monographs on Dosage Forms

ofActive Pharmaceutical Ingredients.
Veterinary Tablets
Veterinary tablets are usually solid, circular cylinders the end
surfaces of which are flat or biconvex and the edges ofwhich
are bevelled except that those weighing 5 g of more may be
elongated or biconical.
Tests
Disintegration (2.5.1). The test may have to .be suitably
modified in the case oflarge tablets; the discs may have to be
omitted because they would otherwise be dislodged from the
disintegration tubes. It may also be necessary to adjust the
volume of the disintegration medium so that the tablet does
not break the surface of the medium at the top of the upstroke, care being taken to apply the minimum practical volume
ofliquid for this purpose. For certain tablets where the diameter
of the tablet may not permit adequate movement of the
disintegration medium, the apparatus and the method should
be suitably modified.
Veterinary Vaccines
. Veterinary Oral Liquids
Vaccines for Veterinary Use
Veterinary oral liquids intended for administration in large

animals may also be called Drenches.

Veterinary Oral Powders are intended for oral administration,
usually after dilution in drinking water or the feed. They may
be in the form of soluble or wettable powders.
Labelling. The label states (I) for single dose containers, the
name and quantity of active medicament(s) per container; (2)
for multiple dose containers, the name and quantity of active
medicament(s) by weight; (3) the name of any added
antimicrobial preservative(s); (4) the directions for use ofthe
preparation.

Veterinary Parenteral Preparations prepared with oily vehicles
are not meant for intravenous administration but are suitable
for intramuscular or subcutaneous use.
Veterinary Parenteral Preparations comply with the appropriate
requirements for Parenteral Preparations (Injections) that are
Vaccines are a heterogeneous class of medicinal products

containing immunogenic substances capable of inducing
specific, active and protective host immunity against
infectious diseases. They may be prepared from bacteria,
viruses, parasites or other organisms or their toxins. Vaccines
may contain live attenuated or avirulent microorganisms or
these may consist of killed or inactivated microorganisms.
Some vaccines consist of antigenic fractions or substances
produced by the same pathogenic organisms but rendered
harmless whilst retaining their irnmunogenecity. Vaccines may
be prepared from one species or from two or more species of
microorganisms.
Vaccines may be prepared by the method described in
the individual monograph or by any other appropriate method
provided the identity of the antigens is maintained and the
preparations are free from microbial contamination and
extraneous agents. Suitable adjuvants may be added
during the preparation of the vaccines. The addition of.
antibiotics during the manufacturing process is normally
restricted to cell culture fluids and other media, egg inocula
and material harvested from skin or other tissues. A suitable
bactericide may be added to sterile and inactivated vaccines.
The final products are distributed aseptically into sterile
2623
IP 2010
GENERAL MONOGRAPHS
containers that are then sealed to exclude extraneous

microorganisms. Unless otherwise indicated in the monograph,
the final vaccine may be filled into single dose or multiple
dose containers; however, inactivated vaccines in multiple
dose containers must invariably contain a bactericide.
Live viral vaccines. Live viral vaccines are prepared using

avirulent or attenuated strains of the specific viruses that are
capable of stimulating immunogenic' response against
pathogenic strains of the same or of antigenically related
viruses.
Bacterial vaccines. Bacterial vaccines are either suspensions

of live or killed bacteria or sterile antigenic extracts or
derivatives of bacteria pathogenic to animals. They may be
simple vaccines prepared from one species or may be combined
or polyvalent vaccines prepared by blending two or more
simple vaccines from different species or strains. Bacterial
vaccines may be prepared from cultures grown on suitable
solid or liquid media. The identity, antigenic potency and purity
of each bacterial culture must be carefully controlled.
Inactivated viral vaccines. Inactivated viral vaccines contain

viruses that have been inactivated by suitable chemical or
physical means in such a way that the preparations retain
adequate immunogenicity or they are suspensions of
immunogenic components of such viruses.
Bacterial vaccines are suspensions of varying degrees of

opacity in colourless or slightly coloured liquids or they may
be freeze-dried so that the water content is not more than
3.0 per cent w/w unless otherwise stated in the individual
monograph. They may be standardised in terms ofinternational
opacity units or, where appropriate, by numbers of live or
killed bacteria determined by viable count or by direct cell
count.
Live bacterial vaccines. Live bacterial vaccines are prepared
from avirulent or attenuated strains of the specific bacteria
that are capable of stimulating immune response against
pathogenic strains of the same or of antigenically related
species of bacteria.
Inactivated bacterial vaccines. Inactivated bacterial vaccines
are either prepared from bacteria or their immunogenic
components that have been inactivated in a suitable way that
they retain adequate immunogenicity.
Bacterial toxoids. Bacterial toxoids are prepared from toxins
by diminishing their toxicity to a very low level or by
completely eliminating it by physical or chemical means whilst
retaining adequate immunising potency. The toxins are
obtained from selected strains of specific microorganisms,
grown in suitable media devoid of agents capable ofinducing
undesirable immunological reactions in animals. Bacterial
toxoids may be liquid or may be prepared by adsorbing on
suitable agents such as aluminium phosphate, aluminium
hydroxide or any other suitable adsorbents. Bacterial toxoids
are clear or slightly opalescent liquids, colourless or slightly
yellow. Adsorbed toxoids may be white or greyish-white
suspensions or paleyellow liquids with sediment at the bottom
ofthe container. Freeze-dried preparations are greyish-white
or yellowish-white powders or pellets.
YiraLvaccines._Viral_vaccines.are.8uspensions-.oLviruses_oL..
prep.araJions.Qbtained Jmm Jissues.orhlood of animals
artificially infected with viruses pathogenic to animals or from
cultures in fertile eggs, or from cell or tissue cultures, they
may be live, inactivated / killed and may be freeze-dried.
Combined vaccines. Combined vaccines consist of two or

more antigens, combined by the manufacturer at the final
forinulation stage or mixed immediately before administration.
Such vaccines are intended to protect against either more
than one disease, or against one disease caused by different
strains or serotypes of the same organism.
Stability. Stability is the ability of a vaccine to retain its
chemical, physical, microbiological and biological properties
within specified limits throughout its shelf1ife.
Adjuvants. Substances that are intended to enhance relevant
immune response and subsequent clinical efficacy of the
vaccine.
Tests
Vaccines comply with the tests prescribed in the individual
monographs including, where applicable, the following:
Aluminium (2.3 .9). Where an aluminium adsorbent has been
used in the vaccine, not more than 1.25 mg ofaluminium (AI)
per single dose, unless otherwise stated.
Calcium (2.3.11). Where a calcium adsorbent has been used
in the vaccine, not more than 1.3 mg ofcalcium (Ca) per single
dose, unless otherwise stated.
Formaldehyde (2.3.20). Where formaldehyde has been used
in the preparation of the vaccine, not more than 0.2 gil offree
formaldehyde is present in the final product, unless otherwise
stated.
Phenol (2.3.36). Where phenol has been used in the
preparation of the vaccine, not more than 2.5 gil is present in
the final product, unless otherwise stated.
Water (2.3.43). For freeze-dried vaccines, not more than
3.0 per cent, unless otherwise stated.
Thiomersal (2.2.12) (2.3.48). Where thiomersal has been used
in the preparation of the vaccine, not more than 0.02 per cent
'!!.~".~_
Extrlli"le()US plltll()gens.UnlessQll1l.'lrWiSl.'l state.ci in.tlle.

individual monograph, live viral vaccines other than those
intended for poultry comply with the following test. The
mixture obtained after neutralisation ofthe vaccine with specific
2624
IP 2010
GENERAL MONOGRAPHS
antiserum does not cause cytopathic effects in cell cultures

known to be sensitive to agents pathogenic for the species in
which the vaccine is intended to be used.
Sterility (2.2.11). Unless otherwise stated in the individual
monograph, use method A. Incubate the media for not less
than 14 days at 30" to 37" in the test for detecting bacteria and
at 20" to 25" in the test for detecting fungi. However, for live
bacterial vaccines growth of the organism from which the
vaccine was prepared is permitted.
The number of containers to be drawn for the test should be I

per cent ofthe containers in a batch, with a minimum on and
a maximum of 10, assuming that the preparation has been
manufactured under appropriately validated conditions
designed to exclude contamination.
Safety test. Unless otherwise stated in the individual
monograph, vaccines other than live viral vaccines intended
for poultry comply with the following test.
Inject at least 2 healthy, susceptible animals of one of the

species in which the vaccine is intended to be used by the
route recommended by the manufacturer for field use. The
quantity to be injected in each animal is twice the appropriate
vaccinating dose. Observe the animals for not less than 7
days. No animal exhibits an abnormal reaction.
Abnormal toxicity. Where stated in the individual monograph
vaccines comply with the following test.
Inject 0.5 ml subcutaneously into each offive mice and 2 ml

intraperitoneally into each of two guinea pigs. If the vaccine
being examined contains an adjuvant, inject 2 ml ofthe vaccine
subcutaneously into each guinea pig. Observe the animals
for 7 days. None of the animals shows significant local or
systemic reaction. If one animal dies or shows signs of ill
health during the observation period repeat the test. None
of the animals of the second group dies or shows signs
of ill health. This test may be omitted if a safety test is
carried out on animals of the species for which the vaccine is
intended.
Potency. Determine the potency of the vaccine using the
method described in the individual monograph. The vaccine
complies with the level of immune response specified in the
monograph. A combined vaccine complies with the level
specified in the respective monographs for each individual
component. Ifthe immunogenicity test (potency test) has been
performed with satisfactory results on representative batch
oflive vaccine from the same seed lot, it may be omitted as a
routine control test during production of other batches of the
vaccine prepared from the same seed lot.
Storage. Store protected from light in a refrigerator (2" to 8"),
unless otherwise directed. Do not freeze. Store freeze-dried
vaccines at a temperature not exceeding 20".
Labelling. The label states (1) the potency ofthe preparation;
(2) the route of administration and dose; (3) the date up to
which the product is expected to remain within specifications;
(4) the storage conditions.
2625
NON BIOLOGICAL VETERINARY MONOGRAPHS

Acepromazine Maleate
Acepromazine Injection
Acepromazine Tablets
Amitraz
LiquidAmitraz Dip
Amitraz Dip Concentrate Powder
Ampicillin and Cloxacillin Intramammary Infusion (Lactating Cow/Buffalo)
Ampicillin and CloxacillinBenzathine Intramammary Infusion (Dry Cow/Buffalo)
Ampicillin Veterinary Oral Powder
AmproliumHydrochloride
Amprolium Hydrochloride and Ethopabate Premix
Amprolium, Ethopabate and Sulphaquinoxaline Premix
Calcium Borogluconate Injection
Calcium Magnesium Borogluconate Injection
Chloral Hydrate
Chloramphenicol Injection
Chlortetracyline Hydrochloride
Chlortetracycline Veterinary Oral Powder
CloxacillinBenzathine
Cloxacillin Benzathine Intramammary Infusion (Dry Cowl Buffalo)
Cloxacillin Sodium IntramammaryInfusion (Lactating Cow/Buffalo)
Dichlofenthion
Dichlorophen
Dichlorophen VeterinaryAerosol
Dichlorophen Tablets
Diethylcarbamazine Injection
Dihydrostreptomycin Sulphate
Dihydrostreptomycin Injection
Dimetridazole
Dimetridazole Premix
2627
2631
2632
2632
2633
2634
2635
2636
2637
2638
2639
2640
2641
2642
2642
2643
2644
2645
2646
2647
2648
2649
2650
2650
2651
2652
2653
2654
2655
2657
2658
2658
2658
Dimetridazole Veterinary Oral Powder

Dinitolrnide
Ethopabate
2659
2660
2660
2661
2662
2663
2663
2664
2665
Furazolidone Veterinary Oral Suspension

Furazolidone Premix
Haloxon
Ivennectin
Ivennectin Injection
Kaolin Veterinary Oral Suspension
Levamisole Injection
Levamisole Hydrochloride Veterinary Oral Solution
2665
2666
2667
2667
2668
2669
2669
2670
2670
2671
2672
2672
2673
2674
2674
2675
2676
2676
2677
Lincomycin Premix
. LithiumAntimonyThiomalate
LithiumAntimonyThiomalate Injection
Magnesium Hypophosphite
MeclofenamicAcid
Mepyramine Injection
Monosulfiram
Monosulfuam Soap
Monosulfuam Solution
Nandrolone Laurate
Nandrolone Laurate Injection
Niclosamide Veterinary Oral Powder
Nitroxynil
Nitroxynil Injection
Oxfendazole
Oxfendazole Veterinary Oral Suspension
Oxyclozanide
Oxyclozanide Veterinary Oral Suspension
Oxyclozanide Premix
.... ~
~~Oxytetfacyclin(rVetefinaryOraJPowder~-
2078--~-~
2679
2680
Pentobarbitone Injection
Progesterone
2628
Progesterone Injection
Promazine Hydrochloride
Promazine Injection
Rafoxanide
Rafoxanide Veterinary Oral Suspension
Ronidazole
Ronidazole Veterinary Oral Powder
Serum Gonadotrophin for Veterinary Use
Serum Gonadotrophin Injection for Veterinary Use
Spectinomycin Hydrochloride
Spectinomycin Injection
Spiramycin
Sulphadiazine and Trimethoprim Injection
Sulphadiazine and Trimethoprim Veterinary Oral Powder
.Sulphadiazine and Trimethoprim Veterinary Oral Suspension
Sulphaquinoxaline
Sulphaquinoxaline Sodium Solution
Sulphathiazole SodilUTI
Thiabendazole Veterinary Oral Suspension
Thiabendazole and Rafoxanide Veterinary Oral Suspension
Thiabendazole Premix
Tylosin
Tylosin Injection
Tylosin Tablets
Tylosin Tartrate
Tylosin Tartrate and Sulphathiazole Sodium Veterinary Oral Powder
2629
2680
2681
2682
2683
2683
2684
2684
2685
2686
2687
2688
2689
2690
2691
2692
2693
2694
2695
2695
2696
2696
2697
2698
2699
2700
2702
IP 2010
ACEPROMAZINE MALEATE
Acepromazine Maleate
2-phenoxyethanol. Shake and use the supernatant liquid.

acepromazine maleate RS in dichloromethane.
HyCOOH
'~
H
COOH
Mol. Wt. 442.5

Acepromazine Maleate is 2-acetyl-1 0-(3dimethylaminopropyl)phenothiazine hydrogen maleate.
Acepromazine Maleate contains not less than 98.5 per cent
and not more than 101.0 per cent of CI9HzzNzOS,C4H404'
Category. Sedative; preanaesthetic.
Dose. Farm animals. By intramuscular or slow intravenous
injection, the equivalent of 50 to 100 mg ofacepromazine per
kg ofbody weight. Dogs and cats. Orally, the equivalent of 1
to 3 mg of acepromazine per kg of body weight; by
intramuscular or intravenous injection, the equivalent of 125
to 250 mg of acepromazine per kg ofbody weight.
(Each 135 mg of acepromazine maleate is approximately
equivalent to 100 mg ofacepromazine).
Identification
Tests BandD may be omitted iftests A, C, E andF are carried
out. Tests A and E may be omitted if tests B, C, D and Fare
carried out.
NOTE -
Carry out the tests in subdued light.
A. Dissolve 20 mg in 2 ml of water, add 3 mI of 2 M sodium

hydroxide, extract with 5 ml of cyclohexane and remove the
solvent under reduced pressure. The residue complies with
the following test.
Compare the spectrum with that obtained with acepromazine
RS or with the reference spectrum of acepromazine.
Impregnate the dry plate by placing it in a tarue containing a

shallow layer ofa mixture of85 volumes ofacetone, 10 volumes
of 2-phenoxyethanol and 5 volumes of polyethyleneglycol
300 so that the plate dips about 5 mm below the surface ofthe
liquid and allow the impregnating solvent to ascend almost to
the top. Use the plate immediately after removing it from the
tank. Apply to the plate 1 III of each solution. After
obtained with the test solution corresponds to the spot in the
chromatogram obtained with the reference solution. A
secondary spot due to maleic acid is also observed in both
chromatograms. Spray the plate with ethanolic sulphuric acid
(10 per cent v/v). The spot in the chromatogram obtained
D. Dissolve 5 mg in 2 ml of sulphuric acid; a yellow colour is
produced which changes to deep orange on warming for
2 minutes.
E. Dissolve 0.2 g in a mixture of 3 ml of water and 2 ml of
5 M sodium hydroxide and shake with three quantities, each
oB ml, of ether. Discard the ether extracts. Add 2 ml of bromine
solution to the aqueous solution, warm in a water-bath for
10 minutes, heat to boiling, cool and add 0.25 ml to a solution
of 10 mg of resorcinol in 3 ml ofsulphuric acid; a bluish-black
colour is produced on hea~ing for 15 minutes in a water-bath.
E Melting range (2.4.21). 136 to 139.
Tests
Solvent mixture. 95 volumes
of methanol and
Mobile phase. A mixture of 75 volumes of hexane, 17 volumes

of 2-butanol and 8 volumes of diethylamine.
B. When examined in the range 230 nm to 360 om (2.4.7), a

0.002 per centw/v solution in 0.1 Mhydrochloric acid, exhibits
maxima at about 244 om and 280 om; absorbance at about 244
om, about 1.1 and at about 280 nm, about 0.82.
Test solution. Dissolve 0.2 g of the substance under examination in 10 ml ofthe solvent mixture.

the plate with kieselguhr G

substance under examination in the solvent mixture.

(40 to 60), 2 volumes of diethylamine and 6 to 8 volumes of

2631
ACEPROMAZINE INJECTION
IP 2010

Sulphated ash (2.3.18) Not more than 0.2 per cent.
acetic anhydride. Titrate with 0.1 M perchloric acid, using
clystal violet solution as indicator. Carry out a blank titration.

CI9HzzNzOS,CJ1404.
Acepromazine Maleate Injection
Acepromazine Injection is a sterile solution ofAcepromazine
Acepromazine Injection contains not less than 92.5 per cent
acepromazine, C19HzzNzOS.
Impregnate the dry plate by placing it in a tank containing a

shallow layer ofa mixture of85 volumes of acetone, 10 volumes
tank. Apply to the plate 1 !J.I of each solution. After
D. To a volume containing 25 mg ofacepromazine add 2 ml of
5 M sodium hydroxide and shake with three quantities, each
oB ml, of ether. Discard the ether extracts. Add 2 ml of bromine
solution to the aqueous solution, warm in a water-bath for
10 minutes, heat to boiling, cool and add 0.25 ml to a solution
of 10 mg of resorcinol in 3 ml ofsulphuric acid; a bluish-black
colour is produced on heating for 15 minutes in a water-bath.
Tests
Usual strengths. The equivalent of200 mg ofacepromazine in

20 ml and of20 mg ofacepromazine in 10 ml.
pH (2.4.24). 4.5 to 5.5.
Identification

NOTE -
eany out the tests in subdued light.
A. To a volume containing 20 mg ofacepromazine, add 2 ml of

water and 3 ml of 2 M sodium hydroxide, extract with two
quantities, each of5 ml, of cyclohexane and remove the solvent
under reduced pressure. The residue complies with the
following test.
B. To 5 mg of the residue obtained in test A, add 2 ml of
sulphuric acid; a yellow colour is produced which changes
to deep orange on warming for 2 minutes.

the plate with kieselguhr G.
2-phenoxyethanol. Shake and use the supernatant liquid.
I?J'l_.~Q1Z!tirzYl, ..:E:.'(trlCt...l .':'()!UII!~_~ol1Jlil1il!g2~ll:lg~i
acepromazine with two quantities, each of 5 ml, of

dichToromethal1e
and use the combIned extracts.
..

Assay. To an accurately measured volume containing 40 mg

ofacepromazine add 5 ml of 1 M sodium hydroxide and extract
with three or more quantities, each of50 ml, of dichloromethane
until the dichloromethane extract is colourless. Wash the
extracts with the same 10 ml of water and filter through a
plug of absorbent cotton previously moistened with
dichloromethane. Evaporate the combined extracts to dryness,
dissolve the residue in 15 ml of acetic anhydride. Titrate with
1 ml of O. 02 M perchloric acid is equivalent to 0.006529 g of
CI9HzzNzOS.
equivalent amount of acepromazine.
.Acepromazme Maleate Tal5lets
Acepromazine Tablets contain not less than 92.5 per cent and
acepromazine, C I9H22N zOS.
2632
IP 2010
AMITRAZ
Tests
Identification

NOTE -
Cany out the tests in subdued light.

acepromazine add 2 ml of water and 3 ml of 2 M sodium
hydroxide. Extract with two quantities, each of 5 ml, of
cyclohexane and remove the solvent under reduced pressure.
B. To 5 mg ofthe residue obtained in test A add 2 ml ofsulphuric
acid; a yellow colour is produced which changes to deep
orange on wanning for 2 minutes.

the plate with laeselguhr G

2-phenm.yethanol. Shake and use the supematent liquid.
Test solution. Extract a quantity ofpowdered tablets containing
20 mg of acepromazine with two quantities, each of 5 ml, of
dichloromethane and use the combined extracts.
Impregnate the dry plate by placing it in a tanIe containing a
shallow layer ofa mixture of85 volumes ofacetone, 10 volumes
tank. Apply to the plate 1 III of each solution. After
D. Dissolve a quantity of the powdered tablets containing
25 mg ofacepromazine as completely as possible in a mixture
.on ml of water and 2 ml of5 M sodium hydroxide and shake
with three quantities, each on ml, of ether. Discard the ether
extracts. Add 2 ml of bromine solution to the aqueous solution,
wann in a water-bath for 10 minutes, heat to boiling, cool and
add 0.25 ml of a solution of 10 mg of resorcinol in 3 ml of
sulphuric acid; a bluish-black colour is produced on heating
for 15 minutes in a water~bath.
Solvent mixture. 95 volumes

of methanol and
Mobile phase. A mixture of 75 volumes of hexane, 17 volumes

of 2-butanol and 8 volumes of diethylamine.
containing 50 mg of acepromazine with 10 ml of
dichloromethane, filter, evaporate to dryness and dissolve
the residue in 5 ml of methanol containing 0.5 per cent v/v of
strong ammonia solution.
with methanol containing 0.5 per cent v/v of strong ammonia
solution.

quantity of the powder containing 60 mg of acepromazine,
. add 5 ml of water and extract with three or more quantities,
each of 50 ml, of dichloromethane until the dichloromethane
extract is colourless. Wash the extracts with the same 10 ml of
water and filter through a plug ofabsorbent corton previously
moistened with dichloromethane. Evaporate the combined
extracts to dryness, dissolve the residue in 15 ml of acetic
anhydride. Titrate with 0.02 M perchloric acid, using Clystal
C19H22N20S.
equivalent amount of acepromazine.
Amitraz
Mol. Wt. 293.41

Amitraz is N,N-di-(2,4-xylyliminomethyl)methylamine.
Amitraz contains not less than 95.0 per cent and not more
than 101.5 per cent of C19H23N3, calculated on the anhydrous
basis.
2633
AMITRAZ
IF 2010
Category. Acaricide.
Dose. The usual recommended concentration in the dip-bath
may vary between 0.005% and 0.1 per cent w/v, depending
upon the host and parasite. Cows, buffaloes, sheep and goats.
0.025 per cent w/v.
Description. A white to buffpowder.
Any secondary spot corresponding to 2,4-dimethylaniline in

intense than the corresponding spot in the chromatogram
Water (2.3.43). Not more than 0.1 per cent, determined on 5 g
and using anhydrous pyridine in place of anhydrous
methanol.
Identification

Compare the spectrum with that obtained with amitraz RS or
with the reference spectrum of amitraz.


Tests

30 volumes of ethyl acetate and 20 volumes of triethylamine.
Reference solution (a). A 0.20 per cent w/v solution of amitraz
RS in toluene.
Internal Standard Solution. A 1.0 per cent v/v solution of

squalane in methyl acetate.
examination in 100 ml of methyl acetate.
examination in 100 ml ofthe internal standard solution.
Reference solution. A 0.8 per cent w/v solution of amitraz RS
in the internal standard solution.
with 3 per cent w/w methyl silicone gum or fluid
(such as OV-l orOV-I0l),
temperature:
column. 250,
flow rate. 30 ml per minute ofthe carrier gas.
Calculate the content of C19H23N3.
Storage. Store in containers which may contain
paraformaldehyde packed in separate sachets as stabiliser.
Reference solution (b). A 0.030 per cent w/v of

2,4-dimethylaniline in toluene.
Impregnate the plate to a depth of about 3.5 cm with a solution Liquid Amitraz Dip Concentrate
prepared by dissolving 35 g of acetamide in 100 ml of methanol, Liquid Amitraz Dip Concentrate contains Amitraz in a suitable
adding 100 ml of triethylamine and diluting to 250 ml with emulsifiable vehicle. It may contain a suitable stabilising agent.
methanol, before standing it in a stream of cold air for about
30 seconds. Immediately apply to the plate, at a level 1 cm Liquid Amitraz Dip Concentrate contains not less than
below the top of the impregnated zone, 2 III of each solution. 90.0 per cent and not more than 110.0 per cent of the stated
After development, dry the plate in air and examine in amount ofamitraz, C19H23N3.
ultraviolet light at 254 nm. Any secondary spot in the Usual strength. 12.5 per cent w/v.
intense than the spot in the chromatogram obtained with Identification
reference solution (a). Expose the plate to the vapours of
hydrochloric acid until the plate smells strongly of acid. A. In the test for Related substances, the principal spot in the
Expose to the vapoursofnitrogendioxide (prepared by the chromatogram obtained with test solution (b) corresponds to
that in the chromatogram-obtained with reference solution (a.).
action of nitric acid on granulatedzinc) for lO minutes, remove -.-.-_.__ _.- _ __ .
. .. - -_ .. __ _-_ _, _
.. _-, '.
the excess ofnitrogen dioxide with air and spray with a 0.5 per B. In the Assay, the principal peak in the chromatogram
cent w/v solution of N-(l-naphthyl)ethylenediamine obtained with the test solution (b) corresponds to the peak in
dihydrochloride in a 50 per cent v/v solution of methano!. the chromatogram obtained with reference solution (b).
....
2634
......
~.
IP 2010
AMITRAZ DIP CONCENTRATE POWDER
Tests


30 volumes ofethyl acetate and 20 volumes of triethylamine.
Test solution (a). Dilute the dip concentrate with toluene to
obtain 5.0 per cent w/v ofAmitraz.
Test solution (b). Dilute the dip concentrate with toluene to
obtain 0.2 per cent w/v ofArnitraz.
Reference solution (a). A 0.20 per cent w/v solution ofamitraz
RS in toluene.
with 3 per cent w/w methyl silicone gum or fluid (such
asOV-lorOV-lOl),
- temperature:
column. 250,
Calculate the content ofC19H23N3.

2,4-dimethylaniline in toluene.
Impregnate the plate to a depth of about 3.5 cm in a solution
prepared by dissolving 35 g ofacetamide in 100 ml ofmethanol,
adding 100 ml of triethylamine and diluting to 250 ml with
30 seconds. Immediately apply to the plate, at a level 1 cm
below the top of the impregnated zone, 2 JlI of each solution.
After development, dry the plate in air and examine in
chromatogram obtained with the test solution (a) is not more
hydrochloric acid until the plate smells strongly of acid.
Expose to the vapours of nitrogen dioxide (prepared by the
action ofnitric acid on granulatedzinc) for 10 minutes, remove
the excess ofnitrogen dioxide with air and spray with a 0.5 per
cent w/v solution of N-(l-naphthyl)ethylenediamine
dihydrochlOJ:ide in a 50 per cent v/v solution of methanol.
the chromatogram obtained with test solution (a) IS not more
Water (2.3.43). Not more than 0.15 per cent w/v, determined in
5 ml of the dip concentrate and using anhydrous pyridine in
place of anhydrous methanol.
Other tests. Complies with the tests stated under Dip
Concentrates.

Arnitraz Dip Concentrate Powder consists of Arnitraz mixed
with suitable wetting, dispersing and suspending agents. It
may contain a suitable stabilising agent.
Amitraz Dip Concentrate Powder contains not less than
amount ofamitraz, C19H23N3.
Usual strengths. 25 per cent w/w and 50 per cent w/w.
Identification
A. Shake a quantity ofthe powder containing 0.1 g ofArnitraz
with 10 ml of acetone for 5 minutes, filter and evaporate the
test.
Compare the spectrum with that obtained with amitraz RS or
with the reference spectrum ofamitraz.
Tests
Test solution (a). Dissolve an accurately measured volume of

the dip concentrate containing 80 mg of Amitraz in 10 ml of
methyl acetate.

30 volumes of ethyl acetate and 20 volumes of triethylamine.
Test solution (b). Dissolve an accurately measured volume of

the dip concentrate containing 80 mg ofArnitraz in 10 ml ofthe
internal standard solution.
Test solution (a). The supernatant liquid obtained by shaking

a quantity ofthe powder containing 0.5 g ofArnitraz with 10 ml
of toluene for 5 minutes and centrifuging the suspension.
2635
AMITRAZ DIP CONCENTRATE POWDER
IP 2010
Test solution (b). The supernatant liquid obtained by shaking

a quantity of the powder containing 20 mg of Amitraz with
10 rn1 of toluene for 5 minutes and centrifuging the suspension.

Calculate the content OfC19H23N3.
Reference solution (a). A 0.20 per cent w/v solution of amitraz

RS in toluene.
Reference solution (b). A 0.030 per cent w/v of 2,4-dimethylaniline in toluene.
Impregnate the plate to a depth of about 3.5 cm in a solution
prepared by dissolving 35 g ofacetamide in 100 ml ofmethanol,
adding 100 ml of triethylamine and diluting to 250 ml with
30 seconds. Immediately apply to the plate, at a level 1 cm
below the top of the impregnated zone, 2 ).tl of each solution.
After development, dry the plate in air and examine in
chromatogram obtained with the test solution (a) is not more
hydrochloric acid until the plate smells strongly of acid.
Expose to the vapours of nitrogen dioxide (prepared by the
action of nitric acid on granulatedzinc) for 10 minutes, remove
the excess ofnitrogen dioxide with air and spray with a 0.5 per
cent w/v solution of N-(l-naphthyl)ethylenediamine
dihydrochloride in a 50 per cent v/v solution of methanol.
Other tests. Complies with the tests stated under Dip
Concentrates.

Test solution (a). Shake a quantity of the powder containing
80 mg ofAmitraz with 10 ml of methyl acetate, centrifuge and
Test solution (b). Shake a quantity of the powder containing
80 mg ofAmitraz with 10 ml ofthe internal standard solution,
centrifuge and use the supernatant liquid.
- a glass column 1.5 m x 4 mm, packed with acid-washed,
with 3 per cent w/w methyLsilicone gum or fluid
asOV-l orOV~I01),
temperature:
column. 250,
Ampicillin and Cloxacillin

Intramammary Infusion (Lactating
Cow/Buffalo)
Ampicillin Sodium and Cloxacillin Sodium Intramamrnary
Infusion (LCIB)
Ampicillin and Cloxacillin Intramammary Infusion (Lactating
CowlBuffalo) is a sterile suspension ofAmpicillin Sodium and
Cloxacillin Sodium in a suitable vehicle containing suitable
suspending agents.
Ampicillin and Cloxacillin Intramammary Infusion (Lactating
Cow/Buffalo) contains not less than 90.0 per cent and not
more than 110.0 per cent of the stated amount of each of
ampicillin, C16H19N304S, and cloxacillin, CI9HISClN30SS.
Usual strength. The equivalent of 75 mg ofampicillin and 200
mg ofcloxacillin.
Identification

6 volumes of glacial acetic acid, 1 volume of I-butanol and
2 volumes of solution A (see below).
Test solution. Extract a quantity of the infusion containing
50 mg of ampicillin with three successive quantities, each of
15 ml, of lightpetroleum ( 120 to 160). Discard the extracts,
wash the residue with 10 ml of ether and dry in a current ofair.
Dissolve the residue in 50 ml of phosphate buffer pH 7.0,
shake well, filter and use the filtrate.
Reference solution. A 0.12 per cent w/v solution of ampicillin
trihydrate RS in phosphate buffer pH 7.0.
Impregnate the plate by spraying it with a 0.1 per cent w/v
solution of disodium edetate in a 5 per cent w/v solution of
sodium dihydrogen phosphate (solution A), allow the plate
to dry in air and heat it at 105 for 1 hour. Apply to the plate
1 ).tl of each solution. After development, dry the plate in air
and heat at 150 for 10 to 15 minutes and spray with a mixture
of I00 volumes ofstarch mucilage, 6 volumes ofglacial acetic
acidand2volumes .0faJpercentw/v.solutionofiodineJna ._
4 percent w/v solution of potassium iodide. Theprincipa,l .
corresponds to the spot in the chromatogram obtained with
2636
IP 2010
AMPICILLIN AND CLOXACILLIN BENZATHINE INTRAMAMMARY INFUSION (DRY COWIBUFFALO)

the plate with silanised silica gel GF254.
Mobile phase. A mixture of 70 volumes of O. 05 M potassium

hydrogen phthalate, 30 volumes of acetone and 1 volume of
formic acid that has been adjusted first to pH 6.0 with 5 M
sodium hydroxide and then to pH 9.0 with 0.1 M sodium
hydroxide.
Test solution. Extract a quantity of the infusion containing
130 mg ofcloxacillin with three successive quantities, each of
15 ml, of lightpetroleum ( 120 to 160). Discard the extracts,
wash the residue with 10 ml of ether and dry in a current ofair.
Dissolve the residue in 50 ml of phosphate buffer pH 7.0,
shake well, filter and use the filtrate.
Reference solution. A 0.28 per cent w/v solution of cloxacillin

sodium RS in phosphate buffer pH 7.0.
dry the plate in air and heat at 150 for 10 to 15 minutes and
spray with a mixture of 100 volumes of starch mucilage,
6 volumes of glacial acetic acid and 2 volumes of a 1 per cent
w/v solution of iodine in a 4 per cent w/v solution of
potassium iodide. The principal spot in the chromatogram
C. Extract a quantity containing 50 mg ofampicillin with three
successive quantities, each of 15 ml, of light petroleum
( 120 to 160). Discard the extracts, wash the residue with
10 ml of ether and dry the residue at 55. The residue produces
an intense, persistent yellowish orange colour when
introduced into a non-luminous flame on a platinum wire
moistened with hydrochloric acid.
Tests
1.5 g using a mixture of 70 volumes of dichloromethane and
30 volumes of anhydrous methanol as the solvent.
Other tests. Complies with the tests stated under
Intramammary Infusions.
Assay. Weigh and mix the contents of 10 containers. Weigh
accurately a quantity ofthe mixed contents containing 50 mg
ofampicillin and extract with three successive quantities, each
of 15 ml, of light petroleum ( 120 to 160) previously
saturated with ampicillin sodium and cloxacillin sodium.
Discard the extracts, wash the residue with ether previously
saturated with ampicillin sodium and cloxacillin sodium, dry in
a current of air, dissolve in water and dilute to 100.0 ml with
water. Centrifuge and use the clear supernatant liquid (solution
B).
For ampicillin - Dilute 2.0 ml of solution B to 50.0 ml with
buffered cupric sulphate solution pH 5.2, transfer 10.0 ml of
the resulting solution to a stoppered test-tube and heat in a
water-bath at 75 for 30 minutes. Cool to room temperature

rapidly, dilute to 20.0 ml with buffered cupric sulphate solution
pH 5.2 and measure the absorbance of the resulting solution
at the maximum at about 320 urn (2.4.7), using as the blank a
solution prepared by diluting 2.0 ml of solution B to 100.0 ml
with buffered cupric sulphate solution pH 5.2.
Calculate the content ofCI6HI9N304S in a. container ofaverage
content from the absorbance obtained by carrying out the
procedure simultaneously using 2.0 ml of a solution prepared
by dissolving 60 mg of ampicillin trihydrate RS in 100.0 ml of
water, diluting to 50.0 ml with bzifferedcupric sulphate solution
pH 5.2 and beginning at the words "transfer 10.0 m!... .." .
For cloxacillin- Dilute 2.0 ml ofsolution B to 100.0 ml with

1 M hydrochloric acid. Measure the absorbance of the
resulting solution at 20 after exactly 12 minutes at the maximum
at about 350 nm (2.4.7), using 1 M hydrochloric acid as the
blank. Calculate the content ofCI9HIsCIN30sS in a container
of average content from the absorbance obtained by carrying
out the procedure simultaneously uSIng 2.0 ml of a solution
prepared by dissolving 0.14 g of cloxacillin sodium RS in
100.0 ml of water.
Labelling. The label states the quantity ofAmpicillin Sodium
in terms ofthe equivalent amount ofampicillin and the quantity
of Cloxacillin Sodium in terms of the equivalent amount of
cloxacillin.
Ampicillin and Cloxacillin Benzathine

Intramammary Infusion (Dry Cowl
Buffalo)
Ampicillin and Cloxacillin Benzathine Intramammary Infusion
(Dry Cow/Buffalo) is a sterile suspension of Ampicillin
Trihydrate and Cloxacillin Benzathine in a suitable vehicle
containing suitable suspending agents.
Ampicillin and Cloxacillin Benzathine Intramammary Infusion
(Dry Cow/Buffalo) contains not less than 90.0 per cent and
not more than 110.0 per cent ofthe stated amounts ofampicillin,
C16H19N304S, and cloxacillin, CI9HISClN30SS.
Usual strength. The equivalent of250 mg of ampicillin and
500 mg ofcloxacillin.
Identification
A. Extract a quantity containing 250 mg of ampicillin with
three quantities, each of 15 ml, of light petroleum ( 120 to
160). Discard the extracts, wash the residue with 10 ml of
ether and dry in a current of air. Shake with 10 ml of
dichloromethane and filter. Keep both the residue and the
filtrate.
2637
AMPICILLIN AND CLOXACILLIN BENZATHINE INTRAMAMMARY INFUSION (DRY COWIBUFFALO)
Wash the residue with two quantities, each of 5 ml, of

dichloromethane and dry in a vaccum desiccator.
On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained
with ampicillin trihydrate RS or with the reference spectrum
ofampicillin trihydrate.
B. Wash the filtrate with two quantities, each of5 ml, of water,
dry the dichloromethane layer with anhydrous sodium
sulphate, filter and dilute the filtrate to 20 ml with
dichloromethane.
On the filtrate determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained
with cloxacillin benzathine RS or with the reference spectrum
ofcloxacillin benzathine.
Tests
1.5 g using a mixture of 70 volumes of dichloromethane and
Intramammary Infusions.
accurately a quantity of the mixed contents containing 60 mg
ofampicillin and extract with three quantities, each on 5 ml, of
light petroleum ( 120 to 160) previously saturated with
ampicillin trihydrate and cloxacillin benzathine. Discard the
extracts, wash the residue with ether previously saturated
with ampicillin trihydrate and cloxacillin benzathine, dry in
a current of air, dissolve in 50 rill of methanol and dilute to
100.0 ml with water. Centrifuge and use the clear supernatant
liquid (solution A).
For ampicillin - Dilute 2.0 ml of solution A to 50.0 ml with

bufferedcupric sulphate solution pH 5.2, transfer 10.0 ml to a
stoppered test-tube and heat in a water-bath at 75 for
30 minutes. Cool to robm temperature rapidly, dilute to 20.0 ml
with buffered cupric sulphate solution pH 5.2 and measure
about 320 urn (2.4.7), using as the blank the unheated buffered
solution of the infusion.
Calculate the content ofCI6HI9N304S in a container ofaverage
content from the absorbance obtained by carrying out the
procedure simultaneously using 2.0 ml of a solution prepared
by dissolving 70 mg of ampicillin trihydrate RS in 100.0 ml of
a 50 per cent v/v solution of methanol, diluting to 50.0 ml with
buffered cupric sulphate solution pH 5.2, and beginning at
the-woI'ds "transferl0~(EllL .."-:----- ---- .--------------
For cloxacillin - Dilute 2.0 ml ofsolution A to 100.0 ml with

1 M hydrochloric acid and measure the absorbance of the
resulting solution at 20 after exactly 12 minutes at the maximum
IP 2010
at about 350 urn, (2.4.7), using 1 M hydrochloric acid as the

blank. Calculate the content OfCI9HlSClN30sS in a container
of average content from the absorbance obtained by carrying
out the procedure simultaneously using 2.0 ml of a solution
prepared by dissolving 0.165 g of cloxacillin benzathine RS
in 100.0 ml of a 50 per cent v/v solution of methanol.
Labelling. The label states the strength of Ampicillin
Trihydrate in terms ofthe equivalent amount ofampicillin and
that ofCloxacillin Benzathine in terms ofthe equivalent amount
ofcloxacillin.

Ampicillin TrihydrateVeterinary Oral Powder
Ampicillin Veterinary Oral Powder is a mixture ofAmpicillin
Trihydrate and Lactose or other suitable diluent.
Ampicillin Veterinary Oral Powder contains not less than
amount ofampicillin, C16H19N304S.
Usual strength. The equivalent of 10 per cent w/w of
ampicillin.
Description. A fine granular powder.
Identification
NOTE

6 volumes of glacial acetic acid, 2 volumes of a 0.1 per cent
w/v solution of disodium edetate in mixed phosphate buffer
pH 4.0 and 1 volume of I-butanol.
Test solution. Shake a quantity of the powder containing
0.1 g of ampicillin with 50 ml ofphosphate buffer pH 7.0 for
15 minutes, filter and use the filtrate.
Reference solution. A 0.2 per cent w/v solution of ampicillin
trihydrate RS in phosphate buffer pH 7. O.
Impregnate the dry plate by placing it in a tank containing a
shallow layer ofa 0.1 per cent w/v solution of disodium edetate
in mixed phosphate buffer pH 4.0, allowing the solvent to
ascend to the top, removing the plate from the tank and
allowing the solvent to evaporate. Use the plate with the flow
of the mobile phase in the direction in which impregnation
was carried out. Before use heat the plate at 100 for Lhour
and allow to cool.. Apply to the plate 1111 of eaoh soll.ltioll.
After development, dry the plate in air and spray with a mixture
of 100 volumes ofa 1 per cent w/v solution ofstarch, 6 volumes
of glacial acetic acid and 2 volumes of a 1 per cent w/v
2638
IP 2010
AMPROLIUM HYDROCHLORIDE
solution of iodine in a 4 per cent w/v solution of potassium

iodide. The principal spot in the chromatogram obtainedwith
the test solution con-esponds to the spot in the chromatogram
B. To a quantity ofthe powder containing 10 mg ofampicillin
add sufficient water to produce 10 ml, shake for 15 minutes
and filter. Place 0.1 ml ofa 0.1 per cent w/v solution of ninhydrin
on a filter paper, dry at 105, superimpose 0.1 ml ofthe solution
of the preparation under examination, heat for 5 minutes at
105 and allow to cool; a mauve colour is produced.
C. Suspend a quantity of the powder containing 10 mg of
ampicillin in 1ml of water and add 2 mlofa mixture of2 ml of
potassium cupri-tartrate solution and 6 ml of water; a
magenta-violet colour is immediately produced.
less than 2.0. The relative retention time with reference to

caffeine for ampicillin is about 0.5.
capacity factor is not more than 2.5, the tailing factor is not
Calculate the content OfC16H19N304S in oral powder.
exceeding 30.
equivalent concentration ofampicillin.
Tests
Uniformity of weight. When supplied in containers intended
for use on one occasion, complies with the test for Uniformity
of weight described under Parenteral Preparations (Powders
for Injection).
Amprolium Hydrochloride
Other tests. Complies with the tests stated under Veterinary

Oral Powders.
el, Hel
Assay. Determine by liquid chromatography (2. 4.14).

NOTE -
Solvent mixture. Mix 10 ml of 1 M monobasic potassium

phosphate and 1 ml of 1 M acetic acid and dilute to 1000 ml
with water.
Test solution. Dissolve an accurately weighed quantity of
powder containing about 100 mg of ampicillin in the solvent
mixture by shaking and mixing if necessary, with the aid of
ultrasound and dilute to 100.0 ml with the solvent mixture.
Reference solution (a). Weigh accurately a suitable quantity
of ampicillin RS, dissolve in the solvent mixture by shaking
and mixing ifnecessary, with the aid ofultrasound to obtain a

solution having a lmown concentration ofabout 1 mg per ml.
Reference solution (b). Dissolve caffeine in reference solution
(a) to obtain a solution containing about 0.12 mg per ml.

microparticles (5 /-lm),
mobile phase: a mixture of 90 volumes of water; 8
volumes of acetonitrile, 1 volume of 1 M monobasic
potassium phosphate and 1 volume of 1 M acetic acid,
injection volume.20 ,.ti.
resolution between the caffeine and ampicillin peaks is not
Cl~19ClN4,HCI
Mol. Wt. 315.2
Amprolium Hydrochloride is hydrochloride salt of

1-[(4-amino-2-propyl-5-pyrimidinyl)methyl]2-methylpyridinium chloride.
Amprolium Hydrochloride contains not less than 97.5 per cent
and not more than 101.0 per cent OfC14H19ClN4,HCI, calculated
on the dried basis.
Category. Coccidiostat.
Dose. Poultry. Usual recommended concentration in feed as
preventive against coccidiosis infection, about 0.0125 per cent
(between 100 and 125 g pertonne offeed).
Description. A white or aln'lOst white powder.
Identification
A. Deten"lline by infrared absorption spectrophotometry (2.4.6).
Compare the spectrum with that obtained with amprolium
hydrochloride RS or with the reference spectrum ofamprolium
hydrochloride.

0.002 per cent w/v solution in 0.1 Mhydrochloric acid exhibits
maxima at about 246 nm and 262 nm; absorbance at about
2639
AMPROLIUM HYDROCHLORIDE
IP 2010
C. To 1 mg add 5 ml of naphthalenediol reagent; a deep violet

colour is produced.

Test solution. Shake continuously for 10 minutes a quantity

containing 10 mg ofEthopabate with 25 ml of acetone that has
been warmed to 50, filter and use the filtrate.
Tests
Picoline. Dissolve 1.5 g in 30 ml of water in a distillation flask,
add 20 ml of a saturated solution of potassium carbonate
sesquihydrate, connect the flask to a ground-glass aerator
extending to the bottom of a lOO-ml graduated cylinder
containing 50 ml of 0.05 M hydrochloric acid and pass air,
which has previously been passed through sulphuric acid
and glass wool, through the system for 60 minutes. To 5 mlof
the hydrochloric acid solution add sufficient 0.05 M
hydrochloric acid to produce 200 ml. Absorbance of the
resulting solution at about 262 nm (2.4.7), not more than 0.52.

solution corresponds to the spot in the chromatogram
Tests
pH (2.4.24). 2.5 to 4.0, determined in a 25 per cent w/v slurry in
carbon dioxide-free water.

on 1.0 g by drying to constant weight at 100 at a pressure not
exceeding 0.7 kPa.
solution. Titrate with 0.1 M perchloric acid, using
titration.
C 1JfI9ClN4,HCl.
Amprolium Hydrochloride and

Ethopabate Premix
Amprolium Hydrochloride and Ethopabate Premix contains
Amprolium Hydrochloride and Ethopabate.
Amprolium Hydrochloride and Ethopabate Premix contains
the stated amounts of amprolium hydrochloride,
CI4HI9CIN4,HCI and ofethopabate, ClzHlsN04.
Usual strength. 25 per cent w/wofAmprolium Hydrochloride
and 1.6 per cent w/w ofEthopabate.
Identification
A. Shake a quantity containing 20 mg of Amprolium
Hydrochloride with 90 ml of methanol and filter. Add 5 ml of
the filtrate to 5 ml of naphthalenediol reagent; a deep violet
colour is produced;
13.J:)eteririiriehY'fliiri::layercnromafograpliY(2)[ITj,coating
NOTE - Prepare the solution immediately before use.
Reference solution. A 0.04 per cent w/v solution of ethopabate

RS in acetone.
Other tests. Complies with the tests stated under Premixes.

Assay. For amprolium hydrochloride - Weigh accurately a
quantity containing 50 mg ofAmprolium Hydrochloride, shake
continuously for 20 minutes with 100.0 ml of a mixture of
2 volumes of methanol and 1volume of water and filter. Dilute
5.0 ml ofthe filtrate to 100.0 ml with the methanol-water mixture.
To 4.0 ml of the resulting solution add lO.O ml of
naphthalenediol reagent, allow to stand for 20 minutes and
obtained by mixing 4.0 ml ofa mixture of2 volumes of methanol
and 1volume of water with 10.0 ml of naphthalenediol reagent
and allowing to stand for 20 minutes. Calculate the content of
CI4HI9ClN4,HCl from the absorbance obtained by carrying out
the procedure simultaneously, using 4.0 ml of a 0.0025 per
cent w/v solution of amprolium hydrochloride RS in a mixture
of2 volumes of methanol and 1 volume of water and beginning
at the words, "To 4.0 ml ofthe resulting solution add 10.0 ml
of...." .
For ethopabate - Weigh accurately a quantity containing

6 mg ofEthopabate, add 75 ml of methanol, shake continuously
for 20 minutes, dilute to 100.0 ml with methanol and filter. To
10.0 ml ofthe filtrate add 10 ml of 1 M sodium hydroxide and
evaporate to dryness. Dissolve the residue in 10.0 ml of water,
heat on a water-bath for 15 minutes, add 10 ml of 2 M
hydrochloric acid, dilute to 100.0 ml with water and filter. To
25.0 ml ofthe filtrate, add 2.5 ml of2 M hydrochloric acid and
5 ml of a 0.1 per cent w/v solution of sodium nitrite prepared
immediately before use. Allow to stand for 3 minutes and add
2.0 . ml. of.a freshlypreparedQ,5 . 12e.L~,(;'l111,':YiYs.QJuti<:>l1,Qf
arl1ploniul11 sulphal11at~. AII()\Vto stal1dJ()!21~inl!t~s, add
N-(l-naphthyl)ethylenediamine dihydrochloride, allow to
stand for 10 minutes and dilute to 50.0 ml with water. Measure
2640
IP 2010
AMPROLIUM, ETHOPABATE AND SULPHAQUINOXALINE PREMIX

about 540 nm (2.4.7), using as the blank a solution obtained
by repeating the procedure with 25 ml of water and beginning
at the words "add 2.5 ml of 2 M hydrochloric acid
".
Calculate the content of C 12H 1SN0 4 from the absorbance
obtained by carrying out the procedure simultaneously, using
10.0 ml of a 0.006 per cent w/v solution of ethopabate RS in
methanol and beginning at the words "add 10 ml of 1 M sodium
hydroxide and evaporate to chyness.....".
Amprolium, Ethopabate and

Sulphaquinoxaline Premix
Amprolium Hydrochloride,
Ethopabate
and
Amprolium, Ethopabate and SulphaquinoxaliI1e Premix

contains Amprolium Hydrochloride, Ethopabate and
Sulphaquinoxaline.
Amprolium, Ethopabate and Sulphaquinoxaline Premix
110.0 per cent of the stated amounts of amprolium hydrochloride, C14H19CIN4,HCI, of ethopabate, C 12H 1SN04, and of
sulphaquinoxaline, C14HIZN40ZS.
Usual strength. 20 per cent w/w ofAmprolium HydrocWoride,

1 per cent w/w of Ethopabate and 12 per cent w/w of
Sulphaquinoxaline
Identification
A. Shake a quantity containing 20 mg of Amprolium
Hydrochloride with 90 ml of methanol and filter. Add 5 ml of
the filtrate to 5 ml of naphthalenediol reagent; a deep violet
colour is produced.

NOTE
Prepare the solution immediately before use.

Test solution. Shake continuously for 10 minutes a quantity
containing 10 mg ofEthopabate with 25 ml of acetone that has
been warmed to 50 0 , filter and use the filtrate.
ethopabate RS in acetone.
sulphaquinoxaline RS in acetone.
chy the plate in air and examine in ultraviolet light at 254 nm.
solution corresponds to the spot in the chromatogram

obtained with reference solutions (a) and (b).
Tests
pH (2.4.24). 2.5 to 4.0, determined in a 25 per cent w/v slurry in
carbon dioxide-fi'ee water.

Assay. For amprolium hydrochloride - Weigh accurately a
quantity containing 50 mg ofAmprolium Hydrochloride, shake
continuously for 20 minutes with 100.0 ml of a mixture of
2 volumes of methanol and 1 volume of water and filter. Dilute
5.0 ml ofthe filtrate to 100.0 ml with the methanol-water mixture.
To 4.0 ml of the resulting solution add 10.0 ml of
naphthalenediol reagent, allow to stand for 20 minutes and
obtained by mixing 4.0 ml ofa mixture of2 volmnes of methanol
and 1 volume of water with 10.0 ml ofnaphthalenediol reagent
and allowing to stand for 20 minutes. Calculate the content of
C14H19CIN4,HCl from the absorbance obtained by carrying out
the procedure simultaneously, using 4.0 ml of a 0.0025 per
cent w/v solution of amprolium hydrochloride RS in a mixture
of2 volumes of methanol and 1 volume of water and beginning
at the words, "To 4.0 ml of the resulting solution add 10.0 ml
of....." .
For ethopabate
Weigh accurately a quantity containilfg
6 mg ofEthopabate, add 75 ml of methanol, shake continuously
for 20 minutes, dilute to 100.0 ml with methanol and filter. To
10.0 ml ofthe filtrate add 10 ml of 1 M sodium hydroxide and
evaporate to dryness. Dissolve the residue in 10.0 ml of water,
heat on a water-bath for 15 minutes, add 10 ml of 2 M
hydrochloric acid, dilute to 100.0 ml with water and filter. To
25.0 ml ofthe filtrate, add 2.5 ml of 2 M hydrochloric acid and
5 ml of a 0.1 per cent w/v solution of sodium nitrite prepared
immediately before use. Allow to stand for 3 minutes and add
ammonium sulphamate. Allow to stand for 2 minutes, add
N-(1-naphthyl)ethylenediamine dihydrochloride, allow to
about 540 nm (2.4.7), using as the blank a solution obtained
by repeating the procedure with 25 ml of water and b<:ginning
at the words "add 2.5 ml of 2 M hydrochloric acid
".
Calculate the content of C 12H 1SN0 4 from the absorbance
10.0 ml of a 0.006 per cent w/v solution of ethopabate RS in
methanol and beginning at the words "add 10 ml of 1 M sodium
hydroxide and evaporate to chyness.....".
For sulphaquinoxaline - Weigh accurately a quantity
containing 40 mg of Sulphaquinoxaline, shake continuously
2641
CALCIUM BOROGLUCONATE INJECTION
IP 2010
for 10 minutes with a mixture of75 ml of water and 4 ml of2 M Tests

sodium hydroxide, dilute to 250.0 ml with water and centrifuge.
To 10.0 ml of the supernatant liquid add 5 ml of 2 M pH (2.4.24). 3.0 to 4.0, determined in a solution diluted if
hydrochloric acid and dilute to 200.0 ml with water. To I 0.0 ml necessary with carpon dioxide-free water to produce a
of the diluted solution add 2.5 ml of 2 M hydrochloric acid solution containing 1.5 percent w/v of calcium.
and 5 ml of a freshly prepared 0.1 per cent w/v solution of Other tests. Complies with the tests stated under Parenteral
sodium nitrite and allow to stand for 3 minutes. Add 5.0 ml of Preparations (Injections).
a freshly prepared 0.5 per cent w/v solution of ammonium
Assay. For calcium - Dilute an accurately measured volume
sulphamate and allow to stand for 2 minutes. Add 5.0 ml of a
containing 45 mg ofcalcium to about 50 ml with water. Titrate
freshly prepared 0.1 per cent w/v solution of
with 0.05 M disodium edetate to within a few ml ofthe expected
N-(I-naphthyl)ethylenediamine dihydrochloride, allow to
end-point, add 4 ml of a 40 per cent w/v solution of sodium
hydroxide and 10 mg of calcon mixture and continue the
. the absorbance of the resulting solution at the maximum at
titration until the colour changes from pink to blue.
about 540 nm (2.4.7), using as the blank the solution obtained
by repeating the procedure with 10 ml of water and beginning 1 ml of 0.05 M disodium edetate is equivalent to 0.002004 g
at the words "add 2.5 ml of 2 M hydrochloric acid......". ofCa.
Calculate the co~tent of Cl4H12N40ZS from the absorbance For boric acid - Dilute an accurately measured volume
obtained by carrying out the procedure simultaneously, using containing 0.1 g of boric acid to 50 ml with water, add 3 g of
10.0 ml ofa 0.0008 per cent w/v solution of sulphaquinoxaline mannitol and titrate with 0.1 M sodium hydroxide using
RS in 0.001 M sodium hydroxide and beginning at the words phenolphthalein solution as indicator.
"To 10.0 ml of the diluted solution add 2.5 ml of
2 M hydrochloric acid.....".
H 3B03
Storage. Store protected from light, at a temperahlre not
exceeding 30.
Calcium Borogluconate Injection is a sterile solution ofCalcium

Gluconate and Boric Acid in Water for Injections. The solution
may contain up to 0.2 per cent w/v of ChIorocresol.
Labelling. The label states (l) the strength in terms of the

equivalent amount of calcium in a suitable dose-volume; (2)
the proportion of boric acid present; (3) the proportion of
chlorocresol, if present.
Calcium Borogluconate Injection contains not less than

amount of calcium, Ca, and boric acid, H 3B03 equivalent to
not more than 2.3 times the stated content of calcium.
Category. Hypocalcaemic.
Dose. All species. By subcutaneous, intravenous or
intraperitoneal injection, the equivalent of 7.5 to 22.5 mg of
calcium per kg ofbody weight.
(Each 100 mg of calcium borogluconate is approximately
equivalent to 7.5 mg ofcalcium).
Usual strength. 25 per cent w/v solution equivalent to 1.9 per

cent w/v ofcalcium (approximately).
Identification
A. Dilute 1 ml with sufficient water to produce a solution
containing about 0.75 per cent wIv ofcalcium and add 0.05 ml
offerrie chloride test solution; an intense yellow or yellowish
~~~~igreei1(501()urisJjfo(luCled:~~ .__
~ _.~~~
~_
~
B. Gives the reactions ofcalcium
salts (2.3.1).
Calcium Magnesium Borogluconate

Injection
Calcium Magnesium Borogluconate Injection is a sterile
solution of Calcium Gluconate, Boric Acid, Magnesium
Hypophosphite and Dextrose in Water for Injections. It may
contain upto 0.2 per cent w/v of Chlorocresol.
Calcium Magnesium Borogluconate Injection contains not
stated amounts of calcium, Ca, of magnesium, calculated as
magnesium hypophosphite, Mg(H zPO z)z,6H zO, and of
dextrose, C6H 1Z0 6, and the content ofboric acid, H 3B03 , is not
more than 2.3 times the stated content of calcium.
Usual strengths. Equivalent to 1.86 per cent w/v; 2.25 per

cent w/v; 3.0
cent w/v of Ca.
_~~~~:...........
.
_. ._..~.. ~
~.~.~.~
Identification
C. To 1 mladdO.15 ml of sulphuric acid and 5 mlofmethanol

and ignite; the mixture bums with a flame tinged with green.
A. Dilute 1 ml with sufficient water to produce a solution

containing about 0.75 per cent wIv ofcalcium and add 0.05 ml
2642
CHLORAL HYDRATE
IP 2010
offerric chloride test solution; an intense yellow or yellowish

green colour is produced.
B. Gives the reactions ofcalcium salts (2.3.1).
C. Gives the reactions ofmagnesium salts (2.3.1).
D. To 1 ml add 5 ml of water, neutralise to pH 7.0 with dilute
ammonia solution and add 5 ml of silver nitrate solution. A
yellow precipitate is produced which does not change colour
on boiling but dissolves on addition of dilute ammonia
solution.
E. To 1 ml add 0.15 ml of sulphuric acid and 5 ml of methanol
and ignite; the mixture bums with a flame tinged with green.
F. To 1 ml add 2 ml of 2 M sodium hydroxide solution and 0.05

ml of copper sulphate solution. The solution is blue and clear.
Heat to boiling. A copious red precipitate is produced.
Tests
pH (2.4.24). 3.0 to 4.0, determined in a solution diluted, if
necessary, with carbon dioxide-free water so as to contain
1.5 per cent w/v ofcalcium.
For boric acid - Dilute a volume containing 0.1 g of boric

acid to 50 ml with water, add 3 g of mannitol and titrate with
0.1 M sodium hydroxide using phenolphthalein solution as
indicator.
1 ml of 0.1 Msodium hydroxide is equivalent to 0.006183 g of
H3B03
F0/' dextrose - Dilute a volume containing 200 mg ofdextrose

to 50 ml with water; add 30 ml of 0.1 M iodine solution and
10 ml of a 5 per cent w/v solution of sodium carbonate and
allow to stand for 20 minutes. Add 15 ml of 1 M hydrochloric
acid and titrate the excess of iodine with 0.1 M sodium
thiosulphate solution using starch solution as indicator. Carry
1 ml of 0.1 M iodine solution is equivalent to 0.009008 g of
dextrose, C6H 120 6
Labelling. The label states (l) the strength in tenns of the
equivalent amount of calcium and magnesium in a suitable
dose-volume; (2) the proportion of boric acid to calcium; (3)
the percentage of any added stabilising agent.
Chloral Hydrate

perml.
Assay. For calcium - Dilute a volume containing 45 mg of
calcium to about 50 ml with water. Add 1 ml of 1 M sodium
hydroxide solution. Titrate with 0.05 M disodium edetate to
within a few ml of the expected end-point, add 5 ml of strong
ammonia-ammonium chloride solution and 10 mg of calcon
mixture as indicator and continue the titration until the colour
changes from pink to blue. Calculate the volume of 0.05 M
disodium edetate consumed by substracting the volume of
0.05 M disodium edetate consumed in the assay for
magnesium.
Ca.
For magnesium - Dilute a volume containing 10 mg of

. magnesium to about 50 ml with water. Add 1 g of ammonium
chloride and 1 g of ammonium oxalate. Neutralise to litmus
paper with dilute ammonia solution and add 5 ml in excess.
Boil for 5 minutes and allow to stand for 1 hour. Filter and
wash the residue with hot water. Collect the filtrate and
washings and add 5 ml of strong ammonia-ammonium
chloride solution. Titrate with 0.05 M disodium edetate using
eriochrome black T mixture as indicator.
1 ml of disodium edetate is equivalent to 0.001216 g of
magnesium or 0.0107894 g of magnesium hypophosphite,
Mg(H2P02)2,6H20.
HO
CI
KCI
HO
CI
~H3Ch02
Mol. Wt. 165.4
Chloral Hydrate is 2,2,2-trichloro-1,1-ethanediol.

Chloral Hydrate contains not less than 98.5 per cent and not
more than 101.0 per cent of the stated amount of chloral
hydrate, C2H3Ch02.
Category. Sedative and hypnotic.
Dose. Cows, buffaloes and horses. By intravenous injection,
60 to 100 mg per kg ofbody weight.
Description. A colourless, transparent crystals; odour,
pungent.
Identification
A. To IOmlofa 10 per centw/v solution in carbon dioxide:free
water (solution A) add 2 ml of 2 M sodium hydroxide. The
mixture becomes cloudy and when heated gives an odour of
chloroform.
B. To 1 ml ofsolution A add 2 ml of sodium sulphide solution;
a yellow colour develops which quickly turns reddish brown
and may yield a red precipitate on standing.
2643
CHLORAL HYDRATE
IP 2010
Tests
Appearance of solution. Solution A is clear (2.4.1) and
colourless (2.4.1).
Heavy metals (2.3.13). 12 ml ofa 5 per cent w/v solution in
water, complies with the limit test for heavy metals, Method D
(20 ppm).
Chlorides (2.3.12). Dissolve 2.5 gin 15 ml with water; the
(100 ppm).

chromatogram obtained with I f.!1 oftest solution corresponds
(a).
C. To 5 ml of a 0.1 per cent w/v solution add a few drops of
silver. nitrate solution; no precipitate is produced. Heat about
50 mg with 3 ml of ethanolic potassium hydroxide solution
on a water-bath for 15 minutes, add 15 mg of decolorising
charcoal, shake and filter. The filtrate gives the reactions of
chlorides (2.3.1).
Tests
Chloral alcoholate. Warm I g with 10 ml of 2 M sodium

hydroxide, filter the upper layer and add 0.05 M iodine
dropwise until a yellow colour is produced. Set aside for
I hour; no yellow precipitate is produced and no smell of
iodoform is perceptible.
Consistence. Chloramphenicol Injection containing 150 mg

per ml passes readily through a 230 hypodermic needle.
Assay. Weigh accurately about 4 g, dissolve in 10 ml of water

and add 40 ml of 1 M sodium hydroxide. Allow the mixture to
stand for exactly 2 minutes and titrate the residual alkali
immediately with 0.5 M sulphuric acid using phenolphthalein
Test solution. Dilute the injection with sufficient ofthe mobile

phase to produce a solution containing 0.030 per cent w/v of
Chloramphenicol.
I ml of 1 M sodium hydroxide is equivalent to 0.1654 g of

GH3Cl30 z.
Chloramphenicol Injection is a sterile suspension of
Chloramphenicol in Water for Injections containing suitable
suspending and stabilising agents.
Chloramphenicol Injection contains not less than 95.0 percent
chloramphenicol, CjjHI2CIzNzOs.
pH (2.4.24). 3.5 to 6.5.
2-Amino-l-(4-nitrophenyl)propane-l,3-diol Determine by
liquid chromatography (2.4.14).
Reference solution. A 0.00225 per cent w/v of 2-amino1-(4-nitrophenyl) propane-l,3-diol RS in the mobile phase.
mobile phase: a filtered and degassed mixture of 85
volumes of 0.012 M sodium pentanesulphonate, 15
volumes of acetonitrile and I volume of glacial acetic
acid,
- injection volume. 20 f.!l.
Usual strengths. 4.5 gin 30 ml; 5.0 g in 50 ml; 11.25 gin 75 ml;
15 gin 100 ml;20 g in 100 mI.

In the chromatogram obtained with test solution the area of
any peak corresponding to 2-amino-I-(4- nitrophenyl)propane-I ,3-diol is not more than the area ofthe peak obtained
Identification

Centrifuge a volume containing 0.15 g of Chloramphenicol;

wash the residue with water and dry over self-indicating silica
gel and then for I hour at 105 0 The dried residue complies
with the following tests.

10 volumes of methanol and I volume of water.
A. Wash 75 mg of the residue with two quantities, each of

10 ml, of light petroleum ( 60 0 to 80 0) and allow to dry. The
residliecompHeswitfdneloHowingtesC

chloramphenicol RS in acetone.
.. ReJe~ence~soiuiion(b).5TfutelmfofrefeieiicesoIuti.on(a)to . .

Compare the spectrum with that obtained with chloramphenicol
RS or with the reference spectrum of chloramphenicol.
Apply to the plate I f.!l and 20 f.!1 of the test solution, I f.!1 of
reference solution (a) and 20 f.!1 ofreference solution (b). After
Test solution. A 1.0 per cent w/v solution of the dried residue
obtained in the test for identification in acetone.
2644
IP 2010
CHLORTETRACYCLINE HYDROCHLORIDE

spot in the chromatogram obtained with reference solution (b).
Assay. To an accurately measured volume containing 0.75 g
ofchloramphenicol add sufficient water to produce 1000.0 ml
and shake until a clear solution is obtained. Dilute 5.0 ml of
this solution to 200.0 ml with water and measure the absorbance
Calculate the content of CIIH12CIzNzOs in the injection taking
Storage. Store protected from light. Do not freeze.
Labelling. The label states (l) the name of any added
suspending agent; (2) that the injection is for intramuscular
injection only; (3) the date after which the contents are not
intended to be used.
OH
o
NH2
CnH23 CINzOg,HCI
Hel
Mol. Wt. 515.4

NOTE -
Usefreshly prepared solution.

Reference solution (a). A 0.05 per cent w/v of chlortetracycline hydrochloride RS in methanol.
w/v each of chlortetracycline hydrochloride RS, tetracycline
hydrochloride RS and metacycline hydrochloride RS in
methanol.
Adjust the pH ofa 10 per cent w/v solution of disodium edetate
to 8.0 with 10M sodium hydroxide and spray this solution
evenly on the plate (about 10 ml for a plate of 100 rom by
200 rom size). Allow the plate to dry in a horizontal position for
at least I hour. Dry the plate in an oven at 100 for I hour
before use. Apply to the plate 1 III of each solution. After
chromatogram obtained with reference solution (a). The test
solution (b) shows three clearly separated spots.
B. To about 2 mg add 5 ml of sulphuric acid; a deep blue
colour develops which becomes bluish green. Add the
solution to 2.5 ml of water; the colour changes to brownish.
Chlortetracycline Hydrochloride is [4S(4a,4aa,5aa,6~, 12aa)]-7-chloro-4- dimethylamino1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxy6-methyl-I,II-dioxo-2-naphthacenecarboxamide

hydrochloride.
Tests
Chlortetracycline Hydrochloride contains not less than

89.5 per cent of chlortetracycline hydrochloride and the sum
of the contents of chlortetracycline hydrochloride and
tetracycline hydrochloride is not less than 94.5 per cent and
not more than 100.5 per cent, calculated on the anhydrous basis.
Dose. Horses, calves and pigs. 10 to 20 mg per kg of body
weight daily. Dogs and cats. 20 to 50 mg per kg ofbody weight
daily. Pigs andpoultry. By mass medication, 130 mg per litre
ofdrinking water.
Cows, buffaloes and horses. By intra-uterine insertion, upto

1 g. Pigs and sheep. By intra-uterine insertion, upto 0.5 g.
Description. Yellow powder.
Identification
pH (2.4.24). 2.3 to 3.3, determined in a 1 per cent w/v solution

in carbon dioxide-ji-ee water prepared by slight heating, if
necessary.
at 20 in a 0.25 per cent w/v solution in water, calculated on
Light absorption. When examined at 460 nm ofa 0.5 per cent
w/v solution in water is not more than 0.40.
Related substances. Carry out the method described under
Assay injecting test solution, reference solutions (e) and (t).
The test is not valid unless the peak in the chromatogram
obtained with reference solution (t) is properly integrated. In
the chromatogram obtained with test solution the area of the
peak corresponding to 4-epichlortetracycline is not more than
the area of the peak corresponding to 4-epichlortetracycline
in the chromatogram obtained with reference solution (e)
2645
CHLORTETRACYCLINE HYDROCHLORIDE
IP 2010
(4 per cent) and the total area of any secondary peaks, other
than the peaks due to tetracycline and 4-epichlortetracycline,
is not more than 25 per cent of the area of the peak
corresponding to 4-epichlortetracycline in the chromatogram
obtained with reference solution (e) (1 per cent). Ignore any
peak with an area smaller than that ofthe principal peak in the
chromatogram obtained with reference solution (f) (0.1 per
cent).
Inject reference solution (d) and adjust the instrument so that

the peak heights correspond to at least 50 per cent of the full
scale deflection of the recorder. If necessary, adjust the
dimethyl sulphoxide content in the mobile phase. The test is
not valid unless the resolution factor between the first peak
(4-epichlortetracycline) and the second (chlortetracycline) is
not less than 2.0 and the symmetry factor for the second peak
Tetracycline hydrochloride. Not more than 8.0 per cent,

calculated on the anhydrous basis and determined as described
under the Assay, injecting separately test solution and
reference solution (e).
relative standard deviation ofthe peak area for chlortetracycline
hydrochloride is not more than 1.0 per cent. Ifnecessary, adjust
the integrator parameters.
heavy metals, Method D (50 ppm), using 2.5 m1 oflead standard
solution (l0 ppm Pb) as the standard.

Water (2.3.43). Not more than 2.0 per ceht ,determinedon 0.3 g.
Calculate the content ofC22H23C1N20s,HCl.
Chlortetracycline Hydrochloride intended for use in the

manufacture of Parenteral Preparations without a further
appropriate procedure for removal of bacterial endotoxins
Bacterial endotoxins (2.2.3). Not more than 1.1 ED per mg.

examination in 100.0 ml of 0.01 Mhydrochloric acid.
Chlortetracycline Hydrochloride intended for use in the

manufacture of Parenteral Preparations without a fitrther
Reference solution (a). A 0.1 per cent w/v solution of appropriate sterilisation procedure complies with the
chlortetracycline hydrochloride RS in 0.01 M hydrochloric following additional requirement.
acid.
Storage. Store protected from light. If it is intended for use in
4-epichlortetracycline hydrochloride RS in 0.01 M
the manufacture of Parenteral Preparations, the container
hydrochloric acid.
should be sterile, tamper-evident and sealed so as to exclude
Reference solution (c). A 0.08 per cent w/v of tetracycline micro-organisms.
hydrochloride RS in 0.01 M hydrochloric acid.
Labelling. The label states (1) the date after which the material
Reference solution (d). Mix 5 ml ofreference solution (a) and is not intended to be used; (2) the storage conditions; (3)
10 ml ofreference solution (b) and dilute to 25 ml with 0.01 M where applicable, that the material is sterile and free from
hydrochloric acid.
Bacterial endotoxins.
Reference solution (e). Mix 5 ml ofreference solution (b) and
5 ml of reference solution (c) and dilute to 50 ml with 0.01 M
hydrochloric acid.
Reference solution (f). Dilute 1 ml ofreference solution (c) to
20 ml with 0.01 M hydrochloric acid and dilute 2.5 ml ofthis
solution to 100 ml with 0.01 M hydrochloric acid.
Chlortetracycline Veterinary Oral

Powder
Chlortetracycline Hydrochloride Veterinary Oral Powder;

Chlortetracycline Soluble Powder
- a stainless steel column 25 cm x 4.6 rom, packed with Chlortetracycline Veterinary Oral Powder is a mixture of
octylsilane groups (5 f.1m),
Chlortetracycline Hydrochloride and Lactose or other suitable
diluent.
- mobile phase: a filtered and degassed mixture of450 ml
of dimeth I suI hoxide 50 ml of 1 M erchloric acid Chlortetracycline Veterinary Oral Powder contains not less
--~---~-~~--~-~and 500 ~~f:;;:~;::~~_:>_~-----~-~_P~~---~-----tJjan90:0 percentand-not more tnannO:O per cent onne
~~
........~
.
stated . amount of chlortetracycline hydrochloride~
- flow rate. 1 m1 per mlllute,
.
,
CnH23 CIN20s,HCI
- injection volume. 20 f.11.
2646
IP 2010
CLOXACILLIN BENZATHINE
Identification
NOTE -
Use freshlY prepared solutions.

Test solution. The supernatant liquid obtained by extracting a
quantity containing 5 mg of Chlortetracycline Hydrochloride
with 10 ml of methanol and centrifuging.
Reference solution (a). A 0.05 per cent w/v of chlortetracycline hydrochloride RS in methanol.
w/v each of chlortetracycline hydrochloride RS, tetracycline
hydrochloride RS and metacycline hydrochloride RS in
methanol.
Adjust the pH ofa 10 per cent w/v solution of disodium edetate
to 8.0 with 10M sodium hydroxide and spray this solution
evenly on the plate (about 10 ml for a plate of 100 mm by
200 mm size). Allow the plate to dry in a horizontal position for
at least I hour. Dry the plate in an oven at 100 for I hour
before use. Apply to the plate I III of each solution. After
B. To a quantity containing 10 mg of Chlortetracycline
Hydrochloride, add 20 ml ofwarm ethanol (95 per cent), allow
to stand for 20 minutes, filter and evaporate to dryness on a
water-bath. Dissolve the residue in sufficient phosphate buffer
pH 7. 6 to produce a 0.1 per cent w/v solution and heat at 100
for I minute; it exhibits a strong blue fluorescence in
ultra-violet light.
Tests
Reference solution (d). Mix 5.0 ml of reference solution (a)

and 10.0 ml ofreference solution (b) and dilute to 25 ml with
0.01 M hydrochloric acid. .
Reference solution (e). Mix 5.0 ml of reference solution (b)
and 5.0 ml of reference solution (c) and dilute to 50 ml with
Reference solution (f). Dilute 1.0 ml ofreference solution (c)
to 20 ml with 0.01 M hydrochloric acid Further, dilute 2.5 ml
of this solution to 100.0 ml with 0.01 M hydrochloric acid.
- column temperature.35,
- mobile phase: a mixture of45 ml of dimethyl sulphoxide,
5 ml of 1 M perchloric acid and 50 ml of water,
Inject reference solution (d). Adjust the instrument so that the
peak heights correspond to at least 50 per cent ofthe full scale
deflection of the recorder. If necessary, adjust the dimethyl
sulphoxide content in the mobile phase. The test is not valid
unless the resolution factor between the first peak
(4-epichlortetracycline) and the second (chlortetracycline) is
not less than 2.0 and the symmetry factor for the second peak
relative standard deviation ofthe peak area for chlortetracycline
hydrochloride is not more than 1.0 per cent.
Calculate the content ofC22H23ClN20g,HCl.
exceeding 30.
Labelling. The label states the strength in terms ofthe concentration of Chlortetracycline Hydrochloride

Oral Powders.
Cloxacillin Benzathine

examination in 100.0 ml of 0.01 M hydrochloric acid.
chlortetracycline hydrochloride RS in 0.01 M hydrochloric
acid.
4-epichlortetracycline hydrochloride RS in 0.01 M
hydrochloric acid.
tetracycline hydrochloride RS in 0.01 M hydrochloric acid.
0
N~\-~;;~:j
~
l
CI
/;
~s
H H
'0
(CI9HlgClN30SS)2,CI6H20N2
CH a
Mol. Wt. 11 12.1
Cloxacillin Benzathine isN,N' -dibenzylethylenediammonium bis-[(6R)-6- {3-(2-chlorophenyl)-5-methylisoxazole-4-carboxamido}penicillanate].
2647
CLOXACILLIN BENZATHINE
IP 2010
Cloxacillin Benzathine contains not less than 92.0 per cent of

(C19HlsClN30SS)Z,CI6HzoNz and not less than 20.0 per cent and
not more than 22.0 per cent of benzathine, C16HzoNz, both
Dose. Dry cowslbuffaloes. By intramammary infusion into each
quarter, alone or in combination with Ampicillin Trihydrate,
the equivalent of 500 mg of cloxacillin as a single dose.
(Each 1.28 g of cloxacillin benzathine is approximately
equivalent to 500 mg ofcloxacillin).
Identification
Compare the spectrum with that obtained with cloxacillin
benzathine RS or with the reference spectrum of cloxacillin
benzathine.
B. Shake 0.1 g with 1 ml of 1 M sodium hydroxide for
2 minutes, add 2 ml of ether, shake for 1 minute and allow to
separate. Evaporate 1 ml of the ether layer to dryness, dissolve
the residue in 2 ml ofglacial acetic acid and add 1 ml of dilute
potassium dichromate solution; a golden yellow precipitate
is produced.
C. Shake 50 mg with 10 ml of water and filter. To 5 ml ofthe
filtrate add a few drops ofsilver nitrate solution; no precipitate
is produced. Heat 50 mg with 2 ml of ethanolic potassium
hydroxide solution on a water-bath for 15 minutes, add 15 mg
of decolorising charcoal, shake and filter. Acidify the filtrate
with 2 M nitric acid; the solution gives reaction A of chlorides
(2.3.1).
Tests
Water (2.3.43). Not more than 5.0 per cent wlw, determined on
0.5g.
Calculate the content of (CI9HlSClN30SS)Z,CI6HzoNz from the

difference obtained by carrying out the procedure
simultaneously using cloxacillin benzathine RS.
For benzathine - Weigh accurately about 1 g, add 30 ml ofa
saturated solution of sodium chloride and 10 ml of5 M sodium
hydroxide, shake well and extract with four quantities, each of
50 ml, of ether. Wash the combined extracts with three
quantities, each of 10 ml, of water, extract the combined
washings with 25 ml of ether and add the extract to the main
ether solution. Evaporate the ether solution to low volume,
add 2 ml of ethanol and evaporate to dryness. To the residue
add 50 ml of anhydrous glacial acetic acid. Titrate with 0.1 M
perchloric acid, using 0.1 ml of 1-naphtholbenzein solution
as indicator. Carry out a blank titration.
1 m1 of 0.1 M perchloric acid is equivalent to 0.01202 g of

C16HzoNz.
Cloxacillin Benzathine intended for use in the manufacture
ofeither parenteral preparations or intramammary infUsions
without a further appropriate sterilisation procedure

Storage. Store protected from moisture. If it is intended for
use in the manufacture of parenteral preparations or
intramammary infusions, the container should be sterile,
tamper-evident and sealed so as to exclude micro-organisms.
Cloxacillin Benzathine Intramammary

Infusion (Dry Cowl Buffalo)
Cloxacillin Benzathine Intramammary Injection; Cloxacillin
Intramammary Infusion (Dry CowIBuffalo); Cloxacillin
Intramammary Infusion (DC/B)
Cloxacillin Benzathine Intramammary Infusion (Dry Cowl

Assay. For cloxacillin benzathine - Weigh accurately about Buffalo) is a sterile suspension of Cloxacillin Benzathine in a
60 mg, add 40 ml of methanol, shake to dissolve, add 25 ml of suitable non-aqueous vehicle containing suitable suspending
1 M sodium hydroxide and allow to stand for 30 minutes. Add agents.
27.5 ml of 1 M hydrochloric acid and sufficient water to
Cloxacillin Benzathine Intramammary Infusion (Dry Cowl
produce 100.0 ml, mix, transfer 20.0 ml of the solution to a
Buffalo) contains not less than 90.0 per cent and not more
stoppered conical flask, add 30.0 ml of 0.01 M iodine, close
than 110.0 per cent of the stated amount of cloxacillin,
the flask with a wet stopper and allow to stand for 15 minutes
C19HISClN30SS.
protected from light. Titrate the excess of iodine with 0.02 M
sodium thiosulphate, using starch mucilage, added towards Usual strength. The equivalent of500 mg ofcloxacillin.
the end ofthe titration, as indicator. Add a further 12 mg ofthe
Identification
substance under examination to 10 ml of water, swirl to
disperse,add30ml of 0.01 Miodine and titrateimmediately Extract a quantity containing1Smg.ofcloxacillin-with three
with 0.02 M sodium thiosulphate, using starch mucilage, quantities, eachofl5 ml,oflightpetroleum(l20.to 16.0).
added towards the end of the titration, as indicator. The Discard the extracts, wash the residue with 10 ml of ether and
difference between the titrations represents the volume of dry in a current ofair. The residue complies with the following
0.01 M iodine equivalent to the total penicillins present.
tests.
2648
IP 2010
CLOXACILLIN SODIUM INTRAMAMMARY INFUSION (LACTATING COW/BUFFALO)
A. Determine by infrared absOlption spectrophotometry (2.4.6).

benzathine RS or with the reference spectrum of cloxacillin
benzathine.
B. Shake 50 mg with 1 ml of 1 M sodium hydroxide for
2 minutes, add 2 ml of ether, shake for I minute and allow to
separate. Evaporate 1 ml ofthe ether layer to dryness, dissolve
the residue in 2 ml ofglacial acetic acid and add 1 ml ofdilute
potassium dichromate solution; a golden yellow precipitate
is produced.
Tests
and using a mixture of 70 volumes of dichloromethane and
Intramamrnary Infusions.
accurately a quantity of the mixed contents containing 80 mg
ofcloxacillin and extract with three quantities, each of 15 ml, of
light petroleum ( 120 to 160) previously saturated with
cloxacillin benzathine. Discard the extracts, wash the residue
with ether previously saturated with cloxacillin benzathine.
Dry in a current ofair, dissolve in 25 ml ofmethanol and dilute
to 50.0 ml with water. Dilute 2.0 ml to 100.0 ml with buffered
cupric sulphate solution pH2.0, transfer 10.0 ml to a stoppered
test-tube and heat in a water-bath at 70 for 20 minutes. Cool
to room temperature rapidly, dilute to 20.0 ml with ethanol and
maximum at about 338 nm (2.4.7), using as the blank 10.0 ml of
the unheated buffered solution of the substance under
examination after dilution to .20.0 ml with ethanol.
suitable non-aqueous vehicle containing suitable suspending

and dispersing agents.
Cloxacillin Sodium Intramammary Infusion (Lactating Cowl
Buffalo) contains not less than 90.0 per cent and not more
than 110.0 per cent of the stated amount of cloxacillin,
CI9HlsClN30sS.
Usual strength. Equivalent of 200 mg ofcloxacillin.
Identification
Extract a quantity containing 75 mg of cloxacillin with three
quantities, each of 15 ml, of light petroleum ( 120 to 160).
Discard the extracts, wash the residue with 10 ml ofether and
dry in a current ofair. The residue complies with the following
tests.
sodium RS or with the reference spectrum ofcloxacillin sodium.
B. Gives reaction A ofsodium salts (2.3.1).
Tests
using a mixture of 70 volumes of dichloromethane and
Intramamrnary Infusions.
Calculate the content of CI9HIsCIN30sS in a container of

average weight from the absorbance obtained by carrying
out the procedure simultaneously using 2.0 ml of a solution
prepared by dissolving 105 mg of cloxacillin benzathine RS
in 50.0 ml of a mixture of equal volumes of methanol and
water.
Test solution. Disperse a quantity of mixed contents of 10

containers containing about 50 mg ofcloxacillin with 15 mlof
petroleum spirit (boiling range 120 to 160), centrifuge
and discard the supernatant liquid. Repeat the extraction with
a further two 15 ml quantities of petroleum spirit (boiling
range 120 to 160). Shake the residue with 20 ml of ether,
centrifuge and dry in a current of air until the solvent get
evaporated. Dissolve the final residue in 50 ml of the mobile
phase. Dilute 5.0 ml ofthis solution to 50.0 ml with the mobile
phase.

equivalent amount of cloxacillin in the sealed container.
Reference solution (a). A 0.011 per cent wIv each ofcloxacillin

sodium RS in the mobile phase.
Reference solution (b). A solution containing 0.01 per cent w/
veach of cloxacillin sodium RS and flucloxacillin sodium
Cloxacillin Sodium Intramammary

Infusion (Lactating Cow/Buffalo)
Cloxacillin Intramammary Injection; Cloxacillin
Intramammary Infusion (Lactating Cow/Buffalo);
Cloxacillin Intramammary Infusion (LC/B)
Cloxacillin Sodium Intramammary Infusion (Lactating Cowl
Buffalo) is a sterile suspension of Cloxacillin Sodium in a
mobile phase: a mixture of25 volumes of acetonitrile
potassium dihydrogen orthophosphate, adjusted to pH
5.0 with 2 M sodium hydroxide,
2649
DICHLOFENTHION
IP 2010

- injection volume.20 /!l.
resolution between the peaks due to cloxacillin and flucoxacillin
Calculate the content ofC,9HlsClN30sS.
1 mg of CI9H17CIN3NaOsS is equivalent to 0.952 mg of
CI9HISClN30SS.
equivalent amount ofcloxacillin.
w/v solution of lithium hydroxide in a mixture of8 volumes of

methanol, 1 volume of diethylene glycol and 1 volume of
water. The principal spot in the chromatogram obtained with
the test solution corresponds to that in the chromatogram
C. Burn 50 mg by the oxygen-flask method (2.3.34), using
20 ml of 1 M sodium hydroxide as the absorbing liquid. The
solution obtained, after acidification with 2 M nitric acid
gives reaction A of chlorides and reaction C of phosphates
(2.3.1).
Tests
Weight per ml (2.4.29). 1.296 to 1.316 g.
Dichlofenthion
Test solution (a). Dissolve 0.3 gof the substance under

Test solution (b). A solution containing 0.3 per cent w/v of
the substance under examination and 0.2 per cent w/v of
methyl stearate (internal standard) in dichloromethane.
Mol. Wt. 315.2

Dichlofenthion is 0-2,4-dichlorophenyl-O,O-diethyl
phosphorothioate.
Dichlofenthion contains not less than 95.0 per cent and not
more than 100.5 per cent ofC IOH I3 Clz03PS.
Category. Insecticide.
Description. A colourless or pale yellow, oily substance.
Identification
dichlofenthion RS or with the reference spectrum of
dichlofenthion.
Reference solution. A solution containing 0.3 per cent w/w of

dichlofenthion RS and 0.2 per cent w/v of methyl stearate
(internal standard) in dichloromethane.
w/w ofphenyl methyl silicone fluid (50 per cent phenyl)
on acid-washed, silanised diatomaceous support (80 to
100 mesh) (such as OV-17),
- temperature:
column 190,
Calculate the content ofC IOH 13 Clz03PS.
Dichlorophen

~~~~djE~{()fenthio,!RSin meth9'101. __ ~
_
OH
OH
CI
CI
~
y
y

5 volumes of 2-butanone.
C I3 H IOClz02
Mol. Wt. 269.1
Dichlorophe:n-ls2,2,:,:methylenebis(4':-chloropiienol).-------
... Apply_to the plate2_/!Lof~eachso1ution.A:ftecdevelopment,

dry the plate in air and spray with a 2 per cent w/v solution of Dich10rophen contains not less than 97.0 per cent and not
4-(4-nitrobenzyl)pyridine in ethyl acetate. Heat the plate at more than 101.0 per cent of C 13H IOC120 2, calculated on the
130 for 10 minutes, allow to cool and spray with a 2 per cent dried basis.
2650
....
__ .....
._~
...
IP 2010
DlCHLOROPHEN VETERINARY AEROSOL

Category. Anthelmintic and fungicide.

Dose. Dogs and cats. As anthelmintic, 200 mg per kg ofbody
weight.
Description. A white or almost white powder; odour, slightly
phenolic.
Identification
absorption maxima at about 245 nm and 304 nm. The
absorbances ofthe solution after further dilution with an equal
volume of 0.1 M sodium hydroxide at these maxima are about
0.65 and 0.27 respectively.
B. Dissolve 0.2 g in 10 ml of 2.5 M sodium hydroxide, cool in
ice and add a solution prepared by mixing I ml of sodium
nitrite solution with a cold solution containing 0.15 ml of
aniline in a mixture of4 ml of water and I ml of hydrochloric
acid; a reddish-brown precipitate is produced.
C. Fuse 0.5 g with 2 g of anhydrous sodium carbonate, cool,

extract the residue with water and filter. The filtrate gives
reaction A ofchlorides (2.3.1).
D. Melting point (2.4.21). about 175.

peak corresponding to 4-dichlorophenol is not more than the
reeference solution (b). The content of 4,4'-dichloro-2,2'(2-hydroxy-4-chloro-m-xylene-a,a-diyl)diphenol in the
substance under examination does not exceed 8.0 per cent
w/w and the sum of the contents of any other impurities,
excluding 4-chlorophenol, is not more than 2.0 per cent w/w.
2-propanol. Titrate with 0.1 M tetrabutylammonium
0.02691 gofC 13H lOCh02'
Labelling. The label states that the substance is intended for
animal treatment only.
Tests
Chlorides (2.3.12). Shake 3.0 g with 6 ml of ethanol (95 per
cent), dilute with water to 100 ml, allow to stand for 5 minutes
cWorides (330 ppm).
Dichloropben Veterinary Aerosol
Sulphates (2.3.17). Shake 1.0 g with 20 ml ofwater for 2 minutes

Dichlorophen Veterinary Aerosol is a solution of Dichlorophen in a suitable solvent to which suitable propellants have
been added. It may contain a suitable dye as a marker.

(2.4.14).
Dichlorophen Veterinary Aerosol contains not less than

amount ofdichlorophen, C13HlOCh02.

Dichlorophen Veterinary Aerosol Spray; Dichlorophen

Veterinary Spray
Usual strengths. 2 per cent w/w; 7 per cent w/w; 7.5 per cent
w/w; 10 per cent w/w.

dichlorophen impurity standard RS in the mobile phase.
Identification

4-chlorophenol in the mobile phase.

octadecylsilane bonded to porous silica (10 /lm) (such
75 volumes of methanol, 25 volumes of water and 1
volume of glacial acetic acid,

Test solution. Solution A obtained in the Assay diluted with
methanol to contain the equivalent of 1 per cent w/v of
Dichlorophen.
dichlorophen RS in methanol.
2651
DICHLOROPHEN VETERJNARY AEROSOL
IP 2010

dry the plate in air and spray with a freshly prepared solution
containing 3.5 per cent w/v ofjerric chloride and 0.25 per
cent w/v ofpotassium jerricyanide. The principal spot in the
chromatogram obtained with the test solution cQrresponds
solution.
Identification
Tests
When examined in the range 220 to 360 urn (2.4.7), the resulting
solution shows absorption maxima at about 245 nm and
304 nm; absorbances at about 245 urn and 304 urn, about 1.3
and 0.54 respectively.

Aerosols.
Assay. Weigh the intact container. Place the container in an
ice-bath for 15 minutes. Make a small hole about 1cm from the
top ofthe body of the container and let the propellant escape.
When the flow ofpropellant stops, enlarge the hole. Transfer
the contents of the container to a tared vessel capable of
being fitted with a reflux condenser. Remove the top of the
container carefully retaining all fragments. Wash the container
with suitable solvents and add the washings to the tared vessel.
Dry the container and reweigh to obtain the net weight ofthe
contents. Heat the contents of the tared vessel under reflux
for 30 minutes, cool and weigh (solution A). Dilute an accurately
measured volume of the resulting solution containing about
0.25 g ofDichlorophen to 100.0 ml with acetone. Dilute 2.0 ml
of this solution to 200.0 ml with ammonia bufferpH 10.9 and
mix. To 10.0 ml ofthe resulting solution add 20 ml of ammonia
buffer pH 10.9 and 2 ml of a freshly prepared 2 per cent w/v
solution of 4-aminophenazone, mix, and add 2 ml ofa freshly
prepared 8 per cent w/v solution of potassium jerricyanide.
Dilute to 50.0 ml with ammonia buffer pH 10.9 and allow to
stand for 15 minutes. Measure the absorbance ofthe resulting
solution at the maximum at about 510 urn (2.4.7), using as the
blank a solution obtained in a similar manner by carrying out
the procedure simultaneously, beginning at the words "To
10.0 ml of the resulting solution....." but omitting the
4-aminophenazone solution. Calculate the weight of
C 13 H IOClzOz in the container from the absorbance obtained by
repeating the; operation using a 0.25 per cent w/v solution of
dichlorophen in acetone beginning at the words "Dilute
2.0m1.....".
Labelling. The label states (1) the weight of Dichlorophen
present in the container; (2) the total weight of contents; (3)
the name and proportion of any added dye.
~Dich:loropherfTabletStontain~~tiotless-th:ati95:0petc-etimtrd
not more than 105:0 percent of the stated amount of

dichlorophen, C 13H IOClzO z.
Usnal strength. 500 mg.

of Dichlorophen with 50 ml of 0.1 M sodium hydroxide for
15 minutes, add sufficient 0.1 M sodium hydroxide to produce
100 ml, centrifuge and dilute a suitable volume of the supernatant liquid with 0.1 M sodium hydroxide to produce a
solution containing 0.002 per cent w/v of Dichlorophen.

of Dichlorophen with a mixture of 5 ml of water and 5 ml of
5 M sodium hydroxide, filter, cool in ice and add a solution
prepared by mixing 1 ml of sodium nitrite solution with a cold
solution containing 0.15 ml of aniline in a mixture of 4 ml of
water and 1 ml of hydrochloric acid; a reddish-brown
C. Fuse a quantity ofthe powdered tablets containing 0.5 g of
Dichlorophen with 2 g of anhydrous sodium carbonate, cool,
extract the residue with water and filter. The filtrate gives
reaction A ofchlorides (2.3.1).
Tests
(2.4.14).

containing 0.50 g ofDichlorophen with 20 ml of methanol for
10 minutes, filter, add 7 ml of water and dilute to 50 ml with the
mobile phase.
Rejerence solution (a). A 1.0 per cent w/v solution of
dichlorophen impurity standard RS in the mobile phase.
4-chlorophenol in the mobile phase.
octadecylsilane bonded to porous silica (10 Ilm) (such
75 volumes of methanol, 25 volumes of water and
1 volume of glacial acetic acid,
I!lj~.Ltll~t~i'lt sollJtillll Jll1~t I~fer~ng~i'lQJutionJ!:~Irl.th~.
chrolTI atogral11 obtaineci with the test solution tIle are~ of the
peak coiresponding to 4-clic:hlorophenol is not more than the
reeference solution (b). The content of 4,4'-dichloro-2,
2652
IP 2010
DIETHYLCARBAMAZINE INJECTION
2'-(2-hydroxy-4-chloro-m-xylene-a,a' -diyl) diphenol in the

substance under examination does not exceed 8.0 per cent
w/w and the sum of the contents of any other impurities,
excluding 4-chlorophenol, is not more than 2.0 per cent w/w.
quantity of the powder containing 0.1 g of Dichlorophen,
shake with 50 ml of 0.1 M sodium hydroxide for 15 minutes
and add sufficient 0.1 M sodium hydroxide to produce
100.0 mi. Centrifuge and dilute 10.0 ml ofthe clear supernatant
liquid to 100.0 ml with 0.1 M sodium hydroxide. Dilute 20.0 ml
ofthis solution to 100.0 ml with 0.1 M sodium hydroxide and
maximum at about 304 urn (2.4.7). Calculate the content of
Ct3HIQCb02 taking 275 as the specific absorbance at 304 urn.
Diethylcarbamazine Citrate Injection.
Diethylcarbamazine Injection is a sterile solution of Diethy1carbamazine Citrate in Water for Injections.
Diethylcarbamazine Injection contains not less than 95.0 per
diethylcarbamazine citrate, CIQH21N30,C6Hg07.
Usual strength. 400 mg in 1 mi.
Identification
A. To a volume containing 0.5 g ofDiethylcarbamazine Citrate
add 2 ml of water and make alkaline with 5 M sodium
hydroxide. Extract with four quantities, each of 5 ml, of
dichloromethane, reserve the aqueous solution for test B,
wash the combined dichloromethane extracts with water and
remove the dichloromethane by evaporation. Add 0.5 ml of
iodoethane to the residue and heat gently under a reflux
condenser for 5 minutes. Remove the excess iodoethane with
a current of air, dissolve the viscous yellow oil in 2 ml of
ethanol (95 per cent) and add, with continuous stirring,
sufficient ether to precipitate the quaternary ammonium salt.
Decant off the ether, dissolve the residue in 2 ml of ethanol
(95 per cent), reprecipitate with ether and dry at 105; the
residue melts at about 152 (2.4.21).
N,N'-Dimethylpiperazine andN-methylpiperazine. Determine

silica gel G.

30 volumes of 2-butanone and 5 volumes of strong ammonia
solution.
Test solution. Dilute a volume ofthe injection with sufficient
methanol to produce a solution containing the equivalent of
5.0 per cent w/v ofDiethylcarbamazine Citrate.
diethylcarbamazine citrate RS in methanol.
N,N'-dimethylpiperazine in methanol.
Reference solution (c). A 0.010 per cent w/v solution
N-methylpiperazine in methanol.
Apply to the plate 10 JlI of each solution. Allow the mobile
phase to rise 12 cm. Dry the plate at 105 and expose it to
iodine vapours for 30 minutes. Any spots corresponding to
N,N'-dimethylpiperazine and N- methylpiperazine in the
chromatogram obtained with the test solution are not more
intense than the spots in the chromatograms obtained with
reference solutions (b) and (c) respectively.
Assay. To an accurately measured volume containing 8 g of
Diethylcarbamazine Citrate add sufficient water to produce
100.0 ml. To 10.0 ml of this solution add 2 ml of5 M sodium
hydroxide and extract with four quantities, each of 25 ml, of
dichloromethane. Wash each extract with the same two
quantities, each of20 ml, of water and with a third quantity if
the second becomes alkaline to phenolphthalein solution.
Extract the combined dichloromethane extracts in succession
with 25.0 mlofO.05 Msulphuricacidand 15 mland 10 ml of
water. Combine the acid and water extracts, remove the
dichloromethane, by wanning, cool and titrate the excess of
acid with 0.1 M sodium hydroxide using bromocresol green
CloH21N30,C6Hg07.
B. Neutralise the aqueous solution obtained in test A with

1 M sulphuric acid, add an excess of mercuric sulphate
solution, boil and add a few drops ofpotassiumpermanganate
solution; a white precipitate is produced.
Tests
pH (2.4.24). 6.0 to 7.0.
2653
DIHYDROSTREPTOMYCIN SULPHATE
IP 2010
Reference solution (a). A 0.10 per cent w/v of

dihydrostreptomycin sulphate RS in water.
Dihydrostreptomycin Sulphate
Reference sol~tion (b). A solution containing 0.10 per cent

w/v of dihydrostreptomycin sulphate RS, 0.1 0 per cent w/v
of neomycin sulphate RS and 0.10 per cent w/v of kanamycin
phase to rise 12 cm. Dry the plate in a current ofwaqn air,
spray it with a mixture of equal volumes of a 0.2 per cent w/v
solution of naphthalene-I,3-diol in ethanol (95 per cent)
and a 46 per cent w/v solution of sulphuric acid and heat at
1500 for 5 to 10 minutes. The principal spot in the chromatogram
B. Dissolve 0.1 g in 2 ml of water and add 1 ml of dilute
sodium hypochlorite solution (3 per cent Cl) and water; a
red colour is produced.
(~IRl1N70d2,3H2S04
Mol. Wt.1461.4
Dihydrostreptomycin sulphate is 0-2-deoxy-2-methylaminod-L-lyxofuranosyl-(1~4)_Nl,W-diamidino-D-streptamine

sulphate.
Dihydrostreptomycin Sulphate contains not less than 95.0
per cent and not more than 102.0 per cent sum of
C 42H 88N 14036S3 and C42H 84N 14036S3, calculated on dried basis.
Dose. All species except cats. By subcutaneous or
intramuscular injection, the equivalent of 10 mg of
dihydrostreptomycin per kg of body weight twice daily.
(Each 12.8 mg of dihydrostreptomycin sulphate is
approximately equivalent to 10 mg of dihydrostreptomycin).
Description. A white or almost white powder; may be
hygroscopic.
Identification
the plate in the following manner. Mix 0.3 g of carbomerwith
240 ml of water, allow to stand with moderate stirring for 1
hour, adjust to pH 7.0 by the gradual addition with constant
shaking of2 M sodium hydroxide and add 30 g of silica gel H.
Spread a uniform layer of the resulting suspension 0.75 mm
thiclc. Heat the plate at 1100 for 1 hour, allow to cool and use
. immediately.

Test solution. Dissolve 1'00 mg of the substance under
C. Dissolve 10 mg in 5 ml of water and add 1 ml of 1 M hydrochlOl'ic acid. Heat in a water-bath for 2 minutes. Add 2 ml of
a 0.5 per cent w/v solution of I-naphthol in 1 M sodium
hydroxide and heat in a water-bath for 1 minute; a violet-pink
colour is produced (distinction from streptomycin).
D. Gives the reactions ofsulphates (2.3.1).
Tests
dioxide-free water is not more intensely .coloured than degree
of about 20 0 for 24 hours, is not more opalescent than
pH (2.4.24). 5.0to 7.0, determined in a25 percentw/v solution.

Specific optical rotation (2.4.22). -83 0 to -91 0, calculated on
the dried basis, determined in a 2 per cent w/v solution in
water.
Sulphate. 18.0 per cent to 21.5 per cent, calculated on the
dried basis.
Dissolve 0.25 gin 100 ml of water, adjust the pH to 11 with
strong ammonia solution and add 10.0 m1 of 0.1 M barium
chloride and 0.5 mg of metalphthalein. Titrate the excess of
barium chloride with 0.1 M disodium edetate, adding 50 ml of
ethanol (95 per cent) when the colour of the solution begins
tochangean(fcontfnuingthe1i1:i:atiori~untirtlieviofet'::1J1ue
colour disappears:
sulphate, S04.
2654
IP 2010
DIHYDROSTREPTOMYCIN INJECTION
Streptomycin. Weigh accurately about 0.10 g and dissolve in

sufficient water to produce 5.0 ml. Add 5.0 ml of 0.2 M sodium
hydroxide and heat for exactly 10 minutes in a water-bath.
Cool in ice for exactly 5 minutes, add 3 ml of a 1.5 per cent
w/v solution offerric ammonium sulphate in 0.25 M sulphuric
acid and sufficient water to produce 25.0 ml, and mix. Exactly
20 minutes after the addition ofthe ferric ammonium sulphate
solution, measure the absorbance of a 2-cm layer at the
maximum at about 525 urn (2.4.7), using as the blank a solution
prepared in the same manner, omitting the substance under
examination. The absorbance is not more than that obtained
by carrying out the procedure simultaneously using 5.0 ml of
a solution prepared by dissolving 10 mg, accurately weighed,
of streptomycin sulphate RS in sufficient water to produce 50
ml and beginning at the words "Add 5.0 ml....", both
absorbances being calculated on the dried basis.
Methanol. Determine by gas chromatography (2.4.13).

Reference solution. A 0.008 per cent w/v of methanol.
a glass column 1.5 to 2.0 m x 2 to 4 mm, packed with
ethylvinylbenzenedivinylbenzene copolymer (150 to
180 mm) porous polymer beads (such as Porapak Q),
temperature:
column 50,
flow rate. 30 to 40 ml per minute ofthe carrier gas.
chromatogram obtained with test solution is not more than
that O;f the peak in the chromatogram obtained with reference
dihydrogen phosphate and dilute to 1000 ml with water,

adjusted to pH 3.0 with 2.25 per cent orthophosphoric
acid,
The relative retention time with reference to dihydrostreptomycin for impurity A is about 0.2, for impurity B is
about 0.8, for streptomycin is about 0.9 and for impurity C is
about 0.95.
Inject the reference solution. Run the chromatogram 1.5 times
the retention time of dihydrostgreptomycin.
Calculate the content ofC42H ggN 14036S3 and ofC 42 Hg4N 14036S3'
Calculate the sum of these contents.
Dihydrostreptomycin Sulphate intended for use in the

requirement.
per mg of dihydrostreptomycin sulphate.

exceeding 30. If it is intended for use in the manufacture of
parenteral preparations or intramammary infusions, the
container should be sterile and sealed so as to exclude
micro-organisms.

Labelling. The label states (1) the number ofUnits per mg; (2)
the name and quantity of any added stabiliser; (3) whether or
not the contents are intended for use in the manufacture of
Parenteral Preparations or intramammary infusions; (4) that
the substance is meant for veterinary use only; (5) the storage
conditions; (6) the date after which the contents are not

dihydrostreptomycin sulphate RS (containing impurities
A,B,C) in 5.0 ml of water.

on 1 g by drying over phosphorus pentoxide at 60 at a
- coloumn temperature. 45,
- mobile phase: mix 4.6 g of anhydrous sodium sulphate,
1.5 g of sodium octanesulphonate, 120 ml of acetonitrile
and 50 ml of 2.72 per cent w/v solution of potassium
Dihydrostreptomycin Sulphate Injection.

Dihydrostreptomycin Injection is a sterile solution of
Dihydrostreptomycin Sulphate in Water for Injections. It is
prepared by dissolving the contents of a sealed container in
the requisite amount of Water for Injections immediately
before use.
2655
DIHYDROSTREPTOMYCIN INJECTION
IP 2010
Dihydrostreptomycin Injection contains not less than

amount of dihydrostreptomycin, CZ1H41N701Z, calculated on
the dried basis.
Usual strength. The equivalent of 250 mg of

dihydrostreptomycin.
Description. A white or almost white powder which yields a
clear, colourless or faintly yellow solution when dissolved in
water.
The injection complies with the tests stated under Parenteral
Preparations (Powders for Injection).
following requirements.
Identification
A. Determine by thin-layer chromatography (2.4.17), prepared
by mixing 0.3 g of cm"bomer with 240 ml of water, allow to
stand with moderate stilTing for 1 hour, adjust to pH 7.0 by the
gradual addition with constant shaking of 2 M sodium
hydroxide and add 30 g of silica gel H. Spread a unifOlID layer
of the resulting suspension 0.75 mm thick. Heat the plate at
110 for 1 hour, allow to cool and use immediately.

Test solutioi? Dissolve 100 mg of the sunbstance under
Reference solution (a). AO.lO per cent w/v of dihydrostreptomycin sulphate RS in water.
w/v of dihydrostreptomycin sulphate RS, 0.10 per cent w/v of
neomycin sulphate RS and 0.10 per cent w/v of kanamycin
phase to rise 12 cm. Dry the plate in a current of warm air,
spray it with a mixture of equal volumes of a 0.2 per cent w/v
solution of naphthalene-I,3-diol in ethanol (95 per cent)
and 46 per cent w/v solution of sulphuric acid and heat at
150 for 5 to 10 minutes. The principal spot in the chromatogram
obtained with test solution corresponds to that in the
B. Dissolve 0.1 g in 2 ml of water and add 1 ml of dilute
I-naphthol solution and 2 ml ofa mixture of equal volumes of
sodium hypochlorite solution (3 percent CO-and water; a
red colour is produced;
C. Dissolve 10 mg in 5 ml of water and add 1 ml of 1 M
hydrochloric acid. Heat in a water-bath for 2 minutes. Add
2 ml ofa 0.5 per cent w/v solution of I-naphthol in 1 Msodium

hydroxide and heat in a water-bath for 1 minute; a violet-pink
colour is produced (distinction from streptomycin).
D. Gives the reactions ofsulphates (2.3.1).
Tests
dioxide-free water is not more intensely coloured than degree
of about 20 for 24 hours, is not more opalescent than
pH (2.4.24). 5.0 to 7.0, determined ill a 25 per centw/v solution.
Specific optical rotation (2.4.22). -83 to-91, calculated on
the dried basis, determined in a 2 per cent w/v solution in
water.
Sulphate. 18.0 per cent to 21.5 per cent, calculated on the
dried basis.
Dissolve 0.25 g in 100 ml of water, adjust the pH to 11 with
strong ammonia solution and add 10.0 ml of 0.1 M barium
chloride and 0.5 mg of metalphthalein. Titrate the excess of
barium chloride with 0.1 M disodium edetate, adding 50 ml of
ethanol (95 per cent) when the colour of the solution begins
to change and continuing the titration until the violet-blue
colour disappears.
sulphate, S04.
Streptomycin. Weigh accurately about 0.1 0 g and dissolve in

sufficient water to produce 5.0 m!. Add 5.0 ml of 0.2 M sodium
hydroxide and heat for exactly 10 minutes in a water-bath.
Cool in ice for exactly 5 minutes, add 3 ml of a 1.5 per cent
w/v solution offerric ammonium sulphate in 0.25 M sulphuric
acid and sufficientwater to produce 25.0 ml, and mix. Exactly
20 minutes after the addition ofthe ferric ammonium sulphate
solution, measure the absorbance of a 2-cm layer at the
prepared in the same manner, omitting the substance under
by carrying out the procedure simultaneously using 5.0 ml of
a solution prepared by dissolving 10 mg, accurately weighed,
of streptomycin sulphate RS in sufficient water to produce
50 ml and beginning at the words "Add 5.0 m!....", both
absorbances being calculated on the dried basis.
l\'lethan~!.l?e_tell.11il1e~yg;~~ cllI:?11J:atogr~E~y ~.4 .13).
Test solution. Dissolve 4.0 g of the substance under examination in 100 ml of water.
Reference solution. A 0.008 per cent w/v of methanol.
2656
IP 2010
DIMETRIDAZOLE
- a glass column 1.5 to 2.0 m x 2 to 4 rom, packed with
ethylvinylbenzene-divinylbenzene copolymer (150 to
180 mm) porous polymer beads (such as Porapak Q),
- temperature:
column 50,
- flow rate. 30 to 40 ml per minute ofthe can'ier gas.
chromatogram obtained with test solution is not more than
that ofthe peak in the chromatogram obtained with reference
solution (0.2 per cent);
Dimetridazole contains not less than 98.0 per cent and not
more than 101.0 per cent ofthe stated amount ofdimetridazole,
C sH 7N 30 2, calculated on the anhydrous basis.
Dose. In drinking water. Pigs. 250 mg per litre. Poultry. 75 to
250 mg per litre. In feed. Pigs. Therapeutic dose, upto 500 g
per tonne; prophylactic dose, upto 200 g per tonne. Pouluy.
75 to 500 g per tonne.
Description. An almost white to brownish yellow powder;
darkens on exposure to light, odourless or almost odourless.
Identification

on 1 g by dlying over phosphorus pentoxide at 60 at a

Compare the spectrum with that obtained with dimetridazole
RS or with the reference spectrum of dimetridazole.
Assay. On the mixed contents often containers cany out the

microbiological assay, Method A or B (2.2.10), and express the
result in Units of dihydrostreptomycin per mg.
B. When examined in the range of230 to 360 nm (2.4.7), a

0.002 per cent w/v solution ill methanol shows a well-defined

requirement.
per mg of dihydrostreptomycin sulphate.
Dihydrostreptomycin Sulphate intelided for use in the

Storage. Store protected from light. Use the injection within
7 days ofthe preparation ofthe solution when stored in a cool
place or within 1 month when stored in a cold place.
Labelling. The label states (1) the strength in tenns of the
equivalent amount of dihydrostreptomycin in a suitable
dose-volume; (2) that the contents are meant for veterinaly
use only; (3) the storage conditions; (4) the date after which
the contents are not intended to be used.
Dimetridazole
C. Dissolve 0.1 g in 20 ml of ether, add 10 ml of a 1 per cent

w/v solution ofpicric acid in ether, induce clystallisation by
scratching the sides of the vessel and allow to stand. Wash
the precipitate obtained with ether and dry at 105; the residue
Tests
2-Methyl-5-nitroimidazole. Determine by thin-layer
GF254.
and 10 volumes of 2-propanol.
in 100 ml of dichloromethane.
Reference solution. A 0.0 I 0 per cent w/v of 2-methyl5-nitroimidazole RS in dichloromethane.
Apply to the plate 5 j..ll of each solution. After development,
illy the plate in air and examine in ultraviolet light at 254 nm.
Any spot cOlTesponding to 2-methyl-5-nitroimidazole in the
reference solution.
Water (2.3.43). Not more than 1.0 percent, determined on 1 g.
CsH7N30 2
Mol. Wt. 141.1
Dimetridazole is 1,2-dimethyl-5-nitro-lH-imidazole.
anhydrous glacial acetic acid. Tiu'ate with 0.1 M perchloric

acid, using crystal violet solution as indicator. eany out a
blank titration.
2657
DIMETRIDAZOLE PREMIX
IP 2010

CSH7N 30 Z
crystallisation and allow to stand. Wash the precipitate

obtained with ether and dry at 105; the residue melts at about
160 (2.4.21).
Tests

Oral Powders.
Dimetridazole Premix contains not less than 95.0 per cent and
dimetridazole, CSH7N 30 Z
Assay. Weigh accurately a quantity containing 0.4 g of

Dimetridazole, transfer to a sintered glass .funnel (porosity
No.4), add 10 ml of dichloromethane, stir for 1 minute, and
apply gentle suction. Repeat the extraction with four further
quantities, each of 10 ml, of dichloromethane. To the combined
dichloromethane extracts add 50 ml of anhydrous glacial acetic
acid previously neutralised to crystal violet solution by the
dropwise addition of 0.1 M perchloric acid. Titrate with
Identification
Mix a quantity containing 0.1 g ofDimetridazole with 20 ml of
ether, shake and filter. To the filtrate add 10 ml of a 1 per cent
w/v solution of picric acid in ether, stir to induce
crystallisation and allow to stand. Wash the precipitate
obtained with ether and dry at 105; the residue melts at about
160(2.4.21).

CSH7N 30 Z
Tests
Dimetridazole, transfer to a sintered glass funnel (porosity
No.4), add 10 ml of dichloromethane, stir for 1 minute, and
apply gentle suction. Repeat the extraction with four further
quantities, each of 10 ml, of dichloromethane. To the combined
dichloromethane extracts add 50 rn1 of anhydrous glacial acetic
acid previously neutralised to clystal violet solution by the
dropwise addition of 0.1 M perchloric acid. Titrate with
CSH7N 30 Z
Dinitolmide
o~ NH~H3
2X
.
OzN
CSH7N30 S
NOz
Mol. Wt. 225.2
Dinitolmide is 3,5-dinitro-2-methylbenzamide.
Dinitolmide contains not less than 98.0 per cent and not more
than 100.5 per cent ofCsH7N 30 s, calculated on the dried basis.
Dose. Poultry. Upto 200 g per tonne of feed.
Description. A cream to light tan powder.
Identification
Dimetridazole Veterinary Oral Powder is a mixture of

Dimetridazole and a suitable water-soluble diluent.
Dimetridazole Veterinary Oral Powder contains not less than
amount ofdimetridazole, CSH 7N 30 Z

Compare the spectrum with that obtained with dinitolmide
RS or with the reference spectrum of dinitolmide.
B. Heat 1 g with 20 ml of 9 M sulphuric acid under a reflux
condenser for 1 hour, cool, add 50 ml of water and filter. The
residue after washing with water and drying at 105 melts at
about205\~.'.~"J.
Identification
MiXiiquaiifitY 6oritiiiiiingo.TgotDimetndazolewlth20 mlof Tests

ether, shake and filter. To the filtrate add 10 ml of a 1 per cent
w/v solution of picric acid in ether, stir to induce
Acid value (2.3.23). Not more than 5.0, detennined on 0.5 g and
using 50 ml of ethanol (95 per cent) as the solvent.
2658
ETHOPABATE
IP 2010


10 volumes of methanol and 5 volumes ofglacial acetic acid.
Reference solution (a). A 0.0125 per cent wIv ofthe substance
under examination in acetone.
Reference solution (b). A 0.0125 per cent w/v of o-toluic acid
in acetone.
Spray with titanium trichloride solution, diluted 5 times with
water, heat at 100 for 5 minutes and spray with ethanolic
dimethylaminobenzaldehyde solution. When viewed under
ultraviolet light at 354 nm the spot in the chromatogram
obtained with reference solution (b) is more intense than any
corresponding spot in the chromatogram obtained with the
test solution. By both methods ofvisualisation any secondary
Assay. Weigh accurately about 0.15 g, dissolve in acetone
and dilute to 50.0 ml. To 10.0 ml of the solution add 10 ml of
glacial acetic acid and 15 ml of a 40 per cent w/v solution of
sodium acetate. Maintain a stream of carbon dioxide through
the flask throughout the determination. Add 25.0 ml of 0.1 M
titanium trichloride and allow to stand for 5 minutes. Add
10 m1 of hydrochloric acid, 10 m1 of water and 1 m1 ofpotassium
thiocyanate solution. Titrate with 0.1 M ferric ammonium
sulphate until the solution becomes first colourless and then
orange. Repeat the operation without the substance under
the amount oftitanium trichloride required.
I ml of 0.1 Mtitanium trichloride is equivalentto 0.001876 g
ofCsH7N30 s.
Ethopabate
Ethopabate contains not less than 96.0 per cent and not more
than 104.0 per cent of ethopabate, CI2HlSN04, calculated on
the dried basis.
Dose. PoultlY. 4 to 8 g per tonne of feed.
Description. A white or pinkish white powder, odourless or
almost odourless.
Identification
A. Detelmine by infrared absorption spectrophotometry (2.4.6).
Compare the spectmm with that obtained with ethopabate RS
or with the reference spectrum of ethopabate.
B. When examined in the range 230 to 360 nm (2.4.7), a
maxima at about 268 nm and atabout 299 nm; absorbance at
about 268 nm, about 1.3 and at about 299 nm, about 0.58.
Tests
Diazotisable substances. Dissolve 0.2 g in 10 ml of
dichloromethane and extract in succession with 100 ml and
90 ml of 0.1 M hydrochloric acid, combine the acid extracts,
wash with 5 ml of dichloromethane, dilute to 200 ml with
0.1 M hydrochloric acid and filter. To 5 ml, add 6 ml of 1 M
hydrochloric acid and 1 ml of a 0.1 per cent w/v solution of
sodium nitrite, mix, and allow to stand for 4 minutes. Add 1 ml
of a 0.5 per cent w/v solution of ammonium sulphamate, mix
and allow to stand for 3 minutes. Add 1.0 ml of a 0.1 per cent
w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride, mix, and allow to stand for 30 minutes. Absorbance
ofthe resulting solution at about 545 nm (2.4.7), not more than
0.70.
Phenolic substances. Dissolve 0.25 g in 15 ml of methanol
and add sufficient methanol to produce 25 ml. To 5 ml add
5 ml ofa 3 per cent w/v solution of anhydrousferric chloride,
mix and allow to stand for 10 minutes. Absorbance of the
resulting solution at about 525 nm (2.4.7), not more than 0.70,
using as the blank a solution prepared by adding 5 ml of a
3 per cent w/v solution of anhydrousferric chloride to 5 ml of
methanol.
on 1.0 g, by drying in an oven at 105 at a pressure not
exceeding 0.7 kPa.
Solvent mixture. Equal volumes of methanol and water.

Mol. Wt. 237.3
Ethopabate is methyI4-acetamido-2-ethoxybenzoate.

examination in 100 ml ofthe solvent mixture. Dilute 1.0 ml of
2659
FURAZOLIDONE VETERINARY ORAL SUSPENSION
IP 2010

ethopabate RS in the solvent mixture.
Reference solution. A 0.5 per cent w/v solution offurazolidone

RS in acetone.

w/v of ethopabate RS and 0.01 per cent w/v of methyl-4acetamido- 2-hydroxybenzoate RS in the solvent mixture.
Apply to the plate 10 f..ll of each solution. After development,

silica particles the surface of which has been modified
with chemically bonded phenyl groups (l0 f..lm),
mobile phase: a mixture oB volumes of acetonitrile, 15
volumes of methanol and 45 volumes of 0.15 M sodium
resolution factor between the peaks due to ethopabate and
methyl-4-acetamido-2 hydroxybenzoate is not less than 1.2.
Calculate the content ofClzH,sN04.
Furazolidone Veterinary Oral

Suspension
Tests
Oral Liquids.

Weigh accurately a quantity of the well-shaken suspension
containing 35 mg ofFurazolidone, add slowly and with stining,
50 ml of dimethylformamide. Warm on a water-bath, with
occasional stilTing, until most ofthe solid is dissolved. Decant
the supernatant liquid and extract the residue further with two
quantities, each of 50 ml, of dimethylformamide, decanting
the supernatant solution. No yellow colour should be visible
in the third extract. Cool the combined dimethylformamide
extracts, add sufficient water to produce 500.0 ml and filter. To
10.0 ml ofthe filtrate add sufficient water to produce 100.0 ml
CSH 7N 30 S taking 754 as the specific absorbance at 367 nm.
Detelmine the weight per ml of the suspension (2.4.29), and
calculate the content of furazolidone, weight in volume.
Furazolidone Veterinary Mixture; Furazolidone Mixture;

Furazolidone Drench
Labelling. The label states that the oral suspension should

be administered undiluted.
Furazolidone Veterinary Oral Suspension is an aqueous

suspension of Furazolidone.
Furazolidone Veterinary Oral Suspension contains not less
than 90.0 per cent and not more than 11 0.0 per cent of the
stated amount of furazolidone, CSH 7N 30 S.
Usual strengths. 5 per cent w/v; 7.5 per cent w/v.
Identification
Furazolidone Premix
Furazolidone Premix contains not less than 95.0 per cent and
furazolidone, CSH7N 3 0 S'
A. Add 0.2 ml to a mixture of 15 ml of dimethy?formamide and

1 ml of 0.5 M ethanolic potassium hydroxide; a blue colour is
produced.
Identification

A. To a mixture of 15 ml of dimethylformamide and 1 ml of

0.5 M ethanolic potassium hydroxide add 5 mg ofthe premix;
a blue colour is produced.
Mobile phase. A mixture of 50 voIumesofdir::lILQrQlngllIqng

and 40 volumes of nitromethane and._, 10 volumes
of methanol.
....
B. Deteriniiie bylliiil=layer clif6mat6gi;apliy(2~4:T7),C6atihg
Test solution. Shake a quantity of the suspension containing

5 mg ofFurazolidone with 1 ml of acetone, allow to stand, and

and 40 volumes of nitromethane and 10 volumes of methanol.
... ,
__
__
__
2660
IP 2010
HALOXON
Test solution. The supernatant liquid obtained by shaking a

quantity of the premix containing 5 mg of furazolidone with
1 ml of acetone.
Reference solution. A 0.5 per cent w/v solution offurazolidone
RS in acetone.

Tests
Assay. Protect the solutions from light througllOut the assay.
Identification
A. Dissolve about 20 mg in 10 ml of dioxan, add 0.5 ml of
0.1 M hydrochloric acid and dilute to 25 ml with methanol.
Dilute 1 ml to 25 ml with methanol.
When examined in the range 230 to 360 nm (2.4.7), the resulting
solution exhibits a maximum at about 290 nm and a less well
defined maximum at about 312 nm. Ratio ofthe absorbance at
about 312 nm to that at about 290 nm, about 1.08.
B. Dissolve 0.1 gin 5 ml of5 M sodium hydroxide with the aid

of warming, cool, acidifY 1 ml of the solution by the addition
of 2 M nitiric acid and add 1 ml of silver nitrate solution, a
white precipitate is formed. The precipitate is soluble in 5 M
ammonia giving a brown solution which exhibits a green
fluorescence when viewed under screened ultraviolet light.
C. Melting range (2.4.21).88 to 93.
Weigh accurately a quantity ofthe premix containing 35 mg of

Furazolidone, add 50 ml of dimethylformamide and shake for
20 minutes. Add sufficient water to produce 500.0 ml and
filter. To 10.0 ml ofthe filtrate add sufficient water to produce
100.0 ml and measure the absorbance ofthe resulting solution
at the maximum at about 367 urn (2:4.7). Calculate the content
ofCsH 7N 30 s taking 754 as the specific absorbance at 367 nrn.
3-Chloro-4-methylumbelliferone. Not more than 2.0 per cent.
Determine the weight per m1, (2.4.29), and calculate the content
offurazolidone, weight in volume.
NOTE
Prepare the solutions immediately before use and
protectedfrom light.
Dissolve 0.20 g in 50 ml of 0.01 M methanolic hydrochloric

acid and dilute 5 ml to 100 ml with 0.01 M methanolic
hydrochloric acid. Measure the fluorescence of the resulting
solution (2.4.5), using an excitation wavelength of about
345 nm and an emission wavelength of about 400 nm and
setting the spectrofluorimeter to zero with 0.01 M methanolic
hydrochloric acid and to 100 with a standard solution prepared
by dissolving 25 mg of 3-chloro-4-methylumbelliferone RS
in sufficient 0.01 M methanolic hydrochloric acid to produce
250 ml (solution A) and diluting 5 m1 to 100 ml with 0.01 M
methanolic hydrochloric acid. Calculate the content of
3-chloro-4-methylumbelliferone from a calibration curve
prepared by measuring the fluorescence of suitable dilutions
of solution A.
Haloxon
C1Jfl4Ch06P
Mol. Wt.415.6
Haloxone is phosphoric acid bis(2-chloroethyl) 3-chloro4-methyl-2-oxo-2H-1-benzopyran-7-yl ester.

Haloxon contains not less than 95.0 per cent and not more
than 100.5 per cent of CI4HI4Cb06P, calculated on the dried
basis
Dose. All species exceptpoultry. 50 mg per kg ofbody weight.
Poultry. 50 to 75 mg per kg ofbody weight.
. Description. A white or almost white powder.
Tests
Acidity. Dissolve 0.1 g in 10 ml of ethanol (95 per cent)
previously neutralised to methyl red solution; the solution
requires for neutralisation not more than 0.1 ml of 0.1 M sodium
hydroxide.
0.7kPa.
acetonitrile to produce 10 ml and record the infrared
absorption ofa 0.2 ImTI layer ofthe solution at the maximum at
about 1155 cm-1(2.4.6). Construct a base line between the
minima at about 1125 cm- Iand 1180 cm- I. Calculate the content
of CI4HI4Cb06P from the absorption obtained by repeating
the procedure using haloxon RS in place of the substance
under examination.
Storage. Avoid contact with metals.
2661
NERMECTIN
IP 2010

Ivermectin
a stainless steel column 25 cm X 4.6 mm, packed with
34 volumes of methanol and 51 volumes of acetonitrile,
Inject reference solution (a). This test is not valid unless

resolution between the component H 2B 1b (first peak) and
component H2B 1n (second peak) is not less than 3.0.
Component B1a R = CH 2 CH 3
Component B1b R = CH 3
C48 H 740 14, H2B 1n
Mol.Wt. 875.1
C47 H n O I4 , H2B 1b
Mol.Wt. 861.1
Ivennectin contains not less than 95.0 per cent and not more
than 102.0 per cent of H2 B 1n + H 2 B 1b, calculated on the
anhydrous and solvent free basis.
The ratio H2BluI(H2Bln + H 2B 1b), determined by liquid
chromatography is not less than 90.0 per cent.
Description. A white crystalline powder, slightly hygroscopic.
Identification
A. Determine by infrared absorption spectrophotometery
ivermectin RS.
Tests

chromatogram obtained with the test solution the impurity
with a relative retention of 1.3 to 1.5 with reference to the
principal peak is not more than 2.5 times the area ofthe principal
(b) (2.5 per cent). The area of any other peak is not more than
with reference solution (b) (LOper cent) and the sum ofall the
secondary peaks is not more than 5 times the area of the
Ethanol and formamide. Ethanol. Not more than 5.0 per cent
and fonnamide. Not more than 3.0 per cent, detelmined by gas
Internal standard solution. Dilute 0.5 ml of propanol to

100 ml with water.
examination in 2.0 ml of m-xylene by heating on a water-bath
at 40 to 50 0 , add 2.0 m1 of water, mix thoroughly and centrifuge.
Remove the upper layer and extract it with 2.0 ml of water.
Discard the upper layer and combine the aqueous layers. Add
1.0 ml ofthe internal standard solution. Centrifuge and discard
any remaining m-xylene.
Appearance ofsolution. A 2.0 per cent w/v solution in toluene

is clear (2.4.1) and not more intensely colored than reference
solution BY57 (2.4.1).
Reference solution (a). Dilute 3.0 g of ethanol to 100 ml with

water.
Specific optical rotation (2.4.22) -17.0 to -20.0, detennined on

a 2.5 per cent w/v solution in methanol.
Reference solution (b). Dilute 1.0 g offormamide to 100 ml

with water.

(2.4.14).
Reference solution (c). Dilute 5.0 rnl of reference solution (a)

and 5 ml ofreference solution (b) to 50.0 m1 with water. Transfer
2.0 ml ofthis solution to a centrifuge tube, add 2 m1 of m-xylene,
mix thoroughly and centrifuge. Remove the upper layer and
extract it with 2.0 ml of water. Discard the upper layer and
combmeiheaqueousTayers.Aa<fl:0mlofthe-Internaistandarc!

Refere-nce -so!lttton .(aTA-O~08-per-ceniw7vsoIUHoiior
lvermeci[n -RS iii methanol.
soli.itioii.-centiifugeaiid dis-cafdiinyremiiiiiirig m~xylene:-Reference solution (d). Dilute 10.0 ml ofreference solution (a)
and 10.0 ml of reference solution (b) to 50.0 ml with water.
2662
IP 2010
KAOLIN VETERINARY ORAL SUSPENSION
Transfer 2.0 ml ofthis solution to a centrifuge tube, add 2 ml of

m-xylene, mix thoroughly and centrifuge. Remove the upper
layer and extract it with 2.0 ml of water. Discard the upper
layer and combine the aqueous layers. Add 1.0 ml ofthe internal
standard solution. Centrifuge and discard any remaining
m-xylene.
a glass column 30 m x 0.53 mm, packed with fused silica
with macrogol20,000 with film thickness 1 mm,
temperature
column 80 increase @ 60 per minute to 240,
injection port 220 and detector 280,
- flow rate. 7.5 ml per minute ofhelium as carrier gas.
Inject 1 III of the test solution and reference solutions (c) and
(d).
Calculate the content of ethanol is not more than 5.0 per cent
andformamide not more than 3.0 per cent.
Heavy metals (2.3 .13). 1 g complies with the limit test for heavy
metals, Method C (20 ppm).
0.5gm.
Calculate the percentage contents ofivermectin (H2Bla + 1:I2Blb)
and the ratio H2BI,/(H2Bla + H 2B 1b).
Tests
Pyrogens (2.2.8). Complies with the test for pyrogens, by
injecting 0.2 mg ofIvermectin per kg body weight ofrabbit.
Test solution. Dilute accurately a volume of the injection

containing 5 mg ofIvermectin to 100 ml with water.
Reference solution. A 0.005 per cent w/v solution of ivermectin
RSin wate/:
- mobile phase: a mixture of9 volumes of methanol and 1
volume of water,
tailing factor is not less than 2.0
Calculate the content of ivermectin in the injection.
Labelling. The label states (1) the strength in mg ofIvermectin
per ml; (2) that the contents are to be used for subcutaneous
use only; (3) the names of any preservatives used.
Ivermectin Injection
Ivermectin Injection is a sterile solution ofIvermectin with or

with out one or more anaesthetics, preservatives and solvents.
Ivermectin Injection contains not less than 90 per cent and
not more than 110 per cent ofH2B 1a, and not more than 5 per
cent of H 2B 1b.
The content ofH2B 'a + H 2B 1b is not less than 95 per cent and
not more than 110 per cent ofthe stated amount ofIvermectin.
Usual strengths.for dogs. 0.2 ml per 33 kg ofbody weight;for
other animals. 1 ml per 50 kg ofbody weight.
Description. A clear, colourless to yellow colour solution.
Identification
When examined in the range 220 nm to 360 nm (2.4.7), a
Kaolin Veterinary Mixture; Kaolin Mixture

Light Kaolin
200 g
Light Magnesium Carbonate
50 g
Sodium Bicarbonate
50 g
Water to produce
1000 ml
Kaolin Veterinary Oral Suspension should be freshly prepared,

unless the Light Kaolin has been sterilised.
Kaolin Veterinary Oral Suspension contains not less than 1.04
per cent w/w and not more than 1.25 per cent w/w ofthe stated
amount of magnesium, Mg and not less than 4.05 per cent wi
wand not more than 4.65 per cent w/w ofthe stated amount of
sodium bicarbonate, NaHC0 3
Usual strengths. All species. 15 to 30 mg per kg of body
weight.
2663
KAOLIN VETERINARY ORAL SUSPENSION
IP 2010
Tests
Acid-insoluble matter. 13.8 to 18.4 per cent wlw, determined
by the following method: Weigh accurately about 3 g, add
15 m1 of water and make acid to litmus paper by the cautious
addition of2 M hydrochloric acid; boil for 5 minutes,replacing
water lost by evaporation, cool and decant the supernatant
layer through a filter. Boil the residue with 20 ml of water and
10 m1 of 2 M hydrochloric acid, cool, filter through the same
filter, and wash the residue with water until the washings are
free from chloride, reserving the filtrate and washings for the
Assay for magnesium. Dry and ignite the residue to constant
weight at red heat.

Oral Liquids.
Assay. For magnesium - Dilute the combined filtrate and
washings reserved in the determination ofadd-insoluble matter
to 100.0 ml with water. To 20.0 ml add 0.1 g of ascorbic acid,
make slightly alkaline to litmus paper with 5 M ammonia and
add 10 ml of triethanolamine, 10 ml of ammonia buffer
pH 10.9 and 1 ml of potassium cyanide solution. Titrate with
0.05 M disodium edetate using eriochrome black T solution
as indicator.
Mg.
For sodium bicarbonate - Weigh accurately about 109, boil
with 100 ml of water for 5 minutes and filter. Boil the residue
with 100 ml of water for 5 minutes and filter. Cool the combined
filtrates and titrate with 0.5 M hydrochloric acid using methyl
orange-xylene cyanol FF solution as indicator. Add 10 ml of
ammonia buffer pH 10.9 and titrate with 0.05 M disodium
edetate using eriochrome black T solution as indicator.
1 ml of 0.5 M hydrochloric acid after subtracting one fifth of

the volume of 0.05 M disodium edetate is equivalent to
0.0420 g ofNaHC03

10. volumes of methanol and 1 volume of strong ammonia
solution.
Test solution. Dilute a volume of the injection to produce a
solution containing 1.0 per cent wlv of Levamisole Hydrochloride in methanol.
Reference solution. A 1.0 per cent wlv of levamisole hydrochloride RS in methanol.
Apply to the plate 1 I.d of each solution. After development,

dry the plate in air and spray with potassium iodoplatinate
B. Dilute a volume of the injection containing 0.75 g of
Levamisole Hydrochloride to 20 m1 with water and add 6 ml of
1 M sodium hydroxide. Extract with 20 ml of dichloromethane,
discard the aqueous layer and wash the dichloromethane layer
with 10 ml of water. Dry by shaking with anhydrous sodium
sulphate, filter and evaporate the solvent at room temperature.
The residue, after drying over phosphorus pentoxide at a
pressure of 1.5 to 2.5 kPa at a temperature not exceeding 40,
C. The injection is laevorotatory.

Tests
pH (2.4.24). 3.3 to 3.7.
2,3-Dihydro-6-phenylimidazo[2,1-b]thiazole hydrochloride.
of methanol and 4 volumes of anhydrous glacial acetic acid.
Test solution. Dilute a volume ofthe injection with methanol
to produce a solution containing 5.0 per cent wlv ofLevamisole
Hydrochloride.
Levamisole Hydrochloride Injection
Levamisole Injection is a sterile solution of Levamisole
Hydrochloride in Water for Injections. It may contain suitable
colouring agents.
Levamisole Injection contains not less than 92.5 per cent and
not more than 107.5 per cent ofthe stated amount oflevamisole
hydrochloride, CIIHI2NzS,HCl.
-Usualstrength. 75-mgin-l ml.
Reference solution. A 0.025 per cent wlv of 2,3-dihydro6-phenylimidazo[2,1-b]thiazole hydrochloride RS in

methanol.

solution. Any spot corresponding to 2,3-dihydro6-phenylimidazo[2, 1-b ]thiazole hydrochloride in the
chromatogram obtained with test solution is not more intense
than the spot in the chromatogram obtained withiefereilce
Identifidition
sohition.


2664
IP 2010
LINCOMYCIN PREMIX
Assay. To an accurately measured volume containing 0.75 g

ofLevamisole Hydrochloride add 50 ml of water and 15 ml of
2 M sodium hydroxide, extract with three quantities, each of
25 ml, 20 ml and 15 ml of dichloromethane, wash the combined
extracts with two quantities, each of 10 ml, of water and discard
the washings. To the clear dichloromethane solution, after
drying with anhydrous sodium sulphate, add 50 ml of
CIIHI2N2S,HCI.
shaking with anhydrous sodium sulphate, filter and allow the

dichloromethane to evaporate at room temperature. The
residue, after drying over phosphorus pentoxide at a pressure
of 1.5 to 2.5 kPa at a temperature not exceedirig 40, melts at
about 59 (2.4.21).
C. The solution is laevorotatory.
Tests
2,3-Dihydro-6-phenylimidazo[2,1-b]thiazole hydrochloride.

of methanol and 4 volumes of anhydrous glacial acetic acid.
Test solution. Dilute a volume of the preparation under
examination with methanol to produce a solution containing
1.0 per cent w/v ofLevamisole Hydrochloride.
Levamisole Hydrochloride Veterinary

Oral Solution
Levamisole Hydrochloride Veterinary Mixture;
Levamisole Veterinary Oral Solution; Levamisole
Veterinary Mixture
Levamisole Hydrochloride Veterinary Oral Solution is an
aqueous solution of Levamisole Hydrochloride containing
suitable stabilising agents.
Levamisole Hydrochloride Veterinary Oral Solution contains
the stated amount oflevamisole hydrochloride, CIIHI2N2S,HCI.
Usual strength. 0.25 per cent w/w; 1.5 per cent w/w.
Identification
A. Detelmine by thin-layer chromatography (2.4.17), coating

solution.
Test solution. Dilute a volume of the preparation under
examination with methanol to produce a solution containing
1.0 per cent w/v ofLevamisole Hydrochloride.
Reference solution. A 1.0 per cent w/v of levamisole hydrochloride RS in methanol.
B. To a quantity containing 0.3 g ofLevamisole Hydrochloride

add 10 ml of water and 6 ml of 1 M sodium hydroxide. Extract
with 20 ml of dichloromethane, discard the aqueous layer and
wash the dichloromethane layer with 10 ml of water. DIy by
Reference solution. A 0.025 per cent w/v of 2,3-dihydro6-phenylimidazo[2,1-b]thiazole hydrochloride RS in

methanol.
Apply to the plate 50 III of the test solution and 10 III of the
reference solution. After development, dry the plate in air and
spray with potassium iodoplatinate solution. Any spot
corresponding to 2,3-dihydro-6-phenylimidazo[2, 1-b]thiazole
hydrochloride in the chromatogram obtained with the test
Oral Liquids.
Levamisole Hydrochloride add 15 ml of 2 M sodium hydroxide,
extract with three quantities each of25 ml, 20 ml and 15 ml of
dichloromethane, wash the combined extracts with two
quantities, each of 10 m1, of water and discard the washings.
To the clear dichloromethane solution, after drying with
anhydrous sodium sulphate, add 50 ml of anhydrous glacial
acetic acid. Titrate with 0.1 M perchloric acid, using
titration.
CIIHI2N2S,HCl.
Lincomycin Premix
Lincomycin Hydrochloride Premix.
Lincomycin Premix contains Lincomycin Hydrochloride.
Lincomycin Premix contains not less than 90.0 per cent and
lincomycin, ClsH34N206S.
2665
LINCOMYCIN PREMIX
IP 2010
Lithium Antimony Thiomalate
Identification
In the Assay, the chromatogram obtained with the test solution
corresponds to the chromatogram obtained with the reference
solution.
Tests
Lincomycin B. Examine test solution as described in the Assay
but increasing the sensitivity by 8 to 10 times while recording
the peak due to the trimethylsilyl derivative oflincomycin B,
which is eluted immediately before the trimethylsilyl derivative
of lincomycin. The area of the peak due to the trimethylsilyl
derivative oflincomycin B, after correction for the sensitivity
factor, is not more than 5 per cent of the area ofthe peak due
to the trimethylsilyl derivative of lincomycin.
(LiOOC-C-CHCOOLi)
H I
3
2
, 9H zO
S-Sb
C12HgLi601zS3Sb,9HzO
Mol. Wt. 766.9
Lithium Antimony Thiomalate contains not less than 15.5 per

cent and not more than 16.5 per cent of Sb and not less than
5.1 per cent and not more than 5.7 per cent ofLi, calculated on
the dried, solvent-free basis.
Category. Anthelmintic against trematodes.
Dose. Cattle. 1 to 1.2 g.
Description. A pinkish white or creamy powder; hygroscopic.
Identification
A. To 0.2 g dissolved in 5 ml of water add 2 ml of hydrochloric

acid and 5 ml ofsodium sulphide solution; a yellowish- orange
precipitate is produced which does not dissolve on addition
of dilute ammonia solution.
Test soli/tion. Dissolve a quantity ofpremix containing about

12 mg of Lincomycin Hydrochloride in 10 ml of the mobile
phase.
B. When moistened with hydrochloric acid and introduced

on a platinum wire it imparts a red colour to a non-luminous
flame.
Reference solution. A 0.12 per cent w/v solution of lincomycin

Tests
- mobile phase: a mixture of 78 volumes a solution
prepared by diluting 13.5 ml of orthophosphoric acid
to 1000 ml of water, adjusted to pH 6.0 with ammonium
hydroxide, 15 volumes of acetonitrile and 15 volumes
of methanol,
tailing factor is not more than 1.3; the column efficiency is not
less than 4000 theoretical plates and relative standard devation
for replicate injections is not more than 2.0. The relative
rentention time with reference to lincomycin for lincomycin B
is about 0.5.
"_""~m
"
_"

equivalent amount of lincomycin.

coloured than reference solution RS3 (2.4.1).
pH (2.4.24). 9.0 to 10.5, determined ina6percentw/v solution
Assay. For antimony - Weigh accurately about 0.50 g , add
35 ml of water and swirl to dissolve. Add 5 g of ammonium
persulphate, 10 ml of sodium hydroxide solution and 3 or
4 glass beads (approximately 0.5 cm diameter). Place a small
funnel in the neck ofthe flask and boil gently for 20 minutes at
such a rate that the volume is not reduced appreciably. Cool,
add through the funnel 0.25 ml of phenolphthalein solution
and sufficient 0.1 M hydrochloric acid until the last trace of
pink colour disappears. Add 25 ml ofa 10 per cent w/v solution
of oxalic acid through the funnel and boil vigorously for
3 minutes. Rinse the funnel, with a small quantity of water,
remove it and add 5 ml of hydrochloric acid and 2 g of
potassium iodide. Allow to stand for 10 minutes and boil until
the solution becomes yellow and shows no further decrease
in colour, but taking care to see that the volume is not reduced
to less than about 30 ml. Cool and remove a small drop ofthe
solution with a sealed capillarymelting-pointtube and add to
starch iodidepaper.Jfabluish colour is produced, add Ldrop
of 0.1 M sodium thiosulphate while swirling and again test
with starch iodide paper. Repeat if necessary until a bluish
colour is no longer produced.
2666
IP 2010
MAGNESIUM HYPOPHOSPHITE
Add 5 g of sodium potassium tartrate, cool to about 15 0 to

20 0 and cautiously add small portions of ~odium bicarbonate
until no further effervescence is produced. Add 2 to 4 g more
of sodium bicarbonate and titrate with 0.1 M iodine until the
first permanent light yellow colour is produced.
1 ml of 0.1 M iodine is equivalent to 0.006088 g ofSb.
For lithium - Weigh accurately about 0.2 g, dissolve in 50 ml

of glacial acetic acid. Titrate with 0.1 M perchloric acid,
using 1 ml of clystal violet solution as indicator. Carry out a
blank titration.
1 ml of 0.1 M perchloric acid is equivalent to 0.000694 g ofLi.
Lithium Antimony Thiomalate Injection

Lithium Antimony Thiomalate Injection is a sterile solution of
Lithium Antimony Thiomalate in Water for Injection containing
a suitable antimicrobial preserVative.
Lithium Antimony Thiomalate Injection contains not less than
amount oflithium antimony thiomalate, C12HgLi6012S3Sb,9H20.
Assay. Dilute 10.0mI with 25 mI ofwater, add 7.5 gofammonium

persulphate and 16 ml of sodium hydroxide solution, boil
gently for 20 minutes, cool and add 0.5 ml ofphenolphthalein
solution. Neutralise the solution with dilute hydrochloric acid
and boil for 3 minutes. Add 50 ml ofa 10 per cent w/v solution
of oxalic acid, 7.5 ml of hydrochloric acid and sufficient
water to make up the volume, if necessary. Add 2 g of
potassium iodide to the hot solution, allow to stand for 10
minutes and boil until it acquires a pale yellow colour (about
10 minutes). Cool and remove the colour by adding 0.1 M
sodium thiosulphate using starch iodide solution as an
external indicator. Add 7.5 g of sodium potassium tartrate and
dilute to 200 ml. Add sodium bicarbonate carefully (avoiding
loss by spurting due to effervescence) till alkaline to litmus
paper and titrate with 0.05 M iodine using 1 ml of starch
solution, added towards the end of the titration, as indicator.
CI2HgLi6012S3Sb,9H20.
HO-P-Mg-P-OH
I
I
OH
OH
Identification
A. Dilute a volume containing 0.2 g of Lithium Antimony
Thiomalate to 5 ml with watel: Add 2 ml of hydrochloric acid
and 5 ml of sodium sulphide solution; a yellowish orange
precipitate is produced which does not dissolve on addition
of dilute ammonia solution.
B. Dilute 0.2 ml ofthe injection under examination to 10 ml
with a 5 per cent w/v solution of sodium potassium tartrate.
To 2 ml ofthe solution add sodium sulphide solution dropwise;
a reddish orange precipitate is produced. The precipitate
dissolves on adding dilute sodium hydroxide solution.
Tests
Appearance of solution. The solution is clear (2.4.1), and not
more intensely coloured than reference solution RS3 (2.4.1).
pH(2.4.24). 9.0 to 10.5.
Mg(H2P02)2,6H20
,6H 20
Mol. Wt. 262.4
Magnesium Hypophosphite contains not less than 98.5 per

cent and not more than 101.0 per cent ofMg(H2P0 2)2,6H20.
Category. Supplement in deficiency conditions; nerve tonic.
Description. Colourless crystals or white crystalline powder.
Identification
A. Gives the reactions of magnesium salts (2.3.1).
B. Dissolve about 50 mg in 5 ml of water and add 0.5 ml of
mercuric chloride solution; a white precipitate is produced.
C. Dissolve about 50 mg in 5 ml of water and acidify with
sulphuric acid. Add 0.5 ml of cupric sulphate solution and
warm; a red precipitate is produced.
Tests
Pyrogens. Complies with the test for pyrogens (2.2.8), using

per 1.5 kg ofthe rabbit's weight, a volume containing 0.012 g
ofLithium Antimony Thiomalate.

(2.4.1) and colourless (2.4.1).
Heavy metals (2.3.13). Dissolve 1.0 g in 20 ml ofwater, add 2 mI
of dilute hydrochloric acid and sufficient water to produce
25 ml. The resulting solution complies with the limit test for
2667
MECLOFENAMIC ACID
IP 2010
Chlorides(2.3.12). To 1 g add 200 ml of water and filter. 10 ml

ofthe filtrate complies with the limit test for chlorides (0.1 per
cent).
Tests
Sulphates (2.3.17). 1 g complies with the limit test for sulphates

(0.015 per cent).
Appearance of solution. A 5.0 per cent w/v solution in 1 M

sodium hydroxide is not more opalescent than reference
suspension OS2 (2.4.1) and is not more intensely coloured

water, add 5 ml of strong ammonia-ammonium chloride
solution and titrate with 0.05 M disodium edetate using 0.1 g
of mordant black II mixture as indicator, until a blue colour is
obtained.

solution in 0.01 M methanolic hydrochloric acid at the
maximum at about 279 nm, not less than 0.400 and not more
than 0.445, and at the maximum at about 335 nm, not less than
0.440 and not more than 0.490.
1 ml of O. 05 M disodium edetate is equivalent to 0.01312 g of

Mg(H2P02)2,6H20.

(2.4.14).

examination in 100 ml of ethanol.
Reference solution (a). A 0.0035 per cent w/v solution of ethyl
meclofenamate RS (internal standard). in ethanol.
Meclofenamic Acid

w/v of the substance under examination and 0.0035 per cent
w/v of ethyl meclofenamate RS (internal standard) in ethanol.
Mol. Wt. 296.2

Meclofenamic acid is N-(2,6-dichloro-3-methylphenyl)
anthranilic acid.
Meclofenamic Acid contains not less than 98.5 per cent and
Ct4HttChN02, calculated on the dried basis.
Category. Anti-inflammatory; analgesic; antipyretic.
Dose. Horses. 2.2 mg per kg of body weight daily, for 5 to 7
days.
Identification
Compare the spectrum with that obtained with mecloftnamic
acid RS or with the reference spectrum ofmeclofenamic acid.
absorption maxima at about 279 ron, and 371 nm; absorbance
at about 279nm, about 0.45, and at about 317nm, about 0.33.
C. Dissolve 25 mg in 15 ml of dichloromethane; the solution

exhibits a
blue fluorescene when examined under
D. Dissolve 1 mg in 2 ml ofsulphuric acid and add 0.05 ml of
0.02 M potassium dichromate; an intense purple colour is
produced, which rapidly fades to purple brown.
octadecasilane bonded to porous silica (10 11m) (such
as Spherisorb ODS),
25 volumes of water and 1 volume of glacial acetic acid,
Inject reference solution (b). The area ofthe peak immediately
preceding the peak due to meclofenamic acid is not more than
one-seventh ofthe area ofthe peak due to the internal standard.
The area of any other peak is not more than the area of the
peak due to the internal standard.
warm ethanol previously neutralised to phenol red solution
and titrate with 0.1 M sodium hydroxide, using phenol red
1 ml ofOIMsodium hydroxide is equivalenHo 0,02962 gof
CI4HttChN02.
2668
IP 2010
MONOSULFlRAM
Mepyramine Maleate Injection; Pyrilamine Maleate
Injection; Pyrilamine Injection
Monosulfiram contains not less than 98.0 per cent and not
more than 101.0 per cent of CIOHzoNzS3, calculated on the
anhydrous basis.
Category. Insecticide.
Mepyramine Inj~ction is a sterile solution of Mepyramine

Description. A yellow or yellowish-brown soft solid; odour,

sulphurous.
Mepyramine Injection contains not less than 95.0 per cent

mepyramine maleate, CI7HZ3N30,C4H404'
Identification
Usual strengths. 25 mg in 1 ml; 50 mg in 1 m!.

Description. Colourless or almost colourless solution.
Identification
A. To a volume containing 0.1 g ofMepyramine Maleate add
2 ml of5 M sodium hydroxide and shake with three quantities,
each on ml, of ether. Warm the aqueous layer in a water-bath
for 10 minutes with 2 ml of bromine solution, heat to boiling,
cool, and add 0.2 ml to a solution oflOmgofresorcinolin3 ml
of sulphuric acid; a blue-black colour develops on heating
for 15 minutes in a water-bath.
E. Dilute a volume containing 20 mg ofmepyramine maleate to

2 ml with water, add 1 ml of cyanogen bromide solution and
5 ml of a 2 per cent w/v. solution of potassium hydrogen
phthalate, mix, allow to stand for 15 minutes and add 1 ml ofa
4 per cent solution of aniline in ethanol (95 per cent); a
Tests

2-cm layer ofa 0.001 per cent w/v solution in methanol shows
a well-defined absorption maximum only at about 281 nm;
absorbance at about 281 nm, about 1.3.
B. Dissolve 0.1 g in a mixture of 0.15 ml of a 1 per cent w/v
solution of cupric sulphate and 5 ml of ethanol (95 per cent),
evaporate on a water-bath and dissolve the residue in
dichloromethane; a deep yellowish brown colour is produced.
C. Boil 0.1 g with 2 M hydrochloric acid; hydrogen sulphide
is evolved which has a characteristic odour and turns filter
paper treated with lead acetate solution, black.
Tests
Fl'eezingpoint (2.4.11). 28.5 to 32.0.
NOTE - Cany out the test in subdued light.

30 volumes of butyl acetate.
pH(2.4.24).5.5t06.5.

examination in 100 ml of ethyl acetate.


disulfiram RS in ethyl acetate.

25 mg of mepyramine maleate add sufficient 0.01 M
hydrochloric acid to produce 100.0 m!. Dilute 10.0 ml ofthis
solution to 100.0 ml with 0.01 Mhydrochloric acid and measure
about 316 nm (2.4.7). Calculate the content ofCI7H23N30,C4~04,

substance under examination in ethyl acetate.
Monosulfiram

the chromatogram obtained with the test solution any
secondary spot is not more intense than the spot in the
chromatogram obtained with reference solution (a) and any
other spot, apart from the principal spot, is not more intense
solution (b).
Sulfrram
H3C,
(CH 3

nitrogen-ji'ee sulphuric acid and carry out the method for the
determination ofnitrogen (2.3.30).
H3C~N I(SyN "-./CH3
S
CIOHzoNzS3
S
Mo!. Wt. 264.5
Monosulflram is bis(diethylthiocarbamoyl)sulphide.

CIQHzoN zS3.
Storage. Store protected fi'om light.
2669
MONOSULFlRAM SOAP
IP 2010
NOTE -Protect the solutionsfrom light throughout the assay.
Monosulfiram Soap.
Monosulfrram Soap contains not less than 5 per cent w/w of
monosulfiram in a toilet soap basis which may be perfumed.
Monosulfrram Soap contains not less than 90.0 per cent and
monosulfiram, CIOHzoNzS3'
Test solution (a). Weigh accurately a quantity of the finely

shredded soap containing aboutO.25 g ofMonosulfiram, shake
for 10 minutes with 50 ml of dimethylformamide, centrifuge
Identification
Test solution (b). Weigh accurately a quantity of the finely

shredded soap containing about 0.25 g ofMonosulfiram, shake
for 10 minutes with 50 ml of dimethylformamide containing
0.125 g ofN-phenylcarbazole (internal standard), centrifuge
In the test for Related substances, the principal spot in the


monosulfiram RS and 0.25 per cent w/v ofN-phenylcarbazole
(internal standard) in dimethy(formamide.
Tests
NOTE -
earlY out the test in subdued light.

Test solution (a). Shake a quantity of the finely shredded
soap containing 20 mg of Monosulfiram with 10 ml of
dichloromethane; filter and wash the filtrate with
dichloromethane. Evaporate the combined filtrate and
washings just to dryness at room temperature in a current of
nitrogen and dissolve the residue in 1 m1 of ethanol
(95 per cent).
Test solution (b). Dilute 0.5 m1 oftest solution (a) to 10 ml with
disulfiram RS in ethanol (95 per cent).
monosulfiram RS in ethanol (95 per cent).
the chromatogram obtained with test solution any spot running
ahead of the principal spot and corresponding in position to
disulfiram is not more intense than the spot in the chromatogram
obtained with reference solution (a) and any spot running
behind the principal spot is not more intense than the spot in
the chromatogram obtained with reference solution (b). Ignore
any~subsldi.aij
spotsduetoTl.1esoap basiswliich~miiY-a1sobe
observed ahead of the principal spot in the clif6iiiat6gram

obtained with test solution (a).
w/w of methyl silicone gum on acid-washed, silanised
diatomaceous support (80 to 100 mesh) (such as SE 30),
temperature:
column 180,
Calculate the content OfCIOHzoNzS3'
Labelling. The label states (1) the proportion ofMonosulfrram
in the preparation; (2) the method of use of the preparation.
Monosulfiram Solution
Monosulfiram Solution is a solution of Monosulfiram in
Ethanol (95 per cent) containing a suitable dispersing agent.
In making Monosulfrram Solution the ethanol (95 per cent)
may be replaced by Industrial Methylated Spirit provided that
the statutory requirements governing the use of Industrial
Methylated Spirit are observed.
Monosulfrram Solution contains not less than 94.0 per cent
monosulfiram, CIOHzoNzS3'
Description. Clear, bright, deep reddish-brown liquid; crystals
from which may deposit slowly at low temperatures but
dissolve on warming. Yields a pale yellow dispersion on
dilution with water.
Identification
In the test for Related substances, the principal spot in the
2670
IP 2010
NANDROLONE LAURATE
Tests
Labelling. The label states (1) the percentage w/w of monosulfiram; (2) the method of use of the preparation.

NOTE
earlY out the test in subdued light.
Nandrolone Laurate

.Test solution (a). Dilute a quantity of the solution under
examination with ethanol (95 per cent) so as to contain of
2.0 per cent w/v of Monosuifiram.
Test solution (b). Dilute 0.5 ml oftest solution (a) to 10 ml with

disulfiram RS in ethanol (95 per cent).
dry the plate in air and examine in ultraviolet light at 254 om. In
running ahead of the principal spot and corresponding in
position to disulfiram is not more intense than the spot in the
chromatogram obtained with reference solution (a) and any'
spot running behind the principal spot is not more intense
solution (b).
Test solution (a). Dilute the solution under examination in

dimethylformamide containing the equivalent of 0.5 per cent
w/v ofMonosuifiram.
Test solution (b). Weigh accurately a quantity of the finely
shredded soap containing about 0.25 g ofMonosulfrram, shake
for 10 minutes with 50 ml of dimethylformamide containing
0.125 g ofN-phenylcarbazole (internal standard), centrifuge
monosuljiram RS and 0.25 per cent w/v ofN-phenylcarbazole
(internal standard) in dimethylformamide.
w/w of methyl silicone gum on acid-washed, silanised
diatomaceous support (80 to 100 mesh) (such as SE 30),
- temperature:
column 1800 ,
inlet port 1800 and detector 280 0 ,
Calculate the content OfCIOHzoNzS3'
C30~S03
Mol. Wt. 456.7
Nandrolone Laurate is 3-oxoestr-4-en-17~-yl-dodecanoate.

Nandrolone Laurate contains not less than 97.0 per cent and
not more than 103.0 per cent ofC30H4s03, calculated on the
dried basis.
Category. Anabolic steroid; androgen.
Dose. All species. By subcutaneous or intramuscular injection,
0.2 to 1 mg per kg of body weight once every two weeks.
Description. A white to creamy white, crystalline powder;
Identification
laurate RS or with the reference spectrum of nandrolone
laurate.
the plate with silica gel GF254, surface of which has been
modified by chemically bonded octadecylsilyl groups (such
as Whatman KC l8F plates).

40 volumes of acetonitrile and 20 volumes of water.
examination in dichloromethane.
Reference solution (a). A 0.5 per cent w/v of nandrolone
laurate RS in dichloromethane.
.
Reference solution (b). A mixture ofequal volumes ofthe test
detectable and heat at 100 0 for 10 minutes. Allow to cool and
examine in ultraviolet light at 254 om. The principal spot in the
chromatogram obtained with the test solution corresponds
2671
NANDROLONELAURATE
IP 2010
solution (a). The test is not valid unless the principal spot in
the chromatogram obtained with reference solution (b) appears
Tests
in a freshly prepared 2 per cent w/v solution in dioxan.
Nandrolone. Determine by thin-layer chromatography (2.4.17),
coating the plate with silica gel G
Mobile phase. A mixture of 70 volumes of n-heptane and

Test solution. Dissolve 1.5 g of the substance under examination in dichloromethane.
Reference solution. A 0.030 per cent w/v of nandrolone RS in
dichloromethane.
detectable, spray with a 10 per cent v/v solution of sulphuric
acid in ethanol (95 per cent), heat at 105 for 30 minutes and
examine in ultraviolet light at 365 nm. Any spot in the
chromatogram obtained with the test solution corresponding
to nandrolone is not more intense than the spot in the
ethanol to produce 100.0 ml and dilute 10.0 ~I to 100.0 ml with
ethanol. Dilute 10.0 ml ofthis solution to 100.0 ml with ethanol
maximum at about 240 urn (2.4.7). Calculate the content of
C30H4S03 taking 380 as the specific absorbance at 240 nm.
by chemically bonded octadecylsilyl groups (such as

Whatman KC 18F plates).

40 volumes of acetonitrile and 20 volumes of water.
Test solution. Dilute a suitable volume with dichloromethane
to produce a solution containing 0.5 per cent w/v of
Nandrolone Laurate .
Reference solution (a). A 0.5 per cent w/v of nandrolone
laurate RS in dichloromethane.
Reference solution (b). A mixture ofequal volumes ofthe test
solution and reference solution (a)
detectable and heat at 100 for 10 minutes. Allow to cool and
chromatogram obtained with the test solution corresponds
solution (a). The test is not valid unless the principal spot in
the chromatogram obtained with reference solution (b) appears
Tests
0.1 g ofNandrolone Laurate add sufficient dichloromethane
to produce 100.0 m!. Dilute 3.0 ml ofthe resulting solution to
50.0 ml with dichloromethane. To 5.0 ml of this solution add
10 ml of isoniazidsolution and sufficient methanol to produce
20.0 m!. Allow to stand for 45 minutes and measure the
380 nm (2.4.7), using as the blank 5 ml of dichloromethane
treated in a similar manner. Calculate the content of C30H4S03
from the absorbance obtained by repeating the procedure
using a suitable quantity of nandrolone RS.
1 mg ofCIsHz60z is equivalentto 0.001664 g ofC30H4s03.

Nandrolone Laurate Injection is a sterile solution ofNandrolone
Laurate in Ethyl Oleate or other suitable ester, in a suitable
fixed oil, or in any mixture ofthese.
Nandrolone Laurate Injection contains not less than 92.5 per
nandrolone 1aurate, C30H4S03'
!!sUl!I~~!!~!!gths:~?IJ1~ i!11!J11;~~~!J1~iIl~!!J1I~ ....

Identification
plate with silica gel GF254, surface ofwhich has been modified

Niclosamide Dispersible Powder for Veterinary Use
Niclosamide Veterinary Oral Powder contains Niclosarrride with
suitable auxiliary substances.
NiclosamideVeterinaryOralPowdercontainsnotless than
97;0 per cent and not more than 103;0 per centofthe stated
amount ofniclosamide, CI3HsCIzNz04'
2672
IP 2010
NITROXYNIL
Identification
Nitroxynil is 4-hydroxy-3-iodo-5-nitrobenzonitri1e.
Heat 50 mg with 5 ml of 1 M hydrochloric acid and 0.1 g of

the filtrate add 0.5 ml of a 1 per cent w/v solution of sodium
nitrite and allow to stand for 10 minutes. Add 2 ml of a 2 per
stand for 10 minutes and add 2 m1 ofa 0.5 per cent w/v solution
of N-(l-naphthyl)ethylenediamine dihydrochloride; a deep
red colour is produced.
Nitroxynil contains not less than 98.0 per cent and not more
than 101.0 per cent ofC7H3lNz0 3, calculated on the illied basis.
Tests
Category. Anthehnintic.
Dose. Cows, buffaloes andsheep. By subcutaneous injection,
10 mg per kg ofbody weight, repeated ifnecessary at intervals
of not less than 28 days.
Description. A yellow to yellowish brown powder.
Identification
2-Chloro-4-nitroaniline. Boil a quantity containing 0.10 g of

Niclosamide with 20 ml of methanol for 2 minutes, cool, add
sufficient 1 M hydrochloric acid to produce 50 ml and filter.
To 10 m1 ofthe filtrate add 0.5 ml ofa 0.5 per cent w/v solution
of sodium nitrite and allow to stand for 10 minutes. Add 1 m1
of a 2 per cent w/v solution of ammonium sulphamate, shake,
allow to stand for 10 minutes and add 1 m1 of a 0.5 per cent
w/v solution of N-(l-naphthyl)ethylenediamine dihydrochloride. The colour produced is not more than that produced
by simultaneously treating 10 mg of 2-chloro-4-nitroaniline
in the same manner.
5-Chlorosalicylic acid. Boil a quantity containing 0.50 g of
Niclosamide with 10 m1 of water for 2 minutes, cool and filter.
To the filtrate add a few drops ofjerric chloride solution; no
red or violet colour is produced.
on 1 g by drying in an oven at 105 for 4 hours.
Oral Powders.
Niclosamide, dissolve in 60 ml of dimethylformamide with the
aid of gentle heat,cool. Titrate with 0.1 M tetrabutylammonium hydroxide, maintaining a stream of nitrogen
through the solution throughout the titration, and determining
titration.
1 m1 of 0.1 M tetrabutylammonium hydroxide is equivalent to
0.03271 g ofC13HsChNz04.
A. Determine by inti'ared absorption spectrophotometry (2.4.6).

Compare the spectrum with that obtained with nitro:>,ynil RS
or with the reference spectrum of nandro10ne nitroxynil.
0.002 per cent w/v solution in 0.01 M sodium hydroxide
exhibits maxima at about 225 nm and at about 271 nm;
absorbance at about 271nm, about 1.3.
C. When heated with sulphuric acid, iodine vapours are
evolved.
Tests
Inorganic iodide. To 0.40 g add 0.35 g ofN-methylglucamine
and 10 m1 of water. Shake to dissolve and add sufficient water
to produce 50 ml. To 10 m1 ofthe resulting solution add 4 ml of
1 M sulphuric acid and extract with three quantities, each of
10 ml, of dichloromethane. Add to the aqueous extract 1 m10f
hydrogen peroxide solution (100 vol) and 1 ml of
dichloromethane, shake for 2 minutes and allow to separate,
Any purple colour in the dichloromethane layer is not more
intense than that obtained by adding 2 ml of a 0.0026 per cent
w/v solution ofpotassium iodide to a mixture of 4 ml of 1 M
sulphuric acid and 8 m1 of water, adding 10 ml of
dichloromethane, shaking for 2 minutes, adding to the
aqueous layer 1 ml of hydrogen peroxide solution (l00 vol)
and 1 ml of dichloromethane, shaking for 2 minutes and
allowing to separate (500 ppm).
Nitroxynil
on 1 g by drying in an oven at 105 for 4 hours.
OH
lyYN0
Assay. Carry out the oxygen flask method for iodine (2.3.34),
using 25 mg.
1 m1 of 0.02 Msodium thiosulphate is equivalent to 0.0009667

g ofC7H3lNz0 3
eN
Mol. Wt. 290.0
2673
NITROXYNIL INJECTION
IP 2010
Oxfendazole
Nitroxynil Injection is a sterile solution oftheN-ethylglucamine

salt ofNitroxynil in Water for Injections.
ClM=
Nitroxynil Injection contains not less than 95.0 per cent and
not more than 105.0 per cent ofthe stated amount ofnitroxynil,
C7H3INZ0 3
H
0
N
;)--N
OCH 3
N
H
Jl
II
Identification
CIsHI3N303S
A. When examined in the range 230 nm to 360 nm (2.4.7), ofthe

final solution obtained in the Assay exhibits a maximum at
about 271 nm.
B. Heat 0.5 ml with 3 ml of sulphuric acid; iodine vapours are
evolved.
Mol. Wt. 315.4
Oxfendazole is methy15-(phenylsulphinyl)2-benzimidazolecarbamate.
Oxfendazole contains not less than 97.0 per cent and not more
than 100.5 per cent of ClsHI3N303S, calculated on the dried
basis.
Tests
pH (2.4.24). 5.0 to 7.0, determined by using a20percentw/v
solution of N-ethylglucamine hydrochloride instead of a
saturated solution ofpotassium chloride as the liquid junction
solution.
Dose. Horses. 10 mg per kg ofbody weight. Cows andbuffaloes.

4.5 mg per kg of body weight. Sheep. 5 mg per kg of body
weight.
Description. A white or almost white powder; odour, slight
and characteristic.
Inorganic iodide. To a volume containing 0.4 g ofNitroxynil

Identification
add 0.35 g ofN-methylglucamine and dilute to 100 ml with
water. To lOm10fthedilutedsolutionadd4mlofl Msulphuric A. Dissolve 0.1 gin 50 ml of methanol, evaporate to a volume
acid and extract with three quantities, each of 10 ml, of ofabout 2 m1, cool, filter, wash the residue with 2 ml of water
dichloromethane. Add to the aqueous extract 1 ml of hydrogen and dry at 105 at a pressure not exceeding 2.7 kPa. The residue
peroxide solution (100 vol) and 1 m1 of dichloromethane, complies with the following test.
shake for 2 minutes and allow to separate. Any purple colour
..
.
in the dichloromethane layer is not more intense than that . Determme by mfrared absorptIOn spectrophotometry (2.4.6).
obtained by adding 2 ml of a 0.0026 per cent w/v solution of Compare the spectrum with that obtained with oxfendazole
potassium iodide to a mixture of 4 ml of 1 M sulphuric acid RS or with the reference spectrum of oxfendazole.
and 8 ml of water, adding 10 ml of dichloromethane, shaking
for 2 minutes, adding to the aqueous layer 1 ml of hydrogen
peroxide solution (loa vol) and 1 m1 of dichloromethane,
shaking for 2 minutes and allowing to separate (500 ppm)
(0.1 per cent w/v of iodide).
1.7 g ofNitroxynil add sufficient O. 01 M sodium hydroxide to
produce 500.0 ml. Dilute 20.0 ml ofthis solution to 500.0 ml
with 0.01 M sodium hydroxide. To 5.0 ml of this solution add
sufficient 0.01 M sodium hydroxide to produce 100.0 ml and
measure the absorbance oLthe resultingsolution.atthe
!l!~)(.jl11lJ.lJ:L~t~1?9lJ.t .271.!1lJ:l(2,4,7),. C!ll(:ml!lt~.l1JeQQl1t~).1JQf
C7H3INZ0 3 taking 660 as the specific absorbance at 271 nm.


0.001 per cent w/v solution in methanol exhibits two maxima
at about 228 nm and about 297 nm; absorbances at about 228
nm, about 1.4 and at about 297 run, about 0.55.
Tests
Solvent mixture. 40 volumes of ethyl acetate and 10 volumes

TestsoliitioYI. .Dissolve 0:SgoftneslibstanceuiiaefexaIfii:
nation in 100m10fso1vent mixture.
substance under examination in solvent mixture.
2674
IP 2010
OXFENDAZOLE VETERINARY ORAL SUSPENSION
Reference solution (b). A 0.0050 per cent w/v solution of methyl

5-phenylthio-1H-benzimidazol-2-yl carbamate RSin solvent
mixture.)
Any spot corresponding to methyl 5-phenylthio-lHbenzimidazol-2-yl carbamate in the chromatogram obtained
with test solution is not more intense than the spot in the
chromatogram obtained with reference solution (b). Any other
on 1 g by drying in an oven at 105 for 2 hours at a pressure
not exceeding 0.7 kPa.
glacial acetic acid, add 3 g of potassium iodide and 1 ml of
acetyl chloride and stir for 10 minutes. Add 50 ml of 1 M
hydrochloric acid and 10 ni.l of dichloromethane and titrate
immediately with 0.1 M sodium thiosulphate, shaking after
each addition, until the dichloromethane layer is colourless.
Repeat the operation omitting the substance under
examination; the difference between the titrations represents
the amount of sodium thiosulphate required.
ofClsH13N303S.
Oxfendazole Veterinary Oral

Suspension
Oxfendazole Veterinary Mixture; Oxfendazole Mixture;
Oxfendazole Oral Suspension
Oxfendazole Veterinary Oral Suspension is an aqueous
suspension of Oxfendazole containing suitable suspending
or dispersing agents.
Oxfendazole Veterinary Oral Suspension contains not less than
amount ofoxfendazole, ClsHJ3N303S.
Usual strengths. 2.265per cent w/v; 9.06 percent w/v.
Identification
Shake a quantity containing 0.1 g ofOxfendazole with 50 ml of
methanol for 15 minutes, centrifuge, evaporate the supernatant
liquid to a volume of about 2 ml, cool, filter and wash the
residue with 2 ml of water and dry at 105 for 1 hour at a
pressure not exceeding 2.7 kPa. The residue complies with the
following tests.
Compare the spectrum with that obtained with oxfendazole
RS or with the reference spectrum of oxfendazole.
0.00 I per cent w/v solution in 1 M hydrochloric acid exhibits
three maxima, at about 226, 284 and 291 nm.
Tests
pH (2.4.24). 4.3 to 5.3.
Solvent mixture. 40 volumes of ethyl acetate and 10 volumes
Test solution. Shake a quantity containing 0.1 g ofOxfendazole
with 20 ml ofsolvent mixture and filter.
50 volumes with the solvent mixture.
Reference solution (b). A 0.0050 per cent w/v solution of methyl
5-phenylthio-1H-benzimidazol-2-yl carbamate RSin solvent
mixture.)
Apply to the plate 20 JlI of each solution. After development,
Any spot corresponding to methyl 5-phenylthio-1Hbenzimidazol-2-yl carbamate in the chromatogram obtained
with test solution is not more intense than the spot in the
chromatogram obtained with reference solution (b). Any other
Oral Liquids.
Assay. Weigh accurately a quantity of the well-mixed
suspension containing about 0.1 g ofOxfendazole and disperse
in 15 ml of water. Add 200 ml of methanol and mix in an
ultrasonic bath for 15 minutes, cool, add sufficient methanol
to produce 500.0 ml and filter. Dilute 2 ml ofthe filtrate to 50 ml
with methanol and measure the absorbance of the resulting
solution at the maximum at about 296 urn (2.4.7). Calculate the
content ofCJsH13N303S taking 550 as the specific absorbance
at 296 urn.
Determine the weight per ml of the suspension (2.4.29), and
calculate the content of oxfendazole, weight in volume.
2675
OXYCLOZANIDE
IP 2010
octadecylsilane bonded to porous silica (5 Jlm) (such as
HypersilODS),
62 volumes of methanol and 38 volumes of water
containing 0.1 per cent v/v of phosphoric acid,
- spectrophotometer set at 300 nm;
Oxyclozanide
CI
~.
CI~NVCI
I a
H
OH
OH
CI
CI
Inject alternatively test solution and the reference solution. In

the chromatogram obtained with test solution the area of any
. secondary peak with a retention time less than that of the
Oxyclozanide is 2,3,5-trichloro-N-(3,5-dichloroprincipal peak is not more than one-third of the area of the
2-hydroxyphenyl)-6-hydroxybenzamide.
Oxyclozanide contains not less than 98.0 per cent and not
solution and the area of any secondary peak with a retention
more than 101.0 per cent of C 13H6ClsN0 3 , calculated on the
time greater than that of the principal peak is not more than
dried basis.
with reference solution.
Mol. Wt. 401.5
Dose. Cows, buffaloes and sheep. 10 to 15 mg per kg of body

weight.
Description. A pale cream to cream-coloured powder.
Identification

Compare the spectrum with that obtained with oxyclozanide
RS or with the reference spectrum of oxyclozanide.
0.003 per cent w/v solution in 1 M methanolic hydrochloric
acid exhibits a maximum only at about 300 nm; absorbance at
about 300 mn, about 0.76.

on 1 g by drying in an oven at 60 at a pressure not exceeding
0.7kPa.
anhydrous pyridine and pass a stream of nitrogen through
the solution for 5 minutes. Titrate with 0.1 M tetrabutylammonium hydrOXide, maintaining a stream of nitrogen
through the solution throughout the titration, determining
titration.
0.02007 gofC 13H6ClsN03
C. Melting range (2.4.21).208 to 211.
Tests
Ionisable chlorine. Dissolve 2 g in 100 ml of methanol, add
10 ml of 1.5 Mnitric acid and titrate with 0.1 Msilver nitrate,
detennining the end-point potentiometrically (2.4.25). Not more
than 1.4 ml is required (0.25 per cent).
Oxyclozanide Veterinary Oral

Suspension

(2.4.14).
Oxyc1ozanide Oral Suspension; Oxyclozanide

Suspension; Oxyc1ozanide Mixture; Oxyc1ozanide
Drench

under examination prepared by dissolving it in a suitable
volume of methanol and slowly diluting with water containing
0.1 per cent v/v of phosphoric acid to give a solution
Oxyc1ozanide Veterinary Oral Suspension is an aqueous

suspension of Oxyclozanide containing suitable suspending
or dispersing agents.
-Oxyelozaiiide VeterinaiyOrar Suspeiisi6ncotitainsnotless
-containing~aboutthesame~proportion-ofmethanol~to~wateras
inthe mobile phase;

Reference solution. Dilute 1 ml oftest solution to 100 ml with
the mobile phase.
than 95.0 per cent and not more than 105:0 per cent of the
stated amount ofoxyclozanide, C 13H 6Cl sN0 3
Usual strength. 3.4 per cent w/v.
2676
IP 2010
OXYCLOZANIDE PREMIX
Identification
In test A for Related substances, the principal spot in the
chromatogram obtained with 10 ml of test solution
Tests
chromatography (2.4.17), coating the plate with silica gel G

(60 0 to 80 0) , 20 volumes of acetone and 5 volumes of glacial
acetic acid.
Test solution. Dilute a quantity with acetone to contain
1.0 per cent w/v of Oxyclozanide, centrifuge and use the
supernatant liquid.
3,5,6-trichloro- 2-hydroxybenzoic acid RS in acetone.
Reference solution (b). A 1.0 per cent w/v solution' of
oxyclozanide RS in acetone.
Apply to the plate 40 III and 10 III of test solution, 4 III of
reference solution (a) and 10 III ofreference solution (b). After
development, dry the plate in air and spray with a 3 per cent
w/v solution of ferric chloride in methanol. In the
chromatogram obtained with 40 III of test solution any spot
corresponding to 3,5,6-trichloro-2-hydroxybenzoic acid RS is
not more intense than that in the chromatogram obtained with
B. Determine by thin.:layer chromatography (2.4.17), coating
Assay. Protect the solutions from light throughout the

procedure. Weigh accurately a quantity containing about
60 mg ofOxyclozanide, add 60 ml of acidified methanol and
boil gently on a water-bath. Shake continuously for 20 minutes,
cool to 2 0 and dilute to 100.0 ml with acidified methanol.
Filter, dilute 5.0 ml of the filtrate to 100.0 ml with acidified
methanol and measure the absorbance ofthe resulting solution
of C 13 H 6 Cl sN0 3 taking 254 as the specific absorbance at
300nm.
calculate the content of oxyclozanide, weight in volume.
Oxyclozanide Premix
Oxyc1ozanide Granules
Oxyclozanide Premix contains Oxyclozanide.
Oxyclozanide Premix contains not less than 92.5 per cent and
oxyclozanide, C 13H6CIsN03
Identification
In test A for Related substances, the principal spot in the
chromatogram obtained with 10 ml of test solution
Tests

solution.
Related substances. A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G
Test solution. Dilute a quantity with acetone to contain

1.0 per cent w/v of Oxyclozanide, centrifuge and use the
supernatant liquid.

(60 0 to 80 0) , 20 volumes of acetone and 5 volumes of glacial
acetic acid.
Reference solution. A 0.040 per cent w/v of 2-amino4,6-dichlorophenol RS in acetone.

Apply to the plate 40 III of test solution and 4 III of reference
solution. After development, dry the plate in air and spray
with lithium and sodium molybdotungstophosphate solution.
In the chromatogram obtained with test solution any spot
corresponding to 2-amino-4,6-dichlorophenol is not more
intense than that in the chromatogram obtained with reference
solution.
Oral Liquids.
Test solution. Extract the finely powdered preparation under

examination with sufficient acetone to produce a mixture
containing 1.0 per cent w/v of Oxyclozanide, centrifuge and
3,5,6-trichloro- 2-hydroxybenzoic acid RS in acetone.
oxyclozanide RS in acetone.
Apply to the plate 40 III and 10 III of test solution, 4 III of
reference solution (a) and 10 III ofreference solution (b). After
development, dry the plate in air and spray with a 3 per cent
2677
OXYCLOZANIDE PREMIX
IP 2010
w/v solution offerric chloride in methanol. In the chromatogram obtained with 40 III of test solution any spot corresponding to 3,5,6-trichloro-2-hydroxybenzoic acid RS is not
more intense than that in the chromatogram obtained with

solution.
Test solution. Extract the fmely powdered preparation under
examination with sufficient acetone to produce a mixture
containing 1.0 per cent w/v of Oxyclozanide, centrifuge and
Reference solution. A 0.040 per cent w/v of 2-amino4,6-dichlorophenol RS in acetone.
Apply to the plate 40 III oftest solution and 4 III of reference
solution. After development, dry the plate in air and spray
with lithium and sodium molybdotungstophosphate solution.
In the chromatogram obtained with test solution any spot
corresponding to 2-amino-4,6-dichlorophenol is not more
intense than that in the chromatogram obtained with reference
solution.

procedure. Weigh accurately a quantity ofthe finely powdered
preparation under examination containing 60 mg of
Oxyclozanide, add 60 ml of acidified methanol and boil gently
on a water-bath. Shake continuously for 20 minutes, cool to 2
and dilute to 100.0 ml with acidified methanol. Filter, dilute
5.0 ml ofthe filtrate to 100.0 ml with acidified methanol and
C 13H 6ClsN03 taking 254 as the specific absorbance at 300 nm.
Labelling. The label states (1) the proportion ofoxyclozanide

in the premix and (2) the method of use of the preparation.
Oxytetracycline Veterinary Oral

Powder
Oxytetracycline Hydrochloride Veterinary Oral Powder;
Oxytetracycline Hydrochloride Soluble Powder;
Oxytetracycline Soluble Powder-----------------OXYtetraCyClIne Yetel"inary Oral Powder is a mixture of
Oxytetracycline Hydrochloride and Lactose or other suitable
diluent.
Oxytetracycline Veterinary Oral Powder contains not less than

amount ofoxytetracycline hydrochloride, CZ2Hz4NzOg,HCl.
Usual strength. 5.6 per cent w/w of Oxytetracycline

Hydrochloride.
Identification
the plate with a substance prepared by mixing 25 g of silica
gel G with 50 ml ofa mixture of2.5 ml ofglycerin and 47.5 ml of
dilute ammonia solution. After spreading the plate, allow it
to stand at room temperature till it is dry (70 to 90 minutes).

dichloromethane and 1 volume of acetone with 25 ml of 0.1 M
ammonia solution.
Test solution. Extract a quantity ofthe oral powder containing
10 mg of Oxytetracycline Hydrochloride with 20 ml of
dry the plate in air, expose to the vapours of ammonia and
reference solution (b) shows three clearly separated spots.
B. To a quantity of the powder containing 0.4 mg of
Oxytetracycline Hydrochloride add 5 ml of a 1 per cent w/v
solution of sodium carbonate, shake and add 2 ml of diazotised
sulphanilic acid solution; a light brown colour is produced.
C. Shake a quantity of the powder containing 100 mg of
Oxytetracyline Hydrochloride with J 0 ml of 2 M nitric acid
and filter. To the filtrate add activated charcoal to decolorise
it and filter again. The filtrate gives the reactions of chlorides
(2.3.1).
Assay; Determine by liquid chromatograpliy (2.4.14).
Test solution. Dissolve a quantity ofthe oral powder containing

about 50 mg ofOxytetracycline Hydrochloride in 100.0 ml of
2678
PENTOBARBITONE INJECTION
IP 2010
0.01 M hydrochloric acid. Dilute 1.0 ml of this solution to

10.0 ml with 0.01 Mhydrochloric acid.
oxytetracycline RS in 0.01 M hydrochloric acid.
Reference solution (b). A 0.1 per cent w/v solution of 4epioxytetracycline RS in 0.01 M hydrochloric acid.
tetracycline hydrochloride RS in 0.01 M hydrochloric acid.
Reference solution (d). Dilute 1.5 ml of a 0.1 per cent w/v
solution of oxytetracycline RS in 0.01 M hydrochloric acid,
1.0 ml ofreference solution (b) and 3.0 ml ofreference solution
(c) to 25.0 ml with 0.01 M hydrochloric acid.
styrene divinylbenzene copolymer (8 to 10 /lm),
mobile phase: to 50 g of 2 methylpropan-2-ol, add 200
ml of water, 60 ml of 0.33 Mphosphate buffer pH 7.5,
50 ml ofl.0 per cent wIv solution oftetrabutylammonium
hydrogen sulphate previously adjusted to pH 7.5 with
2 M sodium hydroxide and 10 ml ofa 0.04 per cent w/v
solution of disodium edetate previously adjusted to
pH 7.5 with 2 M sodium hydroxide and dilute to 1000 ml
with water,
resolution between the peaks due to 4- epioxytetracycline
and oxytetracycline is not less than 4.0, the resolution between
the peak due to oxytetracycline and tetracycline is not less
than 5.0 and the tailing factor of the principal peak due to
oxytetracycline is not more than 1.25.
Calculate the content ofCzzHz4Nz09,HCl in the oral powder.
1mg of CzzHZ~Z09 is equivalent to 1.079 mg ofCzzHz~z09,HCl.
Pentobarbitone Sodium Injection; Pentobarbital Sodium
Injection
Pentobarbitone Injection is a sterile solution ofPentobarbitone
Sodium in a suitable vehicle.
Solutions containing 20 per cent w/v of Pentobarbitone
Sodium in 100-ml and 500-ml containers are also available for
use other than for injection. Such solutions may be coloured

and need not be sterile but must comply with all other
requirements of this monograph.
Pentobarbitone Injection contains not less than 95.0 per cent
pentobarbitone sodium, CIIHI7NzNa03'
Description. A clear, colourless or almost colourless solution.
Identification
Compare the spectrum with that obtained with pentobarbitone
RS or with the reference spectrum of pentobarbitone.
B. The residue obtained in the Assay melts at about 128
(2.4.21 ).
C. When introduced on a platinum wire into a Bunsen burner
flame, a golden yellow colour is imparted to the flame.
Tests
pH (2.4.24). 10.0 to 11.5.
Isomer. To a volume of the injection containing 0.3 g of
Pentobarbitone Sodium diluted, ifnecessary, to 5 ml with water
add 0.3 g of 4-nitrobenzyl bromide dissolved in 10 ml of
ethanol (95 per cent). Heat under a reflux condenser for
30 minutes, cool to 25, scratch the sides of the vessel with a
glass rod ifnecessary to induce crystallisation, filter and wash
the residue with five quantities, each of5 ml, ofwater. Transfer
the residue as completely as possible to a small flask, add
25 ml ofethanol (95 per cent) and heat under a reflux condenser
for 10 minutes. Filter the hot solution, cool to 25 and scratch
the sides of the vessel with a glass rod to induce
crystallisation. Filter and wash the residue with two quantities,
each of5 ml, ofwater and dry at 105 for 30 minutes. The dried
residue melts completely between 136 and 148 (2.4.21).

0.5 g of Pentobarbitone Sodium diluted to 15 ml with water
add 5 ml of2 M hydrochloric acid, extract with 50 ml ofether
and then with successive quantities, each of 25 ml, of ether
extracts with two quantities, each of 5 ml, of water and wash
the combined aqueous extracts with 10 ml of ether. Add the
ether washings to the main ethereal extract, filter and wash the
filter with ether. Evaporate the solvent and dry the residue to
constant weight at 105.
1 g of the residue is equivalent to 1.097 g OfCIIHI7NzNa03'
2679
PROGESTERONE
IP 2010
Progesterone
365 nm. The principal spot in the chromatogram obtained with

the test solution corresponds in position, colour in day light,
fluorescence under ultraviolet light and size to that in the
Tests
in a 1.0 per cent w/v solution in ethanol (95 per cent).
Related substances. Determine by thin layer chromatography
Mol. Wt. 314.5
Progesterone is pregn-4-en-3,20-dione.

Progesterone contains not less than 97.0 per cent and not
more than 103.0 per cent ofCzlH300z, calculated on the dried
basis.
Solvent mixture. 90 volumes of dichloromethane and 10


Dose. By subcutaneous or intramuscular injection. Cows,

buffaloes and horses. 200 to 800 mg per kg of body weight
every 48 hours. Dogs. 3 mg per kg of body weight. By
implantation. Cows, buffaloes andhorses. 200 to 400 mg. Dogs.
25 to 100 mg.

ml withthe solvent mixture.
Apply the plate 5 III of each solution. After development, dry
the plate in air and spray with a saturated solution of
potassium dichromate in sulphuric acid (70 per cent), heat at
1300 for 30 minutes and allow to cool. Any secondary spot in
the chromatogram obtain with test solution is not more intense
than the spot in the chromatogram obtain with reference
solution.
Description. Colourless crystals or a white or almost white

crystalline powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Sulphated ash (2.3.18). Not more than 0.1 per cent.
Compare the spectrum with that obtained with progesterone
RS or with the reference spectrum of progesterone. If the Loss on drying (2.4.19). Not more than 0.5 per cent, determined
0
spectra are not concordant, prepare spectra using 5 per cent on 0.5 g by drying in an oven at 105 for 2 hours.
w/v solutions in chloroform IR.
- Assay. Weigh accurately about 10 mg and dissolve in 100.0 ml
E. Determine by thin-layer chromatography (2.4.17), coating of ethanol (95 per cent). Dilute 5.0 ml of the solution to 50.0
ml with ethanol (95 per cent). Measure the absorbance ofthe
Mobile phase. A mixture of 66 volumes of dichloromethane Calculate the content of CZIH300Z taking 535 as the specific
Solvent mixture. 90 volumes of dichloromethane and 10
.
Progesterone Inj ection
Reference solution. A 0.1 per cent w/v solution ofprogesterone

RS in the solvent mixture.
Progesterone Injection is a sterile solution of Progesterone in

Ethyl Oleate or other suitable ester, in a suitable fixed oil or in
any mixture of these. It may contain suitable alcohols.
Apply to the plate 5 III of each solution. After removal of the

plate, dry the plate in air and examine in ultraviolet light at 254
-nm;-T~eprincipalspot-in.thechromatogramobtained-withthe
testsolutioncorrespondstothatinthe.chromatogramobtained
with the reference solution. Spray the plate with ethanolic
sulphuric acid (20 per cent), heat at 1200 for 15 minutes, allow
to cool and examine in day light and under ultraviolet light at
._.- Progesferone IiijeCtioii-coiifamsl1otTess.tnan92-:-S-pefcenf -
aiidii6Cfiiore tliaril07.5per cent of the stated amount of

progesterone, CZIH300Z.
Usual strengths. 250 mg per ml.
2680
IP 2010
PROMAZINE HYDROCHLORIDE
Identification
Dissolve a volume containing 50 mg ofProgesterone in 8 ml of
lightpetroleum ( 40 to 60) and extract with three quantities,
each of 8 ml, of a mixture of? volumes of glacial acetic acid
and 3 volumes of water until the solution becomes turbid,
allow to stand in ice for 2 hours and filter. The precipitate, after
washing with water and drying at 105, complies with the
following tests.
Compare the spectrum with that obtained with progesterone
RS or with the reference spectrum of progesterone. If the
spectra are not concordant, prepare spectra using 5 per cent
w/v solutions in chloroform JR.

Mobile phase. A mixture of equal volumes of cyclohexane
and light petroleum (40 to 60).
produce 100.0 mI. Dilute 3.0 ml to 50.0 ml with dichloromethane.

To 5.0 ml ofthe solution add 10 ml of isoniazid solution and
sufficient methanol to produce 20.0 ml. Allow to stand for
45 minutes and measure the absorbance of the resulting
blank 5 ml of dichloromethane treated in the same manner.
Calculate the content of CZIH300Z from the absorbance
obtained by repeating the procedure using a 0.003 per cent
w/v solution of progesterone RS in dichloromethane and
beginning at the words "To 5.0 ml of the solution.....".
Storage. Store protected from light. If solid matter separates
on standing, it should be redissolved by heating before use.
Labelling. The label states (1) the composition ofthe solvent;
(2) that the preparation is intended for veterinary use by
subcutaneous or intramuscular injection only.

progesterone RS in the solvent mixture.
was done.
Apply to the plate 2 )..1.1 of each solution. Allow the mobile
the solvent to evaporate, heat at 120" for 15 minutes and spray
Heat at 120" for a nrrther 10 minutes, allow to cool and examine
reference solution (a). The principalspot in the chromatogram
compact spot.
, Hel
C 17HzoNzS,HCl
Mol. Wt. 320.9
Promazine Hydrochloride is 10-(3-dimethylaminopropyl)

phenothiazine hydrochloride.
Promazine Hydrochloride contains not less than 99.0 per cent
and not more than 101.0 per cent ofC 17HzoN zS,HCl, calculated
on the dried basis.
Category. Sedative.
Dose. All species. By intramuscular injection, upto 1 mg per
kg of body weight.
Identification
out.


Compare the spectrum with that obtained with promazine
hydrochloride RS or with the reference spectrum ofpromazine
hydrochloride.

50 mg of Progesterone add sufficient dichloromethane to

0.001 per cent w/v solution in 0.01 Mhydrochloricacid shows
Tests
2681
PROMAZINE INJECTION
IP 2010
an absorption maximum at about 252 nm and a less well-defined

maximum at about 302 nm; absorbance at about 252 nm, about
0.93.
B. To a volume containing 5 mg ofPromazine Hydrochloride

5 minutes; an orange colour is produced.

for 5 minutes; an orange colour is produced.
C. To a volume containing 0.2 g ofPromazine Hydrochloride

add 1 ml of 1 M sodium hydroxide and extract with four
quantities, each of 10 ml, of ether. Wash the combined extracts
with 10 ml of water, remove the ether and dissolve the residue
in 4 ml of methanol. Heat on a water-bath almost to boiling,
immediately add 2 ml ofa boiling 3.5 per centw/v solution of
picric acid in methanol and boil for 2 minutes. Cool in ice,
filter, wash the crystals thrice with methanol, dissolve in 10 ml
of hot methanol and repeat the crystallisation and washing.
The rust-red crystals so obtained, after drying at 105 for
1 hour, melt at about 144 (2.4.21).
D.Meltingrange(2.4.21).I77to 181.
Tests
pH (2.4.24). 4.2 to 5.4, determined in a 5 per cent w/v solution.
acetone. Titrate with 0.1 M perchloric acid, using 3 ml of a
saturated solution of methyl orange in acetone as indicator.
Tests
pH (2.4.24). 4.4 to 5.2.
Related substances. Carry out the test for identification of

related substances in phenothiazines (2.3.5), using mobile
phase A and applying separately to the plate 10 III of each of
the following freshly-prepared solutions.
Promazine Injection
Test solution. Dilute a volume of the injection with sufficient

methanol to produce a solution containing the equivalent of
1.0 per cent w/v ofPromazine Hydrochloride.

C 17HzoNzS,HCl.
Promazine Hydrochloride Injection

Promazine Injection is a sterile solution of Promazine
Hydrochloride in Water for Injections free from dissolved air
and containing suitable buffering and stabilising agents. The
solution is distributed in containers, the air in which is replaced
by nitrogen or other suitable gas.
Promazine Injection contains not less than 95.0 per cent and
not more than 105.0 per cent ofthe stated amount ofpromazine
hydrochloride, C 17HzoNzS,HCl.

test solution is more intense than the spot in the chromatogram
obtained with reference solution (a) and not more than one
such spot is more intense than the spot in the chromatogram
Description. A colourless or almost colourless liquid.

procedure.
Identification
A. To a volume containing 0.1 g ofPromazine Hydrochloride
and extract the mixture with 25 ml of ether. Wash the ether
extract with two quantities, each of 5 ml, of water, dry with
anhydrous sodium sulphate and evaporate the ether. A 10 per

Promazine Hydrochloride, add 5 ml of2 M hydrochloric acid
and sufficient water to produce 1000.0ml. To 10.Omladd lOml
of 0.1 M hydrochloric acid, dilute to 100.0 ml with water and
c~~'Y/Y~QI1.!tionofth~oilyresiduein
c~lorofor111complies
with the following test.
..
_.
--- .. rnax.imumatabout251nm (2.4.'7); Calculatethe .contentof
Cl'iHzoN zS,HCl taking 935 as the specific absorbance at
Determine by infrared absorption spectrophotometry (2.4.6). 251nm.
Compare the spectrum with that obtained with promazine
hydrochloride RS, treated inthe same manner.
2682
IP 2010
RAFOXANIDE VETERINARY ORAL SUSPENSION
Rafoxanide
n ou
~ I
I~NH ~
1

solution.
in 100 ml in dichloromethane.
I~
CI
CI
Reference solution. A 0.0 I0 per cent w/v of rafoxanide RS in

dichloromethane.
OH
I
Mol. Wt. 626.0

Rafoxanide is N-[3-chloro-4-(4-chlorophenoxy)phenyl]2-hydroxy-3,5-diiodobenzamide..
Rafoxanide contains not less than 98.0 per cent and not more
than 101.0 per cent ofC I9 H 1JChI2N0 3, calculated on the dried
basis.
on 1.0 g by drying in an oven at 90 0 at a pressure not exceeding
Assay. To 50 ml ofdioxan add 1 mlofphenolphthaleinsolution,

replace the air in the flask with nitrogen and titrate with 0.1 M
sodium hydroxide. Weigh accurately about 1.25 g, dissolve it
in the mixture and again titrate with 0.1 M sodium hydroxide.
The difference between the titrations represents the amount
of 0.1 M sodium hydroxide required.
Dose. Cows, buffaloes, sheep and goats. 7 to 12 mg per kg of

body weight.
Description. A greyish-white to brown powder.
Identification
Compare the spectrum with that obtained with rafoxanide RS
_ or with the reference spectrum ofrafoxanide.
0.004 per cent w/v solution in 0.1 Mmethanolic hydrochloric
acid shows absorption maxima at about 280 nm and at 335 nm;
absorbance at about 280 nm, about 0.97 and at about 335 nm,
about 0.59.
C. Burn 20 mg by the oxygen-flask method (2.3.34), using 5 ml
of 2 M sodium hydroxide as the absorbing liquid, and dilute
to 25 ml with water. To 5 ml add I ml ofsilver nitrate solution;
a yellow precipitate is produced; add 5 ml of 5 M ammonia,
shake, filter, and acidify the filtrate with nitric acid; a white
D. Shake 10 mg with 10 ml of ethanol (80 per cent) and add
0.1 ml of ferric chloride test solution; a violet colour is
produced.

CI9HIIClzI2N03'
Rafoxanide Veterinary Oral

Suspension
Rafoxanide Suspension; Rafoxanide Veterinary Mixture;
Rafoxanide Mixture
Rafoxanide Veterinary Oral Suspension is an aqueous
suspension ofRafoxanide containing suitable suspending and
dispersing agents and antimicrobial preservatives.
Rafoxanide Veterinary Oral Suspension contains not less than
90.0 per cent and not more than 110.0 per cent ofrafoxanide,
CI9HIIClzI2N03'
E. Melts at about 175 0 (2.4.21).
Identification
Tests
Related substances. Determine bythin-layer chromatography
A. Evaporate a volume containing 0.2 g of Rafoxanide to

dryness on a water-bath and heat the residue over a Bunsen
burner flame; the vapours tum moistened starch-iodidepaper
blue.
NOTE - Carry out the test in subdued light and use freshly
prepared solutions.
B. In addition to the absorbance at about 335 nm, measure the

absorbance at about 280 nm (2.4.7), of the final solution
2683
RONIDAZOLE
IP 2010
obtained in the Assay. The ratio of the absorbance at about

280 nm to that at about 335 nm is 1.59 to 1.69.
C. Melts at about 167 0 (2.4.21).
Tests
Tests
Other tests. Complies with requirements stated under
Veterinary Oral Liquids.
Assay. Weigh accurately a quantity of the well-mixed
suspension containing about 0.12 g of Rafoxanide in a
stoppered 50-ml test tube and add 15 ml of 0.1 M sodium
hydroxide and 15 ml of ether. Shake for 5 minutes and
centrifuge. Remove the ether layer and repeat the extraction
with three further quantities, each of 15 ml, of ethel: Dilute the
combined ether solutions to 250.0 ml with ether and mix. Dilute
5.0 ml of this solution to 100.0 ml with 0.1 Mmethanolic
hydrochloric acid, mix and measure the absorbance of the
resulting solution at about 335 nm (2.4.7). Calculate the content
of CI9HI,ChlzN03 taking 149 as the specific absorbance at
335nill.
Detennine the weight per ml of the suspension (2.4.29), and
calculate the content ofrafoxanide, weight in volume.
Ronidazole

methanol is not more intensely coloured than reference
(1-Methyl-5-nitroimidazol-2-yl)methano .Detennine by thinlayer chromatography (2.4.17), coating the plate with silica
gel GF254.

of methanol and 5 volumes of glacial acetic acid.
in 100 m1 in acetone.
Reference solution. A 0.0050 per cent w/v of (l-methyl-5-nitroimidazol-2-yl)methanol RS in acetone.
test solution corresponding to (1-methyl-5-nitroimidazol2-yl)methanol RS is not more intense than the spot in the
Water (2.3.43). Not more than 0.5 per cent, detennined on 5 g.
Mol. Wt. 200.2
Assay. Weigh accurately about 0.3 g, dissolve in 50 m1 of

acid, detennining the end-point potentiometrically (2.4.25).
Ronidazole is 1-methyl-2-[(carbamoyloxy)methyl]5-nitroimidazole.

C6H gN 40 4
Ronidazole contains not less than 98.5 per cent and not more
than 101.0 per cent ofC6H gN 4 0 4 , calculated on the anhydrous
basis.
Storage. Store proted from light.
Category. Antiprotozoan.
Dose. Pigs. In drinking water, 60 mg per litre ofwater, offered
continuously for 3 days; in feed, 120 mg per kg offeed, offered
continuously for 5 days.
Description. A white to yellowish-brown powder; odourless
or almost odourless.
Identification

Ronidazole Veterinary Oral Powder is a mixture ofRonidazole
with suitable diluents.
Ronidazole Veterinary Oral Powder contains not less than
amount ofronidazole, C6H gN 40 4

Compare the spectrum with that obtained with ronidazole RS
of ronidazole.
Identification
B. When examined in therange 230nmt0360 nm (2A.7),a

0.002 per cent w/v solution in 0.1 M methanolic hydrochloric
acid shows an absorption maximum only at about 270 nm;
absorbance at about 270 nm, about 0.64.
A .. Shake a .quantityQfJbepQWde.LC:Q!1!~iJJj.llgOJgQf
Ronidazole with 10 m1 of acetone for 15 minutes, filter and
evaporate the filtrate to dryness. The residue complies with
the following test.
2684
IP 2010
SERUM GONADOTROPHIN FOR VETERINARY USE

Compare the spectrum with that obtained with ronidazole RS
or with the reference spechllm ofronidazole.
maximum only at about 281 om.
Category. Gonadotrophic honnone.

Dose. By subcutaneous or intramuscular injection. Cows and
she buffaloes. 2 to 4 Units per kg ofbody weight. Pigs. 5 to 15
Units per kg of body weight. Sheep. 15 Units per kg of body
weight. Dogs. 10 to 20 Units per kg of body weight.
Description. A white or pale grey, amorphous powder.
Tests
Identification
(1-Methyl-5-nitroimidazol-2-yl)methanol. Detennine by thinlayer chromatography (2.4.17), coating the plate with silica
gel GF254.
Causes enlargement of the ovaries of immahlre female rats

when administered as directed in the Assay.

of methanol and 5 volumes of glacial acetic acid.
Test solution. Shake a quantity of the powder containing
0.1 g ofRonidazole with 10 ml ofacetone for 15 minutes and
filter.
Reference solution. A 0.0050 per cent w/v of(l-methyl-5-nitroimidazol-2-yl)methanol RS in acetone.
test solution corresponding to (l-methyl-5-nitroimidazol-2-yl)methanol RS is not more intense than the spot in
Oral Powders.
Assay. Weigh accurately a quantity ofpowder containing 2 g
ofRonidazole, dissolve in 450 ml ofwater and add sufficient
100.0 ml with 0.1 M hydrochloric acid and measure the
281 om (2.4.7). Calculate the content ofC6HsN40 4 taking 279
as the specific absorbance at 281 om.
Storage. Store proted from light.
Serum Gonadotrophin for Veterinary

Use
Equine Serum Gonadotrophin for Veterinary Use
Serum Gonadotrophin for Veterinary Use is a dry preparation
of a glycoprotein fraction, obtained from the serum or plasma
of pregnant mares in their 60th to 75th day of pregnancy,
which stimulates the formation of follicles and induces
leutinising activity.
Serum Gonadotrophin for Veterinary Use contains not less
than 1000 Units per mg, calculated on the anhydrous basis.
Tests
80mg.
Assay. Carry outthe biological assay ofserum gonadotrophin
described below.
The potency of serum gonadotrophin for veterinary use is
detennined by comparing its effect in increasing the weight of
the ovaries of immature rats with that of the Standard
Preparation of serum gonadotrophin under the conditions of
the following method of assay.
for serum gonadotrophin, equine, for bioassay, established in
1966, consisting of the freeze-dried active principle from the
serum ofpregnant mares, with lactose (supplied in ampoules
containing 1600 Units), or other suitable preparation the
potency of which has been determined in relation to the
Method
Test animals. Use immahJre female rats ofthe same strain, 21
to 28 days old, differing in age by not more than 3 days and of
approximately equal weights such that the difference between
the heaviest and the lightest rat is not more than 109. Assign
the rats at random to six equal groups of not less than five
animals. If sets of six litter-mates are available, allot one
litter-mate from each set at random to each group and mark
according to the litter.
Procedure. Choose three doses of the Standard Preparation
and three doses of the preparation under examination such
that the smallest dose is sufficient to produce a positive
response in some of the rats and the largest dose does not
produce a maximal response in all of the rats. Use doses in
geometric progression. As an initial approximation total doses
of 8, 12 and 18 Units may be tried although the dose will
depend on the sensitivity of the animals used, which may
vary widely. Dissolve separately the total quantities of the
preparation under examination and ofthe Standard Preparation
2685
SERUM GONADOTROPHIN FOR VETERINARY USE
IP 2010
corresponding to the doses to be used in sufficient of a sterile

saline solution containing I mg of bovine albumin per ml
such that each single dose may be administered by the
injection of6 equally-divided portions, in the same volume of
about 0.2 m!. Store the solutions at a temperature 2 to 8.
Inject subcutaneously into each rat the dose allocated to its
group. Repeat the injections 18,21,24,42 and 48 hours after
the fIrst injection. Kill the rats between 40 hours and 72 hours
after the last injection and remove the ovaries. Remove any
extraneous fluid and tissue and immediately weigh the two
ovaries from each rat.
methods using the combined weight of the two ovaries of
each animal as the response.
Limits oferror - The estimated potency is not less than 80 per

cent and not more than 125 per cent of the stated potency.
The fIducial limits of error (P = 0.95) ofthe estimated potency
are not less than 64 per cent and not more than 156 per cent of
the stated potency.
Serum Gonadotrophin for Veterinary Use intendedfor use in
the manufacture of parenteralpreparations without afitrther
appropriate procedurefor the removal ofpyrogens complies
per kg of the rabbit's weight I ml of a solution in sodium
chloride injection containing 500 Units per m!.


Serum Gonadotrophin Injection for veterinary Use contains
the stated potency.

Usual strength. 1000 Units.
Identification
Causes enlargement of the ovaries of immature female rats
when administered as directed in the Assay.
Tests
Appearance of solution. A solution containing 5000 Units per
ml (solution A) is clear (2.4.1), and colourless (2.4.1).
pH (2.4.24). 6.0 to 8.0, determined on solution A.
Serum Gonadotrophin for "VeterinOly Use intendedfor use in

the manufacture ofparenteralpreparations without afUrther
Water (2.3.43). Not more than 10.0 per cent, determined orr
80 mg.
The potency of serum gonadotrophin for veterinary use is

determined by comparing its effect in increasing the weight of
the ovaries of immature rats with that of the Standard
Preparation of serum gonadotrophin under the conditions of
the following method of assay. -
Storage. Store protected from moisture and light in a refrigerator

(2 to 8). If the contents are sterile, the containers should be
sterile, tamper-evident and sealed so as to exclude
micro-organisms.
Labelling. The label states (I) the number ofUnits per mg; (2)
the total number of Units in the container; (3) the date after
which the material is not intended to be used; (4) the storage
conditions; (5) whether or not it is intended for use in the
manufacture of parenteral preparations.
Serum Gonadotrophin Injection for

Veter!!!!!~~__ !:!~~___________
_
_
SerumGonadotrophin Injection for Veterinary Use is a sterile

material consisting of Serum Gonadotrophin for Veterinary
Use with or without buffering agents and other excipients. It
is fIlled in a sealed container.
Assay. Carry out the biological assay ofserum gonadotrophin

described below.
for serum gonadotrophin, equine, for bioassay, established in
1966, consisting of the freeze-dried active principle from the
serum of pregnant mares, with lactose (supplied in ampoules
containing 1600 Units), or other suitable preparation the
potency of which has been determined in relation to the
Test animals. Use immature female rats ofthe same strain, 21
t028daysold, differingmage_bynotmorethan_3_rlays -and of.
approximatelyequalWflights suchthat thecliff~r~l1cebet\t:len.
the heaviest and the lightest rat is not more than 109. Assign
the rats at random to six equal groups of not less than fIve
animals. If sets of six litter-mates are available, allot one
2686
IP 2010
SPECTINOMYCIN HYDROCHLORIDE
litter-mate from each set at random to each group and mark

according to the litter.
Procedure. Choose three doses of the Standard Preparation
and three doses of the preparation under examination such
that the smallest dose is sufficient to produce a positive
response in some of the rats and the largest dose does not
produce a maximal response in all of the rats. Use doses in
geometric progression. As an initial approximation total doses
of 8, 12 and 18 Units may be tried although the dose will
depend on the sensitivity of the animals used, which may
vary widely. Dissolve separately the total quantities of the
preparation under examination and ofthe Standard Preparation
corresponding to the doses to be used in sufficient of a sterile
saline solution containing 1 mg of bovine albumin per ml
such that each single dose may be administered by the
injection of6 equally-divided portions, in the same volume of
about 0.2 ml. Store the solutions at a temperature 2 to 8.
Inject subcutaneously into each rat the dose allocated to its
group. Repeat the injections 18,21,24,42 and 48 hours after
the first injection. Kill the rats between 40 hours and 72 hours
after the last injection and -remove the ovaries. Remove any
extraneous fluid and tissue and immediately weigh the two
ovaries from each rat.
methods using the combined weight of the two ovaries of
each animal as the response.
Limits oferror - The estimated potency is not less than 80 per

cent and not more than 125 per cent of the stated potency.
The fiducial limits oferror (P = 0.95) ofthe estimated potency
are not less than 64 per cent and not more than 156 per cent of
the stated potency.
per kg of the rabbit's weight 1 ml of a solution in sodium
chloride injection containing 500 Units per ml.
Storage. Store protected from light in a refrigerator (2 to 8) .
Labelling. The label states the number of Units contained in
the sealed container.
Spectinomycin Hydrochloride is [2R(2a,4a~,5a~,6~, 7~,8~,9a,9aa, 1Oa~)]-decahydro-4a,7,9-
trihydroxy-2-methyl-6,8-bis(methylamino)-4H-pyrano[2,3-b]
[1 ,4]benzodioxin-4-one dihydrochloride pentahydrate.
Spectinomycin Hydrochloride contains not less than 95.0 per
cent and not more than 100.5 per cent of CI4H24N207,2HCl,
Dose. Large farm animals and poultlY. By intramuscular or
intravenous injection, the equivalent of 10 to 20 mg of
spectinomycin per kg of body weight daily.
(Each 30 mg ofspectinomycin hydrochloride is approximately
equivalent to 20 mg of spectinomycin).
Identification

Compare the spectrum with that obtained with spectinomycin
spectinomycin hydrochloride.
Tests
pH (2.4.24).3.8 to 5.6, determined in a 10 per centw/v solution.
in a 10 per cent w/v solution within 20 minutes ofpreparation,
Mobile phase. A mixture of 50 volumes of l-propanol,

40 volumes of water, 5 volumes of glacial acetic acid and
5 volumes ofpyridine.
in 100 ml water.
Spectinomycin Hydrochloride

potassium permanganate. Allow the plate to stand for 2 to
chromatogram obtained with the reference solution (1 per
cent).
,2HCI,5H zO
Mol. Wt. 495.4
Sulphated ash (2.3.18). Not more than 1.0 per cent w/v.
2687
SPECTINOMYCIN HYDROCHLORIDE
IP 2010
NOTE - Use the solutions within] hour after preparation.
exceeding 30. Ifthe substance is sterile, the container should

be sterile, tamper~evident and sealed so as to exclude
micro-organisms.
Test solution (a). Take 60 mg of the substance under

examination in a glass-stoppered conical flask, add 10.0 ml of
dimethylformamide and 2.0 ml of hexamethyldisilazane,
shake intennittently for 1 hour and dilute to 20.0 ml with
dimethylformamide.
Test solution (b). Take 60 mg of the substance under
examination in a glass-stoppered conical flask, add 10.0 ml of
a solution containing 0.15 per cent w/v ofphenazone (internal
standard) in dimethylformamide and 2.0 ml of hexamethyldisilazane, shake intermittently for 1 hour and dilute to
20.0 ml with dimethylformamide.
Reference solution. Take 60 mg of the spectinomycin
hydrochloride RS in a glass-stoppered conical flask, add
10.0 ml ofa solution containing 0.15 per cent wIv ofphenazone
(internal standard) in dimethylformamide and 2.0 ml of
hexamethyldisilazane, shake intermittently for 1 hour and
dilute to 20.0 ml with dimethylformamide.
with 3 per cent w/w of phenylmethylsilicone fluid
(50 per cent phenyl),
Labelling. The label states (1) the date after which the material
is not intended to be used; (2) the storage conditions; (3)
whether or not it is intended to be used for manufacture of
parenteral preparations.
Spectinomycin Hydrochloride Injection
Spectinomycin Injection is a sterile material consisting of
Spectinomycin Hydrochloride with or without auxiliary
substances. It is filled in a sealed container.
The injection is constituted by suspending the contents of
the sealed container in the requisite amount of sterile Water
for Injections, immediately before use.
Storage. The constituted suspension should be used

Spectinomycin Injection contains not less than 90.0 per cent
and not more than 110.0 per cent the stated amount of
spectinomycin, CJ4Hz4Nz07.
temperature:
column 200,
Inject the chosen volumes of test solutions (a) and (b). The
test is not valid unless the resolution factor between the peak
due to the internal standard and the principal peak in the
8.0.
Inject alternately test solution (b) and the reference solution.

Usual strength. Equivalent of2 g of spectinomycin.

Dose. All species except adult ruminants. 50 to 100 mg per kg
of body weight.
Identification
Calculate the content ofCI4Hz4NP7,2HCl.
Spectinomycin Hydrochloride intended for use in the

requirement.

Unit per mg determined in a 0.42 per cent w/v solution of
sodium bicarbonate.
Spec.tinolrzyr:irz HydrochlorJc!e .intenclf!clforuf!LrJt.lJ.~

Compare the spectrum with that obtained with spectinomycin
spectinomycin hydrochloride.
Tests
pH (2.4.24). 4.0 to 7.0, determined in a suspension of the
.contents ofasealeocontaiiierin-theYolume6f-theliquid

2688
IP 2010
SPlRAMYCIN

with 3 per cent w/w of phenylmethylsilicone fluid
(50 per cent phenyl),
temperature:
column 200 0 ,
inlet port 200 0 and detector 230 0 ,
Mobile phase. A mixture of 50 volumes of i-propanol,

40 volumes of water, 5 volumes of glacial acetic acid and
5 volumes of pyridine.
Test solution. Prepare a solution containing the equivalent of
1.4 per cent w/v of spectinomycin in water.
Reference solution. Prepare a solution containing the
equivalent of 0.014 per cent w/v ofspectinomycin in water.
potassium permanganate. Allow the plate to stand for 2 to
Inject the chosen volumes oftest solutions (a) and (b). The
test is not valid unless the resolution factor between the peak
due to the internal standard and the principal peak in the
8.0.
Inject alternately test solution (b) and the reference solution.
Calculate the content ofC14H24N207,2HCl.
Storage. Use the injection immediately after preparation but,
O.2g.
in any case, within the period recommended by the

manufacturer provided it is prepared and stored in accordance
with the manufacturer's instructions.

Unit per ml, determined on a solution prepared by dissolving
the contents in a solution containing 0.05 M sodium
bicarbonate in water BET to give a solution containing the
equivalent of 1 mg of spectinomycin per ml (solution A), and
using the maximum valid dilution ofsolution A calculated from
the declared sensitivity of the lysate used in the test.

equivalent amount of spectinomycin.
Spiramycin

pH a
~fJ5:2:lH~CHa
NOTE - Use the solutions within i hour after preparation.

Test solution (a). Weigh and mix the contents of the 10
containers. To an accurately weighed quantity containing
about 60 mg of Spectinomycin Hydrochloride in a
glass-stoppered conical flask, add 10.0 ml of dimethylformamide and 2.0 ml of hexamethyldisilazane, shake
intermittently for 1 hour and dilute to 20.0 ml with dimethylformamide.
Spiramycin I R = H
Spiramycin II R COCHa
Spiramycln III R = COCH 2CHa
CHa
c0
I
I HaCC,
o
H co'
CH a
'''CHO
N-CH a
HO~
.o:::;.-O-.LCHa~O
'OR
OH
LiJ~OH
L CHa
CHa
Mol. Wt. 843.1
Test solution (b). To an accurately weighed quantity containing

about 60 mg of Spectinomycin Hydrochloride in a
glass-stoppered conical flask, add 10.0 ml of a solution
containing 0.15 per cent w/v ofphenazone (internal standard)
in dimethylformamide and 2.0 ml of hexamethyldisilazane,
shake intermittently for 1 hour and dilute to 20.0 ml with
dimethylformamide.
Spiramycin is a mixture comprised primarily ofspiramycin I

produced by Streptomyces ambofacien from soil of
northern France.
Reference solution. TO about 60 mg, accurately weighed, of

spectinomycin hydrochloride RS in a glass-stoppered conical
flask, add 10.0 ml ofa solution containing 0.15 per cent w/v of
phenazone (internal standard) in dimethylformamide and
2.0 ml of hexamethyldisilazane, shake intermittently for
1 hour and dilute to 20.0 ml with dimethylformamide.
Description. A white or slightly yellowish powder; slightly
- a glass column 1.5 m x 4 mm, packed with acid-washed,
Spiramycin contains not less than 3900 Units per mg, calculated
on the dried basis.
Category. Antibactelial.
hygroscopic.
Identification
maximum only at about 232 nm; absorbance at about 232 llID,
about 0.34.
2689
SPlRAMYCIN
IP 2010
B. Dissolve 0.5 g in a mixture of 10 ml of O. 05 M sulphuric acid

and 25 ml of water. Adjust the pH to about 8 by addition of
0.1 M sodium hydroxide and dilute to 50 ml with water. To
5 ml ofthe resulting solution add 2 ml ofa mixture of! volume
of water and 2 volumes of sulphuric acid; a brown colour is
produced.
about 0.41, for impurity A is about 0.45, for impurity D is about

0.5, for impurity G is about 0.66, for impurity B is about 0.73,
for impurity His about 0.87, for spiramycinII is about 1.4, for
spiramycin III is about 2.0 and for impurity E is about 2.5. The
test is not valid unless in the chromatogram obtained with
reference solution (c), the resolution between the peaks due
to impurity A and spiramycin I is not less than 10.0.
Tests
dissolving 0.5 gin 5 ml of methanol and diluting to 100 ml with
carbon dioxide-free water.
Specific optical rotation (2.4.22). -80.0 0 to -85.0 0 , determined
in a 2 per cent w/v solution in 0.2 M acetic acid.
(2.4.14).

Solvent mixtue. 30 volumes of methanol and 70 volumes of
water.
Inject reference solution (b) and the test solution. The area of
secondary peak due to impurity A, B, C, D, E, F, G and H is not
more than the area of principal peak in the chromatogram
secondary peaks is not more than 5 times the area ofprincipal
(b) (10.0 per cent). Ignore any peak with an area less than 0.05
obtained with reference solution (b) (0.1 per cent) and the
peak due to blank, Spiramycin I, II, III.
Sulphated ash (2.3.18). Not more than 0.1 per cent w/v.

on 0.5 g by drying over phosphorus pentoxide at 800 at a

spiramycin RS in the solvent mixture.
Assay. Carry out the microbiological assay of antibiotics

(2.2.10), MethodA.

Reference solution (c). Dissolve 5.0 mg spiramycin RS in 15.0

ml of btif.fer solution pH 2.2 and dilute to 25.0 ml with water,
heat on water-bath at 60 0 for 5 minutes and cool.
- a stainless steel column 25 cm x 4.6 mm, endcapped
polar embedded octadecylsilane amorphous
organosilica polymer (5 Ilm) (polar embedded
octadecylsilane methylsilica (12.5 Ilm),
mobile phase: a mixture of 5.0 volumes of3.48 per cent
solution of dipotassium hydrogen phosphate, adjusted
to pH 6.5 with 2.72 per cent w/v solution ofpotassium
dihydrogen phosphate, 40 volumes of acetonitrile and
=:
Sulphadiazine and Trimethoprim

Injection
Trimethoprim and Sulphadiazine Injection; Co-trimazine
Injection
Sulphadiazine and Trimethoprim Injection is a sterile
suspension in Water for Injections containing Sulphadiazine
and Trimethoprim in the proportion of five parts to one part
respectively.
Sulphadiazine and Trimethoprim Injection contains not less
. stated amounts of sulphadiazine, C IOH ION 40 ZS and of
~j~(~ti!?I1.\,()LU!ll\j. 20
Inject reference solution (a) and (c). Run the chromatogram

three times the retention times ofspiromycin peak. The relative
rentention time with reference to spiramycin I for impurity F is
Usual strengths. 400 mg of Sulphadiazine and 80 mg of

Trimethoprim in 1 ml; 200 mg ofSulphadiazine and 40 mg of
Trimethoprim in 1 ml.
2690
IP 2010
SULPHADIAZINE AND TRlMETHOPRIM VETERINARY ORAL POWDER
Identification
A. When examined in the range 230 mn to 360 nm (2.4.7), the
solution obtained in the Assay for trimethoprim shows an
absorption maximum only at about 271 nm.

15 volumes of dimethylformamide and 5 volumes of water.
Test solution. Add 4 ml of hydrochloric acid to 2.5 ml of the
well-mixed contents of the container and dilute to 50 ml with
1.4 M methanolic ammonia.
Reference solution (a). A 2.0 per cent w/v of sulphadiazine
RS in 1.4 M methanolic ammonia.
Reference solution (b). A 0.4 per cent w/v of trimethoprim RS
in 1.4 M methanolic ammonia.
One ofthe principal spots in the chromatogram obtained with
the test solution corresponds to the principal spot in the
chromatogram obtained with reference solution (a) and the
other corresponds to the principal spot in the chromatogram
C. To 5 ml ofthe filtrate obtained in the Assayfor sulphadiazine
add 10 ml of water and 5 ml of thiobarbituric acid-citrate
buffer. Mix and heat on a water-bath for 30 minutes; a pink
colour is produced.
Tests
pH (2.4.24). 10.0 to 10.5.
Assay. For sulphadiazine- Disperse the trimethoprim evenly
throughout the injection solution by gently inverting the
container several times without foam formation. Transfer an
accurately measured quantity of the injection containing 2 g
of Sulphadiazine to a separating funnel containing 50 ml of
0.1 M sodium hydroxide and extract with two quantities, each
of 100 ml and 50 ml of dichloromethane, washing the extract
with the same 25-ml quantity of 0.1 M sodium hydroxide.
Reserve the combined dichloromethane extracts for the assay
for trimethoprim.
Dilute the combined aqueous solutions and washings to
250.0 ml with water and filter, and dilute 5.0 ml ofthe filtrate to
200.0 ml with water. Dilute 10.0 ml ofthis solution to 100.0 ml
with water. To 3.0 ml ofthe resulting solution add 1 ml of2 M
hydrochloric acid and 1 ml of a 0.1 per cent w/v solution of
sodium nitrite and allow to stand for 2 minutes. Add I ml of a
0.5 per cent w/v solution of ammonium sulphamate and allow
to stand for" 3 minutes. Add 1 ml ofa 0.1 per cent w/v solution
ofN-(l-naphthyl)ethylenediamine dihydrochloride, allow to
stand for 10 minutes, add sufficient water to produce 25.0 ml
C IOH ION 40 2S from the absorbance obtained by carrying out
the procedure simultaneously, using 3.0 ml of a solution
prepared by dissolving 200 mg of sulphadiazine RS in 50 ml
of 0.1 M sodium hydroxide, adding sufficient water to produce
200.0 ml, diluting 5.0 ml to 250.0 ml with water and begilming
at the words "add 1 ml of 2 M hydrochloric acid
".
For trimethoprim - Extract the dichloromethane solution

reserved in the Assay for sulphadiazine with three quantities,
each of 100 ml, 50 ml and 50 ml, of 1 M acetic acid and dilute
the combined extracts to 500.0 m1 with 1 M acetic acid. To 5.0 ml
add 35 ml of 1 M acetic acid and sufficient water to produce
200.0 ml and measure the absorbance ofthe resulting solution
OfC14H1SN403 taking 204 as the specific absorbance at 271 nm.
Labelling. The label states the content of Sulphadiazine and
Trimethoprim in a suitable dose-volume.

Veterinary Oral Powder
Trimethoprim and Sulphadiazine Veterinary Oral Powder;
Sulphadiazine and Trimethoprim Dispersible Powder;
Co-trimazine Veterinary Oral Powder
consists ofSulphadiazine and Trimethoprim in the proportion
of five parts to one part respectively, mixed with suitable
wetting, dispersing and suspending agents.
107.5 per cent of the stated amounts of sulphadiazine,
C IO H ION40 2S, and oftrimethoprim, C14H1SN403.
Usual strength. 10 per cent w/w of Sulphadiazine and 2 per
cent w/w ofTrimethoprim.
Identification

Test solution (aJ. The supernatant liquid obtained by shaking
a quantity of the powder containing 0.2 g of Sulphadiazine
with sufficient 1.4 M methanolic ammonia to produce 100 ml
and centrifuging.
2691
SULPHADIAZINE AND TRIMETHOPRIM VETERINARY ORAL POWDER
Test solution (b). The supernatant liquid obtained by shaking

a quantity of the powder containing 0.2 g of Trimethoprim
with sufficient 1.4 M methanolic ammonia to produce 100 m1
and centrifuging.
sulphadiazine RS in 1.4 M methanolic ammonia.
trimethoprim RS in 1.4 M methanolic ammonia.
dry the plate in air and spray with a 0.1 per cent w/v solution
of 4-dimethylaminobenzaldehyde in a mixture of 1 ml of
hydrochloric acid and 100 ml of ethanol (95 per cent), allow
to dry and spray with dilute potassiUll1 iodobismuthate
solution. The spot in the chromatogram obtained with test
solution (a) having Rr value of about 0.7 corresponds to the
solution (a). The spot in the chromatogram obtained with test
solution (b) having Rr value of about 0.3 corresponds to the
solution (b).
Tests
Oral Powders.
Assay. For sulphadiazine - Weigh accurately a quantity of
the powder containing about 0.125 g ofSulphadiazine, transfer
into a separator containing 20 ml of 0.1 M sodium hydroxide
and extract with four quantities, each of 50 ml, of
dichloromethane. Wash each dichloromethane extract with
the same two quantities, each of 10 ml, of 0.1 M sodium
hydroxide. Combine the aqueous washings and the aqueous
layer from the separator and reserve the combined
dichloromethane extracts for the Assay for trimethoprim.
Dilute the combined aqueous solutions to 250.0 ml with water,
filter and dilute 10.0 m1 ofthe filtrate to 200.0 ml with water. To
2.0 ml ofthe resulting solution add 0.5 ml of 4 Mhydrochloric
acid and 1 ml of a 0.1 per cent w/v solution of sodium nitrite
and allow to stand for 2 minutes. Add 1 ml of a 0.5 per cent
w/v solution of ammonium sulphamate and allow to stand for
3 minutes. Add 1 ml of a 0.1 per cent w/v solution of
stand for 10 minutes. Dilute the solution to 25.0 ml with water
prepared in the same manner using 2 ml of water and begimling
at the words "add 0.5 ml of 4 M hydrochloric acid........".
Calculate the content of C IOH ION 40 2Sfromtheabsorbance
obtained by carrying outthe procedure simultaneously, with
2.0 ml ofa 0.0025 per centw/v solution ofsulphadiazine RSin
0.0005 M sodium hydroxide and beginning at the words "add
0.5 ml of 4 M hydrochloric acid.......".
IP 2010
For trimethoprim - Extract the combined dichloromethane

extracts from the Assay for sulphadiazine with four quantities,
each of 50 ml, of a 5 per cent v/v solution of 6 M acetic acid;
wash the combined aqueous extracts with 5 ml of
dichloromethane, discard the dichloromethane layer and
dilute to 250.0 ml with a 5 per cent v/v solution of 6 Macetic
acid. Dilute 20.0 ml to 100.0 ml with water and determine the
271 nm (2.4.7). Calculate the content ofCI4HIsN403 taking 204

Veterinary Oral Suspension
Sulphadiazine and Trirnethoprim Mixture; Trimethoprim
and Sulphadiazine Veterinary Oral Suspension;
Co-trimazine Oral Suspension; Co-trimazine Mixture
Sulphadiazine and Trimethoprin'-t Veterinary Oral Suspension
is a suspension of Sulphadiazine and Trimethoprim in the
proportion of five parts to one part respectively, containing
suitable suspending and dispersing agents. It may contain
suitable antimicrobial preservatives.
Sulphadiazine and Trimethoprim Veterinary Oral Suspension
110.0 per cent of the stated amounts of sulphadiazine,
C IOH ION40 2S, and oftrimethoprim, C14HISN403'
Usual strengths. 40 per cent w/v of Sulphadiazine and 8 per
cent w/v ofTrimethoprim; 4.55 per cent w/v ofSulphadiazine
and 0.91 per cent w/v ofTrimethoprim.
Identification

Test solution (a). A dilution of the oral suspension in
1.4 M methanolic ammonia containing the equivalent of
0.2 per cent w/v of Sulphadiazine.
Test solution (b). A dilution of the oral suspension in
1.4 M methanolic ammonia containing the equivalent of
0.2 per cent w/v ofTrimethoprim.
sulphadiazine RS in 1.4 M methanolic ammonia.
R-ele,:e-l1ce- -soTiltio-iz-(bTA-o~2- pe-r-cenf w7vsoliltion-6T

iiimidhopriiii RS iii 1. 4M iiiethiJnolicainiJ1oniiJ.
illy the plate in air and spray with a 0.1 per cent w/v solution
2692
SULPHAQUINOXALINE
IP20l0
of 4-dimethylaminobenzaldehyde in a mixture of I ml of
hydrochloric acid and 100 ml of ethanol (95 per cent), allow
to dry and spray with dilute potassium iodobismuthate
solution. The spot in the chromatogram obtained with test
solution (a) having R r value of about 0.7 corresponds to the
solution (a). The spot in the chromatogram obtained with test
solution (b) having Rrvalue of about 0.3 corresponds to the
solution (b).
Detennine the weight per ml of the suspension (2.4.29), and

calculate the contents of sulphadiazine and trimethoprim,
weight in volume.
Labelling. The label states the strength in tenns ofthe amounts
ofSulphadiazine and Trimethoprim.
Sulphaquinoxaline
Tests
Oral Liquids.
Assay. For sulphadiazine - Transfer an accurately weighed
quantity of the oral suspension containing about 0.125 g of
Sulphadiazine, into a separator containing 20 ml of 0.1 M
sodium hydroxide and extract with four quantities, each of
50 ml, of dichloromethane. Wash each dichloromethane extract
hydroxide. Combine the aqueous washings and the aqueous
layer from the separator and reserve the combined
dichloromethane extracts for the Assay for trimethoprim.
Dilute the combined aqueous solutions to 250.0 ml with water,
filter and dilute 10.0 ml ofthe filtrate to 200.0 rnl with water. To
2.0 ml ofthe resulting solution add 0.5 ml of 4 Mhydrochloric
acid and I ml of a 0.1 per cent w/v solution of sodium nitrite
and allow to stand for 2 minutes. Add I ml of a 0.5 per cent
w/v solution of ammonium sulphamate and allow to stand for
3 minutes. Add I ml of a 0.1 per cent w/v solution of
stand for 10 minutes. Dilute the solution to 25.0 ml with water
maximum at about 538 urn (2.4.7), using as the blank a solution
prepared in the same manner using 2 ml of water and beginning
at the words "add 0.5 ml of 4 M hydrochloric acid........".
CI4H12N40ZS
Mol. Wt. 300.3
Sulphaquinoxaline is 4-amino-N-2-quinoxalinylbenzenesulphonamide.
Sulphaquinoxaline contains not less than 98.0 per cent and
not more than 101.0 per cent OfCI4HIZN40ZS, calculated on the
dried basis.
Dose. Poulny. Prophylactic, upto 125 g per tonne offeed for 8
weeks or 300 to 500 mg per litre ofdrinking water.
Description. A yellow colour powder.
Identification
sulphaquinoxaline RS or with the reference spectrum of
sulphaquinoxaline.

0.00 I per cent w/v solution in 0.01 M sodium hydroxide shows
an absorption maximum only at about 252 urn; about 1.1.
C. Dissolve 4mg in 2 ml ofwann 2 M hydrochloric acid. The
solution gives the reaction ofprimary aromatic amines (2.3.1).
Calculate the content of C lOH lON 40 ZS from the absorbance

obtained by carrying out the procedure simultaneously, with
2.0 ml ofa 0.0025 per cent w Iv solution ofsulphadiazine RS in
0.0005 M sodium hydroxide and beginning at the words "add
0.5 ml of 4 Mhydrochloric acid. ......".
Tests
For trimethoprim - Extract the combined dichloromethane

extracts from the Assay for sulphadiazine with four quantities,
each of 50 ml, of a 5 per cent v/v solution of 6 M acetic acid;
wash the combined aqueous extracts with 5 ml of
dichloromethane, discard the dichloromethane layer and
dilute to 250.0 ml with a 5 per cent v/v solution of 6 M acetic
acid. Dilute 20.0 ml to 100.0 ml with water and detennine the
271 nm (2.4.7). Calculate the content OfCl4HlSN403 taking 204
as the specific absorbance at 271 urn.
Heavy metals. Dissolve the residue obtained in the test for

Sulphated ash in I ml of 2 M hydrochloric acid and dilute to
14 ml with water. 12 ml ofthe solution complies with limit test
for heavy metals, Method D (2.3.13) (20 ppm).
Acidity. To 2 g add 100 ml of water, heat at 70 0 for 5 minutes,

cool to 20 0 , and filter. Titrate 50 ml ofthe filtrate to pH 7.0 with
0.1 M sodium hydroxide; not more than 0.2 ml of 0.1 M sodium
hydroxide is required.

2693
SULPHAQUINOXALINE
IP 2010

solution.
examination in 2 ml of 1 M sodium hydroxide and add sufficient
methanol to produce 50 m!.
NI,N'--diquinoxalin- 2-ylsulphanilamide RS in methanol.
sulphanilamide RS in methanol.
detectable and examine in ultraviolet light at 254 nm. Any spot
corresponding to N, N'--diquinoxalin-2-ylsulphanilamide in
the chromatogram obtained with the test solution not more
intense than that of the spot in the chromatogram obtained
with reference solution (a). Any other secondary spot in the
intense than that in the chromatogram obtained by reference
solution (b).
a mixture of equal volumes of 1 M sodium hydroxide and
water. Add 20 ml of glycerin, 20 ml of 9 M sulphuric acid and
5 g of potassium bromide, cool in ice and carry out the nitrite
titration (2.3.31).
C 1JiI2N 40 2S.

Sulphaquinoxaline Sodium Solution is an aqueous solution of
sulphaquinoxaline sodium prepared by the interaction of
Sulphaquinoxaline and Sodium Hydroxide.
Sulphaquinoxaline Sodium Solution contains not less than
amount ofsulphaquinoxaline, C14HI2N402S.
Usual strength. The equivalent of 96 mg of Sulphaquinoxaline
in 1 mI.
n~~~!ip!!c:l!1, A(;l~r,y~llQ~.tQJ:>rQ~.!1sQlu.!iQIh ..
Identification
A. To a volume containing 1 g ofSulphaquinoxaline add 10 m1
of water and 3 ml of 2 M hydr.ochloric acid, filter, wash the
precipitate with water and dry for2 hours at 105. The residue
sulphaquinoxaline RS or with the reference spectrum of
sulphaquinoxaline.
B. Dissolve 4 mg of the residue obtained in test A in 2 ml of
warm 2 M hydrochloric acid. The solution gives the reaction
ofprimary aromatic amines (2.3.1).
C. AcidifY with 6 M acetic acid, filter and evaporate the filtrate
to dryness. The incinerated residue, when moistened with
Bunsen burner flame, gives a yellow colour to the flame.
Tests
solution in carbon dioxide-free water.

solution.
Test solution. Dilute a solution containing 0.20 g of
Sulphaquinoxaline to 50 ml with methanol.
N, N'--diquinoxalin- 2-ylsulphanilamide RS in methanol.

sulphanilamide RS in methanol.
Apply to the plate 5 !-LI of each solution. After development,
detectable and examine in ultraviolet light at 254 nm. Any spot
corresponding to N ,N2-diquinoxalin-2-ylsulphanilamide in the
chromatogram obtained with the test solution not more intense
than that in the chromatogram obtained with reference
solution (a). Any other secondary spot in the chromatogram
obtained with the test solution is not more intense than that
of the spot in the chromatogram obtained with reference
solution (b).
Oral Liquids.
0.48 g ofSulphaquinoxaline add 30 ml water, 20 ml ofglycerin,
20 ml of 9 M sulphuric acid and 5 g of potassium bromide,
.cool.in.ice.and.caiTy.out.the.nitrite.titration(2.3.3.L).-----_ ..
t-ml-ofO.lMsoditltfiTtitriteisequivalenttoO:03003 gof
C 1JiI2N 40 2S,
2694
IP 2010
THIABENDAZOLE VETERINARY ORAL SUSPENSION

equivalent amount of Sulphaquinoxaline in a suitable
dose-volume.
Sulphathiazole Sodium
51
o 0
~5'N-~
I
N
"
Na+
II
-;
22.0 per cent and not more than 27.0 per cent (pentahydrate),
75 ml of water and 10 ml of hydrochloric acid, add 3 g of
potassiuni bromide, cool in ice and carry out the nitrite titration
(2.3.31).
C9HsN3Na02S2.
'\
H2 N ~
Labelling. The label states whether the substance is the

sesquihydrate or the pentahydrate.
C9HsN3Na02S2,11/2 H20
Mol. Wt. 304.3
C9HsN3Na02S2,5H20
Mol. Wt. 367.4
Sulphathiazole Sodium is sodium salt of4-aminoN-2-thiazolylbenzenesulphonamide with five or one and half
molecules ofwater.
Thiabendazole Veterinary Oral

Suspension
Sulphathiazole Sodium contains not less than 99.0 per cent

and not more than 101.0 per cent ofC9HsN3Na02S2' calculated
on the dried basis.
Thiabendazole Oral Suspension; Thiabendazole Mixture;

Thiabendazole Drench
Dose. Pigs. 2.5 g ofthe powder per gallon (4.5 litres) ofdrinking
water offered continuously for 3 days.
Description. A white or yellowish white, crystalline powder or
granules.
Thiabendazole Veterinary Oral Suspension is an aqueous

suspension of Thiabendazole containing suitable suspending
agents and antimicrobial preservatives.
Thiabendazole Veterinary Oral Suspension contains not less
stated amount of thiabendazole, C IOH 7N 3S.
Usual strength. 13.3 percentw/v.
Identification
Compare the spectrum with that obtained with sulphathiazole
sodium RS or with the reference spectrum of sulphathiazole
sodium.
B. Dissolve 1 g in 25 ml of water and add 2 ml of 6 M acetic
acid. Wash the precipitate formed with water and dry for
4 hours at 105. The residue melts at about 201 (2.4.21).
C. The precipitate obtained in test B gives the reaction of

primary aromatic amines (2.3.1).
Tests
pH (2.4.24). 9.0 to 10.0, determined in a 1per cent w/v solution.
Heavy metals. Di!,solve 2.5 g of the substance under examination in 10 ml of water, add 15 ml of 2 M acetic acid, shake
for 30 minutes and filter.
12 ml of this solution complies with the limit test for heavy
metals, Method D (2.3.13) (20 ppm).
Identification

of glacial acetic acid, 8 volumes of acetone and 2 volumes of
water.
Test solution. Add 50 ml of ethyl acetate and 2 ml of glacial
acetic acid to a volume of the well-mixed oral suspension
containing about 0.25 g ofThiabendazole. Shake for 5 minutes,
heat to boiling, cool, shake for a further 15 minutes and filter.
Reference solution. Dissolve 0.25 g of thiabendazole RS in
50 ml of ethyl acetate and add 2 ml of glacial acetic acid.
dry the p.late in air and examine in ultraviolet light at 254 nm.
with reference solution.
Related substances. Complies with test A for related

substances in sulphonamides (2.3.7).
Tests
Loss on drying (2.4.19). Not less than 6.0 per cent and not
more than 10.0 per cent (sesquihydrate) or not less than

Oral Liquids.
2695
THIABENDAZOLE AND RAFOXANIDE VETERINARY ORAL SUSPENSION
Assay. Weigh accurately a quantity ofthe well-mixed oral

suspension containing about 1 g of Thiabendazole, add to
700 ml of 0.1 M hydrochloric acid, shake for 30 minutes, add
sufficient 0.1 M hydrochloric acid to produce 1000.0 ml, mix
and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with 0.1 M
hydrochloric acid. Dilute 5.0 ml to 100.0 ml with 0.1 M
hydrochloric acid and measure the absorbance ofthe resulting
content ofC IOH 7N 3S taking 1230 as the specific absorbance at
302nm.
calculate the content of thiabendazole, weight in volume.
Labelling. The label states that the suspension should be
administered undiluted.
Thiabendazole and Rafoxanide

Veterinary Oral Suspension
Thiabendazole and Rafoxanide Suspension;
Thiabendazole and Rafoxanide Mixture
Thiabendazole and Rafoxanide Veterinary Oral Suspension is
an aqueous suspension of Thiabendazole and Rafoxanide
containing suitable suspending and dispersing agents.
Thiabendazole and Rafoxanide Veterinary Oral Suspension
107.5 per cent ofthe stated amount ofthiabendazole, C IOH 7N 3S,
and not less than 90.0 per cent and not more than 110.0 per
cent ofthe stated amount ofrafoxanide, CI9HIIClzI2N03.
Usual strength. 13.3 per cent w/v ofThiabendazole and 2.27
per cent w/v ofRafoxanide.
Identification
A. Mix a volume containing 20 mg ofThiabendazole with 5 ml
of 0.1 M hydrochloric acid, add 3 mg of 4-phenylenediamine
dihydrochloride, mix, add 0.1 g of zinc powder and allow to
stand for 2 minutes. Add 10 ml ofjerric ammonium sulphate
solution; a deep blue or blue violet colour is produced.
IP 2010
with occasional stirring. Transfer the suspension to a flask,

rinse the vessel with water and add the washings to the flask.
Cool, add sufficient water to produce 1000.0 ml and filter.
Dilute 5.0 ml of the filtrate to 100.0 ml with 0.1 Mhydrochloric
acid and measure the absorbance of the resulting solution at
the maximum at about 302 mn (2.4.7). Calculate the content of
C IOH 7N 3 S taking 1230 as the specific absorbanceat 302 nm.
For rajoxanide - Protect the solutionsfrom light throughout

the determination.
Weigh accurately a volume of the well-mixed suspension
containing about 0.1 g ofRafoxanide in a 500-ml stoppered
flask and add sufficient water to produce 100 ml. Swirl to
disperse, add 20 ml of 1 M hydrochloric acid, mix well and
add 300 ml of ethyl acetate. Shake the mixture for 1 hour, set
aside for separation ofthe immiscible layers and centrifuge a
portion ofth6 ethyl acetate layer. Transfer 15.0 ml ofthe clear
solution to a 50-ml centrifuge tube, add 20 ml of 0.1 M
hydrochloric acid, stopper the tube, shake for 15 minutes,
and centrifuge. Remove and discard the aqueous layer. Repeat
the washing with two quantities, each of 20 ml, of 0.1 M
hydrochloric acid. Evaporate the ethyl acetate solution almost
to dryness in a warm water-bath, passing a stream of nitrogen
over the surface of the liquid. Add 10 ml of water, warm on a
water-bath for 10 minutes, add 5 ml of 1 M sodium hydroxide
and mix. Add 15 ml of ether, shake for 15 minutes, centrifuge,
and remove the ether layer. Repeat the extraction with two
quantities, each of 15 ml, of ether. Evaporate the combined
ether extracts almost to dryness on a warm water-bath, passing
a stream of nitrogen over the surface of.the liquid. Dissolve
the residue in sufficient 0.1 M methanolic hydrochloric acid
to produce 200.0 ml and measure the absorbance of the
Calculate the content ofCI9HIIClzI2N03 from the absorbance
0.1 g of rafoxanide RS and beginning at the words, "add
sufficient water to produce 100 ml... ..".
calculate the content ofthiabendazole and rafoxanide, weight
in volume.
B. In addition to the absorbance at about 335 nm, measure the

absorbance at about 280 nm (2.4.7), of the final solution
obtained in the Assay. The ratio of the absorbance at about
280 nm to that at about 335 nm is 1.59 to 1.69.
Tests
QralLiqllids,
Assay. For thiabendazole = Weigh accurately a volume. of
the well-mixed suspension containing about 85 mg of
Thiabendazole, add 20 ml of water and 9 ml of 0.1 M
hydrochloric acid and warm on a water-bath for 30 minutes
Thiabendazole Premix contains not less than 95.0 per cent and
thiabendazole, C IOH 7N 3S.
centw/w.
.....................
Identification
2696
IP 2010
TYLOSIN

of glacial acetic qcid, 8 volumes of acetone and 2 volumes of
water.
Test solution. To a quantity ofthe premix containing 0.25 g of
Thiabendazole, finely powdered if necessary, add 50 ml of
ethyl acetate and 2 ml of glacial acetic acid, shake for
5 minutes, heat to boiling, cool, shake for a further 15 minutes
and filter.
Reference solution. Dissolve 0.25 g of thiabendazole RS in
50 ml of ethyl acetate and add 2 ml of glacial acetic acid.
Apply to the plate 10 J.ll of each solution. After development,

Tests
Thiabendazole, add 700 ml of 0.1 M hydrochloric acid, shake
for 30 minutes, dilute to 1000.0 ml with 0.1 M hydrochloric
acid and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with
0.1 M hydrochloric acid and measure the absorbance of the
Calculate the content ofC IOH7N3 S, taking 1230 as the specific
Description. Almost white or slightly yellow powder.
Identification
out. Tests D and E may be omitted if tests A, Band Care
carried out.

Compare the spectrum with that obtained with tylosin RS or
with the reference spectrum of tylosin.
(solution A) shows an absorption maximum only at about
C. To 10 ml of solution A add 1 ml of 2 M sodium hydroxide,
heat on a water-bath for 20 minutes and cool. When examined
in the range 250 nm to 430 nm (2.4.7), ofthe resulting solution
shows an absorption maximum only at about 332 nm.
D. In the test for Tylosin A and other tylosins, the retention
time and size ofthe principal peak in the chromatogram obtained
with the test solution are approximately the same as those of
solution (a).
E. Dissolve about 30 mg in a mixture of0.15 ml ofwater, 2.5 ml
of acetic anhydride and 7.5 ml ofpyridine. Allow to stand for
10 minutes; no green colour develops.
Tests
suspension in carbon dioxide-free water.
Tylosin
Heavy metals. To the residue obtained in the test for Sulphated

ash add 2 ml of hydrochloric acid and evaporate slowly to
dryness on a water-bath. Moisten the residue with 0.05 ml of
hydrochloric acid, add 10 ml of boiling water and heat for
10 minutes on a water-bath. Cool and dilute to 25 ml with
water. 12 ml of the solution complies with the limit test for
heavy metals, Method D (2.3.13) (20 ppm).
Mol. Wt. 916.1

Tylosin is a macrolide antibiotic isolatede from a strain of
Stryptomycetes fradiae found in soil from Thailand.
Tylosin has a potency of not less than 900 Units per mg,
calculated on the dried basis. The content of tylosin A is not
less than 80.0 per cent and the sum ofthe contents of tylosin
A, tylosin B, tylosin C and tylosin D is not less than 95.0 per
cent.
Dose. All species except adult ruminants. Orally, 20 t045 mg
per kg ofbody weight daily in divided doses. By intramuscular
injection,.2 to 10 mg per kg ofbody weight.
Tyramine. Dissolve 50 mg in 5 ml of 0.03 M phosphoric acid

in a 25-ml volumetric flask, add 1 ml ofpyridine and 2 ml ofa
saturated solution of ninhydrin in water (approximately 4 per
cent w/v). Close the flask by covering with a piece ofaluminium
foil and heat in a water-bath at 85 for at least 20 minutes. Cool
rapidly and add sufficient water to produce 25 ml. Mix and
measure without delay the absorbance ofthe solution at about
570 nm (2.4.7), using as the blank a solution prepared in a
similar manner but omitting the substance under examination.
The absorbance is not more than that obtained by carrying
out the procedure simultaneously, using 5 ml of a solution in
0.03 M phosphoric acid containing 35 mg of tyramine per ml
and beginning at the words "add 1 ml of pyridine
"
(0.35 percent).
2697
TYLOSIN
IP 2010

on 1.0 g by drying at 60 at a pressure not exceeding 0.7 kPa
for 3 hours.
Tylosin A and other tylosins. Determine by liquid
NOTE- Use freshly prepared solutions.

examination in 100 ml of a mixture of equal volumes of
acetonitrile and water.
Reference solution (a). A 0.02 per cent w/v solution of tylosin
RS in a mixture of equal volumes of acetonitrile and wate/:
w/v each of tylosin A RS and tylosin D RS in a mixture ofequal
volumes of acetonitrile and water.
Nucleosil C18),
60 volumes of 0.85 M sodium perchlorate and
40 volumes of acetonitrile adjusted to pH 2.5 with 1 M
hydrochloric acid,
Inject reference solution (b). If necessary, adjust the molarity
of the sodium perchlorate or increase the temperature of the
column to a maximum of 50 so as to obtain a retention time of
about 12 minutes for tylosin A. The test is not valid unless the
resolution between the peaks due to tylosin A and tylosin D is
at least 2.0.
Inject reference solution (a). The column efficiency, determined
using the peak due to tylosin A, should be not less than
22,000 theoretical plates per metre.
procedure complies with the following additional

requirement.
Storage. Store protected from light. If it is intended for use in
the manufacture of Parenteral Preparations, the container
micro-organisms.
Labelling. The label states (l) the number ofUnits per mg; (2)
the date after which the material is not intended to be used; (3)
the storage conditions; (4) where applicable, that it is suitable
for use in the manufacture ofParenteral Preparations; (5) that
the preparation is intended for veterinary use.
Tylosin Injection
Tylosin Injection is a sterile solution ofTylosin in a mixture of
equal volumes of Propylene Glycol and Water for Injections.
Tylosin Injection contains not less than 95.0 per cent and not
more than 105.0 per cent ofthe stated amount of tylosin. The
content oftylosin A is not less than 80.0 per cent and the sum
ofthe contents oftylosin A, tylosin B, tylosin C and tylosin D
is not less than 90.0 per cent.
Usual strengths. 2.5 gin 50 ml; 20 g in 100 ml.
Description. A pale yellow to amber-coloured solution.
Identification
A. To a volume containing 0.1 g of Tylosin add sufficient

water to obtain a solution containing 0.02 per cent w/v of
Tylosin. To 5 ml of this solution add 10 ml of 0.1 M sodium
hydroxide and extract with 10 ml of dichloromethane. Separate
the dichloromethane layer and extract it with 25 ml of 0.1 M
hydrochloric acid. Discard the dichloromethane layer, wash
the aqueous layer with 3 ml of dichloromethane, discard the
washings and filter. When examined in the range 230 nm to
360 nm (2.4.7), ofthe resulting solution exhibits a maximum
Inject alternatively the test solution and reference solution B. To 10m) of the filtrate obtained in test A add 1 ml of 2 M
(a). The order of elution of the major components of the sodium hydroxide, heat in a water-bath for 20 minutes and
substance under examination is desmycinosy1tylosin, tylosin cool. When examined in the range 250 nm to 430 nm (2.4.7),
. exhibits a maximum only at about 332 nm.
C, tylosin B, tylosin D, tylosin A aldol and tylosin A.
Calculate the percentage content ofcomponents from the areas
of the peaks in the chromatogram obtained with the test
solution by normalisation.
Assay.Canyout tne miciooiologiC~i1-aSs-ayorantioio1iCs
(22.10).
Tylosin intended for use in the manufacture of Parenteral

Preparations without a jiJrther appropriate sterilisation
Tests
Tyramine. Dilute a volume containing 100 mg ofTylosin with
Smlof 0.03MphosphOl-icacidina25~mLvolumetricflask,
add LmL of pyridine and 2 mLof a saturated solution of
ninhydrin in water (approximately 4 per cent w/v). Close the
flask by covering with a piece of aluminium foil and heat in a
water-bath at 85 for at least 20 minutes. Cool rapidly and add
2698
TYLOSIN TABLETS
IP 2010
sufficient water to produce 25 ml. Mix and measure without

delay the absorbance of the resulting solution at about
570 nm (2.4.7), using as the blank a solution prepared in a
similar manner but omitting the preparation under examination.
0.03 M phosphoric acid containing 30 mg of tyramine per ml
and beginning at the words "add 1 ml of pyridine......"
(0.15 percent).

(2.2.10). Calculate the content oftylosin in the injection, taking
each 1000 Units found to be equivalent to I mg of tylosin.
Labelling. The label states that the preparation is intended
for veterinary use by intramuscula.r injection only.

NOTE -
Tylosin Tablets
Use fi-eshly prepared solutions.
Test solution. Dilute the injection with sufficient of a mixture

of equal volumes of acetonitrile and water to produce a
solution containing 0.02 per cent w/v of Tylosin.
RS in a mixture of equal volumes of acetonitrile and wate}:
Tylosin Tablets contain not less than 95.0 per cent and not
more than 105.0 per cent of the stated amount of tylosin. The
content oftylosin A is not less than 80.0 per cent and the sum
ofthe contents oftylosin A, tylosin B, tylosin C and tylosin D
is not less than 90.0 per cent.

Nucleosil CI8),
hydrochloric acid,
column to a maximum of50 so as to obtain a retention time of
resolution between the peaks due to tylosin A and tylosin Dis
at least 2.0.
Inject alternatively test solution and reference solution (a).
The order ofelution ofthe major components ofthe substance
under examination is desmycinosyltylosin, tylosin C, tylosin
B, tylosin D, tylosin A aldol and tylosin A.
.
solution.
Identification
0.2 g ofTylosin with 20 ml of dichloromethane and filter. Dry
the dichloromethane extract by shaking with anhydrous
sodium sulphate, filter and evaporate the filtrate to dryness.
Dry the residue over phosphorus pentoxide at a pressure not
exceeding 0.7 kPa for I hour.
.

0.2 g of Tylosin with two quantities, each of 10 ml, of 0.1 M
hydrochloric acid, filter and dilute the filtrate to 100 ml with
0.1 M hydrochloric acid. Dilute 10 ml ofthe resulting solution
to 50 ml with the same solvent. Dilute 5 ml of this solution
further to 50 ml with the same solvent.
When examined in the range 230 nm to 360 nrn (2.4.7), the
290 nrn; absorbance at about 290 nrn, about 0.94.
C. To 10 ml ofthe final solution obtained in test B add I mlof
2 M sodium hydroxide, heat in a water-bath for 20 minutes
and cool. When examined in the range 250 nrn to 430 nrn (2.4.7),
exhibits a maximum only at about 332 nrn.
Tests
Tyramine. Shake a quantity ofthe powdered tablets containing
50 mg ofTylosin with 5 ml of O. 03 M phosphoric acid. Filter
into a 25-ml volumetric flask, add I ml ofpyridine and 2 ml of
a saturated solution of ninhydrin in water (approximately
4 per cent w/v). Close the flask by covering with a piece of
2699
TYLOSIN TABLETS
IP 2010
aluminium foil and heat in a water-bath at 85 for at least

20 minutes. Cool rapidly and add sufficient water to produce
25 ml. Mix and measure without delay the absorbance of the
solution at about 570 urn (2.4.7), using as the blank a solution
prepared in a similar manner but omitting the substance under
by carrying out the procedure simultaneously, using 5 ml of a
solution in 0.03 M phosphoric acid containing 35 mg of
tyramine per ml and beginning at the words "add 1 ml of
pyridine......" (0.35 per cent).

Assay. Carry out the microbiological- assay -of antibiotics
(2.2.1 0). Calculate the content oftylosin in the tablets, taking
each 1000 Units found to be equivalent to 1 mgoftylosin..
Tylosin Tartrate
o

.CH 3
NOTE -Use freshly prepared solutions.

containing 0.2 g ofTylosin with 50 ml of methanol, filter and
dilute 5 ml of the filtrate to 100 ml with a mixture of equal
Nucleosil C18),
hydrochloric acid,
column to maximum of 50 so as to obtain a retention time of
at least 2.0.
'OH
(C46H77N0 17)2, C~606
CH 3
N-CH3
H OH
"o:::-t--LCH3' ~OH
HOOCXxCOOH
HO H
"
CHO
HO~o
CH3
OH
CH3
Mol. Wt. 1983.3
Tylosin Tartrate is the tartrate ofTylosin, which is a mixture of

antimicrobial macrolides produced by the growth of certain
strains of Streptomyces fradiae or by any other means. It
consists largely of tylosin A tartrate but tartrates of tylosin B
(desmycosin), tylosin C (macrocin) and tylosin D (relomycin)
may also be present.
Tylosin Tartrate contains not less than 800 Units per mg,
calculated on the dried basis. The content of tylosin A is not
less than 80.0 per cent and the sum of the contents of tylosin
A, tylosin B, tylosin C and tylosin D is not less than 95.0 per
cent.
Dose. Pigs andpoulny. The equivalent of2.5 g ofTylosin per
gallon (4.5 litres) ofdrinking water offered continuously for 3
days.
Description. An almost white or slightly yellow, hygroscopic
powder.
Identification
out. Tests D and E may be omitted if tests A, Band Care
carried out.


Compare the spectrum with that obtained with tylosin tartrate
RS or with the reference spectrum of tylosin tartrate.
Inject alternatively the test solution and reference solution

(a). The order of elution of the major components of the
substance-iiilder-examinaIionis-ae-smycinosyltylosin,-ijlosiil
C;ijlosiiiB, tY16siiiD;tY16siri A aldol aridtylbsiii A.
B. When examined in the range 230 urn to 360 nm (2.4.7), a

(sohition A) shows- aii aoso!'ptioii-maximumoiilyiiCaoout
290 iir:ti;aosbtbance at abbut290 nrn; about 0.88.
Calculate the percentage content ofcomponents fi:bm the areas

ofthe peaks in the chromatogram obtained with test solution.
e. To 10 ml of solution A add 1 ml of 2 M sodium hydroxide,

heat on a water-bath for 20 minutes and cool.
2700
IP 2010
TYLOSIN TARTRATE

332nm.

D. In the test for Tylosin A and other tylosins, the retention

time and size ofthe principal peak in the chromatogram obtained
with the test solution are approximately the same as those of
reference solution.
E. Dissolve about 30 mg in a mixture of0.15 ml of water, 2.5 ml
of acetic anhydride and 7.5 ml ofpyridine. Allow to stand for
10 minutes; a green colour develops.
Tests
hydrochloric acid,
Inject reference solution (b). Ifnecessary, adjust the molarity
column to a maximum of50 so as to obtain a retention time of
at least 2.0.
Tyramine. Dissolve 50 nig in 5 ml of 0.03 Mphosphoric acid

in a25-ml volumetric flask, add 1 ml ofpyridine and 2 ml ofa
saturated solution of ninhydrin in water (approximately 4 per Inject alternatively the test solution and the reference solution
cent w/v). Close the flask by covering with a piece ofaluminium (a). The order of elution of the major components of the
foil and heat in a water-bath at 85 for at least 20 minutes. Cool substance under examination is desmycinosyltylosin, tylosin
rapidly and add sufficient water to produce 25 ml. Mix and C, tylosin B, tylosin D, tylosin A aldol and tylosin A.
measure without delay the absorbance ofthe solution at about Calculate the percentage content ofcomponents from the areas
570 nm (2.4.7), using as the blank a solution prepared in a ofthe peaks in the chromatogram obtained with test solution.
similar manner but omitting the substance under examination.
(2.2.10).
0.03 M phosphoric acid containing 35 mg of tyramine per mI Tylosin Tartrate intended for use in the manufacture of
and beginning at the words "add 1 ml of pyridine......" parenteral preparations complies with the above
(0.35 percent).
requirements with the following modification.
on 1.0 g by drying at 60 at a pressure not exceeding 0.7 kPa
for 3 hours.
NOTE -
Use freshly prepared solutions.
Test solution. Dissolve a quantity containing 20 mg oftylosin

in 100 ml of a mixture of equal volumes of acetonitrile and
water.
Nucleosil CI8),
Tyramine. Carry out the procedure described in the test for

Tyramine but using 100 mg in 5 ml of O. 03 Mphosphoric acid.
Measure the absorbance of the solution under the conditions
described in the test. The absorbance is not more than that
obtained by simultaneously carrying out the procedure using
5 ml ofa solution in 0.03 Mphosphoric acid containing 30 mg
of tyramine per ml and beginning at the words '.'add 1 ml of
pyridine......" (0.15 percent).
Tylosin Tartrate intended for use in the manz!facture of

Storage. Store protected from light. Ifit is intended to be used
in the manufacture of parenteral preparations, the container
micro-organisms.
Labelling. The label states (1) the number ofUnits per mg; (2)
the quantity 6fTylosin Tartrate in terms of equivalent amount
oftylosin; (3) the date after which the material is not intended
2701
TYLOSIN TARTRATE AND SULPHATHIAZOLE SODIUM VETERINARY ORAL POWDER
to be used; (4) the storage conditions; (5) where applicable,

that it is suitable for use in the manufacture of parenteral
preparations; (6) that the preparation is intended for veterinary
use.
Tylosin Tartrate and Sulphatbiazole

Sodium Veterinary Oral Powder
Tylosin Tartrate and Sulphathiazole Sodium Veterinary Oral
Powder is a mixture of Tylosin Tartrate and Sulphathiazole
Sodium. It contains 3 parts of Sulphathiazole Sodium for
I part, by weight, of Tylosin.
Tylosin Tartrate and Sulphathiazole Sodium Veterinaly Oral
Powder contains not less than 90.0 per cent and not more than
110.0 per cent of the stated amounts of tylosin and
sulphathiazole sodium sesquihydrate, CgHgNaOzSz,ll/zHzO.
Usual strength. The equivalent of25 g of tylosin as Tylosin

Tartrate and the equivalent of75 g ofsulphathiazole sodium
sesquihydrate as Sulphathiazole Sodium.
Identification
A. Triturate a quantity of the powder containing 0.25 g of
Tylosin with two quantities, each of25 ml, of dichloromethane
and filter. Reserve the dichloromethane-insoluble matter for
test B. Wash the combined filtrates by shaking for I minute
with 20 ml of 0.1 M sodium hydroxide and dry the dichloromethane layer by the addition of anhydrous sodium sulphate.
Evaporate the filtrate to dryness and dry the residue over
phosphorus pentoxide at a pressure not exceeding 0.7 kPa for
I hour. The residue complies with the following test.
B. Dry the dichloromethane-insoluble matter reserved in test
A at 105 for I hour. The residue complies with the following
test.
Compare the spectrum with that obtained with sulphathiazole
sodium RS or with the reference spectrum of sulphathiazole
sodium.
Tests
Sulphonamide-related substances. Determine by thin-layer
chromatography (2.4.17), coating the plate with silica gel H.
mixture.
AmIxture of9volumes of ethano[(95p-er
IP 2010
Test solution. Shake a quantity ofthe powder containing 0.1 g

of sulphathiazole sodium sesquihydrate with 10 ml of the
solvent mixture.
sulphanilamide in the solvent mixture.
dry the plate by heating it'at 105 for 10 minutes and spray
with a 0.1 per cent w/v solution of 4-dimethylaminobenzaldehyde in a mixture of99 volumes of ethanol (95 per
cent) and I volume of hydrochloric acid. Any secondary
with reference solution (0.5 per cent).
Tylosin A and other tylosins. Determine by liquid chromatography (2.4.14).
NOTE -
Usefreshly prepared solutions.
Test solution. Dissolve a quantity containing 20 mg oftylosin

in 100 ml of a mixture of equal volumes of acetonitrile and
watel:
tylosin RS in a mixture of equal volumes of acetonitrile and
watel:
w Iv each of tylosin A RS and tylosin DRS in a mixture ofequal
volumes of acetonitrile and watel:
Nucleosil C18),
hydrochloric acid,
Inject reference solution (b). Ifnecessary, adjust the molarity
column to a maximum of 50 so as to obtain a retention time of
at least 2.0.
using Hie peak due f6tYl6-siii A;.sli6JJIdben6fles-sthan
cent) and 1volume ofsirong ammonlasolittloii
22;000-tliebfeticalplates permtltre:

18 volumes of 10Mammonia.
Inject altematively the test solution and the reference solution

(a). The order of elution of the major components of the
2702
IP 2010
TYLOSIN TARTRATE AND SULPHATHIAZOLE SODIUM VETERINARY ORAL POWDER
substance under examination is desmycinosyltylosin, tylosin

C, tylosin B, tylosin D, tylosin A aldol and tylosin A.
(2.2.10). Calculate the content oftylosin taking each 1000 Units

found to be equivalent to 1 mg of tylosin.

solution by normalisation. In the chromatogram obtained with
the test solution the content of tylosin A is not less than
80.0 per cent and the sum of the contents oftylosin A, tylosin
B, tylosin C and tylosin D is not less than 95.0 per cent.
For sulphathiazole sodium - Weigh accurately a quantity

ofthe powder containing about 0.4 g ofsulphathiazole sodium
sesquihydrate, dissolve in a mixture of 75 ml of water and
10 ml of hydrochloric acid, add 3 g of potassium bromide,
cool in ice and titrate slowly with 0.1 M sodium nitrite, stirring
constantly and determine the end-point potentiometrically
(2.4.25).

Oral Powders.
Assay. For tylosin activity -- Weigh accurately a quantity of
the powder containing about 0.2 g of Tylosin, transfer to a
1OO-ml volumetric flask with three quantities, each of 10 ml, of
methanol, swirl to dissolve and add sufficient sterile
phosphate bufferpH 7. 0 to produce 100.0 ml. Filter and dilute
5.0 ml ofthe filtrate to 100.0 ml with sterile phosphate buffer
pH 7.0. Carry out the microbiological assay of antibiotics
C9HsN3Na02S2' I-:\- H20.

Labelling. The label states the strength of Tylosin Tartrate in
terms of the equivalent amount of tylosin and that of
Sulphathiazole Sodium in terms of the equivalent amount of
sulphathiazole sodium sesquihydrate.
2703
BIOLOGICAL VETERINARY MONOGRAPHS

Anthrax Spore Vaccine, Live
2707
Avian Infectious Bronchitis Vaccine, Inactivated
2707
Avian Infectious Bronchitis Vaccine, Live
2708
Avian Spirochaetosis Vaccine
2709
Blackquarter Vaccine
2710
Canine Contagious Hepatitis Vaccine, Inactivated
2710
Canine Contagious Hepatitis Vaccine, Live
2711
Canine Corona VIrUS Vaccine, Inactivated
2711
Canine Distemper Vaccine, Live
2712
Canine Leptospirosis Vaccine, Inactivated
2713
Canine Parainfluenza VIrUS Vaccine, Live
2713
Canine Parvovirus Vaccine, Inactivated
2714
Canine Parvovirus Vaccine, Live
2715
Multicomponent ClostridiumVaccine, Inactivated
2715
Clostridium Novyi (Type b) Vaccine for Veterinary Use
2716
Clostridium Perfringens Vaccine, Inactivated
2717
Clostridium septicum Vaccine, Inactivated
2719
Duck Pasteurella Vaccine, Inactivated
2721
Duck Plague Vaccine, Live
2722
Egg Drop Syndrome 76 (Adenovirus) Vaccine, Inactivated
2722
Foot-and-Mouth Disease Vaccine, Inactivated
2723
Fowl Cholera Vaccine, Inactivated
2724
Fowl Pox Vaccine, Live
2725
Goat Pox Vaccine, Live
2725
Haemorrhagic SepticaemiaVaccine
2726
Inclusion Body Hepatitis (IBH) Vaccine, Inactivated
2727
Infectious Avian Encephalomyelitis Vaccine, Live
2727
Infectious Bursal Disease Vaccine, Inactivated
2728
Infectious Bursal Disease Vaccine, Live
2729
Infectious ChickenAnemia Vaccine, Inactivated
2730
2705
Infectious ChickenAnemia Vaccine, Live

Infectious Coryza Vaccine
Marek's Disease Vaccine, Live
Peste Des Petits Ruminants Vaccine, Live
Rabies Veterinary Vaccine, Inactivated (Cell Culture)
Ranikhet Disease Vaccine, Inactivated
2730
2731
2732
2732
2733
2735
2736
2737
2737
2738
2739
2740
2740
2741
2741
2742
2742
2745
Ranikhet Disease Vaccine, Live (Lentogenic Strain)

Ranikhet Disease Vaccine, Live (Mesogenic Strain)
Reo Vrrus Vaccine, Inactivated
Reo Vrrus Vaccine, Live
Rinderpest Vaccine, Live
SalmonellaAbortus Equi Vaccine
SalmonellaVaccine, Inactivated
Sheep Pox Vaccine, Live Attenuated
Sterile Diluent for Live Vaccines
Swine Fever Vaccine, Live
Tetanus Veterinary Vaccine
Theileriosis Vaccine, Live
2706
IP 2010
ANTHRAX SPORE VACCINE, LIVE

Anthrax Spore Vaccine, Live consists of a suspension of live
spores of an attenuated, non-capsulated strain of Bacillus
anthracis.
Production
The strain used may either be not lethal to guinea pigs or the
mouse or lethal to guinea pigs but not to the rabbit or lethal to
some rabbits. At the end of growth the spores are suspended
in 50 per cent glycerin saline and counted. The vaccine may
contain an adjuvant.
If the immunogenicity tests have been performed with
satisfactory results on a representative batch of the vaccine
from the seed lot, they may be omitted as a routine control test
during production on other batches of the vaccine prepared
from the same seed lot.
Identification
stated on the label for sheep. Observe the animals for

21 days. Ifmore than two animals die from non-specific causes,
repeat the test.
Ifthe test is carried out in sheep use 5 healthy animals. Inject
subcutaneously or intramuscularly into each sheep weighing
not less than 20 kg, 1/10u] ofthe smallest dose of the vaccine
stated on the label for sheep and observe the animal for
21 days. None ofthe sheep shows any untoward reaction. If
more than 2 animals die from non-specific causes, repeat the
test.
Inject subcutaneously into each vaccinated guinea pig or
rabbit or sheep at least 100 MLD and to each ofthese control
animals 10 MLD of a strain of B.anthracis pathogenic for ~he
species ofanimal used in the test. Observe all the animals for
10 days, all vaccinated animals should survive and all the
controls die from anthrax during the observation period. If a
vaccinated animal dies after the challenge, repeat the test. If
in the second test, a vaccinated animal dies, the vaccine fails
the test.
B. anthracis present in the vaccine is identified by means of Labelling. The label states (1) the strain used for the
morphological, serological test, culture and biochemical tests. preparation of the vaccine; (2) the number of viable spores
perml.
Tests
Safety. Carry out the test on one ofthe species for which the
vaccine is intended. If the vaccine is intended for several
species including the goat, carry out the test on goats.
Administer subcutaneously or intramuscularly to each oftwo
animals having no antibodies against B. anthracis, twice the
dose (example, 2 million spores) stated on the label for the
species used and observe the animals for 10 days. No
abnormal systemic reaction is produced but a mild local
reaction may occur at the site of injection. The severity ofthe
local reaction may vary according to the strain of the B.
anthracis and the adjuvants used in the preparation, but
necrosis does not occur.
Sterility (2.2: 11). Complies with the test for sterility.
Spore count. Detennine the number ofviable spores by plate
count. The number of live spores is not less than 80 per cent
of that stated on the label. The spore count must be not less
than 10 million spores per dose.
Potency. For a strain of B. anthracis which is not lethal to the
guinea pig or the mouse, the test may be carried out in the
guinea pigs. For a strain which is lethal to the guinea pig but
not to the rabbit, the test may be carried out in rabbits. For a
strain which is lethal to some rabbits, carry out the test in
sheep.
If the test is carried out in guinea pigs or rabbits. Inject into

animals (each guinea pig weighing not less than 500 g and
each rabbit weighing not less than 2.5 kg) subcutaneously or
intramuscularly, 1/lOu] of the smallest dose of the vaccine
Avian Infectious Bronchitis Vaccine,

Inactivated
Avian Infectious Bronchitis Vaccine, Inactivated consists of
an emulsion or a suspension of one or more serotypes of
avian infectious bronchitis virus which have been inactivated
in such a manner that the immunogenic activity is retained.
This monograph applies to vaccines intended to protect birds
against drop in egg production or quality; for vaccines also
intended for protection against respiratory signs and
nephropathic symptoms, a demonstration ofefficacy additional
to that described under potency is required.
Production
The virus is propagated in embryonated hen's eggs obtained
from healthy flocks or in suitable cell culture derived from SPF
eggs (2.7.7). The master seed lot complies with the tests for
extraneous agents as described in the General monograph for
Veterinmy Vaccines (2.7.10). The vaccine may contain one or
more suitable adjuvant.
Inactivation
An amplification test for residual live avian infectious

bronchitis virus is carried out on each batch of antigen
immediately after inactivation. The test is carried out in
fertilised hen's eggs from flocks free from specified
pathogens (SPF) or in suitable cell culture derived from SPF
2707
AVIAN INFECTIOUS BRONCIDTIS VACCINE, INACTIVATED
IP 2010
eggs (2.7.7) and the quantity of inactivated virus used is

equivalent to not less than 10 doses of vaccine. No live virus
is detected.
and control chickens and perform haemagglutination inhibition

(ill) test on each serum using 4 haemagglutinating (RA) units
ofantigen and chicken erythrocytes, testing all serum samples
at the same time. The vaccine passes the test if the mean
antibody titre of the vaccinated group is not less than 1:64
and no specific antibody is detected in the control chickens.
Alternatively, serum neutralization test may be carried out in
SPF eggs (2.7.7). Serum neutralization titre should not be less
than 102 neutralization units.
A. In embryonated eggs. For vaccine prepared with embryoadapted strains of virus, inject quantity of inactivated virus
equivalent to 10 doses of vaccine into the allantoic cavity of
ten 9 to ll-day-old fertilized hen eggs from an SPF flock and
incubate. Observe for 5 to 6 days and pool separately the
allantoic fluid from eggs containing live embryos and that
from eggs containing dead embryos, excluding those that die
within the first 24 hours after injection. Examine for
abnormalities in all embryos which die after 24 hours of
inoculation or which survive 5 to 6 days. No death or
abnormality attributable to the vaccine virus occurs.
Inject into the allantoic cavity of each of ten 9 to ll-day-old
fertilized hen eggs from SPF flock, 0.2 m1 ofthe pooled allantoic
fluid from the live embryos and into each of 10 similar eggs
0.2 ml ofthe pooled liquid from the dead embryos and incubate
for 5 to 6 days. Examine for abnormalities in all embryos which
die after 24 hours ofinjection or which survive 5 to 6 days. No
death or abnormality attributable to the vaccine virus occurs.
If more than 20 per cent of the embryos die at either stage
repeat the test from that stage. The vaccine complies with the
test if there is no death or abnormality attributable to the
vaccine virus. Antibiotics may be used to control extraneous
bacterial infection.
B. In cell culture. For vaccine prepared with cell-cultureadapted strains ofvirus, inoculate quantity ofinactivated virus
equivalent to 10 doses of vaccine into suitable cell culture
derived from SPF eggs (2.7.7). Incubate at 36 10 for 7 days.
Make a passage on another set of cell culture derived from
SPF eggs (2.7.7) and incubate at 36 10 for 7 days. None of
the cultures shows signs of infection.
Identification
In susceptible birds, the vaccine stimulates the production of
specific antibodies against each ofthe virus strain incorporated
in the vaccine, detectable by suitable serological method.
Tests
Storage. When stored under the prescribed conditions, the

vaccine may be expected to retain its potency for not less
than 2 years from the date the potency was determined.
preparing the vaccine; (2) the route of administration.
Avian Infectious Bronchitis Vaccine,

Live
Infectious Bronchitis Vaccine, Live, Avian Infectious
Bronchitis Vaccine Living
Avian Infectious Bronchitis Vaccine, Live is a preparation of
one or more suitable strains of avian infectious bronchitis
virus.
Production
The vaccine virus is grown in embryonated hens' eggs or in
cell culture derived from SPF eggs (2.7.7).
Ifthe vaccine virus is grown in embryonated hen's eggs they
are obtained from SPF flock (2.7.7) or in cell culture derived
from SPF flocks (2.7.7).
The production is based on an approved seed lot system.
Each lot of stock seed virus is tested for immunogenicity in
chicken of the same age and source by the method described
under immunogenicity test. If the immunogenicity test has
been performed with satisfactory results on the representative
batch of vaccine from the seed lot, it may be omitted as a
routine control of other batches of the vaccine prepared from
the same seed lot.
Safety. Inject intramuscularly a quantity equivalent to 2 doses

into each often SPF chickens (2.7.7) or healthy susceptible
chickens, 2 to 4 weeks old. Observe the chickens for 14 days.
No abnormal systemic or local reaction is seen.
The master seed lot complies with the tests for extraneous
agents as described in the General monograph for Veterinary
Vaccines (2.7.10).
Potency. Inject one dose by -the rotite stated oii tne label iiito
Identification
eacli6f 10 SPFchickens(2.7:7;table3) ()rhealthy susceptible

chickens, 3 to 4 weeks old. Use 5 similar chickens as controls
and house them together with the vaccinated chickens. After
28 days, collect serum samples from each of the vaccinated
earlY out either the test A or B.
A. Inoculate 0.2 ml undiluted vaccine in the allantoic sac of

SPF embryonated eggs and incubate at 36 10 for 5 to 6 days.
2708
IP 2010
AVIAN SPIROCHAETOSIS VACCINE
Lesions typical of infectious bronchitis (IB) are observed in

the embryos and the allantoic fluid does not agglutinate
chicken erythrocytes.
B. Specific antiserum against the strain or each ofthe strains

of the avian infectious bronchitis virus used in the vaccine
should neutralise corresponding IB virus. When mixed with
specific antiserum, the vaccine no longer infects 9-11 day old
embryonated SPF eggs (2.7.7).
Tests
Mycoplasmas (2.7.9). Complies with the test for mycoplasmas.
Safety. Inject 10 times the dose by the route stated on the
label into each of 10 SPF chickens (2.7.7, table 3) or healthy
susceptible chickens of 5-1 0 days old. Observe the birds for
21 days. Not more than one ofthe vaccinated chickens shows
symptoms of or dies from infectious bronchitis. If during the
period of observation more than 2 of the vaccinated chickens
die from causes not attributable to the vaccine, repeat the
test.
Virus titre. Titrate the vaccine in cell culture derived from
SPF eggs (2.7.7) derived from SPF embryos or by inoculating
into the allantoic sac of SPF embryonated eggs, 9 to 11 days
old. One dose of the vaccine contains not Jess than 103.5
TCIDsolEIDso .
Immunogenicity. Carry out a test for each route of
administration recommended on the label and for each
serotype against which protection is claimed and of the
minimum age stated for vaccination. Administer to each of20
SPF chickens (2.7.7, table 3) or healthy susceptible chickens,
3 to 4 weeks old, for each of the stated routes a volume of
reconstituted vaccine containing a quantity ofvirus equivalent
to the minimum titre stated on the label. Ten additional SPF
chickens (2.7.7, table 3) or healthy susceptible chickens of
same flock for each serotype against which protection is
claimed are used as unvaccinated controls. Three to four
weeks later, administer by eye drop a virulent strain of
bronchitis virus with a titre of at least 103.5 EID so per ml to all
the vaccinated and control birds. Between the fourth to
seventh day after the challenge, take tracheal swabs from
each of the vaccinated and control birds. Place each swab in a
sterile test tube containing 3 ml of tryptose phosphate broth
and antibiotics. Swirl the tubes containing swabs thoroughly
and store at -20 0 pending inoculation into eggs. For each
tracheal swab, inoculate at least 5 chicken embryos, 9 to 11
days old, with 0.2 ml of the broth from each tube into the
allantoic cavity. All the embryos surviving on the third day
after inoculation are used in the evaluation. A tracheal swab is
considered positive for recovery of the virus if any of the
embryos shows typical infectious bronchitis lesions such as

stunting, curling, kidney urates, clubbing down or death
between the fourth and seventh day after inoculation. The
vaccine complies with the test if not less than 80 per cent of
the controls and not more than 20 per cent of the vaccinated
chickens are positive for virus recovery. If less than 80 per
cent ofthe vaccinated chickens are negative for virus recovery
the stock seed is unsatisfactory. The stock seed virus may be
tested for immunogenicity once in 5 years provided it is
maintained under standard conditions of storage of the
bronchitis virus.
than 18 months from the date the virus titre was determined.
The reconstituted vaccine should be used immediately after
preparation.
Labelling. The label/insert states (l) the minimum virus titre
per dose; (2) the dose of vaccine.

Avian spirochaetosis Vaccine is a suspension prepared from
viscera and membranes of developing chicken embryos of
SPF eggs (2.7.7) infected with antigenic strains of Borrelia
anserina, which has been inactivated in a such a manner that
it's immunogenic activity is retained.
Production
Substrate for propagation
The organism is grown in embryonated eggs derived from
SPFflocks.
Identification
Protects chickens against infection with B. anserina.
Tests
Safety. Inject subcutaneously a quantity equivalent to 2 doses
into each of 10 SPF chickens (2.7.7, table 3) or healthy
susceptible chickens of the recommended age at which
vaccine is to be used. Observe the chickens for 14 days, no
abnormal systemic or local reaction is seen.
Potency. Inject at least 10 SPF chickens (2.7.7, table 3) or healthy
susceptible chickens, 8 to 12 week old, with the minimum dose
of vaccine by the route stated on the label. Use 5 chickens of
the same stock as controls. Ten days later challenge all the
chickens intra peritoneally with an adequate dose ofa virulent
culture of B. anserina used to prepare the vaccine or with a
2709
BLACKQUARTER VACCINE
IP 2010
suspension of liver or kidney tissues obtained from infected

chickens. Observe the chickens for 10 days. The vaccinated
chickens do not show any symptoms of the disease and
presence of B. anserina organism in the blood smears of the
vaccinated group. The test is not valid unless the control
chickens show typical symptoms of spirochaetosis with
detection of spirochetes in the blood smears.
Labelling. The label/insert states (1) strain of the bacteria
used; (2) the route of administration.
C. chauvoei infection within 48 hours of challenge ifvirulent

culture was used or within 72 hours ifa spore suspension was
used for the challenge. Repeat the test if 20 per cent ofthe
vaccinated animals die.
On repetition, the vaccine complies with the test if not more
than 10 per cent of the vaccinated animals die within 5 days
and all ofthe control animals die within 48 hours ofchallel).ge
if virulent culture was used or within 72 hours if a spore
suspension was used for challenge.
Labelling. The label states (a) the method of preparation; (b)
the strains of bacteria used to prepare the vaccine.
Canine Contagious Hepatitis Vaccine,

Inactivated
B1ackquarter Vaccine is a culture of suitable strain or strains

of Clostridium chauvoei grown in a suitable anaerobic fluid
medium and rendered sterile and non-toxic by addition of
solution of formaldehyde in such a manner that it retains its
immunizing properties. The vaccine may contain a suitable
adjuvant.
Canine Contagious Hepatitis Vaccine, Inactivated is a

preparation containing canine contagious hepatitis virus
inactivated in such a manner that its immunogenic activity is
retained. It may be a freeze-dried preparation or a liquid
preparation containing a suitable adjuvant.
Identification
Production
Protects susceptible animals against infection with

C. chauvoei.
The virus is propagated in suitable cell culture systems, the

viral suspension is harvested, titrated, inactivated and may
contain a suitable stabilizing solution.
Tests
Safety. Use two healthy susceptible animals of one of the
species for which the vaccine is intended. Inject into each
animal by the recommended route twice the maximum dose
stated on the label. Observe the animals for 7 days. No
significant local or systemic reaction is produced.
Potency. Inject ten healthy adult guinea pigs weighing between
350 and 450 g with a quantity of the product not greater than
the minimum dose and route as stated on the label. Repeat the
inoculation with the same dose of the vaccine after 28 days.
None of the vaccinated guinea pigs shows any systemic
reaction. However a minimal local reaction may be observed
in the animal.
Fourteen days after the second vaccination, inoculate
intramuscularly into each of 10 vacCinated and into each of 5
control guinea pigs with 0.2 ml ofvirulent culture or 25 viable
sp~Ore_SJlsp.eJl:ilonof1;. chau!:Q-.?UIL~per.cent otcalc~um
chloride
_..__._----_.. . _........solution.
..,--_._,_.-
The vaccine complies with the test, if not more than 10 per
cent of the vaccinated guinea pigs die from C. chauvoei
infection within 5 days and all the control animals die from
Identification
When injected into a susceptible dog, the animal develops
specific neutralizing antibodies.
Tests
Water (2.3.43). Not more than 3.0 per cent (for freeze dried
vaccine only).
Safety. Inject each of two healthy dogs, between 8 and 14
weeks old, that have been previously tested and shown to be
free from' canine adenovirus neutralizing antibodies, with twice
the dose and by the route stated on the label. Observe the
animals for 14 days. No abnormal systemic or local reaction
occurs.
Potency. Inject each oftwo healthy susceptible dogs, between
8 and 14 weeks old, that have been previously tested and
shownto-be-free-from-canine~adeno:virus-neutralizing
antibodies, with.the minimum dose and.bY-1he route stated.on

the label. After 14 days inj ect a second dose. Between 14 and
21 days after the second injection collect serum from each
dog separately and examine each sample as described below.
2710
CANINE CORONA VIRUS VACCINE, INACTIVATED
IP 2010
Inactivate the semm by heating at 56 for 30 min and prepare

serial dilutions in a suitable medium. Add to each dilution an
equal volume of serum-virus suspension containing
approximately 102 TCID so . Incubate the mixtures for 1 hour at
37. Add suitable cell culture with minimum offour replicates
for each dilution and incubate at 37 for 4 to 8 days. Examine
each culture for evidence of specific cytopathic effect.
Calculate the antibody titre. The semm from each dog contains
not less than 80 SNso per 0.05 ml.
Labelling. The label states (1) the strain used for the
preparation; (2) the name of any added adjuvant.
each of two control animals of the same stock and weight

range with a quantity of a vimlent strain of canine contagious
hepatitis vims sufficient to cause death or typical signs of the
disease in a susceptible dog. Observe the animals for a further
period of 21 days. The vaccinated animals remain in normal
health and the controls die from hepatitis or show typical
signs of serious infection. If one of the controls does not
show any signs ofthe disease, repeat the test. The vaccinated
animals of the second group remain in nonnal health and the
control animals die from hepatitis or show typical signs of
serious infection.
Canine Contagious Hepatitis Vaccine,

Live
If the potency test has been performed with satisfactory

results on a representative batch of the vaccine from the seed
lot, it may be omitted as a routine control test during production
on other batches of the vaccine prepared from the same seed
lot.
Canine Contagious Hepatitis/Canine Adenovims Vaccine is a

freeze dried preparation containing one or more attenuated
strains of canine adenovims.
Labelling. The label states (1) the minimum vims titre; (2) the
species of the animals in which use of the vaccine is
recommended.
Production
The vims is propagated in a suitable cell cultures, harvested,
titrated and may be mixed with a suitable stabilizing solution.
The stock seed vims should be tested for immunogenicity at
least once in 5 years, if maintained under suitable conditions
of storage.
Identification
The vaccine, mixed with one or more specific antisera ofcanine
adenovims(s), does not produce specific cytopathic effects
in susceptible cell cultures.
Canine Corona Virus Vaccine,

Inactivated
Canine Corona Vims Vaccine, Inactivated is a preparation
containing canine corona vims inactivated in such a manner
that its immunogenic activity is retained. It may be issued as a
liquid or as a freeze-dried preparation to be reconstituted with
a suitable liquid immediately before use. The liquid vaccine
may contain a suitable adjuvant.
Production
Tests
Virus titre. Not less than 10 3 TCID so virus per dose,
determining the titre of the vaccine in a suitable cell culture.
Safety. Inject each oftwo susceptible dogs, between 8 and 14
weeks old, that have been previously tested and shown to be
free from canine adenovims neutralising antibodies, with 10
times the dose and by the route stated on the label. Observe
the animals for 21 days. No abnormal systemic or local reaction
occurs.
The vims is grown in suitable cell culture systems. The vaccine

may contain a suitable adjuvant. Only an inactivated viral
suspension that complies with the requirements mentioned
under final bulk vaccine ofeach batch is used in the preparation
ofthe final vaccine.
Identification
When inoculated into dogs, the vaccine stimulates the
production of specific neutralizing antibodies against canine
corona vims as determined by suitable serological tests.
Tests
Potency. Inject five healthy susceptible dogs, between 8 and

14 weeks old, that have been previously tested and shown to
be free from canine adenovims neutralizing antibodies, with a
quantity ofthe vaccine equivalent to the minimum titre and by
the route stated on the label. Observe the animals for 21 days.
Inject intravenously each of the five vaccinated animals and
vaccine only).
Safety. Inject each of two healthy susceptible dogs, between
8 and 14 weeks old, free from canine corona vims antibodies
with a quantity equivalent to 2 doses by the route stated on
2711
CANINE CORONA VIRUS VACCINE, INACTIVATED
IP 2010
the label. Observe the animals for 14 days. No abnormal

systemic or local reaction occurs.
suitable stabilizing solution. The stock seed virus should be

tested for potency at least once in 5 years, if maintained
under suitable conditions of storage.
Potency. Either ofthe testA or B may be carried out.
Identification
A. Inject each of five healthy susceptible guinea pigs, each

weighing between 350 and 400 g, with halfthe minimum dose
and by the route stated on the label. Repeat the injection after
14 days. Fourteen days after the second injection collect blood
samples and obtain the serum from each guinea pig separately.
Inactivate each serum by heating at 56 for 30 min. Examine
the serum samples for antibodies by the following method.
A. The vaccine infects the chorioallantoic membranes ofSPF

embryonated eggs or suitable cell cultures. This effect is
neutralized by canine distemper antiserum.
Prepare 2-fold serial dilutions of serum in a medium suitable

for canine cells. Add to each dilution an equal volume ofa
virus suspension containing approximately 102 TCID so and
incubate the mixtures at 37 for 1 hour. Inoculate a suitable
volume of canine cells into at least 4 replicates ofserum virus
mixtures and incubate at 37 for 4 days. Examine for evidence
ofspecific cytopathic effects and calculate the antibody titre.
The vaccine complies with the test if the mean antibody titre
is not less than 45 SNsoper 0.05 ml ofserum.
Tests
B. Inject each oftwo healthy susceptible dogs, between 8 and

14 weeks old, having antibody titre less than 6 SNso perO.05 ml
ofserum, with the dose and by the route recommended on the
label. Fourteen days later collect the serum samples from
each dog separately. Inactivate each serum by heating at 56
for 30 min. Examine the serum samples individually for
neutralizing antibodies by the method described in test A.
If one dog fails to respond, i.e., the antibody titre is less than
6 SNso per 0.05 ml ofserum, repeatthetestwith two more dogs
and calculate the mean titres of the three dogs that have
responded. The vaccine complies with the test if the median
antibody titre of the sera is not less than 45 SNso per 0.05 ml.
Labelling. The label states (1) the recommended routes of
administration; (2) that the preparation should be shaken well
before use; (3) that the liquid preparation should not be allowed
to freeze; (4) that the vaccine should be used immediately
after reconstitution.

Canine Distemper'Vaccine, Live is a freeze-dried preparation
of a strain of canine distemper virus that has been attenuated
for dogs and is grown either in SPF embryonated eggs or in
suitable cell cultures.
Production
viral suspension is harvested, titrated and may contain a
B. An injection into susceptible ferrets or dogs does not cause

distemper but immunizes them against the disease. The
vaccine strain is satisfactory with respect to absence of
increase in virulence.

Virus titre. Not less than 103 EIDso/TCID so of the virus per
dose, determining the titre of the vaccine in a suitable cell
culture or SPF eggs (2.7.7).
Extraneous pathogens
A. Mix the vaccine under examination with a mono specific
distemper antiserum. It no longer provokes cytopathic effects
in susceptible cell cultures and shows no evidence of
haemagglutinating or haemadsorbing agents.
B. Use a sufficient number of mice, not less than ten, each
weighing between 11 and 15 g, such that a total of threetenths ofthe dose ofthe vaccine is injected. Inject each mouse
intracerebrally with 0.03 ml of the vaccine. Observe for 21
days. Not more than two mice die within the first 48 hours. If
more than two mice die within the first 48 hours, repeat the
test. From the third day to 21 days after the injection, the mice
do not show any abnormalities attributable to the vaccine.
Mycoplasmas (2.7.8). Complies with the test for freedom from

mycoplasmas.
Safety. Reconstitute the vaccine as recommended on the label
and carry out the following tests.
A. For chicken embryo-adapted vaccine only. Inject 0.3 m1
intracerebrally into each of a group of eight mice, between 3
and 4 weeks old, and 0.5 ml intraperitoneally into each of
another eight mice of the same age group.
Observe both the groups for 7 days. Not more than one mouse
in either group shows any abnormal local or systemic reaction
attributable to the vaccine.
B. Inject each of two susceptible dogs, between 8 and 14
weeks old which do not have antibodies against canine
distemper-vllUswitl110tlmesdose-iinci bytlie-routestated on
the label. Observe the anil11a.lsfor 21 da.ys. None ofthe dogs
shows any abnormal local or systemic reaction.
2712
IP 2010
CANINE PARAINFLUENZA VIRUS VACCINE, LIVE
Potency. Inject each offive susceptible dogs, between 8 and

14 weeks old, that have been previously tested and shown to
be free from distemper virus neutralizing antibodies with a
volume of the reconstituted vaccine containing a quantity of
the virus equivalent to the minimum titre and by the route
. stated on the label. Use another two dogs of the same stock
and age group as unvaccinated controls. Observe the animals
for 21 days. Inject intravenously each of the seven animals
with a quantity of virulent strain of canine distemper virus
sufficient to cause death or produce typical signs ofthe disease
in a susceptible dog. Observe the animals for a further
21 days. The vaccinated animals survive and show no clinical
signs of canine distemper. The test is not valid unless the
control dogs die or show symptoms typical ofcanine distemper.
If one of the control animals does not show any sign of
distemper, repeat the test. The vaccinated animals ofthe second
group remain in normal health and the control animals die
from distemper or show symptoms typical ofcanine distemper~
lot.
preparation; (2) virus titre per dose.
Canine Leptospirosis Vaccine,

Inactivated
Canine Leptospirosis Vaccine (Inactivated) is a suspension
of inactivated whole organisms and/or antigenic extract(s) of
one or more suitable strains of one or more of Leptospira
interrogans serovar canicola, serovar icterohaemorrhagiae
or any other epidemiologically appropriate serovar, inactivated
and prepared in such a way that adequate immunogenicity is
maintained.
Production
The seed material is cultured in a suitable medium; each strain
is cultivated separately. During production, various parameters
such as growth rate are monitored by suitable methods; the
values are within the limits approved for the particular product.
Purity and identity are verified on the harvest using suitable
methods. After cultivation, the bacterial harvests are collected
separately and inactivated by a suitable method. The antigen
may be concentrated. The vaccine may contain an adjuvant.
Inactivation
Carry out a test for inactivation by inoculation on to a specific
medium. Inoculate 1 rnl ofthe vaccine into 100 rnl ofthe medium.
Incubate at 30 for 14 days, subculture into a further quantity

ofthe medium and incubate both media at 30 for 14 days: no
growth occurs in either medium. At the same time, carry out a
control test by inoculating a further quantity of the medium
with the vaccine together with a quantity ofa culture containing
approximately 100 leptospirae and incubating at 30 Growth
of leptospirae occurs within 14 days.
Identification
When administered to experimental animals causes the
appearance of agglutinating antibodies against the serotype
or serotypes used to prepare the vaccine.
Tests
Safety. Use two dogs of the minimum age recommended for
vaccination and which do not have antibodies to the leptospira
serovar(s) present in the vaccine. Administer 2 doses of the
vaccine to each dog by a recommended route. Observe the
animals for 14 days. The animals remain in good health and no
abnormal local or systemic reaction occurs.
Potency. Carry out a separate potency test for each serotype
ifthe vaccine is prepared with different serotypes. Inject each
of five hamsters not more than 3 months old, the animals
being drawn from the same stock, subcutaneously with 1/40
ofthe dose of the vaccine stated on the label for dogs. Use an
equal number of animals of the species used for the test as
controls. After 15 to 20 days inject intraperitoneally into each
of the vaccinated and control animals an adequate dose of a
virulent culture ofleptospirae of the serotype used to prepare
the vaccine or a suspension ofliver or kidney tissue obtained
from animals infected with the serotype used to prepare the
vaccine. Observe the animals for 14 days after the injection.
Not less than four ofthe control animals die showing typical
leptospira infection. Not less than four of the vaccinated
animals remain in good health for not less than 14 days after
the death of the four control animals.
Canine Parainfluenza Virus Vaccine,

Live
Canine Parainfluenza Virus Vaccine, Live is a freeze-dried
preparation of tissue culture fluid containing the cell cultureadapted attenuated canine parainfluenza virus of stock seed
which has been established as pure, safe and immunogenic.
2713
CANINE PARAINFLUENZA VIRUS VACCINE, LIVE
IP 2010
Production
viral suspension is harvested, titrated and may contain a
suitable stabilizing solution.
Identification
When inoculated into dogs, the vaccine stimulates the
production of specific neutralizing antibodies against canine
parainfluenza virus as determined by suitable serological tests.
Tests
Canine Parvovirus Vaccine,

Inactivated
Canine Parvovirus Vaccine, Inactivated is a liquid or freeze
dried preparation ofcanine parvovirus inactivated by a suitable
method so that its immunogenic activity is retained.
Production
The virus is grown in suitable cell culture systems. The virus
may be cloned, purified and concentrated. The vaccine may
contain a suitable adjuvant.
Identification
Virus titre. Not less than 10 TCID so per dose, detelmining

the titre of the vaccine in a suitable cell culture.
Safety. Inject each of two susceptible dogs, between 8 and
14 weeks old, free from canine parainfluenza virus
haemagglutinating antibodies with a dose of the vaccine
reconstituted with the sterile diluent equivalent to 10 times
animals for 14 days. None of the dogs shows any systemic or
local reactions.
When inoculated into dogs, the development of specific

neutralizing antibodies against canine parvovirus can be
demonstrated by suitable serological tests.
Tests
Safety. Inject into each of two healthy susceptible dogs,
between 8 and 14 weeks old, preferably sero negative ones
with a quantity equivalent to 2 doses by the route stated on
the label. Observe the animals for 14 days. No abnormal
systemic or local reaction occurs.
Potency. Use two healthy susceptible dogs, between 8 and 14 Sterility (2.2.11). Complies with the test for sterility.
weeks old, having haemagglutination inhibition antibody titres
Potency. Either of the testA or B may be carried out.
less than 1:4 per 0.1 ml ofserum. Keep equal number of dogs
as unvaccinated controls. Vaccinate each test group dog with A. Inject subcutaneously each of five healthy susceptible
the vaccine as per schedule stated on the label. After 21 days, guinea-pigs, each weighing between 350 and 400g, with half
collect the serum from each dog separately and examine each ofthe dose stated on the label. After 14 days, inject again half
sample as described below. Heat the serum of each animal at ofthe dose stated on the label. Fourteen days after the second
56 for 30 minutes and prepare serial dilutions with saline injection collect blood samples and obtain the serum from
solution. To 0.025 ml ofeach dilution add 0.025 ml ofa canine each guinea pig separately. Inactivate each serum by heating
parainfluenza virus suspension containing 4 haemagglutinating at 56 for 30 min. To 1 volume ofeach serum add 9 volumes of
units. Allow the mixture to stand at room temperature for 30 20 per cent suspension of light kaolin in phosphate buffered
minutes and add 0.05 ml of a suspension of chicken salinepH 7.4. Shake the mixture for 20 minutes and centrifuge
erythrocytes containing 2 x 107 erythrocytes per ml. Allow to at 2,000 rpm for 10 minutes. Collect the supernatant liquid and
stand for 1 hour and note the last dilution of serum that mix with 1 volume of a concentrated suspension ofRhesus
completely inhibits haemugglutination. Calculate the median monkey/pig erythrocytes. Allow the mixture to stand at 4 for
antibody litre of the sera which should not be less than 1:40 60 minutes and centrifuge at 2,000 rpm for 10 minutes and
per 0.025 ml ofthe serum. If one dog fails to respond, repeat collect the supernatant serum obtained in lO-fold dilution.
the test using two more dogs and calculate the result as the . Using each serum, prepare a series of 2-fold dilutions. To
mean of titres obtained from all of three dogs that have 0.025 ml of each of the latter dilutions add 0.025 ml of a
responded which is not less than 1:40 per 0.025 ml of the suspension of canine parvovirus antigen containing
approximately 8 haemagglutinating (RA) units and allow to
serum.
stand at 37 for 30 minutes. Add 0.05 ml or other suitable
quantity of a 1 per cent suspension of Rhesus monkey/pig
results on a representative batch of the vaccine it may be erythrocytes.. containing30_x_106 cellsper-ml-to-atJeastfour
omitted as aroiifine test diiring prodiiCfiononfheofh-er replicates Qfeach dilution.Allow to stand at 4 0 for 90 minutes
oiitcnesof Vacciiiepfepai"ecrfrorrftliesame-seecrIoC --- - --- .
and note the last dilution ofthe serum that completely inhibits
Labelling. The label states (l) the strain used for the haemagglutination. The vaccine complies with the test if the
preparation; (2) the virus titre per dose.
median antibody titre of the sera is not less than 1:80.
2714
IP 2010
MULTICOMPONENT CLOSTRIDIUM VACCINE, INACTIVATED
B. Inject each oftwo healthy susceptible dogs, between 8 and

14 weeks old, having antibody titres less than 4 ND so
(50 percent neutralizing dose) per 0.1 ml of serum, with the
dose and by the route recommended on the label. Fourteen
days later collect the blood/serum samples from each dog
separately. Inactivate each serum by heating at 56 for 30 min.
Examine the serum samples individually for neutralizing
antiboqies as follows:
Prepare 2-fold serial dilutions of serum in a medium suitable
for canine cells. Add to each dilution an equal volume ofvirus
suspension containing approximately 104 TCID so and incubate
the mixtures at 37 for one hour. Inoculate a suitable volume of
canine cells into at least four replicates ofserum virus mixtures
and incubate at 37 for 7 days. Examine for evidence ofspecific
cytopathic effects and calculate the antibody titre. The vaccine
complies with the test if the mean antibody titre is not less
than 32 ND so per 0.1 ml of serum. If one dog fails to respond
repeat the test using two more dogs and calculate the mean
titres of the three dogs that have responded.
stated on the label. Observe the animals for 21 days. No

abnormal systemic or local reaction occurs.
Potency. Inject each of seven dogs, between 8 and 14 weeks
old, free from canine parvovirus haemagglutinating antibodies,
subcutaneously with the minimum dose stated on the label.
Use two dogs of the same stock, weight and age range as
controls. Not less than 21 days after injection of the vaccine,
challenge the vaccinated and control animals through the
oronasal route with a virulent strain of infectious canine
parvovirus. Observe the animals for 14 days. Not less than
five of the vaccinated dogs survive. The test is not valid
unless the control dogs die or show clinical signs of canine
parvovirus infection.

results on a representative batch of the vaccine it may be
omitted as a routine test during production ofthe other batches
of vaccine prepared from the same seed lot.
Labelling. The label states (1) the method of preparation; (2)

the types and strains of virus used to prepare the vaccine.
Labelling. The label states (l) the strain used for the
preparation; (2) virus titre per dose.
Canine Parvovirus Vaccine, Live
Multicomponent Clostridium Vaccine,

Inactivated
Canine Parvovirus Vaccine, Live is a freeze-dried preparation

of a strain of canine parvovirus that is attenuated for the
target species of dogs.
Production
The attenuated virus is grown in suitable cell culture systems.
The viral harvest is titrated and mixed with a suitable stabilizing
solution. The stock seed virus should be tested for
immunogenicity at least once in 5 years, if maintained under
suitable conditions of storage.
Identification
Multicomponent Clostridium Vaccine, Inactivated consists

of five highly antigenic components containing the toxoids
of C. pelfringens type 'B' C. perjringens type 'C', C.
perjringens type 'D', C. oedematiens and C. septicum which
are prepared in double strength and then combined in such a
proportion that would invoke adequate anti-toxin response in
the vaccinated sheep against each antigen incorporated in
the vaccine.
Identification
When inoculated into dogs, the development of specific

neutralizing antibodies against canine parvovirus can be
demonstrated by suitable serological tests.
When injected into susceptible animals, it stimulates the

production ofepsilon and beta antitoxin against C. perjringens
types 'B', 'C' and 'D' and also antitoxins against C. septicum
and toxin of C. oedematiens.
Tests
Tests
Safety. Four sheep each are inoculated with two times the
dose of vaccine subcutaneously and are observed for 7 days
during which period the animals do not show any local or
systemic reaction..
Virus titre. Not less than 128 HA units per dose when tested
using suitable RBC after culturing the virus in a suitable cell
culture.
Safety. Inject each oftwo susceptible dogs, between 8 and 14
weeks old, free from canine parvovirus hemagglutinating
antibodies, a quantity of the vaccine reconstituted with the
sterile diluent equivalent to 10 times the dose and by the route

Potency. Eight sheep each are inoculated with 2 doses of
vaccine subcutaneously at an interval of 21 to 28 days and
bled on 10th day after second inoculation for collection of
2715
CLOSTRIDIUM NOVYI (TYPE B) VACCINE INACTNATED FOR VETERINARY USE
serum for assessing the antitoxin titre against each antigen

incorporated in the vaccine. The post-inoculation serum
contains not less than 5 IU of epsilon antitoxin and 10 units of
beta antitoxins of C. perjringens types 'B' and 'C' and 2.5 IU
of C. septicum antitoxin and 3.5 IU of C. oedematiens antitoxin.
Labelling. The label states (1) the types ofClostridia contained
in the vaccine; (2) the preparation should be shaken before
use.
Clostridium novyi (Type B) Vaccine

Inactivated for Veterinary Use
Clostridium novyi (Type B) Vaccine Inactivated for Veterinary
Use is prepared from a liquid culture of a suitable strain of
Clostridium novyi Type B.
Production
The whole culture or its filtrate or a mixture of the two is
inactivated in such a manner that toxicity is eliminated and
immunogenic activity is retained. Toxoids and/or inactivated
cultures may be treated with a suitable adjuvant, after
concentration if necessary.
Choice of vaccine composition. The vaccine is shown to be
satisfactory with respect to safety and efficacy (2.7.12). For
the latter, it shall be demonstrated that for each target species
the vaccine, when administered according to the recommended
schedule, stimulates an immune response (for example,
induction of antibodies) consistent with the claims made for
the product.
Batch testing
Safety. Administer by a recommended route, to each of 2 sheep
that have not been vaccinated against C. novyi Type B twice
the maximum dose ofthe vaccine stated on the label. Observe
the animals for not less than 14 days. No abnormal local or
systemic reaction occurs.
Residual toxicity. Inject 0.5 ml ofthe vaccine subcutaneously
into each of 5 mice, each weighing between 17 and 22 g.
Observe the animals for 7 days. No abnormal local or systemic
reaction occurs.
Identification
The vaccine stimulates the formation of C. novyi Type B alpha
antitoxin when injected into animals.
Tests
. Sterility (2.2.11). Complies with the test for sterility.
Potency. Inject subcutaneously into each of not less than
IP 2010
10 healthy rabbits, 3 to. 6 months old, a quantity of vaccine

not exceeding the minimum dose stated on the label as the
first dose. After 21 to 28 days, inject into the same animals a
quantity ofthe vaccine not exceeding the minimum dose stated
on the label as the second dose. 10 to 14 days after the second
injection, bleed the rabbits and pool the sera.
The alpha antitoxin titre of the pooled sera is not less than
3.5 ill perml.
The International Unit is the specific neutralising activity for
C. novyi alpha toxin contained in a stated amount of the
International standard, which consists of a quantity of dried
immune horse serum. The equivalence in International Units
of the International standard is stated by the World Health
Organisation.
The potency of the pooled sera obtained from the rabbits is
determined by comparing the quantity necessary to protect
mice or other suitable animals against the toxic effects of a
fixed dose of C. novyi alpha toxin with the quantity of a
reference preparation of Clostridium novyi alpha antitoxin,
calibrated in International Units, necessary to give the same
protection. For this comparison, a suitable preparation of
C. novyi alpha toxin for use as a test toxin is required. The
dose ofthe test toxin is determined in relation to the reference
preparation; the potency of the serum under examination is
determined in relation to the reference preparation using the
test toxin.
Preparation oftest toxin. Prepare the test toxin from asterile
filtrate ofan approximately 3 to 5 day culture in liquid medium
of C. novyi Type B and dry by a suitable method. Select the
test dose ofthe toxin in mice by determining the L+ 10 dose
and the LD so the observation period being 72 hours.
A suitable alpha test toxin contains not less than one L+/
10 dose in 0.05 mg and not less than 10 LD so in each L+/
10 dose.
Determination oftest dose oftoxin. Prepare a solution ofthe
reference preparation in a suitable liquid so that it contains
1 IU per ml. Prepare a solution of the test toxin in a suitable
liquid so that 1 ml contains a precisely known amount such as
1 mg. Prepare mixtures of the solution of the reference
preparation and the solution of the test toxin such that each
mixture contains 1.0 ml of the solution of the reference
preparation (1 IU), one of a series of graded volumes of the
solution of the test toxin and sufficient of a suitable liquid to
bring the total volume to 2.0 ml. Allow the mixtures to stand at
room temperature for 60 min. Using not less than 2 mice, each
weighing between 17 and 22 g, for each mixture, inject a dose
the mice
for72ho lll"s, Ifalltllelllice ~ie, the amountoftoxin present
0.2 ml ofthe mixture is in excess ofthetest dose. Ifnone ofthe
mice dies, the amount oftoxin present in 0.2 ml ofthe mixture
is less than the test dose. Prepare fresh mixtures such that
2716
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CLOSTRIDIUM PERFRINGENS VACCINE, INACTIVATED
2.0 ml of each mixture contains 1.0 ml of the solution of the

reference preparation (1 IU) and one of a series of graded
volumes of the solution of the test toxin separated from each
other by steps of not more than 20 per cent and covering the
expected end-point. Allow the mixtures to stand at room
temperature for 60 min. Using not less than two mice for each
mixture, inject a dose of0.2 ml subcutaneously into each mouse.
Observe the mice for 72 hours. Repeat the determination at
least once and combine the results of the separate tests that
have been made with mixtures of the same composition so
that a series of totals is obtained, each total representing the
mortality due to a mixture of a given composition.
mixture which causes the death ofone halfofthe total number
ofmice injected with it.
Determination of the potency of the serum obtained from
rabbits
Preliminmy test. Dissolve a quantity of the test toxin in a
suitable liquid so that 1 ml contains 10 times the test dose
(solution of the test toxin). Prepare a series of mixtures of the
solution of the test toxin and of the serum under examination
such that each mixture contains 1.0 ml of the solution of the
test toxin, one of a series of graded volumes of the serum
under examination and sufficient of a suitable liquid to bring
the final volume to 2.0 ml. Allow the mixtures to stand at room
temperature for 60 min. Using not less than 2 mice for each
mixture, inject a dose of0.2 rnl subcutaneously into each mouse.
Observe the mice for 72 h. Ifnone ofthe mice dies, 0.2 ml of
the mixture contains more than 0.1 ill. Ifall the mice die, 0.2 rnl
ofthe mixture contains less than 0.1 IU.
Final test. Prepare a series of mixtures of the solution of the
test toxin and ofthe serum under examination such that 2.0 ml
ofeach mixture contains 1.0 ml ofthe solution ofthe test toxin
and one of a series of graded volumes of the serum under
examination, separated from each other by steps of not more
than 20 per cent and covering the expected end-point as
determined by the preliminary test. Prepare further mixtures of
the solution ofthe test toxin and ofthe solution ofthe reference
preparation such that 2.0 ml ofeach mixture contains 1.0 mlof
the solution of the test toxin and one of a series of graded
volumes ofthe solution of the reference preparation, in order
to confirm the test dose of the toxin. Allow the mixtures to
stand at room temperature for 60 min. Using not less than 2
mice for each mixture, proceed as described in the preliminary
test. The test mixture which contains 0.1 ill in 0.2 ml is that
mixture which kills the same or almost the same number of
mice as the reference mixture containing 0.1 ill in 0.2 ml. Repeat
the determination at least once and calculate the average ofall
valid estimates. The test is valid only if the reference
preparation gives a result within 20 per cent of the expected
value.
The confidence limits (P = 0.95) have been estimated to be (a)

85 per cent and 114 per cent when 2 animals per dose are used;
(b) 91.5 per cent and 109 per cent when 4 animals per dose are
used; (c) 93 per cent and 108 per cent when 6 animals per
dose are used.
Labelling. The label states (1) whether the product is a toxoid,
a vaccine prepared from a whole inactivated culture or a
mixture of the two; (2) that the preparation is to be shaken
before use; (3) for each target species, the immunising effect
produced (for example, antibody production, protection
against signs of disease or infection).
Clostridium Perfringens Vaccine,

Inactivated
The vaccines contains inactivated cultures of strains of C.
perfi-ingens type B (Lamb Dysentery Vaccine), type C (Struck
Vaccine) or type D (Enterotoxaemia Vaccine; Pulpy Kidney
Vaccine) or any combination of these types.
Production
The organisms grown in an anaerobic medium, the whole
culture or their filtrates or a mixture ofthe two are inactivated
in such a manner that toxicity is eliminated andimmunogenic
activity is retained. Toxoid and or inactivated cultures may
contain a suitable adjuvant.
Batch tesing
Safety. Inject subcutaneously two healthy susceptible sheep
weighing about 18 kg each or two healthy susceptible rabbits
weighing between 1.5 and 2.0 kg each with twice the dose
stated on the label and observe the animals for 14 days.
reaction occurs.
into each of 5 mice, each weighing 17 to 22 g. Observe the
animals for 7 days. No abnormal local or systemic reaction
occurs.
Potency
C. perji-ingens Type B Vaccine. Inject subcutaneously into

each ofsix healthysusceptible sheep weighing about 18 kg or
ten healthy susceptible rabbits weighing between 1.5 and 2.0
kg with the minimum dose of the vaccine stated on the label.
Repeat the dose after an interval of21 to 28 days. Bleed the
animals 10 to 14 days after the second dose ofthe vaccine and
determine beta antitoxin titres in the pooled serum sample by
2717
CLOSTRIDIUM PERFRINGENS VACCINE, INACTIVATED
IP 2010
testing in mice as per the method described for C. peifringens

Type D Vaccine. Product passes the test ifthe post-inoculation
pooled serum contains not less than 10 ill of beta antitoxins,
and not less than 5 IU ofepsilon antitoxin per milliliter.
C. perfi-ingens Type C Vaccine. Cany out the test for potency

as described for C. perfringens Type B Vaccine.
1 ml of pooled serum contains not less than 10 IU of beta
antitoxin per ml.
C. perjringens Type D (Enterotoxaemia) Vaccine. Cany out

the test as described below.
1 ml of pooled serum contains not less than 5 IU of C.
perfi-ingens epsilon antitoxin per ml.
Identification
International standard for the standard preparations

The International units of the antitoxin is the specific
neutralizing activity of C. pel'fringens epsilon toxin contained
in the stated amountin relation to International standards in
the dried Horse serum.
The International units of the antitoxins is the specific
neutralizing activity for the C. perfringens beta toxin contained
in the stated amount in relation to International standards in
the dried Horse serum.
Test animals
Use healthy mice having body weights such that the difference
between the lightest and heaviest is not more than 5 g.
B. When injected into susceptible animals, the C. perfringens

Type C Vaccine stimulates the production of C. perfi-ingens
beta antitoxin.
Suggested method for preparation of test toxin. Prepare C.

perfringens toxins from sterile supernatants/filtrates of early
cultures of C. perfringens type B, type C or type D. The
supernatants may be purified by precipitation with ammonium
sulphate and the resulting precipitate collected. This may then
be dried over phosPhorus pentoxide at a pressure of 1.5 to
2.5 kPa, powdered and kept dry orre-dissolved and freezedried.
C. When injected into susceptible animals, the C. perfringens

Type D Vaccine stimulates the production of C. perfi-ingens
epsilon antitoxin.
L+ and L+/IO doses. These are the smallest quantities oftoxin

that when mixed respectively with 1Unit ofantitoxin and with
0.1 Unit ofantitoxin and injected intravenously into mice cause
the death of the animals within 72 hours.
A. When injected into susceptible animals, the C. perfringens

Type B Vaccine stimulates the production of C. perfringens
beta and epsilon antitoxins.
Tests
Potency test. Inject subcutaneously into each of at least six
healthy susceptible sheep weighing about 18 kg or ten healthy
susceptible rabbits weighing between 2.0 and 2.5 kg with the
minimum dose of the vaccine stated on the label. Repeat the
dose in each sheep/rabbit after an interval of 21 to 28 days.
Bleed the animals 10 to 14 days after the second inoculation.
The sera of sheep or rabbits are pooled separately and
estimated for the antitoxin levels.
Biological assay of C. perjringens antitoxins
The potency of C. perjringens beta and epsilon antitoxins is
determined by comparing the dose of antitoxin necessary to
protect mice or other suitable animals against the toxic effects
of C. perfringens beta toxin or epsilon toxin with the dose of a
standard preparation of the respective antitoxin necessary to
give the same protection. For this comparison, the standard
preparations of C. perjringens beta antitoxin and C.
perfi-ingens epsilon antitoxin and suitable preparations of C.
peifringens beta and epsilon toxins are required.
'The-test-dose-of-each-toxin-is_establishecLinJelatiolLtoJhe
appropriateStandardpreparationofantitQxin and th~PQtel1-9Y
of antitoxin under examination is then determined in relation
to the appropriate Standard preparation using the appropriate
test toxin.
LD so This is the quantity of toxin that when injected

intravenously into mice causes the death of one-half of the
mice injected within 72 hours.
A suitable C. perji-ingens beta toxin is one that has an L+ dose
in 0.2 mg or less and contains not less than 25 LD so in an
L+dose.
A suitable Clostridium perfringens epsilon toxin is one that
has an L+/1O dose in 0.005 mg or less and contains not less
than 20 LD so in an L+/1 0 dose.
Determination of test dose of C. perjringens beta toxin.
Dissolve a quantity ofdried toxin in a suitable liquid such that
1.0 ml contains a precise amount such as 10 mg. Reconstitute
the standard preparation of C. peljringens beta antitoxin with
a suitable liquid to give a solution containing 5 Units of C.
perjringens beta antitoxin in 1 ml.
Prepare mixtures such that5.0 ml ofeach mixture contains 2.0
ml of the solution of the Standard preparation (10 Units) and
one__ofa_seriesofgrMeJiYQhnJl~!:LQH1~Ql!l:tiQ!!Qf!ht::t()xin
l)iM~ a<::h l11ix1:llre to the same final volume with a suitable
liquid. Allow the mixtures to stand at room temperature,
protected from light, for 30 minutes and then inject a dose of
0.5 ml ofeach mixture intravenously into each ofnot less than
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CLOSTRIDIUM SEPTICUM VACCINE, INACTIVATED
two mice. Observe the mice for 72 hours. Ifall the mice die, the
amount oftoxin present in 0.5 ml ofthe mixture is in excess of
the test dose. If none of the mice dies, the amount of toxin
present in 0.5 ml of the mixture is less than the test dose.
Prepare similar fresh mixtures such that 5.0 ml ofeach mixture
contains 2.0 ml ofthe solution ofthe Standard preparation (l 0
Units) and one of a series of graded volumes of the solution
of tIle toxin, separated from each other by steps of not more
than 20 per cent and covering the expected end-point.
Allow the mixtures to stand at room temperature, protected
from light, for 30 minutes, Inject a dose of0.5 ml ofeach mixture
intravenously into each of not less than two mice. Observe
the mice for 72 hours. Repeat the determinations at least once
and add together the results of the separate tests that have
been made with mixtures ofthe same composition such that a
series of totals is obtained, each total representing the
mortality due to a mixture of a given composition.
mixture that causes the death of one-half of the total number
of mice injected with it within 72 hours.
Determination oftest dose of C perfringens epsilon toxin.

Carry out the method described for the determination oftest
dose of C. peljringens beta toxin with the following
modification. Dissolve a quantity of dried toxin in a suitable
liquid such that 1.0 ml contains a precise amount such as I
mg.
Reconstitute the Standard preparation of C. perji'ingens
epsilon antitoxin with a suitable liquid to give a solution
containing 0.5 Unit in 1 ml (the prepared mixtures will therefore
contain 1 Unit ofthe Standard preparation in 5 ml).
ofmice injected with it within 72 hours.
Determination ofpotency of C perfringens beta antitoxin
Preliminmy test. Dilute the test toxin with a suitable liquid

such that 2.0 ml contains 10 times the test dose. Prepare
solution of the toxin and one of a series of graded volumes of
the preparation under examination. Adjust each mixture to the
same final volume with a suitable liquid. Allow the mixtures to
stand at room temperature, protected from light, for 30 minutes.
Inject a dose of0.5 ml ofeach mixture intravenously into each
ofnot less than two mice and observe the mice for 72 hours. If
all the mice die, 0.5 ml ofthe mixture contains less than 1 Unit
of antitoxin. If none of the mice dies, 0.5 ml of the mixture
contains more than 1 Unit of antitoxin.
Final test. Prepare similar fresh mixtures such that 5.0 ml of

each mixture contains 2.0 ml of the solution of the toxin and
one of a series of graded volumes of the preparation under
than 20 per cent and covering the expected end-point. Prepare

further mixtures of 5.0 ml containing 2.0 ml ofthe solution of
the toxin and graded volumes of the Standard preparation of
C. perjringens beta antitoxin to confirm the test dose of the
toxin. Allow the mixtures to stand at room temperature,
protected from light, for 30 minutes. Inject a dose of0.5 ml of
each mixture intravenously into each of not less than two
mice and observe the mice for 72 hours. The mixture of the
antitoxin under examination which contains 1 Unit of C.
peljNngens beta antitoxin in 0.5 ml is that mixture which causes
the death of the same or almost the same number of mice as
the mixture containing 1 Unit ofthe Standard preparation of
C. peljringens beta antitoxin in 0.5 ml. Repeat the
determinations at least once and calculate the average of all
valid estimates. Estimates are not valid unless the Standard
preparation gives a result within 20 per cent of the expected
value.
Determination ofpotency of C perfringens epsilon antitoxin.

Carry out the preliminary test and final test as described for
the detennination of potency of C. peJjringens beta antitoxin
with the following amendments.
Dilute a quantity ofthe test toxin in a suitable liquid such that
2.0 ml contains 10 times the test dose. Tjle Standard preparation
used in these tests is that of C. peJjringens epsilon antitoxin.
The mixture ofthe antitoxin under examination which contains
0.1 Unit of C. per:tNngens epsilon antitoxin in 0.5 ml is that
mixture which causes the death of the same or almost the
same number ofmice as the mixture containing 0.1 Unit ofthe
Standard preparation of C. perjringens epsilon antitoxin in 0.5
ml.
Limits of error. For the suggested method, the limits oferror
(P = 0.95) have been estimated to be 85 to 114 per cent when
two mice are used per dose, 91.5 to 109 per" cent when four
mice are used per dose, and 93 to 108 per cent when six mice
are used per dose.
Labelling. The label states (a) the type or types of C. perfringens
from which the vaccine has been prepared; (b) whether the
preparation is a toxoid or a vaccine prepared from a whole
inactivated culture or a mixture of the two; (c) that the
preparation is to be shaken before use; (d) for each target
species, the immunising effect produced (for example, antibody
production, protection against signs of disease or infection).
Clostridium Septicum Vaccine,

Inactivated
Clostridium Septicum Vaccine, Inactivated is a suspension of
a culture of a highly toxigenic strain of C. septicum grown in
an anaerobic medium, or a filtrate from such a culture.
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CLOSTRIDIUM SEPTICUM VACCINE, INACTIVATED
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Production
The whole culture or its filtrate or a mixture of the two is
inactivated in such a manner that toxicity is eliminated and
immunogenic activity is retained. Toxoid and/or inactivated
cultures may be treated with a suitable adjuvant.
Batch testing
into each of 5 mice, each weighing between 17 and 22 g.
reaction occurs.
Potency. Inject subcutaneously each of eight healthy
susceptible sheep, between 8 and 12 months old, orten rabbits,
between 3 and 6 months old, with the minimum dose of the
vaccine stated on the label. Repeat the dose after an interval
of21 to 28 days. 10 to 14 days after the second inoculation,
bleed the animals. Pool the sera samples from individual
animals and determine the antitoxin titre by the biological assay
of C. septicum antitoxin described below.
1 ml of serum contains not less than 2.5 Units of C. septicum
antitoxin by biological"assay of C. septicum antitoxin.
The potency of C. septicum antitoxin is determined by
comparing the dose of antitoxin necessary to protect mice or
other suitable animals against the lethal effects of C. septicum
toxin with the dose of a Standard preparation of C. septicum
antitoxin necessary to give the same protection. For this
purpose, the Standard preparation of C. septicum antitoxin
and a suitable preparation of C. septicum toxin for use as a
test toxin are required.
Identification
When injected into healthy susceptible animals, it stimulates
the production of antitoxins to C. septicum.
Tests
The test dose of the toxin is determined in relation to the
Standard preparation ofantitoxin and the potency ofantitoxin
under examination is then determined in relation to the Standard
preparation using the test toxin.
Assay
The .Standard preparation isthe3rdInternational-standard,
established in 1957, consisting of driedhyperimmune horse
serum (supplied in ampoules containing 500 Units) or another
suitable preparation the potency ofwhich has been determined
in relation to the International standard.
Safety. Inject subcutaneously each oftwo healthy susceptible

sheep, between 8 and 12 months old, with twice the dose
stated on the label. Observe the animals for 7 days; none of
the animals shows any systemic or local reaction. Observe
the animals for 14 days.
more than 5 g.
Preparation oftest toxin. Prepare C. septicum toxin by growing
C. septicum in a liquid culture medium, filtering the supernatant
aseptically and precipitating with ammonium sulphate. The
resulting precipitate, which contains the toxin, is collected,
dried over phosphorus pentoxide at a pressure of 1.5 to 2.5
kPa, powdered and kept dry.
Selection of test toxin. Select toxin for use as the test toxin by
L+/5 dose. This is the smallest quantity of the toxin which
when mixed with 0.2 Unit ofantitoxin and injected intravenously
into mice causes the death of the animals within 72 hours.
LD so This is the quantity of toxin which when injected
intravenously into mice causes the death of one-half of the
A suitable C. septicum toxin is one that has an L+/5 dose in 1
mg or less and contains not less than 10 LD so in an L+/5 dose.
Determination of test dose of toxin. Weigh accurately a
quantity of the dried toxin and dissolve it in a suitable liquid
so that 1.0 ml contains a precise Imown amount, such as 4 mg.
Prepare a solution of the Standard preparation in a suitable
liquid such that 1.0 ml contains 1 Unit.
Prepare mixtures such that 5.0 ml ofeach mixture contains 2.0
ml of the solution of the Standard preparation (2 Units) and
one of a series of graded volumes of the solution of the toxin.
Dilute each mixture with a suitable liquid to the same final
volume (5.0 ml). Allow the mixtures to stand at room
temperature, protected from light, for 60 minutes and then
inject a dose of 0.5 ml ofeach mixture intravenously into each
ofnot less than 2 mice. Observe the mice for 72 hours. Ifall the
mice die the amount oftoxin present in 0.5 ml ofthe mixture is
in excess ofthe test dose. Ifnone ofthe mice dies, the amount
of toxin present in 0.5 ml of the mixture is less than the test
dose. Prepare similar fresh mixtures such that 5.0 ml of each
mixture contains 2.0 ml of the solution of the Standard
preparation (2 Units) and one ofa series ofgraded volumes of
the solution ofthe toxin separated from each other by steps of
not more than 20 per cent and covering the expected end-
p()i l1t.
Allow the mixtures. tosta.l1cl at roQIl1J~mP~l"a.tttr~,.protecte<:!
from light, for 60 minutes. Inject a dose of0.5 ml ofeach mixture
intravenously into each of not less than two mice. Observe
the mice for 72 hours. Repeat the determinations at least once
2720
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DUCK PASTEURELLA VACCINE, INACTIVATED
and add together the results of the separate tests that have
been made with mixtures ofthe same composition such that a
series of totals is obtained, each total representing the
mOliality due to a mixture of a given composition.
or a mixture ofthe two; (2) that the preparation is to be shaken

before use; (3) for each target species, the immunising effect.
produced (for example, antibody production, protection
against signs of disease or infection).

ofmice injected within 72 hours.
Determination ofpotency ofthe antitoxin
Preliminmy test. Dilute the test toxin with a suitable liquid

such that 2.0 ml contains 10 times the test dose. Prepare
solution ofthe toxin and one of a series of graded volumes of
the preparation under examination. Adjust each mixture to the
same final volume with a suitable liquid.
fiOln light, for 60 minutes. Inject a dose of0.5 ml ofeach mixture
intravenously into each ofnot less than two mice and observe
the mice for 72 hours. Ifall the mice die, 0.5 ml ofthe mixture
contains less than 0.2 Unit of antitoxin. If none of the mice
dies, 0.5 ml of the mixture contains more than 0.2 Unit of
antitoxin.
Final test. Prepare similar fresh mixtures such that 5.0 ml of

each mixture contains 2.0 ml of the solution of the toxin and
one of a series of graded volumes of the preparation under
than 20 per cent and covering the expected end-point. Prepare
further mixtures of5.0 ml containing 2.0 ml ofthe solution of
the toxin and graded volumes of the standard preparation to
confinn the test dose of the toxin.
fromlight, for 60 minutes. Inject a dose of0.5 ml ofeach mixture
intravenously into each ofnot less than two mice and observe
the mice for 72 hours. The mixture of the antitoxin under
examination which contains 0.2 Unit in 0.5 ml is that mixture
which causes the death ofthe same or almost the same number
of mice as the mixture containing 0.2 Unit of the Standard
preparation in 0.5 ml. Repeat the detenninations at least once
and calculate the average of all valid estimates. Estimates are
not valid unless the Standard preparation gives a result within
20 per cent ofthe expected value.
Limits of error. For the suggested method, the limits of error
(P=0.95) have been estimated to be (a) 85 per cent and 114 per
cent when 2 animals per dose are used; (b) 91.5 per cent and
109 per cent when 4 animals per dose are used; (c) 93 per cent
and 108 per cent when 6 animals per dose are used.
The vaccine passes the test if the pooled serum contains 2.5
IV of C. septicum antitoxins.
Labelling. The label states (1) whether the preparation is a
toxoid or a vaccine prepared from a whole inactivated culture,

Duck Pasteurella Vaccine, Inactivated consists ofan emulsion
or suspension of a virulent strain of Pasteurella multocida
which has been inactivated in such a manner that the
pathogenicity is eliminated and the immunogenic activity is
retained.
Identification
Protects susceptible ducks against infection with P. multocida.
Tests
Safety. Either testA or test B may be carried out.
A. Inject 5 ml subcutaneously into each offour healthy rabbits,
weighing between 1,0 and 1.5 kg. Observe the animals for 7
days. No untoward reaction except slight and transient local
swelling occurs.
B. Inject 5 ml subcutaneously into each oftwo healthy rabbits,

each weighing between 1.0 and 1.5 kg, and 0.5 ml
subcutaneously into each of six mice, each weighing between
25 and 30 g. Observe the animals for 7 days. No untoward
reaction except slight and transient local swelling occurs in
both species of animals.
Potency. Either test A or test B may be carried out.
A. Inject subcutaneously with the minimum dose ofthe vaccine
stated on the label three healthy susceptible ducks, between
4 and 6 weeks old. Use another two ducks of the same stock
and age as unvaccinated controls. Three weeks later, challenge
each of the vaccinated and control ducks, subcutaneously
with 102 mouse LD so viable organisms in 0.2 ml of a suitably
diluted 18-hour broth culture ofthe homologous virulent strain
ofP. multocida. Observe the ducks for 7 days. Not less than
two ofihe vaccinated ducks remain innonnal health and both
the controls die of pasteurellosis.
B. Inject subcutaneously each of six mice, each weighing

between 25 and 30 g, with 0.2 ml of the vaccine under
examination. Use another six mice ofthe same stock and weight
range as unvaccinated controls. Three weeks later, challenge
each of the vaccinated and control mice subcutaneously with
0.2 ml of a suitably diluted 18-hour broth culture of the
homologous virulent strain of P. multocida containing 50
mouse LD so viable organisms. Observe the animals for 7 days.
2721
DUCK PLAGUE VACCINE, LIVE
IP2010
All the vaccinated mice survive. The test is not valid unless
all the control mice die of pasteurellosis during the observation
period.
Labelling. The label states (1) the type of strain; (2) the
recommended age for vaccination.
If potancy test has been performed with satisfactoery results

on arepresentative batch of the vaccine, it may be omitted as
a vaccine test during production on the other batches of
Labelling. The label states (1) the minimum virus titre per
dose; (2) the recommended age of the birds in which the
vaccine is to be used.

Duck Plague Vaccine, Live is a preparation of attenuated
strain of duck plague virus. This monograph applies to
vaccines intended for administration to duck for active
immunisation against duck plague disease.
Egg Drop Syndrome'76 (Adenovirus)

Vaccine, Inactivated
Production
Egg Drop Syndrome'76 (Adenovirus) Vaccine, Inactivated

consists of an emulsion or a suspension of a suitable strain of
egg drop syndrome'76 virus (haemagglutinating avian
adenovirus) which has been inactivated in such a manner that
immunogenic activity is retained.
The vaccine virus is grown in SPF eggs (2.7.7) or in cell

cultures. The master seed lot complies with the tests for
extreneous agents in seed lot (2.7.10).
Egg Drop Syndorme'76 (Adenovirus) Vaccine,
Production
The vaccine virus is grown in embryonated hens' eggs or in

cell cultures obtained from flocks free from specified pathogens
SPF (2.7.7). Ifthe vaccine virus is grown in cell cultures, they
comply with the requirements for cell cultures for production
ofveterinary vaccines. The vaccine virus is filled with suitable
stabilizing agent and freeze dried.
The vaccine strain is propagated in embryonated duck eggs

from healthy flocks or in suitable cell culture derived from SPF
eggs (2.7.7). The master seed lot complies with the tests for
Vetetinary Vaccines (2.7.10).
Identification
Test for inactivation
Protects ducks against duck plague.
The test for inactivation is carried out in fertilized duck eggs

from a flock free from egg drop syndrome '76 virus infection
or hen eggs from a flock free from specified pathogens, or in
suitable cell culture derived from SPF eggs (2.7.7), whichever
is the most sensitive for the vaccine strain; the quantity of
virus used in the test is equivalent to not less than ten doses
of the vaccine. No live virus is detected.
Tests
Safety. Inject subcutaneously each offour healthy susceptible
ducks, between 8 and 12 weeks old and each weighing not
less than 600 g, with 1 ml ofa 1 : 10 dilution ofthe reconstituted
vaccine. Observe the ducks for 14 days. None of the ducks
shows any untoward reaction.
Potency. Inject subcutaneously each of four healthy
susceptible ducks, between 8 and 12 weeks old and each
weighing not less than 600 g, with a volume ofthe reconstituted
vaccine containing a quantity of the virus equivalent to the
minimum dose stated on the label. Fourteen days later,
challenge each ofthe vaccinated ducks and each oftwo control
ducks of the same stock and weight range, subcutaneously
withJO.~ID50-ofvirulentduckplague.virus.-Observetheducks.
. for 14 days. None ofthe vaccinated ducks dies or shows any
clinical symptoms of plague. The test is not valid unless the
control ducks die from duck plague or show typical signs of
serious infection during the observation period.
The vaccine may contain a suitable adjuvant.
Identification,
When inoculated into chicken, the development of specific
neutralizing antibodies against egg drop syndrome '76
(adenovirus) can be demonstrated by suitable serological
tests.
Tests
Safety. Inject each often SPF chickens (2.7.7, table 3) or healthy
susceptible chickens between 2 and 4 weeks old, with two
doses and-6ytlieroutestafed.on theTlioel.Ooservefhe

chicken for 14 days. None ofthe chickerishoWsahy abnormal
local or systemic reaction.
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FOOT-AND-MOUTH DISEASE VACCINE, INACTIVATED
IP 2010
Potency. Inject each oftwenty SPF chickens (2.7.7, table 3) or

healthy susceptible chickens, 3 to 4 weeks old, with the dose
and by the route stated on the label. After 21 days, collect
serum samples from each of the birds as well as ten-control
chickens of the same stock and perform haemagglutination
inhibition test on each serum using 4 haemagglutinating units
of antigen and chicken erythrocytes. The vaccine passes the
potency test ifthe mean antibody titre ofthe vaccinated group
is greater than 1: 128. The test is valid only if no specific
antibody is found in the control chicken.
Labelling. The label/Insert states (1) the strain used for the
preparation; (2) the route of administration.
Foot-and-Mouth Disease Vaccine,

Inactivated
than one infectious particle per 104 litres ofliquid preparation

at the end of the inactivation period.
Tests
Inactivation. A proportion of each batch of bulk inactivated
antigen representing at least 200 doses is tested for freedom
from infectious virus by inoculation in to sensitive cell culture.
A sample of inactivated antigen is concentrated to volumes
adequate for inoculation into cell cultures and it must show
that the concentrated antigen does not interfere with the
sensitivity or reading of the assay. The sample is passaged 3
times at an interval of 24 to 48 hours and inoculated cell
cultures are examined for the presence of foot-and-mouth
disease virus by suitable tests. No cytopathic changes
attributable to foot-and-mouth disease virus replication should
be detected. If infectious foot-and-mouth disease virus is
detected,the bulk antigen is rejected.
FINAL LOT
Foot-and-Mouth Disease Vaccine, Inactivated is a liquid

preparation containing one or more types of foot-and-mouth
disease virus that have been inactivated in such a manner
that its immunogenic activity is retained. Depending on the
number oftypes ofvirus incorporated, the vaccine is described
as monovalent, bivalent, trivalent or polyvalent.
'

containers. The containers are closed so as to avoid
contamination.
released for use.
Production
The virus is grown in suitable cell cultures. The virus is
separated from cellular material by filtration or other suitable
procedures and the virus is inactivated using binary
ethyleneimine (BEl) in suitable conditions. The antigen may
be concentrated and purified. The antigen is used for the
preparation of vaccine. The vaccine contains a suitable
adjuvant.
Only an inactivated antigen suspension that complies with
the requirements mentioned under final bulk vaccine may be
used in the preparation ofthe final lot.
FINAL BULK VACCINE

antigen suspensions.
During inactivation of the virus, samples should be taken at
regular intervals for the purpose of monitoring the rate and
linearity ofthe inactivation process. Virus titre in the samples
is determined by inoculation into sensitive cell culture. The
infectivity of the timed samples is plotted against time, and
the inactivation procedure is not considered to be satisfactory
unless the extrapolation indicates that there would be less
Identification
The serum of a foot-and-mouth disease susceptible animal
that has been immunized with the vaccine neutralizes the types
ofvirus used to prepare the vaccine, when tested by a suitable
method.
Tests
Safety. Use two cattle, not less than six months old, that do
not have antibodies against foot-and-mouth disease virus.
Administer to each animal a double dose ofthe vaccine by the
prescribed route ofadministration stated on the label. Observe
the animals daily for at least 14 days. The vaccine complies
with the test if no animal shows abnormal local or systemic
reactions or dies from causes attributable to the vaccine.
Assay. Use three groups of not less than five cattle per group,
not less than 6 months old, which have never been vaccinated
and are free from antibodies neutralizing the different types of
foot-and-mouth disease virus in the vaccine. Vaccinate the 3
groups by the prescribed route stated on the label. Use
different doses of the vaccine for each group without diluting
2723
FOOT-AND-MOUTH DISEASE VACCINE, INACTIVATED

IP 2010
the vaccine. For example, if 3 ml is one dose, a 1/3 dose of

vaccine would be obtained by injecting 1 ml, and a 1/10 dose
would be obtained by injecting 0.3 ml. Three weeks later,
challenge all the vaccinated animals and a control group of
two cattle susceptible to foot-and-mouth disease, with a
suspension of virus that is fully virulent and ofthe same type
as that used for preparation of the vaccine by inoculating
10,000 ID so (50 per cent bovine infectious dose) intradennally
into two sites into the tongue (O.lml per site). The challenge
ofoil adjuvanted vaccines is effected 28 days post vaccination.
Observe the animals for 8 days and then sacrifice them.
Unprotected animals show lesions at sites other than the
tongue. Protected animals may display lingual lesions. The
test is not valid unless control animals show lesions on at
least three feet. From the number of animals protected in each
group, calculate the PD so content of the vaccine. The potency
ofthe vaccine is expressed as the number of 50 per cent cattle
protective doses (PD so) contained in the dose stated on the
label. The vaccine must contain at least 3 PD so per dose for
cattle.
Alternatively, percentage of protection against generalized
foot infection (PGP) test can be carried out. A group of 16
cattles of six months age which have never been vaccinated
and are free from antibodies neutralizing the different types of
foot-and-mouth disease virus in the vaccine are vaccinated
with a full vaccine dose by the route recommended by the
manufacturer. These animals and a control group of two non- .
vaccinated animals susceptible to foot-and-mouth disease are
challenged three to four weeks after vaccination with a
suspension of virus that is fully virulent and ofthe same type
as that used for preparation of the vaccine by inoculating
10,000 ID50 (50 per cent bovine infectious dose) intradermally
into two sites into the tongue (O.lrnl per site). Observe the
animals for 8 days and then sacrifice them. Unprotected animals
show lesions at sites other than the tongue. Protected animals
may display lingual lesions. The test is not valid unless control
animals show lesions on at least three feet. The vaccine passes
the test if a minimum of 12 animals out of 16 vaccinated are
protected.
Test animals shall be bled on day 0, 21 or 28 days post
vaccination for screening the animals for sero-negative status
and for estimation of the antibody titres post vaccination.
Indirect tests, including post vaccination measurement ofvhus
neutralizing antibodies in cell culture or E~ISA antibodies,
may be used to assess the potency of a vaccine provided that
a statistical evaluation has established a satisfactory
correlation between the results obtained by the test on the
. relevarifV-accii1esefotYpe-and-tliepo~ency-tesrirCcattle.----The description applies to the testing ofa monovalent vaccine.
The potency of polyvalent vaccines may be tested by
challenging each valency as described above.
Labelling. The label states (1) the method ofpreparation; (2)

the serotype of the virus used to prepare the vaccine.

Pasturella multocida Vaccine for Chickens
Fowl Cholera Vaccine, Inactivated is a preparation of suitable
strains of 1 or more serovars of Pasteurella multocida. This
monograph applies to vaccines intended for the active
immunisation of chickens, turkeys, ducks and geese against
fowl cholera infection.
Production
The seed material is inoculated iri a suitable medium. If the
vaccine contains more than 1 strain ofbacterium, the different
strains are grown and harvested separately. The bacterial
harvests are inactivated with suitable agent. The vaccine may
contain suitable adjuvant.
Identification
Protects susceptible chicken against infection with P.
multocida.
Tests
Safety. Administer double dose of vaccine subcutaneously
into each of ten SPF chickens (2.7.7, table 3) or healthy
susceptible chickens of4 to 6 weeks age. Observe the chickens
for 7 days; none of the chicken shows untoward reaction
other than slight transient local swelling.
Potency. Immunize 20 SPF chickens (2.7.7, table 3) or healthy
susceptible chickens 4 to 6 weeks ofage per strain incorporated
in the batch with one dose of vaccine. Give booster dose of
vaccine 15 to 21 days after primary immunization. Keep
unvaccinated healthy susceptible control birds of similar age
10 birds per strain incorporated in vaccine. Challenge the birds
with an appropriate dose of virulent 18 hour old broth culture
of recently bird passaged strain of Pasteurella multocida
that shall kill at least 80 per cent of the unvaccinated
susceptible chickens. Observe birds for 14 days post
challenge. There should be not less than 70 per cent protection
of vaccinated birds and specific mortality of at least 80 per
cent in control.
_"accinemay_he__exp_e_c.ted.J.Q_r~lain iti'!_potency for not les~
tb_a.nfY~!lr~_fl:().rpJl1~li.ll!(ltl1~PQ!.fl!!<::),_'Y~~cl~t~fl11i_Iled.
Labelling. The labellinsett states (1) the serovar(s) used to

prepare the vaccine; (2) the route of administration.
2724
IP 2010
GOAT POX VACCINE, LIVE

Pigeon Pox Vaccine, Live
Fowl Pox Vaccine, Live is a preparation ofa suitable strain(s)
ofpigeon pox vims or fowl pox vims. This monograph applies
to vaccines intended for administration to chickens for active
immunization against avian pox vims.
Production
The vaccine vims is grown in embryonated hens' eggs from
SPF flock (2.7.7) or in cell cultures derived from SPF eggs
(2.7.7) or cell lines. The master seed lot complies with the tests
for extraneous agents as described in the General monograph
for Veterinary Vaccines (2.7.10).
The vaccine vims is grown either in embryonated hens' eggs

from flocks free from specified pathogens SPF (2.7.7) or in
avian cell cultures obtained from flocks free from specified
pathogens SPF (2.7.7) or cell lines.
volume of the reconstituted vaccine containing a quantity of

the vims equivalent to the minimum titre stated on the label.
After 21 days, challenge each chicken by intrafollicular
administration or by scarification with a vimlent strain offowl
poxvims. Observe the birds for 14 days. The vaccinated
chickens survive and show no signs ofdisease except transient
local reactions of fowl pox within 6 days following the
challenge: All control chickens show lesions offowl pox.
results on a representative batch of the vaccine it may be
omitted as a routine test during production ofthe other batches
ofthe vaccine prepared from the same seed lot.
than 18 months from the date the vims titre was detelmined.
preparation.
Labelling. The label/insert states (l) the minimum virus titre;
(2) the dose of vaccine.
Identification
Carry out an immunostaining or neutralization test in cell culture
derived from SPF eggs (2.7.7) to demonstrate the presence of
the vaccine vims or inoculate the vaccine into eggs and notice
the characteristic lesions.

Goat Pox Vaccine, Live is a freeze-dried preparation of an
attenuated strain of goat pox virus propagated in a suitable
cell culture. It is reconstituted immediately before use by a
suitable diluent.
Tests
Production

Safety. Administer 10 doses ofthe vaccine to each often SPF
chickens (2.7.7, table 3) or healthy susceptible chickens 6 to 8
weeks old by the route stated on the label. Observe the birds
for 21 days. No chicken dies from causes attributable to the
vaccine or shows signs of toxicity other than mild, transient,
local reactions. If during the observation period more than
two chickens die from causes not attributable to the vaccine,
repeat the test.
2
Virus titre. Not less than 10 EIDso/TCID so of the vims per

dose, determining the titre by inoculation into the chorioallantoic membrane of SPF embryonated eggs, between 9-11
days old, or one or more route for vims titration depending
upon the strain.
Potency. Carry out a separate potency test for each of the
routes of administration stated on the label. Use not less than
ten SPF chickens (2.7.7, table 3) or healthy susceptible
chickens, 6 to 8 weeks old. Use ten birds from the same flock
and weight range as controls. Administer to each chicken a
The vims is propagated in suitable cell culture. The viral

suspension is harvested, titrated and may be mixed with a
suitable stabilizing agents. The vaccine is then freeze-dried.
Identification
The vaccine protects susceptible animals against goat pox.
Tests
Water (2.3 .43). Not more than 3.0 per cent.
Safety
A. Inoculate 10 doses of the reconstituted vaccine in each

animal of the three species mentioned below by the route
stated against each. Administer 0.2 ml intraperitoneally to each
of six mice and 0.5 ml and 1.0 ml subcutaneously to each of
three guinea pigs and three rabbits respectively. Observe the
animals for 10 days. None of the animals shows an abnormal
reaction.
B. Inject 100 doses of the vaccine contained in 1 ml of the
reconstituted vaccine subcutaneously into each of two
2725
GOAT POX VACCINE, LIVE
IP 2010
susceptible goats, 6 to 8 months old. Observe the goats for 10

days. None of the animals shows abnormalities other than
local erythema of not more than 3 cm in diameter around the
site of injection.
Virus titre. Not less than 103 TCID50 of the virus per dose,
determining the titre in a suitable cell culture or by inoculation
into the chorio-allantoic membrane ofSPF embryonated eggs
(2.7.7),9 to 11 days old.
Potency. Use nine susceptible goats, 8 to 10 months old. Divide
them into three groups of three goats each. Inject
subcutaneously 1I20th of the dose of the vaccine stated on
the label into each goat of one group. Administer a quantity
equivalent to the dose of the vaccine stated on the label into
each goat of the second group. Use the third group as
unvaccinated controls which should be kept along with the
inoculated goats. Observe the animals for 14 days and record
the rectal temperature daily ofeach goat during the observation
period. None of the vaccinated goats shows any thermal
reaction or local or generalised lesion. After 21 days, challenge
the vaccinated and control animals with sufficient quantity of
a virulent goat pox virus by intradermal injection. Observe the
animals for 14 days and record the rectal temperature daily of
each goat during the observation period. None of the
vaccinated goats shows any thermal reaction or local or
generalised lesion. The test is valid only ifthe control animals
develop high fever or show local or generalised lesions. Ifthe
test for potency has been carried out with satisfactory results
on a representative batch of vaccine, this test may be omitted
as a routine control on other batches of vaccine prepared
from the same seed lot, subject-to agreement by the competent
authority.
Labelling. The label states (l) the strain used for the
preparation; (2) the virus titre; (3) the dose and age of
vaccination.
Haemorrhagic Septicaemia Vaccine

Haemorrhagic Septicaemia Vaccine (Inactivated) is a
preparation of Pasteurella multocida. The whole culture is
inactivated by addition of formalin and a suitable adjuvant
(gel or mineral oil).
Identification
The vaccine protects susceptible animals against infection
by P. multocida.
Observe the animals for 5 days; no abnormal systemic reaction

occurs.
Inject two seronegative cattle with twice the maximum dose
stated on the label and observe for 10 days for adverse effects.
Potency. Carry out any of the following three tests.
A. Test on mice. Inject intramuscularly each of fifty mice,
weighing not less than 18 g, with 0.2 ml of the preparation
under examination. Repeat the dose 14 days later. After 7 days
of second vaccine divide the mice into ten groups of five
each. Use another fifty mice ofthe same weight and from the
same stock as controls. Divide the controls also into ten groups
of five each. Challenge the vaccinated and the control mice
with an 8 to l2-hour old broth culture of a virulent strain of P.
multocida in the range of 10-1 to 10- 10 Observe the mice for 5
days and record the number of vaccinated and control mice
found dead in each group. Calculate the median lethal dose
(LD so) for the vaccinated and control mice by standard
methods.
The protection provided by the vaccine is calculated as
number of protection units using following fonnula:
Number ofprotection units = LD so in control animals - LD so in
vaccinated animals.
The vaccine passes the test if it provides minimum protection
of 104 units.
B. Test on rabbits. Inject intramuscularly each ofnot less than
six rabbits, each weighing not less than 2.0 kg, with 2 ml ofthe
vaccine under examination. Use two rabbits ofthe same weight
and of the same stock as controls. After 21 days, challenge
each of the vaccinated rabbits as well as the control rabbits
with an 18 hour old culture of P. multocida containing not less
than 10 LD so ofvirulent organisms. Observe. the animals for 7
days; none of the vaccinated animals dies of pasteurellosis.
The test is not valid unless both the control rabbits die of
pasteurellosis.
C. Test on calves. Inject each of not less than 3 healthy

susceptible calves, weighing not less than 140 kg each with
2 ml of vaccine. Three weeks later, these animals along with
two healthy animals of the same type are challenged
subcutaneously with l8-hours old broth culture of P.
multocida equivalent to at least 50 million mouse minimum
infective dose. Observe the animals for 7 days. Both the
controls should die of pasteurellosis and at least two out of
three vaccinated animals should survive.
Tests
Pofenc)'iscofidiicted-OlfeachseecnotOffof ever,y-fiftlrb-atch
produced from the seed lot: ...
Safety. Inject intraperitoneally into each ofsix mice, weighing

not less than 18 g, with 0.5 ml ofthe vaccine under examination.
Labelling. The label states (1) the type and strains ofbacteria
used to prepare the vaccine; (2) the adjuvant used.
2726
IP 2010
INFECTIOUS AVIAN ENCEPHALOMYELITIS VACCINE, LIVE
when there is 90 per cent protection in vaccinated bird and 80

per cent deaths in unvaccinated controls.
Inclusion Body Hepatitis (IBM)

Vaccine, Inactivated
Hydropericardium Syndrome (HPS)
Inclusion Body Hepatitis (IBH) Vaccine, Inactivated consists
of an emulsion or a suspension ofavian adenovirus(es) which
have been inactivated in such a manner that the immunogenic
activity is retained. The vaccine may contain one or more
suitable adjuvants.
Production
Vaccine virus is multiplied in healthy susceptible chicks or
SPF eggs (2.7.7) or in cell culture derived from SPF eggs (2.7.7).
Vaccines (2.7.10).
Test for Inactivation
To confinn inactivation an amplification test for residual live
IBH/HPS virus is carried out on each batch of antigen
immediately after inactivation or on the final bulle (ifthe vaccine
contains a mixture of inactivated antigens). The test is
conducted on healthy susceptible chickens demonstrated to
be free from antibodies to IBHlHPS virus or in fertilized eggs
derived from specific pathogen free flocks (2.7.7) ifthe vaccine
virus has been propagated in embryos. The quantity of
inactivated virus used in the test is equivalent to not less than
ten doses of the vaccine. No live virus is detected.
B. At least five, 3-6 week old SPF chickens (2.7.7, table 3) or

healthy susceptible chickens are vaccinated with one field
dose of vaccine by intramuscular route. Blood samples are
collected between 3 and 5 weeks and the antibody response
measured by ELISA. The mean antibody titre should be at
least 10 log2 ELISA units.

than 2 years from the date the potency was detennined.
Labelling. The label/insert states (l) strain used for vaccine
production; (2) the route ofadministration.
Infectious Avian Encephalomyelitis

Vaccine, Live
Encephalomyelitis Vaccine Live, Epidemic Tremor
Vaccine Live
Infectious Avian Encephalomyelitis Vaccine, Live is a freezedried preparation of an attenuated strain of infectious avian
encephalomyelitis virus.
Production
Identification
The virus is grown in SPF embryonated eggs (2.7.7) pr in

suitable cell culture derived from SPF eggs (2.7.7). The master
seed lot complies with the tests for extraneous agents as
described in the General monograph for Veterinary Vaccines
(2.7.10).
Protects chickens against infection ofIBH/HPS.
Identification
Tests
Inoculate 0.1 ml of the undiluted reconstituted vaccine into

the yolle sac of SPF embryonated eggs, between 5 to 6 days
old. Keep them in an incubator and transfer to the setter for
hatching. Observe the hatched chickens for 7 days. Not less
than 50 per cent of the chickens show the typical symptoms
characteristic of infectious avian encephalomyelitis-like
weakness or paralysis of legs, sitting posture on hock joints
and tremors.
Safety. Inject subcutaneously a quantity equivalent to 2 doses

into each of 10 SPF chickens (2.7.7, table 3) or healthy
susceptible chickens of the recommended age at which
vaccine is to be used. Observe the chickens for 14 days, no
abnonnal systemic or local reaction is seen.
Tests
A. Inject one dose by the route stated on label into each of20
SPF chickens (2.7.7, table 3) or healthy susceptible chickens
at the age recommended by manufacturer. Use 10 similar
chickens from same source as unvaccinated controls. After
10 days of immunization challenge the birds with 10 per cent
IBH positive infected liver suspension 0.5 ml per bird. Observe
the birds for ten days. The vaccine passes the potency test

Safety. Administer ten SPF chickens (2.7.7, table 3) or healthy
susceptible chickens by ten doses of the vaccine by the
recommended route. Observe the chickens for 21 days. No
chicken develops signs of the disease or dies from causes
2727
INFECTIOUS AVIAN ENCEPHALOMYELITIS VACCINE, LIVE
attributable to the vaccine. Repeat the test if more than two

chickens die from causes not attributable to the vaccine during
the observation period.
IP 2010
Vaccines (2.7.10).
Virus titre. Not less than 102 .5 TCIDsolEIDso of the virus per Inactivation \
dose, determining the titre of the virus in cell culture derived
from SPF eggs (2.7.7) or by inoculation into the yolle sac of . An amplification test for residual live infectious bursal disease
SPF embryonated hen eggs (2.7.7), between 5 to 6 days old.
virus is carried out on each batch ofantigen immediately after
inactivation and the test is carried out in fertilized hen eggs
obtained from SPF flocks (2.7.7) or in suitable cell culture
Potency. Carry out a separate potency test for each of the derived from SPF eggs (2.7.7) or, where chickens have been
routes of administration stated on the label. For each of the used for production of the vaccine, in chickens from a flock
stated routes, use not less than ten SPF chickens (2.7.7, table free from specified pathogens. The quantity of inactivated
3) or healthy susceptible chickens, 3 weeks old. Administer to virus used in the test is equivalent to not less than ten doses
each chicken a volume ofthe reconstituted vaccine containing of the vaccine. No live virus is detected.
a quantity of the virus equivalent to the minimum virus titre
stated on the label. Use ten chickens of the same flock and Test for inactivation
age as controls. After 21 days, challenge each chicken in the
For vaccine prepared with embryo-adapted strains ofthe virus.
vaccinated and control groups with intracerebral injection of
Inject quantity of inactivated virus equivalent to 10 doses of
a suitable quantity ofa virulent avian encephalomyelitis virus.
vaccine into the allantoic cavity or onto the chorio-allantoic
Observe the chickens for another 21 days. Not less than 80
membrane ofthe SPF emblyonated hen eggs, between 9 to 11
per cent of the vaccinated chickens survive or show no signs
days old, and incubate at 361 o. Observe for 6 days and pool
of disease and not less than 70 per cent of the controls die or
separately the allantoic fluid from eggs containing live embryos,
develop signs or paralytic lesions ofavian encephalomyelitis.
and that from eggs containing dead embryos, excluding those
If the potency test has been performed with satisfactory dying from non-specific causes within the first 24 hours after
results on representative batch of the vaccine fi'om the same inoculation.
seed lot, it may be omitted as a routine control test during
Inject into the allantoic cavity ofeach ofthe SPF embryonated
production of other batches of the vaccine prepared from the
hen eggs, between 9 to 11 days old, 0.2 ml of the pooled
same seed lot.
allantoic fluid from the live embryo,S or membrane from the
Storage. When stored under the prescribed conditions, the dead embryos and incubate at 36 I for 6 days. Examine each
vaccine may be expected to retain its potency for not less embryo for lesions of infectious bursal disease. The vaccine
than 18 months from the date the virus titre was determined. complies with the test if there is no evidence of lesions of
The reconstituted vaccine should be used immediately after infectious bursal disease.
preparation.
The test is valid only if not more than 20 per cent of the
Labelling. The labeVinsert states (I) the minimum virus titre; embryos die at either stage ofthe test. Ifmore than 20 per cent
(2) the dose of vaccine.
of the embryos die at either one of the stages of the test,
repeat that stage. In any repeat test, not more than 20 per cent
ofthe embryos die from non-specific causes. Antibiotics may
be used to control extraneous bacterial infection.
Infectious Bursal Disease Vaccine,

Inactivated
Infectious Bursal Disease Vaccine, Inactivated consists of an
emulsion or a suspension of a suitable strain of infectious
bursal disease virus which has been inactivated in such a
manner that immunogenic activity is retained. The vaccine
may contain one or more suitable adjuvant.
Productiou
The virus is propagated in fertilized eggs obtained from
healthy flock or in suitable cell culture derived from SPF eggs
(2.7.7) or in healthy susceptible chicken.
For vaccine prepared with strains of virus not adapted to

embryos. Inject two doses intramuscularly into each oftwenty
chickens, between 14 and 28 days old, complying with the
requirements stated under Test on chicken flocks free from
pathogens for the production and quality control of vaccines
(2.7.7). Four days later, kill ten of the chickens and remove
bursa of fabricius from each chicken,. pool the bursa and
homogenise in an equal volume ofa suitable liquid. Inject 1 ml
oUhe.homogenate .into.. eachofafurther.tenchickensof.the
same flock and age. After 21 days, examine microscopicallY
the bursa of each chicken from the first group and the second
group. No evidence of infectious bursal disease is seen and
no abnormal local reaction develops.
2728
IP 2010
INFECTIOUS BURSAL DISEASE VACCINE, LIVE
For vaccine prepared with cell culture-adapted strains ofthe

virus. The formaldehyde in the test sample is neutralizesd
with sodium metabisulphite. Five ml is tested for the presence
of infective Gumboro Disease vims by inoculation of at least
800 square cm primary or secondary CEF. The cultures are
incubated for 3 to 4 days at a temperature of37. After one
cycle of fi:eezing and thawing the supernatant from these
cultures is passaged onto a fresh CEF cultures. Three to four
days latter this is repeated. Three to four days after final
inoculation the cultures are inspected for CPE. A vital stain
and overlay may be used. If no trace of CPE is detected, the
inactivation of the antigen suspension is accepted to be
completed.
Production
Infectious Bursal Disease Vaccine, Live is a suitable strain of
Infectious Bursal Disease virus. The master seed lot complies
with the tests for extraneous agents as described in the General
monograph for Veterinary Vaccines (2.7.10).
The vaccine virus is grown in embryonated eggs obtained
fi'om SPF flocks or in cell culture derived from SPF eggs (2.7.7)
or susceptible cell lines.
Identification
When mixed with monospecific infectious bursal disease virus

antisenun the vaccine no longer infects susceptible cell culture
.derived from SPF eggs (2.7.7) or embryonated hen eggs, 9 to
Protects susceptible chickens against infectious bursal disease
11 days old.
by producing specific antibodies on inoculation.
Identification
Tests
Tests

Safety. Inject each often healthy chickens, 14 to 28 days old
with twice the minimum vaccinating dose and by one of the
routes stated on the label. Observe the chickens for 14 days.
No abnormal local or systemic reaction is seen.
Potency. Inject each often SPF chickens (2.7.7, table 3) or
healthy susceptible chickens, 3 to 4 weeks old, with the
minimum dose and by the route stated on the label. Use ten
chickens ofthe same flock and age as controls. After 21 days,
collect serum samples from each bird including the ten-control
chickens and perform quantitative agar gel precipitation test
or serum neutralizing test on each serum sample. The mean
antibody titre ofsera in vaccinated group shall be 1:8 by agar
gel diffusion test and 10000 units per ml by serum neutralization
test and there are no IBD specific antibodies in the sera of
control chickens.
Labelling. The label/insert states (1) the type ofstrain; (2) the
route of administration.

Safety. Use not less than ten SPF chickens (2.7.7, table 3) or
healthy susceptible chickens, 10 to 15 days old. According to
the type of viral vaccine strain incorporated in the product Invasive - Moderately invasive- it may be necessary to conduct
the safety test on chicks possessing moderate level ofmaternal
antibodies.
Administer by eye drop to each chicken ten doses of the
vaccine reconstituted so as to obtain a concentration suitable
for the test. Observe the chickens for 21 days. If during the
period of observation more than 2 chickens die from causes
not attributable to the vaccine, repeat the test. The vaccine
complies with the test if non of the chickens shows signs of
the disease, ifno chicken dies from causes attributable to the
vaccine and if 21 days after inoculation of the vaccine, no
chicken shows lesions of the bursa of Fabricius.
Virus titre. Not less than 102 TCIDsolEID so of the virus per
dose. Detennining the titre in cell cultures derived from SPF
embryo or onto the chorio-allantoic membrane of SPF
embryonated hen eggs between 9-11 days old.
- Potency. Use 20 SPF chickens (2.7.7, table 3) or healthy

susceptible chickens 10 to 15 day old. Administer to each
chicken one dose of the vaccine by recommended route. Use
10 chickens of the same flock and age as controls. Fourteen
Infectious Bursal Disease Vaccine, Live days
after immunization challenge chicken of both groups by
Infectious Bursal Disease Vaccine, Live is a freeze dried intraocular route administration of a suitable quantity of
preparation of attenuated strain of infectious bursal disease virulent infectious bursal disease virus. Observe the birds for
(IBD) virus. This monograph applies to vaccines intended for 10 days after challenge. Not more than 4 ofvaccinated chickens
administration to chickens for active immunization.
die or show signs of the infectious bursal disease or on
2729
INFECTIOUS CHICKEN ANEMIA VACCINE, INACTIVATED
IP 2010
histological examination show severe bursal lesions. The test

is not valid unless not less than 50 per cent of the control
birds die or show signs ofIBD and all the surviving controls
show severe bursal lesions on histological examination.
SPF eggs (2.7.7). Incubate at 36 1 0 for 7 days. Make a passage

on another set ofcell culture derived from SPF eggs (2.7.7) or
in cell lines or embryonated SPF eggs (2.7.7) and incubate at
36 10 for 7 days. None of the cultures shows signs of CPE.
Ifat least 90 per cent ofthe follicles show greater than 75 per
cent depletion of lymphocytes, the bird is considered as one
showing severe bursal lesions.
Identification

lot.
preparation.
Labelling. The label/insert states (1) minimum virus titre; (2)
the dose of vaccine.
Infectious Chicken Anemia Vaccine,

Inactivated
Infectious Chicken Anemia Vaccine (ICAV), Inactivated is a
preparation of a suitable strain of chicken anemia virus,
inactivated in such a manner that the immunogenic activity is
retained. This monograph applies to vaccines intended for
administration to chickens for immunization.
Production
The vaccine is grown in embryonated hen's egg obtained
from SPF flocks or in suitable cell culture derived from SPF
eggs (2.7.7) or susceptible cell line. Harvested virus is
inactivated using suitable inactivating agent. The master seed
lot complies with the tests for extraneous agents as described
in the General monograph for Veterinary Vaccines (2.7.10).
Inactivation
An amplification test for residual live chicken infectious anemia
inactivation. The test is carried out in suitable cell culture
derived from SPF eggs (2.7.7) or using susceptible cell lines.
The quantity of inactivated virus used in the test is equivalent
to not less than ten doses of the vaccine. No live virus is
detected.- .. . _ . .
.
Test forlnactivatiou
Inoculate 10 doses ofvaccine virus using suitable cell culture
derived from SPF eggs (2.7.7) or in susceptible cell lines or
In susceptible chicks, the vaccine stimulates the production

Of specific antibodies against vaccine virus detected by
suitable serological tests.
Tests
Safety. Inject a double dose ofvaccine by recommended route
in to each often, 14 to 28 day-old SPF chickens (2.7.7, table 3)
or healthy susceptible chickens. Observe the chickens for 21
days. No abnormal local or systemic reactions occur.
Potency. Carry out a potency test for the route ofadministration
stated on the label. Vaccinate, ten, 21 to 28 day old SPF
chickens (2.7.7, table 3) or healthy susceptible chiekens with
one dose of vaccine. Keep 10 unvaccinated birds of the same
age group as controls. ODserve the birds for 28 days. Collect
serum samples from each bird including the ten-control
chickens. Detect the virus specific antibodies by serological
methods i.e. Enzyme Linked Immunoassay or Virus
Neutralization test. The mean serum neutralization antibody
titre ofsera in vaccinated group shall be 5000 units per ml and
there are no CAV specific antibodies in the sera of control
chickens.
Labelling. The label/insert states (1) strains used for
Infectious Chicken Anemia Vaccine,

Live
Infectious Chicken Anemia Vaccine, Live is a preparation of
a suitable strain of chicken anemia virus. This monograph
applies to vaccines intended for administration to breeder
chicken for active immunization, to prevent excretion ofvirus,
to prevent or reduce transmission through eggs.
Production
Substratefor propagation
Vaccine is grown either in embryonated hen's egg obtained
from SPF flocks (2.7.7) or in cell culture obtained from flocks
free from specified pathogens (2.7.7) or susceptible cell lines.
2730
IP 2010
INFECTIOUS CORYZA VACCINE
Vaccines (2.7.10).
Labelling. The label/insert states (I) strain of virus used; (2)

Identification
The vaccine, diluted if necessary and mixed with a
monospecific chicken anaemia virus (CAV) antiserum, no
longer infects susceptible cell culture derived from SPF eggs
(2.7.7) or egg from SPF flock (2.7.7) into which it is inoculated.
Tests
Infectious Coryza Vaccine is a suspension ofinactivated culture

of suitable strains of one or more serotype/s or preferably
locally prevalent strain/s of Avibacterium (Haemophilus)
paragallinarum in a suitable medium.
Water (2.3.43). Not more than 3.0 per cent. .~
Production

Safety. Use not fewer than 10 SPF chickens (2.7.7, table 3) or
healthy susceptible chickens, not older than the minimum age
recommended for vaccination (2.7.7). Administer by a
recommended route to each chickens 10 doses ofthe vaccine.
Observe the chickens daily for 21 days. The test is not valid
ifmore than 20 per cent ofthe chickens show abnormal clinical
signs or die from causes not attributable to vaccine. The
vaccine complies with the test if no chicken shows notable
clinical signs ofdisease or dies from causes attributable to the
vaccine.
Virus titre. Titrate the vaccine virus by inoculating into
suitable cell lines or eggs from SPF flocks (2.7.7). One dose
vaccine contains not less than 103.0 TCID sol EID so per dose.
Potency. Carry out potency test for each of the routes of
administration stated on the label. Vaccinate ten, 21 to 28 day
old SPF chickens (2.7.7, table 3) or healthy susceptible chickens
with one dose of vaccine. Keep 10 unvaccinated birds of the
same age group as controls. Two to three weeks post
vaccination challenge both the groups by intramuscular route
with 102 CID50 CAV virus. Observe the birds for 14 days.
Bleed individual birds for haematocrit value, thymus atrophy
and bone marrow tissue discolouration.
The vaccine complies with the test if during the observation

period after challenge not less than 90 per cent of the
vaccinated chickens survive and show no notable clinical
signs of disease and/or macroscopic lesions of the bone
marrow and thymus.
It is not necessary to carry out the potency test for each batch
of the vaccine if it has been carried out on a representative
batch using a vaccinating dose containing not more than the
minimum virus stated on the label.

than 18 months from the date the virus titre was detennined.
preparation.
The seed material is inoculated in a suitable medium. If the

vaccine contains more than one strain of bacterium, the
different strains are grown and harvested separately. The
bacterial harvests are inactivated with a suitable agent. The
vaccine may contain suitable adjuvant.
Identification
Protects susceptible chicken against infection with
Avibacterium paragallinarium.
Tests
Safety. Inject double dose of vaccine subcutaneously into
each of10 SPF chickens (2.7.7, table 3) or healthy susceptible
chickens at the minimum age group at which vaccine is
intended. Observe these birds for 7 days; no bird shows
untoward reactions other than slight transient local swelling.
Potency. Inject subcutaneously each of! 0 SPF chickens (2.7.7,
table 3) or healthy susceptible chickens of the minimum age
group at which vaccine is used, with minimum dose stated on
the label. Repeat the vaccination after 2 to 4 weeks. Use 10
healthy chickens of same age group and of same stock as
controls. Two to three weeks later, challenge vaccinated and
control chickens by instillation with 0.2 ml of 18 hour broth
culture of homologous strain of A. paragallinarium diluted
suitably so as to contain I x 106 colony forming units by infra..:
orbital sinus instillation. Observe the chickens for 7 days for
unilateral eye swelling, nasal discharge. There should be not
less than 70 per cent protection of vaccinated birds. The test
is not valid unless 70 per cent of control chickens exhibit
typical symptoms of eye swelling and nasal discharge typical
of infectious coryza.
than 2 years from the date of potency testing.
Labelling. The label/insert states (1) strains used for
2731
MAREK'S DISEASE VACCINE, LIVE
IP 2010

Marek's Disease, Freeze Dried / Cell Associatated Vaccine,
Live is a preparation of a suitable strain or strains of Marek's
Disease Virus (Avian Herpes Virus) or combinations theirof.
Production
The vaccine virus is grown in cell cultures obtained from SPF
(2.7.7) eggs. If the vaccine contains more than one type of
virus, the different types are grown separately. The vaccine
may be freeze-dried or stored in liquid nitrogen.
Vaccines (2.7.10).
Cell culture derived from SPF eggs (2.7.7) obtained from SPF
hens (2.7.7) eggs.
Identification
earlY out either the test A or B.
A. The vaccine on inoculation in susceptible cell cultures

derived from SPF embryos causes cytopathic effects typical
of Marek's Disease virus.
B. When mixed with a specific avian herpes virus antiserum
the vaccine loses its capability to produce cytopathic effects
or plaques in susceptible cell cultures derived from SPF
embryos.
Tests
Water (2.3.43). Not more than 3.0 per cent (For Freeze dried
vaccine only).
Mycoplasams (2.7.9). Complies with the test for mycoplasmas.
Safety. Use ten one-day-old SPF chickens (2.7.7, table 3) or
healthy susceptible chickens. Administer by recommended
route and method to each chicken or chicken embryo 10 doses
of the vaccine. Observe the chicken for 21 days. No chicken
shows persistent clinical signs, dies or, at autopsy, shows
macroscopic lesions from causes attributable to the vaccine.
If during the observation period more than two chickens die
from causes not attributable to the vaccine, repeat the test.
Vaccine containing more than one type ofvllus: For vaccine

containing more than one type of virus, titrate each virus by
inoculation into suitable cell culture derived from SPF eggs
(2.7.7), reading the results by immunostaining using
antibodies. Vaccine complies with the test ifone dose contains
for each vaccine virus not less than 103 PFU ofvirus per dose.
Potency. Carry out a separate potency test for each of the
routes of administration stated on the label. For each of the
stated routes, use not less than thirty susceptible one-dayold SPF chickens (2.7.7, table 3) or healthy susceptible
chickens.
~
Administer each chicken a volume ofthe vaccine containing a

quantity ofthe virus equivalent to the minimum titre stated on
the label. Use thirty chickens of the same flock and age as
controls. After 9 days, challenge each chicken by a suitable
route with a suitable quantity ofvirulentMarek's disease virus.
Observe the birds for 10 weeks. Record the deaths and kill the
survivors to carry out autopsies on both dead and sacrificed
chicken for specific macroscopic lesions of Marek's disease.
For each of the stated routes of administration" the total
number of vaccinated birds that show specific macroscopic
lesions is reduced by not less than 80 per cent as compared
with the control birds and the challenge virus produces specific
macroscopic lesions in not less than 70 per cent ofthe control
birds.
results on representative batch of the vaccine from the same
seed lot, it may be omitted as a routine control test during
production of other batches of the vaccine prepared from the
same seed lot.
than 2 years from the date the virus titrewas detennined. The
reconstituted vaccine should be used immediately' after
preparation.
Labelling. The label states (l) the minimum virus titre; (2) the
dose of vaccine.
The frozen vaccine has to be dispensed in glass ampoules

suitable for liquid nitrogen storage and ifthe above information
cannot be printed on the small size ampoule, the product should
be accompanied by suitable insert which clarifies the
prescribed contents of the labels.

Virus titre. Vaccine containing one type of virus: Titrate the
vaccinevirus.. byinoculationinto.suitable.cellculturederived
from SPEeggs(2.7.7). lithe virus titreis determined in plaque
forming units (PFU), only primary plaques are taken into
consideration. The vaccine complies with the test if one dose
contains not less than 103 PFU per dose.
Peste Des Petits Ruminants Vaccine,

tive---------- ------ -------------- ---Peste Des Petits Ruminants (PPR) Vaccine, Live is a
preparation of a suitable strain ofPPR virus that is attenuated
for sheep and goats.
2732
IP 2010
RABIES VETERINARY VACCINE, INACTIVATED (CELL CULTURE)
Production
The vaccine stTain is grown in suitable cell cultures. The viral
suspension is harvested, mixed with a suitable stabilizing liquid
and freeze-dried.
Batch testing
If the test for potency has been carried out with satisfactory
results on the representative batch of vaccine, this test may
be omitted as a routine control on other batches of vaccine
prepared from the same seed lot, subject to agreement by a
Identification
When injected into the target animals, the vaccine stimulates
the production of specific PPR virus neutralizing antibodies.
Tests
Safety. This test is done in rodents in order to detect any
nonspecific toxicity associated with the product. The test
requires reconstituted vaccine in solvent (mixed contents of
five bottles), six guinea pigs, each weighing 200-250 g; ten
unweaned mice (17-22 g, Swiss line or similar).
Vaccine, 0.5 ml, is injected intramuscularly into a hind limb of
two guinea-pigs, 0.5 ml into the peritoneal cavity oftwo guineapigs, and 0.1 ml into the peritoneal cavity of six mice. Two
guinea-pigs and four mice are kept as uninoculated controls.
The animals are observed for 3 weeks. If one guinea-pig or
two mice die, the test must be repeated. Dead animals undergo
post-mortem examination to ascertain the cause of death. At
the end of 3 weeks of observation, all animals are killed for
post-mortem examination. All the results are recorded. The
vaccine considered to be satisfactory if, during the first or
second test, at least 80 per cent of animals remain in good
health during the period of observation, and no significant
post-mortem lesion is found.
Inject two susceptible goats of one year old free from
antibodies to rinderpest or PPR by subcutaneous route with a
100 times the dose ofvaccine stated on the label. Observe the
animals for 21 days. No sign of illness attributable to PPR is
noticed.
Potency. Use not less than six healthy goats and six healthy
sheep of 1 year old free from antibodies to rinderpest or PPR.
Collect sera from the animals before the time of vaccination
and 3 weeks after vaccination and just before challenge.
Vaccinate two goats and two sheep subcutaneously with one
dose each; vaccinate two goats and two sheep subcutaneously
with 1/1 0 dose each. Keep the remaining animals as the incontact controls. Monitor each animal for clinical signs, in
particular respiratory symptoms and record temperature
measurements daily for three weeks. Three weeks after
vaccination collect sera samples from all vaccinated as well as
control animals and challenge the vaccinated animals and incontact controls group with a suspension of virus containing
either 10 3 LD so pathogenic PPRV or 2.5 ml of a 10% splenic
suspension by subcutaneous route. The animals are observed
for clinical signs and the body temperatures are recorded daily
for two weeks. The vaccine passes the test if all vaccinated
animals resist challenge infection and all the in-contact controls
develop signs ofPPR. The serum neutralization test must be
positive for PPR antibody in vaccinated animal only, in samples
taken three weeks after vaccination.
If the potency test has been performed with satisfactOly
lot.
Labelling. The label states (1) the cell line used for vaccine
manufacture; (2) the virus titre per dose; (3) the recommended
age for vaccination.
Rabies Veterinary Vaccine, Inactivated

(Cell Culture)
Rabies Vaccine for Veterinary Use is a preparation of rabies
fixed virus adapted to and propagated in cell culture and
inactivated by a suitable method. It may be issued as a liquid
containing a suitable adjuvant or as a freeze-dried preparation
to be reconstituted with a suitable liquid immediately before
use.
Production
Virus titre. Not less than 102.5 TCID so per dose.
The vaccine is prepared from virus grown either in suitable

cell lines or in primary cell cultures from healthy animals. The
virus suspension is harvested on one or more occasions within
28 days of inoculation. Multiple harvests from a single
production cell culture may be pooled and considered as a
single harvest. The rabies virus is inactivated by a suitable
method. The vaccine may contain one or more adjuvants.
Extraneous viruses. The reconstituted vaccine when mixed

with specific anti-PPR serum should not produce cytopathic
effects in susceptible cell cultures and the cells should show
no evidence of the presence of haemadsorbing agents.
2733
RABIES VETERINARY VACCINE, INACTIVATED (CELL CULTURE)
Inactivation
A. The test for residual live rabies virus is can-ied out by

inoculation of the inactivated virus into the same type of cell
culture as that used in the production of the vaccine or a cell
culture shown to be at least as sensitive. The quantity of
inactivated virus used in the test is equivalent to not less than
25 doses of the vaccine. After incubation for 4 days, a
subculture is made using trypsinised cells; after incubation
for a further 4 days, the cultures are examined for residual live
rabies virus by an immunofluorescence test. No live virus is
detected.
B. Inject each oftwenty suclding mice, each weighing between
12 and 16 g, intracerebrally with not less than 0.03 ml of the
vaccine or antigen under examination. Observe the animals
for 21 days. None ofthe mice dies or shows any abnormalities
attributable to the vaccine. Ifmore than two mice die within 48
hours, repeat the test.
Identification
When injected into animals, the vaccine stimulates production
of specific neutralising antibodies.
Tests
vaccine only).
Safety. Inject each oftwenty mice, each weighing between 12
and 16 g, intracerebrally with not less than 0.03 ml of the
vaccine under examination. Observe the animals for 21 days.
None ofthe mice dies or shows any abnormalities attributable
to the vaccine. If more than two mice die within 48 hours
repeat the test. If the vaccine is intended for more than one
species including one belonging to the order of Carnivore,
carry out the test in dogs. Otherwise use one of the species
for which the vaccine is intended. Administer, by a
recommended route, a double dose of vaccine to each of
2 animals having no antibodies against rabies virus. Observe
the animals for 14 days. No abnormal local or systemic reaction
occurs.
Potency. The potency of rabies vaccine is determined by
comparing the dose necessary to protect mice against the
clinical effects of the dose of rabies virus defined below,
administered intracerebrally, with the quantity of a reference
preparation, calibrated in International Units, necessary to
pr~~i~e:!~~_~ll:~~l?!_()_t_e~!~()~:
_
Preparation ofthe challenge suspension.Jnoculate.a group
ofmice intracerebrally with the CVS strain ofrabies virus and
when the mice show signs of rabies, but before they die, kill
the mice and remove the brains and prepare a homogenate of
IP 2010
the brain tissue in a suitable diluent. Separate gross particulate

matter by centrifugation and use the supernatant liquid as
challenge suspension. Distribute the suspension in small
volumes in ampoules, seal and store at a temperature below 60. Thaw one ampoule of the suspension and make serial
dilutions in a suitable diluent. Allocate each dilution to a group
of 10 mice and inject intracerebrally into each mouse 0.03ml of
the dilution allocated to its group.. Observe the animals for
14 days and record the number in each group that, between
the fifth and the fourteenth day, develop signs of rabies.
Calculate the ID so of the undiluted suspension.
Determination ofpotency ofthe vaccine
Use in the test, healthy mice about 4 weeks old and from the
same stock. Distribute the mice into at least 10 groups of not
less than 10 mice. Prepare at least three serial dilutions ofthe
vaccine under examination and three similar dilutions of the
reference preparation. Prepare the dilutions such that those
containing the largest quantity of vaccine may be expected to
protect more than 50 per cent of the animals into which they
are injected and those containing the smallest quantities of
vaccine may be expected to protect less than 50 per cent of
the animals into which they are injected. Allocate each dilution
to a different group of mice and inject intraperitoneally into
each mouse 0.5 ml ofthe dilution allocated to its group. Fourteen
days after the injection prepare a suspension of the challenge
virus such that, on the basis of the preliminary titration, it
contains about 50 ID so in each 0.03 mi. Inject intracerebrally
into each vaccinated mouse 0.03 ml ofthis suspension. Prepare
3 suitable serial dilutions ofthe challenge suspension. Allocate
the challenge suspension and the 3 dilutions one to each of
4 groups of 10 unvaccinated mice and inject intracerebrally
into each mouse 0.03 ml of the suspension or one of the
dilutions allocated to its group. Observe the animals in each
group for 14 days. The test is not valid if more than 2 mice of
any group die within the first 4 days after challenge. Record
the numbers in each group that show signs of rabies in the
period 5 days to 14 days after challenge.
examination and the reference preparation the 50 per cent
protective dose lies between the smallest and the largest dose
given to the mice; (b) the titration ofthe challenge suspension
shows that 0.03 ml of the suspension contained at least 10
ID so and not more than 50 ID so ; (c) the confidence limits
(P = 0.95) are not less than 25 per cent and not more than 400
per cent of the estimated potency; (d) the statistical analysis
shows a significant slope and no significant deviations from
linearity or parallelism ofthe dose-response lines.
estlmiie<lpotency is
fh~vacci:necco~npHeswffu-thetestifthe
noflessthiin I-IUliithe'smallesfprescribeddose~- .-. -.
2734
IP 20ID
RANllmETDISEASE VACCINE, INACTNATED
Tests
Newcastle Disease Vaccine, Inactivated
Safety. Inject ten SPF chickens (2.7.7, table 3) or healthy

susceptible chickens ofthe age stated on the label with twice
birds for 21 days. No abnormal local or systemic reactions are
observed.
Ranikhet Disease Vaccine, Inactivated consists ofan emulsion

or a suspension ofa suitable strain ofNewcastle disease virus
(avian paramyxovirus 1) that has been inactivated in such a
manner that immunogenic activity is retained.
Production
A. Inject intramuscularly each often SPF chickens (2.7.7, table

3) or healthy susceptible chickens, between 3 - 4 weeks old,
with a volume of the vaccine equivalent to one-fiftieth of a
dose. Use ten chickens of the same stock and age group as
controls. After 21 days, collect serum samples from each of
the vaccinated and unvaccinated chicken. Perform
haemagglutination inhibition test using the method described
below. Use the positive control serum calibrated against a
Standard preparation of anti-Newcastle disease serum. The
vaccine passes the test if a mean HI titre of the vaccinated
group is equal to or greater than 1: 16 and that of the
unvaccinated controls is equal to or less than 1:4.
The vaccine virus is grown either in embryonated hens' eggs

or in cell culture derived from SPF eggs (2.7.7) or suitable cell
line.
Vaccines (2.7.10).
Inactivation
Inject quantity of inactivated virus equivalent to 10 doses of

vaccine into the allantoic cavity of each of 10 embryonated 9
to 11 days old SPF eggs (2.7.7), and incubate. Observe for
6 days and pool separately the allantoic fluid from eggs
containing live embryos and that from eggs containing dead
embryos, excluding those dying within 24 hours of the
injection. Examine embryos that die after 24 hours ofinjection
for the presence ofNewcastle disease virus. Test the allantoic
fluid from each egg for the presence ofhaemagglutinins using
chicken erythrocytes.
Inject into the allantoic cavity ofeach of 10 SPF eggs (2.7.7), 9
to 11 days old, 0.2 ml ofthe pooled allantoic fluid from the live
embryos and, into each of 10 similar eggs, 0.2 ml ofthe pooled
fluid from the dead embryos and incubate for 5 to 6 days. Test
the allantoic fluid from each egg for the presence of
haemagglutinins using chicken erythrocytes.
The vaccine complies with the test if there is no evidence of
haemagglutinating activity and ifnot more than 20 per cent of
the embryos die at either stage. Ifmore than 20 per cent ofthe
embryos die at one ofthe stages, repeat that stage; the vaccine
complies with the test if there is no evidence of
haemagglutinating activity and not more than 20 per cent of
the embryos die at that stage.
Antibiotics may be used in the test to control extraneous
bacterial infection.
Identification
When injected into susceptible healthy chicken, the vaccine
stimulates the production of specific antibodies against
Newcastle disease virus.
If the HI titre are not satisfactory, carry out the test B.

The Standard preparation is the 1st International reference

preparation, established in 1966, consisting of freeze-dried
chicken serum (supplied in ampoules containing 320 Units),
or another suitable preparation, the potency of which has
been determined in relation to the International reference
preparation.
Suggested method of haemagglutination inhibition test.
Inactivate the serum samples by heating at 56 for 30 minutes.
Add 0.05 ml of saline solution to all the wells in a microtitre
plate and 0.05 ml of the test sera to the first row of wells.
Prepare two-fold dilutions of the serum samples across the
plate. Add 0.05 ml of a suspension ofNewcastle disease virus
containing 4 haemagglutinating units ofinactivated Newcastle
disease virus. Incubate the plate at 4 for one hour. Add 0.05
ml of a 1 per cent suspension of erythrocytes collected from
chicken, between 3-4 weeks old, susceptible to Newcastle
disease.
Incubate the plate at 4 for one hour. It must be ensured that

negative and positive control sera are included in the test.
The positive control serum must show a titre of 300 to 400
Units determined by calibration against the Standard reference
Preparation.
B. Inject intramuscularly each ofthree groups of twenty SPF
chickens (2.7.7, table 3) or healthy susceptible chickens,
between 3 - 4 weeks old, with five fold dilution of vaccine.
Use minimum three dilutions. Allocate a different volume to
2735
RANIKHET DISEASE VACCINE, LIVE (LENTOGENIC STRAIN)
each vaccination group. Vaccinate each chicken by the

intramuscular route with the volume of vaccine allocated to
its group. Maintain not less than 10 chickens as controls.
Challenge each chicken after 21 days by the intramuscular
route with 106 chick LD so of the virulent strain of avian
Paramyxovirus 1. Observe the chickens at least daily for 7 days
after challenge. At the end ofthe observation period, calculate
the PDso by standard statistical methods from the number of
chickens that survive in each vaccinated group without
showing any signs of Newcastle disease during the 7 days.
The vaccine complies with the test if the smallest dose stated
on the label conesponds to not less than 50 PD so and the
lower confidence limit is not less than 35 PDso per dose. Ifthe
lower confidence limit is less than 35 PDso per dose, repeat the
test; the vaccine must be shown to contain not less than 50
PDso in the repeat test. The test is not valid unless all the
control birds die within 6 days of challenge.
than 2 years from the date the potency was detennined.
Labelling. The label/insert states (1) strain of virus used; (2)
the route of administration.
IP 2010
Tests
Safety. For vaccines recommended for use in healthy
susceptible chickens, use not less than 10 SPF chickens (2.7.7,
table 3) or healthy susceptible chickens demonstrated to be
free from antibodies to Newcastle disease virus and of the
youngest age recommended for vaccination. For vaccines
recommended for use only in avian species other than the
chicken, use not less than 10 birds of the species likely to be
most sensitive to Newcastle disease, which do not have
antibodies against Newcastle disease virus and ofthe minimum
age recommended for vaccination. Administer to each bird by
eye-drop, or parenterally if only parenteral administration is
recommended, 10 doses of the vaccine in a volume suitable
for the test. Observe the birds at least daily for 21 days. The
test is not valid if more than 20 per cent of the birds show
abnonnal clinical signs or die from causes not attributable to
the vaccine. The vaccine complies with the test if no bird
shows notable clinical signs of disease or dies from causes
attributable to the vaccine.
Virus titre. Not less than 106TCIDsoIEIDso of the virus per
dose, detennining the titre in suitable cell culture derived from
SPF eggs (2.7.7) or by inoculation into the allantoic cavity of
SPF embryonated eggs, 9 to 11 days old.
Ranikhet Disease Vaccine, Live

(Lentogenic Strain)
Newcastle Disease Vaccine, Live (Lentogenic strain)

Ranikhet Disease Vaccine Live (Lentogenic Strain) is a
preparation ofa suitable strain ofNewcastle diseaselRanikhet
disease virus (avian paral11yxovirus 1). This monograph
applies to vaccines intended for administration to chickens
and/or other avian species for active immunization.
Production
The vaccine virus is grown in embryonated SPF eggs (2.7.7)

or in cell cultures derived from SPF flocks (2.7.7).
Vaccines (2.7.10).
Identification
monospecific Newcastle disease virus antiserum,. no. longer
provokes haemagglutination of chicken red blood cells or
infects embryonated hens' eggs from SPF flock or susceptible
cell culture derived from SPF eggs (2.7.7) into which it is
inoculated.
Potency. Canoy out a potency test for each of the routes of

administration stated on the label. For each ofthe stated routes,
use at least ten SPF chickens (2.7.7, table 3) or healthy
susceptible chickens and of the minimum age recommended
for vaccination.
Administer each chicken with a volume of the reconstituted

vaccine containing a quantity of the virus equivalent to the
minimum titre stated on the label. Use ten chickens of the
same flock and age as controls. After 14 to 21 days, challenge
each chicken by intramuscular injection with lOs LD so, of a
virulent strain of Newcastle disease virus. Observe the
chickens for 14 days. The vaccine complies with the test ifnot
more than two of the vaccinated chickens die or show signs
of disease. The test is valid only if all the control birds die
within 6 days of inoculation of the virulent challenge strain.
on other batches of the vaccine
from the same seed
lot.
than 18 months from the date the virus titre was detennined.
2736
IP 2010
REO VIRUS VACCINE, INACTIVATED

preparation.
Labelling. The labeVinsert states (I) strain of virus used; (2)
Ranikhet Disease Vaccine, Live

(Mesogenic Strain)
Ranikhet Disease Vaccine, Live (Mesogenic Strain) is a
preparation of a suitable strain of Newcastle disease virus
(naturally modified avian Paramyxovirus I). This monograph
applies to vaccines intended for administration to chickens
for active immunization.
Production
use not less than ten SPF chickens (2.7.7, table 3) or healthy
susceptible chickens of the minimum age recommended for
vaccination. Administer each chicken with a volume of the
reconstituted vaccine containing a quantity of the virus
equivalent to the minimum titre stated on the label. Use ten
chickens ofthe same flock and age as controls. After 14 to 21
days, challenge each chicken by intramuscular injection with
105 LD so of a virulent strain of Newcastle disease virus.
Observe the birds for 14 days. The vaccine complies with the
test if not more than two of the vaccinated chickens die or
show signs of disease. The test is valid only if all the control
chickens die within 6 days of inoculation of the virulent
challenge strain.
If the potency test has been performed with satisfactOlY
lot, it may be omitted as a routine control test dming production
lot.
The vaccine virus is grown in embryonated SPF eggs (2.7.7)

or in cell cultures derived from SPF flocks (2.7.7) or susceptible
cell lines. The master seed lot complies with the tests for
Veterinary Vaccines (2.7.10).

preparation.
Identification
Labelling. The label/insert states (1) strain of virus used; (2)


monospecific Newcastle disease virus antiserum, no longer
provokes haemagglutination of chicken red blood cells or
infects embryonated hens' eggs from SPF flock (2.7.7) or
susceptible cell culture derived :IiOln SPF eggs (2.7.7) into
which it is inoculated.
Tests
Safety. Administer fifteen SPF chickens (2.7.7, table3) or healthy
susceptible chickens, 8 to 9 weeks old, with a minimum 10
doses and by the route stated on the label. Observe the
chickens for 21 days. Not morethan 2 chicken show abnonnal
clinical signs or die due to causes attributable to the vaccine.
Ifmore than two chickens die during the period ofobservation
due to causes other than those attributable to the vaccine,
repeat the test.
Virus titre. Not less than 105 TCIDso/EIPso of the virus per
dose, detennining the titre in suitable cell culture derived from
SPF eggs (2.7.7) or by inoculation into the allantoic cavity of
SPF embryonated eggs (2.7.7), between 9-11 days old.
Potency. Carry out potency test for each of the routes of
administration stated on the label. For each ofthe stated routes,
Reo Virus Vaccine, Inactivated

Reo virus vaccine, Inactivated consists of an emulsion or a
suspension of a suitable strain /s of Reo virus which has been
inactivated in such a manner that immunogenic activity is
retained. The vaccine may contain one or more strains and a
suitable adjuvant.
Production
The virus is propagated in fertilized eggs obtained from
healthy flock or in suitable cell culture derived from SPF flocks
(2.7.7) or susceptible cell line.
Vaccines (2.7.10).
Inactivation
An amplification test for residual live infectious avian reo
inactivation and the test is carried out in fertilized SPF hen
eggs or in suitable cell culture derived from SPF eggs (2.7.7).
The quantity of inactivated virus used in the test is equivalent
2737
REO VIRUS VACCINE, INACTNATED
IP 2010
to not less than ten doses of the vaccine. No live virus is

detected.
A. In cell culture derived from SPF eggs (2.7.7). Inoculate 10
doses of vaccine into suitable cell culture derived from SPF
eggs (2.7.7). Incubate at 361 for 7 days. Make a passage on
another set of cell culture derived from SPF eggs (2.7.7) and
incubate at 361 for 7 days. None of the cultures shows
signs ofinfection i.e. CPE.
E. In embryonated eggs. Inject quantity of inactivated virus

equivalent to 10 doses of vaccine into the allantoic cavity of
the SPF embryonated hen eggs, between 9-11 days old, and
incubate at 361 o. Observe for 6 days and pool separately the
allantoic fluid from eggs containing live embryos, and that
from eggs containing dead embryos, excluding those dying
from non-specific causes within the first 24 hours after
inoculation. Inject into the allantoic cavity ofeach ofthe SPF
embryonated hen eggs, between 9-11 days old, 0.2 ml ofthe
pooled allantoic fluid fiom the live embryos or membrane from
the dead embryos and incubate at 361 for 6 days. Examine
each embryo for lesions of Reo virus. The vaccine complies
with the test if there is no evidence of lesions of Reo virus.
The test is valid only if not more than 20 per cent of the
embryos die at either stage ofthe test. Ifmore than 20 per cent
of the embryos die at either one of the stages of the test,
repeat that stage. In any repeat test, not more than 20 per cent
ofthe embryos die from non-specific causes. Antibiotics may
be used to control extraneous bacterial infection.
Identification
In susceptible chickens, the vaccine stimulates the production
of specific antibodies against each of the virus serotypes in
the vaccine detected by virus neutralization.
Tests

Labelling. The label/insert states (l) strains used for
Reo Virus Vaccine, Live

Reo Virus Vaccine, Live is a preparation ofa suitable strain(s)
of Reo virus known to be safe and immunogenic. This
monograph applies to vaccines intended for administration to
chickens for protection against Malabsorption Syndrome and
lor proventriculitis and lor Tenosynovitis in birds.
Production
The vaccine virus is grown in embryonated SPF hens' eggs or
in cell cultures derived from SPF flocks (2.7.7) or suitable cell
line.
Vaccines.
Identification
When mixed with monospecific Reo virus antiserum, the
vaccine no longer induces cytopathic effect in susceptible
cell culture derived from. SPF eggs (2.7.7) or carry out
immunostaining test in cell culture derived from SPF eggs
(2.7.7) to identify the vaccine virus.
Tests
Safety. Inject each often SPF chickens (2.7.7, table 3) or healthy

susceptible chickens, 14 to 28 days old with twice the minimum
vaccinating dose and by one of the routes stated on the label.
Observe the chickens for 14 days. No abnormal local or
systemic reaction should be seen.
Potency. Inject each oftwenty SPF chickens (2.7.7, table 3) or
healthy susceptible chickens, 3 to 4 weeks old, with the
minimum dose and by the route stated on the label. Use ten
chickens ofthe same flock and age as controls. After 21 days,
collect serum samples from each bird including the ten-control.
chickens and perform quantitative agar gel precipitation test
. or-serumneutralization.test.oneach.serum.sample.-'I:he.mean
antibodytitre ofserain vaccinated group shallbe 1: 8 byAgar
gel diffusion test and 10000 units per m1 by serum neutralization
test and there should be no specific antibodies in the sera of
control chicken.

Safety. Final container samples of completed product from
each serial shall be tested as follows:
A. For vaccines intended for use in very young chickens,
each oflO, one day old SPF chickens (2.7.7, table 3) or healthy
tenosynovitis/malabsorption/proventriculitis susceptible
chickens shall be vaccinated with the equivalent of 10 doses
by one method recommended on the label.
E. For vaccines intended for use in older chickens, each of

ten, 4-week-old or older SPF chickens (2.7.7, table 3) or healthy
tenosynovitis susceptible chickens shall be vaccinated with
method
recoilllTIended on
the eqllival~nt of 10 d()s~s. l:>Y one
----c _
. . - _. ._. _ _. _ - th~Tabcl~
The vaccinates shall be observed each day for 21 days. If

unfavourable reactions occur which are attributable to the
product, the serial is unsatisfactory. If unfavorable reactions
2738
RINDERPESTVACClNE,LIVE
IP 2010
occur in more than two vaccinates which are not attributable

to the product, the test is inconclusive and may be repeated.
If the test is not repeated, the serial is unsatisfactory.
such a manner that it remains avirulent but retains its

immunogenicity in cattle. It is reconstituted immediately before
use with a suitable diluent.
Virus titre. Titrate the vaccine in cell cultures derived from

SPF embryos or in SPF eggs (2.7.7). One dose of the vaccine
contains not less than 103 TCID so / EID50 per dose.
Production
SEED LOT
The seed lots should be validated for the following tests:
Potency. Reo susceptible healthy chickens of same age and

from the same source shall be used as test birds. Vaccine
intended for use in very young chickens shall be administered
to chickens of the youngest age for which vaccine is
recommended. Vaccines intended for use in older chickens
shall be administered to 4 weeks or older birds. Ten SPF
chickens (2.7.7, table 3) or healthy susceptible chickens
vaccinates shall be used for each method of administration.
One dose will be injected to vaccinates. Ten chicks shall be'
held as unvaccinated controls.
a) Purity. It should be free from contaminations with viruses,

bacteria, fungi and mycoplasmas;
Potency test of each age group shall be conducted separately.

Twenty one days post vaccination each vaccinate and control
shall be challenged by injecting virulent virus into one foot
pad. The vaccinates & controls shall be observed for 14 days
post challenge. If at least 90 per cent of the controls do not
develop swelling and discolouration in the phalangeal joint
area of injected foot pad typical of infection ofReo virus, the
test is inconclusive and may be repeated. If at least 18 out of
20 vaccinates do not remain free of these signs, disregarding
transient swelling which subsides within 5 days post
challenge, the serial is unsatisfactory.
b) Should not induce any abnormal clinical reaction on

inoculation into rinderpest susceptible cattle;
c) Efficacy. It should induce an immunity to rinderpest in the
susceptible cattle.
CELLS. The primary cells/subcultured cells/continuous cell
lines when used should be free from BVD and other
contaminating viruses.
Identification
A. The vaccine protects cattle against virulent rinderpest virus.
B. The seed and the vaccine must be titrated in a suitable cell
culture system capable of supporting the multiplication ofthe
rinderpest virus.
C. When neutralised with a specific rinderpest antiserum, the
vaccine is no longer capable of protecting cattle against
rinderpest infection.
Tests
The serial is satisfactory when it gives 90 per cent protection

to vaccinated group and 90 per cent controls develop positive . Water (2.3.43). Not more than 3.0 per cent.
Reo virus lesions on challenge.
Mycoplasmas (2.7.4). Complies with the test for absence of
If the potency test has been performed with satisfactory mycoplasmas.
on other batches ofthe vaccine prepared from the same seed
lot.
Extraneous pathogens. Complies with the requirements stated

under Veterinary Vaccines.

The recopstituted vaccine should be used immediately after
preparation.
A. Use four healthy guinea-pigs, each weighing not less than

400 g. Inject two of them intramuscularly and two
intraperitoneally with 0.5 rnl ofthe vaccine under examination.
In addition, inject intraperitoneally each of six mice, each
weighing between 18 and 25 g, with OJ ml of the vaccine.
Observe the animals for 21 days. All th;e animals remain healthy
during the observation period. At the end of the observation
period sacrifice the animals and perform autopsy on each.
None of the animals shows any unusual changes.
Labelling. The labeVinsert states (1) strain of virus used; (2)


Rinderpest Vaccine, Live is a freeze-dried preparation ofa live
attenuated strain of rinderpest virus that has been modified
by adaptation to and propagation in suitable cell cultures in
Safety
B. Inject subcutaneously each of two susceptible cattle, free

from specific antibodies, with a quantity of the vaccine'
containing not less than 100 times the minimum dose stated
on the label, using pooled reconstituted contents of not less
than ten containers taken at random. Observe the animals for
2739
SALMONELLA ABORTUS EQUIVACCINE
IP 2010
21 days. No sign of disease attributable to the vaccine other

than mild transient pyrexia is seen.
Virus titer. Not less than 103 TCID so per dose of cell culture
vaccine determining the virus content of the reconstituted
vaccine in a suitable cell culture system.
Potency. Inject subcutaneously each oftwo susceptible cattle,
free from rinderpest specific antibodies, with a field dose and
1/1Oth of the minimum dose respectively stated on the label,
considering 103 TCIDso ofcell culture vaccine. Use two animals
of the same stock and age as controls. Observe the animals
for 21 days. Challenge intramuscularly each animal with a dose
of not less than 104 IDso of virulent rinderpest virus. Observe
the animals for 14 days. None ofthe vaccinated animals shows
any clinical signs suggestive of Iinderpest. The test is not
valid unless both the control animals develop signs of
rinderpest.
lot, provided the National Regulatory Authority permits.
Labelling. The label states (I) the strain of the virus used;
(2) the number of doses in the container; (3) that the vaccine
should be used immediately after reconstitution.
Potency. Inject each of twelve mice, each weighing not less

than 18 g, subcutaneously with 0.5 ml ofthe preparation under
examination. Use another twelve mice ofthe same weight range
and from the same stock as controls. Three weeks later,
challenge the mice from both groups by injecting
intraperitoneally each animal with 0.5 ml ofa suspension ofan
18-hour old culture containing 10 LD so virulent organisms of
S. abortus equi. Observe the mice for 7 days. The vaccine
passes the test if not less than nine mice of the vaccinated
group survive. The test is not valid unless not less than nine
of the control mice succumb to the challenge.
Labelling. The label states (I) the method of preparation;
(2) the strains of bacteria used to prepare the vaccine.
Salmonella Vaccine, Inactivated

Salmonella Vaccine, Inactivated is a preparation of I or more
suitable strains of 1 or more serovars of Salmonella organism,
inactivated while maintaining adequate immunogenic
properties.
This monograph applies to vaccines intended for the active
immunization of chickens against infection/s of Salmonella
in chickens and reducing Salmonella colonization and fecal
excretion in chickens.
Production
Salmonella abortus equi Vaccine is a suspension of killed

mixture of equal parts of pure fonnalized cultures of smooth
laboratory strains of Salmonella abortus equi.
The seed material is inoculated in a suitable medium. If the

vaccine contains more than I strains ofbacterium, the different
strains are grown and harvested separately. During production,
parameters such as growth rate, purity and identity is verified
on harvests using suitable culture. The bacterial harvests are
inactivated with suitable agent. The vaccine may contain
suitable adjuvant.
Production
Identification
The whole culture or its filtrate or a mixture is inactivated in

such a manner that pathogenecity is eliminated and
immunogenic activity is retained. The inactivated cultures may
be treated with a suitable adjuvant.
Vaccine stimulates production of strain specific antibodies

against Salmonella organisms in susceptible birds.
Identification
Safety:-InjectO:5-mlofthevaccineintraperitoneallyto-eachof
six mice, each weighing not less than 18 g. Observe the mice
for 96 hours, none of the mice dies of salmonellosis.
Safety. Administer double dose of vaccine subcutaneously

into each of ten SPF chickens (2.7.7, table 3) or healthy
susceptible chickens of minimum age recommended for
vaccination. Observe the birds at least for 21 days. The test is
not valid ifmore than 20 per cent ofthe chickens show abnormal
signs or die from causes not attributable to the vaccine. The
vaccine complies with-the test-if-no-chicken-shows-notable
clinical signs ofdisease or dies from causes attributable to the
vaccine.
Salmonella Abortus Equi Vaccine
It protects susceptible animals against infection with

Salmonella abortus equi.
Tests
Tests
2740
IP 2010
STERILE DILUENT FOR LIVE VACCINES
Potency. Carry out separate potency test for each strain of

Salmonella organism incorporated in the vaccine preparation.
Use not less than 10 SPF chickens (2.7.7, table 3) or healthy
susceptible chickens of the minimum age recommended for
vaccination. Administer 1 dose ofvaccine by a recommended
route. Maintain 10 chickens as unvaccinated controls from
the same source and flock used for vaccination for each strain
used in vaccine. Repeat the vaccination with the same dose
and route after 21 days to vaccinated birds. Challenge both
the groups, 2 weeks after last administration of vaccine, by
oral administration to each chicken a sufficient quantity of a
homologous strains of Salmonella organisms that is able to
colonize chickens. Observe the birds daily for 14 days. Collect
fecal samples on 14 th day for detection of presence of
Salmonella organisms by direct plating. The vaccine complies
with the test, if the number of Salmonella organisms in fresh
fecal samples after challenge is significantly lower in
vaccinated birds than in unvaccinated controls.
than 2 years from the date the potency was detennined
Labelling. The label states (1) strains used for preparation;
(2) the route of administration.
Sheep Pox Vaccine, Live Attenuated

Sheep Pox Vaccine, Live Attenuated is a freeze dried
preparation obtained by producing attenuated sheep
poxvirus in a suitable cell culture and mixed with a suitable
stabilizer and freeze dried. The freeze dried vial is reconstituted
with a suitable diluent and used immediately.
Safety. Inoculate not less than 2 sheep of8 to 12 months old,

fi'ee from neutralizing antibodies against sheep pox virus, with
ten times the field dose of the vaccine contained in 1 ml by
subcutaneous route. Observe the animals for 14 days. The
vaccine complies the test if none of the vaccinated animals
show deep necrotic lesion and generalization.
Virus titre. Not less than 10 TCID so of the virus per dose as
determined by the titre of the vaccine in a suitable cell culture
system.
Potency. Administer each of three sheep, between 8 and
12 months old, free from sheep pox neutralizing antibodies,
with the dose of the vaccine and by the route stated on the
label. Use two sheep of the same stock and age as unvaccinated controls. Shave the animals closely on the flank
from the shoulder to the proctodal area. Challenge each animal
after 21 days post-vaccination by inoculating intradennally
with 0.1 ml of a suspension six ten fold dilution of the sheep
pox challenge virus. Make five separate inoculations in a
vertical line for each serial dilution from the anterior to the
posterior of the animals. The titer of the challenge virus is
calculated using a standard statistical method for the
vaccinated and control sheep by the number of pox lesions
observed in each dilution. The titer of the challenge virus is
calculated for the vaccinated and control animals. The vaccine
passes the test ifthere is a difference oflog titer ofmore than
10gIQ2.5.
preparing the vaccine; (2) the virustitre; (3) the minimum dose
and the routes of administration; (4) the volume ofthe liquid
to be used for reconstitution of the vaccine.
Production
The vaccine reconstituted with a suitable liquid and diluted if

necessary to provide a concentration appropriate to the
particular test, complies with the requirements stated under
Veterinary Vaccines with the following modifications.
Sterile diluents are required for reconstitution of freeze dried

and frozen vaccines. The sterile diluents may be a special
liquid solution.
The seed lots used for vaccine preparation must be fi:ee from
extraneous pathogens.
Each diluent batch shall be given a number which shall be

used in records, test reports and final containers.
If the immunogenicity tests have been performed with

satisfactory results on a representative batch of the vaccine
from the seed lot, they may be omitted as a routine control test
during production on other batches of the vaccine prepared
from the same seed lot.
Identification
The vaccine specifically protects sheep against sheep pox.
Tests
Tests
Viral stability. Each batch should be tested for viral stability
by holding two hours after reconstitution of vaccine at
recommended temperature.
pH. Hydrogen Ion concentration shall be determined with a
pH meter which has been standardized with buffer.
Clarity. Each batch should be free from visible particulate
matter.
2741
SWlNEFEVERVACCINE,LNE
IP 2010
Virus titre. Not less than minimum virus titre per dose stated
on the label, determining the titre in a suitable cell culture.

Swine Fever Vaccine, Live is a preparation ofa modified strain
ofclassical swine fever virus, which is devoid ofpathogenicity
for the pig by adaptation either to cell cultures or to the rabbit.
It is prepared immediately before use by reconstitution from
the dried vaccine with a suitable diluent.
Production
The virus is propagated in suitable cell culture. The viral
suspension is harvested, titrated and may be mixed with a
suitable stabilizing agents. The vaccine is then freeze-dried
Identification
LAPINISED VACCINE. Administer 0.5 ml intravenously into
one or more non-immunised rabbits, immunized either with an
identical dose of a vaccine of the same type injected by the
same route between 10 and 60 days before hand or with a
sufficient dose of antiserum administered a few hours before
the injection of the vaccine. Twenty-four hours after the
injection, start recording the temperature of the rabbits in the
mornings and the evenings until the fifth day after the injection.
The immunised rabbits do not exhibit a rise in temperature of
more than 1.5. The test is not valid unless the nonimmunised
rabbitsexhibit a rise in temperature of not less than 1.5.
CELL CULTURE VACCINE. For non-lapinised vaccines
prepared in cell cultures, on administration to pigs immunised
with the vaccine specific neutralizing antibodies develop.
Tests
Test for extraneous pathogens. The vaccine mixed with a mono
specific antiserum does not cause cytopathic effects in
susceptible cell cultures. The cells also show no evidence of
the presence of haemadsorbing agents and the cell-culture
fluids are free of haemagglutinating agents when tested with
.chicken erythrocytes.
Water (2.3.43) Not more than 3.0 per cent.
Safety. Inject intramuscularly 10 times the minimum dose stated
on the label into each of three healthy piglets, between 6 and
7 weeks old, free from swine fever virus antibodies. Observe
the animals for 21 days. Temperature curve should be normal
and animals remain in apparent good health and display normal
growth.
Inject intracerebrally 0.03 ml ofthe vaccine, reconstituted in a
.111ilJ!11erthatLQ111L~I!!8:il1 1l!11 do~~, illtQeach_QJt~Il_111i<::.e,
weighing between llg and l5g. Observe the mice for2l days.
IfIIIore than~tWo mice-die;:Wiiliintheflrst 48 hours repeat the
test. The mice show no abnormalities attributable to the
vaccine within the third and twenty-first day after the injection.

Potency. All the animals are healthy and must have had no
contact with swine fever virus and serologically must be free
from CSF and BVDVantibodies. Use four healthy piglets,
between 6 and 7 weeks old, for each of the 1:50, 1:200 and
1:400 dilutions ofthe vaccine prepared in a suitable diluent or
buffer. Inject intramuscularly 1 ml ofthese dilutions into each
ofthe piglets in respective groups. Use two healthy susceptible
piglets of the same stock and age as control animal group.
After 21 days, inoculate intramuscularly with a sufficient
quantity of the challenge virus in each vaccinated piglet and
in each of the two unvaccinated control animals so that at
least one ofthe two unvaccinated control animals die within 7
to 14 days. Observe the vaccinated animals for 14 days.
Calculate the number of PDso contained in the vaccine by
standard statistical methods from the number ofanimals, which
survive without showing any signs of swine fever. The
vaccine contains not less than 100 PD so per dose. The test is
not valid unless the control animals die within 7 to 14 days
after inoculation. PD so correlation studies with virus titres can
replace the potency test on routine basis.
If the test for potency has been carried out with satisfactory
results on a representative batch of vaccine, this test may be
omitted as a routine control on other batches of vaccine
prepared from the same seed lot, subject-to agreement by the
Labelling. The label states (1) the minimum dose; (2) the
recommended routes of administration; (3) the name of any
added adjuvant.

Tetanus Vaccine for Veterinary Use is a preparation of the
neurotoxin of Clostridium tetani treated in a manner that
eliminates toxicity while maintaining adequate immunogenic
properties.
Production
The C. tetani strain used for production is cultured in a suitable
medium. The toxin is purified and then detoxified or it may be
detoxified before purification. The antigenic purity is
determined in Lf units oftetanus toxoid per milligram ofprotein
and shown to be not less than the value approved for the
particular product.
ChoiCe of vaccme compoSition
The C. tetani strain used in the preparation of the vaccine is
shown to be satisfactory with respect to the production of the
2742
TETANUS VETERJNARYVACCINE
IP 2010
neurotoxin. The vaccine is shown to be satisfactory with

respect to safety and immunogenicity for each species of
animal for which it is intended. As part of the studies to
demonstrate these characteristics, the tests described below
may be used.
Production of antigens. The production ofthe neurotoxin of
C. tetani is verified by a suitable immunochemical method
carried out on the neurotoxin obtained from the vaccine strain
under the conditions used for the production of the vaccine.
Safety. Carry out the test for each recommended route of
administration and species of animal for which the vaccine is
intended; use animals of the minimum age recommended for
vaccination and ofthe most sensitive category of the species.
Use not less than 15 animals, free from antitoxic antibodies for
each test. Administer a double dose ofvaccine to each animal.
Administer a single dose of vaccine to each animal after th~
interval stated on the label. Observe the animals until 14 days
after the last administration. The vaccine complies with the
test if no animal shows abnormal local or systemic signs of
disease or dies from causes attributable to the vaccine.
DETOXIFIED HARVEST
Absence oftoxin and irreversibility oftoxoid. Carry out a test

for reversion to toxicity on the detoxified harvest using 2
groups of5 guinea-pigs, each weighing between 350 to 450 g;
if the vaccine is adsorbed, carry out the test with the shortest
practical time interval before adsorption. Prepare a dilution of
the detoxified harvest so that the guinea-pigs each receive
10 times the amount oftoxoid (measured in Lfunits) that will
be present in a dose ofvaccine. Divide the dilution into 2 equal
parts. Keep one part at 5 3 and the other at 37 for 6 weeks.
Attribute each dilution to a separate group of guinea-pigs
and inject into each guinea-pig the dilution attributed to its
group. Observe the animals for 21 days. The toxoid complies
with the test if no guinea-pig shows clinical signs of disease
or dies from causes attributable to the neurotoxin of C. tetani.
FINAL LOT
containers. The containers are closed so as to avoid
contamination.
Identification
Carry out test A if permitted by the nature of the adjuvant.
Otherwise carry out test B.
A. Separate the toxoid from the adjuvant. For vaccines
adsorbed on aluminium hydroxide, the following treatment is
suitable. Dissolve sufficient sodium ci,trate in the vaccine
under examination to give a 10 per cent w/v concentration.
Maintain at 37 for about 16 hours and centrifuge. The clear
supernatant liquid reacts with a suitable tetanus antitoxin and

B. When inoculated into healthy susceptible animals, the
vaccine stimulates the formation ofantitoxin to the neurotoxin
of C. tetani or protects the animals against the paralytic effects
ofthe toxin.
Tests
Safety. Inject 5 ml ofthe vaccine subcutaneously as two equally
divided doses at separate sites into each of five guinea pigs,
each weighing between 350 and 450g. Observe the guinea
pigs for 21 days. None ofthe animals shows any symptoms of
tetanus or dies from tetanus. If more than one animal dies of
non-specific causes, repeat the test. No animal dies in the
second test.
Sterility (2.2.11). Complies with the test for sterilitY.
Potency. Test A may be omitted if test B is carried out. Test B
may be ommited if test A is carried out.
A. Inject subcutaneously each of ten guinea pigs, each

weighing between 350 and 450 g, with a quantity ofthe vaccine
not more than the minimum dose stated on the label as the
primary dose, and 28 days later with a quantity ofthe vaccine
not more than the minimum dose stated on the label as the
secondary dose. Fourteen days after the second dose, collect
the blood from each guinea pig, pool the sera and determine
the antitoxin titre by the biological assay of C. tetani antitoxin
described below.
1 ml of serum contains not less than 7.5 ill per rnl or, for
vaccine intended for use in equine, not less than 30 ill per ml.
When C. tetani vaccine is presented as a component of a
mixed vaccine intended for use in animals other than equine
and the potency test of the other component or components
normally carried out using rabbits, the potency test described
above may be carried out using ten healthy rabbits, between
3 and 6 months old. 1 m1 of serum contains not less than 2.5
Units.
Biological assay of C. tetani antitoxin
The potency of C. tetani antitoxin is determined by comparing

the dose necessary to protect mice or other suitable animals
against the toxic effects of a fixed dose of C. tetani toxin with
the quantity of a Standard preparation of C. tetani antitoxin
necessary to give the same protection. For this purpose, the
Standard preparation of C. tetani antitoxin and a suitable
preparation of C. tetani toxin are required.
The test dose of the toxin is determined in relation to the
Standard preparation of antitoxin and the potency of the
preparation under examination is then determined in relation
to the Standard preparation using the test toxin.
2743
TETANUS VETERINARYVACCINE
IP 2010
The Standard preparation is the 2nd International standard,

established in 1969, consisting of freeze-dried hyperimmune
horse sernm (supplied in ampoules containing 1400 Units) or
another suitable preparation,the potency of which has been
determined in relation to the International standard.
Suggested method
NOTE
The severity oftetanic paralysis to be regarded as
the end-point is such that the paralysis is readily recognised
but not sufficiently extensive to cause significant suffering.
For human reasons the animals should be examined at least
twice a day and should be killed as soon as the end-point is
reached.
In practice, when using high levels of toxin to determine the
test dose, or when using low levels of antitoxin in the
preliminary and final tests, the development ofparalysis is so
rapid that the defined end-point is usually synchronous with
death. Where death occurs, the combined totals of animals
dying or reaching the paralytic end-point are used in the
calculations.
Preparation oftesttoxin. Prepare C. tetani toxin by growing
C. tetani in liquid culture for 8 to 10 days and then adding
1 volume ofa sterile filtrate ofthe culture to 1 or 2 volumes of
glycerine. Store at 0 or at temperatures slightly below it. The
toxin may be dried by a suitable method.
Selection of test toxin. Select toxin for use as the test toxin by
determining the following quantities:
LP/IO dose (Limes paralyticum). This is the smallest quantity
oftoxin that when mixed with 0.1 Unit ofantitoxin and injected
subcutaneously into mice (or guinea pigs) causes tetanic
paralysis in the animals on or by the fourth day after injection.
Paralytic dose 50. This is the quantity of toxin that when
injected subcutaneously into mice (or guinea pigs) causes
tetanic paralysis in one-half of the animals injected on or
by the fourth day after injection. A suitable toxin is one
that contains not less than lOOO paralytic dose 50 in an
LP/lO dose.
Determination of test dose of toxin. Measure or weigh a
quantity ofthe test toxin and dilute with or dissolve in a suitable
liquid. Reconstitute or dilute the Standard preparation with a
suitable liquid to give a solution containing 0.5 Unit in 1 ml.
Prepare mixtures of the solution of the Standard preparation

and the solution of the test toxin such that each mixture
contains 0.1 UnitQfantitQxID in th~YQIJml~ ~~1~9t~<:l forinje<;tiQI1
Ql1cl()l1tJ gf.Q.serie~Qfgrq~tld \,()lllllJJ~S ()fth.~ s()llltion of the
toxin, separated from each other by steps of not more than
20 per cent and covering the expected end-point. Adjust each
mixture to the same final volume (0.4 to 0.6 ml ifmiceare used
or 4.0 nil ifguinea-pigs are used) with a suitable liquid. Allow

light, for 60 minutes and then inject a dose of the selected
volume of each mixture subcutaneously into each of not less
than 2 animals of the group to which each mixture has been
allocated. Observe the animals for 4 days and record daily the
degree oftetanus developing in each group ofanimals. Repeat
the determination at least once, add together the results of the
separate tests that have been made with mixtures ofthe same
composition such that a series of totals is obtained and
determine the mean values. The test dose of the toxin is the
amount present in that mixture that causes tetanic paralysis in
one-half ofthe total number of animals injected with it. When
the test dose of the test toxin has been determined, a
concentrated solution of the test toxin may be prepared in a
mixture consisting of 1 volume of saline solution and 1 or
2 volumes of glycerine. This concentrated solution may be
stored frozen and diluted as required. The specific activity of
such a solution must be determined at frequent intervals.
Determination ofpotency ofthe antitoxin.
PreliminaJy test. Measure or weigh a quantity of the test

toxin and dilute with or dissolve in a suitable liquid such that
the solution contains 5 test doses per ml. Prepare mixtures of
the solution of the test toxin and the preparation under
examination such that for each mixture the volume selected
for injection contains the test dose oftoxin and one ofa series
of graded volumes of the preparation under examination.
Adjust each mixture to the same final volume with a suitable
liquid. Allow the mixtures to stand at room temperature,
protected from light, for 60 minutes. Inject a dose ofthe selected
volumes of each mixture subcutaneously into each ofnot less
than two animals ofthe group to which each mixture has been
allocated. Observe the animals for 4 days and record daily the
degree of tetanus developing in each group of animals. From
the results select suitable mixtures for the final test.
Final test. Prepare similar fresh mixtures ofthe test toxin and
the preparation under examination such that for each mixture
the volume selected for injection contains the test dose of
toxin and one ofa series ofgraded volumes ofthe preparation
under examination, separated from each other by steps of not
more than 20 per cent and covering the expected end-point as
determined in the preliminary test. Prepare further mixttires
with the same amount oftest toxin and graded volumes ofthe
Standard preparation, centered on 0.1 Unit in the volume
selected for injection to confirm the test dose of the toxin.
Adjust each mixture to the same final volume with a suitable
.liquid,A.ll!:~w1h~aniD.~j:Q~tandatLQQ.1n~mPSl[a1:ll!e-,.P[Qt~c;t.(')<:l
frolJ1light, fo.r6lJ1inutes. Inject a d()se ofthe selected volume

of each mixture subcutaneously into each ofnot less than two
animals ofthe group to which each mixture has been allocated.
Observe the animals for 4 days and record daily the degree of
2744
THEILERIOSIS VACCINE, LIVE
IP 2010
tetanus developing in each group of animals. The mixture of

antitoxin under examination that contains 0.1 Unit in the volume
injected is that mixture which causes tetanic paralysis in the
same, or almost the same number of animals as the mixture
containing 0.1 Unit ofthe Standard preparation in the volume
injected. Repeat the determination at least once and calculate
the average ofall valid estimates. Estimates are not valid unless
the Standard preparation gives a result within 20 per cent of
the expected value.
Limits oferror. For the suggested method, the limits of error

(P = 0.95) have been estimated to be 85 to 114 per cent when
two animals are used per dose, 91.5 to 109 per cent when three
animals are used per dose, and 93 to 108 per cent when six
animals are used per dose.
B. Carry out the biologycal assay ofadsorbed tetanus vaccine
as stated under Tetanus Vaccine (Adsorbed).
This method may only be used for those preparations

for which it has been shown to be suitable and in particular
may not be suitable for vaccine with an oil adjuvant. Where
this alternative method is used the estimated potency is not
less than 150 ill in the smallest dose stated on the label.
Labelling. The label states (1) the name ofthe adjuvant used;
(2) the preparation should be shaken before use.

Theileriosis Vaccine, Live is a lymphoblast cell culture
containing Theileria annulata macroschizonts attenuated by
passage in such a manner that it remains avirulent while it
retains its immunogenicity. The concentrate of the vaccine
may be diluted with a suitable diluent after thawing.
PrOduction
Production is approved by a seed lot system. Each lot of the
seed stock is tested for safety and potency by methods
described below. If the immunogenicity tests have been
performed with satisfactory results on a representative batch

of the vaccine from the seed lot, they may be omitted as a
routine control test during production on other batches of the
Identification
Protects susceptible cattle against theileriosis.
Tests
Safety. Inject each of two healthy susceptible ca.ttle not less
than 9 months old with twice the dose as recommended on the
label. Observe the animals for 30 days. None of the animals
shows systemic reactions other than mild pyrexia and mild
swelling ofsuperficial lymph nodes. No schizonts/piroplasms
should be seen in the blood smears/lymph node smear.
Cell count. Contains not less than 2 million live lymphoblast
cells in each dose.
Potency. Inject each of tlll:ee susceptible cattle not less than
9 months old with the minimum dose by the route stated on
the label. Use two cattle ofthe same stock and age as controls.
After 30 days, challenge each ofthe vaccinated as well as the
control animals with a preparation of gut homogenate ofticks
containing suitable quantity of sporozoites to infect adult
cattle. Observe the animals for 30 days; none ofthe vaccinated
animals shows any abnormal signs. The test is not valid unless
both the control animals show typical signs of theileriosis. If
these tests have been performed with satisfactory results on
a representative batch of the vaccine from the seed lot, they
may be omitted by the manufacturer as a routine control
lot.
Labelling. The label states (1) the number of doses in the
container; (2) the recommended dose; (3) the method of
thawing and reconstihltion; (4) that the reconstituted vaccine
should be used within 3 hours after thawing and
reconstitution.
2745
VETERINARY DIAGNOSTICS MONOGRAPHS
VETERINARY DIAGNOSTICS MONOGRAPHS

Avian MycoplasmaAntigen
2749
BrucellaAbortus ColouredAntigen
2749
BrucellaAbortus Mille Ring TestAntigen, Hematoxylin Stained
2749
BrucellaAbortus Milk Ring TestAntigen, Tetrazoliurn Stained
2750
BrucellaAbortus PlainAntigen
2750
BrucellaAbortus Rose Bengal Plate TestAntigen (Strain 99)
2750
BrucellaAbortus Working Standard Serum
2751
Johnin Purified Protein Derivative
2751
Mallein Purified Protein Derivative
2752
Purified Protein Derivative (PPD), Bovine Tuberculin
2752
SalmonellaAbortus Equi HAntigen
2753
SahnonellaPullorumAntigen
2754
SalmonellaPullorumPlainAntigen
2754
SalmonellaPullorum Positive Serum
2754
2747
IP 2010
BRUCELLA ABORTUS MILK RING TEST ANTIGEN, HEMATOXYLIN STAINED
Avian Mycoplasma Antigen
coloured by the addition of crystal violet and brilliant green,

in phenolised glycerine saline.
Mycoplasma Antigen shall be prepared either from

Mypoplasma gallisepticum or Mycoplasma synoviae, grown
in broth cultures that are inactivated and standardized. Plate
antigen shall be stained with an acceptable dye. Each
intennediate antigen lot shall be tested for purity, density, and
preservative.
Purity. Intennediate antigen lot sample should be free from
extraneous organisms as determined by microscopic
examination and Gram staining.
Density. A 2.5 ml ofsample ofintennediate lot shall be diluted
with 2.5ml ofbuffer solution, fonnulated in the same manner
as the vehicle of the antigen being tested in a modified
Hopkin's tube and then sedimented by centrifugation. If the
packed cell volume ofthe sample is not 1.2 0.4 per cent, the
intennediate antigen lot is unsatisfactory.
Identification
Gives specific agglutination when mixed with the serum of
animals infected with Brucella organisms.
Tests
Assay. Mix 0.5 ml with 4.5 ml ofsaline solution contained in a
Hopkin's graduated tube and centrifuge at 3,000 rpm for
60 minutes. The volume of the packed bacterial cells is not
less than 11 per cent of the total volume.
Storage. As stated under Veterinary Diagnostics.
Labelling. As stated under Veterinary Diagnostics.
Preservative
_Phenol contents of antigen lot shall be 0.250.05 per cent.
A batch offinished product should be tested for Identification,
Homogeneity and Hydrogen Ion Concentration. The batch of
finished product found unsatisfactory for any prescribed test
shall not be released.
Identification
birds infected with M gallisepticum or M synoviae but fails
to react with serum from healthy birds.
Tests
Homogeneity. Antigen shall show no evidence of auto
agglutination or unusual appearance such as presence oflarge
visible particles.
pH (2.4.24). Hydrogen ion concentration shall be detennined
with a pH meter which has been standardized with buffer just
prior to use. The pH of Mycoplasma gallisepticum antigen
shall be 6.00.2. The pH of Mycoplasma synoviae antigen
shall be 7.00.2.
antigen may be expected to retain its potency for not less than
1 year from the date the potency was determined.
Labelling. The label states (l) strains used for preparation;
(2) the dose of test.
Brucella Abortus Milk Ring Test

Antigen, Hematoxylin Stained
Brucella abortus Mille Ring TestAntigen, Hematoxylin Stained
is a suspension of a pure smooth culture ofBrucella abortus
strain 99bacteria stained with hematoxylin and suspended in
saline solution containing 0.5 per cent wlv of phenol, the
reaction of which is adjusted to pH 4.0 with 0.1 M citric acid
or with 0.5 M disodium hydrogen phosphate, as appropriate.
For standardisation, the stained suspension is washed by
centrifugation in a solution containing 6.4 g of sodium
chloride, 1S ml of lactic acid and 4.4 ml of 10 per centw/v
solution of sodium hydroxide in 1,600 ml of distilled water,
the pH ofthe solution being adjusted to 4.0. The washed cells
are resuspended in phenol saline solution and the packed
cell volume of the final product is adjusted to 4 per cent v/v.
Identification
The antigen fonns a blue-coloured ring in the cream layer
when mixed with mille from animals suffering from Brucellosis.
Tests
Diagnostics.
Sensitivity. It has the same sensitivity as that of a standard
antigen when tested by the mille ring test.
Brucella Abortus Coloured Antigen

Brucella abortus Coloured Antigen is a suspension of a pure
smooth culture of Brucella abortus strain 99, which are

2749
BRUCELLA ABORTUS MILK RING TEST ANTIGEN, TETRAZOLIUM-STAINED
IP 2010
Brucella Abortus Plain Antigen
Brucella Abortus Milk Ring Test

Antigen, Tetrazolium-Stained
Brucella abortus Milk Ring Test Antigen, Tetrazolium-Stained
is a suspension of a pure smooth culture of Brucella abortus
strain 99 bacteria stained supravitally with 2,3,5-triphenyltetrazolium chloride in saline solution containing 1 per cent
v/v of glycerin and 1 per cent w/v ofphenol. Smooth strain of
Brucella abortus strain 99 is grown on potato infusion agar
for 48 to 72 hours in roux flasks, at 37. Condensation fluid if
any is pipetted offbefore washing. Each flask is washed with
about 20 ml of normal saline. The pooled washing is filtered
through a gauze and the filtrate is collected in a measuring
cylinder. To every 500 ml ofthe filtrate 1g of2, 3, 5 triphenyltetrazolium chloride is added immediately. The container is
shaken for thirty minutes till the tetrazolium salt is dissolved.
The product is taken out and kept in at 37 for two hours. After
incubation the product is heated at 65 in a water bath for
thirty minutes. It is cooled and centrifuged at 3,000 rpm for
one hour. The supernatant is pipetted off and sediment is
suspended in normal saline containing 1 per cent glycerol
and 1 per cent phenol and filtered through sterile cotton wool.
This forms concentrated antigen.
For the standardisation of the stained antigen, one ml of an
aliquot of the suspension representing the initial undiluted
suspension is taken in each of6 test-tubes to which increasing
quantities (0.6, 0.8, 1.0, 1.2, 1.4 and 1.6 ml) of saline solution
containing 1 per cent v/v of glycerin and Iper cent w/v of
phenol are added. The contents of each tube are then diluted
10-fold with the same diluent and serve as antigen for the tube
agglutination test with the reference standard antiserum. Thus
six sero-reactions will be carried out. During this procedure
the concentrated stained microbial suspension is kept at a
temperature between 2 and 8. The agglutination reactions
are read after 48 hours. The dilution which gives 50 per
cent agglutination with a 1 in 500 final dilution of the
standard antiserum is taken as the final dilution for the
preparation.
Identification
The antigen forms a cherry-red ring in the cream layer when
mixed with milk from animals infected with Brucellosis.
Brucella abortus Plain Antigen is a suspension of a pure

smooth culture of killed Brucella abortus strain
99 bacteria in phenol-saline solution.
Identification
animals infected with Brucella abortus organisms.
Tests
Diagnostics.
Sensitivity. Gives 50 per cent agglutination on incubation at
37 for 20 1 hours with a 1 in 500 dilution of a standard

Brucella antiserum containing 1,000 International Units.
Lahelling. As stated under Veterinary Diagnostics.
Brucella Abortus Rose Bengal Plate

Test Antigen (Strain 99)
Brucella abortus Rose Bengal Plate Test Antigen (Strain 99)
is a suspension of inactivated bacteria from a pure smooth
culture ofBrucella abortus strain 99. The bacteria being stained
with Rose Bengal, in a buffered solution prepared by adding
540 ml of lactic acid to 2,000 ml ofphenol saline solution and
diluting to 6,000 ml.
The antigen is used when an approximate idea of the extent
of the infection in a herd is required to be assessed with
minimum effort and maximum speed or as a screening test to
assess whether an outbreak of abortions is due to Brucellosis.
Identification
Gives the specific agglutination when mixed with the serum of
animals infected with Brucella organisms.
Tests
Tests
pH (2.4.24). 3.6t03.7.
Other-tests.-Complies-with-thetests-statecLunderYeterinary_ Other tests. Complies with the tests stated under Veterinary
Diagnostics.
.-Diagnostics:
Assay. To 0.5 ml quantities taken in each of six Hopkin's

graduated tubes or graduated haematocrit tubes, add 4.5 ml of
saline solution in each tube, mix and centrifuge at 3,000 rpm

2750
JOHNIN PURlFIED PROTEIN DERIVATIVE
IP 2010
for 60 minutes. The packed bacterial cell volume is not less

than 8 per cent.
Identification
Inject intradermally small doses ofthe preparation into suitable
guinea-pigs that have been sensitised with M paratuberculosis; hot, painful oedematous swellings occur at the
sites of inoculation after 48 hours.

Tests
Brucella Abortus Working Standard

Serum
pH (2.4.24). 6.5 to 7.5.

Brucella abortus Working Standard Serum is serum of cattle

infected with Brucella abortus biotype I, or serum raised in
rabbits against smooth cultures of B. abortus strain 99 or
strain 544 which is suitably diluted with healthy cattle serum
orrabbit serum as appropriate. It contains 0.01 per cent w/v of
thiomersal as antibacterial preservative.
The serum is suitably standardised so that a 1 in 500 dilution
gives 50 per cent agglutination in tube agglutination test in
comparison with the Brucella abortus standard serum.
Identification
Gives specific agglutination when mixed with a pure smooth
culture of BnlCelia abortus organisms.
Tests
Abnormal toxicity (2.2.1). Inject 0.5 ml subcutaneously into

each of two guinea-pigs. Observe the animals for 7 days;
none of the guinea pigs shows significant local or systemic
reaction.
Potency. Carry out the biological assay of Johnin Purified
Protein Derivative described below:
Biological assay ofJohnin purified protein derivative
The potency ofJohnin purified protein derivative is determined
by comparing the reactions produced in sensitised guineapigs by intradermal injection of a series of dilutions of the
preparation under examination with those produced by known
concentrations of the Standard preparation.

Diagnostics.
Assay. When tested with standardised Brucella abortus tube
test antigen, gives 50 per cent agglutination at 1 in 500 final
serum dilution in tube agglutination test in comparison with
the Brucella abortus standard serum.

Johnin Purified Protein Derivative is a preparation of a fluid
synthetic medium in which Mycobacterium paratuberculosis
has been grown and which has been freed of the bacilli by
filtration. The active fraction of the filtrate, which is
predominantly protein in nature, is isolated by precipitation,
washed and re-dissolved. It is then distributed in sterile glass
containers and sealed so as to exclude micro-organisms. It
may contain a suitable preservative. It reveals delayed
hypersensitivity in animals sensitised by M paratuberculosis.
Description. A yellowish-brown liquid.
Sterility (2.2.11). Complies with the tests ofsterility, Method A.
The Standard preparation is Johnin purified protein derivative,

maintained by the Indian Veterinary Research Institute,
Izatnagar, or another suitable preparation, the potency ofwhich
has been detennined in relation to the Standard preparation.
Suggested method
Sensitise five guinea-pigs, each weighing between 300 and
450 g, by deep intramuscular injection of 0.5 ml ofa suspension
in saline solution containing 0.1 /lg of moist growth from
solid slants oflive M paratuberculosis. After a period of not
less than 3 weeks carry out the following test. Use two healthy
animals of the same weight range and from the same stock as
controls. Shave the flanks to provide space for not more than
4 injection sites on each side. Prepare 1:500, 1: 1000, 1:2000 and
1:4000 dilutions of each of the Standard preparation and the
preparation under examination in phosphate-buffered saline
pH 7.4 containing 0.005 per cent w/v ofpolysorbate 80. Using
not less than 2 doses of each dilution of the Standard
preparation and the preparation under examination, inject each
dose intradelmally in the same volume (0.1 to 0.2 ml) to the
available sites in a random manner in Latin-square design..
The sensitised guinea-pigs exhibit hot, painful and
oedematous swellings typical of M paratuberculosis at the
sites of injection persisting for not less than 72 hours. The
test is not valid unless the control animals fail to produce
2751
JOHNIN PURIFIED PROTEIN DERIVATIVE
IP 2010
such reactions. With the help of callipers, measure the skin

thickness around the sites of injection, 72 hours after
inoculation.
Calculate the potency using standard statistical methods on
the basis that the skin thickness are directly proportional to
the logarithms of the concentrations of the Johnin Purified
Protein Derivative.
Assay. To 5 ml add 2.5 ml ofwater and 2.5 ml of a 40 per cent
w/v solution oftrichloro acetic acid, mix, allow to stand fodO
minutes and centrifuge for 15 minutes. Discard the supernatant
liquid and dissolve the residue in 0.5 ml of 5 M sodium
hydroxide solution. Transfer the solution to a Kjeldhal flask
with the aid of 6 ml of water. Add about 0.1 g of a mixture of
100 parts of potassium sulphate, 10 parts of cupric sulphate
and 5 parts of selenium dioxide and 1 ml of nitrogen-free
sulphuric acid. Heat until the water evaporates. Continue the
heating until a brown deposit appears. Dissolve the deposit
in 0.5 ml of hydrogen peroxide solution, continue heating
until white fumes appear and boil rapidly for at least 10 minutes.
If a brown deposit again appears add a further 0.5 ml of
hydrogen peroxide solution. Transfer to an ammonia distillation
apparatus with the aidof5 mlofwater and add 5 ml ofa 50per
cent w/v solution of sodium hydroxide to form a lower layer.
Distil for 3 minutes, collecting the distillate in a mixture of5 ml
of a 2 per cent w/v solution of boric acid and 0.05 ml of a
solution containing 0.066 per cent wIv ofmethyl red and 0.033
per cent w/v of bromocresol green in ethanol (95 per cent).
Titrate with 0.00447M sulphuric acid. Repeat the operation
using 5 ml of the water in place of the preparation under
the ammonia liberated by the substance under examination.
For standardisation, four porties previously sensitised with

P. mallei and two healthy ponies are injected intradermopalpebrally with 0.2 ml of the preparation near the rim of the
lower eye-lid of one eye. Typical reaction such as painful
swelling ofthe palpebral tissue with mucopurulent discharge
from the eye of sensitised animals and no such reaction in the
healthy ponies should be seen. A similar test is performed
with the Standard preparation maintained by the Indian
Veterinary Research Institute, Izatnagar. When the reactions
ofthe two preparations are comparable the batch is considered
fit for use.
Mallein Purified Protein Derivative contains not less than
0.95 mg per ml and not more than 1.05 mg per ml ofpurified
protein derivative.
Caution - Mallein Purified Protein Derivative is not
dangerous to humans, but the organism from which it is
prepared is pathogenic to man and may befatal ifan infection
is not treated properly. Treatment should begin promptly if
an infection is suspected.
Description. A yellowish to brown, viscous liquid.

Identification
Inject intradermally small doses ofthe preparation into suitable
guinea-pigs that have been sensitised with killed P. mallei in
an oily adjuvant; hot, tense, painful oedematous swellings
occur at the sites of inoculation after 48 hours.
Tests
pH (2.4.24).6.5 to 7.5.
Phenol (2.3.36), (ifpresent). Not more than 0.5 per cent w/v.
1 ml of 0.00447 M sulphuric addis equivalentto 0.875 mg of

purified protein derivative.
Sterility (2.2.11). Complies with the test of sterility, with

modifications stated under Johnin Purified Protein Derivative.
Abnormal toxicity (2.2.1). Inject 0.5 ml subcutaneously into

each oftwo guinea pigs. Observe the animals for 7 days; none
ofthe guinea pigs shows significant local or systemic reaction.

under Veterinary Diagnostics and also states (1) the total
volume in the container; (2) the name and percentage of any
added preservative.
Assay. Carry out the Assay described under Johnin Purified

Protein Derivative using 2.5 ml.

Mallein Purified Protein Derivative is a preparation of a fluid
synthetic medium in which Pseudomonas mallei
(Burkholderia mallei) has been grown and which has been
freed of the bacilli by filtration. The active fraction of the
-filtrate;whichispredominantly--protein-innature,---isisolated
byprecipitation,washedandre-dissolved.in_phosphate
buffered saline at about neutral pH. It is then distributed in
sterile containers that are inert towards the contents and sealed
so as to exclude micro-organisms.

under Veterinary Diagnostics and also states (1) the total
volume in the container; (2) the name and percentage of any
added preservative.
Purified_.rroteinD~.riY!l.tiyeJPP.n).,_ . _
Bovine-Tuberculin
Purified Protein Derivative (PPD), Bovine Tuberculin is a
preparation of a fluid synthetic medium in which -M bovis
2752
SALMONELLA ABORTUS EQUI H ANTIGEN
IP 2010
AN5 has been grown and which has been freed of the bacilli
by filtration. The final sterile product is distributed in sterile,

tamper-evident glass containers, which are then sealed to
prevent contamination with extraneous microorganisms.
The International Standard for Purified Protein derivative (PPD)

of Mycobacterium bovis Tuberculin was donated to National
Institute for Biological Standards and Control, Potters Bar,
Description. A yellowish-brown viscous liquid, or dry Hertfordshire, UK by Central Diergeneeslcundig, Netherlands.
With effect from I't June 1998, the National Institute for
yellowish-brown powder or pellet. .
Biological Standards and Control (NIBSC), Potters Bar, UK is
the custodian and distributor of this material. Each ampoule
Identification
contains 58,000 International Units of PPD and when
Inject intradermally a range of graded doses at different sites . reconstituted with 1.8 ml of diluting fluid will contain 1 mg
into suitable albino guinea pigs sensitised with tuberculosis. PPD and 32,500 LU. perml.
Depending upon the allergic status of the animal, the
magnitude of dose and specificity of the product, reactions Suggested method
occur at the points of injection as diffused oedematous Sensitise nine guinea-pigs, each weighing not less than 400 g.
swellings with erythema with or without necrosis. When Inject each animal intramuscularly on the medial side ofthigh
similar injections are given to non-sensitised guinea pigs no with 0.0001 mg wet weight of M bovis, live strain of
such reactions occur.
Mycobacterium bovis suspended in 0.5 ml physiological
saline. Test three dilutions of each of 3 preparations. Since it
Tests
is practicable to give only 8 injections to an individual animals,
a balanced incomplete Latin Square design is used, in which a
The preparation, reconstituted if necessary with a suitable
different one ofthe 9 dilution is omitted from each animal. The
liquid and diluted to provide a concentration appropriate
remaining 8 dilutions are allocated to 4 sites.
to the particular test, complies with the requirements stated
under Veterinary Diagnostics with the following Diluting fluid for assay
modifications.
The diluent consists of isotonic phosphate-buffered saline
pH 7.3, containing tween 80 (0.0005 per cent) and is prepared
pH (2.4.24).6.5 to 7.5.
by adding 0,5 ml of 1 per cent w/v solution of tween 80 in
Phenol (ifpresent) (2.3.36). Not more than 0.5 per cent w/v.
distilled water to 1 litre of the following solution:
Other tests. Complies with the tests stated under Veterinary Na2HPO42Hp - 7.60 g; ~PO 4- 1.45 g; NaCl- 4.80 g; distilled
water - 1 litre. Tuberculin diluted 1 in 100, 1 in 500 and 1 in
Diagnostics.
2,500 with 0.1 ml inoculum or 1 in 200, 1 in 1,000 and 1 in
Abnormal toxicity (2.2.1). Inject subcutaneously 0.5 ml ofthe
5,000 ml with 0.2 ml inoculum generally will produce
preparation under exan1ination into each of two guinea-pigs
satisfactory results in guinea pigs.
weighing not less than 250 g. No abnormal effects are produced
Measure the skin thickness at each site at the time of injection
within 7 days.
and after 72 hours. Calculate the results using standard
Potency. Carry out the biological assay of bovine tuberculin statistical methods on the basis that the diameters ofthe lesions
purified protein derivative described below:
are directly proportional to the logarithms of the concentrations of the tuberculin.
Biological assay of bovine tuberculin purified protein
derivative
The potency of bovine tuberculin purified protein derivative
is determined by comparing the reactions produced in
sensitised guinea-pigs by intradermal injection of a series of
dilutions of the preparation under examination with those
produced by known concentrations of the Standard
preparation calibrated in International Units. Sensitise not less
than 9 albino guinea-pigs each weighing between 300 and
450 g by deep intramuscular injection of 0.0001 mg of wet
mass ofliving M bovis of strain AN5 suspended in 0.5 ml of
normal saline solution. Not less than 4 weeks after the
sensitisation of the guinea-pigs, shave their flanks to provide
space for not more than 4 injection sites on each side.
Labelling. The label insert states (1) the number of units per
dose of 0.1 ml or per ml or per mg; (2) the total volume in the
container (for liquid preparation); (3) the name and proportion
of any added substances; (5) the strain used; (6) the storage
conditions; (7) the date after which the contents are not
Salmonella Abortus Equi H Antigen

Salmonella abortus Equi H Antigen is a suspension of killed
organisms derived from a pure smooth culture of actively
motile Salmonella abortus equi.
2753
IP 2010
SALMONELLA PULLORUM ANTIGEN
Identification
animals infected with S. abortus equi organisms.
Tests
buffer just prior to use. The pH of stained antigen shall be

4.6OA. No pH level is specified forpullorum tube antigen but
after dilution, as recommended for use it shall have a pH of8.2
to 8.5.

antigen may be expected to retain its potency for not less than
1 year from the date the potency was determined.
Opalescence ofsuspension. The opalescence ofthe preparation

under examiantion corresponds to Brown's opacity standard
tube No. 2.
Labelling. The label states (1) strains used for preparation;

(2) the dose of test.
Other tests. Complies with the tests stated under Veterinary .

Diagnostics.
Salmonella Pullorum Plain Antigen

Salmonella pullorum Plain Antigen is a suspension of dead
bacterial cells of a pure smooth culture of a suitable strain of
Salmonella pullorum in saline solution containing 0.5 per
cent w/v of phenol.
Salmonella Pullorum Antigen

Salmonella pullorum Antigen is a suspension of a pure
smooth culture of representative strains of Salmonella
pullorum which are of Imown antigenic composition, high
agglutinability, but are not sensitive to negative and nonspecific serum. Each intermediate lot shall be tested for purity,
density, and preservative.
Purity. Intermediate lot sample should be free from extraneous
organisms as determined by microscopic examination and Gram
staining.
Density. The bacterial density shall be 8015 times MacFarland Number 01 for stained antigen and 50 10 times MacFarland Number 01 for tube antigen.
Identification
birds infected with of S. pullorum or S. gallinarium but fails
to react with serum from healthy birds.
Tests
Opalescence ofsuspension. The opalescence ofthe preparation
under examination corresponds to Brown's opacity standard
tube No.1.
Diagnostics.
Preservative
Formalin contents ofthe intermediate lot of coloured antigen

shall be 1.00.2 per cent. Phenol contents of plain antigen
shall be 0.550.5 per cent.
A batch offinished product should be tested for Identification,
Homogeneity and Hydrogen Ion Concentration. The batch of
finished product found unsatisfactory for any prescribed test
shall not be released.
Identification
Salmonella Pullorum Positive Serum

Salmonella pullorum Positive Serum is a liquid or freeze-dried
antiserum raised against. a suitable smooth strain of
S. pullorum in rabbits. The liquid preparation contains
0.01 per cent w/v ofthiomersal or other suitable preservative.
Identification

birds infected with S. pullorum or S. gallinarum but fails to
react with serum from healthy birds.
Gives specific agglutination when mixed with a smooth strain

of S. pullorum and gives a titre 1: 1000 with tube test antigen.
Tests
Tests

Homogeneity. Antigen.shall.show. D-O_evidence.of allto
agglutination or unusual appearance such asp;~s~~c-e~f orIiertests~ ComplieswitliHie-fesfisfiltedunderveteriiiaiy
Diagnostics.
flakes.
pH (2.4.24). Hydrogen ion concentration shall be determined Storage. As stated under Veterinary Diagnostics.
with a pH meter which has been standardized with pH 4.0
2754
EX
2755
IP 2010
INDEX
A
Abacavir Sulphate
148,259,761
Abacavir Oral Solution
761
Abacavir Tablets
762
Abacavir and Lamivudine Tablets
763
Abacavir Sulphate Oral Solution
761
Abacavir Sulphate Tablets
762
Abacavir Sulphate and Lamivudine Tablets
763
Abacavir, Lamivudine and Zidovudine Tablets
765
Abbreviated Statements in Monograph
12,714,1732
Abbreviations and Symbols
703
Abnormal Toxicity
27
ABO Blood Group of Donors, Determination of
248
ABO Blood Group and Rh Group, Determination of
248
ABO Blood-grouping Reagents

Pouitry Vaccines, Test for
Absence of Mycoplasmas, Test for
Absence of Non-Avian Mycoplasmas and
lJreaplasmas, Test for
Absolute Alcohol
248
210
219
1303
Acetaldehyde
563
Acetaldehyde Standard Solution (100 ppm, C2HP)
628
Acetaminophen
1859
Acetaminophen Syrup
1860
Acetaminophen Tablets
1861
Acetate Buffer pH 2.8
560
560
560
560
560
560
560
560
560
560
560
Tris-Acetate Buffer pH 8.5
563
Acetate Buffer Solution
560
Acetate-Edetate Buffer pH 5.5
560
71
Acetates, Tests for
261,772
Absolute Ethanol
1303
Acetazolamide
Absorbent Cotton
1137
Acetazolamide Tablets
773
Absorbent Cotton Wool
1137
Acetic Acid
563
Absorbent Lint
1593
Acetic Acid-Ammonium Acetate Buffer
560
Absorbent Viscose
2046
Acetic Acid, Dilute
563
Acacia
2469
Acetic Acid, Glacial
563,774
Acacia and Acacia Powder

Acarbose
148
148,766
Acetic Acid, Glacial, Anhydrous
563
Acetic Acid Ear Drops
774
Acarbose Tablets
767
Acetic Acid Sp
563
ACD Solution
831
Acetic Acid Sp., Dilute
563
Acebutolol Hydrochloride
148,259,768
Acetic Acid, x M
563
774
563
Acebutolol Hydrochloride Tablets
769
Acetic Acid Otic Solution
Acebutolol Tablets
769
Acetic Anhydride
Aceclofenac
Aceclofenac Tablets
148,260,770
770
Acetic Anhydride-Dioxan Solution
563
Acetic Bromine Solution
570
Acenocoumarol
1777
Acetic-Ammonia Buffer pH 3.7, Ethanolic
560
Acenocoumarol Tablets
1778
Acetic Potassium Iodobismuthate Solution
605
Acetone
563
Acetone, Dry
563
Acetone Solution, Buffered
560
Acepromazine
, Acepromazine Maleate
260
2632
148, 772, 2631
Acepromazine Maleate Injection
2632
Acetone-Dried Ox Brain
598
Acepromazine Maleate Tablets
2632
Acetonitrile
2632
Acetyl Chloride
564
564
Volume 1: i to xxiii and 1 to 704; Volume 2: xxv to xxviii and 705 to 1722; Volume 3: xxix to xxxii and 1723 to 2829
2757
IP 2010
INDEX
71
205
563
Acetyl Groups, Test for

O-Acetyl Groups, Test for
N-Acetylneuraminic Acid
Acetylsalicylic Acid
Acetylsalicylic Acid and Caffeine Tablets
Acetylsalicylic Acid Tablets
Acetylsalicylic Acid Tablets, Soluble
843
564
210
148,775
775
776
589
564
594
Acetyltyrosine Ethyl Ester

Acholeplasma Laidlawii
Aciclovir
Aciclovir Intravenous Infusion
Aciclovir Tablets
Acid Blue 74
Acid Blue 83
Acidified Methanol
Acid Ferric Ammonium Sulphate Solution
584
559
624
Acid Phthalate Buffer pH 2.2 to 4.0

Acid Red 87
Acid Value, Assay for
84
590
Acid-washed Kieselguhr
Aclmowledgements
Acrylamide/bisacrylamide solution
Activated Charcoal
Activated Coagulation Factors
Activated Dimethicone
Activated Polydimethylsiloxane
Activated Zinc
Actuators for Inhalation Preparation
Acyclovir
Acyclovir Intravenous Infusion
Acyclovir Sodium Intravenous Infusion
Acyclovir Tablets
Added Substances, General Notices
564
564
152,573,1043
243
155,1230
1230
621
727
775
775
775
776
11,713,1731
Additions
Adhatoda vasica
Admissions
Adrenaline
564
779
Adrenaline Tartrate
778
84 Adrenaline Tartrate Injection

842 _Adsorbed Diphtheria and Tetanus Vaccine
844 Adsorbed Diphtheria, Tetanus and Pertussis
Vaccine
843
Acetyl Value, Assay for
Acrylamide
Adrenaline Bitartrate, Noradrenaline-free

Adrenaline Injection
2550
XVI11
148,261,777
778
Adrenaline Acid Tartrate

-Adrenaline Acid TartrateInjection---------------------n9
2378
2376
Adsorbed Diphtheria, Tetanus and

2350
Adsorbed Diphtheria, Tetanus, Pertussis (Acellular

Component) and Haemophilus Type b
Conjugate Vaccine
2351

Component) and Hepatitis B (rDNA) Vaccine
2354

Component), Inactivated Poliomyelities Vaccine
and Haemophilus Type b Conjugate Vaccine
2356

Component), Inactivated Poliomyelities Vaccine
2359
Adsorbed Diphtheria, Tetanus, Pertussis and

Poliomyelitis (Inactivated) Vaccine
2361
Adsorbed Diphtheria, Tetanus, Pertussis,

Poliomyelitis (Inactivated) and Haemophilus
Type b Conjugate Vaccine
Adsorbed Pertussis Vaccine (Acellular Component)
Adsorbed Pertussis Vaccine (Acellular, Co-purified)
2364
2366
2369
Adsorbed Diphtheria, Tetanus, Pertussis (Whole Cell),

Hepatitis B (rDNA) and Haemophilus Type b
2379
Conjugate Vaccine
Adsorbed Diphtheria, Tetanus, Pertussis
(Whole Cell) Hepatitis B (rDNA) Vaccine
2382
Adsorbed Diphtheria, Tetanus, Pertussis (Whole Cell)

and Haemophilus Type b Conjugate Vaccine
2384
Adsorbed Diphtheria Vaccine
2387
Adsorbed Hepatitis A (Inactivated) and Hepatitis B

(rDNA) Vaccine
2398
Adsorbed Inactivated Hepatitis A Vaccine
2402
Adsorbed Tetanus Vaccine

Adulasa
Agar
Ajwain ~:.;Alanine
Adrenaline Bitartrate
Albendazole
Adrenaline Bitartrate Injection
2375
Adsorbed Diphtheria and Tetanus Vaccine for

Adults and Adolescents
Adrenaline Acid Tartrate, NOfadrenaliIie::fretf ----
564
148,564,778
779
779
Albtmdazole Tablets
2758
2443
2550
564
-------- -461,-49S,2410
--. --------------564
148, 262, 780
781
IP 2010
Albumin, Bovine
INDEX
564,570
2576
Allantoin
Albumin, Human
2568
Allium Sativum Bulb
Albumin Phosphate Buffer pH 7.2
560
71
Alkaloids, Tests for
Albumin Fraction (Saline), Human
148, 262, 782

2517
Allopurinol
148,263,783
784
Allopurinol Tablets
Albuterol
2083
Albuterol Inhalation Aerosol
2084
Aloes
148,784
Albuterol Sulphate
2085
Alpha Amylase
149,829
(R)-Albuterol Sulphate
1583
Alprazolam
Albuterol Sulphate Injection
2087
Alprazolam Tablets
Albuterol Sulphate Syrup
2088
Alternative Methods, General Notices
Albuterol Sulphate Tablets
2088
Alternative Thioglycollate Medium
Alcohol
1302
Alum
564
Alumina, Anhydrous
564
Alumina, Deactivated
564
Alcohol, Absolute
1302
Alcohol, Dehydrated
1303
Alcohol (95 Per Cent)

Alcohol, General Notices
1304
12
Alcoholic Dimethylaminobenzaldehyde Solution
579
Aldehyde-free Ethanol
582
Aldehyde-free Ethanol (95 per cent)
582
Aldehyde-free Methanol
594
~lginic Acid
148,781
148,263,786
787
11,713,1731
58
Aluminium Acetate Ear Drops
788
Aluminium Acetate Otic Drops
788
Aluminium Acetate Solution
788
Aluminium Chloride, Anhydrous
565
Aluminium Chloride Solution
565
Aluminium Hydroxide, Dried
789
Aluminium Hydroxide Gel
788
Aluminium Hydroxide Gel, Dried
148,789
4-Allyl-2-methoxyphenol
583
Alizarin Red S
622
Alizarin Red S Solution
622
Alizarin S
622
Alizarin S Solution
622
AlleulineAmmonium Citrate Solution
566
Aluminium Nitrate
565
Alleuline Blue Tetrazolium Solution
570
Aluminium Oxide, Anhydrous
564
Alkaline Borate Buffer pH 8.0 to 10.0
560
Aluminium Oxide, Deactivated
564
Alkaline Copper Solution
576
Aluminium Oxide G
565
Alleuline Cupri-Tartrate Solution
603
Aluminium Oxide, Hydrated
789
Aluminum Hydroxide Mixture
788
Aluminium Hydroxide Suspension
788
Aluminium in Adsorbed Vaccines, Limit Tests of
79
Aluminium, Limit Tests of
79
Aluminium Magnesium Silicate
148,789
Alleuline Phosphatase-Conjugated Avidin/Streptavidin

reagent
241
Aluminium Potassium Sulphate
564
Aluminium Potassium Sulphate Dodecahydrate
564
Alkaline Phosphatase Enzyme
601
Aluminium Salts, Tests for
Alkaline Phosphatase Solution
601
Aluminium Standard Solution (2 ppm AI)
628
Alkaline Picric Acid Solution
602
Aluminium Standard Solution (l0 ppm AI)
-628
Alleuline Potassium Mercuri-iodide Solution
605
Aluminium Standard Solution (l ppm NH4)
628
Alleuline Pyrogallol Solution
606
Aluminium Sulphate
Alleuline Sodium Hypobromite Solution
611
Amalaki
Alkaline Sodium Nitroprusside Solution
612
Amantadine
Alleuline Sodium Pickrate Solution
602
Amantadine Hydrochloride
Alleuline Tetrazolium Blue Solution
570
Amantadine Capsules
791
Alleuline Trinitrophenol Solution
602
Amantadine Hydrochloride Capsules
791
71
565
461,496,2471
264
148,791
2759
IP 2010
INDEX
Ambroxol Hydrochloride
148,264,792
3-Aminopropionic Acid
564
3-Amino-7-dimethylamino-2-methylphen
azine Monohydrochloride
626
5-Amino-9-diethylaminobenzo[a]phenoxazinylium Hydrogen Sulphate
626
Aminopyrazolone
565
598
Amidation
2598
Amidone Hydrochloride
1660
Amidone Hydrochloride Injection
1661
Amidone Hydrochloride Linctus
1661
Amidone Hydrochloride Tablets
1662
Amidone Injection
1661
l-Amino-octane
Amidone Linctus
1661
Amiodarone Hydrochloride
Amidone Tablets
1662
Amiodarone Hydrochloride Tablets
803
Amiodarone Tablets
803
148,793
Amikacin
Amitraz
Amikacin Injection
794
Amikacin Sulphate
148,793
Amitraz Dip Concentrate, Liquid
794
Amikacin Sulphate Injection

Amiloride Hydrochloride
148,265,795
Amitriptyline Hydrochloride
148,802
148,265,2633
2634
2635
148, 266, 804
Amiloride Hydrochloride Tablets
796
Amitriptyline Hydrochloride Tablets
805
Amiloride Tablets
796
Amitriptyline Tablets
805
Amines, Primary Aromatic, Tests for
71
1426
Aminoacetic Acid
Amla Juice Powder

Amlodipine Besylate
462,497,2472
148,266, 806
4-Aminoantipyrine
565
4-Aminobenzoic Acid
565
Amlodipine Besylate and Losartan Potassium

and Tablets
p-Aminobenzoic Acid
565
Amlodipine Besylate Tablets
807
N-( 4-Aminobenzoyl)-L-glutamic Acid
565
Amlodipine Tablets
807
Aminocaproic Acid
148,797
S-Amlodipine Besylate
1609
148,267,808
809
Aminocaproic Acid Injection
798
S-Amlodipine Besylate Tablets
Aminocaproic Acid Tablets
798
S-Amlodipine Tablets
809
2-Amino-5-chlorobenzophenone
565
Ammonia
566
7-Aminodesacetoxycephasporanic Acid
565
Ammonia-Ammonium Chloride Buffer
560
4-Amino-2,3-dimethyl-l-phenyl-5-pyrazolone
565
Ammonia-Ammonium Chloride Solution, Strong
565
4-(2-Aminoethyl)phenol
620
Ammonia Buffer pH 9.5
561
2-Amino-2(hydroxymethyl)-1,3-propanediol
620
561
4-Amino-3-hydroxynaphthalene-l-sulphonic Acid
565
561
Aminohydroxynaphthalenesulphonic Acid Solution
565
Ammonia-Cyanide Solution Sp
565
565
a-Amino-b-mercaptopropionic Acid Hydrochloride
577
Ammonia-Cyanide Wash Solution
Amino-2-napthol-4-sulphonic Acid
565
Ammonia-free Water
620
4-Aminophenazone
565
Ammonia Solution, Sp.
566
4-Aminophenazone Solution
565
Ammonia Solution, 18 M
566
4-Aminophenol
565
Ammonia Solution, Dilute
566
p-Aminophenol
565
Ammonia Solution, Iron-free
566
Ammonia Solution Sp., Dilute
566
4-Aminophenol-free Paracetamol
~Aminophylline
~---148,799
. AmmoniaSolution;Strong---------~-~------$66
.A:mmonia, 1 8 M - - - - - - ---566
Aminophylline Injection
800
Aminophylline Tablets
801
Ammonia, x M
565
2-Aminopropane
590
Ariunonia, x M Ethanolic
566
2760
INDEX
IP 2010
Ammonia, x M Methanoloic
566
Ammonium Oxalate
567
Ammoniacal Ethanol, x M
566
Ammonium Oxalate, 0.1 M
567
Ammoniacal Magnesium Sulphate Solution
592
Ammonium Oxalate Solution
567
Ammoniacal Methanol, x M
566
Ammonium Peroxodisulphate
567
Ammoniacal Silver Nitrate Solution
609
Ammonium Persulphate
567
Ammoniated Ruthenium Oxychloride
627
Ammonium Phosphate
567
Ammonium Acetate
566
Ammonium Phosphate, Dibasic
567
Ammonium Acetate, 0.1 M
566
Ammonium Phosphate, Dibasic, 0.2 M
567
Ammonium Acetate Solution
566
Ammonium Polysulphide Solution
567
Ammonium Carbonate
566
Ammonium Pyrrolidinedithiocarbamate
567
Ammonium Carbonate, 2 M
566
Ammonium Pyrrolidinedithiocarbamate Solution
567
Ammonium Carbonate Solution
566
Ammonium Reineckate
567
Ammonium Ceric Nitrate
573
Ammonium Reineckate Solution
567
Ammonium Ceric Nitrate, 0.1 M
631
Ammonium Salts, Tests for
Ammonium Ceric Sulphate
573
Ammonium Standard Solution (1 ppm NH4)
628
Ammonium Ceric Sulphate, 0.1 M
631
Ammonium Sulphamate
567
Ammonium Cerium(IV) Nitrate
573
Ammonium Sulphate
567
Ammonium Cerium(IV) Sulphate
573
Ammonium Sulphide Solution
567
Ammonium Tetramethylenedithiocarbamate
567
Ammonium Chloride
148, 566, 810
71
Ammonium Chloride, 2 M
566
Ammonium Chloride-Ammonium Hydroxide Solution
566
Ammonium Tetrathiocyanatodiaminochromate(II1)
Monhydrate
567
Ammonium Chloride Solution
566
Ammonium Thiocyanate
568
Ammonium Chloride Solution, Dilute
566
Ammonium Thiocyanate, 0.1 M
631
Ammonium Chloride Solution (Nessler's)
566
Ammonium Thiocyanate Solution
568
Ammonium Chloride Solution, Dilute (Nessler's)
566
Ammonium Thiocyanate, xM
568
Ammonium Citrate Solution
566
Ammonium Thioglycollate Solution
568
Ammonium Citrate Solution,Alkaline
566
Ammonium Vanadate
Ammonium Citrate Solution Sp
567
Amodiaquine Hydrochloride
Ammonium Dihydrogen Phosphate
567
Amodiaquine Hydrochloride Tablets
Ammonium Iron(II) Sulphate
584
Amodiaquine Tablets
Ammonium Iron(II) Sulphate,O.l M
632
Amorph.I.Z.S
1502
Ammonium Iron(II1) Sulphate
584
Amorphous Wax
1696
Ammonium Iron(II1) Sulphate,O.l M
632
Amoxicillin and Potassium Clavulanate Injection
817
Ammonium Mercaptoacetate Solution
568
Ammonium Mercurithiocyanate Solution
567
Amoxicillin and Potassium Clavulanate Oral

Suspension
818
Ammonium Metavanadate
568
Amoxicillin and Potassium Clavulanate Tablets
819
Ammonium Molybdate
567
Amoxicillin Capsules
813
Ammonium Molybdate Solution
567
Amoxicillin Injection
814
568
148,810
8Il
8Il
Ammonium Molybdate Solution, Ethanolic
567
Amoxicillin Sodium
267
Ammonium Molybdate-Sulphuric Acid Solution
567
Amoxicillin Sodium Injection
814
Ammonium Nitrate
567
Amoxicillin Oral Suspension
815
Ammonium Nitrate, 0.007 M
567
Amoxicillin Dispersible Tablets
Ammonium Phosphate Monobasic
567
Amoxicillin Trihydrate
817
268,816
Volume 1: ito xxiii and 1 to 704; Volume 2: xxv to xxviii and 705 to 1722; Volume 3: xxix to xxxii and 1723 to 2829
2761
INDEX
IP 2010
Amoxicillin Trihydrate Capsules
813
Anaesthetic Ether
Amoxyicillin Capsules
813
Amoxycillin and Potassium Clavulanate Injection
817
Analytical Procedures, Validation of

Anantmu1a
Amoxycillin and Potassium Clavulanate Oral

Suspension
818
Amoxycillin and Potassium Clavulanate Tablets
819
Andersen Cascade Impactor, Apparatus
Amoxycillin Oral Suspension
815
Amoxycillin Injection
814
Andrographis paniculata
Aneurine Hydrochloride
2513
2209
Aneurine Hydrochloride Injection
2210
Aneurine Hydrochloride Tablets

Anhydrous Acetic Acid
2211
563
Anastrozole
Amoxycillin Sodium
148,812
Amoxycillin Sodium Injection
814
Amoxycillin Dispersible Tablets
817
Amoxycillin Trihydrate
149, 816
156,1305
198
463,499,2474
149,271,830
Anastrozole Tablets
831
733
Anllydrous Alumina
564
Amoxycillin Trihydrate Capsules
813
Anhydrous Aluminium Chloride
565
Amoxycillin Trihydrate Dispersible Tablets
817
Anhydrous Aluminium Oxide

Anhydrous Calcium Chloride
564
571
Amphotericin B
149,268,820
Amphotericin B Injection
821
Anhydrous Citric Acid
575
Amphotericin B Injection, Liposomal
822
Anhydrous Dextrose
577
,A..nhydrous Dibasic Sodium Phosphate
613
Anhydrous Disodium Hydrogen Orthophosphate

Anhydrous Disodium Hydrogen Phosphate
581
581
Anhydrous Ferric Chloride
584
Anhydrous Formic Acid
585
Anhydrous Glacial Acetic Acid

Anhydrous Iron(III) Chloride
563
584
Ampicillin
149,269,822
Ampicillin and Cloxacillin Benzathine Intramammary

Infusion (Dry Cow/Buffalo)
2637
Ampicillin and Cloxacillin Intramammary Infusion

(Lactating Cow/Buffalo)
2636
Ampicillin Capsules
Ampicillin Dispersible Tablets
Ampicillin Injection
Ampicillin Oral Suspension
Ampicillin Sodium
Ampicillin Sodium and Cloxacillin Sodium
Intramammary Infusion (LC/B)
Ampicillin Sodium Injection
823
828
825
827
149,269,824
2636
825
Ampicillin Trihydrate
149,270,828
Ampicillin Trihydrate Veterinary Oral Powder
2638
2638
Amprolium, Ethopabate and Sulphaquinoxaline
Premix
2641
Amprolium Hydrochloride
149,270,2639
Amprolium Hydrochloride and Ethopabate Premix
2640
Amprolium Hydrochloride, Ethopabate and
2641
Amra
462,498,2473
------------------ -----2503
AImita
. 568
Ai'tly1 Acetate
Amyl Alcohol
568
Amylase, Alpha
149
Anhydrous Lactose
1554
Anhydrous Lanolin
2317
Anhydrous Magnesium Perchlorate
592
Anhydrous Methanol
594
Anhydrous Niclosamide
1773
Anhydrous Potassium Carbonate
603
Anhydrous 2-Propanol
606
Anhydrous Propan-2-o1
606
Anhydrous Pyridine
606
Anhydrous Silica Gel
607
Anhydrous Silicon Dioxide
609,2099
Anhydrous Sodium Acetate
609
Anhydrous Sodium Carbonate
610,630
Anhydrous Sodium Phosphate
581,613
Anhydrous Sodium Sulphate

613
Anhydrous- -SodiumSulphite-------- ------------ -------------61-3
Anhydrous T o l u e n e 6 1 8
Aniline
568
Anion Exchange Resin, StroJ:iglYBasic
568
2762
INDEX
IP 2010
Anionic Emulsifying Wax
1274
149,834
Arginine
834
Anisaldehyde
568
L-Arginine
Anisaldehyde Solution
568
Arjuna
463,500,2476
Anisaldehyde Solution, Ethano1ic
568
Arjuna Dry Extract
464,501,2477
Anisaldehyde-Sulphuric Acid Reagent
568
Arsenic Compounds, Test for
71
Anthracene
568
Arsenic, Limit Test for
80
Anthralin
1240
Arsenic Standard Solution (l ppm As)
628
2707
Arsenic Standard Solution (10 ppm As)
628
Anthrone
568
Arsenic Trioxide
Anti-A Blood Grouping Reagent
249
l-o-Arsonopheny lazo-2-naphthol-3-6-disulphonic Acid

618
Sodium Salt
2557
569,630
149,835
Anti A, B (Group 0) Blood Grouping Reagent
249
Arteether
Anti-B Blood Grouping Reagent
249
a-~ Arteether
835
149,271,836
2557
Arteemether
Anti-D (Rh) Immunoglobulin
2558
Artemisia
Anti-D Immunoglobulin for Intravenous Use
2559
Artemisia annua
Anti-D Immunoglobulin Human for Intravenous Use
2559
Artemisinin
149,837
Anti-Human GlobulinSerum
2560
Artesunate
149,272,838
Anti-gas-gangrene (Oedematiens) Serum
2391
Ascending Paper Chromatography
Anti-gas-gangrene (Perfringens) Serum
2392
Ascorbic Acid
Anti-gas-gangrene (Septicum) Serum
2393
L~Ascorbic
Anti-Rh Blood Group Serums
2560
Ascorbic Acid Injection
840
464,2478
2478
136
149,272,569,839
Acid
839
Anticoagulant Citrate Dextrose Solution
831
L-Ascorbic Acid Injection
840
Anticoagulant Citrate Phosphate Dextrose Solution
832
Ascorbic Acid Tablets
Anticoagulant Citrate Phosphate Dextrose

Adenine Solution
840
840
833
Antihaemophilic Fraction, Dried Human
L-Ascorbic Acid Tablets

2563
Ashwagandha
465,502,2479
465,503,2480
Antimicrobial Preservatives, Effectiveness of
27
Asparagus Racemosus Root
Antimony Compounds, Tests for
71
Aspartame
2540
149,841
Antimony Trichloride
568
Aspirin
Antimony Trichloride Reagent
568
Aspirin and Caffeine Tablets
844
Antimony Trichloride Solution
568
Aspirin Tablets
843
843
149,273,842
Antirabic Serum
3435
Aspirin Tablets, Soluble
Antirabies Serum
3435
Assays
Antisera
2349
Assay of Human Anti-D Immunoglobulin Method A
240
19
Assay of Human Anti-D Immunoglobulin Method B

andC
241
107
Assay of Human Coagulation Factor II
243
Apparatus
Apparatus, General Notices
Appearance of Solution
15,717
84
Aprotinin
569
Assay of Human Coagulation Factor VII
244
Aqueous Bromothymol Blue Solution
623
Assay of Human Coagulation Factor VIII
245
Aqueous Calamine Cream
954
Assay of Human Coagulation Factor IX
246
Arachidic Alcohol
569
Assay of Human Coagulation Factor X
247
Assay of Heparin in Coagulation Factors
248
Arachis Oil
569,2475
Volume 1: i to xxiii and I to 704; Volume 2: xxv to xxviii and 705 to 1722; Volume 3: xxix to xxxii and 1723 to 2829
2763
IP 2010
INDEX
Assay ofNitrous Oxide

Assay of Oxygen
Assay of Steroids
Assay ofVitamin A
Assay ofVitamin D
Atazanavir Capsules
Atazanavir Sulphate
Atazanavir Sulphate Capsules
Atenolol
89
90
93
94
96
845
149,844
845
149,273,847
Atenolol Tablets
848
Atomic Absorption Spectrometry
109
Atomic Emission Spectrometry
109
Atomic Weight of Elements, Names, Symbols and
701
Atorvastatin Calcium
149,274,849
850
Atorvastatin Calcium Tablets
Atorvastatin Tablets
850
Atropine Eye Ointment
854
Atropine Injection
853
Atropine Methonitrate
149,851
Atropine Sulphate
149,274,569,852
. Atropine Sulphate Eye Ointmen
854
Atropine Sulphate Injection
853
Atropine Sulphate Tablets
855
Atropine Sulphate and Morphine Sulphate Injection 1707
Atropine Tablets
855
Avian Infectious Bronchitis Vaccine, Inactivated
2707
Avian Infectious Bronchitis Vaccine, Live
2708
Avian Infectious Bronchitis Vaccine Living
2708
Avian Leucosis Viruses, Test for
Avian Live Virus Vaccines-Tests for Extraneous
228
Agents in Batches of Finished Products
2749
Avian Mycoplasma Antigen
Avian Reticuloendotheliosis Virus, Test for
223
2709
Avian Viral Vaccines- Tests for Extraneous
Agents in Seed Lot
Azadirachta indica
Azathioprine
Azathioprine Tablets
221
2524
149,855
856
Azithromycin
149,857
AzithromycinCapsules----------------------858
A:zithr6inyciiiOtal Suspehsi6fl
Azithromycin Tablets
860
861
Azo Violet
622
B
1229
B.A.L.
1229
B.A.L. Injection
2371
Bacillus Calmette-Guerin Vaccine (Freeze-Dried)
149,867
Bacitracin
149,868
Bacitracin Zinc
149,869
Bac10fen
870
Bac10fen Oral Solution
871
Bac10fen Tablets
2489
Bacopa monnieri
28
Bacterial Endotoxins
2546
Balsam ofTolu
569
Barbaloin
569
Barbital
569
Barbitone
561
Barbitone Buffer pH 7.4
561
Barbitone Buffer pH 8.6
561
Barbitone Buffer pH 8.6, Mixed
569
Barbitone Sodium
77
Barbiturates, Identification of
78
Barbiturates, Identification of Related Substance in
72
Barbiturates, Non-nitrogen Substituted, Test for
72
Barbiturates, Test for
569
Barium Chloride
631
Barium Chloride, 0.05 M
569
Barium Chloride Solution
569
Barium Hydroxide
569
Barium Hydroxide, 0.1 M
569
Barium Hydroxide Solution
873
Barium Meal
569
Barium Perchlorate
72
Barium Salts, Tests for
628
Barium Standard Solution (10 ppm Ba)
569,872
Barium Sulphate
873
Barium Sulphate Suspension
625
Basic Blue 9
592
Basic Fuchsin
622
Basic Green 1
592
Basic Magenta
.
--------------------------626
. BasicRed5
Basic Red 9
624
Basic Violet 3
607
Basic Violet 10
2764
INDEX
IP 2010
2548
Basil
Basis ofPhannacopoeia Requirements
149,275,873
Beclomethasone Dipropionate
Benzoic Acid
149,570,630,886
Benzoic Acid Ointment, Compound
887
Benzoic Acid Solution
888
Beclomethasone Dipropionate Inhalation
874
Benzoic and Salicylic Acids Ointment
887
Beclomethasone Inhalation Aerosol
874
Benzoin
888
Beclomethasone Inhalation
874
Benzoin Tincture, Compound
890
Benzophenone
570
601
Beef Heart Infusion Broth
213,220
Beeswax, White
149,875
Benzo[d]pyridazine
Beeswax, Yellow
149,876
4,4-(3H-2, 1-Benzoxathiol-3-ylidene)bis(2,6-
466,2481
Belladonna Leaf
Belladonna Tincture
Bentonite
2484
149,877
Benzalacetone
600
Benzaldehyde
569
Benzalkonium Chloride
877
Benzalkonium Chloride Solution
149,569,878
Benzathine Benzylpenicillin
879
Benzathine Benzylpenicillin Injection
880
Benzathine Benzylpenicillin Injection, Fortified
882
Benzathine Benzylpenicillin Tablets
883
Benzathine Penicillin
4,4'-(3H-2, 1-Benzoxathiol-3-ylidene)bis(6-
149,879
Benzathine Penicillin G
879
Benzathine Penicillin G Injection
880
Benzathine Penicillin G Tablets
883
Benzathine Penicillin Injection
880
Benzathine Penicillin Injection, Fortified
623
bromo-o-cresol)S,S-dioxide
466,504,2483
Belliric Myrobalan
622
dibromo-m-cresol)S,S-dioxide
2483
4,4'-(3H-2, 1-Benzoxathiol-3-ylidene)
623
bis(2,6-dibromophenol)S,S-dioxide
4,4'-(3H-2, I-Benzoxathiol-3-ylidene)bis(2623
bromothymol)S,S-dioxide
4,4'-(3H-2, 1-Benzoxathiol-3-ylidene)di623
o-cresol S,S-dioxide
4,4'-(3H-2, 1-Benzoxathiol-3-ylidene)-di625
m-cresol S,S-dioxide
3H-2, 1-Benzoxathiol-3-ylidene bis-(6-hydroxy-5isopropyl-2-methyl-m-phenylene) methylenenitrilo]

tetraacetic acid S,S-dioxide Tetrasodium salt
626
[3H-2, 1-Benzoxathiol-3-ylidene bis-(6-hydroxy-5methyl-m-phenylene)methylenenitrilo]tetraacetic

acid S,S-dioxide Tetrasodium salt
627
4,4'-(3H-2,1-Benzoxathiol-3-ylidene)diphenol
S,S-dioxide
626
882
4,4'-(3H-2,1-Benzoxathiol-3-ylidene) dithymol
S,S-dioxide
627
Benzathine Penicillin Tablets
883
Benzoyl Chloride
570
Benzene
569
Benzene-1,2-diol
573
N-Benzoyl-L-phenylalanyl-L-valyl-L-arginine
4-nitroanilide Hydrochloride
574
Benzene-1,3-diol
607
Benzene-I,3,5-triol
601
Benzene-I,4-diol
588
Benzethonium Chloride
569
Benzethonium Chloride,O.OO 4 M
631
Benzhexol Hydrochloride
149,275,884
Benzoylmetronidazole
1683
Benzoylmetronidazole Oral Suspension
1684
BenzylA1cohol
Benzyl Benzoate
570,891
150,570,892
Benzyl Benzoate Application
893
(RS)-(2-Benzyl-chloroethyl)-1-methyl-2phenoxyethylamine Hydrochloride
600
Benzhexol Hydrochloride Tablets
885
Benzhexol Tablets
885
Benzyldimethyl-2- {2-[4-(1,1 ,3,3-tetramethylbutyl)

phenoxyl]ethylammonium Chloride Monohydrate
569
Benzidine
569
Benzylpenicillin Injection
896
Benzoates, Tests for

Benzocaine
2,3-Benzodiazine
72
149,276,886
601
Benzylpenicillin Potassium
Benzylpenicillin Sodium
Berberis
150,893
150,570,895
2495,2496
2765
IP 2010
INDEX
Berberis aristata
Betahistine Hydrochloride
2495,2496
150,897
Biperiden Hydrochloride Tablets

Biperiden Tablets
914
914
Betahistine Hydrochloride Tablets
898
Biphasic Insulin Injection
1498
Betahistine Tablets
898
Biphasic Isophane Insulin Injection
1499
Betamethasone
Betamethasone Cream
Betamethasone Dipropionate
150,276,899
903
150,902
(1,1 '-Biphenyl)-4,4;-diamine
569
3,7-bis(dimethylamino)phenothiazin-5-ium chloride
625
Bisacodyl
150,915
Betamethasone Dipropionate Cream
903
BisacodylSuppositories
916
Betamethasone Dipropionate Lotion
904
Bisacodyl Tablets
917
Betamethasone Dipropionate Ointment
905
Bisbenzimide Stock Solution
212
212
Betamethasone Eye Drops
907
Bisbenzimide working solution
Betamethasone Injection
908
Bishop's weed
Betamethasone Lotion
904
Bismuth Carbonate
Betamethasone Ointment
905
Bismuth Compounds, Test for
Betamethasone Sodium Phosphate
150,906
2470
917
72
Bismuth Oxide Nitrate
570
Betamethasone Sodium Phosphate Injection
908
Bismuth Oxynitrate
570
Betamethasone Sodium Phosphate Tablets
Bismuth Subcarbonate
Betamethasone Tablets
909
900
Betamethasone Valerate
150,277,910
Betamethasone Valerate Ointment
911
Bhibhitaki
467,505,2484
Bhibbitaki Aqueous Extract
467,506,2485
Bhringraj
468, 507,2486
BHT
BhuiarnIa
Bicarbonates, Tests for
Bifonazole
Bifonazole Cream
Bile-Tolerant Gram-Negative Bacteria
Biological Assay of Gas-gangrene Antitoxin
(Oedematiens)
941
468, 508, 2488
72
150,912
912
43
2391
150,917
Bismuth Subnitrate
570
Bis(trimethyIsilyl)acetamide
570
N, 0-Bis(trimethylsily1)acetamide
570
Biuret
570
2710
Black pepper
2522
Blank Determinations
630
Bleaching Powder
573
-Bleomycin Injection
919
Bleomycin Sulphate
150,918
Bleomycin Sulphate Injection
919
Blood and Blood Components, Containers for
693
Blood and Blood Components, Sterile Plastic

Containers for
693

(Perfringens)
2392

(Septicum)
2393
Blood Cells, Concentrate ofHuman Red
2562
Biological Assay of Plague Vaccine
2423
Blood Grouping Serums, Anti-Rh
2560
Blood Grouping Serums Anti-D, Anti-C, Anti-E,

Anti-c, Anti-e
2560
2589
Blood and Blood-Related Products, Monographs

Blood and Blood-Related Products, Tests on
Biological Indicators
639
Biological Indicators, Type of
639
2555
235
Biological Methods
Biological Veterinary Monographs
25
2105
Blood, Whole Human
Biotechnology Derived Therapeutic Products
2593
Blue Tetrazolium
570
2591
Blue Tetrazolium Salt
570
Blue Tetrazolium Solution
570
Biotechnology Products, Monographs

Biperiden Hydrochloride
150,913
Blue Litmus Paper
2766
INDEX
IP 2010
Blue Tetrazolium Solution, Alkaline

Boerhaavia diffusa
Boiling Range or Teperature and Distillation Range
570
Bromocriptine Capsules
924
2532
Bromocriptine Mesylate
150,278,923
119
Bromocriptine Mesylate Capsules
924
Borate Buffer, Alkaline, pH range 8.0 to 10.0
560
Bromocriptine Mesylate Tablets
925
Borate Buffer pH 9.0
561
Bromocriptine Tablets
925
Borax
570
Bromomethyl2-Naphthyl Ketone
570
Borax,O.2M
570
Bromophenol Blue
623
150,570,921
Bromophenol Blue Reagent
623
Boric Acid
Boric Acid and Potassium Chloride, 0.2 M
559
Bromophenol Blue Solution
623
Boric Acid Solution
570
Bromophenol Blue Solution, Ethanoloic
623
Boric Buffer pH 9.0
561
Bromophenol Blue Solution, Strong
623
Boron Trifluoride Solution
570
Bromothymol Blue
623
Bovine Albumin
Bromothymol Blue Solution
623
Bovine Euglobulins
583
Bromothymol Blue Solution, Aqueous
623
Bovine Serum Albumin
564
Bronopol
564,570
150,927
Brahmi
469,509,2489
Brucella Abortus Coloured Antigen
2749
Brahmi Extract
469,510,2490
Brucella Abortus Milk Ring Test Antigen,

Hematoxylin Stained
2749
Brucell Abortus Mille Ring Test Antigen,

Tetrazolium-Stained
2750
Brucella Abortus Plain Antigen
2750
Brucella Abortus Rose Bengal Plate Test Antigen

(Strain 99)
2750
Brucella Abortus Working Standard Serum
2751
Brain Heart Infusion
214
Brilliant Green
622
Brilliant Green Solution
622
Brine
610
Bromhexine Hydrochloride
150,277,921
Brornhexine Hydrochloride Tablets
922
Brornhexine Tablets
922
Bromides, Test for
72
150,927
Buclizine Hydrochloride
Budesonide
150,278,928
Brominated Hydrochloric Acid AsT
587
Bromine
570
Bromine, 0.0167 M
570
Bromine, 0.05 M
631
Bromine Solution
570
Bromine Solution, Acetic
570
Bromine Water
570
Buffer type and concentration, Capillary

Electrophoresis for
178
570
Buffered Acetone Solution
560
4-Bromoaniline
570
Buffered Cupric Sulphate Solution pH 2.0
561
p-Bromoaniline
570
561
4 -Bromoaniline Solution
570
561
Bromocresol Blue
622
Buffered Palladium Chloride Solution
561
Bromocresol Green
622
Buffered Sodium Chloride-Peptone Solution pH 7.0
Bromocresol Green Reagent
622
Bumetanide
Bromocresol Green Solution
623
Bumetanide Injection
930
Bromocresol Purple
623
Bumetanide Oral Solution
931
Bromocresol Purple Solution
623
Bumetanide Tablets
932
Bromocresol Purple Solution, Phosphate-buffered
623
Bupivacaine Hydrochloride
(X-
Bromo-2-acetonaphatone
Buffer pH, Capillary Electrophoresis for
178
Buffer (HEPES) Solution pH 7.5
561
Buffer Solution pH 2.5
561
Buffer Solutions
559
Buffer Solutions, Standard
559
46
150,279,930
150,933
2767
IP 2010
INDEX
Bupivacaine Hydrochloride Injection

Bupivacaine Injection
Buprenorphine Hydrochloride
934
934
150,279,935
Cadmium Standard Solution (10 ppm Cd)

Cadmium Sulphate
Caffeine
Buprenorphine Hydrochloride Injection
936
Caffeine, Anhydrous
628
571
150,953
280
Buprenorphine Hydrochloride Tablets
937
Calamine
Buprenorphine Injection
936
Calamine Cream, Aqueous
954
Buprenorphine Tablets
937
Calamine Lotion
955
788
Calamine Ointment
955
150,938
Calamine Prepared
Burow's Solution
Buspirone Hydrochloride
Buspirone Hydrochloride Tablets
Buspirone Tablets
Busulphan
Busulphan Tablets
150,953
953
939
Calciferol
939
Calciferol Capsules
957
150,280,939
150,955
Calciferol Injection
958
940
Calciferol Oral Drops
958
1-Butaneboronic Acid
571
Calciferol Oral Solution
958
1,2-Butanediol
571
Calciferol Solution
958
2,3-Butanedione dioxime
579
Calciferol Tablets
1-Butanesulphonic Acid Sodium Salt
610
Calcitriol
I-Butanol
571
Calcitriol Capsules
961
Butan-l-ol
571
Calcium Acetate
571
2-Butanol
571
Calcium Aspirin Tablets
843
Butan-2-ol
571
2-Butanol Reagent
571
Calcium Carbonate
150,571,961
2-Butanone
571
Calcium Chloride
150,571,962
Butan-2-one
571
Calcium Chloride, Anhydrous
571
Butanoic Acid
571
Calcium Chloride Dihydrate
962
(Z)-But-2-ene-l ,4-dioic Acid
593
Calcium Chloride Dihydrate Injection
963
Butyl Acetate
571
Calcium Chloride Injection
963
n-Butyl Alcohol
571
Calcium Chloride Solution
571
sec-Butyl Alcohol
571
tert-Butyl Alcohol
595
n-Butyl Chloride
571
Butyl Hydroxybenzoate
942
Butylated Hydroxytoluene
1,3-Butylene Glycol
Butylparaben
150,571,941
571
150,942
Butylsilyl Silica Gel
607
Butylsilyl Silica Gel for Chromatography
607
Butyric Acid
571
n- Butyric Acid
571
Calcium Chloride, xM
Calcium Folinate
Calcium Folinate Injection
Calcium Gluconate
959
150,960
2642
571
150,963
964
151,571,965
Calcium Gluconate Injection
965
Calcium Gluconate Tablets
966
Calcium Hydrogen Phosphate
970
Calcium Hydroxide Phosphate
970
Calcium in Adsorbed Vaccines, Limit Test for

Calcium Lactate
Calcium Lactate Tablets
Calcium Levulinate Injection
Cadmium Iodide
571
Calcium Magnesium Borogluconate Injection
Cadmium Iodide Solution
571
Calcium Oxide
2768
80
151,967
967
.. 968
2642
571
INDEX
IP 2010
Calcium Pantothenate
151,969
970
Calcium Phosphate
Calcium Phosphate, Dibasic
151,970
Calcium Phosphate, Tribasic
151,970
Calcium Salt
Calcium Salts, Tests for
Carbamazepine Tablets
979
Carbazole
572
Carbenicillin Disodium
980
Carbenicillin Disodium Injection
981
971
Carbenicillin Injection
981
72
Carbenicillin Sodium
151,282,980
Calcium Standard Solution (10 ppm Ca)
629
Carbenicillin Sodium Injection
981
Calcium Standard Solution (100 ppm Ca), Ethanolic
629
Carbenoxolone
282
Calcium Stearate
151,971
Carbenoxolone Sodium
Calcium Sulphate
151,572
Carbenoxolone Sodium Tablets
982
Carbenoxolone Tablets
982
Calcium Sulphate Dihydrate
572
. Calcium Sulphate Dried
1928
Calcium Sulphate Exsiccated
1928
Calcium Sulphate Solution
572
Calcon
623
Calcon Mixture
623
Calculation ofResults, General Notices
15,717,1735
Camphor
572
dl-1 O-Camphorsulphonic Acid
572
Candida albicans
46
151,981
Carbidopa
151,283,983
Carbimazole
151,283,984
985
Carbimazole Tablets
1893
Carbolic Acid
Carbomers
151,572,986
Carbomer 934P
572
. Carbon Dioxide
572
Carbon Dioxide-free Water

Carbon dioxide detector tube
620
21
Canine Contagious Hepatitis Vaccine, Inactivated
2710
Canine Contagious Hepatitis Vaccine, Live
2711
Canine Corona Virus Vaccine, Inactivated
2711
Carbon monoxide detector hlbe
2712
Carbon Tetrachloride
572
Canine Leptospirosis Vaccine, Inactivated
2713
Carbonate Buffer pH 9.7
561
Canine Parainfluenza Virus Vaccine, Live
2713
Carbonates, Tests for
Canine Parvovirus Vaccine, Inactivated
2714
Carbonylurea
Canine Parvorirus Vaccine, Live
2715
Carboprost Tromethamine
Capacity Factor, Liquid Chromatography
134
Carbon Disulphide
572
21
73
988
Carboprost Tromethamine Injection
989
615
615
281,972
4-Carboxybenzene Sulphonamide
Capecitabine Tablets
973
p-Carboxybenzene Sulphonamide
Capillary Electrophoresis
177
"{-Carboxylation and ~-hydroxylation ofProteins
Capillary Gel Electrophoresis
178
3-Carboxy-4-nitrophenyl Disulphide
581
Capecitabine
.2598
Capillary Isoelectric Focusing
179
Capillary Zone Electrophoresis
177
N-[9-(2-Carboxyphenyl)-6-(diethylamino )-3Hxanthen-3-ylidene]-N-ethylethanaminium Chloride
607
Capreomycin SUlphate
974
9;-(o-carboxypheny1)-6-hydoxy-3H-xanthen-3-one
584
Capreomycin Injection
975
Carboxymethylcellulose
572
Caprylic Acid
598
Carboxymethyl Cellulose, Sodium
989
Capsules
721
Captopril
151,281,976
Carboxymethylcellulose Sodium and

Microcrystalline Cellulose
Captopril Tablets
Carbamazepine
Carbamazepine Extended-release Tablets
977
151,977
978
Carboxypolymethylene
Carica papaya
Carmellose Sodium
2769
1695
572
2528
989
INDEX
IP 2010
Camauba Wax
151,990
Cefixime
Carvedilol
151,991
Cefixime Oral Suspension
Carvedilol Tablets
992
Cefixime Tablets
Casein
572
Cefoperazone Injection
Casein Hydrolysate
572
Cefoperazone Sodium
Casein, Purified
572
Cefoperazone Sodium Injection
Casein Solution
573
Cefotaxime Injection
Casein Soya bean Digest Agar Medium
46
Cefotaxime Sodium
Casein Soya been Digest Broth Medium
46
Cefotaxime Sodium Injection
Cassia angustifolia
2537
Cassia Leaf
2537
Castor Oil
151,2490
Castor Oil, Hydrogenated
158
Castor Wax
2511
Cefpodoxirne Proxetil
151,1018
1020
Cefpodoxime Tablets
Ceftazidime for Injection
Ceftazidime Injection
Ceftfiaxone Sodium
Ceftriaxone Injection
615
1017
Cefpodoxime Proxetil Tablets
573
Cation-Exchange Resin, Styrene-Divinlybenzene,

Strongly Acidic
1017
151,1016
1019
573
614
1016
1019
Catechol Solution
Cation-Exchange Resin, Styrene-Divinlybenzene
1013
1016
151,1014
Cefpodoxime Oral Suspension
Catechol
14,716,1734
1012
Cefpodoxime Proxetil Oral Suspension
Ceftazidime
Category, General Notice
151,1012
Cefuroxirne Axetil
1020
151,1021
1022
1023
151,286,1024
1025
151,286,1026
Catkins (Big)
2530
CefuroximeAxetil Tablets
1027
Catkins (Small)
2531
Cefuroxime Injection
1029
Caustic Potash
604
Cefuroxirne Sodium
Caustic Soda
Cefaclor
2124
151,284,993
Cefuroxime Sodium Injection
151,1028
1029
Cellacefate
1030
Cefaclor Capsules
994
Cellacephate
1030
Cefaclor Oral Suspension
995
Cellulose, 2-Hydroxypropyl Ether
1464
Cellulose, 2-Hydroxypropylmethyl Ether
1465
Cefaclor Sustained-release Tablets

Cefadroxil
Cefadroxil Capsules
997
151,998
999
Cellulose Acetate Electrophoresis

Cellulose Acetate Phthalate
124
151,1030
Cefadroxil Mixture
1000
Cellulose Ethyl Ether
1313
Cefadroxil Monohydrate
284
Cellulose Methyl Ether
1667
Cefadroxil Oral Suspension
1000
Cellulose, Microcrystalline
573
Cefadroxil Tablets
1001
Cell Fixation Buffer
241
1004
Cell Substrates for the Production of Vaccines

for Human Use
207
Cefamandole Injection
Cefamandole Nafate
Cefamandole Nafate Injection
Cefazolin Injection
Cefazolin Sodium
. ... eefazolinSodium Injection
Cefepiirie Hydrochloride
Cefepime Hydrochloride Injection
Cefepime Injection
151,285,1003
1004 Centchroman
1007 Centchroman Hydrochloride
151,285,1005 CeIitchroman Hydrochloride Tablets
---1007 Gentchroman.Tablets
151;1008 Centella asiatica
1010 Cephalexin
1010 Cephalexin Capsules
2770
1821
1821
1822
_--1822
2520
151,287,1031
1032
IP 2010
Cephalexin Dry Syrup

Cephalexin Mixture
Cephalexin Oral Suspension
Cephalexin Tablets
Cephaloridine
Cephaloridine (<x-form)
Cephaloridine (5-form)
Cephaloridine Injection
Cephazolin Injection
Cephazolin Sodium
Cephazolin Sodium Injection
Ceric Ammonium Nitrate
CericAmmonium Nitrate, 0.1 M
Ceric Ammonium Sulphate
Ceric Ammonium Sulphate, 0.1 M
Ceric Sulphate
Cerium(ill) Nitrate
Cerium Sulphate
Cerous Nitrate
Cerous Nitrate Solution
Cetirizine Hydrochloride
Cetirizine Hydrochloride Tablets
Cetirizine Oral Liquid
Cetirizine Syrup
Cetrizine Tablets
Cetostearyl Alcohol
Cetrirnide
Cetrimide Agar Medium
Cetrimide Cream
Cetyl Alcohol
Cetyl Palmitate
Cetyltrimethylammonium Bromide
Changes
Changed Titles of Monographs
Characteristics of Biological Indicators
Charcoal, Activated
Charcoal, Decolorising
Chebulic Myrobalan
Chemical Formulae, General Notices
Chemical Methods
Chicken Flocks free from Specified Pathogens
for the Production and Quality Control of
Vaccines, Test on
Chicken kidney cell, Test in
INDEX
1033
1033
1033
1034
151,1035
287
288
1036
1007
1005
1007
573
631
573
631
573
573
573
573
573
152,288,1037
1039
1039
1039
1039
152,1040
152,573,1041
48
1041
152,1042
152,573,1042
573
XVll
xxiii
642
152,573,1043
573,1043
2508
13,715,1733
69
216
222
Chicken infection anaemia (CIA) Virus, Test for

223
Chloral Hydrate
152,573,2643
Chloral Hydrate Solution
Chlorambucil
573
152,1044
Chlorambucil Tablets
1045
Chloramine
573
Chloramine Solution
573
Chloramine T
Chloramine T Solution
573
573
Chloramphenicol
Chloramphenicol Capsules
152,289,1046
1047
Chloramphenicol Eye Drops
1047
Chloramphenicol Eye Ointment
1048
2644
Chloramphenicol Oral Suspension

Chloramphenicol Palmitate
1050
152,1049
Chloramphenicol Palmitate Mixture
1050
Chloramphenicol Palmitate Oral Suspension
1050
Chloramphenicol Sodium Succinate

Chloramphenicol Sodium Succinate Injection
152,1051
1052
CWorazam
619
Chlorbutol
Chlorcyclizine Hydrochloride
152,1053
1053
Chlordiazepoxide
Chlordiazepoxide Tablets
152, 1054
. 1055
Chlorhexidine
289
Chlorhexidine Acetate
1056
Chlorhexidine Diacetate
Chlorhexidine Gluconate Solution
152
152, 1057
Chlorhexidine Hydrochloride
152, 1058
Chloride Buffer pH 2.0
561
Chloride Standard Solution (5 ppm Cl)
629
Chloride Standard Solution (25 ppm CI)
629
Chlorides, Limit Test for
80
Chlorides, Tests for
73
Chlorinated Lime
Chlorinated Lime Solution
573
573
Chlorine
573
Chlorine Solution
573
4'-Chloroacetanilide
4-Chloroaniline
573
573
p-Chloroaniline
573
4-Chlorobenzenesulphonamide
573
Volume 1: i to xxiii and I to 704; Volume 2: xxv to xxviii and 705 to 1722; Volume 3: xxix to xxxii and 1723 to 2829
2771
IP 2010
INDEX
574
4-Chlorobenzoic Acid
l-Chlorobutane
571
Chlorobutanol
Chlorocresol
8-Chloro-6-(2-chlorophenyl)-1-methyl-4H-[1,2,4]
. triazolo[4,3-a][1,4] benzodiazepine
1053
152,1059
Chlorpheniramine Tablets
Chlorpromazine
Chlorpromazine Hydrochloride
1071
291
152,292,1072
Chlorpromazine Hydrochloride Injection
1073
Chlorpromazine Hydrochloride Tablets
1073
619
ChlorpromaZine Injection
1073
4-Chloro-o-cresol
574
l-Chloro-2,4-dinitrobenzene
574
Chlorpromazine Tablets
Chlorpropamide
Chloroform
152,574,1060
1073
152,292,1074
Chloroform, Ethanol-free
574
Chlorpropamide Tablets
Chloroform IR
574
Chlortetracyline Hydrochloride Veterinary Oral Powder 2646
Chloroform, Prepared
574
Chlortetracyline Soluble Powder
Chlorofonn Water
574
Chlortetracyline Veterinary Oral Powder
Chloroguanide Hydrochloride
1977
Chlorthalidone
Chloroguanide Hydrochloride Tablets
1977
Chlorthalidone Tablets
1075
152,2645
2646
2646
152,293,1076
1077
5-Chloro-8-hydroxyquinoline
574
Chorionic Gonadotrophin
4-Chloro-2-methylphenol
574
Chorionic Gonadotrophin Injection
2-Chloro-4-nitroaniline
574
Chromatograms ofHerbs (LiquidlHPTLC/ Gas)
493
Chloroplatinic Acid
574
574
Chromatographic Siliceous Earth
608
Chromatography, Gas
127
Chromatography, Liquid
Chromatography, Paper
129
135
Chromatography, Size-Exclusion
Chromatography, Thin-Layer
136
137
Chromic Acid Solution
574
Chromic-Sulphuric Acid
574
Chromic-Sulphuric Acid Mixture

Chromium Trioxide
574
574
Chromogenic Substrate
574
Chromotropic Acid
574
Chromotropic Acid Sodium Salt
574
Chloroplatinic Acid Solution

Chloroquine
Chloroquine Phosphate
Chloroquine Phosphate Injection
Chloroquine Phosphate Suspension
Chloroquine Phosphate Tablets
Chloroquine Sulphate
Chloroquine Sulphate Injection
Chloroquine Sulphate Tablets
Chloroquine Syrup
5-Chloroquinolin-8-ol
5-Chlorosalicylic Acid
290
152,1061
1062
1063
1063
152,1064
1065
1066
1066
574
574
Cholecalciferol
Choline Chloride
152,1077
574
Chromotropic Acid Solution

Chymotrypsin
Chlorothiazide
152,1067
Ciclesonide
Chlorothiazide Oral Suspension
1068
Ciclesonide Inhalation
Chlorothiazide Tablets
Chlorotrimethylsilane
1068
619
Cilastatin Ammonium
Cilastatin Sodium
Chloroxylenol
Chloroxylenol Solution
Ghlorpheniraminelnjection
Chlorpheniramine Maleate
152,290,1069 'Cimetidine
1069 Cimetidine Tablets
. 152;291;1070
152, 1079
1080
575
152,1081
152,293,1083
1084
294
152,294,1084
152,295,1086
1087
Cineole, Assay for
-84
Chlorpheniramine Maleate Injection
1071
Cinnamaldehyde
575
Chlorpheniramine Maleate Tablets
1071
Cinnamic Aldehyde
575
2772
IP 2010
Cinnarizine
Cinnarizine Tablets
Ciprofloxacin
Ciprofloxacin Eye Drops
Ciprofloxacin Hydrochloride
Ciprofloxacin Hydrochloride Tablets
Ciprofloxacin Injection
Ciprofloxacin Tablets
Cisplatin
Cisplatin Injection
Citalopram Hydrobromide
Citalopram Hydrobromide Tablets
Citalopram Tablets
Citrate-Cyanide Wash Solution
Citrated Rabbit Plasma
Citrates, Tests for
Citrate Buffer
Citric Acid
Citric Acid, Anhydrous
Citric Acid, 0.1 M
Citric Acid, Iron-free
Citric Acid, Monohydrate
Citric-Molybdic Acid Solution
Citro-phosphate Buffer pH 5.0
Clarithromycin
Clarithromycin Tablets
Clarity ofSolution
Cleaning ofGlassware
Clindamycin Capsules
Clindamycin Hydrochloride
Clioquinol
Clioquinol Cream
Clioquinol Ointment
Clioquinol Tablets
Clioquinol and Hydrocortisone Cream
Clioquinol and Hydrocortisone Ointment
Clobazam
Clobazam Capsules
Clofazimine
INDEX
152,1088
1089
152,295,1090
1094
153,296,1092
1094
1091
1094
153,1095
1096
153,1097
1098
1098
575
602
73
561
153,575,1099
575
575
575
153,1100
575
561
561
561
561
561
153,296,1101
1102
107
639
1104
153,297,1103
153,2027
2028
2029
2030
2031
2032
153,1105
1106
153,297,1107
Clofazimine Capsules
Clomifene Citate
Clomifene Citrate Tablets
Clomifne Tablets
Clomiphene Citate
Clomiphene Citrate Tablets
Clomiphene Tablets
Clomipramine Capsules
Clomipramine Hydrochloride
Clomipramine Hydrochloricde Capsules
Clomophene Tablets
Clonazepam
1108
153,1108
1110
1110
1108
1110
1110
153,1111
1110
1111
111O
153,1112
1113
1114
153,298,1115
Clonidine Hydrochloride
1116
Clonidine Hydrochloride Injection
1117
Clonidine Hydrochloride Tablets
1116
Clonidine Injection
1117
Clonidine Tablets
153,1117
Clopidogrel Bisulphate
1119
Clopidogrel Bisulphate Tablets
1119
Clopidogrel Tablets
691
Closures for Containers of Parenteral Products
Clostridia
46
Clonazepam Injection
Clonazepam Tablets
Clostridium novyi (Type B) Vaccine for

Veterinary Use
2716
Clostridium Perfringens Vaccine, Inactivated
2717
Clostridium Septicum Vaccine, Inactivated
2719
153,298,1120
Clotrimazole
1121
Clotrimazole Cream
1122
Clotrimazole Pessaries
Clotrimazole Vaginal Tablets
Clotting Factor V Solution

Clove Oil
Cloxacillin Benzathine
1122
575
153,2491
153,299,2647
Cloxacillin Benzathine Intramammary

Infusion (Dry CowlBuffalo)
2648
2648
Cloxacillin Benzathine Intramammary Innjection
1124
Cloxacillin Capsules
1125
Cloxacillin Elixir
1125
Cloxacillin Injection
Cloxacillin Intramammary Infusion (Dry CowlBuffalo) 2648
2648
Cloxacillin Intramammary Infusion (DCIB)
2773
IP 2010
INDEX
2649
Cloxacillin Intramammary Injection

Cloxacillin Intramammary Infusion
(Lactating CowlBuffalo)
Codeine Syrup
Cloxacillin Intramammary Infusion (LCIB)

Cloxacillin Oral Solution
Cloxacillin Sodium
Cloxacillin Sodium Capsules
Cloxacillin Sodium Elixir
Cloxacillin Sodium Injection
2649
2649
1125
153,299,1123
1124
1125
1125
Cloxacillin Sodium Intramammary

Infusion (Lactating CowlBuffalo)
Cloxacillin Sodium Syrup
Cloxacillin Syrup
Clozapine
Clozapine Tablets
572
CMC
752
1576
2690
2692
2692
2691
2265
2265
2266
575
575
575
575
575
575
575
575
575
575
Coated Tablets
Co-careldopa Tablets
Co-trimazine Injection
Co-trimazine Oral Suspension
Co-trimazine Mixture
Co-trimazine Veterinary Oral Powder
Co-trimoxazole Mixture
Co-trimoxazole Oral Suspension
Co-trimoxazole Tablets
Cobalt Acetate
Cobalt Chloride
Cobalt Chloride Solution
Cobalt Nitrate
Cobalt Thiocyanate Solution
Cobalt(II) Acetate
Cobalt(II) Chloride
Cobalt(II) Nitrate
Cobaltous Acetate
Cobaltous Chloride
Cobaltous Chloride Colorimetric Solution (CCS)
Coconut Oil
Godeine Phosphate
Codeine Phosphate Hemihydrate
Codeine Phosphate Syrup
Codeine Tablets
Colchicine
Colchicine and Probenecid Tablets
Colchicine Tablets
Coleus
Coleus forskohlii
Coleus Dry Extract
Colloidal Anhydrous Silica
2649
1125
1125
1125
153,1126
1127
Cloxacillin Sodium Oral Solution
Cobaltous Nitrate
Codeine Phosphate Tablets
107
575
2492
.. 153;576,+128
1128
1129
Colloidal Silicon Dioxide
1130
1129
1130
153,300,576,1130
1132
1131
470,511,2492
2492
470,512,2493
2099
167,2099
Colony-fonning Units (CFU), Test for

Colour of Solution
Colourless Solutions
Columbia Agar Medium
Column Efficiency, Liquid Chromatography for
COlmniphora wightii
34
107
108
48
134
2504
Complete Extraction ofAlkaloids
201
Composition of Polysaccharide Vaccines

Compound Benzoic Acid Ointment
205
887
Compound Benzoin Tincture
890
Compound Sodium Chloride and Dextrose Injection

Compound Soidum Chloride and Dextrose
Intravenous Infusion
2115
Compound Sodium Lactate and Dextrose Injection
2115
2117
2117
2125
Compound Sodium Lactate and Dextrose

Injection, Half Strength
2127

Compound Sodium Lactate and Dextrose

Injection, Modified
Compound Sodium Lactate Intravenous Infusion
2128
2129
2129
Compound Sodium Lactate with Dextrose

2125
Compund Sodium Lactate with Dextrose

Intravenous Infusion, Half Strength
2127
Compound Sodium Lactate with Dextrose

Intravenous Infusion, Modified
Compound Sodium Lacate Solution for Irrigation
Concentrate ofHuman Red Blood Cells
Concentrated Glyceryl Trinitrate Solution
Concentrated Human Red Blood Corpuscles
2774
2128
2130
2562
1424
2562
INDEX
IP 2010
Concentrated Hydrochloric Acid
1450
Cortisone Acetate Tablets
1135
Concentrated Nitroglycerin Solution
1424
Cortisone Injection
1134
Concentrated Phosphoric Acid
1909
Cortisone Tablets
1135
Concentrate Platelet
2588
Cotton, Absorbent
1137
Cotton Lint
1593
Concentrated Solutions for Injection
749
Concentrated Solution Erythropoietin
2602
Cotton Wool, Absorbent
1137
Concentrated Solution Filgrastim
2610
Covalent modification of Therapeutic Proteins
2598
Concentrated Solution Interferon Alfa-2
2613
CPD Solution
832
Concentrated Vitamin A and D Solution
2308
CPDA Solution
833
Concentrated Vitamin D Solution
2308
Cream of Magnesia
119
Creams, see also under name ofsubstance
Congealing Range or Temperature
121
Cresol
Congo Red
623
Cresol Red
Congo Red Fibrin
623
Cresol Red Solution
Conductivity
1622
723
153,576, 1138
623
624
Cresol with Soap Solution
153,1139,-
Containers for Blood and Blood Components
693
Croscarmellose Sodium
153,1139
Containers for Pharmaceutical Products
683
Crospovidine
153,1141
Contents of packaged Dosage Forms
193
Cryoprecipitated Antihaemophilic Factor
2562
691
Crude Herbs
2467
23
Cryst. I.Z.S.
1503
681,683,744
Containers
Containers of Parenteral Products, Closures for

Continuous Extraction of Drugs
Coomassie Staining Solution
576
Crystal Violet
624
624
Copper
576
Crystal Violet Solution
Copper Cliloride-Pyridine Reagent
576
Crystalline Insulin
Copper Foil
576
Culture Media
Copper Solution, Alkaline
576
Cupri-Citric Solution
576
Copper Standard Solution
629
Cupric Acetate
576
Copper Standard Solution (10 ppm Cu)
629
Cupric Chloride
576
Copper Sulphate Solution
576
Cupric Chloride-Pyridine Reagent
576
Copper Turnings
576
Cupric Sulphate
576
Copper(I) Chloride
576
Cupric Sulphate, 0.02 M
Copper(II) Acetate
576
Cupric Sulphate Colorimetric Solution (CSS)
Copper(II) Chloride
576
Cupric Sulphate Solution
576
Copper(II) Sulphate
576
Cupric Sulphate Solution, Dilute
576
Copper-Citric Solution
576
Cupric Sulphate Solution pH 2.0, Buffered
561
Corainder Oil
1496
57,210
576,631
107
2494
561
561
Cortisol
1453
Cortisol Acetate
1454
Cupric Sulphate Solution, Weak
576
Cortisol Acetate Eye Ointment
1455
Cupric Sulphate with Pyridine Solution
576
Cortisol Acetate Injection
1456
Cupri-Tartaric Solution
576
Cortisol Hydrogen Succinate
1457
Cupri-Tartrate Solution, Alkaline
603
Cortisol Sodium Succinate Injection
1459
Cuprous Chloride
576
Cortisone Acetate
Cortisone Acetate Injection
153,301,1133
1134
Curcuma longa
Curcumin
Volume 1: i to xxiii and 1 to 704; Volume 2: xxv to xxviii and 705 to 1722; Volume 3: xxix to xXxii and 1723 to 2829
2775
2507
576
IP 2010
INDEX
Cyanocobalamin
153,1142
Cyanocobalamin Injection
D&CRedNo.19
607
578
DeaCtivated Alumina
576
Deactivated Aluminium Oxide
564
564
l-Decanol
577
1143
(2-Cyanoethyl)ether
Cyanogen Bromide Solution
153,301,1144
Cyclizine Hydrochloride
1165
Daunorubicin Injection
Cyclizine Hydrochloride Tablets
1145
Decan-l-ol
577
Cyclizine Tablets
1145
Decolorised Fuchsin Solution
576
Decolorised Magenta Reagent
592
592
576
Decolorised Magenta Solution
592
Decolorised Pararosaniline Solution
598
Cyclohexane
3-Cyclohexylpropionic Acid
Cyclophosphamide
153,302,1145
573,1043
Cyclophosphamide Injection
1146
Decolorising Charcoal
Cyclophosphamide Tablets
1147
Deet
1215
153,1149
Deferoxamine Injection
1169
Cycloserine Capsules
1149
Deferoxamine Mesylate
1167
Cycloserine Tablets
1150
Deferoxamine Mesylate Injection
1169
Dehydrated Alcohol
1303
Cycloserine
Cycohexa-2,5-diene-l A-dione compound with

Benzene-l A-Diol
606
Dehydrated Methanol
594
Cyproheptadine
302
Dehydrated Pyridine
606
153,1151
Cyproheptadine Hydrochloride
Dehydroacetic Acid
154,1165
Cyproheptadine Hydrochloride Syrup
1152
Dehydroemetine Hydrochloride
154,1166
Cyproheptadine Hydrochloride Tablets
1152
Dehydroemetine Hydrochloride Injection
1166
Cyproheptadine Syrup
1152
Dehydroemetine Injection
1166
Cyproheptadine Tablets
1152
Dequalinium Chloride
Cyproterone Acetate
153,303, 1153
Cyproterone Acetate Tablets
1154
Cyproterone Tablets
1154
Cysteine Hydrochloride
577
Cysteine Hydrochloride Solution
1170
Deoxycortone Acetate Injection
1171
l-Deoxy-l-ethylamino-D-Glucitol
583
2-Deoxy-D-Glucose
154
577 Deposition of the emitted dose

153,303,1155 -Depressor Substances
1156 Derivative Spectrophotometry
Cytarabine
Cytarabine Injection
~-Cytosine Arabinoside
~-Cytosine Arabinoside
Injection
1155
Descending Paper Chromatography
1156
Desiccator, General Notices

Description, General Notices
D
Daboia Venom
2458
153,1161
Danazol
Danazol Capsules
1161
154,304,1162
Dapsone
Dapsone Tablets
Daftihafic1faR6Ofs
1163
~-----------------
Danihiridri stems
Daunorubicin Hydrochloride
J:)almorubicin Hydrochloride Injection
154,305,577,1167
Deoxycortone Acetate
471;5-B~-2495-
-- -471,513,2496
154,304,1163
1165
730
33
118
135
12
14,716,1734
Desferrioxamine Injection
1169
Desferrioxamine Mesilate
1167
Desferrioxamine Mesylate
154,305,1167
Desferrioxamine Mesylate Injection
1169
Desoxycorticosterone Acetate
1170
Desoxycorticosterone Acetate Injection
1171
Desoxycortone Acetate
Desoxycortone Acetate Injection --
1171
Destaining Solution
577
Determination ofABO Blood Group and RhGroup
248
2776
INDEX
IP 2010
567
Determination ofABO Blood Group ofBlood Donors
248
Diammonium Orthophosphate
Determination ofHaemoglobin by Photometry
252
Diastase
829
Determination ofRh Group of Donors
251
Diatomaceous Support, Acid-washed, Silanised
577
Determination of Loss on Drying
139
Diatrizoate Sodium
Dexamethasone
Dexamethasone Injection
154, 306, 1171

1176
2119
2120
Diatrizoate Sodium Injection

Diazepam
154,309, 1194
154,306,1175
Diazepam Capsules
1195
Dexamethasone Sodium Phosphate Injection
1176
Diazepam Injection
1196
Dexamethasone Tablets
1173
Diazepam Tablets
1196
Dexamethasone Sodium Phosphate
Dexchlorpheniramine Maleate
154,307,1177
Dexchlorpheniramine Maleate Oral Solution
1179
Dexchlorpheniramine Oral Solution
1179
Dexchlorpheniramine Maleate Tablets
1180
Dexchlorpheniramine Tablets
1180
Dextran 40 Injection
1181
Dextran 40 Intravenous Infusion
1181
1182
1182
1184
1184
Dextrin
Dextromethorphan Hydrobromide
154,1185
154,307,1186
Dextromethorphan Hydrobromide Syrup
1187
Dextro-pantothenyl Alcohol
1856
Dextropropoxyphene Capsules
1188
Dextropropoxyphene Hydrochloride
Dextropropoxyphene Hydrochloride Capsules
Dextropropoxyphene Napsilate
Dextrose
Dextrose, Anhydrous
154,308,1187
1188
154,308,1189
154,1190
577
Dextrose Injection
1191
Dextrose Intravenous Infusion
1191
Detector Tube, Gas

Diacerein
Diacerein Capsules
Diacetyldioxime
21
154,309,1191
1193
579
Diazobenzenesulphonic Acid Solution
577
Diazobenzenesulphonic Acid Solution, Dilute
577
1,4-Diazobicyclo[2.2.2]octane
619
Diazotised Nitroaniline Solution
597
Diazotised Sulphanilic Acid Solution
615
Diazoxide
154,310,1197
1198
Diazoxide Tablets
Dibasic Ammonium Phosphate
567
Dibasic Ammonium Phosphate,0.2 M
567
Dibasic Calcium Phosphate
151,970
Dibasic Potassium Phosphate
581
Dibasic Sodium Phosphate, Anhydrous
613
Dibenzlpyrolle
572
Dibenzo[a,d]cyclohepta-1 ,4-dien-3-one
577
Dibenzosuberone
577
Di-n-butylamine
577
Dibutyl Ether
577
Di-n-butyl Ketone
597
Dibutyl Phthalate
577
Dichlofenthion
154,310,2650
Dichloroacetic Acid
577
Dichloroacetic Acid Solution
577
1,2-Dichloroethane
577
1,2-Dichloroethane, Purified
577
2,7-Dichlorofluorescein
577
5,7-Dichloro-8-hydroxyquinoline
577
Dichloromethane
577
Dichloromethane IR
577
Diagnostic Veterinal)' Products
2747
Dichlorophen .
Dialysis Fluid, Intraperitoneal
1879
2652
Dialysis Solution, Peritoneal
1879
154,2650
Dichlorophen Veterinary Aerosol
2651
1,2-Diaminoethane
583
Dichlorophen Veterinary Aerosol Spray
2651
Diammonium Hydrogen Citrate
577
bichlorophen Veterinary Spray
2651
Diammonium Hydrogen Phosphate
567
2,6-Dichlorophenolindophenol Sodium
Volume 1: i to xxiii and 1to 704; Volume 2: xxv to xxviii and 705 to 1722; Volume 3: xxix to xxxii and 1723 to 2829
2777
578
IP 2010
INDEX
2,6-Dichlorophenolindophenol Solution
578
Diethylaminoethylcellulose
2,6-Dichlorophenolindophenol Standard Solution
578
N,N- Diethylaniline, Limit test of
2,6-Dichloroquinone-4-chloroimide
578
N,N-Diethylaniline
578
5,7-Dichloroquinolin-8-ol
577
Diethylcarbamazine
313
Dichloroquinonechloroimine
578
Diethylcarbamazine Citrate
1200
Diethylcarbamazine Citrate Injection
Diclofenac Injection
Diclofenac Sodium
154,311,1199
578
83
154,314,1212
1214,2653
1213
Diethylcarbamazine Citrate Tablets
Diclofenac Sodium Injection
1200
1214,2653
Diclofenac Sodium Tablets
1200
Diethylcarbamazine Tablets
Diclofenac Tablets
1200
N,N-Diethyl- I ,2-diaminoethane
578
DicIoxaciIlin Capsules
1202
Diethylene Dioxide
580
DicIoxaciIlin Oral Suspension
1203
Diethylene Glycol
578
Dicloxacillin Sodium
154,311,1201
1213
Diethylene Glycol Succinate Polyester
578
DicIoxaciIlin Sodium Capsules
1202
N,N-Diethylethanamine
619
Dicloxacillin Sodium Oral Suspension
1203
Diethyl Ether
582
578
Dicyandiamide
578
N,N- Diethylethylenediamine
Di-2-cyanoethyl Ether
578
Diethyl Phenyl Acetamide
Dicyclohexylamine
578
Diethyl Phthalate
N, N-Dicyclohexylamine
578
Diethylstilboestro I
2156
578
Diethylstilboestrol Tablets
2156
1,3-Dicyclohexylurea
Dicyclomine Hydrochloride
154,312,1204
154
154,1214
154,1215
DiethyItoluamide
Dicyclomine Hydrochloride Injection
1205
Dihydroergocristine Mesylate
154,315
Dicyclomine Hydrochloride Oral Solution
1205
Dihydroergotamine Mesylate
154,315
Dicyclomine Hydrochloride Tablets
1206
2655
Dicyclomine Injection
1205
_Dihydrostreptomycin Sulphate
154,2654
Dicyclomine Oral Solution
1205
Dihydrostreptomycin Sulphate Injection
Dicyclomine Tablets
1206
Dicycloverine Hydrochloride
1204
Dihydroxyspiro[isobenzofuran-l(3H),9' -[9H]
xanthen] 3-one
584
Dicycloverine Hydrochloride Oral Solution
1205
10, II-Dihydro-5H-dibenzo[a,dJ-cycIohepten-5-one
577
Dicycloverine Hydrochloride Tablets
1206
10,Il-Dihydro-5H-dibenz[b,/]azepine
589
1207
9,1 0-dihydro-3,4-dihydroxy-9, 10-dioxo-2anthrancesulphonic acid monosodium salt
1209
Definition, General Notices
154,312,1206
Didanosine
Didanosine Capsules
Didanosine Tablets
3,3',(3,3'-Diemthoxy-4-4'-biphenylylene)bis(2,5diphenyl-2H-tetrazolium Chloride
Digitoxin
Dienestrol
1210
Digitoxin Reagent
Dienestrol Tablets
1210
Digitoxin Standard Solution
Dienoestrol
154,1210
1210
Dienoestrol Tablets
:9iethanolamine
Digol
------------1-54,31J,l-211- Digoxin
DiethanolamineBufferpH-10.0
2,5-Diethoxytetrahydrofuran
Diethylamine
Digitoxin Tablets
- -561
578
-- 578
Bigoxin Injection
Digoxin Paediatric Solution
Digoxin Reagent
2778
622
13,715,1733
Differential Scanning Calorimetry (DSC)

570
2655
175
154,578,1216
578,629
629
1217
578
- - - - - ----155,314,1218-1:219
1220
578
INDEX
IP 2010
Digoxin Standard Solution
629
Dilute Potassium Dichromate Solution
603
Digoxin Tablets
1220
Dilute Potassium Ferricyanide Solution
604
Dihydroergocristine Mesylate
1221
Dilute Potassium Ferrocyanide Solution
604
Dihydroergotamine Mesylate
1223
Dilute Potassium Iodide Solution
605
573
Dilute Potassium Iodobismuthate Solution
605
1,3-Dihydroxybenzene
607
Dilute Potassium Permanganate Solution
605
1,4-Dihydroxybenzene
588
Dilute Sauton's Medium
1,8-Dihydroxy-3-hydroxymethyl-l O-(~-Dglucopyranosyl)anthrone
569
Dilute Silver Nitrate Solution
609
2,5-Dihydroxy-3,6-dinitro-I,4-benzoquinone
596
Dilute Sodium Carbonate Solution
610
3',6'-Dihydroxyfluoran
584
Dilute Sodium Hydroxide Solution
611
1,3-Dihydroxynaphthalene
595
Dilute Stannous Chloride Solution
614
2,7-Dihydroxynaphthalene
595
Dilute Sulphuric Acid
616
4,5-Dihydroxynaphthalene-2,7-disulphonic Acid
574
Diluted Isosorbide Dinitrate
159,1520
5,7-Di-iodo-8-hydroxyquinoline
578
Diluted Isosorbide Mononitrate
159,1524
o-Dihydroxybenzene
Diiodohydroxyquinoline
34
34
Dilute Saution's Solution
155,316,1224
1225
Diiodohydroxyquinoline Tablets
1868
Diluted Potassium Clavulanate
1938
1,5-Di-iodopentane
578
Diluted Sorbide Dinitrate
1520
5,7-Di-iodoquinolin-8-01
578
Diluted Sorbide Nitrate
1520
Di-isopropylamine
578
Dimensions ofHard Gelatin Capsule Shells
Di-isopropyl Ether
579
Dimercaprol
Diloxanide Furoate
155,316,1225
676
155,1229
1229
Dimercaprol Injection
Diloxanide Furoate Tablets
1226
Dimethicone
579,1231
Diloxanide Tablets
1226
Dimethicone, Activated
155,1230
Diltiazem Hydrochloride
155,1227
2,5-Dimethoxybenzaldehyde
579
Diltiazem Hydrochloride Tablets
1228
3,4-Dimethoxybenzoic Acid
620
Diltiazem Tablets
1228
Dimethylamine
579
Dilute Acetic Acid
563
Dimethylamine Solution
579
Dilute Acetic Acid Sp.
563
Dimethyl Phthalate
580
Dilute Ammonia Solution
566
Dimethyl Sulphoxide
580
Dilute Ammonia Solution, Sp.
566
Dimethyl Yellow
624
Dilute Ammonium Chloride Solution (Nessler's)
566
Dimethyl Yellow-Oracet Blue B Solution
624
Dilute Cupric Sulphate Solution
576
Dimethyl Yellow Solution
624
Dilute Diazobenzenesulphonic Acid Solution
577
Dimethyl Yellow-Solvent Blue 19 Solution
624
Dilute Ethanolic Potassium Hydroxide Solution
604
Dimethylacetamide
579
Dilute Formaldehyde Solution
585
4-Dimethylaminoazobenzene
624
587,1450
Dimethylaminobenzaldehyde
579
1460
4-Dimethylaminobenzaldehyde
579
Dilute Hypophosphorus Acid
589
p-Dimethylaminobenzaldehyde
579
Dilute I-Naphthol Solution
Dilute Hydrochloric Acid

Dilute Hydrogen Peroxide Solution
596
4-Dimethylaminobenzaldehyde Solution
579
Dilute Nitric Acid
596
Dimethylaminobenzaldehyde Solution
579
Dilute Phenolphthalein Solution
626
Dimethylaminobenzaldehyde Solution, Alcoholic
579
Dilute Phosphoric Acid
601
Dimethylaminobenzaldehyde Reagent
579
2779
INDEX
IP 2010
Dimethylaminobenzaldehyde Solution, Ethanolic
579
Dip Concentrates
2621
4-Dimethylaminocinnamaldehyde
579
Diphenhydramine Capsules
1233
2-(4-Dimethylaminophenylazo) benzoic Acid
625
Diphenhydramine Hydrochloride
2-(4-Dimethylaminostyryl) quinoline Ethiodide
627
Diphenhydramine Hydrochloride Capsules
Dimethylaniline
579
Diphenoxylate Hydrochloride
2,3-Dimethylaniline
579
Diphenyl
580
155,318,1232
1233
155,318,1234
2,6-Dimethylaniline
579
Diphenylamine
580
N,N-Dimethylaniline
579
Diphenylamine Solution
580
N,N-Dimethylaniline, LimitTestfor
9,10-Diphenylanthracene
580
Diphenylanthracene
580
Dimethylfonnamide Solution (5 per cent v/v)
83
579
579
Diphenylbenzidine
580
2,3-Dimethyl-l-phenyl-3-pyrazolin-5-one
600
N,N' -Diphenylbenzidine
580
Dimethylglyoxime
579
1,5-Diphenylcarbazide
581
3,4-Dimethylphenol
579
Diphenylcarbazide
581
N ,N-Dimethyl-4-phenylenediamine Sulphate
579
Dlphenylcarbazide Solution
581
N,N-Dimethyl-p-phenylenediamine Sulphate
579
1,5-Diphenylcarbazone
581
N,N-Dimethyl-p-phenylenediamine Sulphate Solution
579
Diphenylcarbazone
581
1,4-Dimethylpiperazine
580
Diphenylcarbazone Mercuric Reagent
581
Dimethylpiperazine
Dimethylformamide
580
Diphenylcarbazone-Mercury Reagent
N,N'-DimethyIpiperazine
580
Diphenylhydantoin
1903
581
3-(4,5-Dimethyltruzol-2-yl)-2,5-Diphenyltetrazolihm
Bromide
Diphenylhydantoin Oral Suspension
1907
617
Diphenylhydantoin Sodium
1904
155,317,2657
Diphenylhydantoin Sodium Capsules
1906
2658
Diphenylhydantoin Sodium Injection
1905
2658
Diphenylhydantoin Sodium Tablets
1906
Dimetridazole
Dinitolrnide
155,317,2658
1,5-Diphenylthiocarbazone
581
Diphenylthiocarbazone
581
1,3-Dinitrobenzene
580
Dinitrobenzene Solution
580
Diphosphorus Pentoxide
Dinitrobenzoic Acid
580
Diphtheria Antitoxin
2374
3,5-DinitrobenzoicAcid
580
Diphtheria and Tetanus Vaccine (Adsorbed)
2375
Dinitrobenzoic Acid Solution
580
Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed) 2378
2,4-Dinitrofluorobenzene
584
2,4-Dinitrophenylhydrazine
580
Diphtheria and Tetanus Vaccine (Adsorbed) for

Adults and Adolescents
2376
Dinitrophenylhydrazine
580
Diphtheria, Tetanus and Hepatitis B (rDNA) Vaccine

(Adsorbed)
2350
Diphtheria, Tetanus, Pertussis (Acellular

Component) and Hepatitis B (rDNA) Vaccine
(Adsorbed)
2354
Dinitrophenylhydrazine-Aceto-Hydrochloric
Acid Solution
580
Dinitrophenylhydrazine Reagent
580
Dinitrophenylhydrazine Solution
580
Dioctyl Sodium Sulphosuccinate

580, 1244
DioctylSodium Sulphosuccinate,O;005-M---------------631
1;4~Dioxan580
Dioxan
Dioxyanthranol
580
1240
601
Diphtheria, Tetanus, Pertussis (Acellular

Component) and Haemophilus Tybe b Conjugate
---Vaccine (Adscirbed)
Diphtheria, Tetanus, PertUssis (Acellular Coiiiponeiit)~ .
Inactivated Poliomyelities Vaccine and Haemophilus
Tybe b Conjugate Vaccine (Adsorbed)
2356
2780
INDEX
IP 2010
Diphtheria, Tetanus, Pertussis (Acellular Component)

and Inactivated Poliomyelities Vaccine
2359
Diphtheria, Tetanus, Pertussis and Poliomyelitis
(Inactivated) Vaccine (Adsorbed)
2361
Diphtheria, Tetanus, Pertussis, Poliomyelitis

(Inactivated) and Haemophilus Type b Conjugate
Vaccine (Adsorbed)
2364
Diphtheria, Tetanus, Pertussis (Whole Cell),

Hepatitis B (rDNA) and Haemophilus Type b
2379
Diphtheria, Tetanus, Pertussis (Whole Cell)

Hepatitis B (rDNA) Vaccine (Adsorbed)
2382
Diphtheria, Tetanus, Pertussis (Whole Cell) and

(Adsorbed)
2384
2387
570
Disodium Tetraborate
155,319,1236
Disopyramide
Disopyramide Capsules
1237
Disopyramide Phosphate Capsules
1237
Disopyramide Phosphate Sustained-release Capsules 1238
817
Dispersible Amoxicillin Tablets
817
Dispersible Amoxycillin Tablets
828
Dispersible Ampicillin Tablets
843
Dispersible Aspirin Tablets
752
Dispersible Tablets
189
Dissolution Test
Dissolution Test for Conventional and Prolonged190
release solid dosage forms
Dissolution Test for Prolonged-release dosage fonns
191
Dissolution Test for Modified-release dosage forms
191
Dipicrylamine
586
Dipotassium Hydrogen Orthophosphate
581
Dipotassium Hydrogen Phosphate
581
Dipotassium Hydrogen Phosphate, 0.1 M
581
Dipotassium Sulphate
605
Disintegration Test
187
Disintegration Test for Pessaries and

Suppositories
5,5-DiethylbarbituricAcid
Dithiothreitol
188
Disintegration Test for Capsules
722
Disintegration Test for Compressed Pessaries
189
Dithizone
581
Dithizone Carbon Tetrachloride Solution
581
Dithizone Extraction Solution
581
Dithizone Solution
581
Dithizone Standard Solution
581
Dithranol
155,320,1240
Dithranol Ointment
1240
Divalproex Sodium
2139
Divalproex Sodium Sustained-release Tablets
1241
Divalproex Sustained-release Tablets
1241
DocetaxelInjection
1243
Docetaxel Trihydrate
155,1242
1243
Docetaxel Trihydrate Injection
Docusate Sodium
155,1244
601
Dodecamolybdophosphoric Acid
Dodecyldimethyl-2-phenoxyethylammonium Bromide 581
Domiphen Bromide
581
Domperidone
155,320, 1245
Domperidone Maleate
155, 1246
Domperidone Maleate Tablets
1247
Domperidone Tablets
1247
Donepezil Hydrochloride
155,321,1248
Disintegration Test for Enteric-Coated Tablets
188
Disintegration Test for Tablets and Capsules
187
Disodium (4,4'-biphenylbis-2,2-azo) bis(laminonaphthalene-4-sulphonate)
623
Disodium 4,5-dihydroxynaphthalene-2,7-disulphonate
Disodium Edetate
574
155,581,1234
Disodium Edetate, 0.1 M
632
Disodium Edetate, x M
581
Disodium Edetate Injection
1235
Disodium Hydrogen Citrate
609
Disodium Hydrogen Orthophosphate
581
Disodium Hydrogen Orthophosphate, Anhydrous
581
Disodium Hydrogen Phosphate

Disodium Hydrogen Phosphate, 0.2 M
Disodium Hydrogen Phosphate, Anhydrous
Disodium Hydrogen Phosphate Dodecahydrate
Disodium Hydrogen Phosphate Solution
Disodium Hydrogen Phosphate, xM
Disodium Hydrogen Orthophosphate, Anhydrous
581,2133
559
581
2133
581
581
581
Distilled Water
620
Distilling Range, Temperature or Boiling Range
119
Disulfiram
155,319,1238
Disulfiram Tablets
5,5'-Dithiobis(2-nitrobenzoic acid)
2781
1239
581
569
581
IP 2010
INDEX
Donepezil Hydrochloride Tablets

Donepezil Tablets
L-Dopa
L-Dopa Capsules
L-Dopa Tablets
Dose, General Notice
Dosulepin Capsules
Dosulepin Hydrochloride
Dosulepin Hydrochloride Capsules
Dothiepin Capsules
Dothiepin Hydrochloride
Dothiepin Hydrochloride Capsules
Dotriacontane
Doxepin Capsules
Doxepin Hydrochloride
Doxepin Hydrochloride Capsules
Doxofylline
Doxofylline Tablets
Doxorubicin Hydrochloride
Doxorubicin Hydrochloride Injection
Doxorubicin Injection
Doxycycline Capsules
Doxycycline Hyclate
Doxycycline Hydrochloride
Doxycycline Hydrochloride Capsules
Dragendorff Reagent
Dried Aluminium Hydroxide
DriedAluminium Hydroxide Gel
Dried Calcium Sulphate
1249
1249
1575
1577
1578
14,716,1734
1251
1250
1251
1251
155,321,1250
1251
582
1253
Econazole Nitrate Cream
1267
Econazole Nitrate Pessaries
1268
ECbnazole Pessaries
1268
Econazole Vaginal Tablets
1268
1234
Edetate Disodium
1270
1271
Efavirenz Tablets
Effectiveness ofAntimicrobial Preservatives
27
753
Effervescent Tablets
Egg Drop Syndrome 76 (Adenovirus) Vaccine,
Inactivated
2722
Eglumine
583
155,322,1252
Eicosan-1-o1
1253
155,322,1254
Electrolyte Reagent for the determination of water
569
582
122
177
Electrophoresis
1254 Electrophoresis, Capillary

155,1255 Electrophoresis, Horizontal
1257 Elixirs, see also under name ofsubstance
1257 Ellman's Reagent
2614
744
581
1260
Emblic Myrobalan
1257
Empty Sterile Containers ofPiasticised Poly(vinyl

chloride) for Blood and Blood Components
695
Emtricitabine
155,325,1272
155,323,1257
1260
582
789
148,789
1928
2471
Emtricitabine Capsules
Emulsifying Wax
1273
155,1274
Enalapril Maleate
155,325,1275
Enalapril Maleate Tablets
1275
2727
2563
Encephalomyelitis Vaccine Live
1345
Enoxaparin Injection
Dried Human Antihaemophilic Fraction

Drying and Ignition to constant weight,
General Notices
2563
Enoxaparin Sodium
Ear Drops
Eclipta alba
Econazole Cream
155,324,1269
Efavirenz
Efavirenz Capsules
Dried Factor VIII Fraction

Dried Ferrous Sulphate

Dydrogesterone
Dydrogesterone Tablets
Dynamic Viscosity, Determination of
155,324,1267
Econazole Nitrate
1277
156,1276
Enoxaparin Sodium Injection
12
1279
Enterobacteria Enrichment Broth-Mossel Medium
2721
Enteric Capsules (Gastro-resistant Capsules)
2722
Enteric Capsules
155,323,1261
1262
172
723
2486
1267
47
721
721,722
Enteric-coated Tablets
753
Eosin
624
Eosin Solution
624
Ephedrine Elixir
1281
Ephedrine HydrbchI6rideElixir---
_---1281
Ephedrine Hydrochloride Oral Solution
1281
Ephedrine Hydrochloride Tablets
1282
2782
INDEX
IP 2010
156
Ephedrine Oral Solution
1281
Esomeprazole Magnesium
Ephedrine Tablets
1282
Esomeprazole Magnesium Tablets
1296
Epidemic Tremor Vaccine Live
2727
Esomeprazole Tablets
1294
Epinephrine
777
Essential Vitamins
Epinephrine Bitartrate
778
Epinephrine Tartrate Injection
779
1,8-Epoxy-p-menthane
575
(5R,6S)-4,5-Epoxy-3-methoxy-N-methyl-morphin7-en-6-01- monohydrate
575
85
85
Esters, Assay for
73
Esters, Test for
1296
Estradiol and Norethisterone Acetate Tablets
1296
Estradiol and Norethisterone Tablets
1298
Etacrynic Acid
1298
Etacrynic Acid Tablets
156,329,1298
Ethacrynic Acid
1298
Ethacrynic Acid Tablets
156,329,1299
Ethambutol Hydrochloride
1301
Ethambutol Hydrochloride Tablets
Ethambutol and Isoniazid Tablets
1302
Ethambutol Hydrochloride and Isoniazid Tablets
1302
Ethambutol Tablets
1301
1,2-Ethanediol
583
583
Ethane-1,2-diol
156,582,1303
Ethanol
Ethanol, Aldehyde-free
582
Ethanol, Assay for
99
574
Ethanol-free Chloroform
Ethanol, General Notices
12
201
Ethanol-Soluble Extractive
156,582,1303
Ethanol (95 per cent)
582
Ethanol (95 per cent), Aldehyde-free
Ethanol (x per cent)
582
Ethano1ic Acetic-Ammonia Buffer pH 3.7
560
EthanQlicAmmonia, xM
566
Ethano1ic Ammonium Molybdate Solution
567
Ethanolic Anisaldehyde Solution
568
Ethanolic Bromophenol Blue Solution
623
Ethanolic Calcium Standard Solution (100 ppm Ca)
629
Ethanolic Dimethylaminobenzaldehyde Solution
579
Ethanolic Hydroxylamine Solution
588
588
Ethanolic (60 per cent) Hydroxylamine Solution
Ethanolic (90 per cent) Hydroxylamine Solution
588
Ethanolic Mercuric Bromide Solution
593
596
Ethanolic Ninhydrin Solution
Ethanolic Potassium Hydroxide, 0.1 M
633
604
Ethanolic Potassium Hydroxide Solution
Epsom Salts
1626
Equine Serum Gonadotrophin for Veterinary Use

Ergocalciferol
2685
156,582,955
Ergometrine Injection
1284
Ergometrine Maleate
156,326,1283
Ergometrine Maleate Injection
1284
Ergometrine Maleate Tablets
1285
Ergometrine Tablets
1285
Ergonovine Injection
1284
Ergonovine Maleate
1283
Ergonovine Maleate Injection
1284
Ergonovine Maleate Tablets
1285
Ergonovine Tablets
1285
Ergotamine Injection
1287
Ergotamine Tablets
1288
Ergotamine Tartrate
156,327, 1286
Ergotamine Tartrate Injection
1287
Ergotamine Tartrate Tablets
1288
Eriochrome Black T
624
Eriochrome Black T Mixture
624
.Eriochrome Black T Solution
624
Eriochrome Black T TIiturate
624
Erythromycin
156,327,1290
Erythromycin Stearate
156,328,1291
Erythromycin Stearate Tablets
1292
Elythromycin Tablets
1291
2602
Escherichia coli
Escita10pram Oxalate
45
156,328,1293
Escita10pram Oxalate Tablets
1294
Escita10pram Tablets
1293
Eserine Salicylate
1910
Eserine Salicylate Injection
1911
Esomeprazo1e Magnesium Trihydrate
1295
Ester Value, Assay for
2783
213
IP 2010
INDEX
Ethanolic Potassium Hydroxide Solution, Dilute
604
Ethanolic Potassium Hydroxide, xM
604
Ethanolic Sodium Hydroxide, O.IM
634
611
Ethanolic Sodium Hydroxide, xM
Ethanolic Sulphate Standard Solution (10 ppm S04)
629
616
Ethanolic Sulphuric Acid x per cent
Ethanolic Sulphuric Acid, 0.25 M
635
Ethanolic Sulphuric Acid, x M
616
Ethanolic Thymol Blue Solution
627
1-Etheny 1-2-pyrrolidinone homopolymer
1141
Ether
582
Ether, Anaesthetic
156,1304
Ether, Peroxide-free
582
Ethinylestradiol
330,1306
Ethinylestradiol Tablets
1307
Ethinyloestradiol
156,1306
Ethinyloestradiol Tablets
1307
Ethinyloestradiol and Norgestrel Tablets
1796
Ethionamide
156,330,1308
Ethionamide Tablets
1309
Ethopabate
156,331,2659
Ethopropazine Hydrochloride
156,1309
Ethopropazine Hydrochloride Tablets
1310
Ethopropazine Tablets
1310
Ethosuximide
331,1311
Ethosuximide Capsules
1312
Ethosuximide Oral Solution
1312
Ethosuximide Syrup
1312
p-Ethoxyacetanilide
599
p-Ethoxychrysoidine Hydrochloride
582
Ethoxychrysoidine Hydrochloride Solution
582
4-p- Ethoxyphenylazo-m-phenylenediamine
Hydrochloride
582
Ethyl Acetate
582
Ethyl N-acetyl-L-tyrosinate
564
Ethyl Chloride
156,1314
Ethyl Cyanoacetate
582
Ethyl Iodide
589
Ethyl Oleate
156,1314
Ethylcellulose
.....-.1313
Ethylene Chloride
- -577
Ethylene Glycol
583
Ethylene Glycol MonomethylEther
594
Ethylene Oxide
Ethylenediamine
Ethylenediamine Hydrate
N-Ethylglucamine
N-Ethylglucamine Hydrochloride
583
583
156,1315
583
583
2-EthylhexanoicAcid
2-EthylhexoicAcid
2-Ethyl-2-methylbutanedioicAcid
2-Ethyl-2-methylsuccinicAcid
Ethyloestrenol
Ethyloestrenol Tablets
Etodolac
583
583
583
583
156,332,1315
1317
156,1318
Etodolac Capsules
Etodolac Tablets
Etoposide
1319
1321
156,1322
Etoposide Capsules
Etoposide Concentrate
Etoposide Injection
Eucalyptol
Eucalyptus Oil
1324
1325
1325
575
156,2498
Eugenol
Euglobulins, Bovine
Euglobulins, Human
583
583
583
Eukaryotes
2595
Evaluation ofEfficacy ofVaccines and Immunosera
232
Excipients, General Notices
13,715,1733
xii
Executive Committee
Expert Committees
xii
Expression of Contents, General Notices
12,714,1732
Expression of Concentrations, General Notices 12,714,1732
Exsiccated Calcium Sulphate
Extended Insulin Zinc Suspension
Extraneous Agents in Viral Vaccines
Eye Drops, see also under name ofsubstance
1928
1503
208
724
Eye Ointments, see also under name ofsubstance
725
F
Factor IIa Chromogenic Substrate
Factor Xa Chromogenic Substrate
Famotidine
FamotidineTablets
Fast Blue B Slat
Fehling's Solution
2784
243
247
156,332,1331
...-_ ... -1332
624
603
INDEX
IP 2010
Felodipine
156,1333
Felodipine Sustained-release Tablets
1335
Fenbendazole
156,1336
2536
Fennel
Fiblin
Fibrin Sealant Kit
2566
2610
Film-coated Tablets, For
Fenofibrate
157,1337
Filtration, General Notices
Fentanyl
156,1338
Finasteride
Fentanyl Citrate
156,1339
Finasteride Tablets
Fentanyl Citrate Injection
1340
Flame Photometry
Fentanyl Injection
1340
Fluconazole
584
Ferric Alum
1511
Ferric Ammonium Citrate
584
752
12
157,1350
1351
110
157,333,1353
1353
Fluconazole Capsules
1354
Fluconazole Tablets
Ferric Ammonium Sulphate
584
Flucytosine
Ferric Ammonium Sulphate, 0.1 M
632
Flucytosine Capsules
1357
Ferric Ammonium Sulphate Solution
584
Flucytosine Oral Suspension
1357
FelTic Ammonium Sulphate Solution, Acid
584
Flucytosine Tablets
FelTic Chloride
584
Fludrocortisone Acetate
FelTic Chloride, Anhydrous
584
Fludrocortisone Acetate Tablets
1360
FelTic Chloride Colorimetric Solution (FCS)
107
Fludrocortisone Tablets
1360
Ferric Chloride-FelTicyanide-Arsenite Solution
584
Fluid Thioglycollate Medium
Ferric Chloride Hexahydrate
584
Fluocinolone Acetonide
FelTic Chloride Solution
584
Fluocinolone Acetonide Cream
1362
FelTic Chloride Test Solution
584
Fluocinolone Cream
1362
FelTic Salts, Tests for
73
157,333,1355
1358
157,334,1358
57
157,1361
3'-6'-Fluorandiol
584
584
FelTicyanide-cyanide Reagent
252
Fluorescein
FelToin Solution
624
Fluorescein Eye Drops
FelToin Sulphate Solution
624
Fluorescein Injection
Ferrous Amrnonium Sulphate
584
Fluorescein Sodium
FelTous Ammonium Sulphate, 0.1 M
632
Fluorescein Sodium Eye Drops
1365
Fluorescein Sodium Injection
1366
Fen-ous Fumarate
Fen-ous Fumarate Tablets
Ferrous Gluconate
Ferrous Gluconate Tablets
Ferrous Salts, Tests for
Ferrous Sulphate
Ferrous Sulphate-Citrate Solution
Ferrous Sulphate, Dried
Ferrous Sulphate Solution
FelTous Sulphate Tablets
156,1341
1342
156,1342
1344
73
156,584,1344
584
157,1345
584
1346
1365
1366
157,336,584,1363
Fluorimetly
III
I-Fluoro-2,4-dinitrobenzene
584
9'-Fluoro-l1 b,17b-dihydroxy-17a-methylandrost-4en-3-one
585
Fluorouracil
157,334, 1367
Fluorouracil Injection
1368
Fluoxetine Capsules
1370
F1uoxetine Hydrochloride
157,1369
Fluoxetine Hydrochloride Capsules
1370
Fluoxetine Hydrochloride Oral Solution
1371
1348
Fluoxetine Hydrochloride Tablets
1373
Fexofenadine Hydrochloride Capsules
1348
Fluoxetine Oral Liquid
1371
Fexofenadine Hydrochloride Tablets
1349
Fluoxetine Oral Solution
1371
Fexofenadine Tablets
1349
Fluoxetine Tablets
1373
Fexofenadine Hydrochloride
Fexofenadine Capsules
157,1346
2785
IP 2010
INDEX
Fluoxymesterone
585
Fortified Procaine Penicillin Injection
1971
Fluphenazine
335
2724
2725
Fluphenazine Decanoate
157,335,1374
Fluphenazine Decanoate Ester
1374
Framycetin Sulphate
Fluphenazine Decanoate Injection
1375
Free Formaldehyde, Limit Test for
Fluphenazine Hydrochloride
157,1375
157,1388
Freeze-dried Human Coagulation Factor VIII
Fluphenazine Hydrochloride Injection
1377
Freezing Point
Fluphenazine Hydrochloride Tablets
1377
Freshly prepared, General Notices
Fluphenazine Tablets
1377
Friability of Uncoated Tablets
Flupromazine Hydrochloride
2261
Friar's Balsam
83
2563
122
12
193
890
Flupromazine Hydrochloride Injection
2262
Fructose
Flupromazine Hydrochloride Tablets
2262
d-Fructose
Flupromazine Injection
2262
Fructose Injection
1391
Flupromazine Tablets
2262
Fructose and Sodium Chloride Injection
2114
Flurbiprofen
157,336, 1378
Flurbiprofen Tablets
Flutamide
1379
157,337,1380
Flutamide Capsules
1381
Fructose Intravenous Infusion

Frusemide
338,1390
157,585,1390
1391
157,339,1391
Frusemide Injection
1392
Frusemide Tablets
1393
Fuchsin Basic
592
Fluticasone Propionate Inhalation
1383
Fuchsin Reagent, Decolorised
Fluticasone Propionate Powder for Inhalation
1383
Fuchsin Solution, Deco1orised
592
592
2536
Fumaric Acid
Fluticasone Propionate
157,337,1382
Foeniculum vulgare
Folic Acid
157,585,1384
Folic Acid Tablets

Folin and Ciocalteu Phenol Reagent
1385
600
Foot-and-Mouth Disease Vaccine, Inactivated
2723
Fuming Nitric Acid

Furazolidone
157,1394
596
157,339,1394
Furazolidone Drench
2660
Furazolidone Mixture
2660
Foreign Organic Matter
201
Furazolidone Oral Suspension
1395
Formaldehyde Solution
585
Furazolidone Premix
2660
Formaldehyde Solution, Dilute
585
Furazolidone Tablets
1395
Furazolidone Veterinary Oral Suspension
2660
Formaldehyde Standard Solution (5 ppm CHzO)

Formalin
629
585
Formamide
585
Formic Acid
585
Formic Acid, 15 M
585
Formic Acid, Anhydrous
585
FormolisedPlague Vaccine
2423
Formoterol Fumarate
338
Formoterol Fumarate Dihydrate
157,338,1386
Formoterol Fumarate and Budesonide Powder for
Furazolidone Veterinary Mixture
2660
Furosemide
1391
Furosemide Injection
1392
Furosemide Tablets
1393
Fusidic Acid
157,340,585,1396
Fusidic Acid Mixture
Fortified BenzathineBenzylpenicillin Injection .
882
Gallamine Injection
Fortified Benzathine Penicillin Injection
882
Gallamine Triethiodide
Fortified Benzathine Penicillin G Injection
882
Gallamine Triethiodide Injection
2786
..1402
157,1401
1402
INDEX
IP 2010
Gamma Benzene Hexachloride
1592
Gentamicin Eye Drops
1412
Garcinia
514,2499
Gentamicin Injection
1413
GarciniaAqueous Extract
515,2500
Gentamicin Sulphate
157,1410
1412
Garcinia cambogia
2499
Gentamicin Sulphate Eye Drops
Garlic
2517
Gentamicin SUlphate Injection
1413
Gas Chromatography
127
Giloe
2503
Gas Detector Tubes
21
Ginger
2544
Gas-gangrene Antitoxin (Novyi)
2391
Gitoxin
585
Gas-gangrene Antitoxin (Oedematiens)
2391
Glacial Acetic Acid
Gas-gangrene Antitoxin (Perfringens)
2392
Glacial Acetic Acid, Anhydrous
563
Gas-gangrene Antitoxin (Septicum)
2393
Glass Containers
683
Gas-gangrene Antitoxin, Mixed
2394
Glassware, Cleaning of
639
Gas Liquid Chromatography

Gastric Juice, Artificial
Gatifloxacin
Gatifloxacin Infusion
Gatifloxacin Tablets
Gefitinib
Gefitinib Tablets
127
Glibenclamide
585
Glibenclamide Tablets
157,1402
1403
Glic1azide Tablets
1404
Glimepiride
157,1405
1405
157,585,1406
Gelatin
Gelatin Capsule Shells, Hard
585,1407
Gelatin, Hydrolysed
585
Gelatin, Pancreatic Digest of
585
Gels, see also under name ofsubstance
725
Gemifloxacin Mesylate
157,1408
Gemifloxacin Mesylate Tablets
1409
Gemifloxacin Tablets
1409
Gene Cloning and Protein Expression'
2593
General Body
General Chapters
General Identification Reactions
General Identification Reactions of Ions and
Functional Groups
General Monographs
General Notices, Contents list
General Notices
General Reagents
General Requirements, Vaccines
General Statements
General Techniques used in down-stream
processing of recombinant proteins
General Tests
Gliclazide
x
xvii, 7
71
148,563,774
157,340,1414
1415
157,1416
1417
157,1418
1419
Glimepiride Tablets
Glipizide
157,1420
1421
Glipizide Tablets
Glossary of Symbols, Statistical Analysis
of Results for
661
D-Glucitol
2143
Glucose
1190
D-G1ucose
1190
Glucose Intravenous Infusion
1191
Glutamic Acid
586
Glutaraldehyde Solution
241
1414
G1yburide
Glycerin
157,586,1422
586
Glycerin (85 percent)
1422
Glycerol
71
13,715, 1733
9,709
11,711,1731
563
639,721
2347
11,713,1731
619
Glycerol Triacetate
G1ycery1 Monostearate
157,1423
G1ycery1 Trinitrate Solution, Concentrated
157,1424
Glycery1 Trinitrate Tablets

Glycine
1425
158,586,1426
Glycine Buffer pH 11.3

Glycine Buffer Solution
Glycine Irrigation Solution
Glycollic Acid
2596
637
G1ycosy1ation of Proteins
Glycyrrhetic Acid
2787
561
561
1427
586
2597
586
IP 2010
INDEX
Glycyrrhetinic Acid
~-GlycyrihetinicAcid
586
Haloperidol Oral Drops
586
1436
Haloperidol Oral Solution
1436
2551
Haloperidol Solution
1436
Glyoxal Sodium Bisulphite
586
Haloperidol Tablets
1437
Glyoxaline
589
Haloxon
Glycyn-hiza glabra
48
GN Broth Medium
2725
471,516,2500
Gokhru
2520
GotuKola
ix.
Governing Body
Granulated Tin
618
Granulated Zinc
621,631
Granulocyte Colony Stimulating Factor Solution
2610
158,2661
214
Hanks' Balanced Salt Solution (Modified)
2508
Harad
Hard Capsules
722
Hard Gelatin Capsules
721
Hard Gelatin Capsule Shells
585,1407
Hard Gelatin Capsule Shells, Dimensions of
676
164,1862
Hard Paraffin
Haridra
474,521,2507
Haridra Dry Extract
474,522,2508
1428
Halitaki
475,523,2508
Groundnut Oil
2475
Haritaki Extract
475,524,2510
Guaiphenesin
158,341,586,1429
476,525,2510
Griseofulvin
158,1427
Griseofulvin Tablets
Guanidine Hydrochloride Solution

GuarGum
586
Hartmann's Solution for Injection
2129
158,2501
Hartmann's Solution for In-igation
2130
Gudmar
472,517,2502
Guduchi
472,518,2503
Guggul
2504
Guggul Resin
158,473,519,2504
Gugulipid
158,473,520,2505
Hartmann's Solution with Dextrose for Injection

Heavy Kaolin
1623
160,1619
Heavy Magnesia
Heavy Magnesium Carbonate
Heavy Magnesium Oxide
2125
159,1537
160,592,1626
80
Gugulipid Tablets
2506
Heavy Metals, Limit test for
Gum Acacia
2469
Hedychium spicatum
Gum ofBswellia Sen-ata
2515
Helium
586
Gymnema sylvestre
2502
Heparin
586
2542
248
1440
Heparin in Coagulation Factors, Assay of
Heparin Injection
Haemoglobin by Photometry, Detennination of

Haemolysins
252
35
2395
Haemon-hagic Septicaemia Vaccine
2726
Haldi
2507
Half Strength Compound Sodium Lactate and

Dextrose Injection
Half Strength Compound Sodium Lactate with
2127
2121
Injection
2127
Haloperidol
158,341,1435
1435
158,586,1438
Heparin Sodium Injection

Heparinised Saline Solution
1440
33
Hepatitis A (Inactivated) and Hepatitis B (rDNA)

Vaccine (Adsorbed)
2398
2399
Heptane
586
n-Heptane
586
I-Heptanesulphonic Acid Sodium Salt
611
Herbs and Herbal Products; Monographs
HaffStrel1gthRiIl.ger~LlI.Ctll.teSoil.ltronwJIh-IYextrose--.
Haloperidol Injection
Heparin Sodium
2463
Herbs, Crude
2467
Herbal Formulations
2468
Herbal Prdocuts, Tests on
2788
199
IP 2010
INDEX
Hexadecanoic Acid
1-Hexadecano1
586
1042
Hexadecy1 Hexadecanoate
573
N-hexadecyl-N,N,N-uimethy1ammonium bromides
573
n-HexadecylAlcohol
Hexadecyl Palmitate
895,1042
573
Human Coagulation Factor VII

Human Coagulation Factor VII, Assay of
Human Coagulation Factor VIII (rDNA)
2571
244
2572
Human Coagulation Factor VIII, Assay of

Human Coagulation Factor IX
Human Coagulation Factor IX, Assay of
245
2570
246
Hexamethylenetetramine
586
Human Coagulation Factor X, Assay of
247
Hexamethyl-p-Rosaniline Chloride
2,6,1O,15,19,23-Hexamethyltetracosane
624
613
Human Euglobulins
583
Hexamine
Hexane
586
586
Humanized monoclonal antibodies
n-Hexane
586
Human Normal Albumin
Hexane UV
586
Human Normal Immunoglobulin
Hexanesulphonic Acid Sodium Salt

Hexanitrodiphenylamine
611
586
Human Normal Immunoglobulin for Inuavenous Use
2579
Hexosamines
205

Administration
2579
High Performance Liquid Chromatography

Histamine
129
35
Human Plasma for Fractionation
2586
Human Gamma Globulin
2574
2593
Human Insulin
159,1497
2568
587,2574
2576
2578
Histamine Acid Phosphate
1441
Human Prothrombin Complex
Histamine Acid Phosphate Injection
1441
Human Rabies Vaccine
2336
Human Red Blood Corpuscles, Concentated
2562
Human Red Blood Cells, Concentrate
2562
Histamine Dihydrochloride
Histamine Phosphate
Histamine Phosphate Injection
586
158,586,1441
1441
Hyaluronate Solution
Histidine Monohydroch10ride
587
Hyaluronidase
DL-Histidine Monohydroch10ride
587
Hyaluronidase Injection
Hogweed
Hohnium Oxide
Holmium Perchlorate Solution
Homatropine Eye Drops
587
158,1446
1447
2532
587
Hyaluronidase Solutions, Diluent for
587
Hydralazine Hydrochloride Injection
1449
Hydralazine Injection
1449
1443
Hydralazine Hydrochloride
587
158,343,1448
Homatropine Hydrobromide
Homatropine Hydrobromide Eye Drops
158,342,1442
1443
Hydrated Aluminium Oxide
789
Hydrazine Reducing Mixture
587
Homatropine Methy1bromide
158,342, 1444
Hydrazine Sulphate
587
Homatropine Methylbrornide Tablets

Horizontal Electrophoresis
Host-cell and Vector-derived DNA
HumanAlbumin
Human Albumin Fraction (Saline)
1445
Hydrazine-molybdate Reagent
587
2614
Hydriodic Acid
587
66
587,2568
2576
Hydrochloric Acid
158,587,1450
Hydrochloric Acid, 0.2 M
559
Hydrochloric Acid, 0.5 M Methanolic
632
Human Albumin Solution
2568
Hydrochloric Acid, 1 M
632
Human Anti-D Immunoglobulin
2558
Hydrochloric Acid AsT
587
240
241
1079
Hydrochloric Acid AsT, Brominated
587
Hydrochloric Acid AsT, Stannated
587
Human Anti-D Immunoglobulin Method A

Human Anti-D Immunoglobulin Method Band C
. Human Chorionic Gonadotrophin
Human Coagulation Factor II, Assay of
243
Hydrochloric Acid BET, 0.1 M

Hydrochloric Acid Buffer pH 1.2 to 2.2
2789
29
559
INDEX
IP 2010
HydrochloricAcid, Dilute
587,1450
Hydrochloric Acid, Iron-free

Hydrochloric Acid, xM
587
587
Hydrochloric Acid, xM Methanolic
587
Hydrochlorothiazide
158,343,1451
Hydrochlorothiazide and Losartan Potassium Tablets 1610

Hydrochlorothiazide Tablets
1452
Hydrocortisone
158,344,587,1453
Hydrocortisone Acetate
158,344,1454
Hydrocortisone Acetate Injection
1456
Hydrocortisone Acetate Eye Ointment
1455
Hydrocortisone Eye Ointment
1455
Hydrocortisone Hemisuccinate
158,345,1457
Hydrocortisone Hydrogen Succinate
1457
HydrocOltisone Sodium Succinate
345
Hydrocortisone Sodium Succinate Injection

Hydrocyanic Acid Solution
Hydrofluoric Acid
1459
587
587
158,2511
Hydrogen Peroxide Solution
587
Hydrogen Peroxide Solution, Dilute
587,1460
Hydrogen Peroxide Solution, Strong
1460
Hydrogen Peroxide Solution (l0 Vol)
587
Hydrogen Peroxide Solution (20 Vol)
587,1460
Hydrogen Peroxide Solution (100 Vol)
587,1460
Hydrogen Peroxide Solution (6 per cent)
1460
Hydrogen Peroxide Solution (27 per cent)
1460
Hydrogen sulphide detector tube
21
Hydrogen Sulphide
588
Hydrogen Sulphide Solution
588
Hydroquinone
588
Hydropericardium Syndrome (HPS)
2727
Hydrous Wool Fat
171,2318
Hydroxocobalamin
158,1461
Hydroxocobalamin Injection
1462
2-HydroxyaceticAcid
p-Hydroxyanilinoacetic Acid
588
Hyoscine Butylbromide Tablets

Hyoscine Hydrobromide
607
588
--------
-"",-,._-"u_"---"-""~~"--'~-588
(2-Hydroxyethyl)trimetilyi~a;ninoniulnCI1forlde .....-- -574
1,7-Bis(4-hydroxy-3-methoxyphenyl)-hepta-1 ,6.
dien-3,5-dione
Hyoscine Butylbromide
586
4-Hydroxybenzaldehyde
Hyoscine Hydrobromide Injection

Hyoscine HydrobromideTablets.-.-...Hyoscyamine Injection
Hyoscyamine Oral Solution
576
588
3~-Hydroxy-ll-oxo-18~, 20~-0Iean-12-enoic Acid

586
N-[2-Hydroxy-l,1-bis(hydroxymethyl)ethyl]glycine
619
2-Hydroxy-5-sulphobenzoicAcid
615
1-(2-Hydroxy-5-sulphophenyl)-3-phenyl-5-(2carboxyphenyl)fonnazan
621
3,3-Bis(4-hydroxy-5-isopropy1-2-methylphenyl)
phthalidie
627
Hydroxyl Value, Assay for
85
Hydroxylamine Hydrochloride
588
Hydroxylamine Hydrochloride Reagent
588
Hydroxylamine Hydrochloride Reagent in Ethanol
(60 per cent)
588
Hydroxylamine Hydrochloride Solution
588
Hydroxylamine Hydrochloride Solution Sp.
588
Hydroxylamine Solution
588
Hydroxylamine Solution, Ethanolic
588
Hydroxylamine Solution, Ethanolic (60 per cent)
588
Hydroxylamine Solution, Ethanolic (90 per cent)
588
Hydroxylammonium Chloride
588
p-HydroxyphenylaminoaceticAcid
588
D-d-4-Hydroxyphenylglycine
588
N-(p-Hydroxyphenyl)glycine
588
Hydroxyprogesterone Caproate
1463
Hydroxyprogesterone Caproate Injection
1464
Hydroxyprogesterone Hexanoate
158,346,1463
1464
Hydroxyprogesterone Hexanoate Injection
Hydroxyprogesterone Injection
1464
Hydroxypropyl Cellulose
158,1464
1464
2-Hydroxypropyl Ether Cellulose
Hydroxypropylmethylcellulose
158,1465
1465
2-Hydroxypropylmethylcellulose
8-Hydroxyquinoline
588
5-Hydroxyuracil
588
9-Hydroxyxanthene
620
Hyoscine Butylbromide Injection
2-Hydroxybenzaldehyde
4-Hyai~oxy'coiiinarln
8-Hydroxy-7-iodoquinoline-5-sulphonic Acid
Hyoscyamine Sulphate
2790
158,346,1466
1467
1468
158,588,1469
1470
..--.. . - -__.L47l
.. ..
1473
1474
158,347,588,1472
INDEX
IP 2010
Hyoscyamine Sulphate Injection
1473
Imipramine Hydrochloride Tablets
1488
Hyoscyamine Sulphate Oral Solution
1474
Imipramine Tablets
1488
Hyoscyamine Sulphate Tablets
1474
Immune Human Serum Globulin
2574
Hyoscyam'ine Tablets
1474
Immunochemical Methods
Hypertonic Saline
2116
Immunoglobulin, Nonnal Human
64
589
2349
589
Immunosera
Hypophosphorous Acid, Dilute
589
Implants
749
Hypophosphorous Reagent
589
Impurities
656
Hypophosphorous Acid
Hyprolose
Hypromellose
1464
Impurities, Acceptance Criteria for
658
1465
Impuritiesin Drug Products
657
Impurities in Drug Substances
657
I
Ibuprofen
158,347,1479
IMS
1666
Inactivated Avian Infectious Bronchitis Vaccine
2707
Inactivated Canine Contagious Hepatitis Vaccine
2710
Ibuprofen Cream
1480
Inactivated Canine Corona Virus Vaccine
2711
Ibuprofen Gel
1481
Inactivated Canine Leptospirosis Vaccine
2713
Ibuprofen Tablets
1482
Inactivated Canine Parvovirus Vaccine
2714
Identification, General Notices
14,716,1734
Inactivated Clostridium Perfringens Vaccine
2717
Identification Tests
71
Inactivated Clostridium Septicum Vaccine
2719
Identification of Barbiturates
77
Inactivated Duck Pasteurella Vaccine
2721
Identification of Phenothiazines
77
Identification of Related Foreign Steroids
78
Inactivated Egg Drop Syndrome 76 (Adenovirus)

Vaccine
2716
Inactivated Foot-and-Mouth Disease Vaccine
2723
Identification of Related Substances in

Barbiturates
78
Identification of Related' Substances in

Phenothiazines
78
Identification of Related Substances in

Sulphonamides
78
Idoxuridine
158,348,1483
Idoxuridine Eye Drops
1484
Imatinib Capsules
1485
Imatinib Mesylate
158,1484
Imatinib Mesy1ate Capsules
1485
Inactivated Fowl Cholera Vaccine
2724
Inactivated Hepatitis A Vaccine (Adsorbed)
2402
Inactivated' Hepatitis B Vaccine
2404
Inactivated Inclusion Body Hepatitis (IBH) Vaccine
2727
Inactivated Infectious Bursal Disease Vaccine
2728
Inactivated Infectious Chicken Anemia Vaccine
2730
Inactivated Influenza Vaccine (Split Virion)
2406
Inactivated Influenza Vaccine (Surface Antigen)
2408
Inactivated Influenza Vaccine (Whole Virion)
2410
Inactivated Multicomponent Clostridium Vaccine
2715
Imidazole
589
Inactivated Poliomyelities Vaccine
2426
ImidazoleBufferpH 6.5
561
Inactivated Rabies Veterinary Vaccine (Cell Culture)
2733
Imidazole Buffer pH 7.4
561
Inactivated Ranikhet Disease Vaccine
2735
Imidazole-Mercury Reagent
589
Inactivated Reo Virus Vaccine
2737
Imidazole, Recrystallised
589
Inactivated Tick-Borne Encephalitis Vaccine
2447
Imidazole Solution
589
Inclusion Body Hepatitis (IBH) Vaccine, Inactivated
2727
Iminodibenzyl
589
Indane-1 ,2,3-trione Hydrate
lmipenem
Imipenem and Cliastatin Injection
Imipenem Monohydrate
Imipramine Hydrochloride
158,1486
1487
348
158,349,589,1488
Indapamide
596
158
Indapamide Tablets
Indian Ginseng
1489
2479
Indian Goosebeny
2471
2791
INDEX
IP 2010
Indian Gum
2469
Insulin Preparations
Indian Madder
2521
Insulin, Soluble
1498
ix
740
Insulin Zinc Suspension
1500
Insulin Zinc Suspension (Amorphous)
1502
628
Insulin Zinc Suspension (Crystalline)
1503
622
Insulin Zinc Suspension, Extended
1503
Insulin Zinc Suspension (Mixed)
1500
622
Insulin Zinc Suspension, Prompt
1502
Indigo Carmine
589
Insulins, Assay of
Indigo Carmine Solution
589
Indian Pharmacopoeia Commisssion

Indian Sarsaparilla
2474
Indicator and Test Papers

Indicators
Indicators, General Notices
15,717,1735
Indicators and Indicator Test Papers
Indinavir Capsules
1492
Indinavir Sulphate
158,1491
Indinavir Sulphate Capsules
1492
Indometacin
Indomethacin
158,1494
158,349,1494
101
Interferon Alfa-2- Concentrated Solution
2613
Intramammary Infusions
2621
Intramammary Infusions for Veterinary Use
2621
Intramammary Injections
2621
Intraperitoneal Dialysis Fluid
1879
xvii
Introduction
Indomethacin Capsules
1494
Invert Sugar Injection
Indomethacin Suppositories
1495
Invert Sugar and Sodium Chloride Injection
Indophenol Blue
Industrial Methylated Spirit
625
589,1666
Iodide-free Starch Solution
1505
1506
614
Iodides, Tests for
73
2727
Iodinated Potassium Iodide Solution
605
Infectious Bronchitis Vaccine, Live
2708
Iodinated Zinc Chloride Solution
621
Infectious Bursal Disease Vaccine, Inactivated
2728
Iodine
Infectious Bursal Disease Vaccine, Live
2729
Iodine, 0.05 M
632
Infectious Chicken Anemia Vaccine, Inactivated
2730
Iodine Bromide
589
Infectious Chicken Anemia Vaccine, Live
2730
Iodine Bromide Solution
589
Iodine Monochloride Solution
589
Infectious Avian Encephalomyelitis Vaccine, Live
2731
Infra-red Reference Spectra
255
Infrared Absorption Spectrophotometry

Infrared Spectrophotometry
Infrared Spectrophotometry, Near
lNH
INH Tablets
Inhalation Preparation
Infusions
Injectable Preparations
Injections, see also under name ofsubstance
Insulin
Insulin, Human
Insulin Injection
Insulin Injection, Biphasic
Insuliri Iiijectioii, BiphaSicIsopnane
Insulin Injection, Isophane
Insulin Lente
Insulin, Plain
111
111
113
1515
1516
726
748
746
747
158,1496
159,1497
1498
1498
1499
1499
1500
159,589,1507
Iodine Pentoxide
589
Iodine Solution
590
Iodine Trichloride
Iodine Value, Assay for
590
86
IodinexM
589
Iodochlorhydroxyquin
2027
Iodochlorhydroxyquin Cream
2028
Iodochlorhydroxyquin Ointment
2029
Iodochlorhydroxyquin Tablets
2030
Iodochlorhydroxyquin and Hydrocortisone Cream
2031
Iodochlorhydroxyquin and Hydrocortisone Ointment 2032

Iodochlorhydroxyquinoline
2027
Iodochlorhydroxyquinoline Cream
2028
Iodochlorhydroxyquinoline Ointment
2029
Iodochlorhydroxyquinoline Tablets
2030
Iod~~hl~~hydroxyquinoiineand
Hyclr()c()l1:isolle
Cream
2031
1498
2792
Oiritrrierif
2032
INDEX
IP 2010
Iodoethane
589
Isoprenaline Hydrochloride Injection
1518
Iodoplatinate Reagent
590
Isoprenaline Injection
1518
159,1518
Iodoquinol
1224
Isoprenaline Sulphate
Iodoquinol Tablets
1225
Isoprenaline Sulphate Tablets
Ionagar
IPC Secretariat
213,221
xiii
1519
Isoprenaline Tablets
Isopropyl Alcohol
Ipecac Tincture
476,526,2512
Isopropyl Ether
Ipratropium Bromide
159,350,1508
Isopropyl Myristate
1519
159,606,1520
579
159,590,1515
590
Iproveratril Hydrochloride
2295
Isopropylamine
Iproveratril Hydrochloride Injection
2296
Isoproterenol Hydrochloride
1517
2297
Isoprotrenol Hydrochloride Injection
1518
159,1508
Isoprotrenol Injection
1518
1510
Isoprotrenol Sulphate
1518
1510
Isoprotrenol Sulphate Tablets
1519
159,1511
Isoprotrenol Tablets
1519
1512
Isosorbide Dinitrate
351
Iproveratril Hydrochloride Tablets

Irinotecan Hydrochloride Trihydrate
Irinotecan Hydrochloride Injection
Irinotecan Injection
Iron and Ammonium Citrate
Iron Dextran Injection
Isosorbide Dinitrate, Diluted
Iron-free Ammonia Solution
566
Iron-free Citric Acid
575
Isosorbide Dinitrate Tablets
587
Isosorbide Mononitrate, Diluted
81
Isosorbide Mononitrate Tablets
Iron-free Hydrochloric Acid

Iron, Limit Test for
159,1520
1520
159,1524
1525
Iron Salicylate Solution
590
Isoxsuprine Hydrochloride
Iron Standard Solution (2 ppm Fe)
629
Isoxsuprine Hydrochloride Injection
1528
629
Isoxsuprine Hydrochloride Tablets
1529
629
Isoxsuprine Injection
1528
629
Isoxsuprine Tablets
1529
Iron(III) Chloride
584
Ispaghula Husk
2513
Iron(III) Chloride, Anhydrous
584
Ivennectin
Iron(III) Chloride, Hexahydrate
584
Ivennectin Injection
2663
LZ.S.
1500
568
I.Z.S.,Amorph
1502
588
LZ.S., Cryst.
1503
1514
I.Z.S. (Mixed)
1500
Isapgol Husk
Isoamyl Alcohol
Isobarbituric Acid
Isobutane
2513
Isobutyl Acetate
590
Isobutyl Alcohol
590
Isoelectric Focusing
Isoniazid
180
159,350,590,1515
159,352,1527
159,2662
J
2411
Japanese Encephalitis Vaccine (Human)
139
Jelly Strength
Isoniazid Solution
590
Isoniazid Tablets
1516
Isonicotinylhydrazid
1515
Isonicotinylhydrazid Tablets
1516
Isophane Insulin
1499
Kalmegh
477,527,2513
Isophane Insulin (NPH)
1499
Kalmegh Dry Extract
477,528,2514
Isophane Insulin Injection

Isoprenaline Hydrochloride
1499
159,351,1517
2751
1535
Kanamycin Injection
Kanamycin Acid Sulphate
159,1534
2793
IP 2010
INDEX
Kanamycin Sulphate
159,1533
Kaolin, Heavy
Lactoflavin
2050
Kaolin, Light
159,1537
Lactoflavin Tablets
2052
159,1537
Lactophenol
Kaolin Mixture
2663
Lactose
2663
Lactose Anhydrous
Kaolin Veterinary Mixture
2663
Karl Fischer Reagent

Kerosene
Ketamine Hydrochloride
591
159,355,591,1554
1554
1554
Lactose Monohydrate
590
Lactulose
159,1555
590
Laevulose
585
159,352, 1538
Lamivudine
159,355,1557
Ketamine Hydrochloride Injection
1539
Lamivudine Oral Solution
1558
Ketamine Injection
1539
Lamivudine Tablets
1559
Lamivudine and Tenofovir Tablets
1560
Lamivudine and Tenofovir Disoproxil Fumarate

Tablets
1560
Ketoconazole
159,353,1540
Ketoconazole Tablets
Ketoprofen
1541
159,353,1542
Ketoprofen Capsule
Ketorolac Tromethamine
1542
159,1543
Lamivudine and Zidovudine Tablets
1561
1563
1564
Ketorolac Tromethamine Injection
1544
Lamivudine, Nevirapine and Stavudine Dispersible

Tablets
Ketorolac Tromethamine Tablets
1545
Lamivudine, Nevirapine and Stavudine Tablets
Ketorolac Trometamol
1543
Lamotrigine
Ketorolac Trometamol Injection
1544
Lamotrigine Dispersible Tablets
1567
Ketorolac Trometamol Tablets
1545
Lamotrigine Sustained-release Tablets
1567
Ketotifen Fumarate
159,1546
Ketotifen Hydrogen Fumarate
1546
LansoprazoIe
Lansoprazole Sustained-release Capsules
159,356,1566
159,356,1568
1570
Kieselguhr
590
Lanthanum Nitrate
591
Kieselguhr, Acid-washed
590
Lanthanum Nitrate Solution
591
Kieselguhr for Column Chromatography
590
Lasuna
479,531,2517
Kieselguhr G
590
Lavang
479,2518
Kieselguhr H
591
Lead Acetate
591
Kovac's Reagent
591
Lead Acetate Cotton
591
Kunduru
478,529,2515
Lead Acetate Paper
628
Kutki
478,530,2516
Lead Acetate Solution
591
Lead Compounds, Tests for .
Lead Dioxide
Label, General Notices

Labelling, General Notices
12
16,718,1736
Labetalol
Labetalol Hydrochloride
354
159,354,1551
Lead, Limit Test for
74
591
81
Lead Monoxide
591
Lead Nitrate
591
Lead Nitrate, 0.1 M
632
Labetalol Hydrochloride Tablets
1552
Lead Nitrate Solution
591
Labetalol Tablets
1552
Lead Nitrate Stock Solution
591
Lactated Ringer's and Dextrose InjeCtion, Modifiea -
2128
Lead Oxide
Lactates, Tests for

Lactic Acid
74
159,1553
---- .---- --- .. -591
Lead Subacetate Solution
591
Lead Standard Solution
629
2794
IP 2010
INDEX
Lead Standard Solution (l ppm Pb)
629
Lidocaine Hydrochloride and Dextrose Injection
1586
Lead Standard Solution (2 ppm Pb)
629
Lidocaine Hydrochloride Gel
1586
629
Lidocaine Hydrochloride Injection
1587
629
Lidocaine Injection
1587
Lead Standard Solution (1 00 ppm Pb)
629
Light Kaolin
Lead Standard Solution (0.1 per cent Pb)
629
Light Liquid Paraffin.
Lead Subacetate Solution .
591
Light Liquid Petrolatum
1863
Light Magnesia
1624
Lecithin
159,1571
Legal Notices
v
963
Light Magnesium Oxide
Leucovorin Calcium Injection
964
Light Mineral Oil
159,357,1572
164,598,1863
Light Magnesium Carbonate
Leucovorin Calcium
Levamisole Hydrochloride
159,1537
160,1620
160,592,1624
1863
Light Petroleum (Boiling Range 30 to 160)
599
359
Levamisole Hydrochloride Injection
2664
Lignocaine
Levamisole Hydrochloride Tablets
1573
Lignocaine and Adrenaline Injection
1585
Levamisole Hydrochloride Veterinary Oral Solution
2665
Lignocaine and Dextrose Injection
1586
1586
Levamisole Hydrochloride Veterinary Mixture
2665
Lignocaine Gel
Levamisole Veterinary Oral Solution
2665
Lignocaine Hydrochloride
Levamisole Veterinary Mixture
2665
2664
Lignocaine Hydrochloride and

Adrenaline Bitartrate Injection
1585
Levamisole Tablets
1573
Lignocaine Hydrochloride and Dextrose Injection
1586
Levarterenol Bitartrate
1789
Li~ocaine Hydrochloride Gel
1586
Levarterenol Bitartrate Injection
1790
Lignocaine Hydrochloride Injection
1587
Lignocaine Injection
1587
Levocetirizine Hydrochloride
159,357,1573
Levocetirizine Dihydrochloride Tablets
1574
Limes Flocculationis (Lf)
Levocetirizine Tablets
1574
Limits, General Notices
Levodopa
159,358,1575
160,360,1584
67
15,717,1735
79
Limit Tests
Levodopa and Carbidopa Tablets
1576
Lincomycin Capsules
Levodopa Capsules
1577
Lincomycin Hydrochloride
Levodopa Tablets
1578
Lincomycin Hydrochloride Capsules
1589
Lincomycin Hydrochloride Premix
2665
2665
Levofloxacin Hemihydrate
159,1579
Levofloxacin Infusion
1580
Lincomycin Premix
Levofloxacin Tablets
1580
Linctuses, see also under name ofsubstance
Levonorgestrel
159,358,1581
Levonorgestrel and Ethinyloestradiol Tablets

Levosalbutamol Sulphate
1582
160,359,1583
Linezolid
1589
160,360,1588
744
160,361,1590
Linezolid Tablets
Lindane
1591
160,1592
Linoleic Acid
591
Levothyroxine Sodium
2221
Levothyroxine Sodium Tablets
2222
Linolenic Acid
Levothyroxine Tablets
2222
Lint
1593
Lidocaine and Adrenaline Injection
1585
Lint, Absorbent
1593
Lidocaine and Dextrose Injection
1586
Lint, Cotton
1593
Lint, Unmedicated
1593
Lidocaine Hydrochloride
Lidocaine Hydrochloride and Adrenaline Bitartrate
Injection
. 1584
1585
591
LiposomalAmphotericin B Injection
822
Liposomal Preparations
742
2795
IP 2010
INDEX
Liposomal Injectable Preparations
742
600
2634
Liquid Amitraz Dip Concentrate
129
Liquid Chromatography
493
LiquidlHPTLC/Gas Chromatograms ofHerbs
Liquid Ma1titol
160
164,598,1862
Liquid Paraffin
1863
Liquid Paraffin Emulsion
1863
Liquid Paraffin Oral Emulsion
1862
Liquid Petrolatum
2551
Liquorice Root
160,361,1593
Lisinopril
1594
Lisinopril Tablets
591
Litharge
591
Lithium
Lithium and Sodium Molybdophosphotungstate
592
Solution
160,2666
Lithium Antimony Thiomalate
2667
Lithium Antimony Thiomalate Injection
160,1595
Lithium Carbonate
1596
Lithium Carbonate Tablets
592
Lithium Chloride
592
Lithium Hydroxide
632
Lithium Methoxide,O.l M
592
Lithium Perchlorate
592
Lithium Perchlorate, 0.1 M
Lithium Sulphate
592
Litmus
625
Litmus Paper
628
Litmus Paper, Blue
628
Litmus Paper, Red
628
Litmus Solution
625
2458
Live Yellow Fever Vaccine
Lomustine
160,362,1597
1598
Lomustine Capsules
2530
Long Pepper
1600
Loperamide Capsules
160,1599
Loperamide Hydrochloride
1600
Loperamide Hydrochloride Capsules
-c------ -1601
Lop-eramide HydrochiorideTab1ets
----- -1601
Loperainide Tablets
Liquefied Phenol
Lopinavir
Lopinavir and RitonavirCapsules
160,362,1602
1603
Lopinavir and Ritonavir Tablets
1605
160
Lomoxicam
160,363,1607
Losartan Potassium
Losartan Potassium Tablets
1608
Losartan Potassium and Am10dipine Tablets
1609
Losartan Potassium and Amlodipine Besy1ate Tablets 1609

Losartan Potassium and Hydrochlorothiazide Tablets 1610
Losartan Tablets
1608
Loss on Drying
139
Loss in Ignition
140
34
34
160,1611
1611
1139
Lowenstein-Jensen Medium
Lowenstein-Jensen Solution
Lynoestreno1
Lynestreno1
Lysol
M
MacConkey Agar Medium
47
MacConkey Broth Medium
47
Macrogo1300
602
Macrogol400
602
Macrogol600
602
Macrogol1000
602
Macrogol1500
1929
Macrogo14000
1930
Macrogol6000
1930
Maga1drate
160,1617
Maga1drate Oral Suspension
1618
Magaldrate Suspension
1618
Magaldrate Tablets
1619
Magenta, Basic
592
Magenta Reagent, Deco10rised
592
Magenta Solution, Deco10rised
592
Magnesium
592
Magnesium Acetate
592
Magnesium Carbonate, Heavy
160,1619
Magnesium Carbonate, Light
160,1620
Magnesium Chloride
Magnesium Hydroxide
160,592, 1621
__ ____________________16Q,16n
Magnesium Hydroxide Mixture
1622
Magnesium Hydroxide Oral Suspension
1622
2796
160,2667
INDEX
IP 2010
Magnesium Nitrate
592
2415
2416
Magnesium Oxide, Heavy
160,592,1623
Measles, Mumps and Rubella Vaccine (Live)
Magnesium Oxide, Light
160,592, 1624
Mebendazole
Magnesium Perchlorate
592
Mebendazole Tablets
Magnesium Perchlorate, Anhydrous
592
Mebeverine Hydrochloride
Magnesium Powder
592
Mebeverine Hydrochloride Tablets
Magnesium Salts, Test for

Magnesium Stearate
Magnesium Sulphate
74
160,1625
160,592, 1626
Meclizine Hydrochloride
1638
1638
160,364, 1638
Meclizine Hydrochloride Tablets
1639
1639
633
Meclizine Tablets
Magnesium Sulphate Solution, Ammonical
592
Meclofenamic Acid
160,1626
1636
160,364,1637
Mebeverine Tablets
Magnesium Sulphate, 0.05 M

Magnesium Trisilicate
160,363,1635
160,365,2668
Meclozine Hydrochloride
1638
Magnesium Uranyl Acetate Solution
592
Meclozine Hydrochloride Tablets
1639
Magneson
622
Meclozine Tablets
1639
622
Mecobalamin
Malachite Green G
Malachite Green Solution
Maleic Acid
Malic Acid
34
160,593,1627
Medroxyprogesterone Acetate
Mefenamic Acid
1642
2752
Mefloquine Hydrochloride
161,366,1643
161,366,1645
Megestro1 Acetate
Maltitol
160,1629
Megestrol Acetate Tablets
Mandukaparni
160,365,1641
160,1628
160,2519
Maltodextrin
160,1640
Mefenamic Acid Capsules
Malt Extract
Maltitol, Liquid
160
160,1631
1646
1646
Megestrol Tablets
160,1632
Meglumine
480,532,2520
Meloxicam
Manganese Sulphate
593
Meloxicam Oral Suspension
Manganese(II) Sulphate
593
Melphalan
595
161
1646
161,1647
Mangifera indica
2473
Melphalan Injection
1647
Mango
2473
Melphalan Tablets
1648
Manjistha
480,533,2521
Melting Range or Temperature
Mannitol
160,593,1633
Meningococcal Polysaccharide Vaccine
1634
Mentha
Mannitol Intravenous Infusion
1634
Mentha Oil
D-Mannitol
1633
()-p-Menthan-3-ol
Mannitol Injection
Mannitol Salt Agar Medium
48
Menthol
Mannose
593
()-Menthol
D-Mannose
593
Menthol and Benzoin Inhalation
Manufacture of Drug Products,

Generai Notices
Maricha
Mayer's Reagent
Meanings of Terms, General Notices
140
2417
2523
161,2523
593
161,367,1649
593
1650
593
Menthyl Acetate
13,715,1733
2732
481,534,2522
Meperidine Hydrochloride
1883
Meperidine Hydrochloride Injection
1884
Meperidine Hydrochloride Tablets
1885
605
Mephenterrnine Injection
1651
12,714,1732
Mephentelmine SUlphate
161,1650
2414
Mephenterrnine Sulphate Injection
2797
1651
INDEX
IP 2010
2669
Mepyramine Maleate
Mepyramine Maleate Injection
161,367,1651
2669
Mepyramine Maleate Tablets
1652
Mepyramine Tablets
1652
Mercaptoacetic Acid
Mercaptoacetate Acid Sodium Salt
618
613
2-Mercaptoethanol
593
Mercaptopurine
161,1653
Mercaptopurine Tablets
2-Mercapto-1-methylimidazole
1654
618
Metaphosphoric Acid
N-Methylmethanamine
594
Methy1 N- tosy-L-argininate hydrochloride

Methylpentoses
619
205
579
618
5-Methylpyrimidine-2,4(lH,3H)-dione
161,368,1657
Metformin Hydrochloride
Metformin Hydrochloride Tablets

Metformin Hydrochloride Sustained-release Tablets
Metformin Tablets
1658
1659
1658
369
161,1660
Methadone
Mercuric Acetate
593
Methadone Hydrochloride
Methadone Hydrochloride Injection
Mercuric Acetate Solution
593
Methadone Hydrochloride Linctus
1661
Mercuric Ammonium Thiocyante Solution

Mercuric Bromide
567
593
Methadone Hydrochloride Tablets
1662
Methadone Injection
1661
Mercuric Bromide Solution, Ethanolic
593
Methadone Linctus
1661
Mercuric Chloride
593
Methadone Tablets
1662
p-Methan-3-ol
Methanesulphonic Acid
594
Methanesulphonic Acid, 2 M Methanolic
594
Methanol
Mercuric Chloride, 0.2 M
593
Mercuric Chloride Paper
628
Mercuric Chloride Solution

Mercuric Nitrate
593
593
1661
593
Mercuric Nitrate, 0.02 M
633
Methanol Acidified
594
594
Mercuric Nitrate Solution
593
Methanol, Aldehyde-fi:ee
594
Mercuric Oxide, Nitrogen-free
593
Methanol, xM Ammonical
Mercuric Oxide, Yellow

Mercuric Sulphate
593
593
Methanol, Anhydrous
594
Methanol, Dehydrated
594
Mercuric Sulphate Solution

Mercury
593
593
Methanolic Ammonia, xM
566
Methanolic Hydrochloride Acid, xM
587
594
Mercury Compounds, Tests for
74
Mercury(lI) Acetate
Mercury(lI) Bromide
Methanolic Methanesulphonic Acid, 2 M
593
593
Methanolic Potassium Bicarbonate Solution,

Saturated
Mercury(lI) Chloride
593
Methanolic Silver Nitrate Solution
Mercury(lI) Nitrate
593
Methanolic Sulphuric Acid x per cent
Mercury(lI) Sulphate
593
Methanolic Sulphuric Acid, x M
Meropenem
161,368,1654
Meropenem Injection
Mestranol
1656
161,1657
Metacresol Purple
Metal Containers for Eye Ointments
Metalphthalein~~~---~--~--~----~-
Mefa.hil Yellow
Metanil Yellow Solution

Metaphosphoric-Acetic Acids Solution
~-
~~~~
Methi
Methimazole
Methotrexate
625
Methotrexate Injection
685
Methotrexate Tablets
Methoxamine Hydrochloride
-~-~~---625
~~~~~~~625
625
594
Methoxamine Hydrochloride Injection

Methoxamine Injection
4-Methoxybenzaldehyde
2798
602
609
616
616
481,535,2523
618
161,369,1662
1663
1664
161,3]J}, 16{i4
~.. . .1665
1665
568
IP 2010
INDEX
Methoxyethanol
594
Methylene Blue
625
2-Methoxyethanol
594
Methylene Blue Solution
625
86
Methylene Chloride
577
1-(2'-Methoxyphenylazo)-2-naphthol
627
4,4'-Methylenebis-N-N-dirnethylaniline
595
Methyl Alcohol
594
Methylenebisacrylamide
625
Methyl Caproate
Methoxyl, Assay for
594
Methylergometrine Injection
Methyl Chloroform
619
Methylergometrine Maleate
Methyl Cyanide
564
Methylergometrine Maleate Injection
1670
Methyl n-Decanoate
594
Methylergometrine MaleateTablets
1671
Methyl Dodecanoate
595
Methylergometrine Tablets
1671
Methyl Ethyl Ketone
571
Methylergonovine Injection
1670
Methyl Hexadecanoate
595
Methylergonovine Maleate
1669
Methylergonovine Maleate Injection
1670
1671
1671
Methyl Hydroxybenzoate
1672
1670
161,1669
Methyl Isobutyl Ketone
595
Methylergonovine Maleate Tablets
Methyl Laurate
595
Methylergonovine Tablets
N-Methylmethanamine
579
N-Methylglucamine
595
Methyl Myristate
595
2-Methyl-5-nitroimidazole
595
Methyl Orange
625
Methylparaben
Methyl Orange Solution
625
N-Methylpiperazine
595
Methyl Orange-Xylene Cyanol FF Solution
625
4-Methyl-2-pentanone
595
Methyl Palmitate
595
4-Methylpentan-2-one
595
Methyl Red
625
Methylprednisolone
161,371,1673
Methyl Red-Methylene Blue Solution
625
Methylprednisolone Acetate
161,371,1676
Methyl Red Mixed Solution
625
Methylprednisolone Acetate Injection
1677
Methyl Red Solution
625
Methylprednisolone Tablets
1674
Methyl Salicylate
161,1666
161,1672
3-Methyl-4-nitro-l-(4-nitrophenyl)-5-pyrazolone
602
590
Methyl Stearate
595
2-Methyl-l-propanol
Methyl Tetradecanoate
595
2-Methylpropan-l-ol
590
Methyl Thymol Blue
626
2-Methyl-2-propanol
595
Methylaminophenol Sulphate
594
2-Methylpropan-2-ol
595
4-Methylaminophenol Sulphate
594
2-Methylpropanol
590
Methylaminophenol with Sulphite Solution
594
5-Methylresorcinol
598
Methylated Spirit, Industrial
1666
Metoclopramide Hydrochloride
161,372,1678
851
Metoclopramide Hydrochloride Injection
1679
618
Metoclopramide Hydrochloride Syrup
1680
2-Methylbenzenesulphonamide
618
Metoclopramide Hydrochloride Tablets
1680
4-Methylbenzenesulphonamide
618
Metoclopramide Injection
1679
Metoclopramide Syrup
1680
Metoclopramide Tablets
1680
Methylatropine Nitrate
,Methylbenzene
3-Methylbenzothiazolin-2-one Hydrazone
Hydrochloride
Methylcellulose
Methylcellulose (4000 cps)
Methyldopa
Methyldopa Tablets
594
161,594,1667
594'
Metol
594
Metoprolol
372
161,370,1668
Metoprolol Tablets
1682
1669
Metoprolol Tartrate
161,1681
2799
INDEX
IP 2010
1682
161,373,1682
Metronidazole Benzoate
161,1683
Metronidazole Benzoate Oral Suspension
1684
Metronidazole Injection
1684
Metronidazole Intravenous Infusion
1684
Metronidazole Sterile Suspension
1685
Metronidazole Tablets
1686
Mexiletine Capsules
1688
Mexiletine Hydrochloride
161,1687
Mexiletine Hydrochloride Capsules
1688
Mexiletine Hydrochloride Injection
1689
Mexiletine Injection
1689
Mianserin Hydrochloride
161,373,1689
Mianserin Hydrochloride Tablets
1690
Mianserin Tablets
1690
Miconazole
161,1691
Miconazole Cream
1693
Miconazole Nitrate
161,374,1692
Miconazole Nitrate Cream
1693
Miconazole Pessaries
1694
Miconazole Nitrate Pessaries
1694
Miconazole Tablets
1694
Miconazole Nitrate Vaginal Tablets
1694
Microbial Contamination in nonsterile products
37
Microbial Quality ofPharmaceutical Preparations
677
Microbiological Assay of Antibiotics
49
Microcrystalline Cellulose
161,573,1695
Metoprolol Tartrate Tablets
Mixed Gas-gangrene Antitoxin
Metronidazole
Mixed Phosphate Buffer pH 4.0
Microcrystalline Cellulose and

Carboxymethylcellulose Sodium
Microcrystalline Wax
Mifepristone
Milk of Magnesia
Milk Sugar
Millon's Reagent
Mineral Oil, Light
Mineral Oil, White
Mineral salt solution
Minoxidil
Min()xldil falJl~fs
Miscroscopy, PartiCle size by
Misoprostol
Mixed Barbitone Buffer pH 8.6
1695
162,1696
162
1622
1554
593
1863
1862
34
Mixtures, see also under name ofsubstance
2394
562
562
562
562
562
562
562
562
562
744
Modified Compound Sodium Lactate and

Dextrose Injection
2128

Mixed Phosphate Buffer pH 6.8, 0.2 M
Mixed Phosphate Buffer pH 7.0 with Azide
Mixed Phosphate Buffer pH 7.0,0.067 M
Modified Compound Sodium Lactate with

2128
Modified Lactated Ringer's and Dextrose Injection
2128
Modified Potassium Cupritartrate Solution
576
Modified Potassium Iodobismuthate Solution
605
Modified Release Capsules
721
Modified Release Tablets
753
Molar Solutions
630
Molybdenum(Vl) Oxide
595
Molybdenum Trioxide
595
Molybdic Oxide
595
Mometasone Furoate
162,374,1700
Mometasone Aqueous Nasal Spray
1701
Mometasone Cream
1702
Mometasone Ointment
1702
Monobasic Ammonium Phosphate
567
Monobasic Potassium Citrate
604
Monoclonal Antibodies
2593
Monographs (A to M)
755
Monographs (N to Z)
1737
Monographs, General Notice
13,715,1733
Monographs, General, General Notice
13,715, 1733
Monographs, Individual, General Notice
13,715,1733
Monographs upgraded
Monostearin
Monosulfiram
162, 1697 Monosulfiram Soap

-1698 --Monosulfiram Solution
195 Mofiotliioglyeetbl
162,1699 Montelukast Sodium
561 Montelukast Sodium Tablets
2800
1423
162,2669
2670
-----------2670---162;+703
162,375,1704
1705
IP 2010
INDEX
Montelukast Tablets
1705
1745
Mordant Black II
624
Mordant Black II Mixture
624
Naloxone Hydrochloride Dihydrate
1745
Mordant Black II Solution
624
Naloxone Hydrochloride Injection
1747
Mordant Black 17
623
Naloxone Injection
1747
Mordant Red B
622
Morphine Hydrochloride
595
Naloxone Hydrochloride ,
162,376,1745
162,376,1748
Naltrexone Hydrochloride Tablets
1749
1707
Naltrexone Tablets
1749
Morphine Injection
1708
Name
Morphine SUlphate
162,1706
Morphine and Atropine Injection
Morphine Sulphate and Atropine SUlphate Injection
1707
Morphine Sulphate Injection
1708
Mosapride Citrate Dihydrate
162,1708
Mosapride Citrate Tablets
1709
701
Names, Symbols and Atomic Weights of Elements

Atomic and Molecular Weights,
General Notices
13,715,1733
162,377,1750
750
Nandrolone Laurate
Moulded Suppositories
750
Moving Boundary Electrophoresis
123
Nandrolone Phenpropionate
Moulded Pessaries
713,1731
1751
162,377,2671
2672
162,1751
162,378,1751
Mulethi
2551
Multicomponent Clostridium Vaccine,

Inactivated
2715
Naphazoline Nitrate
Multiple Electrolytes and Dextrose Injection Type I
1710
Naphthalene
595
Multiple Electrolytes and Dextrose Injection Type II
1711
1,3-Naphthalenediol
595
Multiple Electrolytes and Dextrose Injection Type III
1713
Naphthalene-l,3-diol
595
Multiple Electrolytes and Dextrose Injection Type IV
1714
Naphthalene-2,7-diol
595
Multiple Electrolytes and Dextrose Injection Type V
1715
Naphthalenediol Reagent
595
Multiple Electrolytes Injection Type VI
1717
Naphthalenediol Reagent Solution
596
2420
Naphthalenediol Solution
595
162,1718
a-Naphthol
596
Mustine Hydrochloride Injection
1719
~-Naphthol
596
Mustine Injection
1719
I-Naphthol
596
I-Naphthol Solution
596
Mustine Hydrochloride
Mycophenolate Mofetil
Mycophenolate Mofetil Capsules
Nandrolone Phenylpropionate Injection
162,1719
1720
1752
162,1753
I-Naphthol Solution, Dilute
596
Mycoplasma Gallisepticum
210
2-Naphthol
596
Mycoplasma Orale
210
2-Naphthol Solution
596
Mycoplasma Pneumoniae
210
a-Naphtholbenzein
626
210
162,595,1721
l-Naphtholbenzein
626
a-Naphtholbenzein Solution
626
I-Naphtholbenzein Solution
626
a-Naphtholphthalein
626
Mycoplasma Synoviae
Myristic Acid
N
Nalidixic Acid
Nalidixic Acid Tablets.
Nalorphine Hydrochloride Injection
Naphthoresorcinol
595
1743
a-Naphthylamine
596
162,1744
I-Naphthylamine
596
N-(l- Naphthyl)ethane-l ,2-diammonium Dichloride
596
162,375,1743
1745
2801
INDEX
IP 2010
N-(l- Naphthy1)ethy1enediamine Dihydrochloride
Naproxen
596
162,378,1754
Nevirapine
162,380,1770
1771
1755
Nevirapine Tablets
1772
Naproxen Suppositories
1756
Newcastle Disease Vaccine, Inactivated
2735
Naproxen Sustained"re1ease Tablets
1757
Newcastle Disease Vaccine, Live (Lentogenic strain)
2736
Naproxen Tablets
1758
Niacin
1776
Narcotine
1798
Niacin Tablets
1777
Narcotine Linctus
1800
Niacinamide
Niacinamide Tablets
380,1775
Nasal Drops, Solutions and Sprays
743
Nasal Powders
743
Nasal Preparations
743
Nickel Standard Solution (l ppm Ni)

Nickel Sulphate
113
Nickel(II) Sulphate
Near-Infrared Spectrophotometry
1776
629
596
596
162,381,1773
1758
Nebivolol Hydrochloride Tablets
1759
Niclosamide
Niclosamide Anhydrous
Nebivo101 Tablets
1759
Niclosamide Dispersible Powder for Veterinary Use
1773
2672
Niclosamide Tablets
2672
Neem
482,2524
Negligible, General Notices

Ne1finavir Mesylate
12
162,379,1760
1774
Nicotinamide
162,380,1775
1776
162,381,1776
Ne1finavir Mesylate Oral Powder
1761
Ne1finavir Mesylate Tablets
1762
Ne1finavir Tablets
1762
Nicotinic Acid
Neomycin Eye Drops
1764
Nicoumalone
1777
162,382,1777
1765
Nicoumalone Tablets
Nifedipine
1778
162,382,1779

Neomycin Sulphate
162,1763
Neomycin Sulphate Eye Drops
1764
Nifedipine Capsules
Neomycin Sulphate Eye Ointment
1765

Nifedipine Tablets
Neostigmine Bromide
162, 1766
Neostigmine Bromide Tablets
1767
NilgiriOi1
1768
Nikethamide
Neostigmine Methylsu1phate
162,379,1767
1780
1781
1782
2498
162,383,1783
1784
Neostigmine Methylsulphate Injection
1768
Nile Blue A
626
Neostigmine Tablets
1767
Nile Blue A Solution

Ninhydrin
Ninhydrin and Stannous Chloride Reagent
626
596
596
596
596
596
596
Neotame
162,1769
Nessler Cylinders
21
Nessler's Reagent
605
Neuroviru1ence (NVT) for Live Viral Vaccines, Test for
214
Neurovirulence (NVT) for Orval Poliomye1ities Vaccine

(OPV), Test for
214
Neutral Insulin
.1498
--1498
Neutral Insulin Inj ection
Ninhydrin Reagent
Ninhydrin Solution
Ninhydrin Solution, Ethanolic
Ninhydrin-Stannous Chloride Solution
Nitofura1
Nitranilic Acid
1787
-------- --.$96
Neutral Red
626
Nifrilrii1ic Acid Solution
596
Neutral Red Solution
626
Nitrate-free Water
620
559
Nitrate Standard Solution (2 ppm N0 3)
629
Neutralised Phthalate Buffer pH 4.2 to 58
2802
IP 2010
INDEX
Nitrate Standard Solution (l 00 ppm NO)
629
Nitrates, Tests for

Nitrazepam
74
162,383,1784
Nitrazepam Tablets
1785
Nitroso R Salt
597
1-Nitroso-naphthol-3,6-disulphonic Acid
Disodium Salt
597
2-Nitroterephthalate
602
163,597,1788
Nitric Acid
596
Nitrous Oxide
Nitric Acid, 1 M
633
Nitrous Oxide, Assay of
Nitric Acid, Dilute
596
Nitroxynil
Nitric Acid, Fuming
596
2674
Nitric Acid, x M
596
Non-Biological Veterinary Monographs
2627
Nitric Oxide
597
Nonadecanoic Acid
Nitrite Titration, Assay for
88
89
163,384,2673
597
597
Nonan-5-one
597,1789
4-Nitroaniline
597
Noradrenaline Acid Tartrate
p-Nitroaniline
597
Noradrenaline Acid Tartrate Injection
Nitroaniline Solution, Diazotised
597
2-Nitrobenzaldehyde
597
o-Nitrobenzaldehyde
597
Noradrenaline-free Adrenaline Acid Tartrate
564
Nitrobenzene
597
Noradrenaline-free Adrenaline Bitartrate
564
4-Nitrobenzoyl Chloride
597
Noradrenaline Injection
1790
p-Nitrobenzoyl Chloride
597
Norepinephrine Bitartrate
1789
4-Nitrobenzoyl Bromide
597
Norepinephrine Bitartrate Injection
1790
p-Nitrobenzoyl Bromide
597
Norethindrone
1791
4-Nitrobenzyl Chloride
597
Norethindrone Tablets
p-Nitrobenzyl Chloride
597
Norethisterone
4-Nitrobenzyl Chloride Solution
597
4-(4-Nitrobenzyl)pyridine
597
Norfloxacin
Nitrofurantoin
162,1786
Nitrofurazone
1787
162,384,1787
Nitrogen
Nitrogen, Assay for
597
87
1790
163,1789
1790
1792
163,385,1791
1792
163,385,1793
1794
Norfloxacin Tablets
1794
Norgestrel
163,1795
1796
Norgestrel and Ethinyloestradiol Tablets
589
Normal Human Immunoglobulin
Nitrogen for Chromatography
597
Normal Immunoglobulin
2574
Nitrogen-free Mercuric Oxide
593
Normal Serum Reagent
597
Nitrogen-free Sulphuric Acid
616
Nortriptyline
Nitrogen monoxide and nitrogen dioxide detector tube

Nitrogen Mustard
Nitrogen, Oxygen-free
21
386
163,1797
1718 . Nortriptyline Hydrochloride Tablets

597
1797
1797
Nitroglycerin Solution, Concentrated
1424
Noscapine
Nitroglycerin Tablets
1425
Noscapine Hydrochloride
163,386,1798
598
Nitromethane
597
Noscapine Linctus
4-(p-Nitrophenylazo)resorcinol
622
Notices
4-Nitrophenyl Disodium Orthophosphate
597
Novobiocin Sodium
4-Nitrophenyl Disodium Phospahet
597
Nuclear Magnetic Resonance (NMR)
181
597
Nucleic Acids
206
Nitrophenyl Phosphate Solution
1800
163,1800
2803
INDEX
Nucleic Acid Amplification Technique

Nystatin
Nystatin Ointment
Nystatin Pessaries
Nystatin Tablets
Nystatin Vaginal Tablets
IP 2010
'237
163,1801
1802
1803
1803
1803
o
Ocimum sanctum
2548
(9Z, 12Z)-Octadeca-9, 12-dienoic acid
591
(9Z, 12Z, 15Z)-Octadeca-9, 12, 15-trienoicacid
591
(9Z)-Octadec-9-enoic acid
598
Octadecanoic Acid
971
2-0ctanol
598
Octan-2-o1
598
Octanesulphonic Acid Sodium Slat
612
Octanoic Acid
598
Octoxinol
598
Octoxinoll0
598
Octoxyleno19
598
Octylamine
598
n-Octylamine
598
sec-OctylAlcohol
598
(4-tert-Octy lphenoxy)nonaethoxy-ethanol
598
(p- tert-Octy lphenoxy)nonaethoxy-ethanol
598
Oestradiol Benzoate
163,387,598,1807
Oestradiol Benzoate Injection
1807
1807
Official and Official Articles, General Notices 11,713,1731
Official Standards, General Notices
11,713,1731
Ofloxacin
163,387,1808
Ofloxacin Infusion
1809
Ofloxacin Ophthalmic Solution
1809
Ofloxacin Tablets
1810
Oil detector tube
21
Ointments, see also under name ofsubstance
743
Ointments, Eye
725
Ointments, 9Phthalmic
. T>lanziipiiie
Olanzapine
Oleic Acid
Omeprazole
725
163;388;-181-1
.. . 1812
163,598,1812
163,388,1813
Omeprazole Capsules
1814
Omissions
xxiii
163,389,1815
Ondansetron Hydrochloride Injection
1816
Ondansetron Hydrochloride Oral Solution
1818
Ondansetron Hydrochloride Tablets
1819
1816
Ondansetron Orally Disintegrating Tablets
1816
Ondansetron Oral Sulution
1818
Ondansetron Tablets
1819
Opalescence Standards
107
Opalwax
2511
Ophthalmic Drops
724
Ophthalmic Ointments
725
Ophthalmic Preparations, Plastic Containers for
690
Opium
2525
Opium Powder
2527
Optical Rotation and Specific Optical Rotation
143
Oracet Blue B
626
626
Oracet Blue B Solution
744
Oral Drops, see also under name ofsubstance
744
Oral Emulsions, see also under name ofsubstance
Oral Liquids, see also under name ofsubstance
744
Oral Powders, see also under name ofsubstance
745
1820
Oral Solutions, see also under name ofsubstance
744
Oral Suspensions, 536, see also under name
of substance
744
Organic solvents, Capillary Electrophoresis for
178
Orcinol
598
Ormeloxifen Hydrochloride
163,389,1821
Ormeloxifen Hydrochloride Tablets
1822
Omidazole
163,1823
Ornidazole Tablets
1824
1825
1826
Orphenadrine Hydrochloride Tablets
1827
1827
ORSPowd&
1820
Orthophosphates; .Testfor-__- . -~_ --75
Ofth6pli6sphoric A c i d ... .. - ... .. . -1909
Oseltamivir Oral Suspension
2804
1828
1829
IP 2010
Oseltamivir Phosphate
INDEX
163,1.827
1840
Oseltamivir Phosphate Capsules
1828
1836
Oseltamivir Phosphate Oral Suspension
1841
1829
Osmic Acid
598
Osmic Acid Solution
598
Oxytetracycline Hydrochloride Capsules
1840
Osmium Tetroxide
598
Oxytetracycline Hydrochloride Injection
1842
Osmolality
144
xiv
Oxytetracycline Hydrochloride Eye Ointment
1841
Other Participants
Other Tests, General Notices
15,717,1735
163,1838
Oxytetracycline Hydrochloride Veterinary Oral Powder 2678

Oxytetracycline Hydrochloride Soluble Powder
2678
Otic Drops
723
1838
Otic Solutions
723
Oxytetracycline Soluble Powder
2678
Oxalic Acid
598
Oxytetracycline Veterinary Oral Powder
Oxalic Acid and Sulphuric Acid
598
Oxytocin
598
Oxytocin Injection
1845
1846
Oxalic Acid-Sulphuric Acid Reagent

Oxazepam
1830
Oxazepam Tablets
1831
Ox Brain, Acetone-Dried
Oxcarbazepine
Oxfendazole
598
163,390,1832
1833
163,390,2674
Oxfendazole Mixture
2675
Oxfendazole Oral Suspension
2675
Oxfendazole Veterinary Oral Suspension

Oxfendazole Veterinary Mixture
2678
163,598,1842
Packaged Dosage Forms, Contents of
193
2562
Packed Red Cells
163,1853
Paclitaxel
Paclitaxel Injection
1854
2675
Paediatric Digoxin Elixir
1220
2675
Palladium Chloride
598
Oxirane
583
Palladium Chloride Solution, Buffered
561
Oxprenolol
391
Palladous Chloride
598
163,391,1834
Oxprenolol Hydrochloride Tablets
1834
Palmityl Alcohol
Oxprenolol Tablets
1834
PAN
Oxyclozanide
163,392,2676
163,586
Palmitic Acid
1042
626
Pancreatin
163,1855
Oxyclozanide Drench
2676
D-Panthenol
163,1856
Oxyclozanide Granules
2677
Pantoprazole Sodium
163,1856
Oxyclozanide Mixture
2676
Pantoprazole Sodium Sustained-release Tablets
1857
Oxyclozanide Oral Suspension
2676
1857
Oxyclozanide Premix
2677
Pantothenol
1856
Oxyclozanide Suspension
2676
Papain
Oxyclozanide Veterinary Oral Suspension

Oxygen
Oxygen, Assay of
Oxygen 93 Per Cent
Oxygen-Flask Method, Assay for
Oxygen-free Nitrogen
Oxytetracycline
2676
163,598,1835
90
163,1835
91
597
163,1836
163,2528
241
Papain Solution
2525
Papaver somniferum
Papaverine Hydrochloride
598
Paper Chromatography
135
Paper Chromatography, Ascending
136
135
Paper Chromatography, Descending

Paracetamol
163,392,598,1859
VoluIQ.e 1: i to xxiii and 1 to 704; Volume 2: xxv to xxviii and 705 to 1722; Volume 3: xxix to xxxii and 1723 to 2829
2805
INDEX
IP 2010
Paracetamol,4-Aminophenol-free
598
I-Pentanol
599
Paracetamol Oral Solution
1860
Pentan-l-01
599
Paracetamol Syrup
1860
Pentanoic Acid
620
Paracetamol Tablets
1861
Pentazocine (FormA)
393
Paraffin Emulsion, Liquid
1863
Pentazocine (Form B)
Paraffin, Hard
164,1862
Pentazocine
Paraffin, Light Liquid
164,598,1863
Paraffin, Liquid
164,598,1862
Pentazocine Hydrochloride Tablets
Paraffin Ointment
1865
164
164
Paraffin, White Soft

Paraffin, Yellow Soft
Paraldehyde
164,1865
Pentazocine Injection
Pentazocine Lactate
Pentazocine Lactate Injection
Pentazocine Tablets
394
164,1873
164,394,1873
1874
1876
164,395,1875
1876
1874
Pararosaniline Chloride
598
Pentobarbital Sodium
Pararosaniline Hydrochloride
598
Pentobarbital Sodium Injection
2679
Pararosaniline Solution, Decolorised
598
Pentobarbital Sodium Tablets
1877
Parenteral Preparations
746
Particle Size by Microscopy
195
Particulate Contamination
196
Pentobarbitone Sodium Injection
2679
Pentobarbitone Sodium Tablets
1877
1877
Pasturella Multocida Vaccine for Chickens
2724
Patents and Trade Marks
164,1877
2679
164,1877
PBS (Phosphate-Buffered Saline)
241
n-Pentyl Alcohol
PBS-BSA Solution
242
Pepper
2522
2475
Pepper, Long
2530
164,1866
Pepper, Small
2531
Peanut Oil
Penicillamine
Peppermint Oil
D-Penicillamine
1866
1868
Pepsin
1868
Peptide Mapping, Assay for
D-Penicillamine Tablets
Peptide Mapping
599
164,2529
164,599,1878
102
2606
Penicillin G Injection
896
Penicillin G Potassium
893
Perchloric Acid
599
895
PerchloricAcid, 0.1 M
633
Penicillin G Sodium
Penicillin V Potassium
1896
Perchloric Acid, x M
599
Penicillin V Potassium Tablets
1897
Perchloric Acid (60 per cent)
599
Penicillin V Tablets
1897
Periodic-Acetic Acids Solution
599
Periodic Acid Reagent
599
Penicillinase Solution
599
Penicillins and Cephalosporins, Tests for
74
Penicillins, Tests for
74
Peroxide-free Ether
Pentaerythritol Tetranitrate, Dilute

Pentamidine Injection
Pelltaiiiidiiie Isethi6iiate
1868
Peroxide Value, Assay for
1870
Peroxyacetic Acid Solution
1872
Perphenazine
__-164;593~t8'71
1879
582
92
599
164,395,1881
... -1872
Pertussis Vaccine (Acellular Component) (Adsorbed)-2366
I-Pentane
599
Pertussis Vaccine (Acellular, Co-purified) (Adsorbed) 2369
n-Pentane
599
Pertussis Vaccine
Pentami<ilil1e Isethionate Injection
2806
2421
INDEX
IP 2010
Phenol Red Solution
626
Phenol Saline Solution
600
Phenol Solution
Phenol in Vaccines and Antisera, Assay for
600
92
1884
Phenoldisulphonic Acid Solution
600
Pethidine Hydrochloride Tablets
1885
Phenolphthalein
Pethidine Injection
1884
1885
Pessaries, see also under name ofsubstance

Peste Des Petitis Ruminants Vaccine, Live
Pethidine
Pethidine Hydrochloride Injection
Pethidine Tablets
Petroleum Ether
pH Values
Pharmaceutical Methods
Phenacetin
1,10-Phenanthroline
0- Phenanthroline
Phenanthroline Solution
Phenazone
Phenindione
Phenindione Tablets
Pheniramine Maleate
Pheniramine Maleate Injection
Pheniramine Maleate Tablets
Pheniramine Tablets
Phenobarbital
Pheno1;>arbital Sodium
Phenobarbital Sodium Injection
Phenobarbital Sodium Tablets
Phenobarbital Tablets
Phenobarbitone
Pheno~arbitone
Injection
Phenobarbitone Sodium Injection
Cf,-
Phenodiazone
Phenol
Phenol, Liquefied
Phenol Reagent
Phenol Red
Phenol Red Reagent
2732
396
164,396,1883
599
Petroleum, Light (Boiling Ranges 30 to 160)

Petroleum Spirit
Petroleum Wax (Microcrystalline)
750
599
599
1696
164,626,1894
Phenolphthalein Solution
626
Phenolphthalein Solution, Dilute

Phenolphthalein-Thymol Blue Solution
626
626
Phenolsulphonphthalein
626
Phenothiazines, Identification of
77
Phenothiazines, Identification of Related Substances in 78
146 Phenoxyacetic Acid

600
185 Phenoxybenzamine Hydrochloride
600
599 2-Phenoxyethanoic Acid
600
599 2-Phenoxyethanol
600,1895
599 Phenoxyethanol
164,600,1895
600 Phenoxymethylpenicillin Potassium
164,1896
600 Phenoxymethylpenicillin Potassium Tablets
1897
164,1886 Phenoxymethylpenicillin Tablets
1897
1887 . Phentolamine Injection
1899
1889 Phentolamine Mesylate
164,398,1898
164,397,1888 Phentolamine Mesylate Injection
1899
1889 Phenylbis-(4-hydroxynaphthyl)methenol
626
1889 (E)-4-Phenyl-3-buten-2-one
600
1889 (E)-4-Phenylbut-3-en-2-one
600
1890 4-Phenylenediamine Dihydrochloride
600
1891 p-Phenylenediammonium Dichloride
600
1892 Phenylephrine Hydrochloride
164,398,1899
1893 Phenylephrine Hydrochloride Injection
1900
1891 Phenylephrine Injection
1900
164,397,600,1890 Phenylhydrazine
600
1892 Phenylhydrazine Hydrochloride
600
164,1891 Phenylhydrazine Hydrochloride Solution
601
1892 Phenylhydrazine Solution
601
1893 Phenylhydrazine-Sulphuric Acid Solution
601
1891 Phenylhydrazinium Chloride
600
601 Phenylmercuric Acetate
164,1901
164,600,1893 Phenylmercuric Nitrate
164,1901
600 Phenylmethyl Silicone Fluid (50 per cent Phenyl)
601
600 Phenylpropanolamine Hydrochloride
164,1902
626 Phenytoin
164,399,1903
626 Phenytoin Capsules
1906
2807
INDEX
IP 2010
Phenytoin Injection
1905
Phosphates (Orthophosphates), Tests for
1907
Phospholipid
601
Phosphomolybdic Acid
601
1906
Phosphomolybdic Acid Reagent
601
Phenytoin Sodium Injection
1905
Phosphomolybdic Acid Solution
601
Phenytoin Sodium Tablets
1906
Phosphomolybdic Acid Spray Solution
601
Phenytoin Tablets
1906
Phosphomolybdic Reagent
601
Phenytoin Sodium
164,399,1904
Phenytoin Sodium Capsules
Phloroglucinol
601
Pholcodine
164,400,1908
Pholcodine Linctus
1908
Phosphatase Enzyme, Alkaline
601
Phosphatase Solution, Alkaline
601
Phosphomolybdotungstic Reagent, Dilute

Phosphoric Acid
Phosphoric Acid, Concentrated
75
601
165,601,1909
1909
Phosphoric Acid, Dilute
601
Phosphoric Acid, x M
Phosphate Buffer pH 5.8 to 8.0
559
Phosphorus Pentoxide
Phosphate Buffer pH 2.0
562
Phosphotungstic Acid
601
206
601
601
562
Phosphotungstic Acid Solution
601
562
Photoglycine
588
562
Phthalaldehyde
601
Phosphate Buffer pH 4.0, Mixed
562
Phthalaldehyde Reagent
601
Phosphate-Albumin Buffered Saline pH 7.2
560,561
Phosphorus
562
Phthalate Buffer, Acid, pH 2.2 to 4.0
559
562
Phthalate Buffer, Neutralised, pH 4.2 to 5.8
559
562
Phthalate Buffer, pH 4.2 to 5.8
559
562
Phthalazine
601
625
562
Phthalein Purple
Phosphate Buffer pH 6.8,0.2 M Mixed
562
Phyllanthus amarus
562
Physical and Physicochemical Methods
Phosphate Buffer pH 7.0 with Azide, Mixed
562
Physostigmine Injection
Phosphate Buffer pH 7.0, 0.067 M Mixed
562
Phosphate Buffer pH 7.0,0.1 M Mixed
562
Physostigmine Salicylate Injection
Phosphate Buffer pH 7.0,0.1 M
562
Picric Acid
602
562
Picric Acid Solution
602
Phosphate Buffer pH 7.5,0.2 M
562
Picric Acid Solution, Alkaline
602
Phosphate Buffer pH 7.5, 0.33 M Mixed
562
Picrolonic Acid
602
Phosphate Buffer pH 8.0, 0.02 M
562
Picrorhiza kurroa
2516
Phosphate Buffer, 0.025 M Standard
562
Pigeon Pox Vaccine, Live
2725
Phosphate Buffer, 0.05 M
562
Pilocarpine Nitrate
Phosphate Buffer Solution
34
Pimozide
Phosphate-Buffered Bromocresol Purple Solution
623
Pimozide Tablets
Phosphate-Buffered Saline
562
Pindolol
Phospbate~BufferedSaline;
pH6:4----------- --------------562
Phosphate~Bl.lffel'ed Saline, pH 7.4 - - 5 6 3
- Phosphate-Citrate Buffer Solution

Phosphate Standard Solution (5 ppm P0 4)
2488
105
1911
165,1910
1911
165,1911
165,400,1912
1913
165,1914
-Pindolol Tablets
Pioglitazone Hydrochloride
--165,-1916
212
Pioglitazone Hydrochloride Tablets
1917
629
1917
2808
INDEX
IP 2010
Piperacillin
165,401,1918
Piperacillin Intravenous Infusion

Piperazine Adipate
1919
165,401,1920
Piperazine Adipate Tablets
1921
165,402,1921
Piperazine Citrate
Piperazine Citrate Elixir
1922
1922
1922
Piperazine Citrate Oral Solution

Piperazine Hydrate
165,602,1923
165,1924
1924

Piper longum
2531
Piper nigrum
2522
Piper retrofractum
Pippali, Large
2530
482,536,2530
Pippali, Small
483,536,2531
Piracetam
165,1925
Piroxicam
165,402,1926
Piroxicam Capsules
Plague Vaccine
1927
2423
Plain Insulin
1498
Plantago ovata
2513
Plasma, Citrated Rabbit
602
2586
Plasma Protein Fraction

Plasma Protein Fraction, Human
2576
2576
Plasma Protein Solution

Plaster of Paris
2576
165,602,1928
Plastic Containers and Closures
685
Plastic Containers for Parenteral Preparations
686
Plastic Containers for Non-Parenteral Preparations
686
690
Plastic Containers for Ophthalmic Preparations

Platinic Chloride
Platinic Chloride Solution
Pods of Cassia
2588
574
574
2424
2538
2426
2430
Poloxamers
165,1928
Polyacrylamide Rod Gel Electrophoresis

602
602
124

602
602
165, 1929
165, 1930
165,1930
Polyethylene Glycol 20,000
602
Polyethylene Glycol Mono(octylphenyl)ether
598
PolymannuronicAcid
781
1932
Polyoxyethylene 80 Sorbitan Monooleate
1932
Polyoxy135 Castor Oil
1931
Polyoxy140 Hydrogenated Castor Oil
1931
Polysaccharide Vaccines, Composition of
205
Polysorbate 20
165,602,1932
Polysorbate 80
165,602,1932
34
Polysorbate 80 Solution
Polystyrene
257,258
1941
Polyvidone
Polyvinylpyrrolidone
1941
Post-Translational Modifications of Proteins
2597
Potash Alum
564
Potassium Acetate
602
Potassium Antimonate
602
Potassium Antimonate Solution
602
Potassium Bicarbonate
602
Potassium Bicarbonate Solution, Saturated Methanolic 602
Potassium Bisulphate
602
Potassium Bromate
603,630
603
Potassium Bromate, 0.0167 M
Potassium Bromate, 0.0333 M
603
Potas$ium Bromide
603
Potassium Bromide, 0.001 M
603
Potassium Bromide IR
603
Potassium Carbonate
603
Potassium Carbonate, 2 M
603
Potassium Carbonate, Anhydrous
603
Potassium Carbonate Sesquihydrate
603
Potassium Chlorate
603
Potassium Chloride
165,603,1933
559
Potassium Chloride, 0.2 M
Potassium Chloride IR
603
Potassium Chlordie and Dextrose Injection
1934
Potassium Chloride and Dextrose Intravenous
Infusion
1934
2809
IP 2010
INDEX
Potassium Hydrogen ()Tartrate
602
604
Potassium Chlordie in Dextrose Injection
1934
1934
Potassium Hydroxide
604
Potassium Chloride in Dextrose and Sodium Chloride

Injection
1935
Potassium Hydroxide, 0.1 M

Potassium Hydroxide, 0.1 M Ethanolic
633
633
Potassium Chloride, Sodium Chloride and Dextrose

Injection
1935
Potassium Chloride, Sodium Chloride and

1935
Potassium Chloride and Glucose Intravenous

Infusion
Potassium Chloride, Sodium Chloride and Glucose

Potassium Chromate
Potassium Chromate Solution
Potassium Citrate
Potassium Clavulanate Dilute
Potassium Cupri-Tartrate Solution
Potassium Cupritartrate Solution, Modified
Potassium Cyanide
Potassium Cyanide Solution
Potassium Cyanide Solution Sp.
Potassium Dichromate
Potassium Dichromate, 0.0167 M
Potassium Dichromate Solution
Potassium Dichromate Solution, Dilute
Potassium Hydrogen Sulphate
1935
603
603
165,1936
165,1937
1938
603
576
603
603
603
603,630
633
603
603
Potassium Dihydrogen Phosphate
604
604
604
604
Potassium Dihydrogen Phosphate, 0.2 M
559
Potassium Dihydrogen Phosphate, x M
604
604
604
604
604
604
Potassium Dichromate Solution UV

Potassium Dihydrogen Citrate
Potassium Dihydrogen Orthophosphate
Potassium Ferricyanide
Potassium Ferricyanide Solution
Potassium Ferricyanide Solution, Dilute
Potassium Ferrocyanide
Potassium Ferrocyanide Solution
Potassium Ferrocyanide Solution, Dilute
Potassium Hexacyanoferrate(II)
Potassium Hexacyanoferrate(III)
Potassium Hydrogen. Carbonate
Potassium Hydrogen Phthalate
PotassillriiHydr6geilPhthalate, 0.05 M
Potassium Hydrogen Phthalate, 0.2 M
Potassium Hydrogen Phthalate, x M
604
604
604
602
604,630
633
559
604
Potassium Hydroxide, xM Ethanolic
604
Potassium Hydroxide in Ethanol (60 per cent), 0.5 M
634
Potassium Hydroxide Solution

Potassium Hydroxide Solution, Dilute Ethanolic
604
604
604
604
Potassium Hydroxide Solution, Ethanolic

Potassium Hydroxide, x M
Potassium Iodate
Potassium Iodate, 0.05 M
.604,630
634
604
Potassium Iodate Solution

Potassium Iodate, x M
Potassium Iodide
604
165,1939
Potassium Iodide, 1 M
Potassium Iodide and Starch Solution
Potassium Iodide Solution
Potassium Iodide Solution, Dilute
Potassium Iodide Solution, Iodinated
Potassium Iodobismuthate Solution
Potassium Iodobismuthate Solution, Acetic
Potassium Iodobismuthate Solution, Acid
Potassium Iodobismuthate Solution, Dilute
Potassium Iodobismuthate Solution, Modified
Potassium Iodoplatinate Solution
Potassium, Limit Test for
604
604
605
605
605
605
605
605
605
605
605
82
605
605
Potassium Mercuri-iodide Solution, Alkaline
605
Potassium Monoethyl SUlphate
605
Potassium Nitrate
165,605,1940
Potassium Permanganate
634
Potassium Permanganate, 0.02 M
Potassium Mercuri-Iodide Solution
Potassium Permanganate and Phosphoric Acid

Solution
Potassium Pennanganate Solution
Potassium Permanganate Solution, Dilute
Potassium Permanganate Solution, Strong
Potassium Permanganate, x' M
605
605
605
605
605
PotassiurnPeii:l:iahgal1ate~Orthophosphoric
.. . Acid Reagent
Potassium Permanganate-Phosphoric Acid Solution
Potassium Phosphate, Dibasic
2810
. 605
605
581
INDEX
IP 2010
Potassium Pyroantimonate
602
Potassium Salts, Tests for
76
Potassium Sodium Tartrate

Potassium Sorbate
Potassium Sulphate
Potassium Tellurite
Potassium Tetraoxalate
Potassium Thiocyanate
Potassium Thiocyanate Solution
Potassium Trihydrogen Dioxalate
Potentiometric Titration
Povidone
Povidone-Iodine
Powder Fineness
Powders for Inhalation
Powders for Injection
Powders for Liposomal Injection
PPF
PPLOBroth
Pralidoxime Chloride
Pralidoxime Chloride Injection
Pravastatin Sodium
Pravastatin Sodium Tablets
Pravastatin Tablets
Praziquantel
Prazosin
Prazosin Hydrochloride
Prazosin Hydrochloride Tablets
Prazosin Tablets
Precipitated Chalk
Precipitated Sulphur
Prednisolone
Prednisolone Sodium Phosphate Injection
Prednisone
Prednisone Tablets
Preface
Pregabalin
Pregabalin Capsules
613
165
605
605
605
605
605
605
147
165,1941
165,1943
1943
194
736
749
742
2576
214
165,403,1944
1944
165,1945
1946
1946
165,1947
1948
403
165,1949
1950
1950
961
616
165,404,1951
165,1954
165,1955
1956
1952
165,404,1957
1958
vii
166,405,1960
1961
166,1963
2622
Premixes
Preparation and Standarisation of Volumetric Solutions 630
Preparations Veterinary
2621
Prepared Calamine
953
Prepared Chloroform
574
Prepared Toluene
618
Pressurised Metered-dose Preparations
727
Primaquine
405
166,406,1963
Primaquine Phosphate Tablets
1964
Primaquine Tablets
1964
Primary Aromatic Amines, Tests for

Primary Standards
Probenecid
71
630
166,406,1965
Probenecid Tablets
1966
Procainamide
407
166,407, 1966
Procainamide Hydrochloride Injection
1967
Procainamide Hydrochloride Tablets
1968
Procainamide Injection
1967
1968
Procaine and Adrenaline Injection
1969
166,408, 1968
Procaine Hydrochloride and Adrenaline Bitartrate

Injection
1969
Procaine Hydrochloride and Epinephrine Bitartrate

1969
Injection
166,408,1970
Procaine Penicillin
Procaine Penicillin Injection, Fortified
1971
Procaine Penicillin with Benzylpenicillin

Injection
1971
Processed Herbs
2467
Processes for the production of recombinant

therapeutic proteins including mAbs
2594
409
Prochlorperazine
1974
Prochlorperazine Injection
Prochlorperazine Maleate
166,1972
1972
Prochlorperazine Maleate Tablets

166,409,1973
Prochlorperazine Mesylate Injection
1974
1972
1974
Procyclidine Hydrochloride Tablets
1975
Volume 1: i to xxiii and Ito 704; Volume 2: xxv to xxviii and 705 to 1722; Volume 3: xxix to xxxii and 1723 to 2829
2811
INDEX
Production, General Notice
Progesterone
IP 2010
1975
13,715, 1733
166,410,2680
Propranolol
412
166,413,1987
Propranolol Hydrochloride Injection
1988
Progesterone Injectable Suspension
1976
Propranolol Hydrochloride Tablets
1989
Progesterone Injection
2680
Propranolol Injection
1988
Propranolol Tablets
1989
166,410,1977
Proguanil Hydrochloride Tablets
1977
n-Propyl Alcohol
Proguanil Tablets
1977
Propyl Gallate
Prokaryotes
2594
Propyl Hydroxybenzoate
Prolonged-release Tablets
Promazine Hydrochloride Injection
753
166,411,2681
2-Propylamine
Propylene Glycol
2682
Propylene Oxide
Promazine Hydrochloride Tablets
1978
Propylparaben
Promazine Injection
2682
Propylthiouracil
Promazine Tablets
1978
Promethazine
411
166,412,1979
Propyphenazone
Protamine Sulphate
606
166,606,1990
1991
590
166,606,1990
606
166,1991
166,414,1992
1993
166,414,1994
166,1995
Promethazine Hydrochloride Injection
1979
Promethazine Hydrochloride Oral Solution
1980
Prothionamide
Promethazine Hydrochloride Syrup
1980
Promethazine Hydrochloride Tablets
1981
Protein, Assay for
Promethazine Injection
1979
Protein Content
Promethazine Oral Solution
1980
Protriptyline Hydrochloride
Promethazine Syrup
1980
Protriptyline Hydrochloride Tablets
1999
Promethazine Tablets
1981
1999
166,1982
1996
166,1996
1997
103
206
166,415,1998
1982
Provisions Application to Monographs and Test

Methods
12,714,1732
Prompt Insulin Zinc Suspension
1502
Pseudoephedrine
Propanal
1,2-propanediol
606
Pseudoephedrine Hydrochloride
415
166,416,1999
1990
Pseudoephedrine Hydrochloride Syrup
2000
I-Propanol
606
Pseudoephedrine Hydrochloride Tablets
2001
Propan-l-ol
606
2000
2-Propanol
606,1520
2001
Propan-2-ol
606,1520
Pseudomonas aeruginosa
2-Propanol, Anhydrous
606
Psoralen
Propan-2-ol, Anhydrous
606
Pteroy19lutamic Acid
563
Pumice Powder
2-Propanone
Propane
Propellants
Proj:>ioJialdeliyae
Propionic Acid
Propofol
Propofol Injection
1983
45
166,2001
1384
606
Pumice Stone
606
727 Punarnava
483,537,2532
----606- Purified Casein
166;1984 Purified 1,2~Dichlofoethane
---577
166,413,1985 Purified Protein Derivative (PPD), Bovine Tuberculin 2752
1986 Purified Rayon
167,2046
2812
INDEX
IP 2010
Purified Siliceous Earth

Purified Talc
Purified Water
609
2181
620,2314
606
Purine
Pyrantel Embonate
Pyrantel Pamoate
Pyrantel Pamoate Oral Suspension
Pyrazinamide
2002
166,2002
2003
166,416,2004
2005
166,2014
Quinalbarbitone Sodium Tablets
2015
2015
Quinaldine Red
627
Quinaldine Red Solution
627
Quinhydrone
606
2016
Quinidine Bisulphate
Quinidine Sulphate
166,606,2016
Quinidine Sulphate Tablets
2017
Pyridine
606
Quinidine Tablets
2017
Pyridine-Acetic Anhydride Reagent
606
Quinine
606
Pyridine, Anhydrous
606
Quinine Acid Hydrochloride
Pyridine Bromide Solution
606
Quinine Acid Hydrochloride Injection
2023
Pyridine, Dehydrated
606
Quinine Acid Sulphate
2019
Quinine Acid Sulphate Tablets
2020
Pyridoxine Hydrochloride
166,417,2005
.Pyridoxine Hydrochloride Tablets
2006
Quinine Bisulphate
Pyridoxine Tablets
2006
Pyridylazonaphthol
626
Quinine Dibydrochloride
1-(2-Pyridylazo)-2-naphthol
626
Quinine Dihydrochloride Injection
Pyridylazonaphthol Solution
627
Quinine Sulphate
2021
166,2019
2020
167,2021
2023
167, 606, 2025
Pyrilamine Injection
2669
Quinine Sulphate Tablets
2026
Pyrilamine Maleate
1651
Quinine Tablets
2026
Pyrilamine Maleate Injection
2669
Quiniodochlor
Pyrilamine Maleate Tablets
1652
Quiniodochlor Cream
2028
Pyrilamine Tablets
1652
2029
Quiniodochlor Tablets
2030
Pyrimethamine
166,417,606,2007
167,418,2027
2008
Quiniodochlor and Hydrocortisone Cream
2031
Pyrimethamine and Sulphadoxine Tablets
2009
Quiniodochlor and Hydrocortisone Ointment
2032
Pyrimidine-2,4,5-triol
588
Quinol
588
Pyrocatechol
573
Quinolin-8-o 1
588
Pyrogallol
606
Quinoline
606
Pyrogallol Solution, Alkaline
606
Quinoline Solution
607
Pyrogen-free Saline Solution
607
Pyrogens
36
R
607
Rabbit Erythrocyte Suspension
Rabeprazole Sodium
Quantities, General Notices

Quetiapine Fumarate
15,717,1735
166,2013
167,2037
2037
Rabeprazole Sodium Tablets

Rabeprazole Tablets
2037
Quetiapine Fumarate Tablets
2014
Rabies Antiserum
2435
Quetiapine Tablets
2014
Rabies Vaccine (Human)
2436
Rabies Veterinary Vaccine, Inactivated (Cell Culture)
2733
Quicklime
571
Quinalbarbital Sodium
2014
Rafoxanide
Quinalbarbital Sodium Tablets
2015
Rafoxanide Mixture
167,2683
2813
2683
IP 2010
INDEX
2683
Rafoxanide Suspension
Rafoxanide Veterinary Mixture
2683
Rafoxanide Veterinary Oral Suspension
2683
Ramipril
167,418,2038
Ramipril Capsules
2039
Ramipril Tablets
2040
Ramipril and Hydrochlorothiazide Tablets
2041
2735
Ranikhet Disease Vaccine, Live (Lentogenic Strain)
2736
Ranikhet Disease Vaccine, Live (Mesogenic Strain)
2737
Ranitidine HydrochlOlide
167,419,2043
Ranitidine Hydrochloride Injection
2044
Ranitidine Hydrochloride Tablets
2045
2044
Ranitidine Tablets
2045
Rappaport Vassiliadis Salmonella Enrichment
Broth Medium
47
Rauwolfia serpentina Root
2533
Rauwolfia serpentina Powder
484,538,2535
Rauwolfia Serpentina Tablets
539,2535
Raw Opium
2525
Rayon, Purified
167,2046
Reagent, Dragendorff
582
Reagents and Solutions
557
Reagents and Solutions, General Notices
15,717,1735
Reagents, General
563
Recommended Solutions and Culture Media
46
Recrystallised Iodine Pentoxide
589
Red Blood Cells
241
Red Litmus Paper
628
Reducing Mixture
587
Reference Buffer Solutions
146
Reference Data
253
Reference Solution
107
Reference Substances (lPRS)
678
Reference Substances, General Notices
15,717,1735
Refined Sugar
2166
Refractive Index
171
Reinforced Medium for Clostridia Medium
48
R i b o s e - - -----------'----206
Related Foreign Steroids, Identificaiionof
-78
Related Substances in Barbiturates, Identification of
78
Related Substances in Phenothiazines, Identification of 78
Related Substances in Sulphonamides, Identification of 78

Relative Density, Weight Per Millilitre and
174
Relative Density
171
Relative Retntion, Liquid Chromatography for
135
Reo Virus Vaccine, Inactivated
2737
Reo Virus Vaccine, Live
2738
Reserpine
167,419,2047
Reserpine Injection
2048
Reserpine Tablets
2049
Residual Solvents
650
Resolution Factor, Liquid Chromatography for
134
Resorcin
607
Resorcinol
607
Resorcinol Solutions
607
Rh Blood-grouping Reagents
251
Rh Group of Donors, Determination of
251
Rhodamine B
607
Ribavilin
167,420,2050
2051
Ribavirin Solution for Inhalation
2051
Riboflavine
167,420,2052
Riboflavine-5-phosphate (Sodium Slat)
2053
Riboflavine Sodium Phosphate
167,2053
Riboflavine Tablets
2054
l-~-D-Ribofuranosyluracil
620
Rifampicin
167,421,2054
Rifampicin Capsules
2055
2059
Rifampin and Isonicotinylhydrazid Tablets
2059
Rifampicin, Isoniazid and Ethambutol Tablets
2061
Rifampicin, Isoniazid and Pyrazinamide Tablets
2063
Rifampicin, Isonicotinylhydrazid and Ethambutol
2061
Rifampicin, Isonicotinylhydrazid and Pyrazinamide
2063
Tablets
Rifampicin, Isoniazid, Pyrazinamide and Ethambutol
2065
Tablets
Rifampicin, Isonicotinylhydrazid, Pyrazinamide
2065
and Ethambutol Hydrochloride Tablets
2056
Rifampicin Tablets
--2054
Rifampin
2055
Rifampin Capsules
2058
Rifampin Tablets
2814
II
INDEX
IP 2010
2739
Salbutamol Syrup
2088
Ringer-Lactate Solution for Injection
2129
Salbutamol Tablets
2088
Ringer's Injection
2117
Salicylaldehyde
Ringer-Lactate Solution for Irrigation
607
76
2130
Salicylates, Tests for
Ringer-Lactate Solution with Dextrose for Injection
2125
Salicylic Acid
Ringer-Lactate Solution with Dextrose Injection

Half Strength
2127
Saline pH 6.4, Phosphate-buffered
562
Ringer's Solution
2117
Saline pH 7.4, Phosphate-buffered
563
Ritonavir
167,423,607,2089
2116
Saline, Hypertonic
167,421,2066
Saline, Phosphate-buffered
562
Ritonavir Capsules
2067
Saline Solution
607
Ritonavir Tablets
2068
Saline Solution, Heparinised
33
Saline Solution, Pyrogen-free
607
Saline Solution, Sterile
607
Ronidazole
167,422, 2684
2684
592
Rosaniline Hydrochloride
167,2070
Rosiglitazone Maleate Tablets
2070
2070
167,2071
Rosuvastatin Calcium Tablets
2072
2072
Roxithromycin
167,2073
2075
Rubella Vaccine (Livlt)
2439
Rubia cordifolia
2521
Russell's Viper Venom Specific Factor X

Activator (RVV)
247
Henium Oxychloride, Ammoniated
627
Ruthenium Red
627
Ruthenium Red Solution
627
2515
Sallaki Gum
167,424,2090

Inhalation
2091
Salmeterol and Fluticasone Propionate Powder for

Inhalation
.
2091
45
Sahnonella
Salmonella Abortus Equi H Antigen
2753
Salmonella Abortus Equi Vaccine
2740
Salmonella Pullorum Antigen
2754
Salmonella Pullorum Plain Antigen
2754
Salmonella Pullorum Positive Serum
2754
Salmonella Vaccine, Inactivated
2740
Saponification Value, Assay for
93
167,424,2092
167,425,2093
2094
Saquinavir
Saquinavir Mesylate
Saquinavir Mesylate Tablets
2474
Sariva
s
Saccharin
Sarpagandha
47 . Sarpgandha Powder
167,2081 Sarpgandha Tablets
Saccharin Sodium
167,2082
Saccharin, Soluble
2082
486,544,2533
Sabouraud Dextrose Agar Medium with Antibiotic(s)
2535
2535
Salbutamol
167,422,2083
Saturated Methanolic Potassium Bicarbonate

Solution
Saunf
602
484,540,2536
2544
2084
Saunth
Salbutamol Inhalation Aerosol
2084
Sauton's Medium, Dilute
34
Salbutamol Injection
2087
Salbutamol Sulphate
167,423,2085
Saution's Fluid Medium
34
Saution's Solution, Dilute
34
Scientific Body
xi
Salbutamol Sulphate Injection
2087
Salbutamol Sulphate Syrup
2088
Scopolamine Butylbromide
1466
2088
Scopolamine Butylbromide Injection
1467
Salbutamol Sulphate Tablets
Volume 1: i toxxiii and 1 to 704; Volume 2: xxv to xxviii and 705 to 1722; Volume 3: xxix to xxxii and 1723 to 2829
2815
INDEX
IP 2010
Scopolamine Butylbromide Tablets
1468
Silica Gel for Chromatography
607
Scopolamine Hydrobromide
1469
Silica Gel F254
607
Scopolamine Hydrobromide Injection
1470
Silica GelG
607
Scopolamine Hydrobromide Tablets
1471
SilicaGel G/UV254
608
Secnidazole
2095
SilicaGel GF254
608
Secnidazole Tablets
2096
Silica Gel H
608
Secobarbital Sodium
2014
Silica Gel H, Silanised
608
Secobarbital Sodium Tablets
2015
Silica Gel H/UV254
608
Secobarbitone Sodium
2014
Silica Gel HF254
608
Secobarbitone Sodium Tablets
2015
Silica Gel HF254, Silanised
608
607
608
Selenium Dioxide
607
Silica Gel, Self-indicating
Semicarbazide Acetate Solution
607
Silica Gel, Strong Anion-exchange
Semicarbazide Hydrochloride
607
Silicates, Tests for
Senna Dry Extract
486,542,2539
Senna Fruit
2538
Senna Tablets
543,2540
. Senna Leaf
485,2537
Senna Pods
485,541,2538
Serratiopeptidase
2097
2098
Serum Gonadotrophin for Veterinary Use
2685
Serum Gonadotrophin Injection for Veterinary Use
2686
Serum Solution
607
Serum Stock Solution
607
Sesame Oil
607
Shatavari
Shati
487,545,546,2540
487,547,2542
Shell Pessaries
750
Shell Suppositories
750
167,2543
Shellac
Shigella
45
Sialic Acid
Sieves
76
Siliceous Earth, Chromatographic
608
Siliceous Earth, Purified
609
609
609
Silicon Dioxide
Silicon Dioxide, Anhydrous
2099
Silicon Dioxide, Colloidal
609
609
Silicone Oil
SilverAmmonio-Nitrate Solution
Silver Compounds, Test for
Silver Nitrate
76
167,609,2102
Silver Nitrate, 0.1 M
634
Silver Nitrate, x M
Silver Oxide
609
609
609
609
609
609
609
609
Silver Standard Solution (5 ppm Ag)
629
Silver Nitrate-Pyridine Reagent

Silver Nitrate Solution
Silver Nitrate Solution, Arnmonical
Silver Nitrate Solution, Dilute
Silver Nitrate Solution in Pyridine
Silver Nitrate Solution, Methanolic
207
Simethicone
1230
22
Simvastatin
167,426,2103
Silanised Diatomaceous Support, Acid-washed
577
Simvastatin Tablets
Silanised Silica Gel H
608
Single-Dose Preparations, Uniformity ofWeight
Silanised Silica Gel HF254
608
Single-Dose Preparations, Uniformity of Content
192
192
SI Units of Weights and Measures
702
Size-Exclusion Chromatography
136
Sildenafil Citrate
Sildenafil Citrate Tablets
SildenafilTliblefs
167,425,2100
2101
~~~2101~SheepPoxVaccineibiveAttenuated
.. -
2104
2741
Silica Gel, Anhydrous
607
Snake Venom Antiserum
2441
Silica Gel, Butylsilyl
607
Snake Antivenin
2441
2816
INDEX
IP 2010
Snake Antivenom Serum
2441
Snake Venom Antitoxin
2441
Soda Lime
Sodium
Sodium Acetate
Sodium Chloride and Dextrose Intravenous

Infusion, Compound
2115
609
Sodium Chloride and Fructose Injection
2114
609
Sodiium Chloride and Fructose Intravenous Infusion
2114
Sodium Chloride and Frctose Infusion
2114
167,609,2105
Sodium Acetate, Anhydrous
609
Sodium Chloride and Glucose Injection
2113
Sodium Acetate Solution, 0.1 M
609
Sodium Chloride and Glucose Intravenous Infusion
2113
Sodium Acid Citrate
609
Sodium Chloride and Invert Sugar Intravenous

Infusion
1506
Sodium Chloride Hypertonic Injection
2116
Sodium Acid Phosphate
2121
Sodium Acid Sulphite
610
Sodium Alginate
167,2106
610,2116
Sodium Chloride Injection
Sodium Alizarine Sulphonate
622
Sodium Chloride Injection, Compound
2117
Sodium 4-anilinoazobenzene-3-sulphonate
625
Sodium Chloride Intravenous Infusion
2116
Sodium Chloride Irrigation Solution
2118
Sodium Aminosalicylate
167,426,2107
Sodium Aminosalicylate Tablets
2108
Sodium Chloride Solution
Sodium Antimony Gluconate
2136
Sodium Chloride Solution, Compound
2137
Sodium Citrate
Sodium Antimony Gluconate Injection
610
2217
168,610,2118
Sodium Arsenite
609
Sodium Cobaitinitrite
610
Sodium Arsenite, 0.1 M
609
Sodium Cobaitinitrite Solution
610
610
Sodium Cupri-Citrate Solution
576
Sodium I-Decasulphonate
610
Sodium I-Decasulphonate Solution
610
Sodium 2,2-[(diazoamino)di-p-phenylene]bis(6methylbenzothiazole-7-sulphonate)
627
Sodium Arsenite Solution

Sodium Ascorbate
167,2109
Sodium Benzoate
167,2110
Sodium Bicarbonate
167,610,2111
Sodium Bicarbonate, x M
610
SodiumBicarbonate Injection
2111
Sodium Diatrizoate
Sodium Bicarbonate Intravenous Infusion
2111
168,427,2119
2120
Sodium Bicarbonate Solution
610
Sodium Diethyldithiocarbamate
610
Sodium Bismuthate
610
Sodium Diethyldithiocarbamate Solution
610
Sodium Bisulphite
610
Sodium Dihydrogen Phosphate
610
Sodium Bisulphite Solution
610
Sodium Dihydrogen Phosphate Dihydrate
Sodium Butanesulphonate
610
Sodium Dihydrogen Phosphate, x M
610
Sodium Butanesulphonate, xM
610
Sodium 4'-Dimethylaminoazobenzene-4-sulphonate
625
Sodium Carbonate
168,610,2112
Sodium Carbonate, xM
610
610,630
Sodium Carbonate, Anhydrous
Sodium Dodecyl Sulphate, 0.001 M
634
Sodium Dodecyl Sulphate Polyacrylamide Gel

Electrophoresis (SDS-PAGE)
124
610
Sodium Carbonate Solution, Dilute
610
Sodium Fenocyanide
989
Sodium Fluoride
Sodium Carboxymethyl Starch
168,2135
2132
Sodium Disulphite
Sodium Carbonate Solution

Sodium Carboxymethyl Cellulose
168,2121
610
168,611,2122
168,2122
168,610,630,2112
Sodium Fonnate
611
Sodium Chloride and Dextrose Injection
2113
Sodium Fusidate
168,2123
Sodium Chloride and Dextrose Injection, Compound
2115
Sodium Heptanesulphonate
Sodium Chloride and Dextrose Intravenous

Infusion
2113
Sodium Chloride
611
Sodium Heptanesulphonate, 0.025 M
611
Sodium Heptanesulphonate, Monohydrate
611
2817
INDEX
IP 2010
Sodium Hexacyanoferrate(II)
610
Sodium Hexanesulphonate
611
Sodium Hexanesulphonate, 0.03 M
611
Sodium Hexanitritocobaitate(III)
Sodium Hyaluronate
610
611
Sodium Hyaluronate Stock Solution
611
Sodium Hydrogen Carbonate

Sodimn Hydroxide
211
168,611,2124
Sodium Lactate with Dextrose Intravenous Infusion,

Half Strength Compound
2127
Sodium Lactate with Dextrose Intravenous

Infusion, Modified Compound
2128
Sodium Lauryl Sulphate
168,612,2131
Sodium Laury1Sulphate, xM
612
Sodium Mercaptoacetate
613
Sodium Metaarsenite
609
Sodium Hydroxide, 0.1 M Ethanolic
634
168,612,2132
Sodium Hydroxide, 0.2 M
559
Sodium Metaperiodate
613
Sodium Hydroxide, 1M
634
Sodium Methoxide, 0.1 M
635
Sodium Methyl Hydroxybenzoate
2132
Sodium Hydroxide BET, 0.1 M
29
Sodium Hydroxide, Ethanolic

Sodium Hydroxide, Methanolic
611
Sodium Methylparaben
611
611
Sodium Molybdate
Sodium Molybdotungstophosphate Solution
612
612
Sodium Hydroxide Solution
168,2132
612
Sodium Hydroxide Solution, Dilute

Sodium Hydroxide, x M
611
Sodium 1,2-Naphthaquinone-4-sulphonate
611
Sodium Nitrate
612
Sodium Hydroxide, xM Ethanolic
611
Sodium Nitrite
612
Sodium-1-( I-hydroxy-2-naphthylazo)-5-nitro-2naphthol-4-sulphonate
624
Sodium-2-hydroxy-1-(2-hydroxy-1naphthylazo)-naphthalene-4-sulphonate
Sodium Hypobromite Solution
Sodium Hypobromite Solution, Alkaline
Sodium Hypochlorite Solution
Sodium Hypochlorite Solution (3 per cent Cl)
Sodium Hypochlorite Solution (3.5 per cent Cl)
Sodium Hypophosphite
Sodium Hyposulphite
Sodium Hyposulphite Injection
Sodium Indigotindisulphonate
Sodiumlodide
Sodium Lactate and Dextrose Injection, Compound
Sodium Lactate and Dextrose Injection, Half
Stength Compound
Sodium Lactate and Dextrose Injection, Modified
Compound
623
611
611
611
612
612
612
2138
2138
589
612
2125
2127
2128
2125
Sodium Nitrite, 0.1 M
635
Sodium Nitrite Solution
612
Sodium Nitroprusside
612
Sodium Nitroprusside-Carbonate Solution
612
Sodium Nitroprusside Solution
612
Sodium Nitroprusside Solution, Alkaline
612
Sodium Octanesulphonate
612
Sodium Octanesulphonate, 0.02 M
612
Sodium 4-0ctyl Sulphate
612
Sodium Octyl Sulphate
612
Sodium Oxalate
612
Sodium PAS
2107
Sodium PAS Tablets
2108
Sodium Pentacyanonitrosylferrate(III) Dihydrate
612
Sodium 1-Pentanesulphonate
612
Sodium Pentanesulphonate
612
Sodium Perchlorate
613
Sodium Periodate
613
613
613
Sodium Lactate Injection, Compound
2129
Sodium Periodate Solution

Sodium Peroxide
Sodium1fol<::tfolte Intravenous Infusion

Sodium Lactat~fu~~~~~~usI~fu;i~~,
2125
Sodium Phosphate
G()rnR~~nf~~--2129
168,613,2133
SodiU1nPhosphate;-Anhydrous~---~-------.-581,.613.
Sodium Lactate Solution for Irrigation, Compound
2130
Sodium Phosphafe Solution
.. ..~ 613
Sodium Lactate with Dextrose Intravenous

Infusion Compound
Sodium Phosphate, Tribasic
613
2125
Sodium Pickrate Solution, Alkaline
602
2818
IP 2010
INDEX
Sodium Polymannuronate
2106
Sodium Potassium Tartrate
613
Sodium Propyl Hydroxybenzoate
2134
Sodium Propylparaben
168,2134
Sodium Pyrophosphate
613
Sodium Pyrosulphite
Sodium Salicylate
2132
168,427,613,2134
Sodium Salicylate Solution
613
Sodium Salt ofN-Chlorotoulene-p-sulphonamide
573
Sodium Salt of2,6-dichloro-N-(4-hydroxyphenyl)-I,4benzoquinone monoimine

578
Sodium Salts, Tests for
76
Sodium Silicate
613
168,428,2135
168,2136
2137
Soluble Fluorescein
1363
Soluble Insulin
1498
Soluble Phenobarbitone
1891
Soluble Phenobarbitone Injection
1892
Soluble Phenobarbitone Tablets
1893
Soluble Pentobarbitone
1877
Soluble Quinalbarbitone
2014
Soluble Saccharin
2082
Soluble Starch
614
Soluble Tablets
753
Solution, General Notices
12
Solutions, Reagents and
557
Solutions, Standard
628
Solutions, Volumetric
631
Solutions Volumetric Reagents and
630
626
Sodium Sulphate, Anhydrous
613
Solvent Blue 19
Sodium Sulphide
613
Solvent Ether
582
Sodium Sulphide Solution
613
Solvent Yellow 94
584
Sodium Sulphite
613
Sorbic Acid
Sodium Sulphite, Anhydrous
613
Sorbide Dinitrate, Diluted
1520
Sodium Tartrate
613
Sorbide Dinitrate Tablets
1522
Sodium ()Tartrate
613
Sorbide Mononitrate Tablets
1525
Sodium Tetraborate
570
Sorbide Nitrate, Diluted
1520
Sodium Thioglycollate
613
Sorbide Nitrate Tablets
1522
Sodium Thiosulphate
168,613,2138
Sodium Thiosulphate, 0.1 M
635
Sodium Thiosulphate, x M
613
Sodium Thosulphate Injection
2138
Sodium Tungstate
Sodium Valproate
613
168,428,1241,2139
Sodium Valproate Elixir
2140
2140
Sodium Valproate Sustained-release Tablets
1241
SodiumValproate Tablets
2140
Sorbitan Oleate
Sorbitol
168,429,2141
168,2142
168,613,2142
o-Sorbitol
2142
Sorbitol Solution (70 Per cent) (Crystallising)
2144
Sorbitol (70 Per cent) (Crystallising)
2144
Sorbitol (70 Per cent) (Non-crystallising)
2144
Sorbitol Solution (70 Per Cent) (Non-crystallising) 168,2144

Soyabean-Casein Digest Medium
58
Specific Optical Rotation, Optical Rotation
143
168,429,2687
Soft Capsules
722
Spectinomycin Hydrochl.oride
Soft Gelatin Capsules
721
Spectinomycin Hydrochloride Injection
2688
164,1863
2688
164,1864
Spectrophotometry, Infra-red
111
117
Soft Paraffin, White

SoftParaffin, Yellow
Solochrome Black
624
Spectrophotometry, Ultra-violet and Visible
Solochrome Dark Black
623
Spiramycin
Solubility, General Notices
14,716,1734
Spironolactone
Solubility
147
Soluble Aspirin Tablets
843
Squalane
168,2689
168,430,2147
2819
2148
613
INDEX
IP 2010
Sterilisation
Standard solution for the determination of water
559
33
562
628
614
Standard Suspension
107
Storage, General Notices
Standard Hydrochloric Acid AsT
587
614
614
614
614
Storage Containers, General Notices
Standard Buffer Solutions

Standard Histamine Solution
Standard Phosphate Buffer, 0.025 M
Standard Solutions
Stannous Chloride
Stannous Chloride Solution
Stannous Chloride Solution AsT
Stannous Chloride Solution, Dilute
Staphylococcus aureus
Starch
Starch Iodate Paper
Starch-iodide Paper
Starch Iodide Paper
Starch Iodide Solution
Starch Mucilage
Starch, Sodium Carboxymethyl
Starch, Soluble
Starch Solution
Starch Solution, Iodide-free
Starch Substrate
Statement of Content, General Notices
Static Head-Space Gas Chromatography
46
168,614,2543
628
628
628
614
614
2135
614
614
614
614
14,716,1734
129
Stavudine Capsules
Stavudine and Lamivudine Tablets
Stearic Acid
Stearic Anhydride
Stearyl Alcohol
Sterility
56
Steroids, Assay of
93
Stilboestrol
Streptokinase
Streptokinase Bulk Solution
Streptomycin.Sulphate Injection
Streptomycin Sulphate Tablets
168,431,2156
2156
16,718,1736
16,718,1736
168,2157
2617
2159
2162
168,2161
2162
2163
2163
Strong Ammonia Solution
566
Strong Ammonia-Ammonia Chloride Solution
565
608
623
1460
605
Strong Anion-Exchanges Silica Gel

Strong Bromophenol Blue Solution
Strong Hydrogen Peroxide Solution
Strong Potassium Permanganate Solution
Strongly Acidic Styrene-Divinylbenzene
Cation-Exchange Resin
Strongly Basic Anion Exchange Resin
Strontium Chloride
660
Styrene-Divinylbenzene, Cation-Exchange Resin
168,430,2149
2151
2153
2152
168,614,2154
614
168,2155
2741
Styrene-Divinylbenzene, Cation-Exchange Resin,

Strongly Acidic
Statistical Analysis of Results

Stavudine
647
Sterile Plastic Containers for Blood and Blood

Components
693
Sterile Containers ofPlasticised Poly(vinyl chloride)

for Blood and Blood Components Empty
695
615
568
614
614
615
615
Substrate Plasma-Deficient in Clotting Factor V
Substrate Solution
241
168,431,2164
2165
Sucralose
168,432,2165
Sucrose
168,615,2166
SudanRedG
627
627
Sudan Red I
591
Sugar of Lead
2166
Sugar, Refined
terile G<:>I'!!ail1erofPlasticised Poly(vinyl chloride)

Sulfiram
for Blood cont'atmng ana~An1icoa-gi.ilaiirSOli.ili~696~O;:;sulfonation~~
2669
._.. ~_~-_. __.-~~
252.8.
Sterile Saline Solution

Sterile Water for Injection
620
2316

Sulphacetamide Sodium
2820
2168
168,433,2167
IP 2010
INDEX
Sulphacetamide Sodium Eye Drops

SUlphadiazine
2168
Sulphuric Acid, 0.5 M
168,434,2169
Sulphuric Acid, Dilute
616
616
635
2170
Sulphuric Acid, x per cent Ethanolic
Sulphadiazine and Trimethoprim Oispersible Powder
2691
Sulphuric Acid, 0.25 M Ethanolic
635
Sulphadiazine and Trimethoprim Injection
2690
Sulphuric Acid, x per cent Methanolic
616
2692
Sulphuric Acid, Nitrogen-free
616
Sulphuric Acid x per cent
616
Sulphuric Acid, x M
616
Sulphuric Acid, x M Ethanolic
616
Sulphuric Acid, x M Methanolic
616
Sulphuric Acid-Formaldehyde Reagent
616
Sulphadiazine and Trimethoprim Mixture

Sulphadiazine and Trimethoprim Veterinary
Oral Powder
2691
Sulphadiazine and Trimethoprim Veterinary

Oral Suspension
Sulphadoxine
2692
169,434,2170
2009
Sulphadoxine and Pyrimethamine Tablets

Sulphaquinoxaline
432,2693
2694
Sumatriptan
169,2173
2174
Sunthi
488,548,2544
Sulphamethizole
169,435,2171
Sunthi Extract
488,549,2545
SUlphamethoxazole
169,435,2172
Suppositories, see also under name ofsubstance
SulphamicAcid
615
Symmetry Factor, Liquid Chromatography for
4-Sulphamoylbenzoic Acid
615
Synthetic Retinol Concentrate (Oily Form)
SuIphanilamide
615
Synthetic Retinol Concentrate (Powder Form)
750
2164
2165
2742
134
2305
2306
Synthetic Retinol Concentrate (Waterdispersible Form)
2306
Sulphamethoxazole and Trimethoprim

Oral Suspension
2265
Suxamethonium CWoride Injection
Sulphamethoxazole and Trimethoprim Tablets
2266
Suxamethonium Chloride
Sulphanilic Acid
615,631
SUlphanilicAcid Solution, Diazotised
615
Sulphaquinoxaline
169
Synthetic Vitamin A Concentrate (Oily Form)
2305
Sulphate Standard Solution (10 ppm SO4)
629
Synthetic Vitamin A Concentrate (Powder Form)
2306
Sulphate Standard Solution (10 ppm S04)' Ethanolic
629
Synthetic Vitamin A Concentrate (Waterdispersible Form)
2306
Sulphated Ash, Limit Tests for

SUlphates, Limit Tests for
SUlphates, Tests for
Sulphathiazole
Sulphathiazole Sodium
Sulphoethomidine
Sulphomolybdic Acid Solution
4-Sulphamoylbenzoic Acid
82
82
76
Syrups, see also under name ofsubstance
744
2518
Syzigium aromaticum
615
169,436,2695
2170
T
Tables
699
615
Tablets, see also under name ofsubstance .
751
615
Tablets and Capsules, Disintegration Test for

Tablets for Use in the Mouth
187
754
Sulphosalicylic Acid
615
TBS (Tris-Buffered Saline)
241
Sulphur Dioxide
616
Talc
SUlphormethoxine
2170
Sulphur Dioxide, Assay for
94-
Talcum
Sulphur dioxide detector tube
21
Tamoxifen
Sulphur in Organic Compounds, Tests for
76
Tamoxifen Citrate
169,2181
2181
436
169,437,2181
Sulphur, Precipitated
616
Tamoxifen Citrate Tablets
2183
Sulphuric Acid
616
Tamoxifen Tablets
2183
2821
INDEX
Tannic Acid
Tannic Acid Solution
Tannin
Tartaric Acid
L-Tartaric Acid
Tartrates, Tests for

Taxol
Tehnisartan
Tehnisartan Tablets
Temozolomide
Temperatures, General Notices
IP 2010
169,437,2184
616
616
616
169,616,2185
2185
76
1853
169,438,2186
2187
169,438,2193
2193
12
Temperature and Distillation Range or Boiling Range
119
121
140
2191
439,2188
2190
Temperature or Congealing Range

Temperature or Melting Range
Tenofovir Disoproxil Fumarate and Emtricitabine
Tablets
Terazosin Hydrochloride Dihydrate
Terbutaline Inhalation Aerosol
Terbutaline Sulphate Inhalation Aerosol
Terbutaline Sulphate Inhalation
Terbutaline Sulphate Injection
Terbutaline Sulphate Tablets
Terbutaline Tablets
Terminalia aIjuna Bark
Terminalia bellirica
Terminalia chebula
Test Animals, General Notices
Tests for Abnormal Toxicity
Test for Colony-forming Units (CFlJ)
33
Test for Depressor Substances

Test for duck enterities virus
225
Test for duck and goose parvoviruses
226
Test for extraneous agents using embryonated

hens'eggs
Test for egg drops syndrome virus
Test for Marek's Disease Virus
Test for turkey rhinotracheitis virus
Test for Haemolysins
Test for Histamine
Test for Neurovirulence (NVT) for Live Viral Vaccines
Test for Neurovirulence (NVT) for Oral Poliomyelities
Vaccine (OPY)
Test for Pyrogens
Tests for Sterility
Test for Absence of Avian Mycoplasmas in Live

Viral Poultry Vaccines
219
Test for Absence of Mycoplasmas
210
relit foJ' A1:>sence of l\I()l1:~"iflIl.Iv1:ycoplasmasand
221
224
224
224
35
35
214
214
36
56
64
222
Test for Urinary Excretion ofDextrans

Test in Chicken Kidney Cells
17
14,716,1734
Test Methods
2191
169,439,2194
2197
2197
2197
169,440,2195
2197
2197
2197
2198
2198
2476
2484
2508
15,717,1735
27
223
34
Test for Chicken infection anaemia (CIA) virus
Test Methods, General Notices
235
Tests on Blood And Blood-Related Products

Tests on Chicken Flocks free from Specirfied
Pathogens for the Production and Quality Control
of Vaccines
Tests on Herbal Products
Tests on Vaccines
Testosterone
Testosterone Acetate
Test Methods
Tests, General Notices
Tests and Assays, General Notices
Tetanus Antitoxin
Tetrabutylammonium Bromide
Tetrabutylammonium Hydrogen Sulphate
216
199
203
.616
616
169,440,616,2199
2200
17
. 14
14,716,1734
2441
2443
2742
616
616
(jl().
Test for Avian Leucosis Viruses
219
223
TetrabutylammoniumEydroxide...
TetrabutylammoniumHydroxide;O;lM
Test for Avian Reticuloendotheliosis Virus
223
Tetrabutylammonium Iodide
635
616
617
lJreaplasmas
Test for Bacterial Endotoxins
28
Tetrachloroethane
2822
INDEX
IP 2010
1,1,2,2-Tetrachloroethane
617
Thiabendazole and Rafoxanide Mixture
2696
Tetrach10roniethane
572
Thiabendazole and Rafoxanide Suspension
2696
2203
Thiabendazole and Rafoxanide Veterinary

Oral Suspension
2696
2204
Thiabendazole Drench
2695
169,2202
Thiabendazole Mixture
2695
Tetracycline
169,2201
Tetracycline Hydrochloride Capsules
2203
Thiabendazole Oral Suspension
2695
Tetracycline Hydrochloride Eye Ointment
2204
2696
1-Tetradecane
617
2207
n-Tetradecane
617
Thiabendazole Veterinary Oral Suspension
2695
Tetraethylrhodamine
607
Thiacetazone
Tetrahydrofuran
617
Tetramethylammonium Chloride
617
Thiamazole
Tetramethylammonium Hydrogen Sulphate
617
Tetramethylammonium Hydroxide
617
Thiamine Hydrochloride Injection
Tetramethylammonium Hydroxide Solution (1 0 per cent) 617
Thiamine Hydrochloride Tablets
Tetramethylammonium Hydroxide Solution
Thiamine Injection
617
a-(4-(1,1,3,3-Tetramethylbutyl)phenyl]-ro-hydroxypoly
(oxyethylene)
598
595
Tetramethyldiaminophenylmethane
N,N,N',N'-Tetramethyl-p-phenylenediamine
Dihydrochloride
617
N,N,N',N'-Tetramethyl-p-phenylenediammonium
DicWoride
617
Tetramethylethyldiamine
617
1,2,3,4-D-Tetraphenyl-1 ,3-cyclopentadienone
617
1,2,3,4-Tetraphenylcyclopenta1 ,3-dienone
617
Tetrazo1ium Blue
Tetrazolium Blue Solution
169,442,2208
2208
618
169,442,2209
2210
2211
2210
169,443,2212
Thiamine Nitrate
2212
Thiamine Tablets
2211
Thiazole Orange
618
627
Thiazol Yellow
Tick-Borne Encephalitis Vaccine (Inactivated)
2447
Thimerosal
2215
2-(2-Thienyl)acetic acid
618
Thin Layer Chromatograms ofHerbs
459
570
Thin Layer Chromatograms of Herbs and Processed

Herbs
459
570
Thin-Layer Chromatography
137
Tetrazolium Blue Solution, Alkaline
570
Thioacetamide
618
Tetrazolium Bromide
617
Thioacetamide Reagent
618
THAM
620
Thioacetamide Solution
618
617
Thiocolchicoside
Thebaine
Theophylline
2745
169,441,617,2205
169,443,2213
2214
Thioglycerol
1703
799
Thioglycollate Medium, Alternative
58
Theophylline and Ethylenediamine Injection
800
Thioglycollate Medium, Fluid
57
Theophylline and Ethylenediamine Tablets
801
Thioglycollic Acid
Theophylline and Ethylenediamine
618
Theophylline in Dextrose Injection
2206
Thiomersal
2206
Thiomersal, Assay of
103
Therapeutic recombinant rnonoclonalantibodies
2593
Thiopental Injection
2217
Thiopental Sodium
2216
Thermometers
22
Thiabendazole
169,441,2206
63,169,618,2215
Thiopentone
2823
444
INDEX
IP 2010
Thiopentone Sodium
Thiopentone Sodium Injection
2-Thiopeneacetic Acid
Thiosulphates, Tests for
Thiotepa
2217
Tinospora cordifolia
2503
169,2216
Tiotropium Bromide
447
2217
2227
618
Tiotropium Powder for Inhalation
2228
77
169,444,2218
Thiotepa Injection
2219
Titan Yellow
627
Titan Yellow Paper
628
627
Titan Yellow Solution
Thiourea
618
Titanium Dioxide
Thoron
618
Titanium Trichloride
618
Thoronal
618
Titanium Trichloride, 0.1 M
635
Thrombin
618
Titanium Trichloride Solution
618
Thrombokinase Extract
618
Titanium (III) Chloride
618
Thromboplastin Reagent
618
Titanous Chloride
618
Trometamol
620
Titles, General Notice
Thymine
618
Thymol
169,445,618,2220
Thymol Blue
169,2229
13,715,1733
169,2230
Tizanidine Hydrochloride Tablets
2231
2231
627
Tizanidine Tablets
Thymol Blue Solution
627
Tobramycin
Thymol Blue Solution, Ethanolic
627
2233
Thymolphthalein
627
Tobramycin Sulphate Injection
2233
Thymolphthalein Solution
627
<X.- Tocopherol
Acetate
2234
Thymolsulphonaphthalein
627
a,.. Tocopheryl Acetate
2234
Thyroxine Sodium
L-Thyroxine
Sodium
169,2221
169,2232
2221
Tolazamide
Thyroxine Sodium Tablets
2222
Tolazamide Tablets
L-Thyroxine
2222
Tolbutamide
Thyroxine Tablets
2222
Tolbutamide Tablets
Sodium Tablets
170,2234
Tocopheryl Acetate
170,447,2235
2235
170,448,2236
2237
Tiabendazole
2206
Tolnaftate
Tiabendazole Tablets
2207
Tolnafate Cream
2239
Ticarcillin and Clavulanic Acid Injection
2223
Tolnafate Gel
2239
Tillman's Reagent
Timolol
Timolol Eye Drops
Timolol Maleate
170,448,2238
578
Tolnafate Topical Powder
2239
445
Tolnafate Topical Solution
2240
2224
169,446,2224
170,2240
ToluBalsam
170,2546
Timolol Maleate Eye Drops
2224
Toluene
618
Timolol Maleate Tablets
2225
Toluene, Anhydrous
618
Timolol Tablets
2225
Toluene-3,4-dithiol-Zinc Complex,
618
618
Toluene, Prepared
618
Tin, Granulated Sn
618
Toluene-2-sulphonamide
618
fin Standard Solution (5 ppm Sri)
629
Tin
Tinidazole
Tinidazole Tablets
169,446,2226
.2227
Toluene~o-sulphonamide
Toluene-p-sulphonamide
Topiramate
2824
..
, '"
....618
618
170,2242
INDEX
IP 2010
Topiramate Tablets
2243
2260
Trifluoperazine Hydrochloride Injection

Trifluoperazine Hydrochloride Tablets
2260
Topotecan Hydrochloride Injection
2245
2260
Topotecan Injection
2245
Trifluoperazine Tablets
2260
170,2243
Tosylarginine Methyl Ester Hydrochloride
619
Trifluoroacetic Acid
Tosylphenylanyanylehloromethane
619
Triflupromazine Hydrochloride
N- Tosy-L-phenyanylchloromethane
619
Triflupromazine Hydrochloride Injection
2262
L-tosylaminophenethylehloromethyl ketone
619
Triflupromazine Hydrochloride Tablets
2262
Total Ash, Limit Test for
82
619
170,450,2261
2262
174
2262
Total Solids
201
Trihexyphenidyl Hydrochloride
884
Tragacanth
2547
Trihexyphenidyl Hydrochloride Tablets
885
Tramadol Capsules
2246
1,2,3-Trihydroxybenzene
606
170,2245
1,3,5-Trihydroxybenzene
601
Total Organic Carbon in Water
Tramadol Hydrochloride
Tramadol Hydrochloride Capsules
Trandolapril
Travoprost
Travoprost Eye Drops
Triacetin
Triamcinolone
Triamcinolone Acetonide
Triamcinolone Acetonide Injection
Triamterene
Triamterene Capsules
Triazolam
Tribasic Calcium Phosphate
Tribasic Sodium Phosphate
Tribulus terrestris
Tributyl Citrate
2246
2,4,5-Trihydroxypyrimidine
588
170,2247
Trigonella foenum-graecum
2523
2248
170,2250
2251
619
170,449,2252
170,451,2263
Trimethadione
2271
Trimethadione Capsules
2272
Trimethoprim
170,451,2264
Trimethoprim and Sulphadiazine Injection
2690
Trimethoprim and Sulphadiazine Veterinary

Oral Powder
2691
Trimethoprim and Sulphadiazine Veterinary

Oral Suspension
2692

Oral Suspension
2265
Trimethoprim and Sulphamethoxazole Tablets
2266
613
Trimethoprim Tablets
2267
2500
Trimethylehlorosilane
619
170,2253
2255
2252
170,2256
2256
619
151,970
170,2257
2,2,4-Trimethylpentane
619
Tributyl Orthophosphate
619
Trinitrin Tablets
Tributyl Phosphate
619
2,4,6-Trinitrophenol
602
Trichloroacetic Acid
619
Trinitrophenol Solution
602
Trichloroacetic Acid Solution
619
Trinitrophenol Solution, Alkaline
602
1,1 ,I-Trichloroethane
619
Triphenylamine
620
170,2257
Tricine
619
Triprolidine Hydrochloride Tablets
Triethanolamine
619
Triethylamine
Triethyl Citrate
Triethylenediamine
Trifluoperazine
1425
170,452,2268
2268
2268
Tris-Acetate Buffer Solution pH 8.5
563
Tris-Chloride Buffer pH 7.4
563
619
Tris(Hydroxymethyl)aminomethane
620
450
Tris(Hydroxymethyl)aminomethane buffer
solution pH 7.4
563
619
170,449,2258
170,2259
2825
INDEX
Tris(Hydroxymethyl)aminomethane buffer
solution pH 8.1
IP 2010
Undecenoic Acid
170,620,2277
563
Undecylenic Acid
2277
Tris(Hydroxymethyl)methylarnine
620
Uniformity of Content of Capsules
722
Tris(l, 1O-phenanthroline)ferrous Sulphate Complex
624
Uniformity of Content ofInjection
748
Uniformity of Content ofNasal Preparations
743
Uniformity of Content of Pessaries
750
Uniformity of Content of Single-Dose Preparations
192
Uniformity of Content ofTablets
751
Uniformity of delivered dose
728
Uniformity of Weight of Capsules
722
Uniformity ofWeight of Cream
723
Uniformity ofWeight ofNasal Preparations
743
Uniformity of Weight of Pessaries
750
2449
Uniformity ofWeight of Single-Dose Preparation
192
2449
Uniformity ofWeight of Single-Dose Preparation for

Capsules
193
2449
170,453,2272
Uniformity ofWeight of Single-Dose Preparation for

Powders for parenteral use
193
Uniformity of Weight of Tablets
752
Trisodium Citrate
2118
Trisodium Edetate Concentrate for Injection
2269
Trisodium Phosphate
613
613
Tromethamine
620
Trisodium Orthophosphate
Tropicamide
170,452,2270

Troxidone
2270
170,2271
Troxidone Capsules
2272
Trypsin
620
Tuberculin PPD
Tuberculin Purified Protein Derivative
Tuberculin Purified Protein Derivative for
Human Use
Tubocurarine Chloride Injection
2273
2273
Tulasi
Tulasi Dry Extract
93
488,550,2548
Unsaponifiable Matter, Assay for
489,551,2549
Uracil Riboside
620
2507
Uranyl Acetate
620
Turmeric
Tylosin
1593
Unmedicated Lint
170,2697
Urea
170,453,620,2277
2698
Urea Cream
Tylosin Tablets
2699
Urease-active Meal
620
Tylosin Tartrate
170,2700
Uridine
620
Tylosin Injection
Tylosin Tartrate and Sulphathiazole Sodium

Veterinary Oral Powder
2702
Urokinase
Typhoid Paratyphoid A Vaccine
2454
Uronic Acids
_Typhoid Polysaccharide Vaccine
2452
2278
64
Urinary Excretion ofDextrans
Typhoid (Strain Ty 21a) Vaccine, Live (Oral)
2451
Typhoid Vaccine
2455
2455
Typhus Vaccine
2455
Usual Strength, General Notice
170,2279
207
14,716,1734
Tyramine
620
Vaccines and Immunosera for Human Use,

Monographs
2345
Tyrosine
620
Vaccines for Veterinary Use
2623
L-Tyrosine
620
Vaccines: General Requirements
2347
Ultraviolet Ray Lamps
22
Vaccines, Tests on
203
Vaccines--and-Immunosera,Evaluation.ofEffic.a_cy_oi.
232
Valerie Acid
Ultraviolet and Visible Absorption Spectrophotometry
117
n-Valerie Acid
620
Uncoated Tablets
752
Validation ofAnalytical Procedures
198
2826
INDEX
IP 2010
Validation ofNucleic Acid Amplification

Techniques (NAT) for the Detection of
Hepatitis C Virus (HCV) RNA in Plasma Pools:
Guidelines
239
Valproate Injection
2283
Valproate Sodium Injection
2283
ValproicAcid
170,454,2283
2284
Valproic Acid Oral Solution
2285
Valsartan
170,454,2286
Valsartan Tablets
2287
Valsartan and Hydrochlorothiazide Tablets
2287
Valves for Inhalation Preparation
727
170,2289
Vancomycin Capsules
2291
Vancomycin Hydrochloride Capsules
2291
2291
2292
Vanillin
170,620,2294
Vanillin Glacial AceticAcid Reagent
620
Vanillin Sulphuric Acid
620
Varicella Vaccine, Live
2456
Vasaka
489,552,2550
Vasaka Extract
490,553,2550
2294
Veraparnil
455
Verapamil CWoride
2295
Verapamil Chloride Injection
2296
Verapamil Chloride Tablets
2297
171,455,2295
Verapamil Hydrochloride Injection
2296
Verapamil Hydrochloride Tablets
2297
Verapamil Injection
2296
Verapamil Tablets
2297
Veratric Acid
620
Veterinary Aerosols
2622
2622
Veterinary Diagnostics Monographs
2747
Veterinary Oral Liquids
2623
2623
2623
Veterinary Preparations
2621
Veterinary Products, Monographs
2619
2627
Veterinary Products, Non-Biological
2705
Veterinary Products, Biological
2747
Veterinary Products, Diagnostics
2623
Veterinary Tablets
2623
Veterinary Vaccines
2622
Veterinary Sprays
2499
Vilayati Imli
47
Violet Red Bile Glucose Agar Medium
2299
Vinblastine Injection
170,2298
Vinblastine Sulphate
2299
Vinblastine Sulphate Injection
2302
Vinctistine Injection
170,2300
Vinctistine Sulphate
2302
Vincristine Sulphate Injection
170,2303
Vinorelbine Tartrate
2304
Vinorelbine Injection
2304
Vinorelbine Tartrate Injection
1141
1-Vinyl-2-pyrrolidinone homopolymer
2458
Viper Venom
243
Viper Venom Specific Factor II Activator (Ecarin)
2046
Viscose, Absorbent
2046
Viscose Fibre
171
Viscosity
Vitamin A, Assay of
942305
171
Vitamin A Concentrate (Oily Form)
171
Vitamin A Concentrate (Powder Form)
Vitamin A Concentrate (Water-miscible Form)
2306
2306
2307
Vitamins A and D Capsules
2308
Vitamin A and D Solution, Concentrated
2308
Vitamin D Solution, Concentrated
2209
VitarninB I
2210
VitaminB I Injection
2211
VitaminB I Tablets
2052
VitaminB 2
2053
Vitamin B2 Sodium Phosphate
2054
Vitamin B2 Tablets
2005
VitaminB6
1142
VitaminB I2
1143
Vitamin B I2 Injection
839
VitaminC
840
Vitamin C Injection
Volume 1: i to xxiii and 1to 704; Volume 2: xxv to xxviii and 705 to-I722; Volume 3: xxix to xxxii and 1723 to 2829
2827
INDEX
IP 2010
840
Vitamin C Tablets
Vitamin D,Assay of
96
Vitamin D Solution, Concentrated
2308
Wax, Microcrystalline
Weak Cupric Sulphate Solution
576
Weight Per Millilitre and Relative Density
174
Weight per Millilitre, Determination of
174
23
VitaminD2
955
VitaminD 3
1077
Weights and Balances
Vitamin E Acetate
2234
Weights and Measures
22
Volumetric Glassware
Volumetric Reagents and Solutions
630
Volumetric Solutions
631
1696
715,1733
702
Weights and Measures: SI Units
149,875
White Beeswax
White Mineral Oil
1862
1863
White Petroleum Jelly
164,1863
White Soft Paraffin
887
Whitfield's Ointment
Warfarin
Whole Blood (Human)
2589
171,2311
Whole Human Blood
2589
171,2312
Wilson and Blair's BBS Agar Medium
456
Warfarin Sodium
Warfarin Sodium Clathrate
Warfarin Sodium Tablets
2313
Warfarin Tablets
2313
Wash Solution pH 2.5
620
Water
620
Water, Ammonia-free
Water BET
620
12
29
Water, Carbon Dioxide-free
620
Water-bath, General Notices
97
Water, Assay for

Water, Distilled
620
Water for Injection
620
2315
Water for Injection in Bulk
2315
Water for Injections, Sterile
620,2316
Water for Pharmaceutical Use
658
Water, General Notices
12
Water, Nitrate-free
620
47
Wintergreen Oil
1666
Withania somnifera
2479
Wool Fat
171,620,2317
Wool Fat, Hydrous
171,2318
X
Xanthan Gum
171,2321
Xanthen-9-ol
620
Xanthines, Test for
.77
Xanthydrol
620
Xanthydrol Reagent
621
Xylene
621
. 627
Xylene Cyanol FF
XylenolOrange
627
Xylenol Orange Mixture
627
Xylenol Orange Solution
627
Xylenol Orange Triturate
627 .
2,3-Xylidine
579
Water-Miscible Vitamin A Concentate
2306
2,6-Xylidine
579
Water, Purified
2314
Xylometazoline
456
201
Water-SolubIe Extractive
Water vapour detector tube
Wax, Amorphous
Ailioriic EmUlsifying
Wax:
21
1696
.. ~~~~~~_
171,457,2322
Xylometazoline Hydrochloride Nasal Drops
2323
2323
-~~~~~~-~~-1Z74~-o::-X-ylopyranose~~~-~-~~-~~~_
----_.~
2324
Wax, Camauba
Wax, Castor
Wax, Emulsifying
2511
155,1274
D-Xylose
Xyiose-Lysine-Desoxycholate Agar Medium
2828
2324
48
IP 2010
INDEX
Zinc, Granulated
Yasti
490,554,2551
Yasti Dry Extract
191,555,2552
149,876
Yellow Beeswax
Yellow Fever Vaccine, Live
2458
Yellow Mercuric Oxide
593
Yellow Mercury(II) Oxide
593
1864
Yellow Petroleum Jelly
164,1864
Yellow Soft Paraffin
Zinc Oxide
171,2336
Zinc Oxide Cream
2336
Zinc Powder
621
Zinc Salts, Tests for
77
Zinc Shot
621
Zinc and Sodium Carbonate Reagent
621
Zinc Standard Solution (10 ppm Zn)
629
Zinc Standard Solution (25 ppm Zn)
630
Zinc Standard Solution (l 00 ppm Zn)
630
Zinc Stearate
Zinc Sulphate
Zidovudine
Zinc Sulphate, 0.1 M
171,457,2329
621,631
171,2337
171,621,2337
636
Zidovudine Capsules
2330
Zidovudine Injection
2331
Zinc Sulphate Solution
2332
Zinc Sulphate, x M
621
Zidov.pdine Tablets
2333
Zinc Undecenoate
171,622,2338
Zidovudine, Lamivudine and Nevirapine Tablets
2334
2339
621
Zinc Undecylenate
2338
621
Zinc Undecylenate Ointment
2339
Zinc
Zinc, Activated
Zinc, Assay for
'99
ZincAsT
621
Zinc bis(diphenyldithiocarbamate)
621
Zinc Chloride
171,621,2335
Zinc Chloride, 0.05 M
621
Zinc Chloride, O.1M
635
Zinc Chloride-Formic Acid Solution
621
Zinc Chloride Solution
621
Zinc Chloride Solution, Iodinated
621
Zinc Cream
Zinc Dithiol Reagent
Zinc Dust
2336
621
621
2338
621
Zincon
621
Zincon Solution
622
2544
Zingiber officinale
Zirconyl Nitrate
622
Zirconyl Nitrate Solution
622
Zoledronic Acid
171,2339
2340
Zoledronic Acid Injection

Zolpidem Tartrate
171,2341
Zolpidem Tablets
2342
2342
Zolpidem Tartrate Tablets

Zone Electrophoresis
123
Zone Electrophoresis, Capillary
177
2829

Indian Pharmacopoeia 2010 Volume 3

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Indian Pharmacopoeia 2010 Volume 3

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I

2010, Indian Pharmacopoeia Commission

Sixth Edition (6.0)

Designed, produced & published by

The Indian Pharmacopoeia Commission

Price per set:

INDIAN PHARMACOPOEIA 2010

Indian Pharmacopoeia Commission

General Monographs on Dosage Forms

Monographs on Drug substances, Dosage forms and

INDIAN PHARMACOPOEIA 2007

INDIAN PHARMACOPOEIA 2010

Provisions Applicable to Monographs and Test Methods

Weights and Measures

Atomic and Molecular Weights

INDIAN PHARMACOPOEIA 2010

Tests and Assays

Name. The full name or title ofthis book, including addenda

An official substance is a single drug or a drug entity or a

Official Standards. The requirements stated in the

The active pharmaceutical ingredients (drug substances),

Added Substances. An official substance, as distinguished

per cent v/v (percentage, volume in volume) expressing

Label. Any printed packing material, including package inserts

per cent w/v (percentage, weight in volume) expressing

Negligible. A quantity not exceeding 0.50 mg.

per cent v/w (percentage, volume in weight) expressing

provides the method to be followed and that the values

Excipients. Any substance added in preparing an official

monograph. In certain monographs for pharmaceutical

Solubility. Statements on solubility are given in Chapter 2.4.26

In celtain monographs alternative series ofidentification tests

use oflow-actinic glassware, working in a dark room or similar

Unless otherwise stated, comparative tests are carried out

to one decimal place more than the significant figures stated

Where no specific storage directions or limitations are given

Storage. Statements under the side-heading Storage constitute

Storage Containers. The requirements, guidance and

Specific directions are given in some monographs with respect

In general, an article should be packed in a well-closed

Labelling. The labelling of drugs and pharmaceuticals is

INDIAN PHARMACOPOEIA 2010

DRUG SUBSTANCES, DOSAGE FORMS

INDIAN PHARMACOPOEIA 2010

Neomycin Eye Ointment

INDIAN PHARMACOPOEIA 2010

INDIAN PHARMACOPOEIA 2010

NALIDIXIC ACID TABLETS

Reference solution (b). A 0.0008 per cent w/v solution of the

Mol. Wt. 232.2

Apply to the plate 10 III ofeach solution. After development,

Dose. 2 to 4 g daily, in divided doses.

Assay. Weigh accurately about 0.15 g, dissolve in 10 ml of

A. Determine by infrared absorption spectrophotometry (2.4.6).

dichloromethane, add 30 ml of 2-propanol and 10 mlof carbon

(2.4.25) and using a glass electrode as the indicator electrode

C. In the test for Related substances, the principal spot in the

Storage. Store protected from light and moisture.

D. Dissolve 0.1 gin 2 ml of hydrochloric acid and add 0.5 ml

Nalidixic Acid Tablets

NALIDIXIC ACID TABLETS

B. When examined in the range 230 nm to 360 nm (2.4.7), a

Nalorphine Hydrochloride contains not less than 97.0 per cent

Dose. By intravenous injection, 5 mg, repeated twice at three