British Pharmacopoeia 2016 Volume 4

British
Pharmacopoeia
2016
Volume IV
General Notices
Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal Products
Materials for use in the Manufacture of Homoeopathic Preparations
Blood-related Products
Immunological Products
r
Radiopharmaceutical Preparations
Surgical Materials
Incorporating the requirements of the 8th edition of the

European Pharmacopoeia as amended by Supplements 8.1 to 8.5
British Pharmacopoeia 2016
Volume IV
British Pharmacopoeia 2016
Volume IV
The British Pharmacopoeia Commission has caused this British
Pharmacopoeia 2016 to be prepared under regulation 317(1) of the Human
Medicines Regulations 2012 and, in accordance with regulation 317(4), the
Ministers have arranged for it to be published. It has been notified in draft
to the European Commission in accordance with Directive 98/34/EEC.
The monographs of the Eighth Edition of the European Pharmacopoeia
(2013), as amended by Supplements 8.1 to 8.5, published by the Council
of Europe are reproduced either in this edition of the British
Pharmacopoeia or in the associated edition of the British Pharmacopoeia
(Veterinary).
See General Notices
Effective date: 1 January 2016
see Notices
London: The Stationery Office

In respect of Great Britain:
THE DEPARTMENT OF HEALTH
In respect of Northern Ireland:
THE DEPARTMENT OF HEALTH, SOCIAL SERVICES AND
PUBLIC SAFETY
© Crown Copyright 2015
Published by The Stationery Office on behalf of the Medicines and
Healthcare products Regulatory Agency (MHRA) except that:
European Pharmacopoeia monographs are reproduced with the permission
of the Council of Europe and are not Crown Copyright. These are
identified in the publication by a chaplet of stars.
This publication is a ‘value added’ product. If you wish to re-use the
Crown Copyright material from this publication, applications must be made
in writing, clearly stating the material requested for re-use, and the purpose
for which it is required. Applications should be sent to: Dr ร Atkinson,
MHRA, 5th Floor, 151 Buckingham Palace Road, London SW1W 9SZ.
First Published 2015
ISBN 978 011 3230 006

British Pharmacopoeia Commission Office:
MHRA
151 Buckingham Palace Road,
London รพ!พ 9SZ
Telephone: 4-44 (0)20 3080 6561
E-mail: bpcom@mhra.gsi.gov.uk
Web site: http://www.pharmacopoeia.com
Laboratory:
British Pharmacopoeia Commission Laboratory
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Teddington
Middlesex TW11 OLY
Telephone: 4-44 (0)20 8943 8960
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Web site: http://www.pharmacopoeia.com
Contents
Contents of Volume I
FOREWORD
NOTICES
PREFACE
BRITISH PHARMACOPOEIA COMMISSION

EXPERT ADVISORY GROUPS, PANELS OF EXPERTS AND
WORKING PARTIES
CODE OF PRACTICE
MEMBERSHIP
BP Commission, Expert AdGsory Groups, Panels of Experts, Working
Parties
STAFF
British Pharmacopoeia, BP Laboratory, Publisher
INTRODUCTION
Additions, Omissions, Technical Changes, Changes in Title
GENERAL NOTICES
MONOGRAPHS
Medicinal and Pharmaceutical Substances (A - I)
Contents of Volume II
NOTICES
GENERAL NOTICES
MONOGRAPHS
Medicinal and Pharmaceutical Substances (J - Z)
Contents of Volume III
NOTICES
GENERAL NOTICES
MONOGRAPHS
Formulated Preparations: General Monographs
Formulated Preparations: Specific Monographs
rv
Contents of Volume rv
NOTICES
GENERAL NOTICES
MONOGRAPHS
Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal Products

Materials for use in the Manufacture of Homoeopathic Preparations
Radiopharmaceutical Preparations
Surgical Materials
Contents of Volume V
NOTICES
GENERAL NOTICES
INFRARED REFERENCE SPECTRA
APPENDICES
SUPPLEMENTARY CHAPTERS
INDEX
IV-vi
Notices
Monographs of the European Pharmacopoeia are distinguished by a chaplet

of stars against the title. The term European Pharmacopoeia, used without
qualification, means the eighth edition of the European Pharmacopoeia
comprising, unless otherwise stated, the main volume, published in 2014, as
amended by any subsequent supplements and revisions.
Patents In this Pharmacopoeia certain drugs and preparations have been included
notwithstanding the existence of actual or potential patent rights. In so far
as such substances are protected by Letters Patent their inclusion in this
Pharmacopoeia neither conveys, nor implies, licence to manufacture.
Effective dates New and revised monographs of national origin enter into force on
1 January 2016. The monographs are brought into effect under regulation
320(2) of the Human Medicines Regulations 2012.
Monographs of the European Pharmacopoeia have previously been
published by the European Directorate for the Quality of Medicines &
Healthcare in accordance with the Convention on the Elaboration of a
European Pharmacopoeia and have been brought into effect under
European Directives 2001/82/EC, 2001/83/EC and 2003/63/EC, as
amended, on medicines for human and veterinary use.
2016 General Notices IV-
General Notices
IV-2 General Notices 2016
CONTENTS OF THE GENERAL NOTICES
Part I Implementation of Pharmacopoeial Methods

Italic introduction Conventional Terms
European Pharmacopoeia Interchangeable Methods
References to Regulatory Documents
Part II
Italic introduction 1.2 Other Provisions Applying to General
Official Standards Chapters and Monographs
Definition of Terms Quantities
Expression of Standards Apparatus and Procedures
Temperature Water-bath
Weights and Measures Drying and Ignition to Constant Mass
Atomic Weights Reagents
Constant Weight Solvents
Expression of Concentrations Expression of Content
Water Bath Temperature
Reagents 1.3 General Chapters
Indicators Containers
Caution Statements 1.4 Monographs
Titles Titles
Chemical Formulae Relative Atomic and Molecular Masses
Definition Chemical Abstracts Senice (CAS) Registry
Production Number
Manufacture of Formulated Preparations Definition
Freshly and Recently Prepared Limits of Content
Methods of Sterilisation Herbal Drugs
Water Production
Excipients Choice of Vaccine Strain, Choice of
Colouring Agents Vaccine Composition
Antimicrobial Preservatives Potential Adulteration
Characteristics Characters
Solubility Solubility
Identification Identification
Reference Spectra Scope
Assays and Tests First and Second Identifications
Biological Assays and Tests Powdered Herbal Drugs
Reference Substances and Reference Preparations Tests and Assays
Chemical Reference Substances Scope
Biological Reference Preparations Calculation
Storage Limits
Labelling Indication of Permitted Limit of Impurities
Action and Use Herbal Drugs
Crude Drugs; Traditional Herbal and Equivalents
Complementary Medicines Culture Media
Monograph Title Storage
Definition Labelling
Characteristics Warnings
Control Methods Impurities
Homoeopathic Medicines Functionality-related Characteristics of
Unlicensed Medicines Excipients
Reference Standards
Part in
Italic introduction Abbreviations and Symbols
General Notices of the European Pharmacopoeia Abbreviations used in the Monographs on
1.1 General Statements Immunoglobulins, Immunosera and
Quality Systems Vaccines
Alternative Methods Collections of Micro-organisms
Demonstration of Compliance with the 1.6 Units of the International System (SI) used
-A-’rrfJritarA๗iWhMftjTlifA 4V.-A' ๙,’*'•*^ ^.‘•^-.' hj, '■•‘i•พti>e.’PhafniBc0pioera-
Grade of Materials with other Units

General Monographs International System of Units (SI)
Validation of Pharmacopoeial Methods ‘ Notes '‘‘'บ’..
2016 General Notices IV-3
General Notices
Part I
The British Pharmacopoeia comprises the entire text within this publication. The
word ‘official’ is used in the Pharmacopoeia to signify ‘of the Pharmacopoeia3. It
applies to any title, substance, preparation, method or statement included in the
general notices, monographs and appendices of the Pharmacopoeia. The
abbreviation for British Pharmacopoeia is BP.
European Monographs of the European Pharmacopoeia are reproduced in this edition

Pharmacopoeia of the British Pharmacopoeia by incorporation of the text published under
the direction of the Council of Europe (Partial Agreement) in accordance
with the Convention on the Elaboration of a European Pharmacopoeia
(Treaty Series No. 32 (1974) CMND 5763) as amended by the Protocol to
the Convention (Treaty Series No. MISCI6 (1990) CMND 1133). They
are included for the convenience of users of the British Pharmacopoeia. In
cases of doubt or dispute reference should be made to the Council of
Europe text.
* ★ * Monographs of the European Pharmacopoeia are distinguished by a
£ ★ chaplet of stars against the title and by reference to the European
** Pharmacopoeia monograph number included immediately below the
title in italics. The beginning and end of text from the European
Pharmacopoeia are denoted by means of horizontal lines with the symbol
‘Ph Eur3 ranged left and right, respectively.
The general provisions of the European Pharmacopoeia relating to
different types of dosage form are included in the appropriate general
monograph in that section of the British Pharmacopoeia entitled
Monographs: Formulated Preparations. These general provisions apply to
all dosage forms of the type defined, whether or not an individual
monograph is included in the British Pharmacopoeia. In addition, the
provisions of the European Pharmacopoeia General Monograph for
Pharmaceutical Preparations apply to all dosage forms, whether or not an
individual monograph is included in the British Pharmacopoeia.
Texts of the European Pharmacopoeia are governed by the General
Notices of the European Pharmacopoeia. These are reproduced as Part in
of these notices.
I V-4 Lxeneral Notices 2016
Part II
The following general notices apply to the statements made in the monographs of
the British Pharmacopoeia other than those reproduced from the European
Pharmacopoeia and to the statements made in the Appendices of the British
Pharmacopoeia other than when a method, test or other matter described in an
appendix is invoked in a monograph reproduced from the European
Pharmacopoeia.
Official Standards The requirements stated in the monographs of the Pharmacopoeia apply to
articles that are intended for medicinal use but not necessarily to articles
that may be sold under the same name for other purposes. An article
intended for medicinal use that is described by means of an official title
must comply with the requirements of the relevant monograph. A
formulated preparation must comply throughout its assigned shelf-life
(period of validity)- The subject of any other monograph must comply
throughout its period of use.
A monograph is to be construed in accordance with any general
monograph or notice or any appendix, note or other explanatory material
that is contained in this edition and that is applicable to that monograph.
All statements contained in the monographs, except where a specific general
notice indicates otherwise and with the exceptions given below, constitute
standards for the official articles. An article is not of pharmacopoeia! quality
unless it complies with all of the requirements stated. This does not imply
that a manufacturer is obliged to perform all the tests in a monograph in
order to assess compliance with the Pharmacopoeia before release of a
product. The manufacturer may assure himself that a product is of
pharmacopoeial quality, by other means, for example, from data derived
from validation studies of the manufacturing process, from in-process
controls or from a combination of the two. Parametric release in
appropriate circumstances is thus not precluded by the need to comply with
the Pharmacopoeia. The general notice on Assays and Tests indicates that
analytical methods other than those described in the Pharmacopoeia may be
employed for routine purposes.
Requirements in monographs have been framed to provide appropriate
limitation of potential impurities rather than to provide against all possible
impurities. Material found to contain an impurity not detectable by means
of the prescribed tests is not of pharmacopoeial quality if the nature or
amount of the impurity found is incompatible with good pharmaceutical
. practice.
The status of any statement given under the headings Definition,
Production, Characteristics, Storage, Labelling or Action and use is defined
within the general notice relating to the relevant heading. In addition to any
exceptions indicated by one of the general notices referred to above, the
following parts of a monograph do not constitute standards: (a) a graphic or
molecular formula given at the beginning of a monograph; (b) a molecular
weight; (c) a Chemical Abstracts Service Registry Number; (d) any
information given at the end of a monograph concerning impurities known
to be limited by that monograph; (e) information in any annex to a
monograph. Any statement containing the word 'should’ constitutes non

mandatory advice or recommendation.
The expression ‘unless otherwise justified and authorised’ means that the
requirement in question has to be met, unless a competent authority
authorises a modification or exemption where justified in a particular case.
The term ‘competent authority’ means the national, supranational or
international body or organisation vested with the authority for making
decisions concerning the issue in question. It may, for example, be a
licensing authority or an official control laboratory. For a formulated
preparation that is the subject of monograph in the British Pharmacopoeia
any justified and authorised modification to, or exemption from, the
requirements of the relevant general monograph of the European
Pharmacopoeia is stated in the individual monograph. For example, the
general monograph for Tablets requires that Uncoated Tablets, except for
chewable tablets, disintegrate within 15 minutes; for Calcium Lactate
Tablets a time of 30 minutes is permitted.
Many of the general monographs for formulated preparations include
statements and requirements additional to those of the European
Pharmacopoeia that are applicable to the individual monographs of the
British Pharmacopoeia. Such statements and requirements apply to all
monographs for that dosage form included in the Pharmacopoeia unless .
otherwise indicated in the individual monograph.
Where a monograph on a biological substance or preparation refers to a
strain, a test, a method, a substance, etc., using the qualifications ‘suitable’’'-,
or ‘appropriate’ without further definition in the text, the choice of such
strain, test, method, substance, etc., is made in accordance with any
international agreements or national regulations affecting the subject
concerned.
Definition of Terms Where the term ‘about’ is included in a monograph or test it should be
taken to mean approximately (fairly correct or accurate; near to the actual
value). : ■7-’:;
Where the term ‘corresponds’ is included in a monograph or test it
should be taken to mean similar or equivalent in character or quantity.
Where the term ‘similar’ is included in a monograph or test it should be
taken to mean alike though not necessarily identical.
Further qualifiers (such as numerical acceptance criteria) for the above
terms are not included in the BP. The acceptance criteria for any individual
case is set based on the range of results obtained from known reference
samples, the level of precision of the equipment or apparatus used and the
level of accuracy required for the particular application. The user should
determine the variability seen in his/her own laboratory and set in-house
acceptance criteria that he/she judges to be appropriate based on the local
operating conditions.
Expression of Where: the standard for the content of a substance described in a

Standards monograph is expressed in terms of the chemicar formula for that substance
an upper limit exceeding 100% may be stated. Such an upper limit applies
to the result of the assay calculated in terms of the equivalent content of the
specified chemical formula. For example, the statement ‘contains not less
than 99 0% and not more than 101.0% of C20H24N2O25HCF implies that
the result of the assay is not less than 99.0% and not more than 101.0%, -
calculated in terms of the equivalent .content of G20H24N2O2JHGI..
Where the result of an assay or test is required to be calculated with

reference to ±e dried, anhydrous or ignited substance, the substance free
from a specified solvent or to the peptide content, the determination of loss
on drying, water content, loss on ignition, content of the specified solvent
or peptide content is carried out by the method prescribed in the relevant
test in the monograph.
Temperature The Celsius thermometric scale is used in expressing temperatures.
Weights and The metric system of weights and measures is employed; SI Units have
Measures generally been adopted. Metric measures are required to have been
graduated at 20° and all measurements involved in the analytical operations
of the Pharmacopoeia are intended, unless otherwise stated, to be made at
that temperature. Graduated glass apparatus used in analytical operations
should comply with Class A requirements of the appropriate International
Standard issued by the International Organization for Standardization. The
abbreviation for litre is ‘L’ throughout the Pharmacopoeia. In line with
European Directive 80/181/EEC, the abbreviation ‘1’ is also permitted for
use.
Atomic Weights The atomic weights adopted are the values given in the Table of Relative
Atomic Weights 2001 published by the International Union of Pure and
Applied Chemistry (Appendix XXV).
Constant Weight The term ‘constant weight’, used in relation to the process of drying or the
process of ignition, means that two consecutive weighings do not differ by
more than 0.5 mg, the second weighing being made after an additional
period of drying or ignition under the specified conditions appropriate to
the nature and quantity of the residue (1 hour is usually suitable).
Expression of The term ‘per cent’ or more usually the symbol ‘%’ is used with one of four
Concentrations different meanings in the expression of concentrations according to
circumstances. In order that the meaning to be attached to the expression
in each instance is clear, the following notation is used:
Per cent พ/พ (% พ/พ) (percentage weight in weight) expresses the
number of grams of solute in 100 g of product.
Per cent w/v (% w/v) (percentage weight in volume) expresses the
number of grams of solute in 100 mL of product.
Per cent v/v (% v/v) (percentage volume in volume) expresses the
number of millilitres of solute in 100 mL of product.
Per cent v/w (% v/w) (percentage volume in weight) expresses the
number of millilitres of solute in 100 g of product.
Usually the strength of solutions of solids in liquids is expressed as
percentage weight in volume, of liquids in liquids as percentage volume in
volume and of gases in liquids as percentage weight in weight.
When the concentration of a solution is expressed as parts per million
(ppm), it means weight in weight, unless otherwise specified.
When the concentration of a solution is expressed as parts of dissolved
substance in parts of the solution, it means parts by weight (g) of a solid in
parts by volume (mL) of the final solution; or parts by volume (mL) of a
liquid in parts by volume (mL) of the final solution; or 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 of moles of
the stated solute contained in sufficient Purified Water (unless otherwise
stated) to produce 1 litre of solution.
Water Bath The term ‘water bath’ means a bath of boiling water, unless water at some
other temperature is indicated in the text. An alternative form of heating
may be employed providing that the required temperature is approximately
maintained but not exceeded.
Reagents The reagents required for the assays and tests of the Pharmacopoeia are
defined in appendices. The descriptions set out in the appendices do not
imply that the materials are suitable for use in medicine.
Indicators Indicators, the colours of which change over approximately the same range
of pH, may be substituted for one another but in the event of doubt or
dispute as to the equivalence of indicators for a particular purpose, the
indicator specified in the text is alone authoritative.
The quantity of an indicator solution appropriate for use in acid-base
titrations described in assays or tests is 0.1 mL unless otherwise stated in
the text.
Any solvent required in an assay or test in which an indicator is specified
is previously neutralised to the indicator, unless a blank test is prescribed.
Caution Statements A number of materials described in the monographs and some of the
reagents specified for use in the assays and tests of the Pharmacopoeia may
be injurious to health unless, adequate precautions are taken. The principles
of good laboratory practice and the provisions of any appropriate
regulations such as those issued in the United Kingdom in accordance with
the Health and Safety at Work etc. Act 1974 should be observed at all times
in carrying out the assays and tests of the Pharmacopoeia.
Attention is drawn to particular hazards in certain monographs by means
of an italicised statement; the absence of such a statement should not
however be taken to mean that no hazard exists.
Titles Subsidiary titles, where included, have the same significance as the .main
titles. An abbreviated title constructed in accordance with the directions
given in Appendix XXI. A. has the same significance as the main title.
Titles .that are derived by the suitable inversion of words of a main or
subsidiary title,, with the addition of a preposition if appropriate, are also
official titles. Thus, the following are all official titles: Aspirin Tablets,
Tablets of Aspirin; Atropine Injection, Injection of Atropine.
A title of a formulated preparation that includes the full nonproprietary
name of the active ingredient or ingredients, where this is not included, in
the title of the monograph, is also an official title. For example, the title
Promethazine Hydrochloride Oral. Solution has the same significance as
Promethazine Oral Solution and the title Brompheniramine Maleate Tablets
has the same significance as Brompheniramine Tablets.
1 Where the English title at the head of a monograph in the European
Pharmacopoeia is different from that at the head of the text incorporated
into the British Pharmacopoeia, an Approved Synonym has been created-on
the recommendation of the British Pharmacopoeia Commission. Approved
Synonyms have the same significance as the main title and are thus official
iV-เ} General Notices 2016
titles. A cumulative list of such Approved Synonyms is provided in

Appendix XXI B.
Where the names of pharmacopoeial substances, preparations and other
materials occur in the text they are printed with capital initial letters and
this indicates that materials of Pharmacopoeial quality must be used. Words
in the text that name a reagent or other material, a physical characteristic or
a process that is described or defined in an appendix arc printed in italic
type, for example, methanol) absorbance) gas chromatography) and these imply
compliance with the requirements specified in the appropriate appendix.
Chemical Formulae When the chemical composition of an official substance is known or

generally accepted, the graphic and molecular formulae, the molecular
weight and the Chemical Abstracts Service Registry Number are normally
given at the beginning of the 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 statements of
standards of purity and strength and in descriptions of processes of assay, it
is evident from die context that the formulae denote the chemically pure
substances.
Where the absolute stereochemical configuration is specified, die
International Union of Pure and Applied Chemistry (IUPAC) R/S and ElZ
systems of designation have been used. If the substance is an enantiomer of
unknown absolute stereochemistry the sign of the optical rotation, as
determined in the solvent and under the conditions specified in the
monograph, has been attached to the systematic name. An indication of
sign of rotation has also been given where this is incorporated in a trivial
name that appears on an IUPAC preferred list.
All amino acids, except glycine, have the L-configuration unless otherwise
indicated. The three-letter and one-letter symbols used for amino acids in
peptide and protein sequences are those recommended by die Joint
Commission on Biochemical Nomenclature of the International Union of
Pure and Applied Chemistry and the International Union of Biochemistry
and Molecular Biology.
In the graphic formulae the following abbreviations are used:
Me -ch3 Bu1 -CH(CH3)CH2CH3

Et -CH2CH3 Bu” -ch2ch2ch2ch3
Pr‘ -CH(CH3)2 Bu' -C(CH3)3
Pr" -CH2CH2GH3 Ph -c6h5
Bu’ -CH2CH(CH3)2 Ac -coch3
Definition Statements given under the heading Definition constitute an official
definition of the substance, preparation or other article that is the subject of
the monograph. They constitute instructions or requirements and are
mandatory in nature.
Certain medicinal or pharmaceutical substances and other articles are
defined by reference to a particular method of manufacture. 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 certain method, however^ indicates that this is one possible method and
does not imply that other methods are proscribed.
Additional statements concerning the definition of formulated

preparations are given in the general notice on Manufacture of Formulated
Preparations.
Production Statements given under the heading Production draw attention to particular
aspects of the manufacturing process but are not necessarily comprehensive.
They constitute mandatory instructions to manufacturers. 'rhey may relate,
for example, to source materials, to the manufacturing process itself and its
validation and control, to in-process testing or to testing that is to be
carried out by the manufacturer on the final product (bulk material or
dosage form) either on selected batches or on each batch prior to release.
These statements cannot necessarily be verified on a sample of the final
product by an independent analyst. The competent authority may establish
that the instructions have been followed, for example, by examination of
data received from the manufacturer, by inspection or by testing
appropriate samples.
The absence of a section on Production does not imply that attention to
features such as those referred to above is not required. A substance,
preparation or 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 relevant international agreements and
supranational and national regulations governing medicinal products.
Where in the section under the heading Production a monograph on a
vaccine defines the characteristics of the vaccine strain to be used, any test
methods given for confirming these characteristics are provided as examples
of suitable methods, 'rhe use of these methods is not mandatory.
Additional statements concerning the production of formulated
preparations are given in the general notice on Manufacture of Formulated
Preparations.
Manufacture of Attention is drawn to the need to observe adequate hygienic precautions

Formulated in the preparation and dispensing of pharmaceutical formulations. The
Preparations principles of good pharmaceutical manufacturing practice should be
observed.
The Definition in certain monographs for pharmaceutical preparations is
given in terms of the principal ingredients only. Any ingredient, other than
those included in the Definition, must comply with the general notice on
Excipients and the product must conform with the Pharmacopoeial
requirements.
The Definition in other monographs for pharmaceutical preparations is
presented as a full formula. No deviation from the stated formula is
permitted except those allowed by the general notices on Colouring Agents
and Antimicrobial Preservatives. Where additionally directions are given
under the heading Extemporaneous Preparation these are intended for the
extemporaneous preparation of relatively small quantities for short-term
supply and use. When so prepared, no deviation from the stated directions
is permitted. If, however, such a pharmaceutical preparation is
manufactured on a larger scale with the intention that it may be stored,
deviations from the stated directions are permitted provided that the final
product meets the following criteria:
(1) compliance with all of the requirements stated in the monograph;

(2) retention of the essential characteristics of the preparation made strictly
in accordance with the directions of the Pharmacopoeia.
Monographs for yet other pharmaceutical preparations include both a
Definition in terms of the principal ingredients and, under the side-heading
Extemporaneous Preparation, a full formula together with, in some cases,
directions for their preparation. Such full formulae and directions are
intended for the extemporaneous preparation of relatively small quantities
for short-term supply and use. When so prepared, no deviation from the
stated formula and directions is permitted. If, however, such a
pharmaceutical preparation is manufactured on a larger scale with the
intention that it may be stored, deviations from the formula and directions
stated under the heading Extemporaneous Preparation are permitted
provided that any ingredient, other than those included in the Definition,
complies with the general notice on Excipients and that the final product
meets the following criteria:
(1) accordance with the Definition stated in the monograph;
(2) compliance with all of the requirements stated in the monograph;
(3) retention of the essential characteristics of the preparation made strictly
in accordance with the formula and directions of the Pharmacopoeia.
In the manufacture of any official preparation on a large scale with the
intention that it should be stored, in addition to following any instruction
under the heading Production, it is necessary to ascertain that the product
is satisfactory with respect to its physical and chemical stability and its state
of preservation over the claimed shelf-life. This applies irrespective of
whether the formula of the Pharmacopoeia and any instructions given under
the heading Extemporaneous Preparation are followed precisely or
modified. Provided that the preparation has been shown to be stable in
other respects, deterioration due to microbial contamination may be
inhibited by the incorporation of a suitable antimicrobial preservative. In
such circumstances the label states appropriate storage conditions, the date
after which the product should not be used and the identity and
concentration of the antimicrobial preservative.
Freshly and The direction, given under the heading Extemporaneous Preparation, that a
Recently Prepared preparation must be freshly prepared indicates that it must be made not
more than 24 hours before it is issued for use. The direction that a
preparation should be recently prepared indicates that deterioration is likely
if the preparation is stored for longer than about 4 weeks at 15° to 25°.
Methods of The methods of sterilisation used in preparing the sterile materials

Sterilisation described in the Pharmacopoeia are given in Appendix XVIII. For aqueous
preparations, steam sterilisation (heating in an autoclave) is the method of
choice wherever it is known to be suitable. Any method of sterilisation must
be validated with respect to both the assurance of sterility and the integrity
of the product and to ensure that the final product complies with the
requirements of the monograph.
Water The term water used without qualification in formulae for formulated
preparations means either potable water freshly drawn direct from the
public supply and suitable for drinking or freshly boiled and cooled Purified
Water. The latter should be used if the public supply is from a local storage
tank or if the potable water is unsuitable for a particular preparation.
Excipients Where an excipient for which there is a pharmacopoeial monograph is used

in preparing an official preparation it shall comply with that monograph.
Any substance added in preparing an official preparation shall be
innocuous, shall have no adverse influence on the therapeutic efficacy of the
active ingredients and shall not interfere with the assays and tests of the
Pharmacopoeia. Particular care should be taken to ensure that such
substances are free from harmful organisms.
Colouring Agents If in a monograph for a formulated preparation defined by means of a full

formula a specific colouring agent or agents is prescribed, suitable
alternatives approved in the country concerned may be substituted.
Antimicrobial When the term ‘suitable antimicrobial preservative’ is used it is implied that
Preservatives the preparation concerned will be effectively preserved according to the
appropriate criteria applied and interpreted as described in the test for
efficacy of antimicrobial preservation (Appendix XVI C). In certain
monographs for formulated preparations defined by means of a full formula,
a specific antimicrobial agent or agents may be prescribed; suitable
alternatives may be substituted provided that their identity and
concentration are stated on the label.
Characteristics Statements given under the heading Characteristics are not to be

interpreted in a strict sense and are not to be regarded as official
requirements. Statements on taste are provided only in cases where this
property is a guide to the acceptability of the material (for example, a
material used primarily for flavouring). The status of statements on
solubility is given in the general notice on Solubility.
Solubility Statements on solubility given under the heading
Characteristics are intended as information on the approximate solubility at
a temperature between 15° and 25°, unless otherwise stated, and are not to
be considered as official requirements.
Statements given under headings such as Solubility in ethanol express
exact requirements and constitute part of the standards for the substances
under which they occur. •• V
The following table indicates the meanings of the terms used in
statements of approximate solubilities.
Descriptive term Approximate volume of

solvent in millilitres per
gram of solute
very soluble less than 1
freely soluble from 1 to 10
soluble from 1 0 to 30
sparingly soluble from 30 to 100 1
slightly soluble from 100 to 1000
very slightly soluble from 1000 to 10,000
practically insoluble more than 10,000
The term ‘partly soluble’ is used to describe a mixture of which only

some of the components dissolve. ■ ■-1.
Identification The tests described or referred to 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 being examined is in
accordance with the label on the container.
Unless otherwise prescribed; identification tests are carried out at a
temperature between 15° and 25°.
Reference spectra Where a monograph refers to an infrared reference
spectrum; this spectrum is provided in a separate section of the
Pharmacopoeia. A sample spectrum is considered to be concordant with a
reference spectrum if the transmission minima (absorption maxima) of the
principal bands in die sample correspond in position; relative intensities and
shape to those of the reference. Instrumentation software may be used to
calculate concordance with a previously recorded reference spectrum.
When tests for infrared absorption are applied to material extracted from
formulated preparations; strict concordance with the specified reference
spectrum may not always be possible, but nevertheless a close resemblance
between the spectrum of the extracted material and the specified reference
spectrum should be achieved.
Assays and Tests The assays and tests described are the official methods upon which the
standards of the Pharmacopoeia depend. The analyst is not precluded from
employing alternative methods; including methods of micro-analysis, in any
assay or test if it is known that the method used will give a result of
equivalent accuracy. Local reference materials may be used for routine
analysis, provided that these are calibrated against tile official reference
materials. In the event of doubt or dispute, the methods of analysis, the
reference materials and the reference spectra of the Pharmacopoeia are
alone authoritative.
Where the solvent used for a solution is not named, the solvent is
Purified Water.
Unless otherwise prescribed, the assays and tests are carried out at a
temperature between 15° and 25°.
A temperature in a test for Loss on drying, where no temperature range
is given, implies a range of ±2° about the stated value.
Visual comparative tests,, unless otherwise prescribed, are carried out
. using identical tubes of colourless, transparent; neutral glass with a flat
base. The volumes of liquid prescribed are for use with tubes 16 mm in
internal diameter; tubes with a larger internal diameter may be used but the
volume of liquid examined must be increased so that the depth of liquid in
the -tubes is not less than that obtained when the prescribed volume of
liquid and tubes 16 mm in internal diameter are used. Equal volumes of the
liquids to be compared are examined down the vertical axis of the tubes
against a white background or, if necessary, against a black background.
The examination is carried out in diffuse light.
Where a direction is given 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 direction is given that an analytical
operation is to be carried out ‘protected from light’, precautions should be
taken to exclude actinic light by the use of low-actinic glassware, working in
a dark room or similar procedures.
For preparations other than those of fixed strength, the quantity to be
taken for an assay or test is usually expressed in terms of the active
ingj-ejhent. This means tât the quantity of the active ingredient expected to
be present and the quantity of the preparation to be taken are calculated

from the strength stated on the label.
In assays the approximate quantity to be taken for examination is
indicated but the quantity actually used must not deviate by more than
10% from that stated. The quantity taken is accurately weighed or
measured and the result of the assay is calculated from this exact quantity.
Reagents are measured and the procedures are carried out with an accuracy
commensurate with the degree of precision implied by the standard stated
for the assay.
In tests the stated quantity to be taken for examination must be used
unless any divergence can be taken into account in conducting the test and
calculating the result. The quantity taken is accurately weighed or measured
with the degree of precision implied by the standard or, where the standard
is not stated numerically (for example, in tests for Clarity and colour of
solution), with the degree of precision implied by the number of significant
figures stated. Reagents are measured and the procedures are carried out
with an accuracy commensurate with this degree of precision.
The limits stated in monographs are based on data obtained in normal
analytical practice; they take account of normal analytical errors, of
acceptable variations in manufacture and of deterioration to an extent
considered acceptable. No further tolerances are to be applied to the limits
prescribed to determine whether the article being examined complies with
the requirements of the monograph.
In determining compliance with a numerical limit, the calculated result of
a test or assay is first rounded to the number of significant figures stated,
unless otherwise prescribed. The last figure is increased by 1 when the part
rejected is equal to or exceeds one half-unit, whereas it is not modified
when the part rejected is less than a half-unit.
In certain tests, the concentration of impurity is given in. parentheses
either as a percentage or in parts per million by weight (ppm). In
chromatographic tests such concentrations are stated as a percentage
irrespective of the limit. In other tests they are usually stated in ppm unless
the limit exceeds 500 ppm. In those chromatographic tests in which a
secondary, spot or peak in a chromatogram obtained with a solution of the
substance being examined is described as corresponding to a named
impurity and is compared with a spot or peak in a chromatogram obtained
with a reference solution of the same impurity, the percentage given in
parentheses indicates the limit for that impurity. In those chromatographic
tests in which a spot or peak in a chromatogram obtained with a solution of
the .substance being examined is described in terms other than as
corresponding to a named, impurity (commonly, for example, as any (other)
1 secondary spot or peaky. but is compared with a spot or. peak in a
chromatogram obtained with a reference solution of a named impurity, the
percentage given in parentheses indicates an impurity limit expressed in
terms of a nominal concentration of the named impurity. In
chromatographic tests in which a .comparison is made between spots or
peaks in chromatograms obtained with solutions, of different concentrations
of the substance being examined, the percentage given in parentheses
indicates an impurity limit expressed in terms of a nominal concentration of
the medicinal substance itself. In some monographs, in particular those for
certain formulated preparations, the impurity limit is expressed in terms of a
nominal concentration of the active moiety rather than of the medicinal
substance itself. Where necessary for clarification the terms in which the
limit is expressed are stated within die monograph.
In all cases where an impurity limit is given in parentheses, the figures
given are approximations for information only; conformity with die
requirements is determined on the basis of compliance or otherwise with
the stated test.
The use of a proprietary designation to identify a material used in an
assay or test does not imply that another equally suitable material may not
be used.
Biological Assays Methods of assay described as Suggested methods are not obligatory’, but
and Tests when another method is used its precision must be not less than that
required for the Suggested method.
For those antibiotics for which the monograph specifies a microbiological
assay the potency requirement is expressed in the monograph in
International Units (IU) per milligram. The material is not of
pharmacopoeia! quality if the upper fiducial limit of error is less than the
stated potency. For such antibiotics the required precision of die assay is
stated in the monograph in terms of the fiducial limits of error about the
estimated potency.
For other substances and preparations for which the monograph specifies
a biological assay, unless otherwise stated, the precision of die assay is such
that the fiducial limits of error, expressed as a percentage of tire estimated
potency, are within a range not wider than drat obtained by multiplying by
a factor of 10 the square roots of the limits given in the monograph for the
fiducial limits of error about the stated potency.
In all cases fiducial limits of error are based on a probability of 95%
(P = 0.95).
Where the biological assay is being used to ascertain tire purity of the
material, die stated potency means the potency stated on die label in terms
of International Units (IU) or other Units per gram, per milligram or per
millilitre. When no such statement appears on the label, the stated potency
means the fixed or minimum potency required in die monograph. This
interpretation of stated potency applies in all cases except where the
monograph specifically directs otherwise.
Where the biological assay is being used to determine the total activity in
the container, the stated potency means the total number of International
Units (IU) or other Units stated on the label or, if no such statement
appears, the total activity calculated in accordance with the instructions in
the. monograph.
Wherever possible the primary standard used in an assay or test is die
respective International Standard or Reference Preparation established by
the World Health Organization for international use and the biological
activity is expressed in International Units (IU) .
In other cases, where Units are referred to in an assay or test, the Unit
for a particular substance or. preparation is, for the United Kingdom, the
specific biological activity contained in such an amount of the respective
primary standard as the appropriate international or national organisation
indicates-. The necessary information is provided with the primary7 standard.
Unless otherwise directed, animals used in an assay or a test are healdiy
animals, drawn from a uniform stock, that have not previously been treated
with any material that will interfere with the assay or test. Unless otherwise
statetj, guinea-pigs weigh not less than 250 g or, when used in systemic
toxicity tests, not less than 350 g. When used in skin tests they are white or
light coloured. Unless otherwise stated, mice weigh not less than 17 g and
not more than 22 g.
Certain of the biological assays and tests of the Pharmacopoeia are such
that in the United Kingdom they may be carried out only in accordance
with the Animals (Scientific Procedures) Act 1986. Instructions included in
such assays and tests in the Pharmacopoeia, with respect to the handling of
animals, are therefore confined to those concerned with the accuracy and
reproducibility of the assay or test.
Reference Certain monographs require the use of a reference substance, a reference

Substances and preparation or a reference spectrum. These are chosen with regard to their
Reference intended use as prescribed in the monographs of the Pharmacopoeia and
Preparations arc not necessarily suitable in other circumstances.
Any information necessary for proper use of the reference substance or
reference preparation is given on the label or in the accompanying leaflet or
brochure. Where no drying conditions are stated in the leaflet or on the
label, the substance is to be used as received. No certificate of analysis or
other data not relevant to the prescribed use of the product are provided.
The products are guaranteed to be suitable for use for a period of three
months from dispatch when stored under the appropriate conditions. The
stability of the contents of opened containers cannot be guaranteed. The
current lot is listed in the BP Laboratory website catalogue. Additional
information is provided in Supplementary Chapter in E.
Chemical Reference Substances The abbreviation BPCRS indicates a
Chemical Reference Substance established by the British Pharmacopoeia
Commission. The abbreviation CRS or EPCRS indicates a Chemical
Reference Substance established by the European Pharmacopoeia
Commission. Some Chemical Reference Substances are used for the
microbiological assay of antibiotics and their activity is stated, in
International Units, on the label or on the accompanying leaflet and defined
in the same manner as for Biological Reference Preparations.
Biological Reference Preparations The majority of the primary
biological reference preparations referred to are' the appropriate
International Standards and Reference Preparations established by the
World Health Organisation. Because these reference materials are usually
available only in limited quantities, the European Pharmacopoeia has
established Biological Reference Preparations (indicated by the
abbreviation BRP or EPBRP) where appropriate. Where applicable, the
potency of the Biological Reference Preparations is expressed in
International Units. For some Biological Reference Preparations, where an
international standard or reference preparation does not exist, the potency is
expressed in European Pharmacopoeia Units. .
Storage Statements under the side-heading Storage constitute non-mandatory

. advice. The substances and preparations described in the Pharmacopoeia
are to be stored under conditions that prevent contamination and, as far as
possible, deterioration. Unless otherwise stated in the monograph, the
substances and preparations described in the Pharmacopoeia are kept in
well-closed containers and stored at a temperature not exceeding 25°.
Precautions that should be taken in relation to the effects of the
atmosphere, moisture, heat and light are indicated, where appropriate, in
the monographs. Further precautions may be necessary when some

materials are stored in tropical climates or under other severe conditions.
The expression ‘protected from moisture’ means that the product is to be
stored in an airtight container. Care is to be taken when the container is
opened in a damp atmosphere. A low moisture content may be maintained,
if necessary, by the use of a desiccant in the container provided that direct
contact with the product is avoided.
The expression ‘protected from light’ means that the product is to be
stored either in a container made of a material that absorbs actinic light
sufficiently to protect the contents from change induced by such light or in
a container enclosed in an outer cover that provides such protection or
stored in a place from which all such light is excluded.
The expression ‘tamper-evident container’ means a closed container fitted
with a device that reveals irreversibly whether the container has been
opened, whereas, the expression ‘tamper-proof container’ means a closed
container in which access to the contents is prevented under normal
conditions of use. The two terms are considered to be synonymous by the
European Pharmacopoeia Commission.
Labelling The labelling requirements of the Pharmacopoeia arc not comprehensive,

and the provisions of regulations issued in accordance with the
requirements of the territory in which the medicinal product is to be used
should be met.
Licensed medicines intended for use within the United Kingdom must
comply with the requirements of The Human Medicines Regulations 2012
and European Directive 2001/83/EC, Title V (as amended) in respect of
their labelling and package leaflets, together with those regulations for the
labelling of hazardous materials.
Best practice guidance on the labelling and packaging of medicines for
use in the United Kingdom advises that certain items of information are
deemed critical for the safe use of the medicine (see “Best Practice
Guidance on the Labelling and Packaging of Medicines” issued by the
MHRA, 2012). Further information and guidance on the labelling of
medicinal products can be found in Supplementary Chapter I G.
Such matters as the exact form of wording to be used and whether a
particular item of information should appear on the primary label and
additionally, or alternatively, on the package or exceptionally in a leaflet are,
in general, outside the scope of the Pharmacopoeia. When the term ‘label’
is used in Labelling statements of the Pharmacopoeia, decisions as to where
the particular statement should appear should therefore be made in
accordance with relevant legislation.
The label of every official formulated preparation other than those of
fixed strength also states the content of the active ingredient or ingredients
expressed in the terms required by the monograph. Where the content of
active ingredient is required to be expressed in terms other than the weight
of the official medicinal substance used in making the formulation, this is
specifically stated under the heading Labelling. Unless otherwise stated in
the monograph, the content of the active ingredient is expressed in terms of
the official medicinal substance used in making the formulation.
These requirements do not necessarily apply to unlicensed preparations
supplied in accordance with a prescription. For requirements for unlicensed
medicines see the general monograph on Unlicensed Medicines.
Action and Use The statements given under this heading in monographs are intended only
as information on the principal pharmacological actions or the uses of the
materials in medicine or pharmacy. It should not be assumed that the
substance has no other action or use. The statements are not intended to be
binding on prescribers or to limit their discretion.
Crude Drugs; Herbal and complementary medicines are classed as medicines under European
Traditional Herbal Directive 2001183/EC as amended. It is emphasised that, although requirements
and Complementary for the quality of the material are provided in the monograph to assist the
Medicines registration scheme by the UK Licensing Authority, the British Pharmacopoeia
Commission has not assessed the safety or efficacy of the material in traditional
use.
Monograph Title For traditional herbal medicines, the monograph tide
is a combination of the binomial name together with a description of use.
Monographs for the material that has not been processed (the herbal drug)
and the processed material (the herbal drug preparation) are published
where possible. To distinguish between the two, the word ‘Processed’ is
included in the relevant monograph tide.
Definition Under die heading Definition, the botanical name together
with any synonym is given. Where appropriate, for material that has not
been processed, information on the collection/harvesting and/or treatment/
drying of the whole herbal drug may be given. For processed materials,, the
method of processing, where appropriate, will normally be given in a
separate section.
Characteristics References to odour are included only where this is .
highly characteristic. References to taste are not included.
Control methods Where applicable, the control methods to be used in
monographs are:
(a) macroscopical and microscopical descriptions and chemical/
chromatographic tests for identification
(b) tests for absence of any related species
(c) microbial test to assure microbial quality
(d) tests for inorganic impurities and non-specific purity tests, including
extractive tests, Sul fated ash and Heavy metals where appropriate
(e) test for Loss on drying or Water
(f) wherever possible, a method for assaying the active constituent(s) or ■
suitable marker constituent(ร).
The macroscopical characteristics include those features that can be seen
by the unaided eye or by the use of a hand lens. When tw0 species/
subspecies of the same plant are included in the Definition, individual
differences between the two are indicated where possible.
The description of the microscopical characteristics of the powdered drug
includes information bn the dominant or the most specific characters.
Where it is considered to be an aid to identification, illustrations of the
powdered drug may be provided. -
The following aspects are controlled by the general monograph for
Herbal Drugs: they are required to be free from moulds, insects, decay,
animal matter and animal excreta. Unless otherwise prescribed the amount
of foreign matter is not more than 2% พ/พ.-Microbial contamination.should
be minimal.
In determining the content of the active constituents or the suitable

marker substances measurements are made with reference to the dried or
anhydrous herbal drug. In the tests for Acid-insoluble ash, Ash, Extractive
soluble in ethanol, Loss on drying, Sulfated ash, Water, Water-soluble ash
and Water-soluble extractive of herbal drugs, the calculations are made with
reference to the herbal drug that has not been specifically dried unless
otherwise prescribed in the monograph.
Homoeopathic Homoeopathic medicines are classed as medicines under European Directive

Medicines 2001I83IEC as amended. It is emphasised that, although requirements for the
quality of the material are provided in the relevant monograph in order to assist
the simplified registration scheme by the UK Licensing Authority, the British
Pharmacopoeia Commission has not assessed the safety or efficacy of the material
in use.
All materials used for the production of homoeopathic medicines,
including excipients, must comply with European Pharmacopoeia or British
Pharmacopoeia monographs for those materials. Where such European
Pharmacopoeia or British Pharmacopoeia monographs do not exist, each
material used for the production of homoeopathic medicines must comply
with an official national pharmacopoeia of a Member State.
British. Pharmacopoeia monographs for homoeopathic medicines apply to
homoeopathic stocks and mother tinctures only, but may be prefaced by a
section which details the quality requirements applicable to the principle
component where there is no European Pharmacopoeia or British
Pharmacopoeia monograph for the material. These monographs also
include either general statements on the methods of preparation or refer to
specific methods of preparation given in the European Pharmacopoeia.
Homoeopathic stocks and mother tinctures undergo the further process
referred to. as potentisation. Potentisation is a term specific to homoeopathic
medicine and is a process of dilution of stocks and mother tinctures to
produce the final product.
Identification tests are established for the components in homoeopathic
stocks and usually relate to those applied to the materials used in the
production of the homoeopathic stocks. An assay is included for the
principal component(s) where possible. For mother tinctures, an
identification test, usually chromatographic, is established and, where
applicable, an assay for the principle component(ร); where appropriate,
other tests, related to the solvent, , dry matter or known adulterants, are
included.
Specifications have not been set for final homoeopathic products due to
the high dilution used in their preparation and the subsequent difficulty in
applying analytical methodology.
Statements under Crude Drugs; Traditional Herbal and Complementary
Medicines also apply to homoeopathic stocks and mother tinctures, when
appropriate.
Unlicensed The General Monograph for Unlicensed Medicines applies to those

Medicines formulations used in human medicine that are prepared under a
Manufacturer’s ‘Specials’ Licence or prepared extemporaneously under the
supervision of a pharmacist, whether or not there is a published monograph
for the specific dosage form.
An article intended for medicinal use that is described by means of an
official title must comply with the requirements of the relevant monograph.
A formulated preparation must comply throughout its assigned shelf-life

(period of validity). The subject of any other monograph must comply
throughout its period of use.
Unlicensed medicines that are prepared under a Manufacturer’s
‘Specials’ Licence comply with the requirements of the General Monograph
for Pharmaceutical Preparations, the requirements of the General
Monograph for Unlicensed Medicines and, where applicable, the
requirements of the individual monograph for the specific dosage form.
Unlicensed medicines prepared extemporaneously under the supervision
of a pharmacist comply with the requirements of the General Monograph
for Pharmaceutical Preparations, the requirements of the General
Monograph for Unlicensed Medicines and, where applicable, the
requirements of the individual monograph for the specific dosage form.
While it is expected that extemporaneous preparations will demonstrate
pharmacopoeial compliance when tested, it is recognised that it might not
be practicable to carry out the pharmacopoeial tests routinely on such
formulations. In the event of doubt or dispute, the methods of analysis, the
reference materials and the reference spectra of the Pharmacopoeia are
IV-20 General Nonces 2016
Part in
Monographs and other texts of the European Pharmacopoeia that are incorporated
in this edition of the British Pharmacopoeia are governed by the general notices of
the European Pharmacopoeia; these are reproduced below.
GENERAL NOTICES OF THE EUROPEAN
PHARMACOPOEIA
1.1. GENERAL STATEMENTS
The General Notices apply to all monographs and other texts of the
European Pharmacopoeia.
The official texts of the European Pharmacopoeia are published in
English and French. Translations in other languages may be prepared by
the signatory States of the European Pharmacopoeia Convention. In case of
doubt or dispute, the English and French versions are alone authoritative.
In the texts of the European Pharmacopoeia, the word ‘Pharmacopoeia’
without qualification means the European Pharmacopoeia. The official
abbreviation Ph. Eur. may be used to indicate the European
Pharmacopoeia.
The use of the title or the subtitle of a monograph implies that the article
complies with the requirements of the relevant monograph. Such references
to monographs in the texts of the Pharmacopoeia are shown using the
monograph title and reference number in italics.
A preparation must comply throughout its period of validity; a distinct
period of validity and/or specifications for opened or broached containers
may be decided by the competent authority. The subject of any other
monograph must comply throughout its period of use. The period of
validity that is assigned to any given article and the time from which that
period is to be calculated are decided by the competent authority in light of
experimental results of stability studies.
Unless otherwise indicated in the General Notices or in the monographs,
statements in monographs constitute mandator}7 requirements. General
chapters become mandatory when referred to in a monograph, unless such
reference is made in a way that indicates that it is not the intention to make
the text referred to mandatory but rather to cite it for information.
The active substances, excipients, pharmaceutical preparations and other
articles described in the monographs are intended, for human and veterinary
use (unless explicitly restricted to one of these uses).
Quality systems The quality standards represented by monographs are valid only where the
articles in question are produced within the framework of a suitable quality
system. The quality system must assure that the articles consistently meet
the requirements of the Pharmacopoeia.
Alternative methods The tests and assays described are the official methods upon which the
standards of the Pharmacopoeia are based. With the agreement of the
competent authority, alternative methods of analysis may be used for
control purposes, provided that the methods used 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. In the

event of doubt or dispute, the methods of analysis of the Pharmacopoeia are
Demonstration of (1) An article is not of Pharmacopoeia quality unless it complies with all
compliance with the the requirements stated in the monograph. This does not imply that
Pharmacopoeia performance of all the tests in a monograph is necessarily a prerequisite
for a manufacturer in assessing compliance with the Pharmacopoeia
before release of a product. The manufacturer may obtain assurance
that a product is of Pharmacopoeia quality on the basis of its design,
together with its control strategy and data derived, for example, from
validation studies of the manufacturing process.
(2) An enhanced approach to quality control could utilise process analytical
technology (PAT) and/or real-time release testing (including parametric
release) strategies as alternatives to end-product testing alone. Real-time
release testing in circumstances deemed appropriate by the competent
authority is thus not precluded by the need to comply with the
Pharmacopoeia.
(3) Reduction of animal testing: the European Pharmacopoeia is dedicated
to phasing out the use of animals for test purposes, in accordance with
the 3Rs (Replacement, Reduction, Refinement) set out in the European
Convention for the Protection of Vertebrate Animals used for
Experimental and Other Scientific Purposes. In demonstrating
compliance with the Pharmacopoeia as indicated above (1),
manufacturers may consider establishing additional systems to monitor
consistency of production. With the agreement of the competent
authority, the choice of tests performed to assess compliance with the
Pharmacopoeia when animal tests are prescribed is established in such
a way that animal usage is minimised as much as possible.
Grade of materials Certain materials that are the subject of a pharmacopoeial monograph may
exist in different grades suitable for different purposes. Unless otherwise
indicated in the monograph, the requirements apply to all grades of the
material. In some monographs, particularly those on excipients, a list of
functionality-related characteristics that are relevant to the use of the
substance may be appended to the monograph for information. Test
methods for determination of one or more of these characteristics may be
given, also for information.
General Substances and preparations that are the subject of an individual

monographs monograph are also required to comply with relevant, applicable general
monographs. Cross-references to applicable general monographs are not
normally given in individual monographs.
General monographs apply to all substances and preparations within the
scope of the Definition section of the general monograph, except where a
preamble limits the application, for example to substances and preparations
that are the subject of a monograph of the Pharmacopoeia.
General monographs on dosage forms apply to all preparations of the
type defined. The requirements are not necessarily comprehensive for a
given specific preparation and requirements additional to those prescribed
in the general monograph may be imposed by the competent authority.
General monographs and individual monographs are complementary. If

the provisions of a general monograph do not apply to a particular product,
this is expressly stated in the individual monograph.
Validation of The test methods given in monographs and general chapters have been
pharmacopoeial validated in accordance with accepted scientific practice and current
methods recommendations on analytical validation. Unless otherwise stated in the
monograph or general chapter, validation of the test methods by the analyst
is not required.
Implementation of When implementing a pharmacopoeial method, the user must assess

pharmacopoeial whether and to what extent the suitability of the method under the actual
methods conditions of use needs to be demonstrated according to relevant
monographs, general chapters and quality systems.
Conventional terms The term ‘competent authority’ means the national, supranational or
international body or organisation vested with the authority for making
decisions concerning the issue in question. It may, for example, be a
national pharmacopoeia authority, a licensing authority or an official control
laboratory.
The expression ‘unless otherwise justified and authorised’ means that the
requirements have to be met, unless the competent authority authorises a
modification or an exemption where justified in a particular case.
Statements containing the word ‘should’ are informative or advisory.
In certain monographs or other texts, the terms ‘suitable’ and
‘appropriate’ are used to describe a reagent, micro-organism, test method
etc.; if criteria for suitability are not described in the monograph, suitability
is demonstrated to the satisfaction of the competent authority.
Medicinal product (a) Any substance or combination of substances
presented as having properties for treating or preventing disease in human
beings and/or animals; or (b) any substance or combination of substances
that may be used in or administered to human beings and/or animals with a
view either to restoring, correcting or modifying physiological functions by
exerting a pharmacological, immunological or metabolic action, or to
making a medical diagnosis.
Herbal medicinal product Any medicinal product, exclusively
containing as active ingredients one or more herbal drugs or one or more
herbal drug preparations, or one or more such herbal drugs in combination
with one or more such herbal drug preparations.
Active substance Any substance intended to be used in the manufacture
of a medicinal product and that, when so used, becomes an active
ingredient of the medicinal product. Such substances are intended to
furnish a pharmacological activity or other direct effect in the diagnosis,
cure, mitigation, treatment or prevention of disease, or to affect the
structure and function of the body.
Excipient (auxiliary substance). Any constituent of a medicinal product
that.is not an active substance. Adjuvants, stabilisers, antimicrobial
preservatives, diluents, antioxidants, for example, are excipients.
Interchangeable Certain general chapters contain a statement that the text in question IS
methods harmonised with the corresponding text of the Japanese Pharmacopoeia
and/or the United States Pharmacopeia and that these texts are
interchangeable. This implies that if a substance or preparation is found to
comply with a requirement using an interchangeable method from one of

these pharmacopoeias it complies with the requirements of the European
Pharmacopoeia. In the event of doubt or dispute, the text of the European
Pharmacopoeia is alone authoritative.
References to Monographs and general chapters may contain references to documents

regulatory issued by regulatory authorities for medicines, for example directives and
documents notes for guidance of the European Union. These references are provided
for information for users for the Pharmacopoeia. Inclusion of such a
reference does not modify the status of the documents referred to, which
may be mandatory or for guidance.
1.2. OTHER PROVISIONS APPLYING TO GENERAL
CHAPTERS AND MONOGRAPHS
Quantities In tests with numerical limits and assays, the quantity stated to be taken for
examination 's 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 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 substance in the same conditions, the
stated quantity is taken for examination. 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 corresponds
to 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 (for example,
10.0 mL or 0.50 mL), the volume is measured using a pipette, a volumetric. .
flask or a burette, as appropriate; otherwise, a graduated measuring cylinder
or a graduated pipette may be used,. Volumes stated in microlitres are
measured using a micropipette or microsyringe.
It is recognised, however, that in certain cases, the precision with which
quantities are stated does not correspond to the number of significant
figures stated in a specified numerical limit. The weighings and
measurements are then carried out with a sufficiently improved accuracy.
Apparatus and Volumetric glassware complies with Class A requirements of the appropriate
procedures International Standard issued by the International Organisation for
Standardisation. .
Unless otherwise prescribed, analytical procedures are carried out at a
temperature between 15 °C and 25 °C.
Unless otherwise prescribed, comparative tests are carried out using
identical tubes of colourless, transparent, neutral glass with a flat base; the
volumes ;of liquid prescribed- are for use with tubes having an internal
diameter of 16 mm, but tubes with a larger internal diameter may be used
provided the volume of liquid used is adjusted (2.1.5). Equal volumes of
the liquids to be compared are examined down the vertical axis of the tubes
against a white background, or if necessary against a black background. The
examination is carried out in diffuse light.
Any solvent required in a test or. assay in which an indicator is to be used
is previously neutralised to the indicator,.unless a blank test is prescribed.
Water-bath The term ‘water-bath’ means a bath of boiling water unless water at
another temperature is indicated. Other methods of heating may be
substituted provided the temperature is near to but not higher than
100 °C or the indicated temperature.
Drying and ignition The terms ‘dried to constant mass’ and ‘ignited to constant mass’ mean
to constant mass that 2 consecutive weighings do not differ by more than 0.5 mg, the 2nd
weighing following an additional period of drying or of ignition respectively
appropriate to the nature and quantity of the residue.
Where drying is prescribed using one of the expressions ‘in a desiccator’
or ‘in vacuo3) it is carried out using the conditions described in chapter
2.2.32. Loss on drying.
Reagents The proper conduct of the analytical procedures described in the

Pharmacopoeia and the reliability of the results depend., in part, upon the
quality of the reagents used. The reagents are described in general chapter
4. It is assumed that reagents of analytical grade are used; for some
reagents, tests to determine suitability are included in the specifications.
Solvents Where the name of the solvent is not stated, the term ‘solution’ implies a
solution in water.
Where the use of water is specified or implied in the analytical
procedures described in the Pharmacopoeia or for the preparation of
reagents, water complying with the requirements of the monograph Purified
water (0008) is used, except that for many purposes the requirements for
bacterial endotoxins (Purified water in bulk) and microbial contamination
(Purified water in containers) are not relevant. The term ‘distilled water’
indicates purified water prepared by distillation.
The term ‘ethanol’ without qualification means anhydrous ethanol. The
term ‘alcohol’ without qualification means ethanol (96 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 (C2H6O) required.
Expression of In defining content, the expression ‘per cent’ is used according to

content circumstances with one of 2 meanings:
— per cent mini (percentage, mass in mass) expresses the number of
grams of substance in 100 g of’final product;
— per cent V!V (percentage, volume in volume) expresses the number of
millilitres of substance in 100 mL of final product.
The expression ‘parts per million’ (or ppm) refers to mass in mass, unless
otherwise specified.
Temperature Where an analytical procedure describes temperature without a figure, the

general terms used have the following meaning:
— in a deep-freeze: below ”15 °C;
— in a refrigerator: 2 °C to 8 °C;
— cold or cool: 8 °C to 15 °C;
— room temperature: 15 °C to 25 °C.
13. GENERAL CHAPTERS

Containers Materials used for containers are described in general chapter 3.1. General
names used for materials, particularly plastic materials, each cover a range
of products varying not only in the properties of the principal constituent
but also in the additives used. The test methods and limits for materials
depend on the formulation and are therefore applicable only for materials
whose formulation is covered by the preamble to the specification. The use
of materials with different formulations, and the test methods and limits
applied to them, are subject to agreement by the competent authority.
The specifications for containers in general chapter 3.2 have been
developed for general application to containers of the stated category, but in
view of the wide variety of containers available and possible new
developments, the publication of a specification does not exclude the use, in
justified circumstances, of containers that comply with other specifications,
subject to agreement by the competent authority.
Reference may be made within the monographs of the Pharmacopoeia to
the definitions and specifications for containers provided in chapter 3.2.
Containers. The general monographs for pharmaceutical dosage forms may,
under the heading Definition/Production, require the use of certain types of
container; certain other monographs may, under the heading Storage,
indicate the type of container that is recommended for use.
1.4. MONOGRAPHS
Titles Monograph titles are in English and French in the respective versions and
there is a Latin subtitle.
Relative Atomic and The relative atomic mass (y4r) or the relative molecular mass (Afr) is shown,
Mole'cular Masses as and where appropriate, at the beginning of each monograph. The relative
atomic and molecular masses and the molecular and graphic formulae do
not constitute analytical standards for the substances described.
Chemical Abstracts CAS registry numbers are included for information in monographs, where
Service (CAS) applicable, to provide convenient access to useful information for users.
Registry Number CAS Registry Number® is a registered trademark of the American
Chemical Society.
Definition Statements under the heading Definition constitute an official definition of

the substance, preparation or other article that is the subject of the
monograph.
Limits of content พhere limits of content are prescribed, they are those
determined by the method described under Assay.
Herbal drugs In monographs on herbal drugs, the definition indicates
whether the subject of the monograph is, for example, the whole drug or
the drug in powdered form. Where a monograph applies to the drug in
several states, for example both to the whole drug and the drug in
powdered form, the definition states this.
Production Statements under the heading Production draw attention to particular

aspects of the manufacturing process but are not necessarily comprehensive.
They constitute mandatory requirements for manufacturers, unless
otherwise stated. They may relate, for example, to source materials; to the
manufacturing process itself and its validation and control; to in-process
testing; or to testing that is to be carried out by the manufacturer on the

final article, either on selected batches or on each batch prior to release.
These statements cannot necessarily be verified on a sample of the final
article by an independent analyst. The competent authority may establish
that the instructions have been followed, for example, by examination of
data received from the manufacturer, by inspection of manufacture or by
testing appropriate samples.
The absence of a Production section does not imply that attention to
features such as those referred to above is not required.
Choice of vaccine strain, Choice of vaccine composition The
Production section of a monograph may define the characteristics of a
vaccine strain or vaccine composition. Unless otherwise stated, test methods
given for verification of these characteristics are provided for information as
examples of suitable methods. Subject to approval by the competent
authority, other test methods may be used without validation against the
method shown in the monograph.
Potential Due to the increasing number of fraudulent activities and cases of

Adulteration adulteration, information may be made available to Ph. Eur. users to help
detect adulterated materials (i.e. active substances, excipients, intermediate
products, bulk products and finished products).
To this purpose, a method for the detection of potential adulterants and
relevant limits, together with a reminder that all stages of production and
sourcing are subjected to a suitable quality system, may be included in this
section of monographs on substances for which an incident has occurred or
that present a risk of deliberate contamination. The frequency of testing by
manufacturers or by users (e.g. manufacturers of intermediate products,
bulk products and finished products, where relevant) depends on a risk
assessment, taking into account the level of knowledge of the whole supply
chain and national requirements.
This section constitutes requirements for the whole supply chain, from
manufacturers to users (e.g. manufacturers of intermediate products, bulk
products and finished products, where relevant). The absence of this section
does not imply that attention to features such as those referred to above is
not required.
Characters The statements under the heading Characters are not to be interpreted in a
strict sense and are not requirements.
Solubility In statements of solubility in the Characters section, the terms
used have the following significance, referred to a temperature between
15 °C and 25 °C.
Descriptive term Approximate volume of solvent in millilitres

per gran1 of solute
Very soluble less than 1
Freely soluble from 1 to 10
Soluble from 10 to 30
Sparingly soluble . from 30 to 100
Slightly soluble from 100 to 1000
Very slightly soluble from : 1000 to . 10 000
Practically insoluble more than 10 000

The term ‘partly soluble’ is used to describe a mixture where only some
of the components dissolve. The term ‘miscible’ is used to describe a liquid
that is miscible in all proportions with the stated solvent.
Identification Scope The tests given in the Identification section are not designed to give
a full confirmation of the chemical structure or composition of the product;
they are intended to give confirmation, with an acceptable degree of
assurance, that the article conforms to the description on the label.
First and second identifications Certain monographs have
subdivisions entitled ‘First identification’ and ‘Second identification’. The
test or tests that constitute the ‘First identification’ may be used in all
circumstances. The test or tests that constitute the ‘Second identification’
may be used in pharmacies provided it can be demonstrated that the
substance or preparation is fully traceable to a batch certified to comply
with all die other requirements of the monograph.
Certain monographs give two or more sets of tests for the purpose of the
first identification, which are equivalent and may be used independently.
One or more of these sets usually contain a cross-reference to a test
prescribed in the Tests section of the monograph. It may be used to
simplify the work of the analyst carrying out the identification and the
prescribed tests. For example, one identification set cross-refers to a test for
enantiomeric purity while the other set gives a test for specific optical
rotation: the intended purpose of the two is the same, that is, verification
that the correct enantiomer is present.
Powdered herbal drugs Monographs on herbal drugs may contain
schematic drawings of the powdered drug. These drawings complement the
description given in the relevant identification test.
Tests and Assays Scope The requirements are not framed to take account of 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 if common sense
and good pharmaceutical practice require that it be absent. See also below
under Impurities.
Calculation Where the result of a test or assay is required to be
calculated with reference to the dried or anhydrous substance or on some
other specified basis, the determination of loss on drying, water content or
other property is carried out by the method prescribed in the relevant test
in the monograph. The words ‘dried substance’ or ‘anhydrous substance’
etc. appear in parentheses after the result.
Where a quantitative determination of a residual solvent is carried out
and a test for loss on drying is not carried out, the content of residual
solvent is taken into account for the calculation of the assay content of the
substance, the specific optical rotation and the specific absorbance. No
further indication is given in the specific, monograph.
Limits The limits prescribed are based on data obtained in. normal
' analytical practice; they take account of normal analytical errors, of
acceptable variations in manufacture and compounding arid of deterioration
to an extent considered acceptable. No further tolerances are to be applied
to the limits prescribed to determine whether the article being examined
complies with the requirements of the monograph.
In determining compliance with a numerical limit, the calculated result of
a test or assay is first rounded to the number of significant figures stated,
unless otherwise prescribed. The limits, regardless of whether the values are
expressed as percentages or as absolute values, are considered significant to

the last digit shown (for example 140 indicates 3 significant figures). The
last figure of the result is increased by one when the part rejected is equal to
or exceeds one half-unit, whereas it is not modified when the part rejected
is less than a half-unit.
Indication of permitted limit of impurities The acceptance criteria
for related substances are expressed in monographs either in terms of
comparison of peak areas (comparative tests) or as numerical values. For
comparative tests, the approximate content of impurity tolerated, or the
sum of impurities, may be indicated in brackets for information only.
Acceptance or rejection is determined on the basis of compliance or non-
compliance with the stated test. If the use of a reference substance for the
named impurity is not prescribed, this content may be expressed as a
nominal concentration of the substance used to prepare the reference
solution specified in the monograph, unless otherwise described.
Herbal drugs For herbal drugs, the sulfated ash, total ash, water-soluble
matter, alcohol-soluble matter, water content, content of essential oil and
content of active principle are calculated with reference to the drug that has
not been specially dried, unless otherwise prescribed in the monograph.
Equivalents Where an equivalent is given, for the purposes of the
Pharmacopoeia only the figures shown are to be used in applying the
requirements of the monograph.
Culture media The culture media described in monographs and general
chapters have been found to be satisfactory for the intended purpose.
However, the components of media, particularly those of biological origin,
are of variable quality, and it may be necessary for optimal performance to
modulate the concentration of some ingredients, notably:
— peptones and meat or yeast extracts, with respect to their nutritive
properties;
— . buffering substances;
— bile salts, bile extract, deoxycholate, and colouring matter, depending
on their selective properties;
— antibiotics, with respect to their.activity.
Storage The information and recommendations given under the heading Storage do
not constitute a pharmacopoeial requirement but the competent authority
may specify particular storage conditions that must be met.
The articles described in the Pharmacopoeia are stored in such a way as
to prevent contamination and, as far as possible, deterioration. Where
special conditions of storage are recommended, including the type of
container (see section 1.3. General chapters) and limits of temperature, they
•■ are stated in the monograph.
The following expressions are used in monographs under Storage with
the meaning shown.
In an airtight container Means that the product is stored in an airtight
container (3.2). Care is to be taken when the container is opened in a damp
atmosphere. A low moisture content may be maintained, if necessary, by
the use. of a desiccant in the container provided that direct contact with the
product is avoided.
Protected from light Means that the product is stored either in a
container made of a material that absorbs actinic light sufficiently to protect
the contents from change induced by such light, or in a container enclosed
in an outer cover that provides such protection, or is stored in a place from

which all such light is excluded.
Labelling In general, labelling of medicines is subject to supranational and national

regulation and to international agreements. The statements under the
heading Labelling are not therefore comprehensive and, moreover, for the
purposes of the Pharmacopoeia only those statements that are necessary to
demonstrate compliance or non-compliance with the monograph are
mandatory. Any other labelling statements are included as
recommendations. When the term ‘label’ is used in the Pharmacopoeia, the
labelling statements may appear on the container, the package, a leaflet
accompanying the package, or a certificate of analysis accompanying the
article, as decided by the competent authority.
Warnings Materials described in monographs and reagents specified for use in the
Pharmacopoeia may be injurious to health unless adequate precautions are
taken. The principles of good quality control laboratory practice and the
provisions of any appropriate regulations are to be observed at all times.
Attention is drawn to particular hazards in certain monographs by means of
a warning statement; absence of such a statement is not to be taken to
mean that no hazard exists.
Impurities A list of all known and potential impurities that have been shown to be
detected by the tests in a monograph may be given. See also chapter 5.10.
Control of impurities in substances for pharmaceutical use. The impurities are
designated by a letter or letters of the alphabet. Where a letter appears to be
missing, the impurity designated by this letter has been deleted from the list
during monograph development prior to publication or during monograph
revision.
Functionality- Monographs on excipients may have a section on functionality-related

Related characteristics. The characteristics, any test methods, for determination and
Characteristics of any tolerances are not mandatory requirements; they may nevertheless be
Excipients relevant for use of the excipient and are given for information (see also .
section 1.1.General, statements).
Reference Certain monographs require the use of reference standards (chemical

Standards reference substances, herbal reference standards, biological reference
preparations, reference spectra). See also chapter 5.12. Reference standards.
The European Pharmacopoeia Commission establishes the official reference
standards, which are alone authoritative in case of arbitration These
reference standards are available from the European .Directorate for the
Quality of Medicines & Healthcare (EDQAf). Information on the available
reference standards and a batch validity statement can be obtained via the
EDQM website.
1.5. ABBREVIATIONS AND SYMBOLS

/4 Absorbance mp Melting point
per cent
Specific absorbance ..20 Refractive index
A Relative atomic mass Ph. Eur. บ. European Pharmacopoeia Unit
Md Specific optical rotation ppb Parts per billion (micrograms per kilogram)
bp Boiling point ppm Parts per million (milligrams per kilogram)
BRP Biological Reference Preparation R Substance or solution defined under
CRS Chemical Reference Substance 4. Reagents
j20
Relative density’ Retardation factor (sec chapter 2.2.46)
“20
Wavelength Used in chromatography to indicate the ratio
of the distance travelled by a substance to the
HRS Herbal reference standard distance travelled by a reference substance
IU International Unit RV Substance used as a primary standard in
M Molarity’ volumetric analysis (chapter 4.2.1)
Alr Relative molecular mass
Abbreviations used in the monographs on immunoglobulins, immunosera and vaccines
LD50 The statistical!}' determined quantity of a Lo/10 dose The largest quantity of a toxin that, in the
substance that, when administered by the conditions of the test, when mixed with 0.1 IU
specified route, may be expected to cause the of antitoxin and administered by the specified
death of 50 per cent of the test animals within route, docs not cause symptoms of toxicity in
a given period the test animals within a given period
MLP Minimum lethal dose Lf dose The quantity of toxin or toxoid that flocculates
L-r/10 dose The smallest quantity of a toxin that, in the in the shortest time with 1 IU of antitoxin
conditions of the test, when mixed with 0.1 IU CCID50 The statistically determined quantity of virus
of antitoxin and administered by the specified that may be expected to infect 50 per cent of
route, causes the death of the test animals the cell cultures to which it is added
within a given period EID50 The statistically determined quantity of virus
L-r dose The smallest quantity of a toxin that, in the that may be expected to infect 50 per cent of
conditions of the test, when mixed with 1 IU fertilised eggs into which it is inoculated
of antitoxin and administered by the specified ID50 The statistically determined quantity of a virus
route, causes the death of the test animals that may be expected to infect 50 per cent of
within a given period the animals into which it is inoculated
lr/100 dose The smallest quantity of a toxin that, in the PD50 The statistically determined dose of a vaccine
conditions of the test, when mixed with that, in the conditions of the test, may be
0.01 IU of antitoxin and injected expected to protect 50 per cent of the animals
intracutaneously causes a characteristic against a challenge dose of the micro
reaction at the site of injection within a organisms or toxins against which it is active
given period
ED5o The statistically determined dose of a vaccine
Lp/10 dose The smallest quantity of toxin that, in the that, in the conditions of the test, may be
conditions of the test, when mixed with 0.1 IU expected to induce specific antibodies in
of antitoxin and administered by the specified 50 per cent of the animals for the relevant
route, causes paralysis in the test animals vaccine antigens
within a given period
PFU Pock-forming units or plaque-forming units
SPF Specified-pathogen-free.
Collections of micro-organisms
ATCC American Type Culture Collection NCTC National Collection of Type Cultures
10801 University Boulevard Central Public Health Laboratory
Manassas, Virginia 20110-2209, USA Colindale Avenue
C.I.P. Collection de Batteries de 1’Institut Pasteur London NW9 5HT, Great Britain
B.p. 52, 25 rue du Docteur Roux NCYC National Collection of Yeast Cultures
75724 Paris Cedex 15, France AFRC Food Research Institute
IMI International Mycological Institute Colney Lane
Bakeham Lane Norwich NR4 7UA, Great Britain
Surrey TW20 9TY, Great Britain NITE Biological Resource Center
P.
I. Collection Nationale de Culture de Department of Biotechnology
MicrOorganismes (C.N.C.M.) National Institute of Technology and
Institut Pasteur Evaluation
25, rue du Docteur Roux 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba,
75724 Paris Cedex 15, France 292-0818
Japan
NCIMB National Collection of Industrial and Marine
Bacteria Ltd S.S.I. Statens Serum Institut
23 St Machar Drive 80 Amager Boulevard, Copenhagen, Denmark
Aberdeen AB2 1RY, Great Britain
NCPF National Collection of Pathogenic Fungi
London School of Hygiene and Tropical
Medicine
Keppel Street
London wcIE 7HT, Great Britain
1.6. UNITS OF THE INTERNATIONAL SYSTEM (SI) USED IN

THE PHARMACOPOEIA AND EQUIVALENCE WITH OTHER
UNITS
International The International System of Units comprises 3 classes of units, namely base
System Of Units units, derived units and supplementary units1. The base units and their
(SI) definitions are set out in Table 1.6-1.
The derived units may be formed by combining the base units according
to the algebraic relationships linking the corresponding quantities. Some of
these derived units have special names and symbols. The SI units used in
the Pharmacopoeia are shown in Table 1.6-2.
Some important and widely used units outside the International System
are shown in Table 1.6-3.
The prefixes shown in Table 1.6-4 are used to form the names and
symbols of the decimal multiples and submultiples of SI units.
1 The definitions of the units used in the International System are given in the booklet “Le Systeme International d’Unites (SI)” published by
the Bureau International des Poids et Mesures, Pavillion de Breteuil, p-92310 Sevres. ไ^'-',-''.:':
Notes 1. In the Pharmacopoeia, the Celsius temperature is used (symbol โ').

This is defined by the following equation:
where Tq = 273.15 K by definition. The Celsius or centigrade

temperature is expressed in degree Celsius (symbol °C). The unit
‘degree Celsius’ is equal to the unit ‘kelvin’.
2. The practical expressions of concentrations used in the Pharmacopoeia
are defined in the General Notices.
3. The radian is the plane angle between two radii of a circle that cut off
on the circumference an arc equal in length to the radius.
4. In the Pharmacopoeia, conditions of centrifugation are defined by
reference to the acceleration due to gravity (g):
g = 9.806 65 m - ร’'2
5. Certain quantities without dimensions are used in the Pharmacopoeia:

relative density (2.2.5), absorbance (2.2.25), specific absorbance
(2.2.25) and refractive index (2.2.6).
6. The microkatal is defined as the enzymic activity that, under defined
conditions, produces the transformation (e.g. hydrolysis) of 1 micromole
of the substrate per second.
Table 1.6.-1. - SI base units

Quantity* Unit Definition
Name Symbol Name Symbol
m The metre is the length of the path travelled by light in a vacuum during a time
Length z metre
interval of 1/299 792 458 of a second.
Mass m kilogram kg The kilogram is equal to the mass of the international prototype of the kilogram.
The second is the duration of 9 192 631 770 periods of the radiation corresponding
Time t second ร to the transition between the two hyperfine levels of the ground state of the
caesium-133 atom.
Electric current J ampere A The ampere is that constant current which, maintained in two straight parallel
conductors of infinite length, of negligible circular cross-section and placed 1 metre
apart in vacuum would produce between these conductors a force equal to 2 X 10‘7
newton per metre of length.
Thermodynamic T kelvin K The kelvin is the fraction 1/273.16 of the thermodynamic temperature of the triple
temperature point of water.
Amount of substance ท mole mol The mole is the amount of substance of a system containing as many elementary
entities as there are atoms in 0.012 kilogram of carbon-12’.
Luminous intensity candela cd The candela is the luminous intensity in a given direction of a source emitting
monochromatic radiation with a frequency of 540 X 1012 hertz and whose energy
intensity in that direction is 1/683 watt per steradian. ___________ _
• When the mole is used, the elementary entities must be specified and may be atoms, molecules, ions, electrons, other particles or specified groups of
such particles.
Table 1.6.-2. - SI units used in the European Pharmacopoeia and equivalence with other units
Quantity Unit
Name Symbol Name Symbol Expression เท SI Expression in other Conversion of other units into SI units
base units SI units
Wave number y one per metre 1/m m”
Wavelength X micrometre pm 10’6m

nanometre nm 10’m
Area .4, ร square metre m2 nr
Volume V cubic metre mJ m3 1 mL = 1 cm3 = 10 6 m3

Frequency y hertz Hz ร’'
Density p kilogram per cubic kg/m3 kgm’3 1 g/mL = 1 g/cm3 = 103 kgm’3
metre
Velocity V metre per second m/s เท•ร-,
Force F newton N m-kgs’2 1 dyne = 1 gem ■ร'2 - IO’5 N

1 kp = 9.806 65 N
Pressure p pascal Pa m’’-kg-ร’2 N-m’2 1 dyne/cm2 = 10'’ Pa = 10'1 Nm’2
1 atm = 101 325 Pa = 101325 kPa
1 bar = 10s Pa = 0.1 MPa
1 mm Hg = 133.322 387 Pa
] Torr = 133.322 368 Pa
1 psi = 6.894 757 kPa
Dynamic q pascal second "Pa-S m’*kg-s’’ N-s-m"2 1 P.= 10’’Pa-s = 10-‘N-s-m’2
viscosity 1 cP = 1 mPa-s
Kinematic y square metre per nr/s m^s’1 Pa-sm-’-kg’1 . 1 St = 1 cm2-s’1 ■ IO’4 m*s"‘
viscosity second Nm-skg-1
Energy พโ joule J m^kg-s’2 N-m 1 erg - 1 cm^g-s'2 = 1 dynerm - 10’7 J
1 cal = 4.1868 j
Power p watt พ rn^kg-s’3 N-m-s’1 . 1 erg/s = 1 dyne-cm-s'1 =
Radiant flux 10~7 พ ธ IO’7 N-m-s’’ ธ lO-’J-s’1
Absorbed dose D gray Gy m-s Jkg-' 1 rad = 10"2 Gy
(of radiant
energy)
Electric บ volt V m2- kgs’พ1 W-A’’

potential,
electromotive
force
Electric ohm ดุ่ m2- kgs'3A’2 V-A’1
resistance
Quantity of Q coulomb c A-s
electricity
Activity of a .4 becquerel Bq ร’ 1 Ci = 37-10’ Bq = 37-10’ ร-’

radionuclide
Concentration mole per cubic mol/m3 mol-m"3 1 mol/L = 1 M = 1 mol/dm3 = 103 mol-m’3
(of amount of metre
substance),
molar
concentration
Mass p kilogram per cubic kg/m3 kg-m’3 1 g/L ธ 1 g/dm3 = 1 kg-m’3 .
concentration metre
Table 1.6.-3. - Units used with the International System

Quantity Unit Value in SI units
Name Symbol
Time minute min 1 min = 60 ร
hour h 1 h = 60 min = 3600 ร
day d 1 d = 24 h = 86 400 ร
Plane angle degree 0 1° = (n/180) rad
Volume litre L 1 L = 1 dm3 = 10"3 m3
Mass tonne t 11 = 10’ kg
Rotational revolution r/min 1 r/min = (1/60) ร"1

frequency per minute
Table 1.6.4. - Decimal multiples and sub-multiples of units
Factor Prefix Symbol Factor Prefix Symbol
10“ exa E 10-' deci d
10u peta p 10-* centi c
10“ . tera T 10-3 milli m
10’ Sjga G 10-® micro M
106 mega M 10-9 nano ท
103 kilo k 10-12 pico p
102 hecto h 10-15 femto f
พ, deca da 10-18
atto a
Monographs
Formulated Preparations ะ
General Monographs
2016 General Monographs IV-37
FORMULATED PREPARATIONS: ETHICAL CONSIDERATIONS AND GUIDANCE IN

THE PREPARATION OF UNLICENSED
GENERAL MONOGRAPHS PHARMACEUTICAL PREPARATIONS
The underlying principle of legislation for pharmaceutical
preparations is that, subject to specific exemptions, no
pharmaceutical preparation may be placed on the market
without an appropriate marketing authorisation.
Pharmaceutical Preparations * * The exemptions from the formal licensing requirement allow
(Ph. Eur. monograph 2619) *★* the supply of unlicensed products to meet the special needs
Ph Eur ________________________________________________ ’_________
of individual patients. However, when deciding to use an
unlicensed preparation all health professionals involved
INTRODUCTION (e.g. the prescribing practitioners and/or the preparing
This monograph is intended to be a reference source of pharmacists) have, within their area of responsibilities, a duty
standards in the European Pharmacopoeia on active of care to the patient receiving the pharmaceutical
substances, excipients and dosage forms, which are to be preparation.
applied in the manufacture/preparation of pharmaceuticals, In considering the preparation of an unlicensed
but not a guide on how to manufacture as there is specific pharmaceutical preparation, a suitable level of risk assessment
guidance available covering methods of manufacture and is undertaken.
associated controls.
The risk assessment identifies:
It does not cover investigational medicinal products, but — the criticality of different parameters (e.g. quality of active
competent authorities may refer to pharmacopoeial standards substances, excipients and containers; design of the
when authorising clinical trials using investigational medicinal preparation process; extent and significance of testing;
products. stability of the preparation) to the quality of the
DEFINITION preparation; and
Pharmaceutical preparations are medicinal products generally — the risk that the preparation may present to a particular
consisting of active substances that may be combined with patient group.
excipients, formulated into a dosage form suitable for the Based on the risk assessment, the person responsible for the
intended use, where necessary after reconstitution, presented preparation must ensure, with a suitable level of assurance,
in a suitable and appropriately labelled container. that the pharmaceutical preparation is, throughout its shelf
Pharmaceutical preparations may be licensed by the life, of an appropriate quality and suitable and fit for its
competent authority, or unlicensed and made to the specific purpose. For stock preparations, storage conditions and shelf
needs of patients according to legislation. There are 2 life have to be justified on the basis of, for example,
categories of unlicensed pharmaceutical preparations: analytical data or professional judgement, which may be
— extemporaneous preparations, i.e. pharmaceutical based on literature references.
preparations individually prepared for a specific patient or PRODUCTION
patient group, supplied after preparation; Manufacture/preparation must take place within the
— stock preparations, i.e. pharmaceutical preparations framework of a suitable quality system and be compliant with
prepared in advance and stored until a request for a the standards relevant to the type of product being made.
supply is received. Licensed products must comply with the requirements of
In addition to this monograph, pharmaceutical preparations their licence. For unlicensed products a risk assessment as
also comply with the General Notices and with the relevant outlined in the section ‘Ethical considerations and guidance
general chapters of the Pharmacopoeia. General chapters are in the preparation of unlicensed pharmaceutical preparations’
normally given for information and become mandatory when is of special importance, as these products are not previously
referred to in a general or specific monograph, unless such assessed by the competent authority.
reference is made in a way that indicates that it is not the Formulation
intention to make the text referred to mandatory but rather During pharmaceutical development or prior to
to cite it for information. manufacture/preparation, suitable ingredients, processes, tests
Where relevant, pharmaceutical preparations also comply and specifications are identified and justified in order to
with the dosage form monographs (e.g. Capsules (0016), ensure the suitability of the product for the intended
Tablets (0478)) and general monographs relating to purpose. This includes consideration of the properties
pharmaceutical preparations (e.g. Allergen products (1063), required in order to identify whether specific ingredient
Herbal teas (1435), Homoeopathic preparations (1038), properties or process steps are critical to the required quality
Immunosera for human use, animal (0084), Immunosera for of the pharmaceutical preparation.
veterinary use (0030), Monoclonal antibodies for human Active substances and excipients
use (2031), Radiopharmaceutical preparations (0125), Vaccines Active substances and excipients used in the formulation of
for human use (0153), Vaccines for veterinary use (0062)). pharmaceutical preparations comply with the requirements of
Where pharmaceutical preparations are the relevant general monographs, e.g. Substances for
manufactured/prepared using materials of human or animal pharmaceutical use (2034), Essential oils (2098), Extracts
origin, the general requirements of general chapters 5.1.7. (0765), Herbal drugs (1433), Herbal drug preparations (1434),
Viral safety, 5.2.6. Evaluation of safety of veterinary vaccines Herbal drugs for homoeopathic preparations (2045), Mother
and immunosera and 5.2.8. Minimising the risk of transmitting tinctures for homoeopathic preparations (2029), Methods of
animal spongiform encephalophathy agents via human and preparation of homoeopathic stocks and potentisation (2371),
veterinary medicinal products apply, where appropriate. Products offermentation (1468), Products with risk of
transmitting agents of animal spongiform encephalopathies (1483),
FV-38 General Monographs 2016
Products of recombinant DNA technology (0784), Vegetable fatty and/or the immediate container must be assessed. Depending
oils (1579). on the result of this assessment, limits of degradation and/or
In addition, where specific monographs exist, the quality of reaction products arc set and monitored in the
the active substances and excipients used complies with the pharmaceutical preparation. Licensed products require a
corresponding monographs. stability exercise.
Where no specific monographs exist, the required quality Methods used for the purpose of stability testing for all
must be defined, taking into account the intended use and relevant characteristics of the preparation are validated as
the involved risk. stability indicating, i.e. the methods allow the quantification
When physicochemical characteristics of active substances of the relevant degradation products and physical
and functionality-related characteristics (FRCs) of excipients characteristic changes.
(e.g. particle-size distribution, viscosity, polymorphism) are TESTS
critical in relation to their role in the manufacturing process Relevant tests to apply in order to ensure the appropriate
and quality attributes of the pharmaceutical preparation, they quality of a particular dosage form are described in the
must be identified and controlled. specific dosage form monographs.
Detailed information on FRCs is given in general chapter Where it is not practical, for unlicensed pharmaceutical
5.15. Functionality-related characteristics of excipients. preparations, to carry out the tests (e.g. batch size, time
Microbiological quality restraints), other suitable methods are implemented to ensure
The formulation of the pharmaceutical preparation and its that the appropriate quality is achieved in accordance with
container must ensure that the microbiological quality is the risk assessment carried out and any local guidance or
suitable for the intended use. legal requirements.
During development, it shall be demonstrated that the Stock preparations are normally tested to a greater extent
antimicrobial activity of the preparation as such or, if than extemporaneous preparations.
necessary, with the addition of a suitable preservative or The following tests are applicable to many preparations and
preservatives, or by the selection of an appropriate container, are therefore listed here.
provides adequate protection from adverse effects that may Appearance
arise from microbial contamination or proliferation during The appearance (e.g. size, shape and colour) of the
the storage and use of the preparation. A suitable test pharmaceutical preparation is controlled.
method together with criteria for evaluating the preservative
properties of the formulation are provided in general chapter Identity and purity tests
5.1.3. Efficacy of antimicrobial preservation. Where applicable, the following tests arc carried out on the
pharmaceutical preparation:
If preparations do not have adequate antimicrobial efficacy
— identification of the active substance(s);
and do not contain antimicrobial preservatives they are
— identification of specific excipient(s), such as
supplied in single-dose containers, or in multidose containers preservatives;
that prevent microbial contamination of the contents after — purity tests (e.g. investigation of degradation products,
opening. residual solvents (2.4.24) or other related impurities,
In the manufacture/preparation of non-sterile pharmaceutical sterility (2.6J));
preparations, suitable measures are taken to ensure their — safety tests (e.g. safety tests for biological products).
microbial quality; recommendations on this aspect are
Uniformity (2.9.40 or 2.9.512.9.6).
provided in general chapters 5.1.4. Microbiological quality of
non-sterile pharmaceutical preparations and substances for Pharmaceutical preparations presented in single-dose units
pharmaceutical use and 5.ใ.8. Microbiological quality of herbal comply with the test(s) as prescribed in the relevant specific
medicinal products for oral use and extracts used in their dosage form monograph. If justified and authorised, general
preparation. chapter 2.9.40 can be applicable only at the time of release.
Special uniformity requirements apply in the following cases:
Sterile preparations are manufactured/prepared using
— for herbal drugs and herbal drug preparations, compliance
materials and methods designed to ensure sterility and to
with general chapter 2.9.40 is not required;
avoid the introduction of contaminants and the growth of
— for homoeopathic preparations, the provisions of general
micro-organisms; recommendations on this aspect are
chapters 2.9.6 and 2.9.40 are normally not appropriate,
provided in general chapter 5.1.1. Methods of preparation of
however in certain circumstances compliance with these
sterile products.
chapters may be required by the competent authority;
Containers — for single- and multivitamin and trace-element
A suitable container is selected. Consideration is given to the preparations, compliance with general chapters 2.9.6 and
intended use of the preparation, the properties of the 2.9.40 (content uniformity only) is not required;
container, the required shelf-life, and product/container — in justified and authorised circumstances, for other
incompatibilities. Where applicable, containers for preparations, compliance with general chapters 2.9.6 and
pharmaceutical preparations comply with the requirements 2.9.40 may not be required by the competent authority.
for containers (3.2 and subsections) and materials used for
Reference standards
the manufacture of containers (3.1 and subsections).
Reference standards may be needed at various stages for
Stability quality control of pharmaceutical preparations. They are
Stability requirements of pharmaceutical preparations are established and monitored taking due account of general
dependent on their intended use and on the desired storage chapter 5.12. Reference standards.
time.
ASSAY
Where applicable, the probability and criticality of possible Unless otherwise justified and authorised, contents of active
degradation products of the active substance(s) and/or substances and specific excipients such as preservatives are
reaction products of the active substance(s) with an excipient
determined in pharmaceutical preparations. Limits must be

defined and justified.
Suitable and validated methods are used. If assay methods
prescribed in the respective active substance monographs are
used, it must be demonstrated that they are not affected by
the presence of the excipients and/or by the formulation.
Reference standards
See Tests.
LABELLING AND STORAGE
The relevant labelling requirements given in the general
dosage form monographs apply. In addition, relevant
European Union or other applicable regulations apply.
GLOSSARY
Formulation
The designing of an appropriate formula (including materials,
processes, etc.) that will ensure that the patient receives the
suitable pharmaceutical preparation in an appropriate form
that has the required quality and that will be stable and
effective for the required length of time.
Licensed pharmaceutical preparation
A medicinal product that has been granted a marketing
authorisation by a competent authority. Synonym: authorised
pharmaceutical preparation.
Manufacture
All operations of purchase of materials and products,
Production, Quality Control, release, storage, distribution of
medicinal products and the related controls.
Preparation (of an unlicensed pharmaceutical
preparation)
The ‘manufacture’ of unlicensed pharmaceutical preparations
by or at the request of pharmacies or other healthcare
establishments (the term ‘preparation’ is used instead of
‘manufacture’ in order clearly to distinguish it from the
industrial manufacture of licensed pharmaceutical
preparations).
Reconstitution
Manipulation to enable the use or application of a medicinal
product with a marketing authorisation in accordance with
the instructions given in the summary of product
characteristics or the patient information leaflet.
Risk assessment
The identification of hazards and the analysis and evaluation
of risks associated with exposure to those hazards.
Unlicensed pharmaceutical preparation
A medicinal product that is exempt from the need of having
a marketing authorisation issued by a competent authority
but is made for specific patients’ needs according to
legislation.
PhEur
Monographs
Herbal Drugs5 Herbal Drug

Preparations and Herbal
Medicinal Products
Herbal Drugs * * matter is not more than 2 per cent mlm, unless otherwise
prescribed or justified and authorised. An appropriate specific
(Ph. Eur. monograph 1433) *** test may apply to herbal drugs liable to be adulterated.
Herbal Drugs comply with the requirements of the European It may not be possible to perform the test for foreign matter
Pharmacopoeia. These requirements are reproduced below. on a herbal drug that is cut, as described under Definition,
Ph Eur___________ __________________________________________________ for either a specific purpose or for extraction. Under these
circumstances the cut material is presumed to comply with
DEFINITION the test for foreign matter providing that the herbal drug
Herbal drugs are mainly whole, fragmented, or broken prior to cutting was compliant with this test.
plants, parts of plants, algae, fungi or lichen, in an
Loss on drying (2.2.32)
unprocessed state, usually in dried form but sometimes fresh.
Carry out a test for loss on drying, unless otherwise
Certain exudates that have not been subjected to a specific
prescribed or justified and authorised.
treatment are also considered to be herbal drugs. Herbal
drugs are precisely defined by the botanical scientific name Water (2.2.13)
according to the binominal system (genus, species, variety A determination of water may be carried out instead of a test
and author). for loss on drying for herbal drugs with a high essential-oil
content.
Whole describes a herbal drug that has not been reduced in
size and is presented, dried or undried, as harvested; Pesticides (2.8.13)
for example: dog rose, bitter fennel or sweet fennel, Roman Herbal drugs comply with the requirements for pesticide
chamomile flower. residues. The requirements take into account the nature of
Fragmented describes a herbal drug that has been reduced in the plant, where necessary the preparation in which the plant
size after harvesting to permit ease of handling, drying and/or might be used, and where available the knowledge of the
packaging; for example: cinchona bark, rhubarb, passion complete record of treatment of the batch of the plant.
flower. Heavy metals (2.4.27)
Broken describes a herbal drug in which the more-fragile Unless otherwise stated in an individual monograph or unless
parts of the plant have broken during drying, packaging or otherwise justified and authorised:
transportation; for example: belladonna leaf, matricaria — cadmium-, maximum 1.0 ppm;
flower, hop strobile. — lead-, maximum 5.0 ppm;
— mercury-, maximum 0.1 ppm.
Cut describes a herbal drug that has been reduced in size,
other than by powdering, to the extent that the macroscopic Where necessary, limits for other heavy metals may be
description in the monograph of the herbal drug can no required.
longer be applied. When a herbal drug is cut for a specific Where necessary herbal drugs comply with other tests, such
purpose that results in the cut herbal drug being as the following, for example.
homogeneous, for example when cut for herbal teas, it is a Total ash (2.4.16)
herbal drug preparation. Certain cut herbal drugs processed
Ash insoluble in hydrochloric acid (2.8.1)
in this way may be the subject of an individual monograph.
Extractable matter
A herbal drug that complies with its monograph and is
subsequently cut for extraction shall comply in its cut form, Swelling index (2.8.4)
except for its macroscopic description, with the monograph Bitterness value (2.8.15)
for that herbal drug, unless otherwise justified. Aflatoxin B1 (2.8.18)
The term herbal drug is synonymous with the term herbal Where necessary, limits for aflatoxins may be required.
substance used in European Community legislation on herbal
Ochratoxin A (2.8.22)
medicinal products. Where necessary, a limit for ochratoxin A may be required.
PRODUCTION Radioactive contamination
Herbal drugs are obtained from cultivated or wild plants. In some specific circumstances, the risk of radioactive
Suitable collection, cultivation, harvesting, drying, contamination is to be considered.
fragmentation and storage conditions are essential to
Microbial contamination
guarantee the quality of herbal drugs.
Where a herbal drug is used whole, cut or powdered as an
Herbal drugs are, as far as possible, free from impurities such ingredient in a medicinal product, the microbial
as soil, dust, dirt and other contaminants such as fungal, contamination is controlled (5.1.8) Microbiological quality of
insect and other animal contaminations. They are not rotten. herbal medicinal products for oral use and extracts used in their
If a decontaminating treatment has been used, it is necessary preparation or (5.1.4) Microbiological quality of non-sterde
to demonstrate that ±e constituents of the plant are not pharmaceutical preparations and substances for pharmaceutical
affected and that no harmful residues remain. The use of use.
ethylene oxide is prohibited for the decontamination of
ASSAY
herbal drugs.
Unless otherwise prescribed or justified and authorised,
IDENTIFICATION herbal drugs are assayed by an appropriate method.
Herbal drugs are identified using their macroscopic and
microscopic descriptions and any further tests that may be
STORAGE
required (for example, thin-layer chromatography). Protected from light.
________ ___________________________ __________________________ _ Ph Eur
TESTS
Foreign matter (2.8.2)
Carry out a test for foreign matter, unless otherwise
prescribed or justified and authorised. The content of foreign
IV-44 General Monographs 2016
ASSAY
Processed Herbal Drugs Unless otherwise justified and authorised Processed Herbal
DEFINITION Drugs are assayed by an appropriate method.
Processed Herbal Drugs are obtained by subjecting Herbal
Drugs to traditional processing methods.
Processed Herbal Drugs are defined precisely by the
botanical scientific name according to the binomial system
(genus, species, subspecies, variety, and author) and plant Herbal Drug Preparations
part. Monographs for Processed Herbal Drugs may refer to (Ph. Eur. monograph 1434) *1
the relevant monograph for the unprocessed material where
Herbal Drug Preparations comply with the requirements of the
the binomial name is given.
European Pharmacopoeia. These requirements are reproduced
PRODUCTION below.
Processed Herbal Drugs are obtained by subjecting Herbal Ph Eur________________________________________________________________
Drugs to specific types of processing according to traditional
processing methods. These traditional processing methods DEFINITION
have the potential to alter the physical characteristics and/or Herbal drug preparations are homogeneous products
chemical constituents of a Herbal Drug. Traditional obtained by subjecting herbal drugs to treatments such as
processing methods may require the addition of processing extraction, distillation, expression, fractionation, purification,
aids to the herbal drug, for example, honey, vinegar, wine, concentration or fermentation.
milk and salt. The additional processing aids used should be Herbal drug preparations include, for example, extracts,
of a suitable quality or of pharmacopoeial quality where a essential oils, expressed juices, processed exudates, and
monograph exists. The method of traditional processing is herbal drugs that have been subjected to size reduction for
provided under the Production section in individual specific applications, for example herbal drugs cut for herbal
monographs. teas or powdered for encapsulation.
IDENTIFICATION Herbal teas comply with the monograph Herbal teas (1435).
Processed Herbal Drugs are identified using their NOTE The term comminuted used in European Community
macroscopical and, where appropriate, microscopical legislation on herbal medicinal products describes a herbal
descriptions and any further tests that may be required. drug that has been either cut or powdered.
TESTS The term herbal drug preparation is synonymous with the term
A test for foreign matter, Appendix XI D, is carried out, herbal preparation used in European Community legislation
unless otherwise prescribed in the individual monographs. on herbal medicinal products.
________________________________________________________________Ph Eur
A specific appropriate test may be prescribed to detect
potential contaminants in processed herbal drugs.
If appropriate, the Processed Herbal Drugs comply with
other tests, for example, total ash, Appendix XI J, Method II,
ash insoluble in hydrochloric acid, Appendix XI K, Method n, Essential Oils * *
extractable matter, swelling index, Appendix XI c and bitterness
value, Appendix XI N. (Ph. Eur. monograph 2098) ***
The test for loss on drying, Appendix IX D, is carried out on Essential Oils comply with the requirements of the European
Processed Herbal Drugs, unless otherwise prescribed in the Pharmacopoeia; These requirements are reproduced below.
individual monographs. A determination of water by distillation, Ph Eur_______________________________________________________ _______
Appendix IX c, Method II, is carried out for Processed The statements in this monograph are intended to be read in
Herbal Drugs with a high essential oil content. conjunction with individual monographs on essential oils in the
Processed Herbal Drugs comply with the requirements for European Pharmacopoeia. Application of the monograph to other
pesticide residues, Appendix XI L. The requirements take into essential oils may be decided by the competent authority.
account the nature of the Processed Herbal Drugs, where
DEFINITION
necessary the preparation in which the plant might be used,
and where available, the knowledge of the complete record of Odorous product, usually of complex composition, obtained
treatment of the batch of the Processed Herbal Drugs during from a botanically defined plant raw material by steam
cultivation, harvesting and processing. The content of distillation, dry distillation, or a suitable mechanical process
without heating. Essential oils are usually separated from the
pesticide residues may be determined by the method
aqueous phase by a physical process that does not
described in the annex to the general method.
significantly affect their composition.
The risk of contamination of Processed Herbal Drugs by
Essential oils may be subjected to a suitable subsequent
heavy metals must be considered. In an individual
treatment. Thus an essential oil may be commercially known
monograph either a general limit for heavy metals or specific
as being deterpenated, desesquiterpenated, rectified or
limits for individual heavy metal may be required.
‘x’-free.
Where necessary limits for specific toxins, for example — A deterpenated essential oil is an essential oil from which
aflatoxins or ochratoxins, may be applied. monoterpene hydrocarbons have been removed, partially
Where processing is carried out to remove or limit specific or totally.
constituents from the herbal drug a suitable limit test should — A deterpenated and desesquiterpenated essential oil is an
be carried out. essential oil from which mono- and sesquiterpene
In some specific circumstances, the risk of radioactive hydrocarbons have been removed, partially or totally.
contamination is to be considered.
— A rectified essential oil is an essential oil that has been In addition to the system suitability test given in the specific
subjected to fractional distillation to remove certain monograph, it is necessary to check the suitability of the
constituents or modify the content. chromatographic system using the following test, which is to
— An 'x’-free essential oil is an essential oil that has been be carried out periodically within the framework of
subjected to partial or complete removal of one or more performance qualification.
constituents. The chromatogram shown in Figure 2098.-1 is given as an
PRODUCTION example.
Depending on the monograph, the plant raw material may be Reference solution essential oil CRS. If necessary, the reference
fresh, wilted, dried, whole, broken or ground. solution can be diluted with heptane R.
Steam distillation The essential oil is produced by the passage Column’.
of steam through the plant raw material in a suitable — material’, fused silica;
apparatus. The steam may be introduced from an external — size: I = 60 m, 0 = 0.25 mm;
source or generated by boiling water below the raw material — stationary phase: macrogol 20 000 R (0.25 pm).
or by boiling water in which the raw material is immersed. Carrier gas helium for chromatography R.
The steam and oil vapours are condensed. The water and Flow rate 1.5 mL/min.
essential oil are separated by decantation.
Split ratio 1:500. The split ratio/injection volume can be
Dry distillation The essential oil is produced by high- adjusted in order to fit the specific equipment used, provided
temperature heating of stems or barks in a suitable apparatus that the column load stays the same.
without the addition of water or steam. Temperature:
Mechanical process The essential oil, usually known as ‘cold-
pressed’, is produced by a mechanical process without any Time Temperature
heating. It is mainly applied to Citrus fruit and involves (min) __________ m.____________
expression of the oil from the pericarp and subsequent Column 0-15 70
separation by physical means.
15 -100 70 -> 240
In certain cases, a suitable antioxidant may be added to the
essential oil. 100 - 105 240
CHARACTERS Injection port 250
The appearance and the odour of the essential oil is Detector 270
determined.
IDENTIFICATION Detection Flame ionisation.
Essential oils are identified by their gas chromatographic Injection 1 pL.
profile, or failing this, by any other test that may be required
Identification of components Use the chromatogram supplied
(for example, a test by thin-layer chromatography).
with essential oil CRS.
TESTS System suitability: reference solution:
GENERAL TESTS — resolution: minimum 1.5 between the peaks due to linalol
The essential oil complies with the prescribed limits for the and linalyl acetate;
following tests. — signal-to-noise ratio: minimum 100 for the peak due to
Relative density (2.2.5) decanal;
— limits: the percentage content of each of the 9 components
Refractive index (2.2.6) is within the limits stated on the leaflet provided with
Optical rotation (2.2.7) essential oil CRS.
Fatty oils and resinified essential oils (2.5.7) STORAGE
SUPPLEMENTARY TESTS In a well-filled, airtight container, protected from light.
If necessary, the essential oil complies with the prescribed
LABELLING
limits for the following tests.
The label states:
Freezing point {2.2.18) — the scientific name of the plant raw material used;
Acid value {2.5.1) — where applicable, the type and/or the chemotype of the
Peroxide value (2.5.5) essential oil;
— where applicable, the method of production;
Foreign esters (2.5.6) — where applicable, the name and concentration of any
Residue on evaporation (2.5.9) added antioxidant;
Water (2.5.5) — where applicable, additional processing steps that are not
specified under Definition.
Solubility in alcohol {2.8.10)
_______________________________________________________________ PfiEur
Falsification
If appropriate, a test for one or more falsifications may be
carried out by thin-layer chromatography (2.2.27), by gas
chromatography (2.2.25) using a chiral column if necessary,
or by any other suitable method.
Chromatographic profile
Gas chromatography (2.2.25): use the normalisation
procedure.
1. a-pinene 3. hexanol 5. linalol 7. P-caryophyllene 9. benzyl salicylate

2. cineole 4. decanal 6. linalyl acetate 8. eugenol
Figure 2098.-1. - Chromatogram for the test for chromatographic profile of essential oils
Other extracts are not adjusted to a particular content of

Herbal Drug Extracts * * constituents. For control purposes, one or more constituents
Extracts * ** are used as analytical markers. The minimum content for
(Ph Eur monograph 0765) these analytical markers is given in an individual monograph.
Herbal Drug Extracts comply with the requirements of the PRODUCTION
European Pharmacopoeia. These requirements are reproduced Herbal drugs, solvents and other materials used for the
below. preparation of extracts are of suitable quality and where
applicable comply with the requirements of any relevant
Ph Elf.________________________________________________________________
monograph in the European Pharmacopoeia. Where justified,
DEFINITION herbal drugs used for the production of extracts may exceed
Herbal drug extracts are liquid (liquid extraction the limits for heavy metals specified in the monograph Herbal
preparations), semi-solid (soft extracts and oleoresins) or drugs (1433) provided that the resulting extract satisfies the
solid (dry extracts) preparations obtained from Herbal drugs requirements for heavy metals (see Tests).
(1433) using suitable solvents. Different batches of the herbal drug which are compliant
An extract is essentially defined by the quality of the herbal with the relevant monograph, or in the absence of an
drug, by its production process (extraction solvent(s), method individual monograph with other suitable specifications, may
of processing, etc.) and by its specifications. be combined prior to extraction, for example for the purpose
European Pharmacopoeia monographs for extracts cover the of achieving the quantity of herbal drug required for the
genuine (native) extract and, where present, excipients. production process or, in the case of standardised and
quantified extracts, to achieve a certain range of content for
Different types of extract may be distinguished.
one or more constituents in the herbal drug to be extracted.
Standardised extracts are adjusted to a defined content of The herbal drug may also undergo a preliminary treatment,
one or more constituents with known therapeutic activity.
for example, grinding, inactivation of enzymes or defatting.
This is achieved by adjustment of the extract with inert In addition, unwanted constituents (e.g. toxic constituents)
excipients or by blending batches of the extract. or unwanted matter (e.g. insoluble matter) may be removed
Quantified extracts are adjusted to one or more active at a suitable stage in the production process.
markers, die content of which is controlled within a limited,
specified range. Adjustments are made by blending batches
of the extract.
Where solvents are recovered from the production process, range ± 5 per cent to ± 10 per cent taking into account the
such recovered or recycled solvents may be used, provided nature of the extract and the method of assay.
±at the recovery procedures are controlled and monitored to Defined range of content For example, in the monograph
ensure that solvents meet appropriate standards before re-use Frangula bark dry extract, standardised (1214), the content of
or admixture with other approved materials. Water used for assayed constituents is stated as 15.0 per cent to
the production of extracts complies with the requirements of 30.0 per cent. In this case, it is intended that an extract will
the monograph Water for preparation of extracts (2249). consistently be produced to a defined single content selected
Where applicable, miscella (extraction liquors) are from within the defined range taking into account an
concentrated to the intended consistency using suitable acceptable tolerance. Where there is an individual
methods, usually under reduced pressure and at a monograph in the pharmacopoeia for a standardised extract
temperature at which deterioration of the constituents is with a defined range of content, the acceptable tolerance will
reduced to a minimum. Essential oils that have been be stated in the individual monograph (for example, for
separated during processing may be restored to the extracts Frangula bark dry extract, standardised (1214), the acceptable
at an appropriate stage in the production process. Suitable tolerance is stated as ± 10 per cent relative to the declared
excipients may be added at various stages of the production content).
process for technological reasons (for example, as part of the Quantified extracts
drying process or to improve the homogeneity or consistency The content of assayed constituents must be within the
of an extract). For standardised extracts, suitable inert values given in the Definition section of an individual
excipients may also be added to adjust one or more monograph.
constituents to a defined content. For quantified extracts and
‘other’ extracts, the addition of inert excipients to adjust the Other extracts
content of assayed constituents is not permitted. Excipients The content of assayed constituents must not be lower than
are included for technological reasons only, and the the minimum value given in the Definition section of an
manufacturer must declare the content of such excipients as individual monograph. Where justified and authorised, this
a fixed percentage. In some applications, an excipient may be does not preclude the selection of alternative constituents as
added in a narrow percentage range (e.g. silicon dioxide a basis for assay using a corresponding validated analytical
between 0.1-0.5 per cent, to improve flowability of the method, which may be more appropriate to the physical
extract). The proposed range must be justified by the and/or chemical properties of the medicinal product into
manufacturer. Suitable stabilisers, antioxidants and which the extract is to be incorporated. Where alternative
antimicrobial preservatives may be added to extracts where constituents are selected for assay, a suitable minimum value
justified and authorised. for such constituents must be established.
Extraction with a given solvent leads to a typical content of LABELLING
selected constituents in the extracted dry matter; during The label states:
production of standardised and quantified extracts, — the herbal drug used;
purification procedures may be applied that increase the — where applicable, that fresh herbal drug has been used;
content of these selected constituents with respect to the — the form of the extract (for example, liquid, tincture, soft,
expected values; such extracts are referred to as ‘refined’. oleoresin or dry);
— where applicable, that the extract is standardised or
IDENTIFICATION quantified;
Extracts are identified using suitable methods. — for standardised extracts, the defined content of
TESTS constituents with known therapeutic activity;
Where applicable, as a result of analysis of the herbal drug — for quantified extracts, the specified range of content of
used for production and in view of the production process, active markers;
tests for microbiological quality {5.1.4 or 5.1.8), heavy metals — where applicable, that the extract is ‘refined’;
(2.4.27), aflatoxins {2.8.18), ochratoxin A (2.8.22) and — the first solvent or solvents used for extraction (for
pesticide residues {2.8.13) in the extracts may be necessary. example, ethanol 60 per cent VIV)',
Where a test for heavy metals is carried out, the same limits — the name and amount of any excipients present in the
for heavy metals as those given in the monograph Herbal extract (for example, diluents, stabilisers, antimicrobial
drugs (1433) are applicable to extracts unless otherwise stated preservatives, antioxidants);
in an individual extract monograph or unless otherwise — for quantified extracts and ‘other’ extracts, the ratio of the
justified and authorised. quantity of herbal drug to the quantity of genuine (native)
extract {DERgenuinf) expressed on a mass/mass basis for
ASSAY
soft extracts, oleoresins and dry extracts, and on either a
Extracts are assayed by a suitable method, unless otherwise mass/mass or a mass/volume basis for liquid extraction
justified. preparations;
Standardised extracts — where applicable, the percentage of dry residue;
The Definition section of an individual monograph on a — the storage conditions.
standardised extract states the content of the assayed
constituents as either a defined single content or within a
defined range of content.
LIQUID EXTRACTION PREPARATIONS -
Defined single content For example, in the monograph
PRAEPARATIONES FLUIDAE AB
Ipecacuanha liquid extract, standardised (1875), the content of EXTRACTIONS
assayed constituents is stated as 1.80 per cent to Liquid extraction preparations are liquid preparations
2.20 per cent. In this case, the declaration is based on a consisting of a diverse range of products which are described
defined single content of 2.0 per cent with a tolerance of by their extraction solvents, methods of production and drug
+ 10 per cent. The acceptable tolerance is usually within the solvent ratios or drug extract ratios. Included in this range
are products obtained using ethanol, water, glycerol, solvent, or 1 part by mass of herbal drug and 5 parts by mass
propylene glycol and fatty oils as extraction solvents. Liquid or volume of extraction solvent. Alternatively, they may be
(fluid) extracts and tinctures belong to this category and are obtained using either 1 part by mass of herbal drug and
described below. sufficient extraction solvent to produce 10 parts by mass or
LIQUID (FLUID) EXTRACTS - EXTRACTA FLUIDA volume of tincture or 1 part by mass of herbal drug and
DEFINITION sufficient extraction solvent to produce 5 parts by mass or
volume of tincture. Other ratios of herbal drug to extraction
Quantified liquid (fluid) extracts and ‘other’ liquid (fluid)
solvent may be used.
extracts are liquid extraction preparations of which, in
general, 1 part by mass or volume is equivalent to 1 part by Standardised tinctures are only defined by their content of
mass of the dried herbal drug. constituents with known therapeutic activity.
Standardised liquid (fluid) extracts are only defined by then- PRODUCTION
content of constituents with known therapeutic activity. Tinctures are usually prepared by either maceration or
PRODUCTION percolation, using ethanol of a suitable concentration to
extract the herbal drug, or by dissolving a soft or dry extract
Liquid extracts are prepared using ethanol of a suitable
of the herbal drug (which has been produced using the same
concentration and/or water together with, where necessary,
extraction solvent as would be used to prepare the tincture
other substances (e.g. glycerol or ammonia solution) to
by direct extraction) in ethanol of the required concentration.
extract the herbal drug, or by dissolving a soft or dry extract
of the herbal drug (which has been produced using the same The tincture is tested for 2-propanol (2.9.17), with a
extraction solvent as would be used to prepare the liquid maximum of 0.05 per cent v/v, unless assurance of
extract by direct extraction) in either ethanol of the required compliance with this limit is provided by a detailed
concentration or water. knowledge of the ethanol supply chain and the tincture
manufacturing process.
Where the liquid extract contains ethanol, it is tested for
2-propanol (2.9.77), with a maximum of 0.05 per cent VIV3 Except for standardised tinctures, tinctures produced from
unless assurance of compliance with this limit is provided by soft or dry extracts do not contain any excipients other than
a detailed knowledge of the ethanol supply chain and the those that would be present in the tincture prepared by direct
extract manufacturing process. extraction. However, exceptions may be justified in certain
cases such as when the soft extract used to produce the
Except for standardised liquid extracts, liquid extracts
tincture contains stabilisers, antioxidants or antimicrobial
produced from soft or dry extracts do not contain any
preservatives that have been added to ensure its stability.
excipients other than those that would be present in the
liquid extract prepared by direct extraction. However, Tinctures are adjusted, if necessary so that they satisfy the
exceptions may be justified in certain cases such as when the requirements for content of solvent. Tinctures may be filtered
soft extract used to produce the liquid extract contains if necessary.
stabilisers, antioxidants or antimicrobial preservatives that Tinctures are usually clear. A slight sediment may form on
have been added to ensure its stability. standing.
Liquid extracts are adjusted, if necessary, so that they satisfy TESTS
the requirements for content of solvent. Liquid extracts may Relative density (2.2.5)
be filtered, if necessary. Where applicable, the tincture complies with the limits
A slight sediment may form on standing. prescribed.
TESTS Ethanol (2.9.10)
Relative density (2.2.5) The ethanol content complies with the limits prescribed.
Where applicable, the liquid extract complies with the limits Methanol (2.9.11)
prescribed. Maximum 0.05 per cent V/V3 unless otherwise prescribed or
Ethanol (2.9. JO) justified and authorised.
For ethanolic liquid extracts, carry out the determination of Dry residue (2.8.16)
ethanol content. The ethanol content complies with the Where applicable, the tincture complies with the limits
limits prescribed. prescribed.
Methanol (2.9.11) STORAGE
Maximum 0.05 per cent V71Z for ethanolic liquid extracts, Protected from light.
unless otherwise prescribed or justified and authorised.
LABELLING
Dry residue (2.8.16) The label states, in addition to the requirements listed above,
Where applicable, the liquid extract complies with the limits
the ethanol content in per cent VIV.
prescribed.
STORAGE
SOFT EXTRACTS - EXTRACTA SPISSA
Protected from light.
DEFINITION
LABELLING
Soft extracts are semi-solid preparations obtained by
The label states in addition to the requirements listed above,
evaporation or partial evaporation of the solvent used for
the ethanol content in per cent VIV3 where applicable.
production.
TINCTURES - TINCTURAE
TESTS
DEFINITION
Dry residue (2.8.16)
Quantified tinctures and ‘other’ tinctures are liquid extraction The soft extract complies with the limits prescribed.
preparations that are obtained using either 1 part by mass of
herbal drug and 10 parts by mass or volume of extraction
Solvents Extraction solvents

Residual solvents are controlled as described in chapter 5.4, Solvents which are used for the extraction process.
unless otherwise prescribed or justified and authorised. Genuine (native) drug extract ratio (DERgmuin6)
STORAGE See under drug extract ratio (DER) for the explanation of
In an airtight container, protected from light. how the ratio for a DER is designated. A DERgmuine is the
ratio between the quantity of herbal drug used in the
manufacture of an extract and the quantity of genuine
OLEORESINS - OLEORESINA (native) extract obtained. The number (given as the actual
DEFINITION range) written before the colon is the relative quantity of the
Oleoresins are semi-solid extracts composed of a resin in herbal drug; the number written after the colon is the relative
solution in an essential and/or fatty oil and are obtained by quantity of the genuine extract obtained. For example,
evaporation of the solvent(s) used for their production. DERgenuine 2.5-4.5: 1 means that between 2.5 and 4.5 parts
This monograph applies to oleoresins produced by extraction of herbal drug are required to produce 1 part of genuine
and not to natural oleoresins. (native) extract. Where processing aids are added to the
genuine (native) extract to produce, for example, a dry
TESTS extract, the DER and DERgen16ne will have different values;
Water (2.2.13) where a dry extract is produced without the need for any
The oleoresin complies with the limits prescribed. processing aids, the DER and DERgenนน่น. will be identical.
Solvents Oleoresins are usually produced without the need to include
Residual solvents are controlled as described in chapter 5.4, processing aids, therefore, the DER and DERgenuine are
unless otherwise prescribed or justified and authorised. usually identical. For soft extracts and liquid extraction
preparations, where the genuine (native) extract does not
STORAGE
exist without excipients and/or processing aids (e.g. usually
In an airtight container, protected from light.
20-30 per cent of water in soft extracts, ethanolic extraction
solvent in tinctures), the DER and DERgen.น1 1ท6 are identical.
DRY EXTRACTS - EXTRACTA SICCA Genuine (native) herbal drug extract
DEFINITION Refers to the extract without excipients, even if for
Dry extracts are solid preparations obtained by evaporation technological reasons the genuine extract is not available.
of the solvent used for their production. However, for soft extracts and liquid extraction preparations
the genuine extract may contain variable amounts of
Dry extracts usually have a loss on drying of not greater than
5 per cent m/m. Where justified and authorised, a loss on (extraction) solvent.
drying with a different limit or a test for water may be Markers
prescribed. Chemically defined constituents or groups of constituents of
a herbal drug, a herbal drug preparation or a herbal
TESTS
medicinal product which are of interest for control purposes
Loss on drying {2.8.17) independent of whether they have any therapeutic activity.
Where applicable, the dry extract complies with the limits Markers serve to calculate the quantity of herbal drug(s) or
prescribed. herbal drug preparation(s) in the herbal medicinal product if
Water the marker has been quantitatively determined in the herbal
.
(2.5.12) Where a test for loss on drying is not applicable, drug or herbal drug preparation.
the dry extract complies with the limits prescribed. There are 2 categories of markers:
Solvents — active markers are constituents or groups of constituents
Residual solvents are controlled as described in chapter 5.4, which are generally accepted to contribute to the
unless otherwise prescribed or justified and authorised. therapeutic activity;
— analytical markers are constituents or groups of
STORAGE
constituents that serve solely for analytical purposes,
irrespective of any pharmacological or therapeutic activity
which they may be reported to possess.
GLOSSARY - GLOSSA Miscella (extraction liquor)
Constituents with known therapeutic activity Liquid obtained from the extraction process.
Chemically defined substances or groups of substances which Production of tinctures by maceration
are generally accepted to contribute substantially to the A process whereby, unless otherwise prescribed, the herbal
therapeutic activity of a herbal drug, a herbal drug drug to be extracted is reduced to pieces of suitable size,
preparation or a herbal medicinal product. mixed thoroughly with the prescribed extraction solvent and
Drug extract ratio (DER) allowed to stand in a closed container for an appropriate
The ratio between the quantity of herbal drug used in the time, with agitation where required. The residue is separated
manufacture of an extract and the quantity of extract from the extraction solvent and, if necessary, pressed out.
obtained. The number (given as the actual range) written If the residue is pressed, the 2 liquids are combined.
before the colon is the relative quantity of the herbal drug; Production of tinctures by percolation
the number written after the colon is the relative quantity of A process whereby, unless otherwise prescribed, the herbal
the extract obtained. drug to be extracted is reduced to pieces of suitable size and
Drug solvent ratio (DSR) mixed thoroughly with a portion of the prescribed extraction
The ratio between the quantity of herbal drug, expressed in solvent and allowed to stand for an appropriate time.
mass, used in the manufacture of an extract and the quantity The mixture is transferred to a percolator and more
of the first extraction solvent, expressed in mass or volume.
extraction solvent is added until the herbal drug is covered and calculate the average mass of the contents of the
with a layer of extraction solvent. The percolate is allowed to 20 units. Unless otherwise justified, not more than 2 of the
flow slowly from the base of the percolator while extraction 20 individual masses deviate from the average mass by more
solvent is slowly added to the top of the percolator, ensuring than the percentage deviation shown in the table below and
that the herbal drug to be extracted is constantly covered none deviates by more than twice that percentage.
with extraction solvent, until all the extraction solvent has
been added. Percolation continues until the percolate is
Average mass Percentage deviation
recovered. If the residue is pressed, the 2 liquids arc
combined. less than 1.5 g 15 per cent
_______________________________________________________________ Ph Eur 1.5 g to 2.0 g included 10 per cent
more than 2.0 g 7.5 per cent
Tinctures of the British Pharmacopoeia STORAGE

In addition to the requirements for Tinctures of the European
Pharmacopoeia (stated under Extracts), the following statements ------------------------------------------------------------------------------------------------------------ Ph Eur
apply to those tinctures that are the subject of an individual

monograph in the British Pharmacopoeia.
DEFINITION
Certain preparations of the British Pharmacopoeia entitled Instant Herbal Teas * *
Tinctures do not conform strictly to the definition of the
European Pharmacopoeia and consequently application of (Ph. Eur. monograph 2620) ***
some of the above requirements is inappropriate. Any Ph Eur - __________________________________________________________
necessary exceptions are stated in the relevant individual
DEFINITION
monographs.
Instant herbal teas consist of 1 or more herbal drug
preparations (primarily extracts with or without added
essential oils), and are intended for the preparation of an oral
solution immediately before use.
Herbal Teas * * Instant herbal teas may also contain, in addition to herbal
drug preparations, suitable excipients such as maltodextrin
(Ph. Eur. monograph 1435) ***
and added flavourings.
Herbal Teas comply with the requirements of the European Instant herbal teas are presented as a powder or granules and
Pharmacopoeia. These requirements are reproduced below. are usually supplied in bulk form or in sachets.
Ph Eur_______________________________________________________________
The herbal drug preparations used comply with the
DEFINITION appropriate individual European Pharmacopoeia monographs
Herbal teas consist exclusively of one or more herbal drugs or, in the absence of such individual monographs, with the
intended for oral aqueous preparations by means of general monograph Herbal drug preparations (1434) and with
decoction, infusion or maceration. The preparation is other appropriate general monographs, for example
prepared immediately before use. Extracts (0765) or Essential oils (2098).
Herbal teas are usually supplied in bulk form or in bags for IDENTIFICATION
single use. The identity of herbal drug preparations present in instant
The herbal drugs used comply with the appropriate herbal teas is checked by suitable methods.
individual European Pharmacopoeia monographs or in then- TESTS
absence with the general monograph Herbal drugs (1433). General chapter 5.1.8 contains recommendations on the
IDENTIFICATION microbiological quality of extract-containing herbal medicinal
The identity of herbal drugs present in herbal teas is checked products such as instant herbal teas.
by suitable methods such as botanical examinations and/or The proportion of herbal drug preparations present in instant
chromatographic profiles. herbal teas is checked by suitable methods.
TESTS Instant herbal teas in sachets comply with the following test.
Recommendations on the microbiological quality of herbal Uniformity of mass
teas (5.1.8) take into account the prescribed preparation Determine the individual and the average mass of the
method (use of boiling or non-boiling water). contents of 20 randomly chosen units as follows: weigh a
The proportion of herbal drugs present in herbal teas is single full sachet of instant herbal tea, open it without losing
checked by appropriate methods. any fragments. Empty it completely using a brush. Weigh the
empty sachet and calculate the mass of the contents by
Herbal teas in bags comply with the following test:
subtraction. Repeat the operation on the 19 remaining
Uniformity of mass sachets and calculate the average mass of the contents of the
Determine the individual and the average mass of the 20 units. Unless otherwise justified, not more than 2 of the
contents of 20 randomly chosen units as follows: weigh a individual masses deviate from the average mass by more
single full bag of herbal tea, open it without losing any than the percentage deviation shown in the table below and
fragments. Empty it completely using a brush. Weigh the none deviates by more than twice that percentage.
empty bag and calculate the mass of the contents by
subtraction. Repeat the operation on the 19 remaining bags
2016 Agnus Castus Fruir IV-51
Average mass Percentage deviation TESTS

less than 1.5 g 15 per cent Periploca sepium
Thin-layer chromatography (2.2.27).
1.5 g to 2.0 g included 10 per cent
Test solution To 0.3 g of the powdered herbal drug (355)
more than 2.0 g 7.5 per cent
(2.9.12) add 3 mL of methanol R} heat in a water-bath at
60 °C for 1 min and filter.
STORAGE Reference solution Dissolve 5 mg of thymol R and 8 mg of
Protected from light. borneol R in 5 mL of methanol R.
—--- -------------------------------------------------------------------------------------------------- Ph Eur
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
plate R (2-10 pm)].
Mobile phase ethyl acetate R3 methylene chloride R (2:98 VIV).
Application 20 pL [or 1 pL] as bands of 10 mm [or 8 mm].
Acanthopanax Bark * * Development Over a path of 10 cm [or 6 cm].
(Ph. Eur. monograph 2432) *** Drying In air.
Ph Eur______________________________________________________________
Detection Treat with anisaldehyde solution R, heat at 105 °C
for 5 min and examine in daylight.
DEFINITION Results The chromatogram obtained with the test solution
Dried root bark of Eleutherococcus gracilistylus (พ.พ.รทไ.) shows no intense coloured zones above the zone due to
S.Y.Hu var. nodiflorus (Dunn) H.Ohashi {Acanthopanax borneol in the chromatogram obtained with the reference
gracilistylus W.W.Sm.) collected in summer and autumn. solution.
IDENTIFICATION Acanthopanax giraldii
A. The bark occurs in irregular quills, 5-15 cm long, The outer surface of the root bark must not be covered with
0.4-1.4 cm in diameter, about 2 mm thick. The outer surface scaly covering trichomes.
is greyish-brown, with slightly twisted longitudinal wrinkles Loss on drying (2.2.32)
and transverse lenticel-like scars. The inner surface is pale Maximum 12.0 per cent, determined on 1.000 g of the
yellow or greyish-yellow, with fine longitudinal striations. powdered herbal drug (355) (2.9.12) by drying in an oven at
The texture is light, fragile, easily broken. The fracture is 105 °C for 2 h.
irregular, greyish-white. Total ash (2.4.16)
B. Microscopic examination (2.8.23). The powder is greyish- Maximum 12.0 per cent.
white. Examine under a microscope using chloral hydrate Ash insoluble in hydrochloric acid (2.8.1)
solution R. The powder shows the following diagnostic Maximum 2.0 per cent.
characters: cluster crystals of calcium oxalate, 8-64 pm in
Extractable matter
diameter, sometimes included in crystal cells arranged in
Minimum 16.0 per cent.
rows; cork cells, rectangular or polygonal, thin-walled,
sometimes walls of cork cells of older barks unevenly To 2.00 g of the powdered herbal drug (250) (2.9.12) add a
thickened, slightly pitted; fragments of secretory canals mixture of 8 g of water R and 12 g of ethanol (96 per cent) R
containing colourless or pale yellow secretions. Examine and allow to macerate for 2 h, shaking frequently. Filter,
evaporate the filtrate to dryness on a water-bath in vacuo and
under a microscope using a 50 per cent VIV solution of
dry in an oven at 100-105 °C for 2 h. The residue weighs a
glycerol R. The powder shows abundant starch granules,
minimum of 320 mg.
simple, polygonal or subspherical, 2-8 pm in diameter, or
______________________________________________________________ Ph Ear
compound with 2-10 components.
c. Examine the chromatogram obtained in the test for
Periploca sepiutn.
Results See below the sequence of zones present in the
chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint zones may be present
in the chromatogram obtained with the test solution. Agnus Castus Fruit *
Top of the plate
Preparation
Agnus Castus Fruit Dry Extract
Thymol: an orange zone Ph Eli-___ __________________________________________________________
DEFINITION
Borneol: a brown zone Whole, ripe, dried fruit of Vitex agnus-castus L.
A broad pink zone Content
Minimum 0.08 per cent of casticin (C19H18O8; Mr 374.3)
Reference solution Test solution (dried drug).
IDENTIFICATION
A. Agnus castus fruit is oval or almost globular, with a
diameter of up to 5 mm. The persistent calyx is greenish-
grey, finely pubescent, ends in 4-5 short teeth and envelops
2/3 to 3/4 of the surface of the fruit. The blackish-brown
fruit consists of a pericarp that becomes progressively
rV-52 Agnus Castus Fruit 2016
sclerous up to the endocarp. The style scar is often visible. TESTS

Some of the fruits may retain a stalk, about 1 mm long. Foreign matter (2.8.2)
A transverse section of the fruit shows 4 locules, each
Maximum 3.0 per cent.
containing an elongated seed.
B. Microscopic examination (2.8.23). Examine under a
Other species of Vitex3 in particular Vitex negundo L
microscope using Moral hydrate solution R. The powder No fruit of other species with a much greater diameter is
shows the following diagnostic characters: fragments of the present.
outer epidermis of the calyx composed of polygonal cells Total ash (2.4.16)
densely covered with short, bent or undulate, uni-, bi- or tri-
cellular uniseriate covering trichomes; cells of the epicarp
with thick walls and well-marked, large pits; isolated Loss on drying (2.2.32)
glandular trichomes พาth a unicellular stalk and a uni- or Maximum 10.0 per cent, determined on 1.000 g of the
multi-cellular head; layers of parenchyma from the outer part powdered herbal drug (355) (2.9.12) by drying in an oven at
of the mesocarp, some containing brown pigment, others 105 °C for 2 h.
extending into septa; fragments from the inner part of the
mesocarp composed of thin-walled, pitted, sclerenchymatous ASSAY
cells and of typical isodiametric sclerous cells with very thick, Liquid chromatography (2.2.29).
deeply grooved walls and a narrow, stellate lumen; small Test solution Extract 1.000 g of the powdered herbal drug
brown cells of the endocarp; fragments of the testa (355) (2.9.12) with 40 mL of methanol R for 2 min using a
containing areas of fairly large, thin-walled lignified cells with suitable-speed homogeniser. Collect the supernatant liquid
reticulate bands of thickening; numerous fragments of the and filter into a 250 mL flask. Repeat the extraction with a
endosperm composed of thin-walled parenchymatous cells further 40 mL of methanol R, collecting the supernatant
containing aleurone grains and oil droplets. liquid and filtering as before. Rinse the residue carefully with
c. Thin-layer chromatography (2.2.27). a small quantity of methanol R. Combine the methanol
extracts and rinsings and evaporate to dryness in vacuo in a
water-bath at not more than 30 °C. With the aid of
(2.9.12) add 10 mL of methanol R. Heat in a water-bath at
ultrasound, dissolve the residue obtained in methanol R and
60 °C for 10 min. Allow to cool and filter.
dilute to 20.0 mL with the same solvent. Filter the solution
Reference solution Dissolve 0.5 mg of aucubin R and 1 mg of through a membrane filter (nominal pore size 0.45 pm).
agnuside R in methanol R and dilute to 1.0 mL with the same Dilute 2.0 mL of the solution to 10.0 mL with methanol R.
solvent.
Reference solution Suspend a quantity of agnus castus fruit dry
Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel extract HRS corresponding to 0.10 mg of casticin in 7.5 mL
F254 plate R (2-10 pm)]. of methanol R, sonicate for 5 min and dilute to 10.0 mL with
Mobile phase water R3 methanol R3 ethyl acetate R the same solvent. Filter through a membrane filter (nominal
(8:15:77 VIVIV). pore size 0.45 pm).
Application 10 pL [or 8 pL] as bands Of 10 mm [or 8 mm]. Column:
— size: I = 0.125 m, 0 = 4.0 mm;
Development Over a path of 8 cm [or 5 cm].
— stationary phase: end-capped octadecylsilyl silica gel for
Drying In air. chromatography R (5 pm).
Detection treat with anhydrous formic acid R and heat at Mobile phase:
120 °C for 10 min; examine in daylight. — mobile phase A: 5.88 g/L solution of phosphoric acid R;
Results See below the sequence of zones present in the — mobile phase B: acetonitrile R‘3
test solution. Furthermore, other zones may be present in the
chromatogram obtained with the test solution.
Time Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
Top of he plate
0 - 30 70 -> 45 30 -> 55
Agnuside: a blue zone A blue zone (agnuside)
Flow rate 1.0 mL/min.

Aucubin: a blue zone A blue zone (aucubin)
Detection Spectrophotometer at 348 nm.
Reference solution Test solution
Injection 10 pL.
Identification of peaks Use the chromatogram supplied with
agnus castus fruit dry extract HRS and the chromatogram
obtained with the reference solution to identify the peaks due
to penduletin and casticin.
2016 Agnus Castus Fruit Preparations IV-53
System suitability, reference solution: air, then treat with a 50 g/L solution of macrogol 400 R in
— resolution', minimum 1.5 between the peaks due to methanol R, allow to dry and examine in ultraviolet light at
penduletin and casticin. 365 nm.
Calcdate the percentage content of casticin using the Results See below the sequence of fluorescent zones present
following expression: in the chromatograms obtained with the reference solution
and the test solution. Furthermore, other faint fluorescent
zones may be present in the chromatogram obtained with the
Al X 7712 X p X 10 test solution.
A2 X 7711
Top of the plate

A1 = area of the peak due to casticin in the chromatogram
obtained with the test solution;
A2 = area of the peak due to casticin in the chromatogram Caffeic acid: a light blue fluorescent An orange fluorescent zone and a
white or pale yellow fluorescent
obtained with the reference solution; zone, partly superimposed
Hl 1 = mass of the herbal drug used to prepare the test
solution, in grams;
Several light blue fluorescent zones
m2 = mass of agnus castus fruit dry extract HRS used to
prepare the reference solution, in grams; 1 or 2 yellow fluorescent zones
p = percentage content of casticin in agnus castus fruit dry
extract HRS.
A light blue fluorescent zone
______________________________________________________________ Ph Eur
Homoorientin: a yellow fluorescent A yellow fluorescent zone
(homoorientin)
A yellow fluorescent zone
Agnus Castus Fruit Dry Extract *

(Ph. Eur. monograph 2309) **
ASSAY
Ph Eur___________________________________________________________ Liquid chromatography (2.2.29).
DEFINITION Test solution Suspend 0.200 g of the extract to be examined
Dry extract produced from Agnus castus fruit (2147). in 15 mL of methanol R3 sonicate for 5 min and dilute to
Content 20.0 mL with the same solvent. Filter through a membrane
filter (nominal pore size 0.45 pm).
Minimum 0.10 per cent of casticin (C19H18O8; Mr 374.3)
(dried extract). Reference solution Suspend a quantity of agnus castus fruit dry
extract HRS corresponding to 0.10 mg of casticin in 7.5 mL
PRODUCTION of methanol R, sonicate for 5 min and dilute to 10.0 mL with
The extract is produced from the herbal drug by a suitable the same solvent. Filter through a membrane filter (nominal
procedure using ethanol (40-80 per cent V/V). pore size 0.45 pm).
Column:
CHARACTERS
— size: I ■= 0.125 m, 0 = 4.0 mm;
Appearance — stationary phase: end-capped octadecylsilyl silica gel for
Brown, amorphous powder. chromatography R (5 pm).
IDENTIFICATION Mobile phase:
— mobile phase A: 5.88 g/L solution of phosphoric acid R's
— mobile phase B: acetonitrile R;
Test solution Suspend 0.5 g of the extract to be examined in
5 mL of methanol R and sonicate for 15 min. Shake the
mixture vigorously 3 or 4 times during the procedure. Filter.
Reference solution Dissolve 1 mg of caffeic acid R and 4 mg of Time Mobile phase A Mobile phase B
homoorientin 2? in 10 mL of methanol R. (min) (per cent V/V) (per cent V/V)
0 - 30 70 -> 45 30-> 55
Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica
gel F254 plate R (2-10 pm)].
Mobile phase water R, anhydrous formic acid R, toluene R,
tetrahydrofuran R (1:2:8:16 VIVIVIV).
Application 12 pL [or 3 pL] as bands of 10 mm [or 8 mm]. Flow rate 1.0 mL/min.
Development Over a path of 11 cm [or 6 cm]. Detection Spectrophotometer at 348 nm.
Injection 10 pL.
Drying In air.
Detection Treat with a 10 g/L solution of diphenylboric acid agnus castus fruit dry extract HRS and the chromatogram
aminoethyl ester R in methanol R and dry in a current of cold
IV-54 Agrimony 2016
obtained with the reference solution to identify the peaks due

to penduletin and casticin.
รุ)’stem suitability: reference solution:
— resolution: minimum 1.5 between the peaks due to
penduletin and casticin.
Calculate the percentage content of casticin using the
following expression:
Al X 7712 X p X 2
A2 X 7711
A\ = area of the peak due to casticin in the chromatogram

A2 = area of the peak due to casticin in the chromatogram
obtained with the reference solution;
777j — mass of the extract to be examined used to prepare
the test solution, in grams;
ทไ2 = mass of agnus castus fruit dry extract HRS used to
prepare the reference solution, in grams;
p = percentage content of casticin in agnus castus fruit dry
extract HRS.
_______________________________________________________ _______ Ph Eur
Agrimony ; * Figure 1587.-1. - Illustration for identification test B of powdered

(Ph. Eur. monograph 1587) *** herbal drug of agrimony
Ph Elf_______________________________________________________________
DEFINITION
Dried flowering tops of Agrimonia eupatoria L.
parenchyma [Cb], with some of the cells containing calcium
Content
oxalate prisms [Cc]; fragments of lower leaf epidermis in
Minimum 2.0 per cent of tannins, expressed as pyrogallol
surface view [J] with sinuous walls and abundant
(CfcH603; Mr 126.1) (dried drug).
stomata Ja], mostly anomocytic (2.8.3) but occasionally
anisocytic, and glandular trichomes [Jb]; ovoid to
subspherical pollen grains, with 3 pores and a smooth
IDENTIFICATION exine [D]; glandular trichomes with a multicellular, uniseriate
A. The stem is green or, more usually, reddish, cylindrical stalk and a unicellular to quadricellular head [B, Jb];
and infrequently branched. It is covered with long, erect or fragments of the stems [H] with groups of fibres [Ha] and
tangled hairs. The leaves are compound imparipennate with parenchymatous cells, some of which contain cluster crystals
3 or 6 opposite pairs of leaflets, with 2 or 3 smaller leaflets of calcium oxalate [Hb]; small spiral vessels from the
between. The leaflets are deeply dentate to serrate, dark leaflets [G]; fragments of large, spiral or bordered-pitted
green on the upper surface, greyish and densely tomentose vessels from the stem [E].
on the lower face. The flowers are small and form a terminal
c. Thin-layer chromatography (2.2.27).
spike. They are pentamerous and borne in the axils of hairy
bracts, the calyces closely surrounded by numerous terminal Test solution To 2.0 g of the powdered herbal drug (355)
hooked spires, which occur on the rim of the hairy (2.9.12) add 20 mL of methanol R. Heat with shaking at
receptacle. The petals are free, yellow and deciduous. Fruit 40 °C for 10 min. Filter.
bearing obconical receptacles, with deep furrows and hooked Reference solution Dissolve 1.0 mg of isoquercitroside R and
bristles, are usually present at the base of the inflorescence. 1.0 mg of rutin R in 2 mL of methanol R.
B. Reduce to a powder (355) (2.9.12). The powder is
yellowish-green or grey. Examine under a microscope using Plate TLC silica gel plate R.
chloral hydrate solution R. The powder shows the following Mobile phase anhydrous formic acid R} water R} ethyl acetate R
diagnostic characters (Figure 1587.-1): numerous straight or (10:10:80 VIVIV).
bent, unicellular, long, thick-walled (about 500 pm) covering Application 10 pL as bands.
trichomes [Ab, Ca, F], finely warty, and sometimes spirally
marked, often fragmented (F); fragments of the epidermis of Development Over a path of 12 cm.
the stems [A] with stomata [Aa], covering trichomes (Ab) Drying At 100-105 °C.
and glandular trichomes [Ac]; fragment; of upper leaf
Detection Spray the still-warm plate with a 10 g/L solution of
epidermis in surface view [C] with straight walls bearing
diphenylboric acid aminoethyl ester R in methanol R and then
covering tnchomes (Ca). accompanied by palisade
2016 Alchemilla IV-5 5
with a 50 g/L solution of macrogol 400 R in methanol R; allow venation, prominent on the lower surface. The greyish-green
the plate to dry in air for 30 min and examine in ultraviolet or yellowish-green petiole is pubescent, about 1 mm in
light at 365 nm. diameter, with an adaxial groove. The apetalous flowers arc
Results See below the sequence of zones present in the yellowish-green or light green and about 3 mm in diameter.
chromatograms obtained with the reference solution and the The calyx is double with 4 small segments of the epicalyx
test solution. alternating with 4 larger sepals, subacute or triangular. They
are 4 short stamens and a single carpel with a capitate
stigma. The greyish-green or yellowish-green stem is
pubescent, more or less longitudinally wrinkled and hollow.
Top of the plate B. Reduce to a powder (355) (2.9.12). The powder is
greyish-green. Examine under a microscope using chloral
An orange fluorescent zone may hydrate solution R. The powder shows the following diagnostic
be present (quercitroside)
characters: unicellular, narrow trichomes up to 1 mm long
Isoquercitroside: an orange An orange fluorescent zone
fluorescent zone (isoquercitroside) partly tortuous, acuminate, and bluntly pointed at the apex,
An orange fluorescent zone
with thick lignified walls, somewhat enlarged and pitted at
(hyperoside) the base; fragments of leaves with 2 layers of palisade
Rutin: an orange fluorescent zone An orange fluorescent zone (rutin) parenchyma, the upper layer of which is 2-3 times longer
Reference solution Test solution than the lower layer and with spongy parenchyma, containing
scattered cluster crystals of calcium oxalate, up to 25 pm in
diameter; leaf fragments in surface view with sinuous or wavy
epidermal cells, the anticlinal walls unevenly thickened and
beaded, anomocytic stomata (2.8.3); groups of vascular tissue
TESTS and lignified fibres from the petioles and stems, the vessels
Loss on drying (2.2.22) spirally thickened or with bordered pits; occasional thin
walled conical trichomes, about 300 pm long; thin-walled
Maximum 10.0 per cent, determined on 1.000 g of the parenchyma containing cluster crystals of calcium oxalate;
powdered herbal drug (355) (2.9.72) by drying in an oven at spherical pollen grains, about 15 pm in diameter, with
105 °C for 2 h. 3 distinct pores and a granular exine; occasional fragments of
Total ash (2.4.16) the ovary wall with cells containing a single crystal of calcium
oxalate.
ASSAY
Tannins (2.8.14) (2.9.12) add 5 mL of methanol R and heat in a water-bath at
Use 1.000 g of the powdered herbal drug (180) (2.9.12). 70 °C under a reflux condenser for 5 min. Cool and filter.
_______________________________________________________________ Ph Eur
Reference solution Dissolve 1.0 mg of caffeic acid R and 1.0 mg
of chlorogenic acid R in 10 mL of methanol R.
Plate TLC silica gel plate R.
Mobile phase anhydrous formic acid R, water R) ethyl acetate R
(8:8:84 VIVIV).
Application 20 pL of the test solution and 10 pL of the
reference solution, as bands.
Alchemilla * * Development Over a path of 10 cm.
(Ph. Eur. monograph 1387) *** Drying At 100-105 °C for 5 min.
Ph Elt____________________________________________ _ Detection Spray with a 10 g/L solution of diphenylboric acid
aminoethyl ester R in methanol R. Subsequently spray with a
DEFINITION
50 g/L solution of macrogol 400 R in methanol R. Allow to dry
Whole or cut, dried, flowering, aerial parts of Alchemilla in air for about 30 min. Examine in ultraviolet light at
vulgaris L. sensu latiore. 365 nm.
Content Results See below the sequence of the zones present in the
Minimum 6.0 per cent of tannins, expressed as pyrogallol chromatograms obtained with the reference solution and the
(C6H6O3; 126.1) (dried drug). test solution. Furthermore, other fluorescent zones may be
present in the chromatogram obtained with the test solution.
IDENTIFICATION
A. The greyish-green, partly brownish-green, radical leaves
which are the main part of the drug are reniform or slightly
semicircular with a diameter generally up to 8 cm, seldom up
to 11 cm and have 7 to 9, or 11 lobes and a long petiole.
The smaller, cauline leaves, which have a pair of large
stipules at the base, have 5-9 lobes and a shorter petiole or
they are sessile. The leaves are densely pubescent especially
on the lower surface and have a coarsely serrated margin.
Young leaves are folded with a whitish-silvery pubescence;
older leaves are slightly pubescent and have a finely meshed
IV-56 Aloes 2016
Top of the plate Top of the plate
2 red fluorescent zones Aloe emodin: a yellow fluorescent

(chlorophyll)
Caffeic acid: a light blue florescent 1 or 2 intense light blue fluorescent
zone zones
One or several intense green or
greenish-yellow fluorescent zones
Chlorogenic acid: a light blue An intense yellow or orange

fluorescent zone fluorescent zone
Barbaloin: an orange fluorescent zone An orange fluorescent zone
(barbaloin)
A bluish-white fluorescent zone
A bluish-green fluorescent zone

TESTS
Loss on drying (2.2.32) Reference solution Test solution
Maximum 10.0 per cent, determined on 1.000 g of the
powdered herbal drug (355) (2.9.12) by drying in an oven at
105 °C for 2 h. Results B See below the sequence of zones present in the
Total ash (2.4.16) chromatograms obtained with the reference solution and the
Maximum 12.0 per cent. test solution. Furthermore, other faint zones may be present
ASSAY in the chromatogram obtained with the test solution.
Tannins (2.8.14)
Use 0.50 g of the powdered herbal drug (355) (2.9.12).
__ _____________________ _ ______________________________________Ph Eur
Top of the plate
Aloe emodin: a violet zone
Barbados Aloes * **
Curasao Aloes **
Barbaloin: a brown zone A brown zone (barbaloin)
(Ph. Eur. monograph 0257)
A violet zone
Preparation
Standardised Aloes Dry Extract
Pn Eur______ _ _______________________________________________________ Reference solution Test solution
DEFINITION
Concentrated and dried juice of the leaves of Aloe barbadensis TESTS
Mill. Loss on drying (2.2.32)
Content Maximum 12.0 per cent, determined on 1.000 g of the
Minimum 28.0 per cent of hydroxyanthracene derivatives, powdered herbal drug by drying in an oven at 105 °C.
expressed as barbaloin (C21H22O9; Mr 418.4) (dried drug). Total ash (2.4.16)
CHARACTERS Maximum 2.0 per cent.
Appearance Cape aloes
Dark brown masses, slightly shiny or opaque with a Thin-layer chromatography (2.2.27).
conchoidal fracture, or brown powder. Test solution To 0.25 g of the powdered herbal drug add
Solubility 20 mL of methanol R and heat to boiling in a water-bath.
Partly soluble in boiling water, soluble in hot ethanol Shake for a few minutes and decant the solution. Store at
(96 per cent). about 4 °C and use within 24 h.
IDENTIFICATION Reference solution Dissolve 2 mg of aloe emodin R and 2 mg of
Examine the chromatograms obtained in the test for Cape barbaloin R in methanol R and dilute to 1 mL with the same
aloes. solvent.
Results A See below the sequence of zones present in the Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
chromatograms obtained with the reference solution and the plate R (2-10 pm)].
test solution. Furthermore, other faint fluorescent zones may Mobile phase water R} methanol R, ethyl acetate R
be present in the chromatogram obtained with the test (13:17:100 VIVIV).
solution. Application 10 gL [or 2 gL] as bands of 20 mm [or 8 mm].
Drying In air.
Detection A Examine in ultraviolet light at 365 nm.
2016 Cape Aloes IV-57
Results A The chromatogram obtained with the test solution CHARACTERS

shows no blue fluorescent zones above the orange fluorescent Appearance
zone due to barbaloin. Dark brown masses tinged with green and having a shiny
Detection B Treat with a 100 g/L solution of potassium conchoidal fracture, or greenish-brown powder.
hydroxide R in methanol R, heat at 110 °C for 5 min and Solubility
examine in daylight. Partly soluble in boiling water, soluble in hot ethanol
ASSAY (96 per cent).
Carty out the assay protected from bright light. IDENTIFICATION
Introduce 0.300 g of the powdered herbal drug (180) A. Examine the chromatograms obtained in the test for
(2.9.72) into a 250 mL conical flask. Moisten with 2 mL of Barbados aloes.
methanol Ry add 5 mL of water R warmed to about 60 °C, Results The chromatogram obtained with the test solution
mix, then add a further 75 mL of water R at about 60 °C shows in the central part a yellow fluorescent zone
and shake for 30 min. Cool, filter into a volumetric flask, (barbaloin) similar in position to the zone due to barbaloin in
rinse the conical flask and filter with 20 mL of water Ry add the chromatogram obtained with the reference solution and
the rinsings to the volumetric flask and dilute to 1.0 L with in the lower part 2 yellow fluorescent zones (aloinosides A
water R. Transfer 10.0 mL of this solution to a 100 mL and B) and 1 blue fluorescent zone (aloesine).
round-bottomed flask containing 1 mL of a 600 g/L solution B. Shake 1 g of the powdered herbal drug with 100 mL of
offerric chloride R and 6 mL of hydrochloric acid R. Heat in a boiling water R. Cool, add 1 g of talc R and filter. To 10 mL
water-bath under a reflux condenser for 4 h, with the water of the filtrate add 0.25 g of disodium tetraborate R and heat to
level above that of the liquid in the flask. Allow to cool, dissolve. Pour 2 mL of the solution into 20 mL of water R.
transfer the solution to a separating funnel, rinse the flask A yellowish-green fluorescence appears which is particularly
successively with 4 mL of water Ry 4 mL of 1 M sodium marked in ultraviolet light at 365 nm.
hydroxide and 4 mL of water R and add the rinsings to the
C. To 5 mL of the filtrate obtained in identification test B
separating funnel. Shake the contents of the separating funnel
add 1 mL of freshly prepared bromine water R. A yellow
with 3 quantities, each of 20 mL) of ether R. Wash the
precipitate is formed. The supernatant is not violet.
combined ether layers with 2 quantities, each of 10 mL, of
water R. Discard the washings and dilute the organic phase to TESTS
100.0 mL with ether R. Evaporate 20.0 mL of the solution Barbados aloes
carefully to dryness on a water-bath and dissolve the residue Thin-layer chromatography (2.2.27).
in 10.0 mL of a 5 g/L solution of magnesium acetate R in Test solution To 0.25 g of the powdered herbal drug add
methanol R. Measure the absorbance (2.2.25) at 512 nm 20 mL of methanol R and heat to boiling in a water-bath.
using methanol R as the compensation liquid. Shake for a few minutes and decant the solution. Store at
Calculate the percentage content of hydroxyanthracene about 4 °C and use within 24 h.
derivatives, expressed as barbaloin, using the following Reference solution Dissolve 25 mg of barbaloin R in methanol R
expression: and dilute to 10 mL with the same solvent.
Plate TLC silica gel G plate R.
A X 19.6 Mobile phase water Ry methanol R, ethyl acetate R
m (13:17:100 VIVIV).
Application 10 pL, as bands of 20 mm by maximum 3 mm.
i.e. taking the specific absorbance of barbaloin to be 255. Development Over a path of 10 cm.
/4 = absorbance at 512 nm;
Drying In air.
m = mass of the substance to be examined, in grams.
Detection Spray with a 100 g/L solution of potassium
STORAGE hydroxide R in methanol R. Heat at 110 °C for 5 min and
In an airtight container. examine in ultraviolet light at 365 nm.
______________________________________________________________ Ph Eur Results The chromatogram obtained with the test solution
shows no violet fluorescent zone just below the zone due to
barbaloin.
Cape Aloes ** ** powdered herbal drug by drying in an oven at 105 °C.
(Ph. Eur. monograph 0258) *** Total ash {2.4.16)
Preparation Maximum 2.0 per cent.
Standardised Aloes Dry Extract ASSAY
Ph Eur______________________________________________________________ Cany out the assay protected from bright light.
Introduce 0.400 g of the powdered herbal drug (180)
DEFINITION
(2.9.72) into a 250 mL conical flask. Moisten with 2 mL of
Concentrated and dried juice of the leaves of various species
methanol Ry add 5 mL of water R warmed to about 60 °C,
of Aloey mainly Aloe ferox Miller and its hybrids.
mix, then add a further 75 mL of water R at about 60 °C
Content and shake for 30 min. Cool, filter into a volumetric flask,
Minimum 18.0 per cent of hydroxyanthracene derivatives, raise the conical flask and filter with 20 mL of water Ry add
expressed as barbaloin (C21H22O9; Mr 418.4) (dried drug). the rinsings to the volumetric flask and dilute to 1000.0 mL
with water/?. Transfer 10.0 mL of this solution to a 100 mL
IV-58 Aloes Preparations 2016
round-bottomed flask containing 1 mL of a 600 g/L solution Mobile phase water R, methanol R, ethyl acetate R
of ferric chloride R and 6 mL of hydrochloric acid R. Heat in a (13:17:100 VIVIV).
water-bath under a reflux condenser for 4 h, with the water Application 10 pL [or 2 pL] as bands of 20 mm [or 8 mm].
level above that of the liquid in the flask. Allow to cool,
transfer the solution to a separating funnel, rinse the flask
successively with 4 mL of water R, 4 mL of 1 M sodium Drying In air.
hydroxide and 4 mL of water R and add the rinsings to the Detection A Examine in ultraviolet light at 365 nm.
separating funnel. Shake the contents of the separating funnel Results A See below the sequence of zones present in the
with 3 quantities, each of 20 mL, of ether R. Wash the chromatograms obtained with the reference solution and the
combined ether layers with 2 quantities, each of 10 mL, of test solution. Furthermore, other faint fluorescent zones may
water R. Discard the washings and dilute the organic phase to be present in the chromatogram obtained with the test
100.0 mL with ether R. Evaporate 20.0 mL of the solution solution.
carefully to dryness on a water-bath and dissolve the residue
in 10.0 mL of a 5 g/L solution of magnesium acetate R in Top of the plate
methanol R. Measure the absorbance (2.2.25) at 512 nm Aloe emodin: a yellow
using methanol R as the compensation liquid. fluorescent zone
Calculate the percentage content of barbaloin from the
following expression: A blue fluorescent zone
A X 19.6
A blue fluorescent zone
Barbaloin: an orange An orange fluorescent An orange fluorescent

i.e. taking the specific absorbance of hydroxyanthracene fluorescent zone zone (barbaloin) zone (barbaloin)
derivatives, as barbaloin, to be 255.
A ะ= absorbance at 512 nm,
m = mass of the substance to be examined in grams.
A bluish-white A bluish-white
fluorescent zone fluorescent zone
STORAGE
A bluish-green
In an airtight container. fluorescent zone
______________________________________________________________ Ph Eur Reference solution Test solution Test solution
(Barbados aloes) (Cape aloes)
Detection B Treat with a 100 g/L solution of potassium

hydroxide R in methanol R, heat at 110 °C for 5 min and
Standardised Aloes Dry Extract ★ * examine in daylight.
Results B See below the sequence of zones present in the
(Ph. Eur. monograph 0259) * **
Ph Elf_______________________________________________________________ test solution. Furthermore, other faint zones may be present
DEFINITION in the chromatogram obtained with the test solution.
Standardised dry extract prepared from Barbados aloes (0257)
Top of the plate
or Cape aloes (0258)3 or a mixture of both.
Aloe emodin: a violet
Content
19.0 per cent to 21.0 per cent of hydroxyanthracene
derivatives, expressed as barbaloin (C21H22O9; Mr 418.4),
adjusted if necessary (dried extract).
PRODUCTION
The extract is produced from the herbal drug by a suitable
procedure using boiling water. Barbaloin: a brown zone A brown zone A brown zone
(barbaloin) (barbaloin)
CHARACTERS
A violet zone
Appearance
Brown or yellowish-brown powder.
Solubility
Sparingly soluble in boiling water. Reference solution Test solution Test solution
(Barbados aloes) (Cape aloes)
IDENTIFICATION
TESTS
Test solution To 0.25 g of the extract to be examined add Loss on drying (2.8.17)
20 mL of methanol R and heat to boiling in a water-bath. Maximum 4.0 per cent m/m.
Shake for a few minutes and decant the solution. Store at
about 4 °C and use within 24 h.
Total ash (2.4.16)
Reference solution Dissolve 2 mg of aloe emodin R and 2 mg of
barbaloin R in methanol R and dilute to 1 mL with the same ASSAY
solvent. Carry out the assay protected from bright light.
Plate TEC silica gel plate R (5-40 pm) [or TLC silica gel Introduce 0.400 g of the extract to be examined into a
plate R (2-10 pm)]. 250 mL conical flask. Moisten with 2 mL of methanol R, add
2016 Amomum Fruit IV-5 9
5 mL of water R warmed to about 60 °C, mix, add a further longitudinally channelled and scaly, and bears numerous soft
75 mL of water R at about 60 °C and shake for 30 min. spiny protuberances, sometimes branched. The apex bears a
Cool, filter into a volumetric flask, rinse the conical flask and prominent stylopodium and the base shows the scar of the
the filter with 20 mL of water R3 add the rinsings to the stalk. The pericarp is thick and hard. The seeds are
volumetric flask and dilute to 1.0 L with water R. Transfer agglomerated into 3 relatively small masses, each consisting
10.0 mL of this solution to a 100 mL round-bottomed flask of about 2-24 seeds, separated by incomplete septa.
containing 1 mL of a 600 g/L solution of ferric chloride R and The seeds are small, 1.5-2 mm in diameter, polyhedral with
6 mL of hydrochloric acid R. Heat in a water-bath under a rounded edges, reddish-brown or blackish-brown on the
reflux condenser for 4 h, with the water level above that of surface, finely wrinkled and covered with a pale brown,
the liquid in the flask. Allow to cool, transfer the solution to transparent, membranous aril; the endosperm is whitish.
a separating funnel, rinse the flask successively with 4 mL of B. A. villosum. Microscopic examination (2.8.23).
water R, 4 mL of 1 M sodium hydroxide and 4 mL of water R, The powder is greyish-brown. Examine under a microscope
and add the rinsings to the separating funnel. Shake the using chloral hydrate solution R. The powder shows the
contents of the separating funnel with 3 quantities, each of following diagnostic characters: fragments of epicarp
20 mL, of ether R. Wash the combined ether layers with consisting of brownish-orange polyhedral cells containing
2 quantities, each of 10 mL, of water R. Discard the microcrystals and prisms of calcium oxalate, clearly visible in
washings and dilute the organic layer to 100.0 mL with polarised light and sometimes long covering trichomes, about
ether R. Evaporate 20.0 mL carefully to dryness on a water 250 pm long, usually unicellular, straight or bent, with
bath and dissolve the residue in 10.0 mL of a 5 g/L solution slightly and regularly thickened walls; fragments of mesocarp
of magnesium acetate R in methanol R. Measure the composed of thin-walled polygonal cells and round oil cells,
absorbance (2.2.25) at 512 nm using methanol R as the with orange to dark brown contents; vascular bundles
compensation liquid. consisting of vessels, mainly spiral, and fibres with thick and
Calculate the percentage content of hydroxyanthracene pitted walls; fragments of the outer testa consisting of a layer
derivatives, expressed as barbaloin, using the following of cells, fusiform in surface view, with slightly and regularly
expression: thickened walls, usually accompanied by a layer of
rectangular or polyhedral cells, perpendicular to the previous
A X 19.6 layers, and sometimes by underlying oil cells; brownish-red
m fragments of the inner testa, in surface view, composed of
very regularly polyhedral cells with heavily thickened walls
i.e. taking the specific absorbance of barbaloin to be 255. and a punctiform lumen; brownish-red fragments of the inner
A = absorbance at 512 nm; testa, in transverse section, composed of palisade cells with
m = mass of the substance to be examined, in grams. strongly thickened inner and lateral walls. Examine under a
microscope using a 50 per cent VIV solution of glycerol R.
—___________________________________________________________ Ph Eur
The powder shows numerous fragments composed of sub-
rectangular or irregular cells, filled with aggregates of small
starch granules; some of these cells also contain small prisms
of calcium oxalate; a few aggregates of small starch granules
are also present.
Amomum fruit * **
A longiligulare Microscopic examination (2.8.23). The powder
(Ph. Eur. monograph 2554) *** is greyish-brown. Examine under a microscope using chloral
Ph Eur______________________________________________________________ hydrate solution R. The powder shows the following diagnostic
characters: fragments of epicarp consisting of brownish-
DEFINITION orange, polyhedral or elongated cells containing microcrystals
Dried, whole or fragmented, peeled or unpeeled ripe fruit of and prisms of calcium oxalate, clearly visible in polarised
Amomum villosum Lour, or Amomum longiligulare T.L.Wu. light; fragments of mesocarp composed of thin-walled
Content polygonal cells and occasional round oil cells with orange to
— essential oil-, minimum 30 mL/kg for A. villosum dark brown contents; vascular bundles composed of spiral or
(anhydrous drug) and minimum 10 mL/kg for reticulate vessels and fibres with thick and distinctly pitted
A. longiligulare (anhydrous drug); walls; fragments of the aril consisting of elongated, very thin
— bornyl acetate (C12H20O2; Mr 196.3): minimum walled cells, some of which contain prisms and microcrystals
30.0 per cent of the essential oil. that are clearly visible in polarised light; fragments of the
outer testa consisting of a layer of cells, fusiform in surface
IDENTIFICATION
view, with slightly and regularly thickened walls, usually
A. A. villosum. The fruit is an indehiscent capsule, ovoid or
accompanied by a layer of rectangular or polyhedral cells
ellipsoidal, indistinctly 3-ridged, up to 2 cm long and 1.5 cm
with brown contents, perpendicular to the previous layers;
in diameter. The outer surface is brown and covered with
brownish-red fragments of the inner testa, composed of very
soft spiny protuberances. The apex bears the remains of the
thick-walled and very regularly polyhedral cells, in surface
perianth and the base usually bears a stalk. The pericarp is
view; brownish-red fragments of the inner testa, in transverse
thin and soft. The seeds are agglomerated into 3 masses,
section, composed of rectangular or palisade cells with
each consisting of 5-25 seeds, separated by whitish septa. heavily thickened inner and lateral walls. Examine under a
The seeds are hard, irregularly polyhedral, 2-3 mm in microscope using a 50 per cent VIV solution of glycerol R.
diameter, reddish-brown or blackish-brown on the surface, The powder shows numerous fragments of the endosperm
finely wrinkled and covered with a pale brown, transparent, with sub-rectangular or polyhedral cells, filled with small
membranous aril; the endosperm is whitish-grey. starch granules aggregated into masses and free aggregates of
A longiligulare The fruit is an indehiscent capsule, long, ovoid starch granules.
or ellipsoidal, distinctly 3-ridged, up to 2 cm long and
1.2 cm in diameter. The outer surface is brown,
IV-60 Amomum Fruit 2016
c. Thin-layer chromatography (2.2.27). TESTS

Test solution To 1 g of the powdered herbal drug (355) Water (2.2.13}
(2.9.72) add 5 mL of a mixture of equal volumes of toluene R Maximum 120 mUkg, determined by distillation on 20.0 g
and xylene R. Sonicate for 10 min. Centrifuge and use the of the powdered herbal drug (355) (2.9.72).
supernatant. Total ash (2.4.16}
Reference solution Dissolve 10 pL of bornyl acetate R, 10 pL of Maximum 9.0 per cent.
cineole R and 10 mg of borneol R in 1 mL of toluene R. ASSAY
Plate TLC silica gel plate R (2-10 pm). Essential oil (2.8.12}
Mobile phase ethyl acetate R, toluene R (7:93 V!V}. Use 10.0 g of the herbal drug reduced to a coarse powder
Application 5 pL as bands of 8 mm. (1400) (2.9.72) immediately before the assay, a 500 mL
Development Over a path of 6 cm. round-bottomed flask, 200 mL of water R as the distillation
liquid and 0.5 mL of trimethylpentane R in the graduated
Drying In air.
tube. Distil at a rate of 3-3.5 mUmin for 5 h.
Detection A Examine in ultraviolet light at 366 mn.
Bornyl acetate
Results A See below the sequence of zones present in the Gas chromatography (2.2.28}: use the normalisation
chromatogram obtained with the test solution. The reference procedure.
solution shows no spots at 366 nm. Furthermore, other faint
Test solution Dilute a volume of the essential oil-
fluorescent zones may be present in the chromatogram
trimethylpentane mixture obtained in the assay of essential oil
obtained with the test solution.
corresponding to 150 pL of the essential oil in heptane R and
Top of the plate
dilute to 10.0 mL with the same solvent.
Reference solution (a) Dissolve 25 mg of camphor R in
A faint blue fluorescent zone
heptane R, add 25 pL of bornyl acetate R and dilute to 5.0 mL
— with heptane R.
1 or 2 red fluorescent zones
Reference solution (b) Dilute 5 pL of bornyl acetate R to
100.0 mL with heptane R. Dilute 1.5 mL of the solution to
10.0 mL with heptane R.
A red fluorescent zone Column:
1 or 2 red fluorescent zones — material: fused silica;
— size: z = 60 m, 0 = 0.25 mm;
Reference solution Test solution — stationary phase: macrogol 20 000 R (film thickness
0.25 pm).
Detection B Treat with anisaldehyde solution R, heat at Carrier gas helium for chromatography R.
100-105 °C for 3 min and examine in daylight. Flow rate 0.9 mUmin.
Results B See below the sequence of zones present in the Split ratio 1:50.
Temperature:
in the chromatogram obtained with the test solution.
Time Temperature
1 Top of the plate
(min) (°C)
A reddish-brown zone Column 0 - 60 60 -> 210
Injection port 230
A bluish-violet zone Detector 250
Bornyl acetateะ a greyish-brown A greyish-brown zone (bornyl

acetate)
Detection Flame ionisation.
Injection 1 pL.
Identification of peaks Use the chromatogram obtained with
reference solution (a) to identify the peaks due to camphor
and bornyl acetate.
1,8-Cineole: a bluish-violet zone Relative retention With reference to camphor (retention
time = about 26 min): bornyl acetate = about 1.1.
A greyish-brown zone
System suitability: reference solution (a):
Borneol: a greyish-brown zone at A greyish-brown zone (borneol) — resolution: minimum 5 between the peaks due to camphor
the border between the middle at the border between the middle and bornyl acetate.
and lower thirds and lower thirds
Calculate the percentage content of bornyl acetate. Disregard
any peak due to the solvent or with an area less than the area
of the principal peak in the chromatogram obtained with
A bluish-violet zone reference solution (b) (0.05 per cent).
LABELLING
The label states the species present.
1 or 2 bluish-violet zones
___________________________________________________ _ Ph Eur
2016 Anethum Graveolens Sowa Fruit IV-61
Anethum Graveolens Sowa Fruit Top of the plate

DEFINITION
A faint brown band
Anethum Graveolens Sowa Fruit is the dried ripe fruit of
Anethum graveolens L. Sowa Group.
A brown band (dillapiole) A brown band (dillapiole)
Content
It contains not less than 3.0% v/w of essential oil calculated A purple band (carvone) A purple band (carvone)
with reference to the anhydrous drug.
IDENTIFICATION A grey band
A. The dried fruits usually occur as separate mericarps,
pedicels normally absent; broadly oval, highly compressed
dorsally, about 4 mm long, 2 to 3 mm broad, with 5 dorsal
ridges, each mericarp exhibiting 3 pale brown dorsal ridges,
the two lateral ridges elongated into characteristic Solution (1) Solution (2)
membranous wings; surface glabrous; remnants of the
stylopod at the apex; commissural surface flat, often with
attached, paler brown carpophore; vittae visible as two
darker, arc-shaped, longitudinal bands. TESTS
Apiole
B. Reduce to a powder (355). The powder is pale brown.
Carry out the method for gas chromatography,
Examine under a microscope using chloral hydrate solution.
Appendix in B, using the following solutions.
The powder contains numerous fragments of the epicarp,
with cuticular striations and infrequent anomocytic stomata; (1) Use the oil retained in the Assay of Essential oil.
parquetry layer of endocarp in surface view; endosperm of (2) 1.0% w/v of apiole in toluene.
oval to rectangular thick-walled cells containing oil globules (3) 0.05% w/v of apiole in toluene.
and aleurone grains with embedded microrosette crystals of (4) 0.05% v/v of p-myrcene and 0.8% v/v of limonene in
calcium oxalate; fragments of yellowish-brown septate vittae; toluene.
parenchyma of mesocarp consisting of elongated, lignified,
reticulately thickened cells; sclereids of mesocarp thick-walled CHROMATOGRAPHIC CONDITIONS
with few pits. (a) Use a fused silica capillary column (30 m X 0.53 mm)
c. Carry out the method for thin-layer chromatography, bonded with a 1 pm film thickness, polyethylene glycol 20,000
Appendix III A, using the following solutions. (DB-Wax is suitable).
(1) Add lOmL of methanol (70%) to 0.5 g of the powdered (b) Use helium as the carrier gas at 1.5 mL per minute.
drug (355), mix and place in an ultrasonic bath for (c) Use the gradient conditions described in the table.
30 minutes. Filter (a 0.45-pm PTFE is suitable) into a (d) Use a flame ionisation detector maintained at a
10 mL volumetric flask and dilute to 10 mL with temperature of 260°.
methanol (70%). (e) Inject 1 pL of each solution at a temperature of 250°.
(2) 0.05% w/v each of carvone and dillapiole in methanol
(70%).
Time Temperature Comment
CHROMATOGRAPHIC CONDITIONS (min)
(a) Use high-performance silica gel 60 F254 plates (Merck
0—5 60 isothermal
silica gel 60 F254 HPTLC plates are suitable).
(b) Use the mobile phase as described below. 5 — 68 60 — 250 linear gradient
(c) Apply 10 pL of each solution as 6 mm bands. 68 — 75 250 isothermal
(d) Develop the plate to 8 cm.
(e) After removal of the plate, dry in air, spray with vanillin
reagent, heat the plate at 110° until the coloured bands
SYSTEM SUITABILITY
appear and examine in daylight.
The test is not valid unless, in the chromatogram obtained
MOBILE PHASE
with solution (4), the resolution factor between the peaks due
2 volumes of acetic acid, 10 volumes of ethyl acetate and to P-myrcene and limonene, is at least 4.5.
88 volumes of toluene. In the chromatogram obtained with solution (4), the
SYSTEM SUITABILITY substances elute in the following order. P-myrcene and
The test is not valid unless the chromatogram obtained with limonene.
solution (2) shows two clearly separated bands. The signal-to-noise ratio of the peak due to apiole in solution
(3) is at least 3.
CONFIRMATION
The chromatogram obtained with solution (1) shows a LIMIT
purple band corresponding in position and colour to the In the chromatogram obtained with solution (1) the area of
band due to carvone in the chromatogram obtained with any peak due to apiole is not more than the area of the peak
solution (2); a brown band corresponding in position and due to apiole in the chromatogram obtained with
colour to the band due to dillapiole and other bands as solution (3).
shown in the table. Other bands may be present. Water
Not more than 10.0% v/w, Appendix IX c, method n using
30 g of powdered drug (1400).
IV-62 Angelica Archangelica Root 2016
Total Ash occasionally branched roots often with incompletely

Not more than 8.0%, Appendix XI J, Me±od II. encircling, transverse ridges. The apex sometimes shows
Chromatographic profile remnants of stem and leaf bases. The fracture is uneven.
Carry out the method for gas chromatography, The transversely cut surface shows a greyish-white, spongy,
Appendix III B, using the following solutions. distinctly radiate bark, in which the secretory channels are
visible as brown spots, and a bright yellow or greyish-yellow
(1) Use the oil retained in the Assay of Essential oil.
wood which, in the rhizome, surrounds the greyish or
(2) 0.4% w/v each of limonene, dihydrocarvone, carvone and brownish-white pith.
dillapiole in toluene.
B. Microscopic examination (2.5.25). The powder is
(3) 0.05% v/v of p-myrcene and 0.8% v/v of limonene in brownish-white. Examine under a microscope using chloral
toluene hydrate solution R. The powder shows the following diagnostic
CHROMATOGRAPHIC CONDITIONS characters (Figure 1857.-1): fragments of cork consisting of
The chromatographic procedure described under the test for several layers of thin-walled, greyish-brown or reddish-brown
Apiole may be used. cells, in surface view [C] or in transverse section [E]; large,
yellowish-brown secretory channels, whole or fragmented, in
SYSTEM SUITABILITY
transverse section [A] or in longitudinal section [F];
The test is not valid unless, in the chromatogram obtained fragments of medullary rays, 2 or 4 cells wide [G]; fragments
with solution (3), the resolution factor between the peaks due of xylem [B] consisting of lignified vessels with reticulate
to p-myrcene and limonene is at least 4.5. thickening [Ba] occurring singly or in small groups, and
In the chromatogram obtained with solution (2), the unlignified parenchyma in which some of the cells associated
substances elute in the following order: limonene, with the vessels are collenchymatously thickened. Examine
dihydrocarvone isomer 1, dihydrocarvone isomer 2, carvone under a microscope using a 50 per cent VIV solution of
and dillapiole. glycerol R. The powder shows numerous, simple starch
In the chromatogram obtained with solution (3), the granules 2-4 pm in diameter, free or included in parenchyma
substances elute in the following order: P-myrcene and cells [D].
limonene.
Calculate the content of limonene, dihydrocarvone, carvone
and dillapiole by normalisation. Disregard the peak due to
toluene.
Limits:
— limonene: 15.0 to 28.0%,
— sum of dihydrocarvone isomers 1 and 2: 5.0 to 30.0%,
— carvone: 20.0 to 45.0%,
— dillapiole: 15.0 to 35.0%.
Disregard any peak with an area less than 0.025 times the
area of the peak due to carvone in the chromatogram
obtained with solution (2).
ASSAY
Essential oil
Carry out the method for Essential OUS in Herbal Drugs,
Appendix XI E using 18 g of freshly prepared powdered drug
(1400) with 250 mL of water as the distillation liquid. Distil
at a rate of 2 to 3 mL per minute for 2 hours using 0.50 mL
of toluene in the graduated tube. Measure the quantity of
essential oil distilled and use in the tests for Apiole and
Chromatographic profile.
Angelica Archangelica Root * *

Ph Eur____________ __ _________________________________________________
DEFINITION
Whole or cut, carefully dried rhizome and root of Angelica
archangelica L. (syn. A. officinalis Hofim.). Figure 1857.-1. - Illustration for identification test B of powdered
herbal drug of angelica archangelica root
Content
Minimum 2.0 mL/kg of essential oil (dried drug). c. Examine the chromatograms obtained in the test for other
species of Angelica, Levisticum and Ligusticum described in the
CHARACTERS European Pharmacopoeia.
Bitter taste. Results A See below the sequence of zones present in the
IDENTIFICATION chromatograms obtained with the reference solution and the
A. The rhizome is greyish-brown or reddish-brown, with test solution. Furthermore, other faint fluorescent zones may
transversely annulated thickenings. The base bears greyish- be present in the chromatogram obtained with the test
brown or reddish-brown, cylindrical, longitudinally furrowed, solution.
2016 Angelica Dahurica Root IV-63
Top of the plate Loss on drying (2.2.32)

(Z)-Ligustilide: a bluish-white Maximum 10.0 per cent, determined on 1.000 g of the
fluorescent zone powdered herbal drug (355) (2.9.12) by drying in an oven at
105 °C for 2 h.
Osthole: a blue fluorescent zone A blue fluorescent zone Total ash (2.4.16)
Imperatorin ะ a whitish fluorescent A whitish fluorescent zone
A blue fluorescent zone Maximum 2.0 per cent.
ASSAY
Essential oil (2.8.12)
3 blue fluorescent zones
Reduce the herbal drug to a powder (500) (2.9.12) and
Reference solution Test solution immediately use 40.0 g for the determination. Use a 2 L
round-bottomed flask, 10 drops of liquid paraffin R3 500 mL
of water R as distillation liquid and 0.50 mL of xylene R in
Results B See below the sequence of zones present in the the graduated tube. Distil at a rate of 2-3 mL/min for 4 h.
_____________________________________________________________ Ph Eur
test solution. Furthermore, other faint quenching zones may
be present in the chromatogram obtained with the test
solution.
Top of the plate Angelica Dahurica Root * \

(Z)-Ligustilide: a blue fluorescent (Ph. Eur. monograph 2556) ***
Ph Elf_____________________________________________________________
Osthole: a quenching zone

DEFINITION
A quenching zone.
Dried, whole or fragmented root, with rootlets removed, of
Imperatorin: a quenching zone A quenching zone Angelica dahurica (Hofim.) Benth. & Hook.f. ex French. &
A quenching zone Sav. collected in summer or autumn.
Content
Minimum 0.08 per cent of imperatorin (C16H14o4; Mr
Several quenching zones 270.3) (dried drug).
Reference solution Test solution IDENTIFICATION
A. The non-fragmented drug consists of conical roots, about
10-25 cm long and 1.5-2.5 cm in diameter. The root crown,
TESTS more or less quadrangular, is obtuse and shows stem scars on
Other species of Angelica.) Levisticum and Ligusticuni prominences. It tapers to the tip. The outer surface is
described in the European Pharmacopoeia brownish-grey or yellowish-brown and clearly striated
Thin-layer chromatography (2.2.27). longitudinally, showing scars of the secondary roots and
Test solution To 1 g of the freshly powdered herbal drug lenticel-like transverse protuberances, some of them arranged
(355) (2.9.12) add 4 mL of heptane R, close and sonicate for in 4 longitudinal rows. The texture is compact, hard and
5 min. Centrifuge the mixture and use the supernatant. heavy. The fracture, white or whitish grey and mealy, is
Reference solution Dissolve 1 mg of imperatorin R3 1 mg of marked with concentric striations. The cambium occurs as a
(Z)-ligustilide R and 1 mg of osthole R in 10 mL of brown ring. Very many brown dots, corresponding to a
methanol R. transverse section of the secretory canals, are visible in the
cortical part.
Plate TLC silica gel F254 plate R (2-10 pm).
Mobile phase glacial acetic acid R3 ethyl acetate R3 toluene R
yellowish-white. Examine under a microscope using chloral
(1:10:90 VIVIV).
hydrate solution R. The powder shows the following diagnostic
Application 4 pJL as bands of 8 mm. characters: reticulate lignified vessels, free or in groups of 2
Development Over a path of 6 cm. or 3 and accompanied by ligneous parenchyma cells with fine
Drying In air. celldose walls; numerous fragments of parenchyma with
Detection A Examine in ultraviolet light at 365 nm. ovoid cells; a few orange cork fragments, consisting of several
layers of superimposed cells; secretory canals, usually broken,
Residts A The chromatogram obtained with the test solution
with yellow or pale brown contents and oil droplets. Examine
shows no zone at the position of (Z)-ligustilide in the
under a microscope using a 50 per cent v/v solution of
chromatogram obtained with the reference solution.
glycerol R. The powder shows very many starch granules
Detection B Examine in ultraviolet light at 254 nm. varying in size from 5 to 25 (im; some are simple and
Results B The chromatogram obtained with the test solution rounded, others consist of 2-8 elements, but most are
shows no zone at or just below the position of (Z)-ligustilide polyhedral, either due to compound granules breaking up or
in the chromatogram obtained with the reference solution. to compression in the cells.
Foreign matter (2.8.2) c. Examine the chromatograms obtained in the test for other
Maximum 5 per cent of leaf bases and stem bases, maximum officinal species of Angelica) Levisticum and Ligusticuni.
5 per cent of discoloured pieces and maximum 1 per cent of Results A See below the sequence of zones present in the
other foreign matter. chromatograms obtained with the reference solution and the
IV-64 Angelica Dahurica Root 2016
test solution. Furthermore, other faint fluorescent zones may TESTS

be present in the chromatogram obtained with the test Other officinal species of Angelica, Levisticum and
solution. Ligusticum
Top of the plate Test solution To 1 g of the powdered herbal drug (355)
(Z)-Ligustilide: a bluish-white
(2.9.72) add 4 mL of heptane R, close and sonicate for 5 min.
fluorescent zone Centrifuge the mixture and use the supernatant.
Reference solution Dissolve 1 mg of imperatorin R, 1 mg of
A whitish fluorescent zone
(Z)-ligustilide R and 1 mg of osthole R in 10 mL of
methanol R.
Osthole: a blue fluorescent zone A blue fluorescent zone
Imperatorin: a whitish fluorescent A whitish fluorescent zone Mobile phase glacial acetic acid R, ethyl acetate R, toluene R
(imperatorin)
(1:10:90 PZJW).
Application 4 pL as bands of 8 mm.
A whitish fluorescent zone Development Over a path of 6 cm.
Reference solution Test solution Drying In air.
Results B See below the sequence of zones present in the Results A The chromatogram obtained with the test solution
chromatograms obtained with the reference solution and the shows No bluish-white fluorescent zone at the position of
test solution. Furthermore, other faint quenching zones may (Z)-ligustilidc in the chromatogram obtained with the
be present in the chromatogram obtained with the test reference solution.
solution, In particular in the lower third of the Detection B Examine in ultraviolet light at 254 nm.
chromatogram, below the zone due to imperatorin. Results B The chromatogram obtained with the test solution
shows no blue fluorescent zone at the position of
Top of the plate (Z)-ligustilidc in the chromatogram obtained with the
(Z)-Ligustilide: a blue fluorescent
reference solution; the chromatogram obtained with the test
zone solution shows no quenching zone at die position of osthole
in the chromatogram obtained with the reference solution.
A quenching zone
Detection c Treat with a 10 per cent VIV solution of sulfuric
acid R in methanol R, heat at 100 °C for 5 min and examine
Osthole: a quenching zone in daylight.
Imperatorin: a quenching zone A quenching zone (imperatorin) Results c The chromatogram obtained with the test solution
shows no violet zone at the position of osdiole in the
105 °C for 2 h.
Results c See below the sequence of zones present in the Total ash (2.4.76)
chromatograms obtained with the reference solution and the Maximum 6.0 per cent.
test solution. Furthermore, other faint zones may be present Ash insoluble in hydrochloric acid (2.5.7)
in the chromatogram obtained with the test solution. Maximum 1.5 per cent.
ASSAY
Top of the plate
Liquid chromatography (2.2.29).
2 prominent reddish zones Test solution Disperse 0.400 g of the powdered herbal drug
(Z)-Ligusti!ide: a grey zone
(355) (2.9.72) in 45 mL of methanol R and sonicate for 1 h.
Cool and dilute to 50.0 mL with methanol R. Filter through a
membrane filter (nominal pore size 0.45 pm).
A faint blue zone Reference solution (a) Dissolve 5.0 mg of imperatorin CRS in
methanol R and dilute to 50.0 mL with the same solvent.
Osthole: a violet zone
Dilute 1.0 mL of the solution to 10.0 mL with methanol R.
Imperatorin: a reddish-grey zone A yellow and violet double-zone
Reference solution (b) Disperse 80 mg of Angelica dahurica
root HRS in 9 mL of methanol R and sonicate for 1 h. Cool
and dilute to 10 mL with methanol R. Filter through a
A prominent violet zone
A yellow zone Precolumn'.
Reference solution Test solution — size'. I = 4 mm, 0 = 4.0 mm;
— stationary phase: octadecylsilyl silica gel for chromatography R
(5 pm).
Column:
— size: I = 0.125 m, 0 = 4.0 mm;
(5 pm).
2016 Angelica Pubescens Root IV-65
Mobile phase’. scars and transverse lenticel-like protuberances. The fracture

— mobile phase A’, water R3 shows greyish-yellow bark, with abundant brown dots due to
— mobile phase B: acetonitrile R13 secretory canals; the cambium ring is brown and the wood is
greyish-yellow or yellowish-brown.
Time Mobile phase A Mobile phase B B. Microscopic examination (2.8.23). The powder is
(per cent V/V) (per cent V/V) yellowish-brown or brown. Examine under a microscope
0 - 15 45 55 using chloral hydrate solution R. The powder shows the
15 - 33 45 -> 5 55 -> 95 following diagnostic characters: fragments of lignified vessels
up to 90 gm in diameter with spiral or reticulate thickenings,
33 - 35 5 95
free or in groups of 2 or 3; fragments of phloem parenchyma
with fine, sinuous fusiform cells, about 7-38 gm in diameter,
Flow rate 1.0 mLAnin. with slightly thickened walls and fine, oblique criss-cross
Detection Spectrophotometer at 210 nm. striations; orange-brown cork fragments, consisting of several
layers of superimposed, somewhat polyhedral cells in surface
Injection 20 gL.
view; secretory canals, usually broken, with yellow or pale
Identification of peaks Use the chromatogram supplied with brown contents and droplets of essential oil. Examine under
Angelica dahurica root HRS and the chromatogram obtained a microscope using a 50 per cent VIV solution of glycerol R.
with reference solution (b) to identify the peak due to The powder shows numerous small, rounded or ovoid,
phellopterin. simple starch granules, about 10 gm in size, with a
Relative retention With reference to imperatorin (retention punctiform hilum that is visible on the largest granules; a few
time = about 5 min): phellopterin = about 1.1. starch granules consisting of 2-10 components are also
System suitability: reference solution (b): present.
— resolution: minimum 1.5 between the peaks due to c. Examine the chromatograms obtained in the test for other
imperatorin and phellopterin. officinal species oi Angelica 3 Levisticum and Ligusticum.
Calculate the percentage content of imperatorin using the Results A See below the sequence of zones present in the
following expression: chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint fluorescent zones may
.41 X 7712 X p be present in the chromatogram obtained with the test
A2 X m\ X 10 solution.
A1 = area of the peak due to imperatorin in the Top of the plate

chromatogram obtained with the test solution;
(Z)-Ligustilide ะ a bluish-white A bluish-white fluorescent zone
A2 = area of the peak due to imperatorin in the fluorescent zone
chromatogram obtained with reference solution (a);
m1 = mass of the herbal drug to be examined used to
A very faint whitish zone
prepare the test solution, in grams;
m2 = mass of imperatorin CRS used to prepare reference Osthole: a blue fluorescent zone A prominent blue fluorescent
solution (a), in grams; zone (osthole)
p = percentage content of imperatorin in imperatorin Imperatorin: a whitish fluorescent A whitish fluorescent zone (may
zone be missing)
CRS.
______________________________________________________________ Ph Eur
Angelica Pubescens Root * *

(Ph. Eur. monograph 2557) *** Results B See below the sequence of zones present in the
Ph Eur______________________________________________________________
DEFINITION be present in the chromatogram obtained with the test
Dried root, without rootlets, of Angelica pubescens Maxim, solution.
f. biserrata R.H.Shan et C.Q.Yuan, collected in early spring
before sprouting, or in the end of autumn when stem and Top of the plate
leaves wither. (Z)-Ligustilide: a blue fluorescent A faint quenching zone
Content zone
Minimum 0.50 per cent of osthole (CisH16O3; Mr 244.3)
(dried drug). Osthole: a quenching zone A quenching zone (osthole)
IDENTIFICATION A blue fluorescent zone

A. The taproot is more or less cylindrical, branching rapidly A quenching zone (may be
Imperatorin: a quenching zone
into 2-3 or more principal roots at the lower part; the whole missing)
is about 5-30 cm long. The root crown is enlarged, with
transverse, annulated wrinkles and measures about
2 or 3 quenching zones
0.5-4.5 cm in diameter; it shows the remains of stems, leaves
or buds. The greyish-brown or dark brown outer surface is Reference solution Test solution
longitudinally wrinkled and shows slightly prominent rootlet
TV-66 Angelica Root 2016
Results c See below the sequence of zones present in the ASSAY

chromatograms obtained with the reference solution and the Liquid chromatography (2.2.29).
test solution. Furthermore, other faint zones may be present Test solution Disperse 0.500 g of the powdered herbal drug
in the chromatogram obtained with the test solution. (355) (2.9.72) in 18 mL of methanol R and sonicate for
30 min. Cool and dilute to 20.0 mL with methanol R.
Top of the plate Mix and filter. Dilute 5.0 mL of the filtrate to 20.0 mL with
methanol R.
A prominent reddish zone
Reference solution (a) Dissolve 5.0 mg of osthole CRS in
(Z)-Ligustilideะ a grey zone methanol R and dilute to 100.0 mL with the same solvent.
Reference solution (b) Disperse 0.250 g of Angelica pubescens
root HRS in 9 mL of methanol R and sonicate for 30 min.
Osthole: a violet zone A violet zone (osthole)
Cool and dilute to 10.0 mL with methanol R. Mix and filter.
Imperatorin ะ a grey zone A violet zone (may be missing) Dilute 5.0 mL of the filtrate to 20.0 mL with methanol R.
Column:
— size: 7 = 0.125 m, 0 = 2.0 mm;
A prominent violet zone
(4 pm).
Reference solution Test solution Mobile phase water R, acetonitrile R (40:60 VIV).
TESTS Injection 10 pL.
Other officinal species of Angelica, Levisticum and
Retention time Osthole = about 8 min.
Ligusticum
System suitability: reference solution (b):
— resolution: minimum 1.5 between the peak due to osthole
Test solution To 1 g of the powdered herbal drug (355) and peak 2; use the chromatogram supplied with Angelica
(2.9.72) add 4 mL of heptane R, close and sonicate for 5 min. pubescens root HRS to identify peak 2.
Centrifuge the mixture and use the supernatant.
Calculate the percentage content of osthole using the
Reference solution Dissolve 1 mg of imperatorin R, 1 mg of following expression:
(Z)-ligustilide R and 1 mg of osthole R in 10 mL of
methanol R. Al X 7712 X p X 0.8
Plate TLC silica gel F254 plate R (2-10 pm). A2 X 7711
Mobile phase glacial acetic acid R, ethyl acetate R, toluene R
(1:10:90 F/P7F). A1 = area of the peak due to osthole in the chromatogram
Application 4 pL as bands of 8 mm. obtained with the test solution;
Development Over a path of 6 cm. A2 = area of the peak due to osthole in the chromatogram
obtained with reference solution (a);
Drying In air.
nil = mass of the herbal drug to be examined used to
Detection A Examine in ultraviolet light at 365 nm. prepare the test solution, in grams;
Residts A The chromatogram obtained with the test solution m2 = mass of osthole CRS used to prepare reference
shows no intense whitish fluorescent zone directly above the solution (a), in grams;
position of osthole and no blue fluorescent zone just below p = percentage content of osthole in osthole CRS.
the position of imperatorin in the chromatogram obtained
_______________________________________________________________ Ph Ear
with the reference solution.
Detection B Examine in ultraviolet light at 254 nm.
Residts B The chromatogram obtained with the test solution
shows no blue fluorescent zone corresponding to the zone
due to (Z)-ligustilide in the chromatogram obtained with the Angelica Sinensis Root
reference solution. DEFINITION
Detection c Treat with a 10 per cent VIV solution of sulfuric Angelica Sinensis Root is the dried whole root of Angelica
acid R in methanol R, heat at 100 cc for 5 min and examine sinensis (Oliv.) Diels. (Angelica polymorpha Maxim, var.
in daylight. sineทรis Oliv.). The dried root consists of the top (uppermost
Results c The chromatogram obtained with the test solution part), main body and small lateral roots (tails).
shows no zone corresponding to the zone due to It is collected in late autumn, removed from rootlets and
(Z)-ligustilide in the chromatogram obtained with the dried.
reference solution. It contains not less than 0.1% of Z-ligustilide (C12H1462)1
Loss on drying (2.2.52) calculated with reference to the dried material.
IDENTIFICATION
powdered herbal drug (355) (2.9. 72) by drying m an oven at
A. The whole root is yellowish-brown to brown, up to 25 cm
105 cc for 2 h. long, irregularly cylindrical with 3 to 5 or more branch roots
Total ash (2.4.16) arising from the lower end. The upper part is 1.5 to 5 cm in
Maximum 8.0 per cent. diameter, annulated on the surface and rounded at the apex
Ash insoluble in hydrochloric acid (2.8.1) which may show purple or yellowish-green remains of stems
Maximum 3.0 per cent. and leaves; the surface of the remainder of the main root is
2016 Angelica Root IV-67
strongly longitudinally wrinkled and has pale, transverse for 2 to 3 hours in 200 mL of water, collecting the distillate
lenticels; the branch roots are 0.3 to 1 cm in diameter in the in suitable glassware. Extract the oily drops on top of the
upper part, twisted and tapering towards the base, the outer distillate with 5 mL of toluene.
surface is strongly striated and has few rootlet scars. (2) 0.2% w/v each of coumarin and eugenol in toluene.
B. Reduce to a powder (355). The powder is pale yellowish (3) 0.1% w/v of Z-ligustilide CRS in acetonitrile.
to buff. Examine under a microscope using chloral hydrate
(4) 0.1% w/v of benzyl alcohol in toluene.
solution. The powder shows brown fragments of cork
composed of thin-walled cells; abundant thin-walled (5) 0.1% w/v of (-)-carvone in toluene.
parenchyma from the secondary cortex, phloem and (6) 0.1% w/v of octanoic acid in toluene.
medullary rays, some of the phloem cells fusiform with (7) 0.1% w/v of 3^propylidenephthalide in toluene.
slightly thickened walls; lignified vessels in groups of 2 or 3
CHROMATOGRAPHIC CONDITIONS
associated with small celled and pitted xylem parenchyma;
the vessels are up to 80 pm in diameter and have reticulate (a) Use a fused silica capillary column (50 m X 0.32 mm)
or scalariform thickening. Examine under a microscope using bonded with a film (1.05 pm) of 5% phenyl/95%
50% v/v of glycerol. The powder shows small groups of single dimethylpolysiloxane (HP 5 is suitable).
starch granules, spherical to ovoid, up to about 8 pm in (b) Use helium as the carrier gas at 1 mL per minute.
diameter. (c) Use an oven maintained at an initial temperature of 40°
c. Carry out the method for thin-layer chromatography, increasing linearly to 220c at a rate of 5° per minute, then
Appendix III A, using the following solutions. maintained at 220°.
(1) Add 4 mL of heptane to 1.0 g of the powdered drug, mix (d) Use a split injection system having a split ratio of
with the aid of ultrasound for 5 minutes and filter (use a 1:20 maintained at 250°.
0.22 pm membrane filter). (e) Use a flame ionisation detector maintained at a
(2) 0.1% w/v of linoleic acid in methanol. temperature of 250°.
(3) 0.1% w/v of ferulic acid in methanol. (f) Inject 1 pL of each solution.
(4) 0.1% w/v of TAigustilide CRS in methanol. (g) Record the chromatograms for a sufficient length of time
to elute all the peaks in the chromatogram obtained with
solution (1) (55 minutes may be suitable).
(a) Use a silica gel F254 precoated plate (Merck silica gel 60
SYSTEM SUITABILITY
F254 HPTLC plates are suitable).
(b) Use the mobile phase as described below.
with solution (2), the resolution between coumarin (eluting
(c) Apply 10 pL of solution (1) and 5 pL of solutions (2) to at approximately 34 minutes) and eugenol (eluting at
(4), as bands. approximately 37 minutes) is at least 3.0.
CONFIRMATION
(e) Remove the plate, allow to dry' in a stream of warm air
In the chromatogram obtained with solution (1):
for 5 minutes or until the solvents are completely removed.
— there are no peaks corresponding to the principal peaks in
Examine under ultraviolet light (254 nm). Spray the plate with
the chromatograms obtained with solutions (4), (5), (6)
methanolic sulfuric acid (5%), heat at 105° for 3 minutes and
and (7);
examine in daylight.
— there is a peak corresponding to the principal peak in the
MOBILE PHASE chromatogram obtained with solution (3).
1 volume of formic acid, 10 volumes of ethyl acetate and Loss on drying
90 volumes of toluene. When dried at 100° to 105° for 2 hours, loses not more than
SYSTEM SUITABILITY 12.0% of its weight. Use 1 g.
When examined under ultraviolet light (254 nm) the violet Total ash
band with an Rf value of approximately 0.7 the Not more than 7.0%, Appendix XI J, Method n.
chromatogram obtained with solution (4) corresponds in Acid-insoluble ash
colour and position to that in the chromatogram obtained Not more than 2.0%, Appendix XI K.
with solution (1). A band with an Rf value of approximately
Ethanol-soluble extractive
0.23 in the chromatogram obtained with solution (3)
Not less than 45%, Appendix XI Bl.
corresponds in position to a band in the chromatogram
obtained with solution (1). Other bands may be present in ASSAY
the chromatogram obtained with solution (1). Carry out the method for liquid chromatography,
Appendix in D, using the following solutions.
CONFIRMATION
(1) Finely powder not less than 5.0 g of the drug being
When sprayed with methanolic sidfuric acid (5%) the
examined. Transfer 0.5 g of the powder into a 25 mL
chromatogram obtained with solution (1) shows three spots
volumetric flask and add 20 mL of methanol, place in an
with similar Rf values to the spots in the chromatograms
ultrasonic bath (maintained at a low temperature by adding
obtained with solutions (2), (3) and (4). Other spots may be
ice to the bath) for 100 minutes, equilibrate to ambient
present in the chromatogram obtained with solution (1).
temperature and dilute to volume with methanoL Centrifuge
TESTS the solution at 5000 rpm for 5 minutes or until a clear
Lovage root (Levisticum officinale) supernatant is obtained. Filter through a 0.45-pm filter.
Carry out the method for gas chromatography, (2) 0.025% w/v of Z-ligustilide CRS in acetonitrile.
Appendix in B, using the following solutions.
(1) Extract approximately 20 g of the coarsely powdered
drug in a 500 mL round-bottomed flask by hydrodistillation
IV-68 Angelica Sinensis Root 2016
CHROMATOGRAPHIC CONDITIONS Content

(a) Use a stainless steel column (15 cm X 4.6 mm) packed Minimum 0.050 per cent of trans-ferulic acid (C10H10o4;
with octadecylsilyl silica gel for chromatography (5 pm) (Hypersil Mr 194.2) (dried drug).
ODS is suitable). IDENTIFICATION
(๖) Use isocradc elution and the mobile phase described A. Taproot branching rapidly into 10 or more conical
below. principal roots; the whole is about 15-25 cm long.
(c) Use a flow rate of 1.0 mL per minute. The annulated root crown is about 1.5-4 cm in diameter;
(d) Use a detection wavelength of 350 nm. its blunt, rounded tip shows the yellowish-green remains of
stems and petioles of leaves. The outer surface is light
(e) Inject 10 pL of each solution.
brownish-yellow or dark browท, lumpy, irregularly striated
MOBILE PHASE longitudinally and shows scars of secondary roots and
A mixture of 8 volumes of water and 12 volumes of transversal lenticel-like markings. The branching roots have a
acetonitrile. thick upper part (0.3-1 cm in diameter) and a thin lower
SYSTEM SUITABILITY part. They are frequently twisted and show few scars of
secondary roots. The texture is friable. The fracture,
Inject solution (2) not less than five times. The test is not
yellowish-white or yellowish-brown, shows a thick bark with
valid unless the relative standard deviation of the peak areas
some clefts and numerous brown dots due to secretory
of the Z-ligustilide peak is not more than 3.0%, the relative
canals. The cambium occurs as a yellowish-brown ring.
standard deviation of the retention times of the Z-ligustilide
The wood is light coloured.
peak is not more than 3.0%. The column efficiency,
determined on the Z-ligustilide peak, is not less than 5000 The fragmented roots occur as long strips about 1.5-2 mm
theoretical plates. The symmetry factor, determined on the thick, 1.5-4 cm wide at the root crown and 10-15 cm long.
Z-ligustilide peak, is not more than 1.3. B. Microscopic examination (2.8.23). The powder is
Inject solution (1). The test is not valid unless the resolution yellowish-white. Examine under a microscope using chloral
factor between the Z-ligustilide peak and the closest peak hydrate solution R. The powder shows the following diagnostic
(relative retention about 0.9 with respect to Z-ligustilide) is characters: reticulate or scalariform lignified vessels up to
not less than 1.5. 80 pm in diameter, free or in groups of 2 or 3 and
accompanied by ligneous parenchyma cells with thick walls;
DETERMINATION OF CONTENT numerous fragments of parenchyma with ovoid cells; orange
Using the retention time and the peak area from the cork fragments, consisting of several layers of superimposed
chromatograms obtained with solution (2), locate and cells, more or less rectangular in surface view; very small
integrate the peak due to Z-ligustilide in the chromatogram calcium oxalate prisms, visible in polarised light, in the cork;
obtained with solution (1). rare secretory canals, usually broken, with orange-yellow
Calculate the content of Z-ligustilide in the sample using the contents, up to 170 pm in diameter. Examine under a
declared content of Z-ligustilide (C12H14o2) in microscope using a 50 per cent v/v solution of glycerol R: the
Z-ligustilide CRS and the following expression: powder shows small (less than 10 pm), simple, rounded or
ovoid starch granules, usually included in parenchyma cells,
A. ๓2 V. 100 c. Examine the chromatograms obtained in the test for other
officinal species of Angelica, Levisticum and Ligusticum.
a/v/m.xpxioo-d
Results A See below the sequence of zones present in the
A1 = Area of the peak due to Z-ligustilide in the test solution. Furthermore, other faint fluorescent zones may
chromatogram obtained with solution (1). be present in the chromatogram obtained with the test
A2 = Area of the peak due to Z-ligustilide in the solution.
chromatogram obtained with solution (2).
m 1 = Weight of the drug being examined in mg.
Top of the plate
โท2 = Weight of Z-ligustilide CRS in mg.
V1 = Dilution volume of solution (1) in mL. (Z)-Ligustilideะ a bluish-white A prominent bluish-white
v2 = Dilution volume of solution (2) in mL. fluorescent zone fluorescent zone ((Z)-ligustilide)
p = Percentage content of c 12H1402 in Z-ligustilide CRS.

d = Percentage loss on drying of the herbal drug being Osthole: a blue fluorescent zone
examined.
Imperatorin ะ a whitish fluorescent
STORAGE
Angelica Sinensis Root should be protected from moisture.
Processed Angelica Sinensis Root *****

(Angelica Sinensis Root, Ph Eur monograph 2558) k chromatograms obtained with the reference solution and the
Ph Eur_________________________________________________________________
DEFINITION solution.
Smoke-dried, whole or fragmented root, with rootlets
removed, of Angelica sinensis (Oliv.) Diels collected in late
autumn.
2016 Aniseed IV-69
Top of the plate Reference solution (b) In order to prepare cis-ferulic acid in
(Z)-Ligustilide: a blue fluorescent A prominent blue fluorescent
situ, introduce 2 mL of reference solution (a) into a
zone zone ((Z)-ligustilide) transparent vial and expose to ultraviolet light at 254 nm for
A faint quenching zone about 60 min.
Column:
— size: z = 0.150 m, 0 = 2.0 mm;
Osthole: a quenching zone A faint quenching zone — stationary phase: octadecylsilyl silica gel for chromatography R
Imperatorin: a quenching zone (4 pm);
— temperature: 35 °C.
Mobile phase acetonitrile R, 0.085 per cent VIV solution of
phosphoric acid R (17:83 VIV).
Reference solution Test solution Flow rate 0.23 mUmin.
Injection 10 pL.
TESTS
Retention time tram-ferulic acid = about 13 min; cis-ferulic
Other officinal species of Angelica, Levisticum and acid = about 14 min.
Ligusticum
Test solution To 1 g of the powdered herbal drug (355) tram-ferulic acid and cis-ferulic acid.
(2.9.72) add 4 mL of heptane R, close and sonicate for 5 min.
Calculate the percentage content of tram-ferulic acid using
Centrifuge and use the supernatant.
the following expression:
Reference solution Dissolve 1 mg of (Z)-ligustilide R, 1 mg of
imperatorin R and 1 mg of osthole R in 10 mL of methanol R. Al X 7ท2 X p
Plate TLC silica gel F254 plate R (2-10 pm). A2 X 7711 X 5
Mobile phase glacial acetic acid R, ethyl acetate R,.toluene R
(1:10:90 VIVIV). A1 ะ= area of the peak due to tram-ferulic acid in the
Application 4 pL as bands of 8 mm. chromatogram obtained with the test solution;
A2 = area of the peak due to tram-ferulic acid in the
Development Over a path of 6 cm.
Drying In air. nil = mass of the herbal drug to be examined used to
Results A The chromatogram obtained with the test solution m2 = mass of ferulic acid CRS used to prepare reference
shows no intense blue fluorescent zone at or below the solution (a), in grams;
position of osthole in the chromatogram obtained with the p — percentage content of tram-ferulic acid in ferulic acid
reference solution. CRS.
Detection B Examine in ultraviolet light at 254 nm. _____________________________________________________________ Ph Eur
Results B The chromatogram obtained with the test solution

shows no quenching zone at or below the position of
imperatorin in the chromatogram obtained with the reference
solution.
Aniseed *****
Maximum 12.0 per cent, determined on 1.000 g of the Anise ***
powdered herbal drug (355) (2.9.72) by drying in an oven at (Ph. Eur. monograph 0262)
105 °C for 2 h. When Powdered Aniseed is prescribed or demanded,
Total ash (2.4.16) material complying with the requirements below, with the
Maximum 7.0 per cent. exception of Identification test A and the test for Foreign
Ash insoluble in hydrochloric acid (2.5.7) matter, shall be dispensed or supplied.
Maximum 2.0 per cent. Ph Elf----------------------------------------------------------------------------------------------- ----------
ASSAY DEFINITION
Liquid chromatography (2.2.29). Whole, dry cremocarp of Pimpinella anisum L.
Test solution Disperse 0.200 g of the powdered herbal drug Content
(355) (2.9.72) in 20.0 mL of a 70 per cent VIV solution of Minimum 20 mL/kg of essential oil (anhydrous drug).
methanol R in a conical flask, stopper tightly and weigh. Heat CHARACTERS
under a reflux condenser for 30 min, cool and weigh again.
Reminiscent odour of anethole.
Compensate the loss of solvent with a 70 per cent VIV
solution of methanol R, mix well and allow to stand. Filter the The fruit is a cremocarp and generally entire; a small
fragment of the thin, rigid, slightly curved pedicel is
supernatant through a membrane filter (nominal pore size
0.45 pm); use the filtrate. frequently attached.
Reference solution (fl) In a brown-glass volumetric flask, IDENTIFICATION
dissolve 10.0 mg of ferulic acid CRS in a 70 per cent P7I7 A. The cremocarp is ovoid or pyriform and slightly
solution of methanol R and dilute to 100.0 mL with the same compressed laterally, yellowish-green or greenish-grey,
solvent. 3-5 mm long and up to 3 mm wide, surmounted by a
stylopod with 2 short, reflexed stylar points. The mericarps
IV-70 Star Anise 2016
are attached by their tops to the carpophore with a plane Reference solution Dissolve 3 J1L of anethole R and 40 pL of
commissural surface and a convex dorsal surface, the latter olive oil 7? in 1 mL of toluene R.
being covered with short, warty trichomes visible using a Plate TLC silica gel GF254 plate R.
lens; each mericarp shows 5 primary ridges, running
Mobile phase toluene R.
longitudinally, comprising 3 dorsal ridges and 2 lateral ridges,
non-prominent, and lighter in colour. Application 2 pL and 3 jiL of the test solution, then 1 pL,
2 pL and 3 pL of the reference solution, at 2 cm intervals.
greenish-yellow or brownish-green. Examine under a Development Over a path of 10 cm.
microscope using chloral hydrate solution R. The powder Drying In air.
show's ±e following diagnostic characters (Figure 0262.-1): Detection A Examine in ultraviolet light at 254 nm.
fragments of epicarp in surface view [D] with a striated Results A The chromatograms show a quenching zone
cuticle, occasional anomocytic stomata (2.8.3) [Da], bases of (anethole) in the central part against a light background.
covering trichomes [De] and whole covering trichomes [Db],
Detection B Spray with a freshly prepared 200 g/L solution of
mostly unicellular, sometimes curved, with a blunt apex and
phosphomolybdic acid R in ethanol (96 per cent) R, using
a wart}' cuticle; isolated fragments of covering trichomes [E];
10 mL for a 200 mm square plate, and heat at 120 °C for
fragments [H] of numerous narrow, branched vittae [Ha],
5 min.
often accompanied by elongated cells of the commissural
surface [Hb]; fragments of testa [B] consisting of a layer of Results B The spots due to anethole appear blue against a
brown, polyhedral, thin-walled cells; fragments of endosperm yellow background. In the chromatogram obtained with 2 pL
[G] containing oil droplets [Ga], aleurone grains and small of the test solution, the spot due to anethole is intermediate
cluster crystals of calcium oxalate [Gb]; oblong sclereids from in size between the corresponding spots in the
the mesocarp [C] or the commissural surface of the fruit; chromatograms obtained with 1 pL and 3 pL of the reference
bundles of short sclerenchymatous fibres [A] from the solution. The chromatograms obtained with the test solution
carpophore and the pedicel [Ab], accompanied by vessels show in the lower third a blue spot (triglycerides) similar in
with spiral or annular thickening [Aa, F]. position to the spot in the lower third of the chromatograms
obtained with the reference solution (triglycerides of olive
oil).
TESTS
Water (2.2.13)
Maximum 70 mUkg, determined on 20.0 g of the powdered
herbal drug.
Total ash (2.4.16)
ASSAY
Use 10.0 g of the herbal drug reduced to a coarse powder
immediately before the determination, a 250 mL round-
bottomed flask, and 100 mL of water R as the distillation
liquid. Place 0.50 mL of xylene R in the graduated tube.
Distil at a rate of 2.5-3.5 mUmin for 2 h.
______________________________________________________________ Ph Eur
Star Anise *****

Preparation
Concentrated Anise Water
Ph Eur.----- -------------------------------------------------------------------------------- ------------------
DEFINITION
Dried composite fruit of Illicium verum Hook.f.
Figure 0262.-1. - Illustration for identification test B of powdered
Content
herbal drug of aniseed
— minimum 70 mL/kg of essential oil (anhydrous drug),
c. Thin-layer chromatography (2.2.27). — minimum 86.0 per cent of tram-anethole in the essential
Test solution Shake 0.10 g of the powdered herbal drug oil.
(1400) (2.9.12) with 2 mL of methylene chloride R for 15 min.
CHARACTERS
Filter and carefully evaporate the filtrate to dryness on a
The fruit carpels are brown.
water-bath at 60 °C. Dissolve the residue in 0.5 mL of
Odour of anethole.
toluene R.
2016 Star Anise IV-71
IDENTIFICATION ventrally turned hook; follicles with a profile fitting into a

A. The fruit generally consists of 8 developed, one-seeded rectangle; pedicels more than 5 cm long; seedless fruits; seeds
follicles, each 12-22 mm long and 6-12 mm high, radially either very flat or almost spherical.
arranged around a short, central, blunt-ending columella. B. Thin-layer chromatography (2.2.27).
In some fruits 1 or 2 follicles may be missing, but their Test solution To 2.0 g of the powdered herbal drug (355)
position is clearly visible. Each follicle is boat-shaped or boot (2.9.72) add 10 mL of methanol R and heat under a reflux
shaped, with a greyish-brown dorsal surface showing rough condenser in a water-bath at 60 °C for 5 min. Allow to cool
markings and lateral surfaces bearing scars from the and filter.
neighbouring follicles. One or more follicles are split open
along the ventral suture, exposing a single, lenticular, shiny, Reference solution Dissolve 1 mg of caffeic acid R, 1 mg of
reddish-brown seed about 8 mm in diameter. The markings chlorogenic acid R, 2.5 mg of quercitrin R, 2.5 mg of rutin R
and 2.5 mg of hyperoside R in 10 mL of methanol R.
on the dorsal surface are not visible from the ventral surface.
Some of the follicles (1-3) may be imperfectly developed. Plate TLC silica gel plate R (2-10 pm).
Isolated follicles, pedicels and seeds may be present. Mobile phase anhydrous formic acid R, glacial acetic acid R,
B. Reduce to a powder (355) (2.9. 72). The powder is water R, ethyl acetate R (11 ะ 11ะ 26:100 VIVIVIV).
reddish-brown. Examine under a microscope using chloral Application 5 pL as bands.
hydrate solution R. The powder shows the following diagnostic Development Over a path of 6 cm.
characters: brown epicarpal cells, polygonal in surface view, Drying In a current of warm air.
with a strongly striated cuticle and occasional anomocytic
Detection Spray with a 10 g/L solution of diphenylboric acid
stomata (2.5.5); fragments of the endocarp with long
aminoethyl ester R in methanol R and then with a 50 g/L
palisade-like cells; fragments of the mesocarp with large
solution of macrogol 400 R in methanol R; after 30 min,
parenchymatous cells, vessels, oil-containing cells and groups
examine in ultraviolet light at 365 nm.
of stone cells; fragments of the seed testa with palisade-like,
sclerified, strongly pitted, yellow cells up to 200 pm long; Results The chromatogram obtained with the test solution
fragments of the columella and the fruit stalk with strongly shows no brownish-yellow fluorescent zone at or above the
and irregularly thickened, star-shaped stone cells about position of the zone due to quercitrin in the chromatogram
400 pm long and 150 pm wide; rhomboidal or’rectangular obtained with the reference solution. No yellow fluorescent
crystals of calcium oxalate. zone is seen at or above the position of the zone due to
caffeic acid in the chromatogram obtained with the reference
c. Examine the chromatograms obtained in test B for Illicium
solution. No brownish-yellow fluorescent zone is seen directly
anisatum (= I. religiosum) and certain other Illicium spp.
above the zone due to hyperoside in the chromatogram
Results See below the sequence of the zones present in the obtained with the reference solution.
test solution. Furthermore, other weaker zones may be Water (2.2.13)
present in the chromatogram obtained with the test solution. Maximum 100 mL/kg, determined by distillation on 20.0 g
of the powdered herbal drug (355) (2.9.72).
Total ash (2.4.16)
Top of the plate
ASSAY
Caffeic acid: a light blue Essential oil (2.8.12)
fluorescent zone Use a 250 mL round-bottomed flask and 100 mL of water R
Quercitrin: a brownish-yellow as the distillation liquid. Immediately before the
fluorescent zone
determination, reduce 50.0 g of the drug to a coarse powder
A brownish-yellow fluorescent
(1400) (2.9.12) and mix. Further reduce about 10.0 g of this
mixture to a finer powder (710) (2.9.12). Use 2.50 g of the
powder for the determination. Introduce 0.50 mL of xylene R
A greenish fluorescent zone into the graduated tube. Distil at a rate of 2-3 mL/min for
Hyperoside: a brownish-yellow A brownish-yellow fluorescent 2 h.
fluorescent zone
trans-Anethole
Chlorogenic acid: a light blue Gas chromatography (2.2.25): use the normalisation
fluorescent zone
procedure.
A green fluorescent zone
Test solution Dilute the mixture of essential oil and xylene R
Rutin: a brownish-yellow A brownish-yellow fluorescent
obtained in the assay of essential oil to 5.0 mL with xylene R
fluorescent zone
by rinsing the apparatus.
Reference solution To 1.0 mL of xylene R add 20 pL of
estragole R3 20 mg of ซ.-terpineol R and 60 pL of anethole R.
TESTS Column'.
Illicium anisatum (=ะ I. religiosum) and certain other — material', fused silica;
Illicium spp — size: I = 30 m, 0 = 0.25 mm;
A. Adulteration with Illicium anisatum or certain other — stationary phase: macrogol 20 000 R.
Illicium spp. is indicated by the presence of fruits mainly Carrier gas helium for chromatography R.
consisting of more than 8 follicles; fruits either smaller than Flow rate 1.0 mL/min.
2.5 cm or greater than 3.5 cm; follicles with the suture edged
split ratio 1:100.
with a thickening extending to the neighbouring follicle, or
with dorsal markings visible from the ventral surface; follicles
somewhat undulate and ending in a fine beak or a small,
IV-72 Star Anise Oil 2016
Temperature: Top of the plate
Time Temperature A quenching zone, partly separated

(°C) Anethole: a quenching zone A very strong quenching zone
Column 0-5 60 (anethole)
5 - 80 60 -> 210
80 - 95 210 Anisaldehyde: a quenching zone A quenching zone (anisaldehyde)
Injection port 200
Detector 220 Reference solution Test solution

Detection B Spray with methyl 4-acetylbenzoate reagent R and
Injection 1 pL.
heat at 100-105 °C for 10 min; examine the still hot plate in
Elution order Order indicated in the preparation of the daylight within 10 min.
reference solution.
System suitability: reference solution: chromatograms obtained with the reference solution and the
— resolution: minimum วิ between the peaks due to estragole test solution. Furthermore, other zones may be present in the
and a-terpineol. chromatogram obtained with the test solution.
Use the retention times from the chromatogram obtained
with the reference solution locate the components of the
Top of the plate
reference solution in the chromatogram obtained with the
test solution. A violet-brown zone, not fully
separated
Calculate the percentage content of rrans-anethole. Disregard
Anethole: a brown zone A very strong brown zone
any peak due to the solvent or with an area less than (anethole)
0.05 per cent of the area of the principal peak in the
Anisaldehyde: a yellow zone A yellow zone (anisaldehyde)
_______________________________________________________________ Ph Eur
Linalol: a grey zone A grey zone (linalol)
Star Anise Oil * ** Reference solution Test solution

PhEir_______________________________________________________________
B. Examine the chromatograms obtained in the test for
chromatographic profile.
DEFINITION
Results The characteristic peaks in the chromatogram
Essential oil obtained by steam distillation from the dry ripe
obtained with the test solution are similar in retention time to
fruits of niicium verum Hook.f.
those in the chromatogram obtained with the reference
CHARACTERS solution.
Appearance TESTS
Clear, colourless or pale yellow liquid.
Relative density (2.2.5)
IDENTIFICATION 0.979 to 0.985.
First identification B Refractive index (2.2.6)
Second identification A 1.553 to 1.556.
A. Thin-layer chromatography (2.2.27). Freezing point (2.2.18)
Test solution Dissolve 1 g of the substance to be examined in 15 °C to 19 °C.
toluene R and dilute to 10 mL with the same solvent. Fenchone
Reference solution Dissolve 10 pL of linalol R, 30 pL of Gas chromatography (2.2.28) as described in the test for
anisaldehyde R and 200 pL of anethole R and in toluene R and chromatographic profile with the following modifications.
dilute to 15 mL with the same solvent. Dilute 1 mL of this Test solution Dissolve 400 pL of the substance to be examined
solution to 5 mL with toluene R. in 2.0 mL of hexane R.
Plate TLC silica gel F254 plate R- Reference solution (a) Dilute 10 pL of fenchone 7? to 1.2 g with
Mobile phase ethyl acetate R} toluene R (7:93 V!V). hexane R.
Application 5 pL as bands of 10 mm (for normal TLC plates) Reference solution (b) Dilute 100 pL of reference solution (a)
or 2 pL as bands of 10 mm (for fine particle TLC plates). to 100 mL with hexane R.
Development Over a path of 15 cm (for normal TLC plates) System suitability: reference solution (b):
or over a path of 6 cm (for fine particle size plates). — signal-to-noise ratio: minimum 10 for the principal peak.
Drying In air. Limit:
— fenchone: maximum 0.01 per cent.
Results A See below the sequence of zones present in the Pseudoisoeugenyl 2-methylbutyrate
chromatograms obtained with the reference solution and the Gas chromatography (2.2.28) as described in the test for
test solution. Furthermore, other zones may be present in the chromatographic profile with the following modifications.
chromatogram obtained with the test solution. Test solution The substance to be examined.
2016 Star Anise Oil IV-73
2 5
4
6
3
1 น__ JLllui
1. linalol 3. a-terpineol 5. /rans-anethole 7. foeniculin
2. estragole 4. ๘ร-anethole 6. anisaldehyde
Figure 2108.-1. - Chromatogram for the test for chromatographic profile of star anise oil
Reference solution (a) Dilute 10 mg of the test solution to Reference solution To 1.0 mL of hexane R, add 20 pL of
1.000 g with hexane R. Dilute 0.5 mL of this solution to linalol R3 20 pL of estragole R} 20 pL of a-terpineol R3 60 pL
100 mL with hexane R. of anethole R and 30 pL of anisaldehyde R.
Reference solution (b) Pseudoisoeugenyl 2-methylbutyrate for peak Column:
identification CRS. — material: fused silica,
System suitability: — size: I = 30 m, 0 = 0.25 mm,
— the chromatogram obtained with reference solution (b) is — stationary phase: macrogol 20 000 R (film thickness
similar to the chromatogram provided with 0.25 pm).
pseudoisoeugenyl 2-methylbutyrate for peak Carrier gas helium for chromatography R.
identification CRS. Flow rate 1.0 mUmin.
— signal-to-noise ratio: minimum 10 for the principal peak in Split ratio 1:100.
the chromatogram obtained with reference solution (a).
Temperature:
Limit Locate the peak due to pseudoisoeugenyl
2-methylbutyrate by comparison with the chromatogram
provided with pseudoisoeugenyl 2-methylbutyrate for peak Time Temperature
(min) CC)
identification CRS.
Column 0-5 60
— pseudoisoeugenyl 2-methylbutyrate: maximum 0.01 per cent.
5-80 60 -> 210
Fatty oils and resinified essential oils (2.5.7)
It complies with the test for fatty oils and resinified essential 80-95 210
oils. Injection port 200
Chromatographic profile Detector 220
procedure.
Test solution Dissolve 200 pL of the substance to be examined Detection Flame ionisation.
in 1.0 mL of hexane R. Injection 0.2 pL.
IV-74 Anise Oil 2016
Elution order Order indicated in the composition of the test solution. Furthermore, other zones may be present in the
reference solution; record the retention times of these chromatogram obtained with the test solution.
substances.
System suitability: reference solution:
Top of the plate
— resolution', minimum 1.5 between the peaks due to
estragole and a-terpineol. Anethole: a quenching zone A very strong quenching zone
(anethole)
Using the retention times determined from the
chromatogram obtained with the reference solution, locate
the components of the reference solution in the A quenching zone
chromatogram obtained with the test solution and locate Anisaldehyde: a quenching zone A quenching zone (anisaldehyde)
cfr-anetholc and foeniculin using the chromatogram shown in
Figure 2108.-1 (disregard any peak due to hexane).
Determine the percentage content of these components. Reference solution Test solution
The percentages are within the following ranges:
— linalol'. 0.2 per cent to 2.5 per cent,
— estragole: 0.5 per cent to 6.0 per cent, Detection B spray with methyl 4-acetylbenzoate reagent R and
— a-terpmeol: maximum 0.3 per cent, heat at 100-105 °C for 10 min; examine the still hot plate in
— cis-anethole: 0.1 per cent to 0.5 per cent, daylight within 5 min.
— trans-anethole: 86 per cent to 93 per cent, Results B See below the sequence of zones present in the
— anisaldehyde: 0.1 per cent to 0.5 per cent, chromatograms obtained with the reference solution and the
— foeniculin: 0.1 per cent to 3.0 per cent. test solution. Furthermore, other zones may be present in the
STORAGE chromatogram obtained with the test solution.
At a temperature not exceeding 25 °C.
_______________________________________________________________Ph Eur Top of the plate
A violet-brown zone (monoterpene

hydrocarbons) (solvent front)
Anethole: a brown zone A very strong brown zone
(anethole), distinctly separated
Anise Oil * *
Aniseed Oil *** A grey zone
(Ph. Eur. monograph 0804) Anisaldehyde: a yellow zone A yellow zone (anisaldehyde)
Preparation
Concentrated Anise Water
Linalol ะ a grey zone A grey zone (linalol)
Ph Ew_______________________________________________________________
A grey zone
DEFINITION
Essential oil obtained by steam distillation from the dry ripe Reference solution Test solution
fruits of Pimpinella anisum L.

CHARACTERS B. Examine the chromatograms obtained in the test for
Appearance chromatographic profile.
Clear, colourless or pale yellow liquid. Results The characteristic peaks in the chromatogram
IDENTIFICATION obtained with the test solution are similar in retention time to
First identification B those in the chromatogram obtained with the reference
Second identification A solution.
A. Thin-layer chromatography (2.2.27). TESTS
Test solution Dissolve 1 g of the substance to be examined in Relative density (2.2.5)
toluene R and dilute to 10 mL with the same solvent. 0.980 to 0.990.
Reference solution Dissolve 10 pL of linalol R, 30 pL of Refractive index (2.2.6)
anisaldehyde R and 200 pL of anethole R in toluene R and 1.552 to 1.561.
dilute to 15 mL with the same solvent. Dilute 1 mL of this Freezing point (2.2.18)
solution to 5 mL with toluene R. 15 °C to 19 °C.
Plate TLC silica gel F254 plate R- Fenchone
Mobile phase ethyl acetate R, toluene R (7:93 VIV). Gas chromatography (2.2.28) as described in the test for
Application 5 pL as bands of 10 mm (for normal TLC plates) chromatographic profile with the following modifications.
or 2 pL as bands of 10 mm (for fine particle size plates). Test solution Dissolve 400 pL of the substance to be examined
Development Over a path of 15 cm (for normal TLC plates) in 2.0 mL of hexane R.
or over a path of 6 cm (for fine particle size plates). Reference solution (a) Dilute 10 pL offenchone R to 1.2 g with
Drying In air. hexane R.
Detection A Examine in ultraviolet light at 254 nm. Reference solution (b) Dilute 100 pL of reference solution (a)
to 100 mL with hexane R.
chromatograms obtained with the reference solution and the System suitability: reference solution (b):
— signal-to-noise ratio: minimum 10 for the principal peak.
1. linalol 3. a-terpineol 5. irans-anethole 7. pseudoisoeugenyl 2-methylbutyrate
2. estragole 4. cis-anethole 6. anisaldehyde
Figure 0804.-1. - Chromatogram for the test for chromatographic profile of anise oil
Limit'. Column'.
— fenchone', maximum 0.01 per cent. — material', fused silica,
Foeniculin — size'. / = 30 m, 0 = 0.25 mm,
Gas chromatography (2.2.25) as described in the test for — stationary phase: macrogol 20 000 R (film thickness
chromatographic profile with the following modifications. 0.25 pm).
Test solution The substance to be examined. Carrier gas helium for chromatography R.
Reference solution (a) Dilute 10 mg of the test solution to Flow rate 1.0 mL/min.
1.000 g with hexane R. Dilute 0.5 mL of this solution to Split ratio 1:100.
100 mL with hexane R. Temperature:
Reference solution (b) Foeniculin for peak identification CRS.
System suitability'. Time Temperature
— the chromatogram obtained with reference solution (b) is (min) (°C)
similar to the chromatogram provided with foeniculin for Column 0-5 60
peak identification CRS, 60 -4 210
5-80
— signal-to-noise ratio', minimum 10 for the principal peak in
the chromatogram obtained with reference solution (a). 80-95 210
Limit Locate the peak due to foeniculin by comparison with Injection port 200
the chromatogram provided with foeniculin for peak Detector 220
identification CRS.
— foeniculin'. maximum 0.01 per cent.
Fatty oils and resinified essential oils (2.5.7) Detection Flame ionisation.
It complies with the test for fatty oils and resinified essential Injection 0.2 pL.
oils. Elution order Order indicated in the composition of the
Chromatographic profile reference solution. Record the retention times of these
Gas chromatography (2.2.25): use the normalisation substances.
procedure. System suitability: reference solution:
Test solution Dissolve 200 pL of the substance to be examined — resolution: minimum 1.5 between the peaks due to
in 1.0 mL of hexane R. estragole and a-terpineol.
Reference solution To 1.0 mL of hexane R, add 20 j.lL of Using the retention times determined from the
linalol R} 20 pL of estragole R, 20 pL of a-terpineol R, 60 pL chromatogram obtained with the reference solution, locate
of anethole R and 30 pL of anisaldehyde R. the components of the reference solution in the
IV-76 Anise Preparations 2016
chromatogram obtained with the test solution and locate about 8-10 mm long, are green with yellowish-green external
cis-anethole and pseudoisoeugenyl 2-methylbutyrate using the hairs visible under a lens. The receptacle, about 6 mm in
chromatogram shown in Figure 0804.-1 (disregard any peak diameter, is convex, alveolate and covered with hairs.
due to hexane). Its periphery bears about 20 ligulate florets 20-30 mm long;
Determine the percentage content of these components. the disc bears a greater number of tubular florets about
The percentages are within the following ranges: 15 mm long. The ovary, 4-8 mm long, is crowned by a
— linalol: maximum 1.5 per cent, pappus of whitish bristles 4-8 mm long. Some brown
— estragole. 0.5 per cent to 5.0 per cent, achenes, crowned or not by a pappus, may be present.
— a-terpineol: maximum 1.2 per cent, IDENTIFICATION
— cis-anethole: 0.1 per cent to 0.4 per cent, A. The involucre consists of elongated oval bracts with acute
— trans-anethole: 87 per cent to 94 per cent, apices; the margin is ciliated. The ligulate floret has a
— anisaldehyde: 0.1 per cent to 1.4 per cent, reduced calyx crowned by fine, shiny, whitish bristles,
— pseudoisoeugenyl 2-methylbutyrate. 0.3 per cent to bearing small coarse trichomes. The orange-yellow corolla
2.0 per cent. bears 7-10 parallel veins and ends in 3 small lobes.
STORAGE The stamens, with free anthers, are incompletely developed.
At a temperature not exceeding 25 °C. The narrow, brown ovary bears a stigma divided into
2 branches curving outwards. The tubular floret is
________________________________________________________________ Ph Eur
actinomorphic. The ovary and the calyx are similar to those
of the ligulate floret. The short corolla has 5 reflexed
triangular lobes; the 5 fertile stamens are fused at the
anthers.
Concentrated Anise Water B. Microscopic examination (2.8.23). Separate the capitulum
DEFINITION into its different parts. Examine under a microscope using
Anise Oil or Star Anise Oil 20 mL chloral hydrate solution R. The powder shows the following
Ethanol (90 per cent) 700 mL diagnostic characters (Figure 1391.-1): the epidermises of the
Water Sufficient to produce 1000 mL bracts of the involucre [L, M, o, Q] have stomata [Lb, Oa,
Qa] and trichomes, more abundant on the outer (abaxial)
Extemporaneous preparation surface. There are several different types of trichomes:
The following directions apply. uniseriate multicellular covering trichomes, varying in length
Dissolve the Anise Oil or Star Anise Oil in the Ethanol from 50-500 pm, particularly abundant on the margins of the
(90 per cent) and add gradually, with vigorous shaking after bract, whole [La] or fragmented [P]; secretory trichomes with
each addition, sufficient Water to produce 1000 mL. uni- or biseriate multicellular stalks and with multicellular,
Add 50 g of previously sterilised Purified Talc, or other globular heads, about 300 pm long, abundant on the outer
suitable filtering aid, allow to stand for a few hours, shaking surface of the bract [Qb]; secretory trichomes with
occasionally, and filter. multicellular stalks and with multicellular, globular heads,
The water complies with the requirements stated under Aromatic about 80 pm long, abundant on the inner surface of the
Waters and with the following requirements. bract, in surface view [Ob] or in side view [Ma].
The epidermis of the ligulate corolla [C, G, H, J] consists of
TESTS lobed or elongated cells covered by a striated cuticle [Ga], a
Ethanol content few stomata and trichomes of different types: covering
60 to 64% v/v, Appendix vni F. trichomes, with very sharp ends, whose length may exceed
Weight per mL 500 pm, consisting of 1-3 proximal, thick-walled cells and
0.898 to 0.908 g, Appendix V G. 2-4 distal, thin-walled cells [C, Hb]; secretory trichomes with
biseriate multicellular heads in surface view [Gb] or in side
view [Ja]; secretory trichomes with multicellular stalks and
multicellular globular heads [K]. The ligule ends in rounded
papillose cells [Ha]. Fragments of the epidermis of the
Arnica Flower * * ovary [A, B, D] are covered with trichomes of 2 types:
(Ph. Eur. monograph 1391) * ** secretory trichomes with short stalks and multicellular
globular heads, in surface view [Aa] or in side view [Da];
Preparation twinned covering trichomes usually consisting of
Arnica Tincture 2 longitudinally united cells, with common pitted walls, in
Ph Eur -------- -______________ surface view [Ab] or in side view [Ba]; their ends are sharp
DEFINITION and sometimes bifid. The epidermises of the calyx consist of
Whole or partially broken, dried flower-heads of Arnica elongated cells bearing short, unicellular, covering trichomes
montana L. pointing towards the upper end of the bristle [E]. The pollen
grains have a diameter of about 30 pm, are rounded, with a
Content spiny exine, and have 3 germinal pores [F, N].
Minimum 0.40 per cent mlm of total sesquiterpene lactones,
expressed as dihydrohelenalin tiglate (dried drug).
c. Examine the chromatograms obtained in the test for
Calendula officinalis L. - Heterotheca inuloides Cass.
CHARACTERS Results The chromatogram obtained with the test solution
Aromatic odour. shows, in the middle, a fluorescent blue zone corresponding
The capitulum, when spread out, is about 20 mm in to the zone due to chlorogenic acid in the chromatogram
diameter and about 15 mm deep, and has a peduncle 2-3 cm obtained with the reference solution; it shows, above this
long. The involucre consists of 18-24 elongated lanceolate
bracts, with acute apices, arranged in 1-2 rows: the bracts,
2016 Arnica Flower IV-77
zone, 3 fluorescent yellowish-brown or orange-yellow zones,

and above these 3 zones a fluorescent greenish-yellow zone
due to astragalin; the zone located below the astragalin zone
is due to isoquercitroside; the zone located just below this
zone is due to luteolin-7-glucoside; it also shows a fluorescent
greenish-blue zone below the zone due to caffeic acid in the
TESTS
Calendula officinalis L. - Heterotheca inuloides Cass
(2.9J2) add 10 mL of methanol R. Heat in a water-bath at
60 °C for 5 min with shaking. Cool and filter.
Reference solution Dissolve 2.0 mg of caffeic acid R, 2.0 mg of
chlorogenic acid R and 5.0 mg of rutin R in methanol R and
dilute to 30 mL with ±e same solvent.
Mobile phase anhydrous formic acid R, water R3 methyl ethyl
ketone R, ethyl acetate R (10:10:30:50 VIVIVIV).
Application 15 pL as bands.
Drying In air for a few minutes.
Detection spray with a 10 g/L solution of diphenylboric acid
aminoethyl ester R in methanol R, and then with a 50 g/L
solution of macrogol 400 R in methanol R’3 heat at 100-105 °C
for 5 min, allow to dry in air and examine in ultraviolet light
at 365 nm.
Results The chromatogram obtained with the reference
solution shows in the lower part an orange-yellow fluorescent
zone due to rutin, in the middle part a fluorescent zone due
to chlorogenic acid and in the upper part a light bluish
fluorescent zone due to caffeic acid; the chromatogram
obtained with the test solution does not show a fluorescent
orange-yellow zone corresponding to the zone due to rutin in
the chromatogram obtained with the reference solution, nor
does it show a zone below this.
105 °C for 2 h.
Total ash {2.4.16)
ASSAY
Internal standard solution Dissolve immediately before use
0.010 g of santonin CRS, accurately weighed, in 10.0 mL of
methanol R.
Test solution Introduce 1.00 g of the powdered herbal drug
(355) {2.9.12) into a 250 mL round-bottomed flask, add
50 mL of a mixture of equal volumes of methanol R and
water R and heat under a reflux condenser in a water-bath at
50-60 °C for 30 min, shaking frequently. Allow to cool and
filter through a paper filter. Add the paper filter, cut into
pieces, to the residue in the round-bottomed flask, add
50 mL of a mixture of equal volumes of methanol R and
Figure 1391.-1. - Illustration for identification test B of powdered water R and heat under a reflux condenser in a water-bath at
herbal drug of arnica flower 50-60 °C for 30 min, shaking frequently. Repeat this
procedure twice. To the combined filtrates add 3.00 mL of
the internal standard solution and evaporate to 18 mL under
reduced pressure. Rinse the round-bottomed flask with
IV-78 Arnica Preparations 2016
water R and dilute, with the washings, to 20.0 mL. Transfer

the solution to a chromatography column about 0.15 m long Arnica Tincture
and about 30 mm in internal diameter containing 15 g of (Ph. Eur. monograph 1809) *
kieselguhr for chromatography R. Allow to stand for 20 min.
Ph Eur______________________________________________________________
Elute with 200 mL of a mixture of equal volumes of ethyl
acetate R and methylene chloride R. Evaporate ±e eluate to DEFINITION
dryness in a 250 mL round-bottomed flask. Dissolve the Tincture produced from Arnica flower (1391).
residue in 10.0 mL of methanol R and add 10.0 mL of Content
water R. Add 7.0 g of neutral aluminium oxide R, shake for Minimum 0.04 per cent of sesquiterpene lactones expressed
120 ร, centrifuge at 5000 g for 10 min and filter through a as dihydrohelenalin tiglate (€20H26(ว5; Mr 346.42).
paper filter. Evaporate 10.0 mL of the filtrate to dryness.
Dissolve the residue in 3.0 mL of a mixture of equal volumes PRODUCTION
of methanol R and water R and filter. The tincture is produced from the herbal drug by a suitable
procedure using 10 parts of ethanol (60-70 per cent K/l7) for
Column:
1 part of drug.
— size: I = 0.12 m, 0 = 4 mm;
— stationary phase: octadecylsilyl silica gel for chromatography R CHARACTERS
(4 pm). Appearance
Mobile phase: Yellowish-brown liquid.
— mobile phase A: water R) IDENTIFICATION
— mobile phase B: methanol R'} Examine the chromatograms obtained in the test for
Calendula officinalis - Heterotheca inuloides.
Time Mobile phase A Mobile phase B Chromatogram obtained with the test solution:
(min)(per cent V/V)(per cent V/V) — in the middle, a fluorescent blue zone corresponding to
0 -3 62 38 the zone due to chlorogenic acid in the chromatogram
3 - 20 62 -> 55 obtained with the reference solution;
— above this zone, 3 fluorescent yellowish-brown to orange
20 - 30 55
yellow zones, and above these 3 zones a fluorescent
30 - 55 55 -> 45 greenish-yellow zone corresponding to astragalin; the zone
55 -> 100 located below the astragalin zone corresponds to
isoquercitrin; the zone located just below this zone
57 - 70 0 100
corresponds to luteolin-7-glucoside;
70 - 90 62 38 — a fluorescent greenish-blue zone below the zone due to
caffeic acid in the chromatogram obtained with the
reference solution.
TESTS
Calendula officinalis - Heterotheca inuloides
Injection 20 pL loop injector. Thin-layer chromatography (2.2.27).
Calculate the percentage content of total sesquiterpene Test solution The tincture to be examined.
lactones, expressed as dihydrohelenalin tiglate, using the
Reference solution Dissolve 2.0 mg of caffeic acid R, 2.0 mg of
chlorogenic acid R and 5.0 mg of nai'ท R in methanol R and
dilute to 30.0 mL with the same solvent.
5ls X c X V X 1.187 X 100
Ss X m X 1000 plate R (2-10 pm)].
Mobile phase anhydrous formic acid R, water R, methyl ethyl
5ls = area of all peaks due to sesquiterpene lactones ketone R, ethyl acetate R (10:10:30:50 VIVIVIV).
appearing after the santonin peak in the
Application 30 pL [or 8 pL] as bands.
1ร,s = area of the peak due to santonin in the Development Over a path of 15 cm [or 8 cm].
chromatogram obtained with the test solution; Drying At 80-105 °C.
m = mass of the herbal drug to be examined, in Detection Spray the plate whilst still hot with a 10 g/L
grams; solution of diphenylboric acid aminoethyl ester R in methanol R
c = concentration of santonin in the internal standard and then with a 50 g/L solution of macrogol 400 R in
solution used for the test solution, in milligrams methanol R’i heat 5 min at 100-105 °C, allow the plate to dry
per millilitre; in air and examine in ultraviolet light at 365 nm.
V = volume of the internal standard solution used for Results The chromatogram obtained with the reference
the test solution, in millilitres; solution shows in the lower part an orange-yellow fluorescent
1.187 ะ= peak correlation factor between dihydrohelenalin zone (rutin), in the middle part a fluorescent zone due to
tiglate and santonin. chlorogenic acid and in the upper part a light bluish
________ ______________________________________ Ph Eur fluorescent zone (caffeic acid). The chromatogram obtained
with the test solution does not show any fluorescent orange
yellow zone corresponding to rutin in the chromatogram
obtained with the reference solution and no zone below the
zone corresponding to rutin.
2016 Artichoke Leaf IV-79
Ethanol {2.9.10) m = mass of the tincture to be examined, in grams;

The final ethanol concentration is not less than 90 per cent c = concentration of santonin in the internal standard
of that of the initial extraction solvent. solution used to prepare the test solution, in
Methanol and 2-propanol {2.9.11) milligrams per millilitre;
Maximum 0.05 per cent P7E of me±anol and maximum V = volume of the internal standard solution used to
0.05 per cent v/v of 2-propanol. prepare the test solution, in millilitres;
1.187 = peak correlation factor between dihydrohelenalin
Dry residue {2.8.16)
tiglate and santonin.
_____________________________________________________________ Ph Eur
ASSAY
Liquid chromatography {2.2.29).
Internal standard solution Dissolve immediately before use
0.010 g accurately weighed of santonin CRS and 0.02 g of
butyl 4-hydroxybenzoate R in 10.0 mL of methanol R. Artichoke Leaf * *
Test solution In a round-bottomed flask introduce 5.00 g of (Ph. Eur. monograph 1866) ***
the tincture to be examined, add 2.00 mL of the internal
standard solution and 3 g of anhydrous aluminium oxide R-, Preparation
shake for 120 ร and filter through a filter paper. Rinse the Artichoke Leaf Dry Extract
round-bottomed flask and filter with 5 mL of a mixture of Ph Eur______________________________________________________________
equal volumes of methanol R and water R and filter. DEFINITION
Evaporate the filtrate to dryness. Dissolve the residue in Whole or cut, dried leaf of Cynara carduncidus L.
2.0 mL of a mixture of 20 volumes of water R and
(syn. c. scolymus L.).
80 volumes of methanol R and filter through a membrane
filter (nominal pore size 0.45 pm). Content
Minimum 0.7 per cent of chlorogenic acid (C16H18O9;
Reference solution Dissolve 0.02 g of methyl
Mr 354.3) (dried drug).
4-hydroxybenzoate R and 0.02 g of ethyl 4-hydroxybenzoate R
in methanol R and dilute to 10.0 mL with the same solvent. IDENTIFICATION
Column'. A. The entire leaf may be up to 70 cm long and 30 cm wide.
— size'. / = 0.12 m, 0 = 4 mm; The lamina is deeply lobed in the upper part to within
— stationary phase', end-capped octadecylsilyl silica gel for 1-2 cm of the petiole on either side, in the lower part the leaf
chromatography R (5 pm);
— temperature'. 20 °C.
Mobile phase’.
— mobile phase A', water R;
— mobile phase B: methanol R;

0-3 62 38
3 - 20 62 -> 55 38 -> 45
20 - 30 55 45
30 - 55 55 -» 45 45 -> 55
Flow rate 1.2 mUmin.

Detector spectrophotometer at 225 nm.
Injection 20 pL.
Relative retention With reference to santonin (retention
time = about 9.5 min): butyl 4-hydroxybenzoate = about
4.6.
System suitability, reference solution:
— resolution', minimum 5 between the peaks due to methyl
4-hydroxybenzoate and ethyl 4-hydroxybenzoate.
Calculate the percentage of lactone sesquiterpenes, expressed
as dihydrohelenalin tiglate, using the following expression:
Fl X c X V X 1.187
F2 X m X 10
Fl ะะ=area of all peaks appearing between the peaks due

to santonin and butyl 4-hydroxybenzoate in the
F2 = area of the peak due to santonin in the Figure 1866.-1. - Illustration for identification test B ofpowdered
chromatogram obtained with the test solution; herbal drug of artichoke leaf
IV-80 Artichoke Leaf 2016
becomes pinnate; all the segments have markedly dentate Test solution To 2.0 g of the powdered herbal drug (1000)
margins and taper at the apex. Spines are absent. The upper (2.9.12) add 20 mL of ethanol (60 per cent V/V) R. Allow to
surface of the lamina is green with a fine covering of whitish stand for 2 h with occasional stirring. Filter.
hairs, the lower surface is pale green or white and densely Reference solution Dissolve 5 mg of luteolin-7-glucoside R and
tomentose with long, tangled hairs. The petiole and main 5 mg of chlorogenic acid CRS in methanol R and dilute to
veins are flat on the upper surface, prominently raised and 10 mL with the same solvent.
longitudinally ridged on the lower surface, with conspicuous
hairs on both surfaces. plate R (2-10 pm)].
B. Reduce to a powder (1000) (2.9.12). The powder is Mobile phase anhydrous formic acid R) glacial acetic acid R3
greenish-grey. Examine under a microscope using chloral water R> ethyl acetate R (11:11:27:100 VIVIVIV).
characters (Figure 1866.-1): fragments of the epidermises of Application 10 pL [or 2 pL] as bands of 10 mm [or 8 mm].
the lamina, in surface view; the upper epidermis [F] is Development Over a path of 13 cm [or 6 cm].
composed of cells with straight or slightly sinuous walls [Fa], Drying In air.
accompanied by palisade parenchyma [Fb]; the lower Detection Heat at 100 °C for 5 min; treat the warm plate with
epidermis [C] is composed of more sinuous-walled cells; a 10 g/L solution of diphenylboric acid aminoethyl ester R in
abundant anomocytic stomata (2.8.3) on both surfaces [D] methanol R followed by a 50 g/L solution of macrogol 400 R
and multicellular, uniseriate covering trichomes in felted in methanol R’i examine in ultraviolet light at 365 nm.
masses, the majority fragmented [Ca] with a short stalk Results See below the sequence of fluorescent zones present
composed of several cells and a very long, narrow and in the chromatograms obtained with the reference solution
frequently curled terminal cell, others consisting of 4-6 and the test solution. Furthermore, other fluorescent zones
cylindrical cells; very occasional glandular trichomes with a may be present in the chromatogram obtained with the test
short stalk and a uniseriate or biseriate head (surface view solution.
[E], transverse section [Ba]); abundant fragments of covering
trichomes [G]; fragments of the lamina (transverse
section [B]); abundant fragments of vascular tissue from the
petiole and veins [A].
2016 Artichoke Leaf Preparations IV-81
Top of the plate m\ = mass of the herbal drug to be examined in the test
solution, in grams;
m2 = mass of chlorogenic acid CRS in the reference
solution, in grams;
Luteolin-7-glucoside: a yellow or A yellow or orange fluorescent p = percentage content of chlorogenic acid in chlorogenic
orange fluorescent zone zone (luteolin-7-glucoside) acid CRS.
Chlorogenic acid: a light blue A light blue fluorescent zone
fluorescent zone (chlorogenic acid)
TESTS
Artichoke Leaf Dry Extract * *
Total ash (2.4.16) (Ph. Eur. monograph 2389) ***
Maximum 20.0 per cent. Ph Eur_____________________________________________________________
Loss on drying (2.2.32) DEFINITION
Maximum 12.0 per cent, determined on 1.000 g of the Dry extract produced from Artichoke leaf (1866).
105 °C for 2 h. Content
Minimum 0.6 per cent of chlorogenic acid (Cl6H18O9;
ASSAY
Mr 354.3) (dried extract).
Test solution To 0.500 g of the powdered herbal drug (1000) PRODUCTION
(2.9.12) add 50.0 mL of methanol R and heat under a reflux The extract is produced from the herbal drug by a suitable
condenser on a water-bath at 70 °C for 1 h. Centrifuge and procedure using water of minimum 80 °C.
transfer the supernatant to a 200 mL volumetric flask. CHARACTERS
Repeat the procedure and dilute to 200.0 mL with water R. Appearance
Reference solution Dissolve 5.0 mg of chlorogenic acid CRS in Light brown or brown, amorphous powder.
50.0 mL of methanol R. Transfer 5.0 mL of this solution to a IDENTIFICATION
volumetric flask, add 5 mL of methanol R and dilute to Thin-layer chromatography (2.2.27).
20.0 mL with water R.
Test solution Dissolve 1.0 g of the extract to be examined in
Column'.
10 mL of ethanol (60 per cent V/V) R. Sonicate for 5 min and
— size'. I = 0.25 m, 0 = 4.6 mm;
filter.
— stationary phase', end-capped octadecylsilyl silica gel for
chromatography R (5 pm); Reference solution Dissolve 5 mg of luteolin-7-glucoside R and
— temperature: 40 °C. 5 mg of chlorogenic acid R in 10 mL of methanol R.
Mobile phase: Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
— mobile phase A: phosphoric acid R, water R (0.5:99.5 V/V)'3 plate R (2-10 pm)].
— mobile phase B: phosphoric acid R, acetonitrile R Mobile phase anhydrous formic acid R, glacial acetic acid Rj
(0.5:99.5 1Z/P); water Rj ethyl acetate R (11:11:27:100 V/V/V/V).
(min)(per cent V/V)(per cent V/V)
0-1 92 8 Drying In air.
1 - 20 92 -> 75 8 -> 25 Detection Heat at 100 °C for 5 min; spray the warm plate
with a 10 g/L solution of diphenylboric acid aminoethyl ester R
20 - 33 75 25
in methanol R followed by a 50 g/L solution of macrogol
33 - 35 75 -» 0 25 -> 100 400 R in methanol Rj examine in ultraviolet light at 365 nm.
Results See below the sequence of fluorescent zones present
Flow rate 1.2 mUmin. in the chromatograms obtained with the reference solution
Detection Spectrophotometer at 330 nm. and the test solution. Furthermore, other fluorescent zones
Injection 25 pL. may be present in the chromatogram obtained with the test
System suitability Test solution: solution.
— the chromatogram obtained is similar to the
chromatogram shown in Figure 1866.-2; Top of le plate
— resolution: minimum 2.0 between the peak due to A light blue fluorescent zone
chi orogenic acid and the subsequent peak (peak 2).
Calculate the percentage content of chlorogenic acid using
Luteolin-7-glucoside: a yellow or A yellow or orange fluorescent zone
the following expression: orange fluorescent zone (luteolm-7-glucoside)
Al X 7ท2 X p
Chlorogenic acid ะ a light blue A light bine fluorescent zone
A2 X mi
(chlorogenic acid)
fluorescent zone
A1 = area of the peak due to chlorogenic acid in the

chromatogram obtained with the test solution; Reference solution Test solution
chromatogram obtained with the reference solution;
IV-82 Ash Leaf 2016
TESTS ทไ2 = mass of chlorogenic acid CRS used to prepare

Loss on drying (2.8.17) reference solution (a), in milligrams;
Maximum 6.0 per cent. p = percentage content of chlorogenic acid in
Total ash (2.4.16) chlorogenic acid CRS.
Maximum 30.0 per cent. ____ ______ ____________________________________________________ Ph Eur
ASSAY
Solvent mixture methanol R3 water R (30:70 V/V).
Test solution Dissolve 30.0 mg of the extract to be examined Ash Leaf * *
in the solvent mixture and dilute to 25.0 mL with the solvent (Ph. Eur. monograph 1600) ***
mixture.
Ph Eur______________________________________________________________
Reference solution (a) Dissolve 5.0 mg of chlorogenic acid CRS
in 50.0 mL of methanol R. Transfer 5.0 mL of this solution DEFINITION
to a volumetric flask, add 5 mL of methanol R and dilute to Dried leaf of Fraxinus excelsior L. or Fraxinus angustifolia Vahl
20.0 mL with water R. (syn. Fraxinus oxyphylla M. Bieb) or of hybrids of these
Reference solution (b) Dissolve 30 mg of the artichoke leaf diy 2 species or of a mixture.
extract HRS in the solvent mixture and dilute to 25.0 mL Content
with the solvent mixture. Minimum 2.5 per cent of total hydroxycinnamic acid
Column-. derivatives, expressed as chlorogenic acid (C10H18O9;
— size'. I = 0.25 m, 0 = 4.6 mm; Mt 354.3) (dried drug).
— stationary phase', octadecylsilyl silica gel for chromatography R IDENTIFICATION
(5 gm); A. The leaf consists of leaflets that are sometimes detached
— temperature-. 40 °C. and separated from the rachis. The leaflet is about 6 cm long
Mobile phase’. and 3 cm wide. Each leaflet is subsessile or shortly petiolate,
— mobile phase A: phosphoric acid R3 water R (0.5:99.5 VIV)-3 oblong, lanceolate, somewhat unequal at the base, acuminate
— mobile phase B: phosphoric acid R, acetonitrile R at the apex, with fine, acute teeth on the margins; the upper
(0.5:99.5 VIV)'3 surface is dark green and the lower surface is greyish-green.
The midrib and secondary veins are whitish and prominent
on the lower surface.
(min)(per cent V/V)(per cent V/V) B. Microscopic examination (2.8.23). The powder is greyish-
0-1 92 8 green. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic
1 - 20 92 -> 75 8 -> 25
characters (Figure 1600.-1): fragments of the upper
20 - 33 75 25 epidermis of the lamina in surface view [B], with some of the
33 - 35 75 0 25 -> 100 cells showing cuticular striations, accompanied by underlying
palisade parenchyma [Ba]; fragments of the lower epidermis
in surface view [A] consisting of cells covered by fine
Flow rate 1.2 mUmin. cuticular striations [Aa], numerous anomocytic stomata
Detection Spectrophotometer at 330 nm. (2.8.3) [Ab] and rare peltate glandular trichomes with a
Injection 25 |1L. unicellular stalk and a glandular head composed of radiating
cells [Ac]; fragments of lamina in transverse section [F] with
2 layers of palisade parenchyma [Fa], spongy
— peak-to-valley ratio: minimum 2.5, where Hp = height
parenchyma [Fb] and, occasionally, glandular trichomes
above the baseline of the peak immediately after the peak
embedded in the epidermis [Fc]; occasional multicellular,
due to chlorogenic acid and Hv = height above the
uniseriate, conical covering trichomes composed of cells with
baseline of the lowest point of the curve separating this
thick striated walls, either on an epidermis [C] or
peak from the peak due to chlorogenic acid;
fragmented [D]; fragments of vascular tissue from the
leaflets [E] composed of spiral vessels [Ea], short fibres [Eb]
chromatogram supplied with the artichoke leaf dry
and sometimes palisade parenchyma [Ec]; fragments of
extract HRS. vascular tissue from the veins [G] composed of fibres [Ga],
Calculate the percentage content of chlorogenic acid using sometimes accompanied by cells with thick, pitted walls from
the following expression: the meddiary rays [Gb].
Al X 7ท2 X p X 0.125 Fraxinus omus.
A2 X 7ท1 Results See below the sequence of zones present in the
A} = area of the peak due to chlorogenic acid in the test solution. The intensity of the zones present in the
chromatogram obtained with the test solution; chromatogram obtained with the test solution may vary
A2 = area of the peak due to chlorogenic acid in the depending on the presence of F. excelsior3 F. angustifolia, then-
chromatogram obtained with reference solution hybrids or their concentration in a mixture. Furthermore,
(a); other fluorescent zones may be present in the chromatogram
พ1 = mass of the extract to be examined used to obtained with the test solution.
prepare the test solution, in milligrams;
2016 Astragalus Mongholicus Root IV-83
Top of the plate Drying In air.

Detection Heat at 100 °C for 3 min; treat the still-warm plate
(acteoside)
in methanol R-, dry in air; treat with a 50 g/L solution of
Chlorogenic acid: a light blue A light blue fluorescent zone may
macrogol 400 R in methanol R3 dry in air; examine in
fluorescent zone be present (chlorogenic acid) ultraviolet light at 365 nm.
Results The chromatogram obtained with the test solution
does not show any intense light blue fluorescent zones in the
upper third of the chromatogram.
Rutin: an orange fluorescent zone An orange fluorescent zone
(rutin)
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Test solution (a) To 0.300 g of the powdered herbal drug
(355) (2.9.12) add 95 mL of ethanol (50 per cent V/V) R. Boil
in a water-bath under a reflux condenser for 30 min. Allow
to cool and filter. Rinse the filter with 5 mL of ethanol
(50 per cent V/V) R. Combine the filtrate and the rinsings in
a volumetric flask and dilute to 100.0 mL with ethanol
(50 per cent V/V) R.
Test solution (b) To 1.0 mL of test solution (a) in a test tube,
add 2 mL of 0.5 M hydrochloric acid3 2 mL of a solution
prepared by dissolving 10 g of sodium nitrite R and 10 g of
sodium molybdate R in 100 mL of water R, then add 2 mL of
dilute sodium hydroxide solution R and dilute to 10.0 mL with
water R3 mix.
Immediately measure the absorbance (2.2.25) of test
solution (b) at 525 nm, using as compensation liquid a
solution prepared as follows: mix 1.0 mL of test solution (a),
2 mL of 0.5 M hydrochloric acid3 2 mL of dilute sodium
hydroxide solution R and dilute to 10.0 mL with water R.
Calculate the percentage content of total hydroxycinnamic
acid derivatives, expressed as chlorogenic acid, using the
A X 5.3
m
A = absorbance at 525 nm;

m = mass of the herbal drug to be examined, in grams.
______________________________________________________________ Ph Eur
herbal drug of ash leaf
TESTS
Maximum 3.0 per cent of stems and maximum 2.0 per cent
Astragalus Mongholicus Root * *
of other foreign matter. (Ph. Eur. monograph 2435) ***
Fraxinus omus Ph Elf______________________________________ ________________________
DEFINITION
Test solution To 1 g of the powdered herbal drug (355) Whole, dried root of 2Astragalus mongholicus var. mongholicus
(2.9.12) add 20 mL of methanol R. Stir with a magnetic (syn. Astragalus membranaceus Bunge var. mongholicus (Bunge)
stirrer for 10 min. Filter. P.K. Hsiao) and Astragalus mongholicus var. dahuricus (DC.)
Reference solution Dissolve 5 mg of rutin R and 5 mg of Podlech (syn. Astragalus membranaceus Bunge), freed from
chlorogenic acid R in 10 mL of methanol R. rootlets and rootstock, collected from spring to autumn.
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel Content
plate R (2-พ pm)]. Minimum 0.040 per cent of astragaloside IV (C41H68O14;
Mobile phase anhydrous formic acid R3 water R, ethyl acetate R Mr 785) (dried drug).
(10:10:80 V/V/V).
IDENTIFICATION
Application 10 pL [or 4 pL] as bands of 10 mm [or 8 mm]. A. Cylindrical, often with branches, upper part relatively
Development Over a path of 10 cm [or 6 cm]. thick, 30-90 cm long and 1-3.5 cm in diameter. Externally
IV-84 Astragalus Mongholicus Root 2016
pale brownish-yellow or pale brown, with irregular, Detection B Treat with anisaldehyde solution R. Heat at 100 °C
longitudinal wrinkles or furrows. Texture hard and tenacious; for 3 min. Examine in ultraviolet light at 366 nm.
uneasily broken, fracture highly fibrous and weakly Results B See below the sequence of zones present in the
(cultivated origin) or strongly starchy (wiId origin), bark chromatograms obtained with the reference solution and the
yellowish-white, wood pale yellow, with radiate striations and test solution. Furthermore, other faint zones may be present
fissures; the central region is dark brown and in older roots in the chromatogram obtained with the test solution.
may be broken down to form a hollow surrounded by
fragments of disintegrating tissue.
Top of the plate
A violet zone
yellowish-white. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic Daidzein: a pale blue zone
characters: fibres, in bundles or scattered, 8-30 pm in
A violet zone
diameter, thick-walled with longitudinal fissures on the
surface, the primary’ walls often separated from the secondary
walls, both ends often broken or tassel-like, or slightly A violet zone
truncated; colourless or orange vessels with closely arranged
bordered pits; cork fragments consisting of several layers, Daidzin: a pale blue zone A brown zone
often accompanied by collenchymatous phelloderm; stone 5 brown zones
cells occasionally visible, rounded, oblong or irregular,
slightly thick-walled. Examine under a microscope using a
50 per cent VIV solution of glycerol R: ±e powder shows Reference solution Test solution
small, rounded or ovoid starch granules, usually simple or
sometimes 2- or 3-compound, about 5 pm in diameter.
TESTS
c. Thin-layer chromatography (2.2.27). Foreign matter (2.5.2)
Test solution Heat 3 g of the powdered herbal drug (355) Maximum 5 per cent.
(2.9.72) with 50 mL of methanol R for 50 min under reflux Loss on drying (2.2.52)
and then filter. Evaporate the filtrate under reduced pressure
to dryness and take up the residue in 1 mL of water R. Apply powdered herbal drug (355) (2.9J2) by drying in an oven at
the solution to a 6 mL solid phase extraction column 105 °C for 3 h.
containing octadecylsilyl silica gel for chromatography R
previously conditioned with 3 mL of methanol R and then Total ash (2.4.16)
with 3 mL of water R. Wash the column with 15 mL of Maximum 5.0 per cent.
water R followed by 15 mL of a 30 per cent VIV solution of Ash insoluble in hydrochloric acid (2.5./)
methanol R. Discard the washings. Elute with 20 mL of Maximum 1.0 per cent.
methanol R and collect the eluate. Evaporate the eluate under ASSAY
reduced pressure to dryness and take up the residue with
2 mL of methanol R.
Test solution Weigh 4.0 g of the powdered herbal drug (355)
Reference solution Dissolve 10.0 mg of daidzin R and 5.0 mg
(2.9.72) into a Soxhlet type extractor and add 40 mL of
of daidzein R in 5.0 mL of methanol R.
methanol R. Macerate overnight. Add again 40 mL of
Plate TLC silica gel 7*254 plate R (2-10 pm). methanol R. Heat under a reflux condenser for 4 h. Evaporate
Mobile phase water R, methanol R} ethyl acetate R to dryness. Dissolve the residue in 10 mL of water R, heating
(10:13.5:100 VIV/V). slightly if necessary. Shake with 4 quantities, each of 40 mL,
Application 3 pL as bands of 8 mm. of butanol R saturated with water R. Combine the butanol
Development Over a path of 7 cm. extracts and wash with 2 quantities, each of 40 mL, of
ammonia R. Discard the ammonia layers and evaporate the
Drying In air. butanol layers to dryness. Dissolve the residue in 5 mL of
Detection A Examine in ultraviolet light at 254 nm. water R and cool. Apply the solution to a solid phase
Results A See below the sequence of zones present in the extraction column containing 1 g of octadecylsilyl silica gel for
chromatograms obtained with the reference solution and the chromatography R previously washed with 5 mL of methanol R
test solution. Furthermore, other faint zones may be present and 5 mL of water R. Wash the column with 20 mL of
in the chromatogram obtained with the test solution. water R and 20 mL of ethanol (25 per cent VIV) R. Elute with
25 mL of ethanol (70 per cent VIV) R. Evaporate the eluate to
dryness. Dissolve the residue in 5.0 mL of methanol R.
Top of the plate
Reference solution (a) Dissolve 10.0 mg of
A blue fluorescent zone astragaloside IV CRS in methanol R and dilute to 10.0 mL
Daidzeinโ a quenching zone with the same solvent.
A quenching zone
Reference solutions (b)3 (c)3 (d) Dilute reference solution (a) to
obtain 3 reference solutions of astragaloside IV, the
concentrations of which span the expected value in the test
A quenching zone solution.
Reference solution (e) Dissolve 5.0 mg of ginsenoside Rbl R in
Daidzin ะ a quenching zone A quenching zone
5 mL of methanol R and dilute to 10.0 mL with reference
solution (a).
Reference solution Test solution Column'.
— size'. I ะะะ 0.25 m, 0 ะ= 3.2 mm;
2016 Atractylodes Lancea Rhizome IV-85
stationary phase', octadecylsilyl silica gel for chromatography R Content

(3 pm); Minimum 14 mL/kg of essential oil (anhydrous drug).
IDENTIFICATION
Mobile phase:
A. The whole rhizome is curved, irregular, nodular and
— mobile phase A: water Rj
cylindrical, 3-10 cm long and 1-3 cm in diameter.
— mobile phase B: acetonitrile Rj
The external surface is transversally wrinkled, dark greyish-
Time Mobile phase A Mobile phase B brown or yellowish-brown; it shows numerous rounded
(min) (per cent V/V) (per cent V/V) protuberances and large circular stem scars and smaller root
0-5 90 10 scars.
5 - 10 90 -> 80 10-» 20 The fragmented rhizome occurs in slices with a highly
variable diameter (1-4 cm) and a thickness of about 0.5 cm.
10 - 20 80 -> 75 20 -» 25 The external surface is wrinkled, dark greyish-brown or
20 - 30 75 -> 67 25 -» 33 yellowish-brown and shows numerous scars. The transverse
30 - 40 67 -» 65 33-» 35
section is pale yellow or brownish-yellow, consisting of
fibrous tissues scattered with particularly abundant orange oil
40 - 50 65 -> 40 35 -» 60 cavities appearing as dots.
50 - 55 40 60 B. Microscopic examination (2.8.23). The powder is
brownish-yellow. Examine under a microscope using chloral
Flow rate 0.5 mUmin. hydrate solution R. The powder shows the following diagnostic
Detection Evaporative light-scattering detector; the following characters: fragments of orange cork, with polyhedral cells,
settings have been found to be suitable; if the detector has often accompanied by subrectangular or ovoid sclereids with
different setting parameters, adjust the detector settings so as very thick channelled walls from the phelloderm; isolated,
to comply with the system suitability criterion: ovoid or subrectangular sclereids with very thick, channelled
— carrier gas: air; walls and a narrow lumen, variable in shape (20-80 pm in
— flow rate: 1.5 mUmin; diameter); fragments of parenchyma with polyhedral or
— evaporator temperature: 50 °C. subrectangular cells containing small needle-shaped crystals
Injection 20 pL of the test solution and reference of calcium oxalate (5-30 pm) clearly visible in polarised light;
solutions (b), (c), (d) and (e). fragments of fibres in bundles, with heavily thickened and
slightly pitted walls (40 pm in diameter) and a narrow
Relative retention With reference to ginsenoside Rbl
lumen, very often associated with xylem vessels; fragments of
(retention time = about 33.6 min):
short, reticulate or pitted vessels, usually included in
astragaloside IV = about 1.05.
parenchyma with thin-walled cells; fragments of oil glands
System suitability: with thin-walled cells and granular orange-brown contents
— resolution: minimum 4.0 between the peaks due to and orange oil droplets. Examine under a microscope,
astragaloside rv and ginsenoside Rbl in the without heating, using glycerol R: the powder shows pieces of
chromatogram obtained with reference solution (e). inulin, free or included in parenchyma cells.
Establish a calibration curve with the logarithm of the c. Examine the chromatograms obtained in the test for
concentration (mg/mL) of reference solutions (b), (c) and (d) Atractylodes macrocephala.
(corrected by the declared percentage content of
astragaloside IV CRS) as the abscissa and the logarithm of the
corresponding peak area as the ordinate. Calculate the
percentage content of astragaloside IV using the following
expression:
10A X 0.5
Top of t le plate
m
P-Caryophyllene: a pink zone A pink or violet zone
A = logarithm of the concentration corresponding to the
An orange zone may be present
astragaloside rv peak in the chromatogram
obtained with ±e test solution, determined from
the calibration curve; An intense greyish-green zone
m ะ= mass of the herbal drug to be examined used to
prepare the test solution, in grams. A very faint violet zone
_______________________________________________________ _______ Ph Eur
Bornyl acetate: a brown zone
Several violet zones
Atractylodes Lancea Rhizome * Reference solution Test solution
(Ph Eur monograph 2559)

PhEir_________________— TESTS
Atractylodes macrocephala
DEFINITION
Dried, whole or fragmented rhizome of Atractylodes
lancea (Thunb.) DC. (syn. Atractylodes chinensis (Bunge) Test solution Introduce 0.5 g of the powdered herbal drug
Koidz.) with the roots removed, collected in spring and (355) (2.9.12) into a centrifuge tube, add 2 mL of
autumn.
IV-86 Atractylodes Rhizome, Largehead 2016
methanol R and stopper the tube. Sonicate at 25 °C for hydrate solution R. The powder shows the following diagnostic
15 min and centrifuge. characters: fragments of orange cork, with polyhedral cells;
Reference solution Dissolve 10 mg of P-caiyophyllene R and fragments of parenchyma with polyhedral or subrectangular
10 mg of bornyl acetate R in 5 mL of methanol R. cells, many of which contain small needle-shaped crystals of
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel calcium oxalate (10-32 pm) clearly visible in polarised light;
sclereids, isolated or in small groups, with very thick,
plate R (2-10 pm)].
channelled walls, variable in shape (35-65 pm in diameter);
Mobile phase ethyl acetate R} heptane R (5:95 VIV).
fragments of fibres, isolated or in bundles, with moderately
Application 5 pL [or 3 pL] as bands of 10 mm [or 6 mm]. thickened and slightly pined walls (40 pm in diameter);
Development In an unsaturated tank, over a path of 10 cm [or fragments of short, reticulate or pitted vessels, usually
6 cm]. included in parenchyma with thin-walled cells; fragments of
Drying In air. oil glands with thin-walled cells and granular orange-brown
Detection Treat with anisaldehyde solution R and heat at contents. Examine under a microscope, without heating,
105-110 ๐c for 5-10 min; examine in daylight. using glycerol R', the powder shows numerous pieces of inulin,
free or included in parenchyma cells.
shows an intense greyish-green zone in the middle third. c. Examine the chromatograms obtained in the test for
Atractylodes lancea.
In the case of a substitution by Atractylodes macrocephala, no
intense greyish-green zone is present in the middle third. Results See below the sequence of zones present in the
Water (2.2.75)
Maximum 100 mL/kg, determined on 20.0 g of the
powdered herbal drug (355) (2.9.12).
Total ash (2.4.76)
Maximum 7.0 per cent. Top of the plate
Ash insoluble in hydrochloric acid (2.8.1) 0-Caryophyllene: a pink zone A pink or violet zone
Maximum 1.0 per cent. An orange zone
ASSAY
A very faint violet zone
Use 15.0 g of freshly powdered herbal drug (710) (2.9.12)5 a
500 mL round-bottomed flask, 200 mL of water R as the
distillation liquid and 0.50 mL of xylene R in the graduated
Bornyl acetate: a brown zone
tube. Distil at a rate of 2-3 mL/min for 2 h.
A very faint violet zone
Ph Eur
Several faint violet zones
Largehead Atractylodes Rhizome * * D. To 0.5 g of the powdered herbal drug (355) (2.9.12) add
(Ph Eur monograph 2560) * ** 5 mL of ethanol (96 per cent) R, heat in a water-bath at 60 °C
Ph Eur_______________________________________________________________
for 2 min and filter. To 1 mL of the filtrate add 0.25 mL of
a solution freshly prepared as follows: dissolve 5 mg of
DEFINITION vanillin R in 0.5 mL of ethanol (96 per cent) R} to this
Dried, whole or fragmented rhizome of Atractylodes solution add 0.5 mL of water R and 3 mL of hydrochloric
macrocephala Koidz. with the roots removed, collected in acid R. Shake immediately; a red or reddish-purple colour
winter when the lower leaves of the plant turn yellow and the develops and persists.
upper leaves become fragile.
TESTS
Content Atractylodes lancea
Minimum 9 mUkg of essential oil (anhydrous drug). Thin-layer chromatography (2.2.27).
IDENTIFICATION Test solution Introduce 0.5 g of the powdered herbal drug
A. The whole rhizome is irregularly shaped, 3-13 cm long (355) (2.9.12) into a centrifuge tube, add 2 mL of
and 1.5-7 cm in diameter. Externally yellowish-grey or dark methanol R and stopper the tube. Sonicate at 25 °C for
brown, with small knob-like protrusions, interrupted 15 min and centrifuge.
longitudinal wrinkles and grooves. Reference solution Dissolve 10 mg of fi-caryophyllene R and
The fragmented rhizome occurs in slices with a highly 10 mg of bornyl acetate R in 5 mL of methanol R.
variable diameter (1-7 cm) and a thickness of about 0.5 cm. Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
The external surface is wrinkled or grooved, more or less plate R (2-10 pm)].
dark yellowish-brown with numerous root scars.
Mobile phase ethyl acetate R) heptane R (5:95 VIV).
The transverse section is pale yellow, consisting of tissues
with wide spaces between them and scattered with many Application 5 pL [or 3 pL] as bands of 10 mm [or 6 mm].
orange oil cavities appearing as dots that are particularly Development In an unsaturated tank, over a path of 10 cm [or
abundant in the external tissues. 6 cm].
The fracture is hard and fibrous. Drying In air.
B. Microscopic examination (2.8.23). The powder is Detection Treat with anisaldehyde solution R and heat at
browiush-yellow. Examine under a microscope using chloral 105-110 °C for 5-10 min; examine in daylight.
2016 Azadirachta Indica Leaf IV-87
Results The chromatogram obtained with the test solution CHROMATOGRAPHIC CONDITIONS
shows no greyish-green zone in the middle third, above the (a) Use a silica gel 60 or high-performance silica gel 60
very faint violet zone. precoated plate [Merck silica gel 60 HPTLC plates are
Water (2.2.13) suitable].
Maximum 100 mL/kg, determined on 20.0 g of the (b) Use the mobile phase as described below.
powdered herbal drug (710) (2.9.12). (c) Apply as bands 5 pL of each solution.
Total ash (2.4.16) (d) Develop the plate to 15 cm [or 7 cm].
(e) After removal of the plate, spray with vanillin reagent, heat
Ash insoluble in hydrochloric acid (2.8.1) the plate at 100° for 3 minutes and examine in daylight.
MOBILE PHASE
ASSAY 3 volumes of hexane and 7 volumes of ethyl acetate.
SYSTEM SUITABILITY
Use 15.0 g of freshly powdered herbal drug (710) (2.9.12), a
500 mL round-bottomed flask, 200 mL of water R as the The test is not valid unless the chromatogram obtained with
distillation liquid and 0.50 mL of xylene R in the graduated solution (2) shows three clearly separated spots.
tube. Distil at a rate of 2-3 mUmin for 2 h. CONFIRMATION
---------- —------------------- ------------------------------------------------------------------------Ph Eur In the chromatogram obtained with solution (1), a black
band with an Rf value of approximately 0.15 corresponding
in position to a brown band in the chromatogram for
solution (2) is obtained. A black band with an Rf value of
approximately 0.3 corresponding in position to the indigo
Azadirachta Indica Leaf band in the chromatogram for solution (2) is obtained for
Nimba Leaf salannin. An indigo band with an Rf value of approximately
DEFINITION 0.6 corresponding in position to a purple band in the
chromatogram for solution (2) is obtained for P-sitosterol.
Azadirachta Indica Leaf is the dried leaf of Azadirachta indica
A. Juss.
It contains not less than 1.0% of tetranortriterpinoids, Top of the plate
expressed as salannin, calculated with reference to the dried
drug.
IDENTIFICATION
A. Leaflets thin and fragile, ovate to lanceolate, 3 to 10 cm Purple band P-sitosterol: a purple
long and 1 to 2.5 cm wide, curved with a serrate margin; band
base markedly asymmetrical, apex acuminate and terminating
in a fine point; upper surface dark brownish-green, lower
surface paler with distinct midrib and lateral veins running to
the margins; both surfaces glabrous. Fragments of the rachis
may be present; these are pale brown, slender, up to about
Black band Salannin: an indigo
10 cm long, cylindrical with faint longitudinal striations and
band
bearing alternating pairs of scars where the leaflets were
attached.
B. Reduce to a powder (355). The powder is green. Examine Black band Azadirachtin: a brown
under a microscope using chloral hydrate solution. The powder band
shows fragments of the epidermis composed of thin-walled Solution (1) Solution (2)
tangentially elongated cells with abundant anomocytic
stomata, Appendix XI H; abundant fragments of single
layered palisade and thin-walled parenchymatous cells of the
spongy mesophyll present, some with associated vessels; some TESTS
fragments display rosette crystals of calcium oxalate often in Foreign matter
rows. Not more than 2%, Appendix XI D.
c. Carry out the method for thin-layer chromatography, Loss on drying
Appendix in A, using the following solutions. When dried for 2 hours at 105°, loses not more than 10.0%
(1) Add 30 mL of methanol to approximately 5 g of of its weight. Use 1 g.
powdered herbal drug, mix thoroughly by hand and with the
Ash
aid of ultrasound for 30 minutes. Centrifuge at 3000 rpm for
Not more than 10.0%, Appendix XI J, method n.
5 minutes and collect the clear supernatant liquid. Repeat the
extraction twice, combine the supernatant liquid and dilute Water-soluble extractive
to 100 mL with methanol. Filter approximately 30 mL of the Not less than 20.0%, Appendix XI B2.
solution through a 0.45-pm filter and use the filtrate. ASSAY
(2) 0.025% w/v each of azadirachtin, salannin CRS and Carry out the method for liquid chromatography.
P-sitosterol in methanol. Appendix in D, using the following solutions.
(1) Add 30 mL of methanol to approximately 5 g of
powdered herbal drug, mix thoroughly by hand and with the
aid of ultrasound for 30 minutes. Centrifuge at 3000 rpm for
IV-88 Bacopa Monnieri 2016
5 minutes and collect the clear supernatant liquid. Repeat the V] = dilution volume of solution (1),
extraction twice, combine the supernatant liquid and dilute V2 = dilution volume of solution (2),
to 100 mL with methanol. Filter approximately 30 mL of the p = percentage content of salannin in salannin CRS,
solution through a 0.45-pm filter and use the filtrate. d = percentage loss on drying of the herbal drug being
(2) 0.0025% w/v of salannin CRS and 0.001% w/v of examined.
จ&adirachtin-A CRS in methanol.
STORAGE
CHROMATOGRAPHIC CONDITIONS Azadirachta Indica Leaf should be protected from moisture.
(a) Use a stainless steel column (15 cm X 2.1 mm) packed
with octadecylsilyl silica gel for chromatography (5 pm)
(Spherisorb ODS1 is suitable).
(b) Use gradient elution and the mobile phase described
below.
Bacopa Monnieri
(c) Use a flow rate of 0.5 mL per minute. DEFINITION
Bacopa Monnieri is the dried aerial parts of Bacopa monnieri
(d) Use a column temperature of 30°.
(L.) Wettst.
(e) Use a detection wavelength of 217 nm.
It contains not less than 1.0% พ/พ of bacopa saponins,
(f) Inject 10 pL of each solution. expressed as bacopaside II (047แ7608), calculated with
When the chromatograms are recorded under the prescribed reference to the dried drug.
conditions the retention time of the peak due to azadirachtin-
A is about 15 minutes and the retention time of the peak due
IDENTIFICATION
to salannin is about 22 minutes. A. Pieces of herb, consisting mainly of stem and leaf; buff or
greenish brown, angular stems, 1 to 2 mm in diameter and
MOBILE PHASE 10 to 30 cm long, nodes prominent, often showing sprouting
Mobile phase A 0.1 volume of trifluroacetic acid and rootlets and with numerous ascending branches; greenish
100 volumes of water. leaves sessile or short petioled, fleshy, glabrous on the upper
Mobile phase B 0.1 volume of trifluroacetic acid and surface, simple, opposite, decussate, 0.6 to 2.5 cm long and
100 volumes of acetonitrile. 3 to 8 mm wide, reniform, spathulate or oblanceolate,
margin entire or, rarely, dentate. If present, flowers are
axillary and solitary, on peduncles usually longer than the
Time Mobile phase A Mobile phase B Comment
leaves; corolla up to 1 cm long, five lobed, oblong, obtuse;
(Minutes) (% v/v) (% v/v)
fruit capsule ovoid-acuminate or slightly beaked at the apex,
0-10 90->70 10->30 linear gradient glabrous, up to 5 mm long.
10-25 70->30 30->70 linear gradient B. Reduce to a powder (355). The powder is greenish or
25-40 30 70 isocratic yellowish-brown. Examine under a microscope using chloral
40-45 30->90 70->10 linear gradient hydrate solution. The powder shows fragments of the
epidermis, with a thin striated cuticle, multicellular glandular
45-50 90 10 re-equilibration
trichomes, anomocytic stomata; numerous xylem vessels with
reticulate thickening. Examine under a microscope using
SYSTEM SUITABILITY 50% v/v of glycerol in water. Starch granules are present,
The test is not valid unless, in the chromatogram obtained usually simple, round or ovoid, 4 to 14 pm in diameter,
with solution (2): without a visible hilum.
the symmetry factor of the peak due to azadirachtin-A is at c. Carry out the method for thin-layer chromatography,
most 1.2; Appendix in A, using the following solutions.
the symmetry factor of the peak due to salannin is at most 1.4. (1) Add 30 mL of methanol (70%) to 5.0 g of the powdered
herbal drug, heat on a water-bath under reflux for
DETERMINATION OF CONTENT
30 minutes, cool, centrifuge at 3000 rpm for 5 minutes and
Calculate the total content of tetranortriterpinoids, expressed decant the supernatant liquid. Repeat the extraction
as salannin, from the sum of the areas of the peaks eluting procedure with a further two 30-mL quantities of methanol
from three minutes before to three minutes after the Combine the supernatant liquid, dilute to 100 mL
retention time of salannin and from the declared content of with methanol (70%) and filter (0.45 pm PTFE is suitable).
salannin in salannin CRS using the following expression: (2) 0.05% w/v of bacopaside II CRS in methanol (70%).
(3) 0.05% w/v each of bacopaside I CRS and bacopaside
A] m2 V] 100 11 CRS in methanol (70%).
A?x m, x p x 100 -d CHROMATOGRAPHIC CONDITIONS
(a) Use silica gel 60 precoated plates or high-performance
silica gel 60 (Merck silica gel 60 plates are suitable).
A] = combined areas of the peaks in the chromatogram (b) Use the mobile phase as described below.
obtained with solution (1) with retention times from
(c) Apply 20 pL [or 10 pL] of each solution, as bands.
three minutes before to three minutes after the
retention time of peak due to salannin in the (d) Develop the plate to 15 cm [or 8 cm].
chromatogram obtained with solution (2), (e) After removal of the plate, dip in anisaldehyde solution Rl)
A2 = area of the peak due to salannin in the chromatogram heat in an oven at 105° for 5 minutes and examine in
obtained with solution (2), daylight.
m 1 = weight of the drug being examined in mg,
m2 == weight of salannin CRS in mg,
2016 Barbary Wolfberry Fruit FV-89
MOBILE PHASE
(d) Use a column temperature of 30°.
10 volumes of 1 % v/v of formic acid, 20 volumes of methanol (e) Use a detection wavelength of 205 nm.
and 70 volumes of ethyl acetate.
(f) Inject 20 pL of each solution.
SYSTEM SUITABILITY (g) Record the chromatograms for 75 minutes.
The test is not valid unless the chromatogram obtained with
MOBILE PHASE
solution (3) shows two clearly separated bands.
315 volumes of acetonitrile and 685 volumes of 0.71% w/v
CONFIRMATION anhydrous sodium sulfate, previously adjusted to pH 2.3 with
The chromatogram obtained with solution (1) shows a dark sulfuric acid.
band with an Rf value of approximately 0.4 corresponding to When the chromatograms are recorded under the prescribed
the band obtained with bacopaside II in the chromatogram conditions the retention time of bacopaside II is about
obtained with solution (3), a lighter band with an Rf value of 36 minutes. The retention times relative to bacopaside n are:
approximately 0.3 corresponding to the band obtained with bacoside A3, about 0.9; bacopaside X, about 1.2;
bacopaside I in the chromatogram obtained with solution (3). bacopasaponin c, about 1.3; bacopaside I, about 1.4.
Bands with Rf values of approximately 0.2 and 0.8 are also
SYSTEM SUITABILITY
present. Other bands may be present in solution (1).
Top of the plate to bacoside A3 and bacopaside n is at least 1.5 and the
resolution factor between the peaks due to bacopaside X and
bacopasaponin c is at least 2.4.
unknown: dark band
Calculate the total content of bacopa saponins (bacoside A3,
bacopaside II, bacopaside X, bacopasaponin c and
bacopaside I), expressed as bacopaside II from the
chromatograms obtained, and using the declared content of
bacopaside II: dark band bacopaside II: dark band bacopaside II: dark band
bacopaside II in bacopaside II CRS.
bacopaside 1: light band bacopaside I: light band
unknown: light band
Barbary Wolfberry Fruit * *

Solution (1) Solution (2) Solution (3) (Ph. Eur. monograph 2612) ***
Ph Eur_____________________________________________________________
TESTS DEFINITION
Foreign matter Dried, whole, ripe fruit of Lycium barbarum L.
Not more than 1.0%, Appendix XI D. IDENTIFICATION
Loss on drying A. The berry is elliptical, fusiform or ovoid and frequently
Not more than 11.0%, Appendix IX D. Use 1 g. flattened, about 6-20 mm long and 3-10 mm in diameter.
Ash The apex of the fruit shows a ring-shaped scar of the nectar
Not more than 13.0%, Appendix XI J, Method II. bearing base of the style and the base of the fruit bears the
whitish to light brown remnants of the cut stalk. The external
Water-soluble extractive surface is orange-red or dark red. The pericarp is fleshy,
Not less than 15.0%, Appendix XI B2. wrinkled, soft and viscous. It contains 20-50 hard, flat,
ASSAY subreniform seeds, bent upwards. Each pale yellow or
Carry out the method for liquid chromatography, yellowish-brown seed is about 1.7 mm long and 1.5 mm
Appendix III D, using the following solutions. wide.
(1) Reduce to a powder (355). To 5.0 g of the powder, add B. Microscopic examination {2.8.23). The powder is orange-
30 mL of methanol (70%) and heat under reflux for red or reddish-brown. Examine under a microscope using
30 minutes. Allow to cool, centrifuge and collect the chloral hydrate solution R. The powder shows the following
supernatant liquid. Repeat the extraction twice with two diagnostic characters: fragments of the epicarp with polygonal
further 30-mL quantities of methanol (70%). Combine the or elongated cells, about 60 pm in diameter, with straight or
three supernatant liquids, dilute to 100 mL with methanol slightly wavy walls, covered with a thick cuticle, with distinct,
(70%) and filter (0.45 pm P ILE is suitable). more or less parallel striations; fragments of the mesocarp
(2) 0.05% w/v of bacopaside II CRS in methanol (70%). with thin-walled subpolygonal cells containing reddish-orange
or brownish-red spherical granules and microsphenoidal
(3) 0.05% w/v of bacoside A CRS in methanol (70%).
crystals of calcium oxalate; fragments of the seeds, with the
CHROMATOGRAPHIC CONDITIONS testa consisting of greenish-yellow sclereids with thin external
(a) Use a stainless steel column (25 cm X 4.6 mm) packed walls and deeply lobed, irregularly thickened, striated, radial
with end-capped octadecylsilyl silica gel for chromatography and internal walls; fragments of endosperm containing oil
(5 pm) (Phenomenex Luna C18 is suitable). droplets; fragments of vascular tissue with narrow, spiral or
(b) Use isocratic elution and the mobile phase described annular vessels.
below. c. Thin-layer chromatography (2.2.27).
(c) Use a flow rate of 1.0 mL per minute.
IV-90 Holy Basil Leaf 2016

(2.9.72) add 7 mL of water R. Sonicate for 10 min and
Holy Basil Leaf
centrifuge. Prepare a ready-to-use sample preparation DEFINITION
cartridge containing 0.50 g of octadecylsilyl silica gel (50 pm) Holy Basil Leaf is the dried leaves of Ocimum tenuiflorum
using 3 mL of methanol R, drying with a stream of air, then Linn.(syn. Ocimum sanctum). The leaf content is not less
using 3 mL of water R. The flow rate does not exceed than 70% of the material harvested.
6 mUmin. Apply 4 mL of the supernatant to the top of the
IDENTIFICATION
cartridge. Wash the cartridge twice with 1 mL of a mixture
A. Pieces of herb consisting mainly of whole or fragmented
of 1 volume of methanol R and 9 volumes of พater R. Elute
leaf, petiole and stem, with some flower parts, mainly calyx.
the cartridge with 1 mL of methanol R; collect the eluate and
Leaves simple, opposite, petiolate, ovate or elliptical with an
use it as the test solution.
acute or obtuse apex, up to 5 cm long and 3.5 cm wide,
Reference solution Dissolve 1 mg of scopoletin R in 10 mL of green or purple, with an entire or serrated margin and
methanol R. Dilute 1 mL of the solution to 10 mL with pubescent on both surfaces. Fragments of petiole and stem
methanol R to obtain solution (a). Dissolve 1 mg of rutin R in twisted, hairy, purplish-brown or dark green-black,
5 mL of solution (a). subquadrangular, petiole thin up to 3 cm long, stem
Plate TLC silica gel F254 plate R (2-10 pm). herbaceous or woody and fibrous, thicker and highly
Mobile phase anhydrous formic acid R, glacial acetic acid R, branched. Fragments of calyx, if present, membranous,
water R, ethyl acetate R (11:11:27:100 VIVIVIV). veined, 3 to 4 mm long, ovoid or campanulate bi-lipped with
Application 2 pL as bands of 8 mm. upper lip broadly obovate and shortly apiculate, lower lip
longer with two short lateral and two larger central
mucronate teeth. Corolla about 4 mm long, pubescent; fruit
Drying In air. consisting of 4 nutlets enclosed in a calyx, each nutlet sub-
Detection Heat at 100 °C for 3 min; treat the still-warm plate globose, slightly compressed, nearly smooth; pale brown or
with a 5 g/L solution of diphenylboric acid aminoethyl ester R in reddish with a small black hilum and each with one seed.
ethyl acetate R, then treat with a 50 g/L solution of macrogol Seed rounded to ovoid brown, about 0.1 cm long.
400 R in methylene chloride R', examine in ultraviolet light at B. Reduce to a powder. The powder is brownish-green to
365 run after 5 min. greenish-brown. Examine under a microscope using chloral
Results See below the sequence of zones present in the hydrate solution. The powder shows the following
chromatograms obtained with the reference solution and the characteristics: leaf tissue fragments with polygonal, more or
test solution. Furthermore, other faint zones may be present less straight walled epidermal cells and occasional diacytic
in the chromatogram obtained with the test solution. stomata; covering trichomes elongated, uniseriate with three
to seven cells; glandular trichomes of two types: (a)
unicellular base with small, rounded unicellular or bicellular
Top of the plate
head; (b) unicellular base with enlarged oval head composed
Scopoletin: a bright blue A blue fluorescent zone (scopole of eight radiating cells. Fragments of leaf midrib and petiole
fluorescent zone tin)
have thin walled ovoid cells and underlying collenchyma, up
to four cells in depth. Some fragments from calyx, seeds and
stems may also be seen. Calyx fragments are paler in colour
and have characteristic epidermal cells with deeply sinuous
walls, elongated over the veins and occasional covering and
Rutin: an orange fluorescent zone An orange fluorescent zone glandular trichomes of the same type as the leaf tissues. Seed
(rutin) fragments consist of closely packed cells with thick wavy
3-4 blue fluorescent zones walls and blackish contents and closely packed polygonal
parenchyma. Stem fragments consist of narrow, closely
packed fibres, vessels with spiral thickening, some pitted
vessels and closely packed parenchyma merging with
Reference solution Test solution collenchyma.
Partially clear a second mount with chloral hydrate solution,
then carefully remove the chloral hydrate solution whilst
TESTS retaining the powder. Irrigate the powder thoroughly with a
Loss on drying (2.2.22) 10 % v/v alcoholic solution of phloroglucinol, allow the
Maximum 11.0 per cent, determined on 2.000 g of the solution to evaporate, and then add 1 to 2 drops of
powdered herbal drug (355) (2.9.12) by drying in an oven at hydrochloric acid. Mix, then mount in a 50% v/v solution of
105 °C for 2 h. glycerol. The large, eight celled glandular trichomes are
Total ash (2.4.16) stained red due to the presence of eugenol. Lignified fibres
Maximum 5.0 per cent. and vessels also stain red.
c. Carry out the method for thin-layer chromatography,
Extractable matter
Appendix in A, using the following solutions.
To 2.00 g of the powdered herbal drug (355) (2.9.12) add (1) Add 5 mL of methanol to approximately 500 mg of the
50.0 g of water R, boil under reflux for 1 h, compensate the powdered herbal drug in a centrifuge tube. Mix using a
vortex mixer briefly and then with the aid of ultrasound for
loss of water and filter. Evaporate 25.0 g of the filtrate to
10 minutes. Centrifuge at 2500 rpm for 10 minutes and use
dryness on a water-bath and dry in an oven at 105 °C for
the supernatant.
3 h. The residue weighs a minimum of 0.55 g.
______________________________________________ Ph Eur
(2) 0.016% w/v each of rutin, hyperoside and rosmarinic acid in
methanol.
2016 Holy Basil Leaf LV-91
CHROMATOGRAPHIC CONDITIONS (e) After removal of the plate, dry in air for 5 minutes.
(a) Use as the coating octadecylsilyl silica gel for HPTLC Dip in anisaldehyde solution and heat at 100° for 3 minutes.
(Merck silica gel HPTLC plates are suitable). Examine under white light and ultraviolet light (366 nm).
(b) Use the mobile phase as described below. MOBILE PHASE
(c) Apply 10 J1L of solution (1) and 2 pL of solution (2) as 15 volumes of ethyl acetate and 85 volumes of toluene.
8 mm bands.
SYSTEM SUITABILITY
(e) After removal of the plate, dry in air for 5 minutes and solution (3) shows two clearly separated bands and the band
heat at 100° for 3 minutes. Whilst the plate is hot dip the due to methyleugenol in the chromatogram obtained with
plate into a 0.5% w/v solution of diphenylboric acid amino ethyl solution (2) has an Rf of approximately 0.6.
ester in ethyl acetate, dry and dip in a 5% w/v solution of
CONFIRMATION
polyethylene glycol 400 in dichloromethane. Examine under
ultraviolet light (366 nm). When examined under white light the chromatogram
obtained with solution (1), as shown in the table, shows a
MOBILE PHASE
pink band that elutes above the band due to methyleugenol
1 volume of formic acid, 1 volume of water and 15 volumes of in the chromatogram obtained with solution (2) and a grey
ethyl acetate. band corresponding to the grey band due to urosolic acid
SYSTEM SUITABILITY obtained with solution (3). A grey-green band corresponding
The test is not valid unless, the chromatogram obtained with to the band due to eugenol in the chromatogram obtained
solution (2) shows three clearly separated bands of which two with solution (3) may be present. A grey-green band
are orange bands with Rf values of approximately 0.1 (rutin) corresponding to the band due to methyl-eugenol in the
and 0.2 (hyperoside). chromatogram obtained with solution (2) may be present.
Other bands may be present.
CONFIRMATION
The chromatogram obtained with solution (1) shows a strong Top of the plate
yellow-orange fluorescent band that elutes between the 2
orange bands due to rutin and hyperoside in the
chromatogram obtained with solution (2). Several other AfMM
Aywy^b«1 («^๙)
fluorescent bands will be present as shown in the table.
Other fluorescent bands may be present.
Top of the plate Solution (1) Solution (2) Solution (3)
A red fluorescent band
A turquoise-blue fluorescent band A turquoise-blue fluorescent band

(rosmarinic add)
When examined under ultraviolet light (366 nm) the
Two blue fluorescent bands may be present chromatogram obtained with solution (1) shows the bands as
shown in the table. Other bands may be present.
Two turquoise fluorescent bands may be
An orange fluorescent band (hyperoside)
A yellow-orange fluorescent band

An orange fluorescent band (rutin)
Solution (1) Solution (2)
D. Carry out the method for thin-layer chromatography,

Appendix m A, using the following solutions.
(1) Add 5 mL of methanol to approximately 500 mg of the
When examined under both white light and ultraviolet light
powdered herbal drug in a centrifuge tube. Mix using a
(366 nm) at least one band due to either eugenol or methyl
vortex mixer briefly and then with- the aid of ultrasound for
eugenol should be present in the chromatogram obtained
10 minutes. Centrifuge at 2500 rpm for 10 minutes and use
with solution (1).
the supernatant.
(2) 0.04% w/v of methyleugenol in methanol. TESTS
Loss on Drying
(3) 0.02% w/v of ursolic acid and 0.04% w/v of eugenol in
When dried at 100° to 105° for 2 hours, loses not more than
methanol.
10% of its weight. Use 1 g.
Foreign matter
(a) Use as the coating octadecylsilyl silica gel for HPTLC Not more than 2% of foreign matter.
(Merck silica gel HPTLC plates are suitable).
Total ash
(b) Use the mobile phase as described below. Ignite for 18 hours at 450°. Not more than 17.7%,
(c) Apply 10 pL of solution (1) and 2 |1L each of solutions Appendix XI J, Method I.
(2) and (3) as 8 mm bands. Acid insoluble ash
(d) Develop the plate to 7 cm. Not more than 6.0%, Appendix XI K, Method II. Use a
second ignition temperature set at 900° for 1 hour (to ensure
full combustion of the filter paper).
IV-92 Bearbeny Leaf 2016
Bearberry Leaf ** **
Uva Ursi ***
Ph Eur___________________________________________________
DEFINITION
Whole or fragmented, dried leaf of Arctostaphylos uva-
ursi (L.) Spreng.
Content
Minimum 7.0 per cent of anhydrous arbutin (C12H16O7;
Afr 272.3) (dried drug).
IDENTIFICATION
A. The leaf, shiny and dark green on the adaxial surface,
lighter on the abaxial surface, is generally 7-30 mm long and
5-12 mm wide. The entire leaf is obovate with smooth
margins, somewhat reflexed downwards, narrowing at ±e
base into a short petiole. The leaf is obtuse or retuse at its
apex. The lamina is thick and coriaceous. The venation,
pinnate and finely reticulate, is clearly visible on both
surfaces. The adaxial surface is marked with sunken veinlets,
giving it a characteristic grainy appearance. Only the young
leaf has ciliated margins. Old leaves are glabrous.
B. Microscopic examination (2.8.23). The powder is green,
greenish-grey or yellowish-green. Examine under a
microscope using chloral hydrate solution R. The powder
shows the following diagnostic characters (Figure 1054.-1):
fragments of adaxial epidermis in surface view [A] showing
thick and irregularly pitted polygonal cells [Aa] usually Figure 1054.-1. - Illustration for identification test B of powdered
accompanied by palisade parenchyma [Ab]; fragments of herbal drug of bearbeny leaf
adaxial epidermis in transverse section [G], showing straight
walled cells [Ga] covered by a thick smooth cuticle [Gb], and Results See below the sequence of zones present in the
accompanied by palisade parenchyma [Gc] consisting of 3 or chromatograms obtained with the reference solution and the
4 layers of cells of unequal lengths, some of which contain test solution. Furthermore, other blue or brown zones may
numerous prisms of calcium oxalate [Gd]; fragments of be present in the chromatogram obtained with the test
abaxial epidermis, in surface view [B, E], showing solution.
anomocytic stomata {2.8.3) [Ba] surrounded by 5-11
subsidiary’ cells, scars of hair bases [Ea], and accompanied by
Top of the plate
spongy parenchyma [Eb]; groups of lignified fibres from the
pericycle [D]; fragments of the vascular system [F] consisting
of pined vessels [Fa] and fibres [Fb] accompanied by rows of
cells containing prisms of calcium oxalate [Fc]; oil droplets
Gallic acid: a brownish zone A brownish zone
are present in the parenchymatous cells; occasional fragments
of conical, unicellular covering trichomes [C].
A brown zone
(2.9.72) add 5 mL of a mixture of equal volumes of
methanol R and water Ry and heat under a reflux condenser
for 10 min. Filter whilst hot. Wash the flask and the filter Arbutin: a blue zone An intense blue zone (arbutin)
with a mixture of equal volumes of methanol R and water R
and dilute to 5 mL with the same mixture of solvents. Reference solution Test solution
Reference solution Dissolve 50 mg of arbutin R and 25 mg of

gallic acid R in methanol R and dilute to 20.0 mL with the
TESTS
same solvent.
Foreign matter {2.8.2)
Plato TLC silica gel plate R (5-40 pm) [or TLC silica gel
Maximum 5 per cent of stems and maximum 3 per cent of
plate R {2-10 pm)]. other foreign matter.
Mobile phase anhydrous formic acid Ry water Ry ethyl acetate R
Leaves of different colour
(6:6:88 VIVIV). Maximum 10 per cent, determined in the same manner as
Application 10 pL [or 2 pL] as bands of 15 mm [or 8 mm]. foreign matter {2.8.2).
Development Over a path of 15 cm [or 6 cm]. Loss on drying {2.2.32)
Drying At 105-110 °C until the mobile phase has evaporated. Maximum 10.0 per cent, determined on 1.000 g of the
Detection Treat with a 10 g/L solution of powdered herbal drug (355) (2.9.72) by drying in an oven at
dichloroquinonechlorimide R in methanol Ry then treat with a 105 °C for 2 h.
20 g/L solution of anhydrous sodium carbonate R.
2016 Belamcanda Chinensis Rhizome IV-93
Total ash (2.4.16) Content

Maximum 5.0 per cent. Minimum 0.10 per cent of irisflorentin (C2oH1808;
assay Mr 386.4) (dried drug).
Liquid chromatography (2.2.29). IDENTIFICATION
Test solution In a 100 mL flask with a ground-glass neck, A. The whole rhizome is nodular, rounded, about 3-10 cm
place 0.800 g of the powdered herbal drug (250) (2.9.12). long and 1-2 cm in diameter, irregular, more or less
Add 20 mL of water R and heat under a reflux condenser on branched, with numerous annular striations; there are
a water-bath for 30 min. Allow to cool and filter the liquid crateriform, annular stem scars on the upper surface and
through a plug of absorbent cotton. Add the absorbent small roots about 2-3 mm in diameter on the lower surface.
cotton to the residue in the 100 mL flask and extract with The longitudinally fragmented rhizome is found as pieces
20 mL of water R under a reflux condenser on a water-bath about 2-8 cm long, 2 cm wide and 1 cm thick; stem scars
for 30 min. Allow to cool and filter through a paper filter. and root fragments are present. The orange-brown or dark
Combine the filtrates and dilute to 50.0 mL with water R. brown outer surface is the same colour as the fracture.
Filter through a paper filter. Discard the first 10 mL of the The central parenchyma has a pitted appearance due to the
filtrate. numerous primary vascular bundles. The texture is hard.
Reference solution (a) Dissolve 50.0 mg of arbutin CRS in the The fracture is granular.
mobile phase and dilute to 50.0 mL with the mobile phase. B. Microscopic examination (2.8.23). The powder is orange
Reference solution (b) Dissolve 2.5 mg of hydroquinone R in the brown or dark brown. Examine under a microscope using
mobile phase and dilute to 10.0 mL with the mobile phase. chloral hydrate solution R. The powder shows the following
To 5.0 mL of the solution, add 2.5 mL of reference diagnostic characters: rare fragments of brown cork with
solution (a) and dilute to 10.0 mL with the mobile phase. superimposed polyhedral cells; numerous, somewhat rounded
Column'. parenchyma cells with irregularly thickened and pitted walls,
granular and oily contents, with some of the cells containing
— size'. I — 0.25 m, 0 = 4 mm;
a very large calcium oxalate prism up to 250 pm long and
— stationary phase', base-deactivated octadecylsilyl silica gel for
about 50 pm in diameter; very numerous free calcium oxalate
chromatography R (5 pm).
prisms, usually broken; reticulate or pitted lignified vessels.
Mobile phase methanol R, water R (10:90 VII7). Examine under a microscope using a 50 per cent v/v
Flow rate 1.2 mL/min. solution of glycerol R. The powder shows very numerous
Detection Spectrophotometer at 280 nm. rounded or ovoid starch granules, 3-15 pm in diameter,
Injection 20 pL. simple or rarely compound with 2-5 components.
The punctiform hilum is sometimes visible. The starch
granules are free and very often included in the
— resolution: minimum 4.0 between the peaks due to arbutin
parenchymatous cells.
and hydroquinone.
c. Examine the chromatograms obtained in the test for Iris
Calculate the percentage content of arbutin using the
tectorum Maxim.
Fl X 7722 X p
Fz X mi
F] = area of the peak due to arbutin in the chromatogram

Top of the plate
F2 = area of the peak due to arbutin in the chromatogram
obtained with reference solution (a); Coumarin: a quenching zone A quenching zone
m2 = mass of arbutin CRS used to prepare reference Irisflorentin: a quenching zone A quenching zone (irisflorentin)
solution (a), in grams;
p = percentage content of arbutin in arbutin CRS.
_______________________________________________________________ Ph Eur
Belamcanda Chinensis Rhizome ***** Results B See below the sequence of zones present in the
(Ph. Eur. monograph 2561) *** chromatograms obtained with the reference solution and the
Ph Eur_____________ _________________________________________________
DEFINITION solution.
Dried, whole or fragmented rhizome of Iris domestica (L.)
Goldblatt et Mabb. (syn. Belamcanda chinensis (L.) DC.),
collected in early spring while the plant is budding or in late
autumn while the aerial part is withering, with roots
removed.
IV-94 Belamcanda Chinensis Rhizome 2016
Top of the plate Reference solution (a) Dissolve 5.0 mg of irisflorentin CRS in
ethanol (70 per cent VIV) R and dilute to 50.0 mL with the
same solvent. Dilute 5.0 mL of the solution to 50.0 mL with
Coumarin: a faint dark blue A faint blue fluorescent zone ethanol (70 per cent VIV) R.
fluorescent zone
Reference solution (b) Disperse 0.10 g of belamcanda chinensis
rhizome HRS in 10 mL of ethanol (70 per cent VIV) R in a
A black zone 50 mL centrifuge tube. Sonicate for 30 min. Mix and
A broad blue fluorescent zone centrifuge for 5 min. Transfer the supernatant to a 25 mL
volumetric flask. Add to the residue 10 mL of ethanol
Irisflorentin ะ a blue fluorescent A blue fluorescent zone
(irisflorentin)
(70 per cent VIV) R and sonicate for 30 min. Filter and add
the filtrate to the same volumetric flask. Dilute to 25.0 mL
with ethanol (70 per cent V/V) R and mix. Filter through a
Reference solution Test solution Column'.
— size'. I = 0.25 m, 0 = 4.6 mm;
— stationary phase', octadecylsilyl silica gel for chromatography R
TESTS (5 pm).
Iris tectorum Maxim Mobile phase'.
Thin-layer chromatography (2.2.27). — mobile phase A: 0.05 per cent v/v solution of phosphoric
Test solution To 0.5 g of the powdered herbal drug (355) acid R;
(2.9.72) add 5 mL of methanol R and sonicate for 10 min. — mobile phase B: acetonitrile R'y
Centrifuge and filter.
Reference solution Dissolve 1 mg of irisflorentin R and 1 mg of Time Mobile phase A Mobile phase B
coumarin J? in 4 mL of methanol R. (per cent V/V) (per cent V/V)
Plate TLC silica gel F254 plate R (2-10 pm). 0-5 82 18
Mobile phase glacial acetic acid R, cyclohexane R> ethyl acetate R 5 - 20 82 -> 80 18 -> 20
(1:20:80 VIVIV). 20 - 30 80 -> 67 20 -» 33
Application 4 pL as bands of 8 mm. 67 -> 60
30 - 50 33 -> 40
50 - 65 60 -> 47 40 -» 53
Drying In air.
Results A The chromatogram obtained with the test solution Flow rate 1.0 mUmin.
shows no quenching zone between the zones due to Detection Spectrophotometer at 266 nm.
coumarin and irisflorentin in the chromatogram obtained Injection 10 pL.
with the reference solution. No quenching zones are present Identification of peaks Use the chromatogram obtained with
in the lower third of the chromatogram obtained with the test reference solution (a) to identify the peak due to irisflorentin;
solution. use the chromatogram supplied with belamcanda chinensis
Detection B Examine in ultraviolet light at 365 nm. rhizome HRS and the chromatogram obtained with reference
Results B The chromatogram obtained with the test solution solution (b) to identify the peaks due to tectoridin and
shows no pale blue fluorescent zone above the zone due to peak 2 (unknown).
coumarin in the chromatogram obtained with the reference Retention time Irisflorentin = about 54 min.
solution. System suitability: reference solution (b):
Loss on drying (2.2.32) — resolution: minimum 1.5 between the peak due to
Maximum 10.0 per cent, determined on 1.000 g of the tectoridin and peak 2.
powdered herbal drug (355) (2.9.72) by drying in an oven at Calculate the percentage content of irisflorentin using the
105 °C for 2 h. following expression:
Total ash (2.4.16)
Maximum 7.0 per cent. Al X 7ท2 X p
Ash insoluble in hydrochloric acid (2.8.1) A2 X 7721 X 20
ASSAY Al = area of the peak due to irisflorentin in the
Liquid chromatography (2.2.29). chromatogram obtained with the test solution;
A2 = area of the peak due to irisflorentin in the
Test solution Disperse 0.100 g of the powdered herbal drug
chromatogram obtained with reference solution
(355) (2.9.72) in 10 mL of ethanol (70 per cent VIV) R in a
(a);
50 mL centrifuge tube. Sonicate for 30 min. Mix and
centrifuge for 5 min. Transfer the supernatant to a 25 mL
volumetric flask. Add to ±e residue 10 mL of ethanol m2 = mass of irisflorentin CRS used to prepare reference
(70 per cent V/V) R and sonicate for 30 min. Filter and add
the filtrate to the same volumetric flask. Dilute to 25.0 mL
p = percentage content of irisflorentin in irisflorentin
with ethanol (70 per cent VIV) R and mix. Filter through a CRS.
Ph Eur
2016 Belladonna Leaf LV-95
Belladonna Leaf ** **
Belladonna Herb ***
Preparations
Prepared Belladonna
Standardised Belladonna Leaf Dry Extract
Belladonna Tincture
When Belladonna Herb, Belladonna Leaf or Powdered
Belladonna Herb is prescribed, Prepared Belladonna shall be
supplied.
Ph Elf___________
DEFINITION
Dried leaf or dried leaf and flowering, and occasionally fruit
bearing, tops of Atropa belladonna L.
Content
Minimum 0.30 per cent of total alkaloids, expressed as
hyoscyamine (C17H23NO3; Mr 289.4) (dried drug).
The alkaloids consist mainly of hyoscyamine together with
small quantities of hyoscine (scopolamine).
CHARACTERS
Slightly nauseous odour.
IDENTIFICATION
A. The leaves are green or brownish-green, slightly darker on
the upper surface, often crumpled and rolled and partly
matted together in the drug. The leaf is petiolate and the
lamina is acute and decurrent. The margin is entire. Figure 0221.-1. - Illustration for identification test B of powdered
The flowering stems are flattened and bear at each node a herbal drug of belladonna leaf
pair of leaves unequal in size, in the axils of which occur
singly the flowers or occasionally fruits. The flowers have a c. Shake 1 g of the powdered herbal drug (180) (2.9.22)
gamosepalous calyx and campanulate corolla. The drug may with 10 mL of 0.05 M sulfuric acid for 2 min. Filter and add
contain fruits, as globular berries, green or brownish-black to the filtrate 1 mL of concentrated ammonia R and 5 mL of
and surrounded by the persistent calyx with widely spread water R. Shake cautiously with 15 mL of ether R, avoiding
lobes. formation of an emulsion. Separate the ether layer and dry
B. Microscopic examination {2.8.23}. The powder is dark over anhydrous sodium sulfate R. Filter and evaporate the ether
green. Examine under a microscope using chloral hydrate in a porcelain dish. Add 0.5 mL of fuming nitric acid R and
solution R. The powder shows the following diagnostic evaporate to dryness on a water-bath. Add 10 mL of
characters (Figure 0221.-1): fragments of the lamina showing acetone R and, dropwise, a 30 g/L solution of potassium
sinuous-walled epidermal cells with striated cuticle [A, C] hydroxide R in ethanol (96 per cent) R. A deep violet colour
and part of the underlying palisade parenchyma [Aa] develops.
associated with the upper epidermis [A]; numerous D. Examine the chromatograms obtained in the
stomata [Ca] more frequent on the lower epidermis [C], chromatography test.
anisocytic and also some anomocytic (2.5.3); multicellular, Results The principal zones in the chromatograms obtained
uniseriate covering trichomes with a smooth cuticle [F], with the test solution are similar in position, colour and size
glandular trichomes with unicellular heads and multicellular, to the principal zones in the chromatograms obtained with
uniseriate stalks [D] or with multicellular heads and the same volume of the reference solution.
unicellular stalks [B]; parenchyma cells including rounded
cells, some of which contain microsphenoidal crystals of TESTS
calcium oxalate [E]; annularly and spirally thickened Chromatography
vessels [K]. The powdered herbal drug may also show: fibres Thin-layer chromatography (2.2.27).
and reticulately thickened vessels from the stems; Test solution To 0.6 g of the powdered herbal drug (180)
subspherical pollen grains, 40-50 Jim in diameter, with {2.9.12} add 15 mL of 0.05 M sulfuric acid, shake for 15 min
3 germinal pores, 3 furrows and an extensively pitted and filter. Wash the filter with 0.05 M sulfuric acid until
exine [H]; fragments of the corolla with a papillose 20 mL of filtrate is obtained. To the filtrate add 1 mL ๙
epidermis [J] or bearing numerous covering or glandular concentrated ammonia R and shake with 2 quantities, each of
trichomes of the types previously described [L]; fragments of 10 mL, of peroxide-free ether R. If necessary, separate by
the brownish-yellow testa consisting of irregularly sclerified centrifugation. Dry the combined ether layers over anhydrous
cells [G]. sodium sulfate R} filter and evaporate to dryness on a water
bath. Dissolve the residue in 0.5 mL of methanol R.
Reference solution Dissolve 50 mg of hyoscyamine sulfate R in
9 mL of methanol R. Dissolve 15 mg of hyoscine
hydrobromide R in 10 mL of methanol R. Mix 1.8 mL of the
IV-96 Belladonna 2016
hyoscine hydrobromide solution and 8 mL of the chloroform R. Combine the chloroform layers, add 4 g of
hyoscyamine sulfate solution. anhydrous sodium sulfate R and allow to stand for 30 min with
Plate TLC silica gel G plate R. occasional shaking. Decant the chloroform and wash the
Mobile phase concentrated ammonia R, water R, acetone R sodium sulfate with 3 quantities, each of 10 mL, of
(3:7:90 VIVIV). chloroform R. Add the washings to the chloroform extract,
evaporate to dryness on a water-bath and heat in an oven at
Application 10 pL and 20 pL, as bands of 20 mm by 3 mm,
100-105 °C for 15 min. Dissolve the residue in a few
leaving 1 cm between the bands.
millilitres of chloroform R, add 20.0 mL of 0.01 M sulfuric acid
Development Over a path of 10 cm. and remove the chloroform by evaporation on a water-bath.
Drying At 100-105 °C for 15 min; allow to cool. Titrate the excess of acid with 0.02 M sodium hydroxide using
Detection A Spray Hath potassium iodobismuthate solution R2, methyl red mixed solution R as indicator.
using about 10 mL for a plate 200 mm square, until the Calculate the percentage content of total alkaloids, expressed
orange or brown zones become visible against a yellow as hyoscyamine, using the following expression:
background.
Results A The zones in the chromatograms obtained with the 57.88 X (20 - ท)
test solution are similar in position (hyoscyamine in the lower (100 — d) X m
third, hyoscine in the upper third of the chromatograms) and
colour to the bands in the chromatograms obtained with the d = loss on drying, as a percentage;
reference solution. The zones in the chromatograms obtained ท = volume of 0.02 M sodium hydroxide, in millilitres;
with the test solution are at least equal in size to ±e m = mass of the powdered herbal drug, in grams.
corresponding zones in the chromatogram obtained with the ______ _________________________________________________________ Ph Eur
same volume of the reference solution. Faint secondary zones
may appear, particularly in the middle of the chromatogram
obtained with 20 pL of the test solution or near the stalling
point in the chromatogram obtained with 10 pL of the test
solution.
Prepared Belladonna ******
Detection B spray with sodium nitrite solution R until the Prepared Belladonna Herb ***
coating is transparent; examine after 15 min. (Ph. Eur. monograph 0222)
Results B The zones due to hyoscyamine in the Ph Eur________________________________________________________________
test solution change from brown to reddish-brown but not to DEFINITION
greyish-blue (atropine) and any secondary zones disappear. Belladonna leaf powder (180) (2.9.12) adjusted, if necessary,
by adding powdered lactose or belladonna leaf powder with a
Foreign matter (2.8.2) lower alkaloidal content.
Maximum 3 per cent of stems with a diameter greater than
5 mm. Content
0.28 per cent to 0.32 per cent of total alkaloids, expressed as
Total ash (2.4.16) hyoscyamine (A4r 289.4) (dried drug).
CHARACTERS
Maximum 4.0 per cent. Slightly nauseous odour.
ASSAY IDENTIFICATION
A. The powder is dark green. Examine under a microscope,
a) Determine the loss on drying (2.2.32) on 2.000 g of the
powdered herbal drug (180) (2.9.72), by drying in an oven at using chloral hydrate solution R. The powder shows the
following diagnostic characters: fragments of leaf lamina
105 °C.
showing sinuous-walled epidermal cells, a striated cuticle and
b) Moisten 10.00 g of the powdered herbal drug (180) numerous stomata predominantly present on the lower
(2.9.12) with a mixture of 5 mL of ammonia R, 10 mL of epidermis (anisocytic and also some anomocytic) (2.8.3)',
ethanol (96 per cent) R and 30 mL of peroxide-free ether R and multicellular uniseriate covering trichomes with smooth
mix thoroughly. Transfer the mixture to a suitable percolator, cuticle, glandular trichomes with unicellular heads and
if necessary with the aid of the extracting mixture. Allow to multicellular, uniseriatc stalks or with multicellular heads and
macerate for 4 h and percolate with a mixture of 1 volume of unicellular stalks; parenchyma cells including rounded cells
chloroform R and 3 volumes of peroxide-free ether R until the containing microsphenoidal crystals of calcium oxalate;
alkaloids are completely extracted. Evaporate to dryness a annular and spirally thickened vessels. The powdered herbal
few millilitres of the liquid flowing from the percolator, drug may also show the following: fibres and reticulately
dissolve the residue in 0.25 M sulfuric acid and verify the thickened vessels from the stems; subspherical pollen grains,
absence of alkaloids using potassium tetraiodomercurate 40-50 pm in diameter, with 3 germinal pores, 3 furrows and
solution R. Concentrate the percolate to about 50 mL by an extensively pitted exine; fragments of the corolla, with a
distilling on a water-bath and transfer it to a separating papillose epidermis or bearing numerous covering or
funnel, rinsing with peroxide-free ether R. Add a quantity of glandular trichomes of the types previously described;
peroxide-free ether R equal to at least 2.1 times the volume of brownish-yellow seed fragments containing irregularly
the percolate to produce a liquid of a density well below that sclerified and pitted cells of the testa. Examined in glycerol
of water. Shake the solution with no fewer than 3 quantities, (85 per cent) R, the powder may be seen to contain lactose
each of 20 mL, of 0.25 M sulfuric acid, separate the 2 layers crystals.
by centrifugation if necessary and transfer the acid layers to a
B. Shake 1 g with 10 mL of 0.05 M sulfuric acid for 2 min.
2"d separating funnel. Make the acid layer alkaline with Filter and add to the filtrate 1 mL of concentrated ammonia R
ammonia R and shake with 3 quantities, each of 30 mL, of
and 5 mL of water R. Shake cautiously with 15 mL of
2016 Belladonna Preparations IV-97
ether R, avoiding formation of an emulsion. Separate the Ash insoluble in hydrochloric acid {2.8.1)
ether layer and dry over anhydrous sodium sulfate R. Filter and Maximum 4.0 per cent.
evaporate the ether in a porcelain dish. Add 0.5 mL of
fuming nitric acid R and evaporate to dryness on a water-bath. ASSAY
Add 10 mL of acetone R and, dropwisc, a 30 g/L solution of a) Determine the loss on drying {2.2.32) on 2.000 g by
potassium hydroxide R in ethanol (96 per cent) R. N deep violet drying in an oven at 105 °C.
colour develops. b) Moisten 10.00 g with a mixture of 5 mL of ammonia R,
c. Examine the chromatograms obtained in the test 10 mL of ethanol (96 per cent) R and 30 mL of peroxide-free
Chromatography. ether R and mix thoroughly. Transfer the mixture to a
suitable percolator, if necessary with the aid of the extracting
Results The principal zones in the chromatograms obtained mixture. Allow to macerate for 4 h and percolate with a
with the test solution are similar in position, colour and size mixture of 1 volume of chloroform R and 3 volumes of
to the principal zones in the chromatogram obtained with the peroxide-free ether R until the alkaloids are completely
same volume of the reference solution. extracted. Evaporate to dryness a few millilitres of the liquid
TESTS flowing from the percolator, dissolve the residue in 0.25 M
Chromatography sulfuric acid and verify the absence of alkaloids using
Thin-layer chromatography (2.2.27). potassium tetraiodoniercurate solution R. Concentrate the
Test solution To 0.6 g of the drug to be examined add 15 mL percolate to about 50 mL by distilling on a water-bath and
of 0.05 M sulfuric acid, shake for 15 min and filter. Wash the transfer it to a separating funnel, rinsing with peroxide-free
filter with 0.05 M sulfuric acid until 20 mL of filtrate is ether R. Add a quantity of peroxide-free ether R equal to at
obtained. To the filtrate add 1 mL of concentrated ammonia R least 2.1 times the volume of the percolate to produce a
and shake with 2 quantities, each of 10 mL, of peroxide-free liquid of a density well below that of water. Shake the
ether R. If necessary, separate by centrifugation. Dry the solution with no fewer than 3 quantities, each of 20 mL, of
combined ether layers over anhydrous sodium sulfate R, filter, 0.25 M sulfuric acid, separate the 2 layers by centrifugation if
necessary and transfer the acid layers to a 2nd separating
and evaporate to dryness on a water-bath. Dissolve the
residue in 0.5 mL of methanol R. funnel. Make the acid layer alkaline with ammonia R and
shake with 3 quantities, each of 30 mL) of chloroform R.
Reference solution Dissolve 50 mg of hyoscyamine sulfate R in Combine the chloroform layers, add 4 g of anhydrous sodium.
9 mL of methanol R. Dissolve 15 mg of hyoscine sulfate R and allow to stand for 30 min with occasional
hydrobromide R in 10 mL of methanol R. Mix 1.8 mL of the shaking. Decant the chloroform and wash the sodium sulfate
hyoscine hydrobromide solution and 8 mL of the with 3 quantities, each of 10 mL, of chloroform R. Add the
hyoscyamine sulfate solution. washings to the chloroform extract, evaporate to dryness on a
Plate TLC silica gel G plate R. water-bath and heat in an oven at 100-105 °C for 15 min.
Mobile phase concentrated ammonia R, water R, acetone R Dissolve the residue in a few millilitres of chloroform R, add
(3:7:90 VIVIV). 20.0 mL of 0.01 M sulfuric acid and remove the chloroform
Application 10 pL and 20 |1L of each solution, as bands of by evaporation on a water-bath. Titrate the excess of acid
20 mm by 3 mm, leaving 1 cm between each band. with 0.02 M sodium hydroxide using methyl red mixed
solution R as indicator.
Calcdate the percentage content of total alkaloids, expressed
Drying At 100-105 °C for 15 min; allow to cool.
as hyoscyamine, using the following expression:
Detection A spray with potassium iodobismuthate solution R2,
using about 10 mL for a plate 200 mm square, until orange 57.88 X (20 - ท)
or brown zones become visible against a yellow background.
(100 -d)xm
Results A The zones in the chromatograms obtained with the
test solution are similar in position (hyoscyamine in the lower d = loss on drying as a percentage;
third, hyoscine in the upper third) and colour to those in the ท ะ= volume of 0.02 M sodium hydroxide used, in
chromatograms obtained with the reference solution; millilitres;
the zones in the chromatograms obtained with the test m = mass of the herbal drug used, in grams.
solution are at least equal in size to the corresponding zones
in the chromatogram obtained with the same volume of the STORAGE
reference solution; faint secondary zones may appear, In an airtight container.
particularly in the middle of the chromatogram obtained with ______________________________________ _ ______________________ Ph Eur
20 pL of the test solution or near the point of application in

the chromatogram obtained with 10 pL of the test solution.
Detection B Spray with sodium nitrite solution R until the
coating is transparent and examine after 15 min. Standardised Belladonna Leaf Dry ** **
Results B The zones due to hyoscyamine in the
chromatograms obtained with the test solution and the
Extract *****
reference solution change from brown to reddish-brown but (Ph. Eur. monograph 1294)
not to greyish-blue (atropine), and any secondary zones PhEir--------------------------------------------------- --------------------- —----------------------------
disappear. DEFINITION
Loss on drying (2.2.52) Standardised dry extract obtained from Belladonna
Maximum 5.0 per cent, determined on 1.000 g by drying in leaf (0221).
an oven at 105 °C. Content
Total ash {2.4.16) 0.95 per cent to 1.05 per cent of total alkaloids, expressed as
Maximum 16.0 per cent. hyoscyamine (C17H23NO,; M, 289.4) (dried extract).
IV-98 Belladonna Preparations 2016
PRODUCTION Detection A treat with potassium iodobismuthate solution R2,

The extract is produced from the herbal drug by a suitable until orange or brown zones become visible against a yellow
procedure using ethanol (70 per cent VIV). background.
CHARACTERS Results A The zones in the chromatogram obtained with the
Appearance test solution are similar in position (hyoscyamine in the lower
Brown or greenish, hygroscopic powder. third, hyoscine in the upper third) and colour to those in the
chromatogram obtained with the reference solution. Other
IDENTIFICATION faint zones may be present in the chromatogram obtained
A. Thin-layer chromatography (2.2.27). with the test solution.
Test solution To 1 g of the extract to be examined add Detection B treat with sodium nitrite solution R until the coating
5.0 mL of methanol R. Shake for 2 min and filter. is transparent and examine after 15 min.
Reference solution. Dissolve 1.0 mg of chlorogenic acid R and Results B The zones due to hyoscyamine in the
2.5 mg of rutin R in 10 mL of methanol R. chromatograms obtained with the test solution and the
Plate TLC silica gel plate R. reference solution change from orange or brown to reddish-
Mobile phase anhydrous formic acid R, water R, methyl ethyl brown but not to greyish-blue (atropine).
ketone R, ethyl acetate R (10:10:30:50 VIVIVIV). Loss on drying {2.8.17)
Application 20 pL as bands. Maximum 5.0 per cent.
Development Over a path of 15 cm. ASSAY
Drying At 100-105 °C. At each extraction stage it is necessary to check that the
Detectioti treat the warm plate with a 10 g/L solution of alkaloids have been completely extracted. If the extraction is
diphenylboric acid aminoethyl ester R in methanol R, then treat into the organic phase this is done by evaporating to dryness
with a 50 g/L solution of macrogol 400 R in methanol R; allow a few millilitres of the last organic layer, dissolving the
to dry in air for 30 min and examine in ultraviolet light at residue in 0.25 M sulfuric acid and verifying the absence of
365 run. alkaloids using potassium tetraiodomercurate solution R. If the
Results The chromatograms obtained with the reference extraction is into the acid aqueous phase, this is done by
taking a few millilitres of the last acid aqueous phase and
solution and the test solution show in the central part a light
verifying the absence of alkaloids using potassium
blue fluorescent zone (chlorogenic acid) and in the lower part
a yellowish-brown fluorescent zone (rutin); furthermore, the tetraiodomercurate solution R.
chromatogram obtained with the test solution shows a little Disperse 3.00 g in a mixture of 5 mL of ammonia R and
above the start a yellowish-brown fluorescent zone and 15 mL of water R. Shake with no fewer than 3 quantities,
directly above that a yellow fluorescent zone, and a yellow or each of 40 mL, of a mixture of 1 volume of methylene
yellowish-brown fluorescent zone between the zone due to chloride R and 3 volumes of peroxide-free ether R until the
rutin and the zone due to chlorogenic acid. Further zones alkaloids are completely extracted. Concentrate the combined
may be present. organic layers to about 50 mL by distilling on a water-bath
and transfer the resulting liquid to a separating funnel,
rinsing with peroxide-free ether R. Add a quantity of peroxide
atropine.
free ether R equal to at least 2.1 times the volume of the
Results The principal zones in the chromatogram obtained liquid to produce a layer having a density well below that of
with the test solution are similar in position and colour to the water. Shake the resulting solution with no fewer than
principal zones in the chromatogram obtained with the 3 quantities, each of 20 mL, of 0.25 M sulfuric acid until the
reference solution. alkaloids are completely extracted. Separate the layers by
TESTS centrifugation, if necessary, and transfer the acid layers to a
Atropine 2nd separating funnel. Make the combined acid layers
Thin-layer chromatography (2.2.27). alkaline with ammonia R and shake with no fewer than
Test solution To 0.20 g of the extract to be examined add 3 quantities, each of 30 mL, of methylene chloride R until the
10.0 mL of 0.05 M sulfuric acid, shake for 2 min and filter. alkaloids are completely extracted. Combine the organic
Add 1.0 mL of concentrated ammonia R and shake with layers, add 4 g of anhydrous sodium sulfate R and allow to
2 quantities, each of 10 mL, of peroxide-free ether R. stand for 30 min with occasional shaking. Decant the
If necessary, separate by centrifugation. Dry the combined methylene chloride and wash the sodium sulfate with
ether layers over about 2 g of anhydrous sodium sulfate R, filter 3 quantities, each of 10 mL, of methylene chloride R. Combine
and evaporate to dryness on a water-bath. Dissolve the the organic extracts and evaporate to dryness on a water
bath. Heat the residue in an oven at 100-105 °C for 15 min.
residue in 0.5 mL of methanol R.
Dissolve the residue in a few millilitres of methylene chloride R,
evaporate to dryness on a water-bath and again heat the
9 mL of methanol R. Dissolve 15 mg of hyoscine residue in an oven at 100-105 °C for 15 min. Dissolve the
hydrobromide R in 10 mL of methanol R. Mix 1.8 mL of the
residue in a few millilitres of methylene chloride R, add
hyoscine hydrobromide solution and 8 mL of the 20.0 mL of 0.01 M sulfuric acid and remove the methylene
hyoscyamine sulfate solution. chloride by evaporation on a water-bath. Titrate the excess of
Plate TLC silica gel plate R. acid with 0.02 M sodium hydroxide using methyl red mixed
Mobile phase concentrated ammonia R, water R, acetone R solution R as indicator.
(3:7:90 VIVIV). Calculate the percentage content of total alkaloids, expressed
Application 20 pL as bands. as hyoscyamine, using the following expression:
57.88 X (20 - ท)
Drying Kt 100-105 °C for 15 min; allow to cool.
100 X 7ท
2016 Belladonna Preparations IV-99
w = volume of 0.02 M sodium hydroxide used, in millilitres; Results A See below the sequence of zones present in the
m = mass of the extract to be examined, in grams. chromatograms obtained with the reference solution and the
------------------------- ------------------ - ----------------------------------------------------------- Ph Eur
test solution. Faint secondary zones may appear, particularly
in the middle of the chromatogram obtained with 40 pL of
the test solution or near the point of application in the
chromatogram obtained with 20 pL of the test solution.
Belladonna Tincture ***** Top of the plate

(Standardised Belladonna Leaf Tincture, **★ Hyoscine: a brownish-orange A brownish-orange zone
Ph Eur monograph 1812) zone (hyoscine)
PfiEir__________ ___________________________________________________
DEFINITION
Tincture produced from Belladonna leaf (0221).
Content
Hyoscyamine: a brownish-orange A brownish-orange zone
0.027 per cent to 0.033 per cent of total alkaloids, calculated zone (hyoscyamine)
as hyoscyamine (C17H23NO3; Afr 289.4). The alkaloids Faint secondary zones
consist mainly of hyoscyamine together with small quantities
of hyoscine. Reference solution Test solution
PRODUCTION
The tincture is produced from 1 part of the powdered herbal TESTS
drug (355) (2.9.72) and 10 parts of Atropine
ethanol (70 per cent VIV) by a suitable procedure. Thin-layer chromatography (2.2.27).
IDENTIFICATION Test solution To 15.0 mL of the tincture to be examined add
A. Thin-layer chromatography (2.2.27). 15 mL of 0.05 M sulfuric acid. Filter. Add 1 mL of
Test solution Evaporate to dryness 10.0 mL of the tincture to concentrated ammonia R to the filtrate and shake with
be examined in a water-bath at 40 °C under reduced 2 quantities, each of 10 mL, of peroxide-free ether R. Separate
pressure. Dissolve the residue in 1.0 mL of methanol R. by centrifugation if necessary. Dry the combined e±er layers
over anhydrous sodium sulfate R. Filter and evaporate to
Reference solution Dissolve 1.0 mg of chlorogenic acid R and
dryness on a water-bath. Dissolve the residue in 0.5 mL of
2.5 mg of rutin R in 10 mL of methanol R.
methanol R.
Mobile phase anhydrous formic acid R, water R, methyl ethyl 9 mL of methanol R. Dissolve 15 mg of hyoscine
ketone R, ethyl acetate R (10:10:30:50 VIVIVIV). hydrobromide R in 10 mL of methanol R. Mix 1.8 mL of the
Application 40 pL as bands. hyoscine hydrobromide solution and 8 mL of the
Development Over a path of 15 cm. hyoscyamine sulfate solution.
Drying At 100-105 °C. Plate TLC silica gel plate R.
Detection Spray the warm plate with a 10 g/L solution of Mobile phase concentrated ammonia R, water R, acetone R
diphenylboric acid aminoethyl ester R in methanol R; (3:7:90 V/V/V).
subsequently spray the plate with a 50 g/L solution of Application 20 pL and 40 pL of each solution, as bands.
macrogol 400 R in methanol R; allow the plate to dry in air for Development Over a path of 10 cm.
30 min and examine in ultraviolet light at 365 nm. Drying At 100-105 °C for 15 min.
Results See below the sequence of zones present in the Detection A Spray with potassium iodobismuthate solution R2.
Detection B Spray with sodium nitrite solution R until ±e plate
test solution. Furthermore, other fluorescent zones may be
present in the chromatogram obtained with the test solution. is transparent. Examine after 15 min.
Results B The zones due to hyoscyamine in the
chromatograms obtained with the test solution and the
Top of he plate
reference solution change from brownish-orange to reddish-
brown but not to greyish-blue (atropine) and any secondary
Chlorogenic acid: a light blue A light blue fluorescent zone zones disappear.
fluorescent zone (chlorogenic acid) Ethanol (2.9.10)
A yellow or yellowish-brown 64 per cent VIV to 69 per cent VIV.
fluorescent zone
ASSAY
Evaporate 50.0 g of the tincture to be examined to a volume
Rutin ะ a yellowish-brown A bluish-grey fluorescent zone
of about 10 mL. Transfer quantitatively to a separating
fluorescent zone
funnel, with the minimum volume of alcohol
(70 per cent VIV) R. Add 5 mL of ammonia R and 15 mL of
A yellowish-brown fluorescent water R. Shake with not fewer than 3 quantities each of
40 mL of a mixture of 1 volume of methylene chloride R and
Reference solution Test solution 3 volumes of peroxide-free ether R, carefully to avoid emulsion,
until the alkaloids are completely extracted. Combine the
B. Examine the chromatograms obtained in the test for organic layers and concentrate the solution to a volume of
atropine, detection A. about 50 mL by distilling on a water-bath. Transfer the
IV-100 Siam Benzoin 2016
resulting solution quantitatively to a separating funnel, rinsing be present in the chromatogram obtained with the test
with peroxide-free ether R. Add a quantity of peroxide-free solution.
ether R equal to at least 2.1 times ±e volume of the solution
to produce a layer having a density well below that of water.
Top of the plate
Shake the resulting solution with not fewer than 3 quantities
each of 20 mL of 0.25 M sulfuric acid until the alkaloids are
completely extracted. Separate the layers by centrifugation if
Methyl cinnamate: a very
necessary and transfer the layers to a separating funnel. Make prominent quenching zone
the combined layers alkaline with ammonia R and shake with Benzoic acid: a quenching zone A quenching zone (benzoic acid)
not fewer than 3 quantities each of 30 mL of methylene
chloride R until the alkaloids are completely extracted. Cinnamic acid: a prominent
quenching zone
Combine the organic layers, add 4 g of anhydrous sodium
sidfate R and allow to stand for 30 min with occasional
shaking. Decant the methylene chloride and filter. Wash the A quenching zone
sodium sulfate with 3 quantities each of 10 mL of methylene
A very prominent quenching
chloride R. Combine the organic extracts, evaporate to zone
dryness on a water-bath. Heat the residue in an oven at Vanillin: a quenching zone A quenching zone (vanillin)
Series of unresolved zones
millilitres of methylene chloride R, evaporate to dryness on a including a quenching zone
water-bath and heat the residue in an oven at 100-105 °C for
15 min again. Dissolve the residue in a few millilitres of
methylene chloride R. Add 20.0 mL of 0.01 M sulfuric acid and
remove the methylene chloride by evaporation on a water
TESTS
bath. Titrate the excess of acid with 0.02 M sodium hydroxide
Styrax benzoin
using methyl red mixed solution R as indicator.
A. To 0.2 g of the finely powdered herbal drug add 10 mL
Calculate the percentage content of total alkaloids, expressed of ethanol (96 per cent) R. Shake vigorously until almost
as hyoscyamine, using the following expression: completely dissolved and filter. Place 5 mL of the filtrate in a
test-tube and add 0.5 mL of a 50 g/L solution of feme
57.88 X (20 - ท) chloride R in ethanol (96 per cent) R. A green colour is
100 X m produced. No yellow colour is produced.
B. Thin-layer chromatography (2.2.27).
ท = volume of 0.02 M sodium hydroxide used, in millilitres,
Test solution Sonicate 0.2 g of the finely powdered herbal
m = mass of the herbal drug used, in grams.
drug in 5 mL of ethanol (96 per cent) R and filter. Collect the
_______________________________________________________________Ph Eur filtrate.
Reference solution Dissolve 20 mg of benzoic acid R, 10 mg of
trans-cinnamic acid R, 4 mg of vanillin R and 20 mg of methyl
cinnamate R in 10 mL of ethanol (96 per cent) R.
Siam Benzoin ***\ Plate TLC silica gel F2<M plate R.
Mobile phase glacial acetic acid Rj di-isopropyl ether R, hexane R
(Ph. Eur. monograph 2158) *** (10:40:60 VIVIV).
Preparation Application 10 pL as bands.
Siam Benzoin Tincture Development Over a path of 12 cm.
Ph Elf_______________________________________________________________
Drying In air.
DEFINITION Detection Examine in ultraviolet light at 254 nm.
Resin obtained by incising the trunk of Styrax tonkinensis Results The chromatogram obtained with the test solution
(Pierre) Craib ex Hartwich. shows no zone in the same position as the zone due to
Content cinnamic acid in the chromatogram obtained with the
45.0 per cent to 55.0 per cent of total acids, calculated as reference solution.
benzoic acid (C7H6O2; Mr 122.1) (dried drug). Matter insoluble in ethanol
CHARACTERS Maximum 5 per cent.
Characteristic odour of vanillin. To 2 g of the powdered herbal drug add 25 mL of ethanol
(90 per cent VtV) R. Boil until almost completely dissolved.
IDENTIFICATION
Filter through a previously tared sintered-glass filter (16)
A. Siam benzoin occurs as opaque, granular, rounded or
(2.1.2) and wash with 3 quantities, each of 5 mL, of boiling
ovoid masses (tears), varying in size from a few millimeters
ethanol (90 per cent VtV) R. Heat the glass filter and its
up to 3 cm, separated or sometimes agglomerated together
contents in an oven at 100-105 °C for 2 h. Weigh after
by a reddish-brown, transparent resin. Individual tears are
cooling.
yellowish-white to reddish externally with a waxy, whitish
fracture which becomes reddish on exposure to air. Loss on drying (2.2.32)
Maximum 5.0 per cent, determined on 2.00 g of the coarsely
B. Examine the chromatograms obtained in test B for Styrax
powdered herbal drug by drying in vacuo for 4 h.
benzoin.
Results See below the sequence of the zones present in the
Total ash (2.4.16)
chromatograms obtained with the reference solution and the Maximum 2.0 per cent.
2016 Sumatra Benzoin IV-lOl
assay TESTS
Place 0.750 g of the finely powdered herbal drug in a Sumatra benzoin tincture
250 mL borosilicate glass flask and add 15.0 mL of 0.5 M Thin-layer chromatography (2.2.27).
alcoholic potassium hydroxide. Boil under a reflux condenser on Test solution The tincture to be examined.
a water-bath for 30 min. Allow to cool and rinse the
Reference solution Dissolve 20 mg of benzoic acid R, 10 mg of
condenser with 20 mL of ethanol (96 per cent) R. Titrate the
trans-cinnamic acid R) 4 mg of vanillin R and 20 mg of methyl
excess of potassium hydroxide with 0.5 M hydrochloric acid.
cinnamate R in 20 mL of ethanol of the same concentration
Determine the end-point potentiometrically (2.2.20). Carry
as that used for the production of the tincture.
out a blank titration.
Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel
1 mL of 0.5 M alcoholic potassium hydroxide is equivalent to
F254 plate R (2-10 pm)].
61.05 mg of benzoic acid (C7H6O2).
Mobile phase glacial acetic acid R) di-isopropyl ether R, hexane R
----------------------------------------------------- --------------------------------------------------- Ph Eur
(10:40:60 VIVIV).
Drying In air.
Siam Benzoin Tincture ** ** Detection Examine in ultraviolet light at 254 nm.
(Ph. Eur. monograph 2157) * ** Results The chromatogram obtained with the test solution
Ph Elf __ _________________ does not show any zone in the same position as the zones
due to cinnamic acid and methyl cinnamate in the
DEFINITION chromatogram obtained with the reference solution.
Tincture produced from Siam benzoin (2158).
Ethanol (2.9.10)
Content 95 per cent to 105 per cent of the content stated on the
Minimum 5.0 per cent m/m of total acids, calculated as label.
benzoic acid (C7H602; Mr 122.1).
ASSAY
PRODUCTION Place 3.50 g in a 250 mL borosilicate glass flask and add
The tincture is produced from 1 part of the drug and 5 pans 15.0 mL of 0.5 M alcoholic potassium hydroxide. Boil under a
of ethanol (75 per cent VIV to 96 per cent VIV) by a suitable reflux condenser on a water-bath for 30 min. Allow to cool
procedure. and rinse the condenser with 20 mL of ethanol
CHARACTERS (96 per cent) R. Titrate the excess of potassium hydroxide
Appearance with 1 M hydrochloric acid) determining the end-point
Orange-yellow liquid. potentiometrically (2.2.20). Carry out a blank titration.
It has a characteristic odour of vanillin. 1 mL of 0.5 M alcoholic potassium hydroxide is equivalent to
IDENTIFICATION
_____________________________________________________________ Ph Elf
A. Place 10 mL in a test tube; add 0.5 mL of a 50 g/L
solution of ferric chloride R in ethanol (96 per cent) R. A green
colour is produced.
Sumatra benzoin tincture. Sumatra Benzoin *
Results See below the sequence of the zones present in the Benzoin **
chromatograms obtained with the reference solution and the (Ph. Eur. monograph 1814)
Preparations
Benzoin Inhalation
solution.
Compound Benzoin Tincture
Sumatra Benzoin Tincture
Top of the plate
Ph Elf---------------------------------------------------------------------------------------------------------
DEFINITION
Methyl cinnamate: a very Resin obtained by incising the trunk of Styrax benzoin
prominent quenching zone
Dryander.
Benzoic acid: a quenching zone A quenching zone (benzoic acid)
Content
Cinnamic acid: a prominent 25.0 per cent to 50.0 per cent of total adds, calculated as
quenching zone
benzoic add (C7H6O2; Mr 122.1) (dried drug).
IDENTIFICATION
A quenching zone
A. Sumatra benzoin occurs as creamy white, rounded to
A very prominent quenching ovoid tears, which may be embedded in a dull greyish-brown
zone or reddish-brown matrix. It is hard and brittle and the
Vanillin: a quenching zone A quenching zone (vanillin) fractured surface is dull and uneven.
Series of unresolved zones B. Examine the chromatograms obtained in test B for Styrax
including a quenching zone tonkinensis.
Reference solution Test solution Results See below the sequence of quenching zones present in
the chromatograms obtained with the reference solution and
IV-102 Benzoin Preparations 2016
the test solution. Furthermore, other faint quenching zones Results The chromatogram obtained with the test solution
may be present in the chromatogram obtained with the test shows 2 faint zones in the same positions as the dark zones
solution. due to benzoic acid and vanillin in the chromatogram
obtained with the reference solution.
Top of the plate Matter insoluble in ethanol
A very intense dark zone
To 2.0 g of the powdered herbal drug add 25 mL of ethanol
(90 per cent V/V) R. Boil until almost completely dissolved.
Methyl cinnamate: a very intense Filter through a tared sintered-glass filter (16) (2.1.2) and
dark zone wash with 3 quantities, each of 5 mL, of boiling ethanol
Benzoic acid: a dark zone A very weak dark zone (benzoic (90 per cent V/V) R. Heat the glass filter and its contents in
acid) an oven at 100-105 °C for 2 h. Allow to cool and weigh.
Cinnamic acid: an intense dark A very intense dark zone
(cinnamic acid) Loss on drying (2.2.32)
coarsely powdered herbal drug by drying in vacuo for 4 h.
A dark zone
Total ash (2.4.16)
A very intense dark zone Maximum 2.0 per cent.
A dark zone ASSAY
Vanillin: a dark zone A very weak dark zone (vanillin) Place 0.750 g of the finely powdered herbal drug in a
250 mL borosilicate glass flask and add 15.0 mL of 0.5 M
Series of unresolved zones
alcoholic potassium hydroxide. Boil under a reflux condenser on
including 2 dark zones
a water-bath for 30 min. Allow to cool and rinse the
Reference solation Test solution
condenser with 20 mL of ethanol (96 per cent) R. Titrate the
excess of potassium hydroxide with 0.5 M hydrochloric acid,
determining the end-point potentiometrically (2.2.20). Carry
TESTS
out a blank titration.
Dammar gum
Thin-layer chromatography (2.2.27). 1 mL of 0.5 M alcoholic potassium hydroxide is equivalent to
Test solution Dissolve 0.2 g of the drug to be examined with
_______________________________________________________________ Ph Eur
gentle heating in 10 mL of etitanol (90 per cent V/V) R and
centrifuge.
Plate TLC aluminium oxide G plate R.
Mobile phase light petroleum R4, ether R (40:60 V/V).
Application 5 J1L. Sumatra Benzoin Tincture * *
Development Over a path of 10 cm. (Ph. Eur. monograph 1813) ***
Drying In air. Ph Eur___________________________________________________________ ___
Detection Spray with anisaldehyde solution R and heat at
DEFINITION
100-105 °C for 5 min.
Tincture produced from Sumatra benzoin (1814).
Results The chromatogram obtained does not show any
prominent spot with an Rp between 0.4 and 1.0. Content
Minimum 4.0 per cent ni/ni of total acids, calculated as
Sty rax tonkinensis benzoic acid (C7H6O2; Afr 122.1).
A. To 0.2 g of the finely powdered herbal drug add 10 mL
of ethanol (96 per cent) R. Shake vigorously until almost PRODUCTION
completely dissolved and filter. Place 5 mL of the filtrate in a The tincture is produced from 1 part of the drug and 5 parts
test-tube and add 0.5 mL of a 50 g/L solution of ferric of ethanol (75 per cent P7I7 to 96 per cent V/V) by a suitable
chloride R in ethanol (96 per cent) R. A yellowish, slightly procedure.
green colour is produced. CHARACTERS
B. Thin-layer chromatography (2.2.27). Appearance
Test solution Sonicate 0.2 g of the finely powdered herbal Orange-yellow liquid.
drug in 5 mL of ethanol (96 per cent) R and filter. Collect the IDENTIFICATION
filtrate. Examine the chromatograms obtained in the test for Siam
Reference solution Dissolve 20 mg of benzoic acid R, 10 mg of benzoin tincture.
trans-cinnamic acid R, 4 mg of vanillin R and 20 mg of methyl Results See below the sequence of quenching zones present in
cinnamate R in 10 mL of ethanol (96 per cent) R. the chromatograms obtained with the reference solution and
Plate TLC silica gel F254 plate R (5-40 pm) [°r TLC sihca รel the test solution. Furthermore, other faint quenching zones
F254 plate R (2-10 pm)]. may be present in the chromatogram obtained with the test
Mobile phase glacial acetic acid R, di-isopropyl ether R, hexane R solution.
(10:40:60 v/v/v).
Drying In air.
Detection Examine in ultraviolet light at 254 nm.
2016 Benzoin Preparations IV-103
Top of the plate

Compound Benzoin Tincture
A very intense dark zone
Friars’ Balsam
DEFINITION
Methyl cinnamate ะ a very intense A dark zone Barbados Aloes or Cape Aloes 20 g
dark zone
Benzoic acid: a dark zone
Prepared storax of commerce 100 g
A very weak dark zone (benzoic
acid) Sumatra Benzoin crushed 100 g
Cinnamic acid: an intense dark A very intense dark zone (cinnamic Ethanol (90 per cent) Sufficient to produce 1000 mL
acid)
Extemporaneous preparation
The following directions apply.
A dark zone
Macerate the Barbados Aloes or Cape Aloes, ±e prepared
A very intense dark zone storax and the Sumatra Benzoin with 800 mL of Ethanol
A dark zone
(90 per cent) in a closed vessel for not less than 2 days,
shaking occasionally, filter and pass sufficient Ethanol
Vanillin: a dark zone A very weak dark zone (vanillin) (90 per cent) through the filter to produce 1000 mL.
Series of unresolved dark zones The tincture complies with the requirements for Tinctures stated
Reference soludon Test soludon under Extracts and with the following requirements.
Content of total balsamic acids
Not less than 4.5% w/v, calculated as cinnamic acid,
TESTS C9H8O2.
Siam benzoin tincture
TESTS
Ethanol content
Test solution The tincture to be examined. 70 to 76% v/v, Appendix vm F, Method in.
Reference solution Dissolve 20 mg of benzoic acid R, 10 mg of Dry residue
trans-cinnamic acid R, 4 mg of vanillin R and 20 mg of methyl 15 to 19% w/v.
cinnamate R in 20 mL of ethanol of the same concentration
as that used for the production of the tincture. Relative density
0.880 to 0.910, Appendix V G.
F254 plate R (2-10 pm)]. ASSAY
Mobile phase glacial acetic acid R, di-isopropyl ether R, hexane R Carry out the Assay described under Benzoin Inhalation
(10:40:60 VIVIV). using 10 mL of the tincture. Each mL of 0.1m sodium
hydroxide KS is equivalent to 14.82 mg of total balsamic
acids, calculated as cinnamic acid, C9H8O2.
Drying In air.
does not show zones due to benzoic acid and vanillin that are
Benzoin Inhalation
more intense than the corresponding zones in the Benzoin Inhalation Vapour
chromatogram obtained with the reference solution. DEFINITION
Ethanol (2.9.10) Benzoin Inhalation is an inhalation vapour, solution.
95 per cent to 105 per cent of the content stated on the Sumatra Benzoin crushed 100 g
label. Prepared storax of 50 g
commerce
ASSAY Ethanol (96 per cent) Sufficient to produce 1000 mL
Place 3.50 g in a 250 mL borosilicate glass flask and add
15.0 mL of 0.5 M alcoholic potassium hydroxide. Boil under a In making Benzoin Inhalation, Ethanol (96 per cent) may be
replaced by Industrial Methylated Spirit1.
reflux condenser on a water-bath for 30 min. Allow to cool
and rinse the condenser with 20 mL of ethanol Extemporaneous preparation
(96 per cent) R. Titrate ±e excess of potassium hydroxide The following directions apply.
with 1 M hydrochloric acid, determining the end-point Macerate the crushed Sumatra Benzoin and the prepared
potentiometrically (2.2.20). Carry out a blank titration. storax with 750 mL of Ethanol (96 per cent) for 24 hours.
1 mL of 0.5 M alcoholic potassium hydroxide is equivalent to Filter and pass sufficient Ethanol (96 per cent) through the
61.05 mg of benzoic acid (C7H6O2). filter to produce 1000 mL
—1 1__________________________________________________________ Ph Eur The inhalation complies with the requirements stated under
Preparations for Inhalation and with the following requirements.
Not less than 3.0% w/v, calculated as cinnamic add,
C9H802.
Total solids
9.0 to 12.0% w/v when determined by drying at 105 for
4 hours, Appendix XI A. Use 2 mL.
IV-104 Berbens Aristata 2016
ASSAY (80%). Combine the supernatants and dilute to 20 mL with

Boil 10 mL with 25 mL of 0.5m ethanolic potassium hydroxide methanol (80%).
under a reflux condenser for 1 hour. Evaporate the ethanol, (2) 0.04% w/v each of berberine chloride BPCRS and palmatine
disperse the residue in 50 mL of hot water, cool, add 80 mL chloride in methanol (80%)).
of water and 1.5 g of magnesium sulfate dissolved in 50 mL of
water. Mix thoroughly and allow to stand for 10 minutes.
Filter, wash the residue on the filter with 20 mL of water, (a) Use as the coating silica gel F254.
acidify the combined filtrate and washings with hydrochloric (b) Use the mobile phase as described below.
acid and extract with four 40-mL quantities of ether. Discard (c) Apply 20 pL of each solution as 6 mm bands.
the aqueous solution, combine the ether extracts and extract (d) Develop the plate to 15 cm.
with successive quantities of 20, 20, 10, 10 and 10 mL of
(e) After removal of the plate, dry in air and examine under
sodium hydrogen carbonate solution, washing each aqueous
extract with the same 20 mL of ether. Discard the ether ultraviolet light (254 nm).
layers, carefully acidify the combined aqueous extracts with MOBILE PHASE
hydrochloric acid and extract with successive quantities of 30, 10 volumes of anhydrous formic acid, 10 volumes of water and
20, 20 and 10 mL of chloroform, filtering each extract through 80 volumes of ethyl acetate.
anhydrous sodium sulfate supported on absorbent cotton. Distil
SYSTEM SUITABILITY
the chloroform from the combined filtrates until 10 mL
remains and remove the remainder in a current of air. The test is not valid unless the chromatogram obtained with
Dissolve the residue, with the aid of gentle heat, in 10 mL of solution (2) shows two clearly separated bands.
ethanol (96%), previously neutralised to phenol red solution, CONFIRMATION
cool and titrate with 0.1m sodium hydroxide vs using phenol The chromatogram obtained with solution (1) shows a
red solution as indicator. Each mL of 0.1m sodium hydroxide principal yellow band corresponding in colour and position to
PS is equivalent to 14.82 mg of total balsamic acids, the band obtained for berberine chloride in solution (2), a
calculated as cinnamic acid, C9H8O2. yellow band corresponding in colour and position to the
band obtained for palmatine in solution (2) and several other
1 Statutory regulations governing the use of Industrial Methylated Spirit must
bands as shown in the table. Other bands may be present.
be observed.
Top of the plate
Berberis Aristata
DEFINITION
Berberis Aristata is the dried, cut stem of Berberis aristata Yellow band (berberine chloride) Yellow band (berberine chloride)
Faint yellow band
DC.
Yellow band (palmatine) Yellow band (palmatine)
It contains not less than 1.4% of berberine (C2qH19NO5),
calculated with reference to the dried material.
IDENTIFICATION Purple band
A. The cut pieces of stem are subcylindrical, often branched Solution (1) Solution (2)
and somewhat swollen at the nodes, from about 15-20 mm
diameter and varying in length. The bark is soft, about TESTS
4-8 mm thick, with a yellowish brown outer surface, finely
d-T etrahydropalmatine
wrinkled longitudinally or deeply furrowed, peeling off in
Carry out the method for liquid chromatography,
places and exposing the inner dark yellow wood. Fracture
Appendix III D, using the following solutions.
short in the region of the bark, hard and fibrous in the wood.
(1) To 0.5 g of powdered sample, add 400 mL of a mixture
B. Reduce to a powder. The powder is yellowish brown.
of equal volumes of acetonitrile and 0.1% v/v orthophosphoric
acid. Mix with the aid of ultrasound for 40 minutes and
The powder contains numerous fragments of xylem, the
allow to cool. Dilute to 500 mL with the mobile phase and
vessels reticulately and spirally thickened, some tracheids;
filter through a 0.45-pm filter.
thick-walled, short, spindle-shaped, lignified, yellowish fibres
of the phloem and xylem with a wide lumen; stone cells (2) 0.01% w/v each of palmatine chloride and berberine
elongated with thick, pitted walls, some containing a single chloride BPCRS in the mobile phase.
calcium oxalate crystal, normally present in groups; (3) 0.01% w/v of D-tetrahydropalmatine hydrochloride in the
parenchyma cells of the medullary rays, some with yellow- mobile phase.
brown contents, single prism crystals of calcium oxalate, or CHROMATOGRAPHIC CONDITIONS
simple starch granules; cork cells yellowish-brown, thin (a) Use a stainless steel column (15 cm X 4.6 mm) packed
walled; numerous scattered starch grains and calcium oxalate with end-capped octadecylsilyl silica gel for chromatography
crystals. (5 pm) (Phenomenex Luna C18 is suitable).
c. Carry out the method for thin-layer chromatography, (b) Use isocratic elution and the mobile phase described
Appendix in A, using the following solutions. below.
(1) Add 4 mL of methanol (80%) to 250 mg of the powdered (c) Use a flow rate of 1.2 mL per minute.
herbal drug (180) in a centrifuge tube. Mix with the aid of
(d) Use an ambient column temperature.
ultrasound for 10 minutes. Centrifuge at 3000 rpm for
5 minutes and collect the clear supernatant. Repeat the (e) Use a detection wavelength of 235 nm.
extraction twice with a further two 2-mL portions of methanol (f) Inject 10 pL of each solution.
2016 Bilberry IV-105
When the chromatograms are recorded under the prescribed d = percentage loss on drying of the herbal drug being
conditions the retention times relative to palmatine (retention examined.
time = about 8 minutes) are berberine chloride = about 1.1;
D-tetrahydropalmitine = about 1.6. STORAGE
Berbens Aristata should be protected from moisture.
MOBILE PHASE
27 volumes of acetonitrile and 73 volumes of a 1.36% w/v

solution of potassium dihydrogen orthophosphate.
SYSTEM SUITABILITY Dried Bilberry ** *
(Dried Bilberry Fruit, Ph Eur monograph 1588) ***
to palmatine and berberine chloride is at least 2.0. Ph Eur _ ___________________________________________________________
CONFIRMATION DEFINITION
In the chromatogram obtained with solution (1), there are no Dried ripe fruit of Vaccinium myrtillus L.
peaks corresponding to the peak due to D-tetrahydropalmitine Content
in the chromatogram obtained with solution (3). Minimum 1.0 per cent of tannins, expressed as pyrogallol
Loss on drying (C6H603; Mr 126.1) (dried drug).
When dried for 2 hours at 105°, loses not more than 10.0% CHARACTERS
of its weight, Appendix IX D. Use 1 g. Sweet and slightly astringent taste.
Total Ash IDENTIFICATION
Not more than 3.0%, Appendix XI J, Method II. A. Dried bilberry is a dark blue, subglobular, shrunken berry
ASSAY about 5 mm in diameter, with a scar at the lower end and
Carry out the method for liquid chromatography. surmounted by the persistent calyx, which appears as a
Appendix in D, using the following solutions. circular fold and the remains of the style. The deep violet,
(1) To 0.5 g of powdered sample, add 400 mL of a mixture fleshy mesocarp contains numerous small, brown, ovoid
of equal volumes of acetonitrile and 0.1% v/v orthophosphoric seeds.
acid. Mix with the aid of ultrasound for 40 minutes and B. Reduce to a powder (355) (2.9.72). The powder is violet
allow to cool. Dilute to 500 mL with the mobile phase and brown. Examine under a microscope using chloral hydrate
filter through a 0.45-pm filter. solution R. The powder shows: violet-pink sclereids from the
(2) 0.01% w/v each of palmatine chloride and berberine endocarp and the mesocarp, usually aggregated, with thick,
chloride BPCRS in the mobile phase. channelled walls; reddish-brown fragments of the epicarp
consisting of polygonal cells with moderately thickened walls;
brownish-yellow fragments of the outer seed testa made up of
The chromatographic conditions described under the test for elongated cells with U-shaped thickened walls; clusters and
D-tetrahydropalmatine may be used. prisms crystals of various size of calcium oxalate.
MOBILE PHASE c. Thin-layer chromatography (2.2.27)
27 volumes of acetonitrile and 73 volumes of a 1.36% w/v Test solution To 2 g of the powdered herbal drug (355)
solution of potassium dihydrogen orthophosphate. (2.9.12) add 20 mL of methanol R. Shake for 15 min and
SYSTEM SUITABILITY filter.
The test is not valid unless, in the chromatogram obtained Reference solution Dissolve 5 mg of chrysanthemin R in 10 mL
with solution (2), the resolution factor between the peaks due of methanol R.
to palmatine and berberine chloride is at least 2.0. Plate TLC silica gel plate R.
DETERMINATION OF CONTENT Mobile phase anhydrous formic acid R, water R, butanol R
Using the retention time and the peak area from the (16:19:65 VIVIV).
chromatogram obtained with solution (2), locate and Application 10 pL, as bands.
integrate the peak due to berberine chloride in the Development Over a path of 10 cm.
chromatogram obtained with solution (1). Calculate the
Drying In air.
content of berberine in the sample using the declared content
of berberine in berberine chloride BPCRS and the following Detection Examine in daylight.
expression: Results See below the sequence of ±e zones present in the
chromatograms obtained with the reference and test
A. m, ¥1100 solutions.
v/m.xpx 100-d
Top of the plate
A] = area of the peak due to berberine in the A violet-red zone of low Intensity
chromatogram obtained with solution (1),
Chrysanthemin: a violet-red A principal violet-red zone
A2 = area of the peak due to berberine in the
zone
chromatogram obtained with solution (2), A compact set of other principal
mi = weight of the drug being examined in mg, zones ะ
m2 = weight of berberine chloride BPCRS in mg, - a violet-red zone
V! ะ= dilution volume of solution (1) in mL,
- several violet-blue zones
v2 ะะะ dilution volume of solution (2) in mL)
p = percentage content of berberine in berberine chloride Reference solution Test solution
BPCRS,
IV-106 Bilberry 2016
TESTS Top of the plate

A violet-red zone
powdered herbal drug by drying in an oven at 105 °C for Chrysanthemin: a violet-red A principal violet-red zone
2 h.
A compact set of other principal
Total ash (2.4.16) zones:
Maximum 5.0 per cent. - a violet-red zone
ASSAY - several violet-blue zones
Tannins (2.8.14)
------------------------------------------------------------------------------ ---------------------- ------ Ph Eur
TESTS
Total ash (2.4.16)
Fresh Bilberry ****** Loss on drying (2.2.32)
80.0 per cent to 90.0 per cent, determined on 5.000 g of the
(Fresh Bilberry Fruit, Ph Eur monograph 1602) * ** freshly crushed drug by drying in an oven at 105 °C.
Preparation ASSAY
Fresh Bilberry Fruit Dry Extract, Refined and Standardised Crush 50 g extemporaneously. To about 5.00 g of the
Ph Eur_______________________________________________________________ crushed, accurately weighed drug, add 95 mL of methanol R.
DEFINITION Stir mechanically for 30 min. Filter into a 100.0 mL
Fresh or frozen, ripe fruit of Vaccinium myrtillus L. volumetric flask. Rinse the filter and dilute to 100.0 mL with
methanol R. Prepare a 50-fold dilution of this solution in a
Content 0.1 per cent VIV solution of hydrochloric acid R in methanol R.
Minimum 0.30 per cent of anthocyanins, expressed as
Measure the absorbance (2.2.25) of the solution at 528 nm,
cyanidin 3-O-glucoside chloride (chrysanthemin,
using a 0.1 per cent VIV solution of hydrochloric acid R in
C2]H2iC!On; Afr 484.8) (dried drug).
methanol R as the compensation liquid.
CHARACTERS Calculate the percentage content of anthocyanins, expressed
Sweet and slightly astringent taste. as cyanidin 3-O-glucosidc chloride, using the following
IDENTIFICATION expression:
A. The fresh fruit is a blackish-blue globular berry about
5 mm in diameter. Its lower end shows a scar or, rarely, a A X 5000
fragment of the pedicel. The upper end is flattened and 718 X m
surmounted by the remains of the persistent style and of the
calyx, which appears as a circular fold. The violet, fleshy 718 = specific absorbance of cyanidin 3-O-glucoside
mesocarp includes 4 to 5 locules containing numerous small, chloride at 528 nm;
brown, ovoid seeds. A = absorbance at 528 nm;
B. The crushed fresh fruit is violet-red. Examine under a m = mass of the substance to be examined in grams.
microscope using chloral hydrate solution R. It shows violet
STORAGE
pink sclereids from the endocarp and the mesocarp, usually
aggregated, with thick, channelled walls; reddish-brown When frozen, store at or below -18 °C.
fragments of the epicarp consisting of polygonal cells with _______________________________________________________________ Ph Eur
moderately thickened walls; brownish-yellow fragments of the

outer layer of the testa composed of elongated cells with
U-shaped thickened walls; cluster crystals of calcium oxalate,
Refined and Standardised Fresh * *
Test solution To 5 g of the freshly crushed drug, add 20 mL
of methanol R. Stir for 15 min and filter. Bilberry Fruit Dry Extract *****
Reference solution Dissolve 5 mg of chrysanthemin R in 10 mL (Ph. Eur. monograph 2394)
of methanol R. Ph Eur______________________________________________________________
Plate TLC silica gel plate R. DEFINITION

Mobile phase anhydrous formic acid R, water R, butanol R Refined and standardised dry extract produced from Bilberry
(16:19:65 VIVIV). fruit, fresh (1602).
Application 10 pL, as bands. Content
Development Over a path of 10 cm. 32.4 per cent to 39.6 per cent of anthocyanins, expressed as
Drying In air. cyanidin 3-O-glucoside chloride [chrysanthemin
(C21H21CIO11; Mt 484.8)] (dried extract).
Detection Examine in daylight.
Results See below the sequence of the zones present in the PRODUCTION
chromatograms obtained with the reference solution and the The extract is produced from the herbal drug by a suitable
procedure using ethanol (96 per cent VIV) or methanol
test solution.
(minimum 60 per cent VIV). Refinement may be performed
by ion-exchange chromatography.
2016 Bilberry Fruit Preparations IV-107
CHARACTERS with the solvent mixture. Dilute 2.0 mL of this solution to

Appearance 100.0 mL with dilute phosphoric acid R.
Dark reddish-violet, amorphous, hygroscopic powder. Reference solution (b) Dissolve 0.1250 g of bilberry dry
IDENTIFICATION extract CRS in the solvent mixture and dilute to 25.0 mL
First identification B with the solvent mixture. Dilute 5.0 mL of this solution to
20.0 mL with dilute phosphoric acid R.
Second identification A
Column".
A. Thin-layer chromatography (2.2.27).
— size". I = 0.250 m, 0 = 4.6 mm;
Test solution Dissolve 0.10 g of the extract to be examined in — stationary phase: octadecylsilyl silica gel for chromatography R
25 mL of methanol R. Stir for 15 min and filter. (5 pm);
Reference solution Dissolve 2 mg of chrysanthemin R and 2 mg — temperature: 30 °C.
of myrtillin R in 5 mL of methanol R. Mobile phase:
Plate TLC plate coated with cellulose for chromatography R — mobile phase A: anhydrous formic acid R, water R
(5-40 pm) [or TLC plate coated with cellulose for (8.5:91.5 F7F);
chromatography R (2-10 pm)]. — mobile phase B: anhydrous formic acid R> acetonitrile R)
Mobile phase". methanol R, water R, (8.5:22.5:22.5:41.5 VIVIVIV)’,
— mobile phase A ะ hydrochloric acid R, acetic acid R,
water R (3:15:82 V/V/Vfi (min) (per cent V/V) (per cent V/V)
— mobile phase B: water R, acetic acid R (40:60 พF). 0 - 35 93 -> 75 7 -» 25
35 - 45 75 -> 35 25 -> 65
Development A Over a path of 10 cm [or 6 cm] with mobile
phase A. 45 - 46 35 -> 0 65 -> 100
Drying A In warm air. 46 - 50 0 100
Development B Over a path of 10 cm [or 6 cm] with mobile

phase B. Flow rate 1.0 mUmin.
Drying B In air. Detection spectrophotometer at 535 run.
Detection Examine in daylight. Injection 10 pL.
Results See below the sequence of zones present in the Identification of peaks Use the chromatogram supplied with
chromatograms obtained with the reference solution and the bilberry dry extract CRS and the chromatograms obtained with
test solution. Furthermore, other faint zones may be present reference solutions (a) and (b) to identify the peaks due to
in the chromatogram obtained with the test solution. the anthocyanins and the anthocyanidins.
Retention times The retention times and the elution order of
Top of the plate the peaks are similar to those shown in the chromatogram
(Figure 2394.-1).
A violet-red zone — peak-to-valley ratio: minimum 2.0, where Hp = height
above the baseline of the peak due to cyanidin 3-O-
Chrysanthemin: a violet-red zone A violet-red zone
(chrysanthemin)
galactoside (peak 3) and Hv = height above the baseline
of the lowest point of the curve separating this peak from
Myrtillin: a violet-red zone A violet-red zone (myrtillin)
the peak due to delphinidin 3-O-arabinoside (peak 4).
Calculate the percentage content of total anthocyanidins,
Reference solution Test solution expressed as cyanidin chloride, using the following
expression:
B. Liquid chromatography (2.2.29) as described in the test Al X 7ท2 X 100 X p
for total anthocyanidins. 7ท1 X A2 X 1250
The characteristic anthocyanin peaks (peaks 1-8, 10-15
and 17) in the chromatogram obtained with the test solution A1 = sum of the areas of the peaks due to the
are similar in their retention times to those in the anthocyanidins (peaks 9, 16, 18-20) in the
chromatogram obtained with reference solution (b). chromatogram obtained with the test solution;
TESTS A2 = area of the peak due to cyanidin chloride (peak
Loss on drying (2.8.17) 16) in the chromatogram obtained with reference
Maximum 4.5 per cent. solution (a);
m1 = mass of the extract to be examined used to
Total ash (2.4.16) prepare the test solution, in grams;
Maximum 2.0 per cent. m2 = mass of cyanidin chloride CRS used to prepare
Total anthocyanidins reference solution (a), in grams;
Liquid chromatography (2.2.29). Maintain the solutions at p = percentage content of cyanidin chloride in
4 °C cyanidin chloride CRS.
Solvent mixture hydrochloric acid R, methanol R (2:98 V!V). Limits Not more than 1.0 per cent of total anthocyanidins,
Test solution Dissolve 0.1250 g of the extract to be examined expressed as cyanidin chloride.
in the solvent mixture and dilute to 25.0 mL with the solvent
mixture. Dilute 5.0 mL of this solution to 20.0 mL with ASSAY
Liquid chromatography (2.2.29) as described in the test for
dilute phosphoric acid R.
total anthocyanidins with the following modification.
Reference solution (a) Dissolve 10.0 mg of cyanidin
chloride CRS in the solvent mixture and dilute to 25.0 mL Injection Test solution and reference solution (b).
IV-108 Birch Leaf 2016
1. delphinidin 3-Ogalactoside chloride 11. petunidin 3-O-arabinoside chloride
2. myrtillin (delphinidin 3-O-glucoside chloride) 12. peonidin 3-O-glucoside chloride
3. cyanidin 3-O-galactoside chloride 13. malvidin 3-O-galactoside chloride
4. delphinidin 3-O-arabinoside chloride 14. peonidin 3-O-arabinoside chloride
5. chrysanthemin (cyanidin 3-O-glucoside chloride) 15. malvidin 3-O-glucoside chloride
6. petunidin 3-O-galactoside chloride 16. cyanidin chloride
7. cyanidin 3-O-arabinoside chloride 17. malvidin 3-O-arabinoside chloride
8. petunidin 3-O-glucoside chloride 18. petunidin chloride
9. delphinidin chloride 19. peonidin chloride
10. peonidin 3-O-galactoside chloride 20. malvidin chloride
Figure 2394.-1. - Chromatogram for the assay of refined and standardised fresh bilberry fruit dry extract
Calculate the percentage content of total anthocyanins,

expressed as cyanidin 3-O-glucoside chloride, using the
Birch Leaf *
following expression: (Ph. Eur. monograph 1174) ***
Al X m2 X p Ph Eur_______________________________________________________________
mi X >12 DEFINITION
A\ = sum of the areas of the peaks due to the Whole or fragmented, dried leaves of Betula pendula Roth
anthocyanins (peaks 1-8, 10-15 and 17) in the and/or Betula pubescent Ehrh. as well as hybrids of both
chromatogram obtained with the test solution; species.
A2 = area of the peak due to cyanidin 3-O-glucoside Content
chloride (peak 5) in the chromatogram obtained Minimum 1.5 per cent of flavonoids, expressed as hyperoside
with reference solution (b); (C21H20O12; Afr 464.4) (dried drug).
= mass of the extract to be examined used to
IDENTIFICATION
A. The leaves of both species are dark green on the adaxial
m2 =ะ mass of bilberry dry extract CRS used to prepare
surface and lighter greenish-grey on the abaxial surface; they
reference solution (b), in grams;
show a characteristic dense reticulate venation. The veins are
p ะะะ percentage content of cyanidin 3-O-glucoside
light brown or almost white.
chloride in bilberry dry extract CRS.
Ph Eur
2016 Birch Leaf IV-109
long, usually 100-200 |im, numerous on the margin of the

lamina [F] or on the epidermises, in surface view [H].
Test solution To 1 g of the powdered herbal drug (355)
(2.9.12) add 10 mL of methanol R and shake. Heat on a
water-bath at 60 °C for 5 min. Cool and filter the solution.
Reference solution Dissolve 1 mg of chlorogenic acid R, 1 mg of
caffeic acid R, 2.5 mg of hyperoside R and 2.5 mg of rutin R in
10 mL of methanol R.
Mobile phase anhydrous formic acid R3 water R, methyl ethyl
Application 10 gL as bands.
Drying In a current of warm air.
Detection Treat with a 10 g/L solution of diphenylboric acid
aminoethyl ester R in methanol R; subsequently treat with a
50 g/L solution of macrogol 400 R in methanol R’> allow to dry
in air for 30 min and examine in ultraviolet light at 365 nm.
solution shows 3 zones in its lower half: in increasing order
of Rf, a yellowish-brown fluorescent zone (rutin), a light blue
fluorescent zone (chlorogenic acid) and a yellowish-brown
fluorescent zone (hyperoside), and in its upper third, a light
blue fluorescent zone (caffeic acid). The chromatogram
obtained with the test solution shows 3 zones similar in
position and fluorescence to the zones due to rutin,
chlorogenic acid and hyperoside in the chromatogram
obtained with the reference solution. The zone due to rutin is
Figure 1174.-1. - Illustration for identification test B of powdered very faint and the zone due to hyperoside is intense. It also
herbal drug of birch leaf shows other yellowish-brown faint fluorescent zones between
the zones due to caffeic acid and chlorogenic acid in the
The leaves of B. pendula are glabrous and show closely chromatogram obtained with the reference solution. Near the
spaced glandular pits on both surfaces. The leaves of solvent front, the red fluorescent zone due to chlorophylls is
B. pendula are 3-7 cm long and 2-5 cm wide; the petiole is visible. In the chromatogram obtained with the test solution,
long and the doubly dentate lamina is triangular or rhomboid between this zone and the zone due to caffeic acid in the
and broadly cuneate or truncate at the base. The angle on chromatogram obtained with the reference solution, there is a
each side is unrounded or slightly rounded, and the apex is brownish-yellow zone due to quercetin.
long and acuminate.
TESTS
The leaves of B. pubescens show few glandular trichomes and Foreign matter (2.8.2)
are slightly pubescent on both surfaces. The abaxial surface Maximum 3 per cent of fragments of female catkins and
shows small bundles of yellowish-grey trichomes at the maximum 3 per cent of other foreign matter.
branch points of the veins. The leaves of B. pubescens are
slightly smaller, oval or rhomboid and more rounded. They
are more roughly and more regularly dentate. The apex is
neither long nor acuminate.
105 °C for 2 h.
greenish-grey. Examine under a microscope using chloral Total ash (2.4.16)
hydrate solution R. The powder shows the following diagnostic Maximum 6.0 per cent.
characters (Figure 1174.-1): numerous fragments of the ASSAY
lamina, in surface view, with straight-walled, adaxial Stock solution In a 100 mL round-bottomed flask introduce
epidermal cells accompanied by underlying palisade 0.200 g of the powdered herbal drug (355) (2.9.12), 1 mL of
parenchyma [E] and cells of the abaxial epidermis a 5 g/L solution of hexamethylenetetramine R, 20 mL of
surrounding anomocytic stomata (2.8.3) [G]; large, free, acetone R and 2 mL of hydrochloric acid Rl. Boil the mixture
glandular trichomes usually measuring 100-120 |im [D]; under a reflux condenser for 30 min. Filter the liquid
fragments of the lamina in transverse section [B], showing through a plug of absorbent cotton into a 100 mL flask.
glandular trichomes on the epidermises [Ba], heterogeneous, Add the absorbent cotton to the residue in the round-
asymmetrical mesophyll containing cluster crystals [Bb] and bottomed flask and extract with 2 quantities, each of 20 mL,
prisms [Be] of calcium oxalate; fragments of spongy of acetone Ri each time boiling under a reflux condenser for
parenchyma [A] accompanied by crystal sheaths [Aa] and 10 min. Allow to cool to room temperature, filter the liquid
cells containing cluster crystals of calcium oxalate [Ab]; through a plug of absorbent cotton then through a filter
fragments of vessels and sclerenchyma fibres [C]. paper into the volumetric flask, and dilute to 100.0 mL with
If B. pubescens is present, the powder also contains unicellular acetone R by rinsing the flask and filter. Introduce 20.0 mL of
covering trichomes with very thick walls, about 80-600 |im the solution into a separating funnel, add 20 mL of water R
rV-110 Bistort Rhizome 2016
and extract the mixture with 1 quantity of 15 mL and then thick-walled fibres [Eb, Jb]; free fragments of vessels [C]; free
3 quantities, each of 10 mL, of ethyl acetate R. Combine the fibres [F]; fragments of parenchyma [D] with rounded cells
ethyl acetate extracts in a separating funnel, wash with พith slightly thickened walls; fragments of collenchyma [K].
2 quantities, each of 50 mL, of water R) and filter the extract Examine under a microscope using a 50 per cent VIV
over 10 g of anhydrous sodium sulfate R into a 50 mL solution of glycerol R. The powder shows rounded or ovoid
volumetric flask and dilute to 50.0 mL with ethyl acetate R. starch granules, simple, about 5-12 pm in diameter, free or
Test solution To 10.0 mL of the stock solution add 1 mL of included in parenchyma cells [A].
aluminium chloride reagent R and dilute to 25.0 mL with a
5 per cent VIV solution of glacial acetic acid R in methanol R.
Compensation liquid Dilute 10.0 mL of the stock solution to
25.0 mL with a 5 per cent VIV solution of glacial acetic
acid R in methanol R.
Measure the absorbance (2.2.25) of the test solution after
30 min, by comparison with the compensation liquid at
425 nm.
Calculate the percentage content of flavonoids, expressed as
hyperoside, using the following expression:
A X 1.25
i.e. taking the specific absorbance of hyperoside to be 500.

----------------------------------------------------------------------------------------------------------- Ph Eur
Bistort Rhizome ★ *
Ph Eur_______________________________________________________________
DEFINITION
Whole or fragmented, dried rhizome of Persicaria bistorta (L.)
Samp. (syn. Polygonum bistorta L.) without adventitious roots.
Content
(C6H6O3; Mr 126.1) (dried drug).
herbal drug of bistort rhizome
CHARACTERS c. Thin-layer, chromatography (2.2.27).
The whole rhizome is up to 13 cm long and 2.5 cm in
diameter. The remnants of the roots are not longer than
(2.9.12) add 10 mL of a mixture of equal volumes of
1 cm and are about 1 mm in diameter.
methanol R and water R3 heat on a water-bath at about 65 °C
IDENTIFICATION for 30 min and filter.
A. The whole rhizome, reddish-brown or blackish-brown, is Reference solution Dissolve 5 mg of fructose R and 5 mg of
thick, twisted, and turned back on itself. Its outer surface catechin R in 5 mL of methanol R.
shows transverse striations and blackish spots. It is flattened Plate TLC silica gel plate R (2-10 pm).
and somewhat depressed on the upper surface, convex on the
Mobile phase water R, anhydrous formic acid R) ethyl acetate R
lower surface. It shows adventitious root scars on the surface.
(5:10:85 VIVIV).
The fracture, pinkish-beige, shows an elliptical zone of
whitish pits corresponding to the vessels. The drug may also Application 2 pL as bands.
be obtained as more or less cylindrical fragments about Development Over a path of 7 cm.
0.3 cm in diameter and up to 1 cm long, with a reddish- Drying In air.
brown outer surface, marked by adventitious root scars and a Detection Treat with anisaldehyde solution R and heat at
pinkish-beige fracture. 100-105 °C for 5 min; examine in daylight.
B. Microscopic examination (2.8.23). The powder is reddish- Results See below the sequence of zones present in the
brown. Examine under a microscope using chloral hydrate chromatograms obtained with the reference solution and the
solution R. The powder shows the following diagnostic test solution. Furthermore, other faint zones may be present
characters (Figure 2384.-1): very numerous cluster crystals of in the chromatogram obtained with the test solution.
calcium oxalate, 15-65 pm in diameter, either free [G] or
included in parenchyma cells [Da]; rare cork fragments in
side view [B] or in surface view [H]; vascular bundles in
longitudinal section [E] or in transverse section [J] including
small pined vessels [Ea, Ja] accompanied by finely pitted,
2016
Black Cohosh IV-111
Top of the plate

Catechin ะ a brown zone A brown zone (catechin)
A brown zone
A violet zone
A brown zone
An orange zone
Fructose: a green zone A green zone (fructose)
TESTS
Paris polyphylla Sm. or Paris quadrifolia L
Microscopic examination {2.8.23}. Examine under a
microscope using chloral hydrate solution R. The presence of
raphides of calcium oxalate, free or in bundles, indicates
adulteration by the rhizome of
p. polyphylla Sm. var. yunnanensis (Franch.) Hand.-Mazz.
or p. polyphylla Sm. var. chinensis (Franch.) H.Hara or
p. quadrifolia L.
Loss on drying {2.2.32}
powdered herbal drug (355) {2.9.12} by drying in an oven at
105 °C.
Total ash {2.4.16}
Ash insoluble in hydrochloric acid (2.8.1} herbal drug of black cohosh
ASSAY wide outer bark, a dark brown cylinder, in which the central
Tannins {2.8.14} region is composed of 3-6 lighter wedges of vascular tissue
Use 1.000 g of the powdered herbal drug (180) {2.9.12}. united at the centre and separated by broad, non-lignified
------------------------------ - -------------------------------------------------------------------------- PhEur
medullary rays.
Fragmented drug More or less angular, irregular pieces of the
rhizome and cylindrical pieces of the roots. The hard, homy
rhizome fragments usually show a dark brown surface
Black Cohosh corresponding to the outer surface and several frequently
striated, light brown surfaces corresponding to the section.
(Ph Eur monograph 2069) The dark brown, more or less cylindrical root fragments are
Ph Elf_______________________ wrinkled longitudinally. The lighter coloured transverse
DEFINITION section shows a distinct cambium line separating a thick
outer bark from a central region composed of 3-6 wedges of
Dried, whole or fragmented rhizome and root of Actaea
vascular tissue united at the centre and separated by broad
racemosa L. (syn. Cimicifuga racemosa (L.) Nutt.).
medullary rays.
Content
B. Microscopic examination {2.8.23}. The powder is light
Minimum 1.0 per cent of triterpene glycosides, expressed as
brown. Examine under a microscope using chloral hydrate
monoammonium glycyrrhizate (C42H65NO16; Afr 840) (dried
drug).
characters (Figure 2069.-1): fragments of the epidermis of
IDENTIFICATION the rhizome, with brown polygonal cells [A]; numerous
A. Whole drug. The rhizome is dark brown, hard, fragments of parenchyma consisting of rounded cells, with
subcylindrical and somewhat knotted; 1.5-2.5 cm in diameter slightly and regularly thickened walls with small triangular
and 2-15 cm long; it shows numerous closely arranged, spaces between them [H]; groups of short vessels with closely
upright or curved branches each terminating in the remains arranged bordered pits [C, J] sometimes accompanied by
of a bud or in a circular, cup-shaped scar. The fracture is finely pitted fibres [Ja]; fragments of the parenchyma of the
homy, the transverse section shows a thin outer bark pith of the rhizome with thick-walled and channelled ovoid
surrounding a ring of numerous pale, narrow wedges of cells [F]; a few fragments of the phloem containing long
vascular tissue alternating with darker medullary rays and a isolated sclereids [D]; fragments of the dermal tissue of the
large central pith. Roots attached to the lower surface of the roots (surface view [E], longitudinal section [B]), consisting
rhizome are usually broken off, leaving circular scars. of brown cells covered by a dark brown cuticle [Ba].
The roots are dark brown, 1-3 mm in diameter, brittle, Examine under a microscope using a 50 per cent VIV
nearly cylindrical or obtusely quadrangular and longitudinally solution of glycerol R. The powder shows abundant starch
wrinkled; the fracture is short; the transverse section shows a granules, spherical or polygonal, simple, 5-10 pm in
IV-112 Black Cohosh 2016
Table 2069.-1. - Application scheme

Track 1 2 3 4 5 6 7
Application 2 2 2 2 2
2
volume (pL)
Reference Reference Reference Reference Test

Solution Test solution Blank
solution (a) soludon (b) solution (a) solution (b) solution
After development, the plate is cut along track 4 (blank). Tracks 1-3 are used for detection of a substitution by c. americana, c. foetida, c. dahurica or
c. heracleifolia (detection A), tracks 5-7 for identification c (detection B).
Table 2069.-2. - Application scheme
Track 1 2 3 4 5 6 า 8 9
Application 20
20 2 2 20 - 20 2 2
volume (pL)
Reference Reference Reference Reference Reference Reference

Solution Test solution Blank Test solution
solution (a) solution (b) solution (c) solution (a) solution (b) solution (c)
After development and examination for detection of c. americana (detection A), the plate is cut along track 5 (blank). Tracks 1-4 are used for detection of
adulteration with c. foetida (detection B), tracks 6-9 for detection of adulteration with c. heracleifolia and/or c. dahurica (detection C).
diameter, or 2 or 3 (rarely up to 6) compound; some Foreign matter (2.8.2)

granules have a slit-shaped hilum [G]. Maximum 5 per cent.
c. Examine the chromatograms obtained in the test for Total ash (2.4.16)
substitution by Cimicifiiga americana Michx., c. foetida L., c. Maximum 10 per cent.
dahurica (Turcz.) Maxim, or c. heracleifolia Kom. Ash insoluble in hydrochloric acid (2.8.1)
Results B Use the chromatograms supplied with Actaea Maximum 5 per cent.
racemosa HRS and the chromatogram obtained with reference
Substitution by Cimicifuga americana Michx., c.
solution (a) to identify the bands corresponding to A.
foetida L., c. dahurica (Turcz.) Maxim, or c.
racemosa.
heracleifolia Kom.
See below the sequence of zones present in the Thin-layer chromatography (2.2.27).
chromatograms obtained with reference solutions (a) and (b)
and the test solution. Furthermore, other faint zones may be
(2.9.12) add 10 mL of ethanol (50 per cent VIV) R and shake
present in the chromatograms obtained with reference
well. Sonicate for 10 min and centrifuge. Use the
solution (a) and the test solution.
supernatant.
Reference solution (a) To 0.50 g of Actaea racemosa HRS add
Top of the plate
10 mL of ethanol (50 per cent VIV) R and shake well.
Sonicate for 10 min and centrifuge. Use the supernatant.
Reference solution (b) Dissolve 2 mg of actein R in methanol R
and dilute to 10 mL with the same solvent.
Actein ะ a brown zone Actein ะ a brown A brown zone (actein)
Mobile phase anhydrous formic acid R, ethyl formate R} toluene R
23-Epi-26- A brown zone (23-epi- (20:30:50 VIVIV).
deoxyactein: a 26-deoxyactein) Application 2 pL as bands of 8 mm (see Table 2069.-1).
brown zone
Drying In air.
— the Rp value of the zone due to actein is between 0.35
and 0.40 (detection B).
A violet zone
Results A The chromatogram obtained with the test solution
A brown zone A brown zone does not show any quenching zones more intense than those
in the chromatogram obtained with reference solution (a)
between Rp value 0.2 and Rp value 0.35.
Reference Reference
Test solution Detection B Treat with a 10 per cent VIV solution of sulfuric
solution (a) solution (b)
acid R in methanol R‘3 heat at 100 °C for 5 min; allow to cool
to room temperature and examine in daylight.
TESTS
Maximum 12 per cent, determined on 1.000 g of the
105 °C for 2 h.
2016 Black Cohosh IV-113
Adulteration with Ciniicifuga americana Michx., c. Top of the plate

foetida L., c. dahurica (Turcz.) Maxim, and/or c.
heracleifolia Kom.
Thin-layer chromatography (2.2.27) as described in the test Actein: a weak Actein: a weak A weak whitish
whitish zone whitish zone zone (actein)
for substitution by Cimicifuga americana Michx., c. foetida L.J
c. dahurica (Turcz.) Maxim, or c. heracleifolia Kom., with
the following modifications. A bluish zone A bluish zone
Reference solution (c) Dissolve 2 mg of cimifugin R in

methanol R and dilute to 10 mL with the same solvent. Cimifugin ะ
Application 2 |1L of reference solutions (b) and (c), 20 pL of a bright FLUORESCENT
fluorescent zone ZONE
the test solution and reference solution (a), as bands of (CIMIFUGIN)
8 mm (see Table 2069.-2). A brownish A brownish
A bluish zone A bluish zone
the Rf value of the zone due to actein is between 0.35
and 0.40 (detections B and C). A FLUORESCENT
ZONE
Results A Absence of more than 10 per cent of c. americana.
Reference Reference Reference c. foetida (5 per
Compare the chromatogram supplied with Actaea solution (a) solution (b) 1 solution (c) cent)
racemosa HRS for c. americana and the chromatograms
obtained with the test solution and reference solution (a). Detection c Dissolve 8 g of antimony trichloride R in 200 mL
The chromatogram obtained with the test solution does not of methylene chloride R-, treat with this solution and heat at
show any quenching zone at Rf value 0.3 (zone presented in 120 °C for 10 min; examine in ultraviolet light at 365 nm.
capitals in the chromatogram of c. americana, see below).
Results c Absence of more than 5 per cent of c. heracleifolia
The presence of this zone in the chromatogram obtained
and/or c. dahurica.
with the test solution indicates adulteration with c. americana
at a level greater than 10 per cent. Compare the chromatogram supplied with Actaea
racemosa HRS for c. heracleifolia and c. dahurica and the
chromatograms obtained with the test solution and reference
Top of the plate solutions (a) and (b). The chromatogram obtained with the
test solution does not show any bright fluorescent zone just
above the zone due to actein (zone presented in capitals in
the chromatogram of c. heracleifolia or c. dahurica, see
A weak zone A weak zone below). The presence of this zone in the chromatogram
2 weak zones 2 weak zones obtained with the test solution indicates adulteration with c.
heracleifolia and/or c. dahurica at a level greater than
A weak zone A weak zone
5 per cent.
____ A DARK ZONE

Top of the plate
A weak zone A weak zone
A dark zone A dark zone A BRIGHT FLUORESCENT

ZONE
A dark zone A dark zone
Actein: a weak Actein: a weak A weak brownish zone
brownish zone brownish zone (actein)
Reference solution (a) c. americana (10 per cent)
A brownish zone A brownish zone

Detection B Dissolve 4.5 g of boric acid R in 150 mL of
A bluish zone A bluish zone
anhydrous ethanol R (solution A); dissolve 5 g of oxalic acid R
in 50 mL of anhydrous ethanol R (solution B); combine
solutions A and B and mix well; treat the plate with this c. heracleifolia
freshly prepared solution and heat at 120 °C for 5 min; Reference Reference (5 per cent) and/or
solution (a) solution (b) c. dahurica (5 per
________ ____________
Results B Absence of more than 5 per cent of c. foetida.
Compare the chromatogram supplied with Actaea
racemosa HRS for c. foetida and the chromatograms obtained ASSAY
with the test solution and reference solutions (a), (b) and (c). Liquid chromatography (2.2.29).
The chromatogram obtained with the test solution does not Test solution Introduce 4.00 g of the powdered herbal drug
show any intense fluorescent zone between Rf value 0.03 (355) (2.9.12) into a 200 mL screw-cap bottle. Add 50.0 mL
and Rf value 0.06 or at the same position as the bright of a mixture of equal volumes of methanol R and water R.
fluorescent zone in the chromatogram obtained with Sonicate for 45 min and shake for 15 min. Filter through a
reference solution (c) (zones presented in capitals in the membrane filter (nominal pore size 0.45 |im).
chromatogram ofc. foetida, see below). The presence of 1 or Reference solution (a) Dissolve 10.0 mg of Actaea racemosa for
both zones in the chromatogram obtained with the test assay CRS (containing monoammonium glycyrrhizate) in
solution indicates adulteration with c. foetida at a level methanol R with the aid of ultrasound and dilute to 10.0 mL
greater than 5 per cent. with the same solvent.
IV-114 Blackcurrant 2016
Reference solution (b) Dilute 5.0 mL of reference solution (a) A = logarithm to base 10 of the concentration of each
to 10.0 mL with methanol R. peak in the chromatogram obtained with the test
Reference solution (c) Dilute 2.0 mL of reference solution (a) solution, determined from the calibration curve;
to 10.0 mL with methanol R. m ะะะ mass of the herbal drug to be examined used to
Reference solution (d) Dilute 1.0 mL of reference solution (a) prepare the test solution, in grams.
to 20.0 mL with methanol R. Calculate the percentage content of triterpene glycosides by
Reference solution (e) Dissolve 500 mg of Actaea racemosa dry taking the sum of the percentage contents of peaks 1 to 12.
extract for system suitability HRS in methanol R and dilute to _____ _ _______________________________________________________ Ph Eur
10.0 mL with the same solvent; sonicate and filter through a
Column'.
— size: I = 0.25 m, 0 = 4.6 mm;
— stationary' phase: octadecylsilyl silica gel for chromatography R Black Currant
(5 gm). Preparation
Mobile phase: Black Currant Syrup
— mobile phase A: 0.1 per cent VIV solution of anhydrous DEFINITION
formic acid R in water R; Black Currant consists of the fresh ripe fruits of Ribcs nigrum
— mobile phase B: 0.1 per cent VIV solution of anhydrous L., together with their pedicels and rachides.
formic acid 7? in a mixture of equal volumes of
acetonitrile R and methanol Rj CHARACTERISTICS
Odour, strong and characteristic.
Time Mobile phase A Mobile phase B Macroscopical Berries: globose, ranging in diameter from
(per cent V/V) (per cent V/V) about 7 to 15 mm, occurring in pendulous racemes; epicarp
0 - 40 50 20 50 -> 80 shiny black externally, enclosing a yellowish green translucent
pulp containing numerous flattened ovoid seeds, about
40 - 41 20 -> 5 80 95
2.5 mm long, 1.25 mm wide and 1 mm thick; berry crowned
41 -44 5 95 with withered remains of five-cleft calyx; pedicels thin, up to
about 10 mm long, attached to a rachis of variable length.
Flow rate 1.0 mUmin. Microscopical Epicarp: glands yellow, disc-shaped, roughly
circular or broadly elliptical, varying in diameter from about
Detection Evaporative light-scattering detector; the following
140 to 240 pm, each consisting of a single layer of cells
settings have been found to be suitable; if the detector has
attached in the centre to the epicarp by means of a short,
different setting parameters, adjust the detector settings so as
multiseriate stalk. Calyx: trichomes unicellular, blunt-ended
to comply with the system suitability criterion for the signal-
with thin, crooked walls, about 10 to 14 pm wide and
to-noise ratio:
averaging about 350 pm in length. Seed: testa with pigment
— carrier gas: nitrogen R;
layer composed of small cells with horseshoe-shaped wall
— flow rate: 0.8 mlVmin;
thickenings as seen in cross section, each cell containing one
— evaporator temperature: 100 °C;
or two prismatic crystals of calcium oxalate; endosperm cells
— nebuliser temperature: 60 °C.
with irregularly thickened walls.
Injection 10 pL.
Actaea racemosa dry extract for system suitability HRS and the
chromatogram obtained with reference solution (e) to identify
the peaks to be quantified. Black Currant Syrup
System suitability: DEFINITION
— signal-to-noise ratio: minimum 4.0 for the peak due to Black Currant Syrup is prepared either from the clarified
monoammonium glycyrrhizate in the chromatogram juice of Black Currant or from concentrated black currant
obtained with reference solution (d); juice of commerce. It contains a suitable antioxidant.
— peak-to~valley ratio: minimum 3, where Hp = height above Permitted food grade colours may be added.
the baseline of peak 4 and Hv = height above the baseline
of the lowest point of the curve separating this peak from
PRODUCTION
peak 5 in the chromatogram obtained with reference It is prepared by dissolving 700 g of Sucrose either in
solution (e). 560 mL of clarified juice, previously diluted with Water to a
weight per mL of 1.045 g, or in 560 mL of a solution of the
Establish a calibration curve with the logarithm to base 10 of
same weight per mL prepared from the concentrated juice of
the concentration (in milligrams per millilitre) of reference
commerce and Water, and adding to this solution sufficient
solutions (a), (b), (c) and (d) (corrected by the assigned
Benzoic Acid to give a final concentration of not more than
percentage content of monoammonium glycyrrhizate in
800 ppm, or sufficient Sodium Metabisulfite or other suitable
Actaea racemosa for assay CRS) as the abscissa and the
sulfite to give a final concentration of not more than
logarithm to base 10 of the corresponding peak area as the 350 ppm of sulfur dioxide.
ordinate.
The syrup complies with the requirements stated under Oral
Calculate the percentage content of each peak using the
Liquids and with the following requirements.
Content of ascorbic acid1, C6H8o6
Not less than 0.055% พ/พ.
10x X 5
2016 Blackcurrant Leaf IV-115
TESTS irregularly thickened walls [Ba], numerous anomocytic

Sulfur dioxide stomata (2.8.8) [Bb] accompanied by spongy
Not more than 350 ppm, Appendix IX B. parenchyma [Be]; fragments, in surface view [C] or in
Weight per mL transverse section [E], of the upper epidermis [Ca, Ea],
1.27 to 1.30 g, Appendix V G. accompanied by palisade parenchyma [Cb, Eb]; cluster
crystals of calcium oxalate up to 30 pm in diameter,
ASSAY
isolated [F] or included in parenchymatous cells [Bd, Cc,
Mix 5 g with 25 mL of a freshly prepared 20% w/v solution Ec].
of metaphosphoric acid, add 20 mL of acetone and dilute to
100 mL with water. To four 3 mL quantities of this solution
add 0.4, 0.5, 0.6 and 0.7 mL, respectively, of double-strength
standard 2,6-dichlorophenolindophenol solution, mix well by
agitation with a fine stream of carbon dioxide, add 3 mL of
chloroform, agitate for a further 15 seconds, examine the
solutions against a white background and select the two that
are on either side of the end point (that is, one colourless
and one pink). Prepare a further six solutions as directed
above, but adding to the first an amount of dye solution
equal to that added to the selected colourless solution,
successively increasing this volume by 0.02 mL increments in
the second to the fifth solutions and adding to the sixth
solution a volume equal to that added to the selected pink
solution. Select the solution exhibiting the faintest pink
colour. Each mL of double-strength standard
2,6-dichlorophenolindophenol solution added to this solution is
equivalent to 0.200 mg of C6H8O6.
STORAGE
Black Currant Syrup should be kept in a well-filled container
and protected from light.
Black Currant Syrup contains, in 10 mL, about 7.5 mg of
ascorbic acid.
’The requirement for Content of ascorbic acid docs not apply when Black
Currant Syrup is used as a flavouring agent for pharmaceutical purposes.
Blackcurrant Leaf * *
(Ph. Eur. monograph 2528) *** herbal drug of blackcurrant leaf
PhEir______________________________________________________________ c. Thin-layer chromatography (2.2.27).
DEFINITION Test solution To 1 g of the powdered herbal drug (355)
Dried leaf of Ribes nigrum L. (2.9.72) add 10 mL of methanol R. Heat in a water-bath at
60 °C for 10 min with occasional stirring. Allow to cool.
Content
Filter.
Minimum 1.0 per cent of flavonoids, expressed as
isoquercitroside (C21H20012; Mr 464.4) (dried drug). Reference solution Dissolve 5 mg of isoquercitroside R and 5 mg
of rutin J? in 10 mL of methanol R.
IDENTIFICATION
A. The leaf is simple. The lamina may be up to 10 cm long plate 7? (2-10 pm)].
and 12 cm wide and shows 3 (rarely 5) rounded triangular
Mobile phase anhydrous formic acid R, water R, ethyl acetate R
lobes, dentate or crenate on the margins, with the median
lobe being the largest. The light-brown midrib and secondary (10:10:80 VIVIV).
veins are very visible on the lower surface, and form a Application 10 pL [or 5 pL] as bands of 10 mm [or 8 mm].
characteristic network through numerous anastomoses. Development Over a path of 10 cm [or 6 cm].
The rigid, light-brown petiole shows a very distinct gutter on Drying At 100-105 °C for 10 min.
the upper part and its length is equal to half the length of the Detection Treat with a 10 g/L solution of diphenylboric acid
lamina. aminoethyl ester R in methanol R, then with a 50 g/L solution
B. Microscopic examination (2.5.25). The powder is of macrogol 400 R in methanol R; allow to dry in air for about
brownish-green. Examine under a microscope using chloral 30 min, then examine in ultraviolet light at 365 nm.
hydrate solution R. The powder shows the following diagnostic Results See below the sequence of zones present in the
characters (Figure. 2528.-1): curved, unicellular covering chromatograms obtained with the reference solution and the
trichomes, with moderately thickened, slightly verrucose test solution. Furthermore, other fluorescent zones may be
walls [D]; orange-yellow, globular or ovoid glandular present in the chromatogram obtained with the test solution.
trichomes, lacking a visible stalk, with a multicellular head up
to 200 pm in diameter, in surface view [A]; fragments of the
lower epidermis, in surface view [B], composed of cells with
IV-116 Black Horehound 2016
Top of the plate System suitability: reference solution (c) ะ

— resolution: minimum 3.0 between the peaks due to rutin
and isoquercitroside.
A green zone
Disregard limit The area of the principal peak in the
Isoquercitroside: an orange zone An orange zone (mainly chromatogram obtained with reference solution (d).
isoquercitroside)
Calculate the percentage content of total flavonoids,
A light blue zone
expressed as isoquercitroside, using the following expression:
Al X 7712 X p
Rutin ะ an orange-yellow zone An orange-yellow zone (rutin) A2 X 7711 X 5

A1 = sum of the areas of the peak due to rutin and all
peaks eluting after the peak due to rutin in the
TESTS chromatogram obtained with the test solution;
Foreign matter (2.8.2) A2 = area of the peak due to isoquercitroside in the
Maximum 3 per cent. chromatogram obtained with reference solution
Loss on drying (2.2.52) (a);
Maximum 10.0 per cent, determined on 1.000 g of the m1 = mass of the herbal drug to be examined used to
powdered herbal drug (355) (2.9.72) by drying in an oven at prepare the test solution, in grams;
105 °C for 2 h. m2 = mass of isoquercitroside CRS used to prepare
reference solution (a), in grams;
Total ash (2.4.16)
p = percentage content of isoquercitroside in
isoquercitroside CRS.
ASSAY
______________________________________________________________ Ph Eur
(355) (2.9.72) in 10 mL of an 80 per cent VIV solution of
methanol R. Heat under a reflux condenser in a water-bath at
60 °C for 30 min. Sonicate for 15 min. Allow to cool, dilute
Black Horehound * \
to 20.0 mL with an 80 per cent VIV solution of methanol R. (Ph Eur monograph 1858) * **
Filter through a membrane filter (nominal pore size
Ph Eur_______________________________________________________________
0.45 pm).
Reference solution (a) Dissolve 5.0 mg of isoquercitroside CRS DEFINITION
in an 80 per cent VIV solution of methanol R and dilute to Dried flowering tops of Ballota nigra L.
100.0 mL with the same solution. Content
Reference solution (b) Dissolve 5.0 mg of rutin R in methanol R Minimum 1.5 per cent of total orf/io-dihydroxycinnamic acid
and dilute to 100.0 mL with the same solvent. derivatives, expressed as acteosidc (C29H360 15; 7Vfr 625)
(dried drug).
Reference solution (c) Dilute 10.0 mL of reference solution (a)
to 20.0 mL with reference solution (b). IDENTIFICATION
Reference solution (d) Dilute 1.0 mL of reference solution (a) A. The stems are conspicuously 4-angled, longitudinally
to 20.0 mL with an 80 per cent VIV solution of methanol R. striated, dark green or reddish-brown and more or less
Column'. pubescent. The leaves are greyish-green, pctiolate, the lamina
— size'. I = 0.25 m, 0 = 4 mm; ovate or orbicular, 2-4 cm wide, the margin irregularly
— stationary phase', end-capped octadecylsilyl silica gel for crenate, and cuneate or cordate at the base; both surfaces are
chromatography R (5 pm); covered with abundant whitish hairs; the venation is pinnate,
— temperature: 30 °C. prominent on the lower surface, slightly depressed on the
upper. The flowers are sessile or very shortly pedicellate, the
Mobile phase:
calyx is infundibuliform, densely pubescent, with 10
— mobile phase A: 0.05 per cent VIV solution of trifluoroacetic
prominent ribs and 5 subequal, broadly ovate teeth;
acid Rj
the corolla, with a tube slightly shorter than the calyx tube, is
— mobile phase B: acetonitrile R)
purple and bilabiate, the upper lip pubescent on the outer
surface and the lower lip with 3 lobes, the middle of which is
notched.
0 - 45 97 -> 60 3 -> 40 B. Microscopic examination (2.8.23). The powder is greyish-
green and slightly flocculent. Examine under a microscope
using chloral hydrate solution R. The powder shows the
Flow rate 1.0 mlVrnin. following diagnostic characters (Figure 1858.-1): numerous
Detection spectrophotometer at 350 nm. long, uniseriate, multicellular covering trichomes consisting of
Injection 10 pL. 4 or more cells, thickened and swollen at the junctions, with
Identification of peaks Use the chromatogram obtained with slightly lignified and pitted walls, free [C] or on an epidermis
reference solution (a) to identify the peak due to in transverse section [Ea]; fewer glandular trichomes, usually
isoquercitroside and the chromatogram obtained with on epidermises, in transverse section [E, F, G] ะ some with a
reference solution (b) to identify the peak due to rutin. unicellular or multicellular stalk and a globose, uni- or
bicellular head [Ga], others with a unicellular stalk and a
Retention time Rutin = about 28 min;
multicellular head in surface view [Ac] or in transverse
isoquercitroside = about 29 min.
section [Eb], others with a unicellular stalk and an 8-celled
2016 Black Horehound IV-117
head of lamiaceous type, in surface view [Ad] or in transverse Results See below the sequence of zones present in the
section [Fa]; fragments of the adaxial leaf epidermis [B] with chromatograms obtained with the reference solution and the
cells with sinuous walls, accompanied by cells of the palisade test solution. Furthermore, other fluorescent zones may be
parenchyma, most containing fine, needle-shaped crystals present in the chromatogram obtained with the test solution.
[Ba]; fragments of the abaxial leaf epidermis [A] bearing
numerous stomata, the majority anomocytic (2.8.ร) [Aa] but Top of the plate
some diacytic [Ab]; fragments of the epidermis of the corolla A reddish fluorescent zone
composed of polygonal cells, those of the inner epidermis of
A faint yellow fluorescent zone
the lips papillose [H] and those of the inner epidermis of the
tube bearing uni- or bicellular covering trichomes in a stellate A light blue fluorescent zone
arrangement [K]; pollen grains subspherical with 3 pores and (caffeoylmalic acid)
a smooth exine [D]; fragments from the stem (G) with A greenish-blue fluorescent zone
(acteoside)
groups of collenchymatous cells [Gb] and lignified vessels,
with annular or spiral thickenings [J]. A yellowish-brown fluorescent
zone (luteolin 7-lactate)
Chlorogenic acid: a light blue
fluorescent zone
A greenish-blue fluorescent zone
(forsythoside B)
Rutin: an orange-yellow 2 greenish-blue fluorescent zones
fluorescent zone (arenarioside)
(luteolin 7-lactate glucoside).
A faint greenish-blue fluorescent
zone (ballotetroside).
TESTS
105 °C for 2 h
Total ash (2.4.16)
ASSAY
Stock solution Place 1.000 g of the powdered herbal drug
(355) (2.9.12) in a flask. Add 90 mL of ethanol
(50 per cent V/V) R. Heat under a reflux condenser on a
water-bath for 30 min. Allow to cool and filter, collecting the
filtrate in a 100 mL volumetric flask. Rinse the flask and the
filter with 10 mL of ethanol (50 per cent V/V) R. Add the
rinsings to the filtrate and dilute to 100.0 mL with ethanol
Test solution Into a 10 mL volumetric flask, introduce
successively, with shaking after each addition, 1.0 mL of the
stock solution, 2 mL of 0.5 M hydrochloric addy 2 mL of a
Figure 1858.-1. - Illustration for identification test B of powdered solution containing 100 g/L of sodium nitrite R and 100 g/L of
herbal drug of black horehound sodium molybdate Ry and 2 mL of dilute sodium hydroxide
c. Thin-layer chromatography (2.2.27). solution Ry and dilute to 10.0 mL with พater R.
Test solution To 2 g of the powdered herbal drug (355) Compensation liquid Into a 10 mL volumetric flask, introduce
(2.9.12) add 100 mL of methanol R. Heat on a water-bath 1.0 mL of the stock solution, 2 mL of 0.5 M hydrochloric add
under a reflux condenser for 30 min. Allow to cool. Filter. and 2 mL of dilute sodium hydroxide solution Ry and dilute to
Evaporate the filtrate under reduced pressure until a volume 10.0 mL with water R.
of about 10 mL is obtained. Measure immediately the absorbance (2.2.25) of the test
Reference solution Dissolve 1 mg of chlorogenic acid R and solution at 525 nm, by comparison with the compensation
2.5 mg of rutin R in 10 mL of methanol R. liquid.
Plate TLC silica gel plate R. Calculate the percentage content of total ortho-
Mobile phase anhydrous formic acid Ry glacial acetic acid Ry dihydroxycinnamic acid derivatives, expressed as acteoside,
water Ry ethyl acetate R (7.5:7.5:18:67 V/V/V/V). using the following expression:
Application 20 |1L as bands. A X 1000
Development Over a path of 15 cm. 185 X m
Drying In air.
i.e. taking the specific absorbance of acteoside to be 185.
Detection Spray with a solution containing 10 g/L of
y4 = absorbance at 525 nm;
diphenylboric acid aminoethyl ester R and 50 g/L of macrogol m = mass of the substance to be examined, in grams.
400 R in methanol Ry allow to dry in a current of warm air;
________________ ___________ __ _____________________ Ph Eur
examine in ultraviolet light at 365 nm after 30 min.
FV-118 Bogbean Leaf 2016
Bogbean Leaf ** ** TESTS

(Ph. Eur. monograph 1605) *** Maximum 10.0 per cent, determined on 1.000 g of the
PhEir______________________________________________________________ powdered herbal drug (355) (2.9.12) by drying in an oven at
105 °C for 2 h.
DEFINITION
Total ash (2.4.16)
Dried, entire or fragmented leaf of Menyanthes trifoliata L.
CHARACTERS
Bitterness value (2.8.15)
Very bitter and persistant taste. Minimum 3000.
IDENTIFICATION ________________ _____________________________________________ Ph Eur
A. The leaf is long-petiolated, trifoliate, with long sheaths
from the base; the petiole is up to วิ mm in diameter and
strongly striated longitudinally. The lamina is divided into
equal leaflets, sessile, obovate up to 10 cm long and up to
5 cm wide, with an entire, occasionally sinuous margin with Boldo Leaf * *
brownish or reddish hydathodes and a spathนlate base; it is
(Ph. Eur. monograph 1396) *★*
glabrous, dark green on the upper surface and paler green on
the lower surface, with a wide, whitish, finely striated Preparation
prominent midrib. Boldo Leaf Dry Extract
B. Reduce to a powder (355) (2.9.12). The powder is Ph Eur_______________________________________________________________
yellowish-green. Examine under a microscope using chloral DEFINITION
hydrate solution R. The powder shows fragments of upper Whole or fragmented dried leaf of Peumus boldus Molina.
epidermis with polyhedral cells and thin wavy walls;
fragments of lower epidermis with sinuous walls; anomocytic Content
stomata (2.8.3), on both surfaces, with the subsidiary cells Minimum 0.1 per cent of total alkaloids, expressed as
showing radiating striations; epidermal cells from the veins boldine (C19H21NO4; Mr 327.4) (anhydrous drug).
straight walled and papillose; fragments of mesophyll CHARACTERS
parenchyma with large intercellular spaces (aerenchyma); Characteristic odour, especially when rubbed.
irregular cells with rare sclereids; fragments of spiral or
IDENTIFICATION
annular vessels.
A. The leaf is oval or elliptical usually 5 cm long with a short
c. Thin-layer chromatography (2.2.27). petiole, an obtuse or slightly emarginate or mucronate apex
Test solution To 1.0 g of the powdered herbal drug (355) and an equal and rounded base; the margin is entire and
(2.9.12) add 10 mL of methanol R. Heat, with stirring, in a slightly undulate and the thickened edges are more or less
water-bath at 60 °C for 5 min. Allow to cool and filter. revolute. The lamina is greyish-green, thick, tough and
Evaporate to dryness under reduced pressure in a water-bath brittle. The upper surface is rough with numerous prominent
at 60 CC. Dissolve the residue in 2.0 mL of methanol R. small protuberances and a depressed venation. The lower
Reference solution Dissolve 5 mg of loganin R in 15 mL of surface is finely pubescent, with the protuberances less well-
methanol R. marked, and a prominent, pinnate venation.
Plate TLC silica gel plate R. B. Reduce to a powder (355) (2.9.12). The powder is
Mobile phase water R, methanol R, ethyl acetate R greyish-green. Examine under a microscope using chloral
(8:15:77 P7P7F)- hydrate solution R. The powder shows fragments of the upper
epidermis and underlying hypodermis with straight or slightly
Application 30 pL, as bands.
sinuous thickened and beaded walls, those of the lower
Development Over a path of 15 cm. epidermis with numerous stomata surrounded
Drying In air. by 4-7 subsidiary cells; solitary, bifurcated or stellate
Detection Spray with vanillin reagent R. Heat in an oven at clustered unicellular covering trichomes with more or less
100-105 °C for 10 min. Examine in daylight. thickened and lignified walls; fragments of the lamina
Results See below the sequence of the zones present in the showing a two-layered palisade; debris of the spongy
chromatograms obtained with the reference and test mesophyll including numerous large, rounded oil cells and
solutions. Furthermore, other zones are present in the parenchyma containing fine needle-shaped crystals; thick
chromatogram obtained with the test solution. walled fibres and lignified, pitted parenchymatous cells
associated with vascular tissue from the veins.
Top of the plate

Test solution Mix 1.5 g of the powdered herbal drug (355)
(2.9.12) and 5 mL of methanol R and sonicate for 10 min.
An intense blue zone Filter the supernatant through a 3 cm X 0.5 cm column of
cellulose for chromatography Rl. Use the first 1 mL of the
Loganine ะ a greyish-violet zone A violet to greyish-violet zone
eluate as the test solution.
A grey to greyish-blue zone Reference solution Dissolve 2 mg of boldine R and 10 mg of
A brownish zone hyoscine hydrobromide R in 5 mL of methanol R.
plate R (2-10 pm)].
Mobile phase diethylamine R, methanol R, toluene R
(10:10:80 VIVIV).
2016 Boldo Leaf IV-119
Top of the plate
A yellowish-brown zone
Hyoscine: a pale brown zone
A brown zone
A brown zone
Boldine : a brown zone A brown zone (boldine)
Several zones
TESTS
Maximum 40 mL/kg (anhydrous drug).
Use 10.0 g of the freshly fragmented drug, a 1000 mL flask
and 300 mL of water R as the distillation liquid. Distil at a
rate of 2-3 mUmin for 3 h.
Maximum 4 per cent of twigs and maximum 2 per cent of
other foreign matter.
Water (2.2.13)
Maximum 100 mL/kg, determined by distillation of 20.0 g of
the powdered herbal drug (355) (2.9.72).
A. Fragment of the lamina, G. Fragment of the lamina, in Total ash {2.4.16)
in surface view, showing the transverse section, showing the Maximum 13.0 per cent.
upper epidermis (Aa), upper epidermis (Ga),
hypodermis with thickened hypodermis (Gb), palisade ASSAY
and beaded walls (Ab), and parenchyma (Gc) and spongy Alkaloids
palisade parenchyma (Ac) parenchyma (Gd) containing oil Liquid chromatography (2.2.29).
cells (Ge) Test solution To 1.000 g of the powdered herbal drug (355)
B and c. Lower epidermis H. Spongy parenchyma {2.9.12) add 50 mL of dilute hydrochloric acid R. Shake in a
with stomata surrounded by containing fine needle-shaped water-bath at 80 °C for 30 min. Filter, take up the residue
4-7 subsidiary cells crystals and oil cells (Ha) with 50 mL of dilute hydrochloric acid R and shake in a water
D. Unicellular covering J. Vascular tissue with fibres bath at 80 °C for 30 min. Filter and repeat the operation
trichome, solitary once on the residue obtained. Filter. Combine the cooled
E and F. Unicellular filtrates and shake with 100 mL of a mixture of
covering trichomes, stellate equal volumes of ethyl acetate R and hexane R. Discard the
clustered organic layer. Adjust the aqueous layer to pH 9.5 with dilute
Figure 1396.-1. - Illustration of powdered herbal drug of boldo ammonia Rl. Shake successively with 100 mL, 50 mL and
leaf (see Identification B) 50 mL of methylene chloride R. Combine the lower layers and
evaporate to dryness under reduced pressure. Dissolve the
residue in the mobile phase and dilute to 10.0 mL with the
Application 40 pL [or 6 pL] of the test solution and 20 pL
mobile phase.
[or 2 pL] of the reference solution, as bands of 15 mm [or
8 mm]. Reference solution Dissolve 12 mg of boldine CRS in the mobile
phase and dilute to 100.0 mL with the mobile phase. Dilute
1.0 mL of this solution to 10.0 mL with the mobile phase.
Drying In air.
Column'.
Detection Spray with potassium iodobismuthate solution R2, dry — size'. I = 0.25 m, 0 = 4.6 mm;
for 5 min in air and spray with sodium nitrite solution R; — stationary phase', octadecylsilyl silica gel for chromatography R
examine in daylight after 30 min. (5 pm).
Results See below the sequence of zones present in the Solution A Mix 0.2 mL of diethylamine R and 99.8 mL of
chromatograms obtained with the reference solution and the acetonitrile R.
test solution. Solution B Mix 0.2 mL of diethylamine R and 99.8 mL of
water R and adjust to pH 3 with anhydrous formic acid R.
Mobile phase Solution A, solution B (16:84 VIV).
Injection 20 pL.
IV-120 Boldo Leaf Preparations 2016
Relative retention With reference to boldine (retention Mobile phase diethylamine R, methanol Ry toluene R
time = about 6 min): isoboldine = about 0.9; isocorydine (10:10:80 VIVIV).
N-oxide = about 1.8; laurotetanine = about 2.2; Application 20 |1L [or 3 J1L], as bands of 15 mm [or 8 mm].
isocorydine = about 2.8; N-methyllaurotetanine =ะ about 3.2.
Additional peaks may be present.
Drying In air.
System statability' Test solution:
— resolution', minimum 1 between the peaks due to Detection Spray with potassium iodobismuthate solution R2y
isoboldine and boldine. allow to dry in air for 5 min and spray with sodium nitrite
solution R'y examine in daylight after 30 min.
Calculate the percentage content of total alkaloids expressed
as boldine using the following expression: Results See below the sequence of zones present in the
(£>h) X m2 X p test solution. Furthermore, other faint zones may be present
A? X 7ท1 X 100 in the chromatogram obtained with the test solution.
nil = mass of the herbal drug to be examined, in Top of the plate

grams;
m2 = mass of boldine CRS in the reference solution, in
grams; A yellowish-brown zone
YA 1 = sum of the areas of the peaks due to the 6 An orange-yellow zone
alkaloids identified in the chromatogram obtained
Hyoscine: a pale brown zone
with the test solution;
A2 = area of the peak due to boldine in the An orange zone
chromatogram obtained with the reference An orange zone
solution;
p = percentage content of boldine in boldine CRS.
Boldine: a brown zone A brown zone (boldine)
_______________________________________________________________ Ph Eur
Several orange zones
Boldo Leaf Dry Extract

ASSAY
Ph Eur_________________________________
Test solution To 1.000 g of the extract to be examined add
DEFINITION 50 mL of dilute hydrochloric acid R and sonicate for 10 min.
Extract produced from Boldo leaf (1396). Transfer to a separating funnel and wash with 10 mL of a
Content mixture of equal volumes of ethyl acetate R and hexane R.
— for aqueous extracts', minimum 0.5 per cent of total Adjust the aqueous phase to pH 9.5 with dilute ammonia Rl.
alkaloids, expressed as boldine (C]9H2]NO4; Mr 327.4) After cooling, shake successively with 100 mL, 50 mL, and a
(dried extract); further 50 mL of methylene chloride Ry taking care not to form
— for hydroalcoholic extracts', minimum 1.0 per cent of total an emulsion. Evaporate the combined lower layers to dryness
alkaloids, expressed as boldine (C19H21NO4; Mr 327.4) under reduced pressure. Dissolve the residue in the mobile
phase and transfer the solution to a volumetric flask. Rinse
(dried extract).
and dilute to 10.0 mL with the mobile phase.
PRODUCTION Reference solution Dissolve 12.0 mg of boldine CRS in the
The extract is produced from the herbal drug by a suitable mobile phase and dilute to 100.0 mL with the mobile phase.
procedure using either hot water at not less than 65 °C or a
Column'.
hydroalcoholic solvent equivalent in strength to e±anol
— size'. I -= 0.25 m, 0 = 4.6 mm;
(45-75 per cent VIV).
CHARACTERS (5 pm).
Appearance Solution A Mix 0.2 mL of diethylaniine R with 99.8 mL of
Brown or greenish-brown, hygroscopic powder. acetonitrile R.
IDENTIFICATION Solution B Mix 0.2 mL of diethylamine R with 99.8 mL of
Thin-layer chromatography (2.2.27). water R and adjust to pH 3 with anhydrous formic acid R.
Test solution To 0.5 g of the extract to be examined add Mobile phase Solution A, solution B (16:84 VIV).
1 mL of hydrochloric acid R and 20 mL of water R. Sonicate Flow rate 1.5 mL/min.
for 10 min. Transfer the liquid to a separating funnel and Detection Spectrophotometer at 304 nm.
make alkaline with 2 mL of dilute ammonia Rl. Shake with
Injection 20 J1L.
2 quantities, each of 20 mL, of methylene chloride R.
Evaporate the combined organic layers to dryness. Dissolve Relative retention With reference to boldine (retention
time = about 6 min): isoboldine = about 0.9; isocorydine
the residue in 1 mL of methanol R.
N-oxide = about 1.8; laurotetanine = about 2.2;
Reference solution Dissolve 2 mg of boldine R and 10 mg of isocorydine = about 2.8; N-methyllaurotetanine = about 3.2.
hyoscine hydrobromide R in 5 mL of methanol R. Additional peaks may be present.
Plate TLC silica gel plate R (5-40 Jim) [or TLC silica gel
plate R (2-10 pm)].
2016 Buckwheat Herb IV-121
System suitability Test solution: striated walls; fragments of the lower epidermis of the lamina
resolution: minimum 1.0 between the peaks due to [C] with thin-walled polygonal cells, numerous stomata [Ca]
isoboldine and boldine. and rare glandular trichomes with a biseriate stalk and a
Calculate the percentage content of total alkaloids, expressed globular head usually composed of 8 cells [Cb]; fragments of
as boldine, using the following expression: mesophyll [F] with narrow, annular or spiral vessels [Fa] and
of spongy parenchyma, numerous cells of which contain
(ร^!) X 7ท2 X p cluster crystals of calcium oxalate, varying in diameter
A2 X mi X 10 (25-100 pm) [Fb], smaller prismatic crystals of calcium
oxalate [Fc], occurring scattered in the mesophyll and also in
the parenchyma of the stem; fragments of lignified tissue [H]
= sum of the areas of the peaks due to the 6
with bordered-pitted [Ha], reticulate or annular [Hb] vessels
alkaloids identified in the chromatogram obtained
with the test solution; and thin-walled, pitted fibres [He]; occasional fragments of
the corolla with a papillose epidermis [E]; spherical or ovoid
= area of the peak due to boldine in the
pollen grains, about 50 pm in diameter, with a pitted exine
chromatogram obtained with the reference
solution; and 3 furrows [G].
mi = mass of the extract to be examined used to
m2 = mass of boldine CRS used to prepare the reference
solution, in grams;
p = percentage content of boldine in boldine CRS.
—————-- ----------------------------------------------------------------------------- Ph Eur
Buckwheat Herb
PAftr______________________
DEFINITION
Whole or fragmented aerial parts of Fagopyrum esculentum
Moench, collected in the early flowering period prior to
fruiting and dried immediately.
Content
Minimum 3.0 per cent of rutin (C27H30016; Mr 611) (dried
drug).
IDENTIFICATION
A. The stem is cylindrical, hollow, finely ridged
longitudinally, about 2-6 mm in diameter, brownish-green or
reddish, with few branches and thickened at the internodes;
the leaves are arranged spirally and have membranous,
sheathing stipules; the surface is glabrous except in the region
of the stipules, where short, white hairs may occur.
The leaves are dark green, paler on the lower surface, up to
7 cm wide and 11 cm long, saggitate or cordate, almost Figure 2184.-1. - Illustration for identification test B of powdered
pentagonal with 2 widely rounded lobes; the lower leaves are herbal drug of buckwheat herb
petiolate, the upper leaves sessile or amplexicaul; the lamina
is glabrous and the margin finely sinuate and fringed with
minute, reddish-brown projections; similar projections occur Test solution To 0.5 g of the powdered herbal drug (355)
on the veins on the upper surface. The inflorescence is a (2.9.12) add 5.0 mL of methanol R and heat in a water-bath
cymose panicle, the individual flowers 1-2 mm long and at 60 °C under a reflux condenser for 10 min. Cool and
6 mm in diameter with 5 free, white or reddish petals. filter.
B. Microscopic examination (2.8.23). The powder is dark Reference solution Dissolve 10 mg of hyperoside R and 10 mg
green. Examine under a microscope using chloral hydrate of rutin R in 10 mL of methanol R.
solution R. The powder shows the following diagnostic Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
characters (Figure 2184.-1): fragments of the epidermis of plate R (2-10 pm)].
the stem, in surface view [D], composed of elongated cells Mobile phase anhydrous formic acid R, water Ri ethyl acetate R
showing striations on the outer walls [Da] and anomocytic (10:10:80 VIVIV).
stomata (2.8.3) [Db]; fragments of the upper epidermis of Application 20 pL [or 5 pL] as bands of 15 mm [or 8 mm].
the lamina, in surface view [B], consisting of polygonal cells
covered by a striated cuticle [Ba] and anomocytic stomata
[Bb], often accompanied by palisade parenchyma [Be]; Drying At 100-105 °C.
fragments of the epidermis of the leaf margins [A] and of the Detection treat with a 10 g/L solution of diphenylboric acid
epidermis covering the veins, often showing ovoid or rounded aminoethyl ester R in methanol R, subsequently treat with a
papilla-like projections, often reddish, with thickened and 50 g/L solution of macrogol 400 R in methanol R; allow to dry
IV-122 Greater Burnet Root 2016
in air for about 30 min and examine in ultraviolet light at Flow rate 1.0 mL/min.
365 nm. Detection Spectrophotometer at 350 nm.
Results See below the sequence of zones present in the Injection 10 J1L.
— elution order, order indicated in the composition of
present in the chromatogram obtained with the test solution. reference solution (b), when the chromatogram is
recorded in the prescribed conditions; -
Top of the plate
— resolution: minimum 3 between the peaks due to
2 red zones troxerutin and quercitrin.
1-2 light blue zones
chromatogram obtained with reference solution (a), locate
the peak due to rutin in the chromatogram obtained with the
An orange zone test solution.
An orange zone
Calculate the percentage content of rutin using the following
expression:
Hyperoside: an orange zone 2 blue zones
Al X 7712 X p
A2 X 7721
Rutin ะ an orange-yellow zone An orange-yellow zone (rutin)
Reference solution Test solution A1 = area of the peak due to rutin in the chromatogram
A2 = area of the peak due to rutin in the chromatogram
TESTS
Loss on drying (2.2.32) m1 = mass of the herbal drug to be examined used to
m2 = mass of rutoside trihydrate CRS used to prepare
105 °C for 2 h.
Total ash (2.4.16) p = percentage content of rutin in rutoside trihydrate CRS.
______________________________________________________________ Ph Eur
ASSAY
(2.9.12)
, add 30 mL of an 80 per cent VIV solution of Greater Burnet Root * **
methanol R. Heat the mixture under a reflux condenser in a
water-bath at 60 °C for 30 min, then extract ±e mixture in (Sanguisorba Root, Ph Eur monograph 2385) ***
an ultrasonic bath for 15 min. Allow to cool, dilute to Ph Eur__________________________________________________________ ____
50.0 mL with an 80 per cent VIV solution of methanol R and
DEFINITION
filter.
Whole or fragmented, dried underground parts of
Reference solution (a) Dissolve 25.0 mg of rutoside
Sanguisorba officinalis L. without rootlets.
trihydrate CRS in an 80 per cent VIV solution of methanol R
and dilute to 50.0 mL with the same solvent. Content
Reference solution (b) Dissolve 20.0 mg of troxerutin R and
(C6H603; Afr 126.1) (dried drug).
5.0 mg of quercitrin R in an 80 per cent VIV solution of
methanol R and dilute to 50.0 mL with the same solvent. CHARACTERS
Column: The adventitious roots are about 5-25 cm long and up to
— size: I = 0.125 m, 0 = 4 mm; 2 cm in diameter.
— stationary phase: octadecylsilyl silica gel for chromatography R IDENTIFICATION
(5 tun); A. The whole drug consists of the rhizome, often ramified,
— temperature: 30 °C. thick, short, fusiform or cylindrical and the adventitious roots
Mobile phase: whose surface is reddish-brown or blackish-brown, with
— mobile phase A: mix 50 volumes of acetonitrile R and longitudinal striations, sometimes with transverse fissures,
950 volumes of water R adjusted to pH 2 with phosphoric and showing rootlet scars.
acid R; It may also be found as more or less cylindrical fragments up
— mobile phase B: mix 95 volumes of water R adjusted to to 2 cm long or elliptical or irregular discs. The fracture is
pH 2 with phosphoric acid R and 905 volumes of light-coloured and very fibrous.
acetonitrile R‘,
B. Reduce to a powder (355) (2.9.12). The powder is light
yellowish-brown. Examine under a microscope using chloral
(per cent V/V) (per cent V/V)
(min)
94 6
characters: numerous, whole or fragmented phloem fibres,
0-6
usually isolated, narrow, sometimes more than 500 |im long
6 - 16.5 94 -> 85 6-» 15
and often rough-walled; calcium oxalate cluster crystals, free
16.5 - 22 85 -> 76 15->24 or inside parenchyma cells; a few reticulate lignified vessels;
24 -> 41
rare cork fragments. Examine under a microscope using a
22 - 25 76 ■> 59
50 per cent VIV solution of glycerol R. The powder shows
2016 Butcher’s Broom IV-123
rounded or ovoid starch granules, single or in groups of 2-4; TESTS

the diameter of a component granule may reach 30 pm. Loss on drying (2.2.32)
Some starch granules are found in the parenchyma cells or in Maximum 12.0 per cent, determined on 1.000 g of the
cells of the medullary rays. powdered herbal drug (355) (2.9.72) by drying in an oven at
c. Thin-layer chromatography (2.2.27). 105 °C.
Test solution To 2.0 g of the powdered herbal drug (355) Total ash (2.4.16)
(2.9.72) add 50 mL of zuater R and boil under a reflux Maximum 10.0 per cent.
condenser for 30 min. Cool the solution and centrifuge for Ash insoluble in hydrochloric acid (2.8.1)
10 min. Shake the supernatant with 2 quantities, each of Maximum 2.0 per cent.
15 mL, of di-isopropyl ether R saturated with hydrochloric ASSAY
acid R. Combine the ether layers. Evaporate to dryness and Tannins (2.8.14)
dissolve the residue in 1.0 mL of methanol R. Filter through a Use 0.500 g of the powdered herbal drug (180) (2.9.72).
polypropylene syringe filter (nominal pore size 0.45 pm). ________________________________________ PhEar
Reference solution Dissolve 5 mg of gallic acid R and 20 mg of
resorcinol R in 20 mL of methanol R.
Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel Butcher’s Broom *****
F254 plate R (2-10 pm)].
Mobile phase anhydrous formic acid R, ethyl acetate R, toluene R
(10:30:60 VIVIV). PhEur_____________________________________________________________
Application 10 pL [or 4 pL] as bands. DEFINITION

Development Over a path of 10 cm [or 6 cm]. Dried, whole or fragmented underground parts of Ruscus
Drying In air. aculeatus L.
Content
Minimum 1.0 per cent of total sapogenins, expressed as
Results A See below the sequence of quenching zones present ruscogenins [mixture of neoruscogenin (C27H40O4;
in the chromatograms obtained with the reference solution Mr 428.6) and ruscogenin (C27H42O4; Mr 430.6)] (dried
and the test solution. Furthermore, other faint quenching drug).
test solution.
IDENTIFICATION
A. The rhizome consists of yellowish, branched, articulated,
somewhat knotty pieces, cylindrical or subcortical, about
Top of the plate 5-10 cm long and about 5 mm thick. The surface is marked
A quenching zone with thin annuladons about 1-3 mm wide, separated from
one another; rounded scars of the aerial stems are present on
the upper surface. On the lower surface numerous roots, or
Resorcinol: a quenching zone their scars, occur; the roots are about 2 mm in diameter and
similar in colour to the rhizome. The outer layer is easily
A quenching zone
detached, revealing a yellowish-white, very hard central
cylinder.
Gallic acid: a quenching zone A quenching zone (gallic acid) B. Reduce to a powder (355) (2.9.72). The powder is
yellowish. Examine under a microscope using chloral hydrate
A quenching zone solution R. The powder shows the following diagnostic
A quenching zone characters (Figure 1847.-1): groups of sclereids of the
rhizome, with variously-shaped cells, rounded, elongated or
rectangular; the walls are moderately thickened and distinctly
beaded, with large, rounded or oval pits [F, G, L, p, Q];
Detection B Spray with a 10 g/L solution of ferric chloride R in fragments of the endodermis composed of a single layer of
anhydrous ethanol R and heat at 100-105 °C for 15 min; irregularly thickened cells [K]; groups of rounded
examine in daylight. parenchymatous cells, thickened at the comers, with small,
Results B See below the sequence of the zones present in the triangular intercellular spaces [D, E, N]; thin-walled
chromatograms obtained with the reference solution and the parenchyma Q] with some cells containing raphides of
test solution. Furthermore, other faint zones may be present calcium oxalate [C]; groups [H] of thick-walled fibres [Ha]
in the chromatogram obtained with the test solution. and small vessels, up to about 50 pm in diameter, the walls
showing numerous small, slit-shaped pits [A, Hb]; rare
fragments of dermal tissue of the root [B]; raphides of
, Top of the plate calcium oxalate, isolated [M].
Resorcinol: a brown zone
(355) (2.9.72) and 50 mL of dilute hydrochloric acid R into a
A blackish-blue zone 100 mL flask with a ground-glass neck. Heat on a water-bath
under a reflux condenser for 40 min. Allow to cool and
extract the unfiltered mixture with 3 quantities, each of
Gallic acid: a blackish-blue zone A blackish-blue zone (gallic acid) 25 mL, of methylene chloride R. Combine the organic
A blackish-blue zone solutions and dry over anhydrous sodium sulfate R. Filter and
evaporate to dryness. Dissolve the residue in 5 mL of
methanol R.
IV-124 Butcher’s Broom 2016
TESTS
Maximum 5 per cent.
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
(2.9.12) add 60 mL of anhydrous ethanol R, 15 mL of water R
and 0.2 g of potassium hydroxide R. Extract on a water-bath
under a reflux condenser for 4 h. Allow to cool and filter into
a 100 mL volumetric flask. Rinse the extraction flask and the
residue in the filter with 3 quantities, each of 10 mL, of
anhydrous ethanol R and add the rinsings to the volumetric
flask. Dilute to 100.0 mL with anhydrous ethanol R. Introduce
25.0 mL of this solution into a round-bottomed flask fitted
to a rotary evaporator and evaporate to dryness. Dissolve the
residue in 10 mL of butanol R and add 3 mL of hydrochloric
acid R1 and 8 mL of water R. Heat on a water-bath under a
reflux condenser for 1 h. Allow to cool and transfer to a
100 mL volumetric flask. Rinse the round-bottomed flask
with 3 quantities, each of 20 mL, of methanol R. Add the
Figure 1847.-1. - Illustration for identification test B of powdered rinsings to the volumetric flask and dilute to 100.0 mL with
herbal drug of butcher's broom methanol R.
Reference solution Dissolve 5.0 mg of ruscogenins CRS in
Reference solution Dissolve 1 mg of ruscogenins CRS and 1 mg 100 mL of methanol R.
of stigmasterol R in methanol R and dilute to 5 mL with the Column:
same solvent. — size: I = 0.25 m, 0 = 4.6 mm;
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel — stationary phase: octadecylsilyl silica gel for chromatography R
plate R (2-10 pm)]. (5 pm).
Mobile phase:
Mobile phase methanol R, methylene chloride R (7:93 V!V).
— mobile phase A: water Ry
Application 10 pL [or 4 pL] as bands. — mobile phase B: acetonitrile Rl’y
Drying In air.
Detection Spray with vanillin reagent R3 dry in an oven at (min) (per cent V/V) (per cent V/V)
100-105 °C for 1 min and examine in daylight. 0 - 25 40 60
Results See below the sequence of zones present in the 25 - 27 40 -> 0 60 -> 100
27 - 37 0 100
test solution. Furthermore, other weak zones may be present
Top of the plate Detection Spectrophotometer at 203 nm.
Several zones of various colours Injection 20 pL.
Stigmasterol: a violet zone
Identification ofpeaks Use the chromatogram supplied with
ruscogenins CRS and the chromatogram obtained with the
reference solution to identify the peaks due to neoruscogenin
and ruscogenin.
Relative retention With reference to neoruscogenin (retention
Ruscogenins: a yellow zone A yellow zone (ruscogenins)
time = about 16 min): ruscogenin = about 1.2.
Several zones of various colours System suitability: reference solution:
neoruscogenin and ruscogenin.
Calculate the percentage content of sapogenins, expressed as
ruscogenins (neoruscogenin and ruscogenin), using the
following expressionะ
2016 Calendula Flower IV-125
Al X 777.2 X 4 X P! A3 X 7712 X 4 X p2
A2 X 7711 A4 X 7711
A1 = area of the peak due to ruscogenin in the

A2 = area of the peak due to ruscogenin in the
A3 = area of the peak due to neoruscogenin in the
A4 ะะะ area of the peak due to neoruscogenin in the
WI = mass of the herbal drug to be examined used to
m2 = mass of niscogenins CRS used to prepare the
reference solution, in grams;
Pl = percentage content of ruscogenin in niscogenins CRS',
p2 = percentage content of neoruscogenin in niscogenins
CRS.
--------------------------------------------------------------------------- Ph Eur
Calendula Flower
Ph Eur_______________________________________________
DEFINITION
Whole or cut, dried, and fully opened flowers that have been
detached from the receptacle of the cultivated, double
flowered varieties of Calendula officinalis L. Figure 1297.-1. - Illustration for identification test B of powdered
Content herbal drug of calendula flower
Minimum 0.4 per cent of flavonoids, expressed as hyperoside
(C2]H2o012; Mr 464.4) (dried drug). Reference solution Dissolve 1.0 mg of caffeic acid R, 1.0 mg of
IDENTIFICATION chlorogenic acid R and 2.5 mg of rutin R in 10 mL of
methanol R.
A. The ligulate florets consist of a yellow or orange-yellow
ligule, about 3-5 mm wide and about 7 mm in the middle Plate TLC silica gel plate R.
part, with a 3-toothcd apex and a hairy, partly sickle-shaped, Mobile phase anhydrous formic acid R, water R, ethyl acetate R
yellowish-brown or orange-brown tube with a projecting style (10:10:80 VIVIV).
and a bifid stigma occasionally with a partly bent yellowish- Application 20 pL of the test solution and 10 |1L of the
brown or orange-brown ovary. The tubular florets, about reference solution, as bands.
5 mm long, are present and consist of the yellow, orange-red Development Over a path of 10 cm.
or reddish-violet 5-lobed corolla and the yellowish-brown or
Drying At 100-105 °C.
orange-brown tube, hairy in its lower part, mostly with a
partly bent yellowish-brown or orange-brown ovary. Detection Spray the still-warm plate with a 10 g/L solution of
spray with a 50 g/L solution of macrogol 400 R in methanol R;
allow to dry in air for 30 min and examine in ultraviolet light
at 365 nm.
characters (Figure 1297.-1): fragments of epidermises of the
corolla [C, F, KJ containing light yellow oil droplets, some Results The chromatogram obtained with the reference
with fairly large anomocytic stomata (2.8.3) [Fa, Ka]; solution shows in the lower part a yellowish-brown
covering trichomes biseriate, multicellular and conical [G], fluorescent zone (rutin), in the middle part a light bluish
usually fragmented, and glandular trichomes with a fluorescent zone (chlorogenic acid) and in the upper part a
multicellular stalk [E], very abundant on the base of the light bluish fluorescent zone (caffeic acid).
corolla [D]; fragments of parenchyma of the corolla [B] The chromatogram obtained with the test solution shows a
containing prisms and very small cluster crystals of calcium yellowish-brown fluorescent zone corresponding in position
to the zone due to rutin in the chromatogram obtained with
oxalate [Ba, Da] and small vessels [Bb]; spherical pollen
grains up to about 40 |im in diameter with a sharply spiny the reference solution, below and directly above it, it shows a
yellowish-green fluorescent zone and a light bluish
exine and 3 germinal pores [A, J]; occasional fragments of
fluorescent zone corresponding to the zone due to
the stigmas with short, bulbous papillae [H].
chlorogenic acid in the chromatogram obtained with the
c. Thin-layer chromatography (2.2.27). reference solution, a yellowish-green fluorescent zone above it
Test solution Mix 1.0 g of the powdered herbal drug (500) and a light bluish fluorescent zone shortly below the zone
(2.9.12) and 10 mL of methanol R and heat on a water-bath due to caffeic acid in the chromatogram obtained with the
under a reflux condenser for 10 min. Cool and filter.
IV-126 Capsicum 2016
reference solution. Furthermore, other zones may be present

Capsicum * *
TESTS (Ph. Eur. monograph 1859) ***
Foreign matter (2.8.2) Preparations
Maximum 5 per cent of bracts and maximum 2 per cent of Refined and Quantified Capsicum Oleoresin
other foreign matter. Standardised Capsicum Tincture
Loss on drying (2.2.32) Ph Eur___________________________________ __________________________
powdered herbal drug (500) (2.9.12) by drying in an oven at DEFINITION
105 ๐c for 2 h. Dried ripe fruits of Capsicum annuum L. var. minimum
(Miller) Heiser and small-fruited varieties of Capsicum
Total ash (2.4.16) frutescens L.
Content
ASSAY Minimum 0.4 per cent of total capsaicinoids, expressed as
Stock solution Into a 100 mL round-bottomed flask introduce capsaicin (C]8H27NO3; Mr 305.4) (dried drug).
0.800 g of the powdered herbal drug (500) (2.9.12), 1 mL of
a 5 g/L solution of hexamethylenetetramine R, 7 mL of
CHARACTERS
hydrochloric acid R1 and 20 mL of acetone R. Boil the mixture Extremely pungent taste.
under a reflux condenser for 30 min. Filter the liquid IDENTIFICATION
through a plug of absorbent cotton into a 100 mL volumetric A. The fruit is yellowish-orange or reddish-brown, oblong
flask. Add the absorbent cotton to the residue in the round- conical with an obtuse apex, about 1-3 cm long and up to
bottomed flask and extract with 2 quantities, each of 20 mL, 1 cm in diameter at the widest part, occasionally attached to
of acetone R, each time boiling under a reflux condenser for a 5-toothed inferior calyx and a straight peduncle. Pericarp
10 min. Allow to cool to room temperature, filter the liquid somewhat shrivelled, glabrous, enclosing about 10-20 flat,
through a plug of absorbent cotton, then filter the combined reniform seeds 3-4 mm long, cither loose or attached to a
acetone solution through a filter-paper into the volumetric reddish dissepiment.
flask, and dilute to 100.0 mL with acetone R by rinsing the B. Microscopic examination (2.8.23). The powder is orange.
flask and filter. Introduce 20.0 mL of this solution into a Examine under a microscope using chloral hydrate solution R.
separating funnel, add 20 mL of water R and extract the The powder shows the following diagnostic characters
mixture with 1 quantity of 15 mL and then with 3 quantities, (Figure 1859.-1): fragments of the epicarp, in surface view,
each of 10 mL, of ethyl acetate R. Combine the ethyl acetate with cells often arranged in rows of 5 to 7 [E], thick-wallcd
extracts in a separating funnel, rinse with 2 quantities, each when close to the peduncle [B] and with a cuticle uniformly
of 50 mL, of water R, filter the extract over 10 g of anhydrous striated [A]; fragments of the pericarp, in transverse section
sodium sulfate R into a 50 mL volumetric flask and dilute to [D], showing the epicarp covered by a thick cuticle [Da] and
50.0 mL with ethyl acetate R. parenchymatous cells frequently containing droplets of red
Test solution To 10.0 mL of the stock solution add 1 mL of oil, occasionally containing microsphenoidal crystals of
aluminium chloride reagent R and dilute to 25.0 mL with a calcium oxalate [Db]; fragments of endocarp [C] with
5 per cent v/v solution of glacial acetic acid R in methanol R. characteristic island groups of sclerenchymatous cells [Ca],
Compensation liquid Dilute 10.0 mL of the stock solution to the groups being separated by thin-walled parenchymatous
25.0 mL with a 5 per cent v/v solution of glacial acetic cells [Cb]; fragments of the seeds having an episperm
acid R in methanol R. composed of large, greenish-yellow, sinuous-walled sclereids
Measure the absorbance (2.2.25) of the test solution after with thin outer walls and strongly and unevenly thickened
30 min, by comparison with the compensation liquid at radial and inner walls which are conspicuously pitted [G];
425 run. endosperm parenchymatous cells with drops of oil and
aleurone grains, 3-6 pm in diameter [H]; occasional
fragments from the calyx having an outer epidermis with
anisocytic stomata (2.8.3) [J], an inner epidermis with no
stomata and many glandular trichomes with uniseriate stalks
A X 1.25 and multicellular heads [N], and a mesophyll [L] with many
m idioblasts containing prisms of calcium oxalate [La] or
microsphenoidal crystals of calcium oxalate [Lb]; prisms [K]
i.e. taking the specific absorbance of hyperoside to be 500. or clusters [M] of calcium oxalate, isolated; annularly and
A = absorbance at 425 am; spirally thickened vessels [F].
m = mass of the herbal drug to be examined, in grams. c. Thin-layer chromatography (2.2.27).
________________________________________________________________ Ph Eur Test solution To 0.50 g of the powdered herbal drug (500)
(2.9.12) add 5.0 mL of ether R, shake for 5 min and filter.
Reference solution Dissolve 2 mg of capsaicin R and 2 mg of
dihydrocapsaicin R in 5.0 mL of ether R.
Plate TLC octadecylsilyl silica gel plate R.
Mobile phase water R, methanol R (20:80 V/V).
Drying In air.
2016 Capsicum IV-127
— stationary phase: base-deactivated end-capped phenylsilyl silica

gel for chromatography R (5 pm);
Mobile phase acetonitrile Rl> 1 g/L solution of phosphoric acid R
(40:60 V!V}.
Injection 10 pL.
Run time 1.2 times the retention time of dihydrocapsaicin.
Elution order Nordihydrocapsaicin, nonivamide, capsaicin,
dihydrocapsaicin.
System suitability Reference solution (a):
nonivamide and capsaicin.
Calcdate the percentage content of nonivamide with
reference to the total capsaicinoid content, using the
Al X ไท2 X Pl X 100
>เ2 X 7ท1 X c
Al = area of the peak due to nonivamide in the

A2 = area of the peak due to nonivamide in the
chromatogram obtained with reference solution (b);
Figure 1859.-1. - Illustration for identification test B of powdered prepare the test solution, in grams;
herbal drug of capsicum ni2 = mass of nonivamide CRS used to prepare reference
solution (b), in grams;
p 1 = percentage content of nonivamide in nonivamide
Detection treat with a 5 g/L solution of
CRS\
dichloroquinonechlorimide R in methanol R, and expose to
c = percentage content of total capsaicinoids, as
ammonia vapour until blue zones appear. Examine in
determined in the assay.
daylight.
Limit:
— nonivamide: maximum 5.0 per cent of the total
capsaicinoid content.
chromatogram obtained with the test solution. Foreign matter {2.8.2}
Fruits of c. annuum L. var. longum (Sendtn.) are absent.
powdered herbal drug (500) {2.9.12} by drying in an oven at
Capsaicin ะ a blue zone A blue zone (capsaicin) 105 °C for 2 h.
Dihydrocapsaicin ะ a blue zone A blue zone (dihydrocapsaicin) Total ash {2.4.16}
ASSAY
Reference solution Test solution Liquid chromatography (2.2.29) as described in the test for
nonivamide.
TESTS Calculate the percentage content of total capsaicinoids (C),
expressed as capsaicin, using the following expression:
Nonivamide
(A3 4- As 4- As) X 7714 X p2 X 2
(2.9.12} add 100 mL of methanol R. Allow to macerate for
A4 X m3
30 min. Place in an ultrasonic bath for 15 min. Filter into a
A3 = area of the peak due to capsaicin in the
100 mL volumetric flask, rinse the flask and filter with
methanol R3 then dilute to 100.0 mL with the same solvent.
Reference solution (a) Dissolve 10.0 mg of capsaicin CRS and chromatogram obtained with reference solution (a);
2.0 mg of nonivamide CRS in methanol R and dilute to A5 = area of the peak due to dihydrocapsaicin in the
50.0 mL with the same solvent. chromatogram obtained with the test solution;
Reference solution (b) Dissolve 4.0 mg of nonivamide CRS in A$ = area of the peak due to nordihydrocapsaicin in the
methanol R and dilute to 100.0 mL with the same solvent. chromatogram obtained with the test solution;
Column'. m3 = mass of the herbal drug to be examined used to
— size: I = 0.25 m, 0 = 4.6 mm; prepare the test solution, in grams;
IV-128 Capsicum Oleoresin 2016
พ4 = mass of capsaicin CRS used to prepare reference Reference solution (a) Dissolve 10.0 mg of capsaicin CRS and
solution (a), in grams; 2.0 mg of nonivamide CRS in methanol R and dilute to
p2 = percentage content of capsaicin in capsaicin CRS. 50.0 mL with the same solvent.
--------------------------------------------------------------------------------------------------------- Ph Eur Reference solution (b) Dissolve 4.0 mg of nonivamide CRS in
Column:
— size: I = 0.25 m, 0 = 4.6 mm;
— stationary phase: base-deactivated end-capped phenylsilyl silica
Refined and Standardised * * gel for chromatography R (5 pm);
Capsicum Oleoresin ***** — temperature: 30 °C.
(Ph. Eur. monograph 2336) Mobile phase acetonitrile RL 1 g/L solution of phosphoric acid R
Ph Eur______________________________________________________________
(40:60 VIV).
DEFINITION
Refined and standardised oleoresin produced from
Capsicum (1859). Injection 10 pL.
Content Run time 1.2 times the retention time of dihydrocapsaicin.
12.0 per cent to 18.0 per cent m/m of total capsaicinoids, Elution order Nordihydrocapsaicin, nonivamide, capsaicin,
expressed as capsaicin (C18H27NO3; Afr 305.4). dihydrocapsaicin.
PRODUCTION Relative retention With reference to capsaicin (retention
time = about 19 min): nordihydrocapsaicin = about 0.9;
The oleoresin is produced from the herbal drug by an
appropriate procedure, using ethanol (minimum nonivamide = about 0.95; dihydrocapsaicin = about 1.3.
90 per cent V/V). System suitability Reference solution (a):
CHARACTERS nonivamide and capsaicin.
Appearance
Calculate the percentage content of nonivamide with
Red or brown mobile extract.
reference to the total capsaicinoid content, using the
IDENTIFICATION following expression:
Al X 7ท2 X Pl X 100
Test solution Dissolve 50 mg of the oleoresin to be examined
in 5 mL of ether R. A2 X mi X c
Reference solution Dissolve 2 mg of capsaicin R and 2 mg of A1 = area of the peak due to nonivamide in the
dihydrocapsaicin R in 5 mL of ether R. chromatogram obtained with the test solution;
Plate TLC octadecylsilyl silica gel plate R (ว-40 pm) [or A2 = area of the peak due to nonivamide in the
TLC octadecylsilyl silica gel plate R (2-10 pm)]. chromatogram obtained with reference solution
Mobile phase water R3 methanol R (20:80 VIV). (b);
m1 = mass of the oleoresin to be examined used to
Development Over a path of 12 cm [or 6 cm]. 1ท2 = mass of nonivamide CRS used to prepare reference
Drying In air. solution (b), in grams;
Detection Treat with a 0.25 g/L solution of p] = percentage content of nonivamide in nonivamide
dichloroquinonechlorimide R in ethyl acetate R, expose to CRS>
ammonia vapour until blue zones appear. Examine in c = percentage content of total capsaicinoids, as
daylight. determined in the assay.
Results See below the sequence of zones present in the Limit:
chromatograms obtained with the reference solution and the — nonivamide: maximum 5.0 per cent of the total
test solution. Furthermore, other zones may be present in the capsaicinoid content.
chromatogram obtained with the test solution. Water (2.5.72)
Maximum 8.0 per cent, determined on 5.00 g.
Top of the plate ASSAY
Capsaicin ะ a blue zone
nonivamide.
A blue zone (capsaicin)
Calculate the percentage content of total capsaicinoids (C),
Dihydrocapsaicin: a blue zone A faint blue zone (dihydrocapsaicin)
(A3 4- A5 + A6) X m3 X P2 X 2
Reference solution Test solution A4 X mi

TESTS chromatogram obtained with the test solution;
Nonivamide A4 = area of the peak due to capsaicin in the
Liquid chromatography (2.2.29). chromatogram obtained with reference solution
Test solution Dissolve 0.300 g of the oleoresin to be examined (a);
in 60 mL of methanol R and dilute to 100.0 mL with the A5 = area of the peak due to dihydrocapsaicin in the
same solvent. chromatogram obtained with the test solution;
2016 Capsicum Soft Extract, Standardised IV-129
•^6 = area of the peak due to nordihydrocapsaicin in the TESTS

chromatogram obtained with the test solution; Nonivamide
W1 = mass of the oleoresin to be examined used to Liquid chromatography (2.2.29).
prepare the test solution, in grams; Test solution Stir the extract to be examined until
w3 = mass of capsaicin CRS used to prepare reference homogeneous, heating, if necessary, to not more than 60 °C.
solution (a), in grams; Disperse 0.350 g of the homogeneous extract in 35 mL of a
p2 = percentage content of capsaicin in capsaicin CRS. mixture of water R and propanol R (40:60 VIV). Shake for
______________________ PhEur 30 min and dilute to 50.0 mL with propanol R. Dilute
25.0 mL of the solution to 50.0 mL with the mobile phase
and filter through a membrane filter (nominal pore size
0.45 pm).
Capsicum Soft Extract, ;* *; Reference solution (a) Dissolve 2.0 mg of nonivamide CRS in
Standardised ***** the mobile phase and dilute to 25.0 mL with the mobile
phase (solution A). Dissolve 8.0 mg of capsaicin CRS in a
mixture of 5.0 mL of solution A and 45 mL of the mobile
PhEur. _____________
phase. Dilute to 100.0 mL with the mobile phase.
DEFINITION Reference solution (b) Dissolve 8.0 mg of nonivamide CRS in
Standardised soft extract produced from Capsicum (1859). the mobile phase and dilute to 100.0 mL with the mobile
Content phase. Dilute 5.0 mL of the solution to 100.0 mL with the
2.0 per cent to 2.4 per cent of total capsaicinoids, expressed mobile phase.
as capsaicin (C18H27NO3; Mr 305.4). Column:
— size: I = 0.25 m, 0 = 4.6 mm;
PRODUCTION — stationary phase: base-deactivated end-capped phenylsilyl silica
The extract is produced from the herbal drug by a suitable gel for chromatography R (5 pm);
procedure using ethanol (80 per cent VIV). — temperature: 30 GC.
The content of total capsaicinoids in the extract is Mobile phase acetonitrile R1, 1 g/L solution of phosphoric acid R
determined and adjusted, if necessary, to the value specified (40:60 VIV).
by adding a suitable inert excipient, for example liquid
glucose.
CHARACTERS
Injection 10 pL.
Appearance
Run time 1.2 times the retention time of dihydrocapsaicin.
Reddish-brown, glutinous matter.
Elution order Nordihydrocapsaicin, nonivamide, capsaicin,
IDENTIFICATION dihydrocapsaicin.
Relative retention With reference to capsaicin (retention
Test solution To 0.25 g of the extract to be examined add time = about 19 min): nordihydrocapsaicin = about 0.9;
10 mL of a mixture of tvater R and propanol R (40:60 VIV}. nonivamide ะ= about 0.95; dihydrocapsaicin = about 1.3.
Shake for 5 min. Filter, if necessary.
Reference solution Dissolve 2 mg of capsaicin R and 1 mg of — resolution: minimum 1.5 between the peaks due to
dihydrocapsaicin R in 5 mL of methanol R. nonivamide and capsaicin.
Plate TLC octadecylsilyl silica gel plate R (5-40 pm) [or Calcdate the percentage content of nonivamide with
TLC octadecylsilyl silica gel plate R (2-10 pm)]. reference to the total capsaicinoid content, using the
Mobile phase water R) methanol R (20:80 VIV). following expression:
Development Over a path of 12 cm [or 6 cm]. Al X 7ท2 X Pl X 5
Drying In air. A2 X mi X c
Detection Treat with a 0.25 g/L solution of
dichloroquinonechlorimide R in ethyl acetate R, expose to Al = area of the peak due to nonivamide in the
ammonia vapour until blue zones appear. Examine in chromatogram obtained with the test solution;
daylight. A2 = area of the peak due to nonivamide in the
Results See below the sequence of zones present in the chromatogram obtained with reference solution
chromatograms obtained with the reference solution and the (b);
test solution. Furthermore, other zones may be present in the m1 = mass of the extract to be examined used to
chromatogram obtained with the test solution. prepare the test solution, in grams;
m2 = mass of nonivamide CRS used to prepare reference
Top of the plate solution (b), in grams;
Pl = percentage content of nonivamide in nonivamide
CRS-,
Capsaicin: a blue zone A blue zone (capsaicin)
C = percentage content of total capsaicinoids, as
Dihydrocapsaicin: a blue zone A blue zone (dihydrocapsaicin) determined in the assay.
Limit:
— nonivamide: maximum 5.0 per cent of the total
capsaicinoid content.
IV-130 Capsicum Preparations 2016
Dry residue (2.8.16) Detection treat with a 0.25 g/L solution of

Minimum 70.0 per cent พ/พ, determined on 2.00 g. dichloroquinonechlorimide R in ethyl acetate R, expose to
ASSAY ammonia vapour until blue zones appear. Examine in
daylight.
nonivamide. Results See below the sequence of zones present in the
Calculate the percentage content of total capsaicinoids (C),
(A3 + A5 + A6) X 7ท3 X p2

Top of the plate
A4 X 7711
A} = area of the peak due to capsaicin in the Capsaicin: a blue zone A blue zone (capsaicin)
Dihydrocapsaicin: a blue zone A faint blue zone (dihydrocapsaicin)
A4 = area of the peak due to capsaicin in ±e
(a); Reference solution Test solution
A5 = area of the peak due to dihydrocapsaicin in the
Af) = area of the peak due to nordihydrocapsaicin in the TESTS
chromatogram obtained with the test solution; Nonivamide
พ1 = mass of the extract to be examined used to Liquid chromatography (2.2.29).
Test solution Dilute 50.0 g of the tincture to be examined to
พ3 = mass of capsaicin CRS used to prepare reference
100.0 mL with methanol R.
p2 = percentage content of capsaicin in capsaicin CRS. Reference solution (a) Dissolve 10.0 mg of capsaicin CRS and
2.0 mg of nonivamide CRS in methanol R and dilute to
______________________________________________________________ Ph Eur 50.0 mL with the same solvent.
Reference solution (b) Dissolve 4.0 mg of nonivamide CRS in
Column'.
Standardised Capsicum Tincture ***** — size'. I = 0.25 m, 0 = 4.6 mm;
— stationary phase', base-deactivated end-capped phenylsilyl silica
(Ph. Eur. monograph 2337) *** gel for chromatography R (5 pm);
Ph Elf_______________________________________________________________ — temperature'. 30 °C.
DEFINITION Mobile phase acetonitrile R13 1 g/L solution of phosphoric acid R
Standardised tincture produced from Capsicum (1859) or (40:60 V/V).
Refined and standardised capsicum oleoresin (2336). Flow rate 1.0 mUmin.
Content Detection Spectrophotometer at 225 nm.
90 per cent to 110 per cent of the nominal content of total Injection 10 pL.
capsaicinoids, expressed as capsaicin (C18H27NO3; Run time 1.2 times the rentention time of dihydrocapsaicin.
Mt 305.4), stated on the label, which is between Elution order Nordihydrocapsaicin, nonivamide, capsaicin,
0.020 per cent พ/พ and 0.060 per cent m/m. dihydrocapsaicin.
PRODUCTION System suitability Reference solution (a):
The tincture is produced from the herbal drug or oleoresin — resolution: minimum 1.5 between the peaks due to
and ethanol (70 per cent v/v to 85 per cent V/V) by an nonivamide and capsaicin.
appropriate procedure. Calculate the percentage content of nonivamide with
CHARACTERS reference to the total capsaicinoid content, using the
Appearance following expression:
Yellowish-orange or reddish-orange liquid.
Al X 7712 X Pl X 100
IDENTIFICATION
Thin-layer chromatography (2.2.27). A2 X 7ท1 X c
Test solution Shake 10 mL of the tincture to be examined
with 10 mL of hexane R. Allow to separate and use the lower A1 = area of the peak due to nonivamide in the
layer. chromatogram obtained with the test solution;
Reference solution Dissolve 1 mg of capsaicin R and 1 mg of A2 = area of the peak due to nonivamide in the
dihydrocapsaicin R in 5 mL of ether R. chromatogram obtained with reference solution
(b);
Plate TLCoctadecylsilyl silica gel plate R (5-40 pm) [or
พ1 ะ= mass of the tincture to be examined used to
TLC octadecylsilyl silica gel plate R (2-10 pm)].
Mobile phase water R, methanol R (20:80 V/V). พ2 = masร of nonivamide CRS used to prepare reference
Application 20 pL [or 2 pL] as bands of 15 mm [or 8 mm]. solution (b), in grams;
Development Over a path of 12 cm [or 6 cm]. pl = percentage content of nonivamide in nonivamide
Drying In air. CRS,
2016 Caraway IV-131
c ะ= percentage content of total capsaicinoids, as B. Reduce to a powder (355) (2.9.12). The powder is
determined in the assay. yellowish-brown. Examine under a microscope using chloral
Limit: hydrate solution R. The powder shows the following diagnostic
nonivamide: maximum 5.0 per cent of the total characters: fragments of the secretory cells composed of
capsaicinoid content. yellowish-brown or brown, thin-walled, polygonal secretory
Ethanol (2.9./0) cells, frequently associated with a layer of thin-walled,
transversely elongated cells, 8-12 pm wide; fragments of the
95 per cent to 105 per cent of the content stated on the
label. epicarp with thick-walled cells and occasional anomocytic
stomata (2.8.3)', numerous endosperm fragments containing
Methanol and 2-propanol (2.9.//) aleurone grains, droplets of fatty oil and microcrystals of
Maximum 0.05 per cent VIV of methanol and maximum calcium oxalate in rosette formation; spiral vessels
0.05 per cent VIV of 2-propanol. accompanied by sclerenchymatous fibres; rarely some fibre
ASSAY bundles from the carpophore; groups of rectangular to sub-
Liquid chromatography (2.2.29) as described in the test for rectangular sclereids from the mesocarp with moderately
nonivamide. thickened and pitted walls may be present.
Calculate the percentage content of total capsaicinoids (C), c. Thin-layer chromatography (2.2.27).
expressed as capsaicin, using the following expression: Test solution Shake 0.5 g of the powdered herbal drug (710)
(2.9.12) with 5.0 mL of ethyl acetate R for 2-3 min. Filter
(A3 + A5 + As) X m4 X P2 X 2 over 2 g of anhydrous sodium sulfate R.
A4 X m3 Reference solution Dissolve 2 pL of carvone R and 5 pL of olive
oil R in 1.0 mL of ethyl acetate R.
A3 = area of the peak due to capsaicin in the Plate TLC silica gel plate R.
chromatogram obtained with the test solution; Mobile phase ethyl acetate R, toluene R (5:95 VIV).
A4 ะะะ area of the peak due to capsaicin in the Application 20 pL of the test solution and 10 pL of the
chromatogram obtained with reference solution reference solution, as bands.
(a); Development Over a path of 10 cm.
A5 = area of the peak due to dihydrocapsaicin in the
chromatogram obtained with the test solution; Drying In air.
A6 ะ= area of the peak due to nordihydrocapsaicin in the Detection A Examine in ultraviolet light at 254 nm.
chromatogram obtained with the test solution; Results A The chromatograms obtained with the test solution
= mass of the tincture to be examined used to and with the reference solution show a quenching zone
prepare the test solution, in grams; (carvone) in the central part against a light background.
m4 = mass of capsaicin CRS used to prepare reference Detection B Spray with anisaldehyde solution R and, while
solution (a), in grams; observing, heat at 100-105 °C for 2-4 min; examine in
p2 = percentage content of capsaicin in capsaicin CRS. daylight.
---------- ----------------------------------------------------------------------------------------------- Ph Eur Results B The zones due to carvone are dark orange-brown;
the chromatogram obtained with the test solution shows
above the zone due to carvone a violet zone similar in
position and colour to the zone due to triglycerides of olive
oil in the chromatogram obtained with the reference solution;
Caraway ★ * the chromatogram obtained with the test solution shows
(Caraway Fruit, Ph Eur monograph 1080) *** close to the solvent front a weak violet zone due to terpene
hydrocarbons and in the lower part some weak, mostly violet-
When Powdered Caraway is prescribed or demanded, greyish and brownish zones.
material complying with the appropriate requirements below
and containing not less than 2.5% v/w (25 mUkg) of TESTS
essential oil shall be dispensed or supplied. Water (2.2.13)
Ph Eur______________________________________________________________
powdered herbal drug.
DEFINITION Total ash (2.4.16)
Whole, dry mericarp of Carum carvi L. Maximum 7.0 per cent.
Content ASSAY
Minimum 30 mI7kg of essential oil (anhydrous drug).
CHARACTERS Use 10.0 g of drug reduced to a powder (710) (2.9.12)
Odour reminiscent of carvone. immediately before the determination, a 500 mL round-
bottomed flask, 200 mL of water R as the distillation liquid,
IDENTIFICATION
and 0.50 mL of xylene R in the graduated tube. Distil at a
A. The fruit is a cremocarp of almost cylindrical shape. It is
rate of 2-3 mL/min for 90 min.
generally 3-6.5 mm long and 1-1.5 mm wide. The mericarps,
____________ ___________ Ph Eur
usually free, are greyish-brown or brown, glabrous, mostly
sickle-shaped, with both ends sharply terminated. Each bears
5 prominent narrow ridges. When cut transversely the profile
shows an almost regular pentagon and 4 vittae on the dorsal
surface and 2 on the commissural surface may be seen with a
lens.
IV-132 Caraway Oil 2016

Caraway Oil ** ** chromatographic profile.
(Ph. Eur. monograph 1817) *** Results The characteristic peaks in the chromatogram
PhEir______________________________________________________________ obtained with the test solution are similar in retention time to
DEFINITION
solution.
Oil obtained by steam distillation from the dry fruits of
Canon carui L. TESTS
CHARACTERS
0.904 to 0.920.
Appearance
Clear, colourless or yellow liquid. Refractive index (2.2.6)
1.484 to 1.490.
IDENTIFICATION
Optical rotation (2.2.7)
First identification B
-r 65° to + 81°.
Acid value (2.5.7)
A. Thin-layer chromatography (2.2.27). Maximum 1.0, determined on 5.00 g.
Test solution Dissolve 40 pL of the substance to be examined
in 1.0 mL of toluene R.
Reference solution Dissolve 10 pL of carvone R and 5 pL of procedure.
carveol R in 1.0 mL of toluene R.
Test solution Dissolve 0.200 g of the substance to be
Plate TLC silica gel F25.\ plate R (5-40 pm) [or TLC silica gel examined in heptane R and dilute to 10.0 mL with the same
plate R (2-10 pm)]. solvent.
Mobile phase ethyl acetate R, toluene R (5:95 VIV). Reference solution (a) Dissolve 5 |.lL of p-myrcene R, 80 pL of
Application 10 pL [or 2 pL] as bands. limonene R, 5 pL of dihydrocarvone R, 100 pL of carvone R
Development Over a path of 10 cm [or 5 cm]. and 5 pL of carveol R in heptane R and dilute to 10.0 mL
Drying In air. with the same solvent.
Detection A Examine in ultraviolet light at 254 nm. Reference solution (b) Dissolve 10 pL of carvone R in heptane R
and dilute to 10 mL with the same solvent. Dilute 0.1 mL of
Results A See below ±e sequence of zones present in the
this solution to 10 mL with heptane R.
Column:
— material: fused silica,
— size: z = 30 m, 0 =ะ 0.53 mm,
— stationary phase: macrogol 20 000 R, (film thickness 1 pm).
Top of the plate Carrier gas helium for chromatography R.
Carvone; a quenching zone A quenching zone (carvone) Split ratio 1:50.
Temperature:

Time Temperature
(min) (°C)_____________
Column 0-5 60
Detection B Spray with anisaldehyde solution R and heat at
100-105 °C for 5-10 min. Examine immediately in daylight. 5 - 68 60 -> 250
Results B See below the sequence of zones present in the 68 - 75 250
Injection port 250
test solution. Furthermore, several zones of weak intensity are
present, particularly in the lower third, in the chromatogram Detector 260
Top of the plate Injection 1.0 pL.
A reddish-violet zone Elution order Order indicated in the composition of reference
solution (a). Record the retention times of these substances.
A reddish-violet zone — resolution: minimum 4.5 between the peaks due to
Carvone: a red to orange-brown An intense red to orange-brown P-myrcene and limonene.
zone (carvone)
Carveol; a reddish-violet zone A reddish-violet zone (carveol) the components of the reference solution in the
A violet-blue zone
Limits:
Reference solution Test solution — P-myrcene: 0.1 per cent to 1.0 per cent,
— limonene: 30.0 per cent to 45.0 per cent,
— trans-dihydrocarvone: maximum 2.5 per cent,
2016 Cardamom Oil LV-133
— carvone'. 50.0 per cent to 65.0 per cent, Macroscopical Fruit: a trilocular inferior capsule, up to about
— trans-carveol: maximum 2.5 per cent. 2 cm long, ovoid or oblong, dull green to pale buff, plump or
disregard limit', the area of the peak in the chromatogram slightly shrunken, obtusely triangular in cross section, nearly
obtained with reference solution (b). smooth or longitudinally striated. Seeds in each loculus in
Chiral purity two rows, forming an adherent mass attached to the axile
Gas chromatography (2.2.28). placenta. Seed: pale to dark reddish brown, about 4 mm long
and 3 mm broad, irregularly angular, marked with six to
Test solution Dissolve 20 mg of the substance to be examined
in heptane R and dilute to 10.0 mL with the same solvent. eight transverse wrinkles, with a longitudinal channel
containing the raphe, each seed enveloped by a colourless,
Reference solution Dissolve 10 mg of (—)-carvone R and 10 mg membranous aril. Transversely cut surface of seed showing a
of carvone R1 in heptane R and dilute to 10.0 mL with the brown testa, white starchy perisperm, grooved on one side,
same solvent.
yellowish endosperm and a paler embryo.
Column'.
Microscopical Seed: aril composed of flattened, thin-walled,
— material', fused silica,
parenchymatous cells. Testa composed of the following
— size: z = 30 m, 0 = 0.25 mm,
layers: (i) outer epidermis of thick-walled, narrow, axially
— stationary phase: modified p-cyclodextrin for chiral elongated cells; (ii) a layer of collapsed parenchyma subjacent
chromatography R1 (film thickness 0.25 pm). to the outer epidermis; (iii) a single layer (two or three layers
Carrier gas helium for chromatography R. near the raphe) of large, thin-walled, rectangular cells
Flow rate 2.0 mL/min. containing volatile oil; (iv) two or three layers of parenchyma;
Split ratio 1:30. (v) layers of thin-walled, flattened cells; (vi) distinctive
Temperature: sclerenchymatous layer of closely packed brown, thick-walled
cells, each with a bowl-shaped cavity in the upper part
containing a warty silica body; (vii) inner layer consisting of
Time Temperature flattened cells. Perisperm: cells thin-walled, packed with
(min) (°C) numerous starch granules up to 6 pm in diameter and, in a
Column 0 - 80 50 -> 170
small cavity, one to seven prisms of calcium oxalate about
Injection port 230 10 to 30 pm long. Endosperm parenchymatous, thin-walled,
Detector 230
with a granular hyaline mass of protein in each cell. Embryo:'
cells small, containing aleurone grains.
TESTS
Detection Flame ionisation. Foreign matter
Injection 1 pL. Of the fruit, not more than 1.0%; of the separated seeds, not
System suitability: reference solution: more than 3.0%, Appendix XI D.
— resolution: minimum 2.4 between the peaks due to Volatile oil
(-)-carvone (1st peak) and carvone R1 (2nd peak). In the seeds, not less than 4.0% v/w, Appendix XI E,
Calculate the percentage content of the (-)-carvone from the Method I. Use 20 g of the unground seeds and distil for
following expression: 5 hours.
Acid-insoluble ash
-41 -1-- X 100 Of the seeds, not more than 3.5%, Appendix XI K.
Al + A2 Ash
Of the seeds, not more than 6.0%, Appendix XI J.
A1 = area of the peak due to (—)-carvone,
A2 = area of the peak due to carvone Rl.
Limit:
— (-)-carvone: maximum 1 per cent. Cardamom Oil
STORAGE Preparations
At a temperature not exceeding 25 °C. Aromatic Cardamom Tincture
___________________ i_______________ Ph Eur Compound Cardamom Tincture
DEFINITION
Cardamom Oil is obtained by distillation from crushed
Cardamom Fruit.
Cardamom Fruit CHARACTERISTICS
In making preparations of Cardamom, only the seed is used. A clear, colourless or pale yellow liquid, visibly free from
The seed is removed from the fruit, immediately powdered water; odour, that of Cardamom Fruit.
or bruised and used immediately in making the preparation.
TESTS
Cardamom seed, after removal from the fruit, should not be
Ester value
stored.
90 to 156, Appendix X c.
DEFINITION Optical rotation
Cardamom Fruit consists of the dried, nearly ripe fruit of +20° to +40°, Appendix V F.
Elettaria cardamomum Maton var. minuscula Burkill.
Refractive index
CHARACTERISTICS 1.461 to 1.467, Appendix V E.
Odour and taste of the seeds, strongly aromatic.
IV-134 Cardamom Preparations 2016
Solubility in ethanol 0.1m sodium hydroxide KS is equivalent to 9.210 mg of

Soluble, at 20°, in 6 volumes of ethanol (70%) 3 glycerol. Calcdate the percentage v/v of glycerol, taking its
Appendix X M. weight per mL to be 1.260 g.
Weight per mL Relative density
0.917 to 0.940 g, Appendix V G. 0.925 to 0.937, Appendix V G.
STORAGE
Cardamom Oil should be kept in a well-filled container and
protected from light.
Cascara * *
Aromatic Cardamom Tincture Preparation
DEFINITION Standardised Cascara Dry Extract
Cardamom Oil 3 mL When Powdered Cascara is prescribed or demanded,
Caraway Oil 10 mL material complying with the requirements below with the
Cinnamon Oil 10 mL exception of Identification test A and the test for Foreign
Clove Oil 10 mL matter shall be dispensed or supplied.
Strong Ginger Tincture 60 mL Ph Eur_______ __ ____________________________________________________
Ethanol (90 per cent) Sufficient to produce 1000 mL
DEFINITION
The tincture complies with the requirements for Tinctures stated Dried, whole or fragmented bark of Rhaninus purshiana DC.
under Extracts and with the following requirements. (syn. Frangula purshiana (DC.) A.Gray).
TESTS Content
Ethanol content Minimum 8.0 per cent of hydroxyanthracene glycosides of
84 to 87% v/v, Appendix vni F, Method in. which minimum 60 per cent consists of cascarosidcs, both
Relative density expressed as cascaroside A (C27H32O14; Afr 580.5) (dried
0.825 to 0.845, Appendix V G. drug).
IDENTIFICATION
A. The bark occurs in slightly channelled or nearly flat
pieces, usually 1-5 mm in thickness, usually varying greatly in
Compound Cardamom Tincture length and width. The outer surface is grey or dark greyish-
brown and shows occasional lenticels that are orientated
DEFINITION
transversally. It is usually more or less completely covered by
Cardamom Oil 0.450 mL
a whitish coat of lichens, epiphytic moss and foliaceous
Caraway Oil 0.400 mL
liverwort. The inner surface is yellow or reddish-brown or
Cinnamon Oil 0.225 mL
almost black with fine longitudinal striations; it turns red
Cochineal, in moderately coarse powder 7g
when treated with alkali. The yellow fracture is short and
Glycerol 50 mL
granular in the outer part and somewhat fibrous in the inner
part.
Extemporaneous preparation B. Microscopic examination {2.8.23}. The powder is
The following directions apply. yellowish-brown. Examine under a microscope using chloral
Moisten the Cochineal with a sufficient quantity of Ethanol hydrate solution R. The powder shows the following diagnostic
(60 per cent) and prepare 900 mL of tincture by percolation, characters (Figure 0105.-1): bundles [A] of partly lignified
Appendix XI F. Add the Cardamom Oil, the Caraway Oil, phloem fibres [Aa], accompanied by crystal sheaths
the Cinnamon Oil and the Glycerol and sufficient Ethanol containing prisms of calcium oxalate [Ab] and sometimes
(60 per cent) to produce 1000 mL; mix. Filter, if necessary. including meddiary rays [Ac]; isolated sclereids [G] or
The tincture complies with the requirements for Tinctures stated groups of sclereids [B] accompanied by crystal sheaths [Ba];
under Extracts and with the following requirements. isolated cluster crystals [C] or prisms [E] of calcium oxalate;
parenchymatous cells [F, H] containing a yellow substance
TESTS that becomes deep red when treated with alkali, sometimes
Ethanol content accompanied by cells containing cluster crystals of calcium
52 to 57% v/v, Appendix vm F, Method III. oxalate [Ha]; cork cells, in surface view [D] or in transverse
Glycerol section [J], associated with parenchyma, some cells of which
4.5 to 5.5% v/v when determined by the following method. contain cluster crystals of calcium oxalate [Ja]; frequently
Dilute 20 mL to 100 mL with water. To 20 mL of this epiphytes [K], which may be liverworts, entire or in
solution add 100 mL of water and 1 g of activated charcoal fragments, having a lamina 1 cell thick without a midrib and
and boil under a reflux condenser for 15 minutes. Filter and composed of isodiametric cells, or leaves of mosses, having a
wash the filter and charcoal with sufficient water to produce lamina 1 cell thick composed of elongated cells and
150 mL. Add 0.25 mL of bromocresol purple solution and possessing a midrib several cells thick.
neutralise with 0.1m sodium hydroxide or 0.05m sulfuric acid to
the blue colour of the indicator. Add 1.4 g of sodium periodate
and allow to stand for 15 minutes. Add 3 mL of propane-132-
diol, shake and allow to stand for 5 minutes. Add 0.25 mL of
bromocresol purple solution and titrate with 0.1m sodium
hydroxide vs to the same blue colour. Each mL of
2016 Cascara IV-135

(2.9.72) add 5 mL of ethanol (70 per cent VIV) R and heat to
boiling. Cool and centrifuge. Decant the supernatant
immediately and use within 30 min.
Reference solution Dissolve 20 mg of barbaloin R in ethanol
(70 per cent VIV) R and dilute to 10 mL with the same
solvent.
Plates TLC silica gel plate R (2 plates).
Mobile phase water R, methanol R, ethyl acetate R
(13:17:100 V/V/V).
A. Application: 10 pL as bands.
Drying In air for 5 min.
Detection spray with about 10 mL of a 50 g/L solution of
potassium hydroxide R in ethanol (50 per cent VIV) R and heat
at 100-105 °C for 15 min; examine immediately after
heating.
solution shows, in the central part, a reddish-brown zone due
to barbaloin; examine in ultraviolet light at 365 nm; the zone
due to barbaloin shows intense yellowish-brown fluorescence;
in the chromatogram with the test solution, no zone with
orange-brown fluorescence is seen between the zone due to
barbaloin and the zones due to cascarosides.
B. Application: 10 pL of the test solution, as a band.
Drying In air for not more than 5 min.
Detection Spray immediately with a 5 g/L solution of
nitrotetrazolium blue R in methanol R and examine
Figure 0105.-1. - Illustration for identification test B of powdered immediately.
herbal drug of cascara
Results No violet or greyish-blue zones appear.
c. Examine the chromatograms obtained in test A for Other Foreign matter (2.8.2)
species of Rhamnus, anthrones. Maximum 1 per cent.
Results The chromatogram obtained with the test solution Loss on drying (2.2.32)
shows several reddish-brown zones with different intensities: Maximum 10.0 per cent, determined on 1.000 g of the
there are 4 faint zones, 3 being situated at about the mid powdered herbal drug (180) (2.9.72) by drying in an oven at
point of the chromatogram and 1 in the lower third and 105 °C for 2 h.
there is a strong zone in the upper third of the Total ash (2.4.16)
chromatogram. Examine in ultraviolet light at 365 nm. Maximum 7.0 per cent.
The chromatogram obtained with the test solution shows ASSAY
several zones with the same fluorescence, situated above and
Cany out the assay in 24 h, protected from bright light.
particularly below (cascarosides) that due to barbaloin in the
chromatogram obtained with the reference solution. Stir 1.00 g of the powdered herbal drug (180) (2.9.72) into
100 mL of boiling water R and continue boiling and stirring
D. Heat 0.2 g of the powdered herbal drug (180) (2.9.72)
for 5 min. Allow to cool, dilute to 100.0 mL with water R}
with 50 mL of water R on a water-bath for 15 min. Allow to
shake, filter and discard the first 20 mL of filtrate. Transfer
cool and filter. To 10 mL of the filtrate add 20 mL of
10.0 mL of the filtrate to a separating funnel, add 0.1 mL of
hydrochloric acid R1 and heat on a water-bath for 15 min.
7 M hydrochloric acid and shake with 2 quantities, each of
Allow to cool, transfer to a separating funnel and shake with
20 mL, of a mixture of 1 volume of ether R and 3 volumes of
3 quantities, each of 20 mL, of ether R. Reserve the aqueous
hexane R. Wash the combined organic extracts with 5 mL of
layer (solution A). Combine the 3 ether extracts and shake
water R) discard the organic layer and return the rinsings to
with 10 mL of dilute ammonia R2. The aqueous layer the aqueous layer. Shake the combined aqueous layers with
becomes reddish-violet. Transfer solution A to a small flask,
4 quantities, each of 30 mL, of ethyl acetate R freshly
add 5 g of ferric chloride R and heat on a water-bath for saturated with water R (to 150 mL of ethyl acetate R add
30 min. Allow to cool, transfer to a separating funnel and 15 mL of water R} shake for 3 min and allow to stand) on
shake with 15 mL of ether R. Wash the ether layer with each occasion allowing separation to take place until the
10 mL of water Ry discard the aqueous layer and shake the organic layer is clear. Combine the ethyl acetate extracts.
ether layer with 5 mL of dilute ammonia R2. A red colour Use the aqueous layer for the assay for cascarosides and the
develops in the aqueous layer. organic layer for the assay for hydroxyanthracene glycosides
TESTS other than cascarosides.
Other species of Rhamnus\ anthrones Hydroxyanthracene glycosides other than cascarosides
Thin-layer chromatography (2.2.27). Transfer the organic layer to a suitable flask and remove the
solvent by distillation, evaporating almost to dryness.
IV-136 Cascara Preparations 2016
Dissolve the residue in 0.3-0.5 mL of methanol R and transfer Content

to a volumetric flask, rinsing the 1st flask with warm water R 90 per cent to 110 per cent of the nominal content of
and adding the rinsings to the methanolic solution. Allow to hydroxyanthracene glycosides, expressed as cascaroside A
cool and dilute to 50.0 mL with water R. Transfer 20.0 mL (C27H32OH; Mr 580.5), stated on the label; minimum
of this solution to a 100 mL round-bottomed flask with a 60 per cent of the hydroxyanthracene glycosides are
ground-glass neck and containing 2 g offerric chloride R and cascarosides, expressed as cascaroside A. The nominal
12 mL of hydrochloric acid R. Attach a reflux condenser and content of hydroxyanthracene glycosides is within the range
place the flask in a water-bath so that the level of the water is 8.0 per cent to 25.0 per cent m/m (dried extract).
above that of the liquid in ±e flask and heat for 4 h. Allow
PRODUCTION
to cool, transfer the solution to a separating funnel and rinse
The extract is produced from the herbal drug by an
the flask successively with 3-4 mL of 1 M sodium hydroxide
appropriate procedure using either boiling water or a
and 3-4 mL of water Rj adding the rinsings to ±e separating
hydroalcoholic solvent at least equivalent in strength to
funnel. Shake the contents of the separating funnel with
ethanol (60 per cent VIV}.
3 quantities, each of 30 mL, of a mixture of 1 volume of
ether R and 3 volumes of hexane R. Wash the combined CHARACTERS
organic layers with 2 quantities, each of 10 mL, of water R Appearance
and discard the rinsings. Dilute the organic layer to Brown, free-flowing powder.
100.0 mL with the mixture of ether and hexane. Take IDENTIFICATION
20.0 mL, evaporate carefully to dryness on a water-bath and
dissolve the residue in 10.0 mL of a 5 g/L solution of
magnesium acetate R in methanol R. Measure the absorbance Test solution To 0.2 g of the extract to be examined add
(2.2.25) at 440 nm and 515 nm using methanol R as the 5 mL of ethanol (70 per cent VIV) R and heat to boiling. Cool
compensation liquid. If the ratio of the absorbance at and centrifuge. Decant the supernatant solution immediately
515 nm to that at 440 nm is less than 2.4, the assay is and use within 30 min.
invalid. Reference solution Dissolve 20 mg of barbaloin R and 2 mg of
Calculate the percentage content of hydroxyanthracene emodin R in ethanol (70 per cent VIV) R and dilute to 10 mL
glycosides other than cascarosides, expressed as with the same solvent.
cascaroside A, using the following expression: Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
plate R (2-10 pm)].
A X 6.95 Mobile phase water R) methanol R, ethyl acetate R
m (13:17:100 V/V/V).
i.e. taking the specific absorbance to be 180. Development Over a path of 10 cm [or 6 cm].
Cascarosides hydroxide R in ethanol (50 per cent VIV) R and heat to
Dilute the aqueous layer to 50.0 mL with water R. Treat 100-105 °C for 15 min; examine in ultraviolet light at
20.0 mL of this solution as described above in the assay of 365 nm.
hydroxyanthracene glycosides other than cascarosides.
Measure the absorbance (2.2.25) of the test solution at
440 nm and 515 nm. If the ratio of the absorbance at
test solution.. Furthermore, other zones may be present in the
515 nm to that at 440 nm is less than 2.7, the assay is
invalid.
Calculate the percentage content of cascarosides, expressed
as cascaroside A, using the following expression: Top of the plate
Emodin : a red fluorescent zone A faint red fluorescent zone

A X 6.95
i.e. taking the specific absorbance to be 180. Barbaloin: a yellowish-brown A yellowish-brown fluorescent zone
A = absorbance at 515 nm; fluorescent zone
m = mass of the substance to be examined, in grams. A blue fluorescent zone
___________ ________ __________ _ _______________________________ Ph Eur
An intense yellowish-brown
fluorescent zone
3 yellowish-brown fluorescent
Standardised Cascara Dry Extract ******* zones
(Ph Eur monograph 1844) *

Preparation
Cascara Tablets
Ph Eur________________________ _ __________________________________ ____ TESTS
DEFINITION
Standardised dry extract obtained from Cascara (0105). Maximum 5.0 per cent.
2016 Cascara Preparations IV-137
ASSAY If the ratio of the absorbance at 515 nm to that at 440 nm is

Carry out the assay within 24 h, protected from bright light. less than 2.7, the assay is invalid.
To 0.500 g of the extract to be examined add 80 mL of Calculate the percentage content of cascarosides, expressed
ethanol (70 per cent V/V) R. Shake, and allow to stand in the as cascaroside A, using the following expression:
dark for at least 8 h. Dilute to 100.0 mL with ethanol
(70 per cent V/V) R. Shake and filter, discarding the first A X 6.95
20 mL of filtrate. Transfer 10.0 mL of the filtrate to a
separating funnel, add 0.1 mL of 1 M hydrochloric acid and
shake with 2 quantities, each of 20 mL, of a mixture of i.e. taking the specific absorbance to be 180.
1 volume of ether R and 3 volumes of hexane R. Wash the A = absorbance at 515 nm;
combined organic extracts with 5 mL of water R. Discard the m = mass of the substance to be examined, in grams.
organic layer and return the rinsings to the hydroalcoholic
layer. Shake with 4 quantities, each of 30 mL, of ethyl LABELLING
acetate R freshly saturated wi± water R (prepared as follows: The label states the nominal content of hydroxyanthracene
to 150 mL of ethyl acetate R add 15 mL of water R, shake for glycosides, expressed as cascaroside A.
3 min and allow to stand), on each occasion allowing the _____________________________________________________________ Ph Eur
layers to separate until the organic layer is clear. Combine
the ethyl acetate extracts. Use the aqueous layer for the assay
of cascarosides and the organic layer for the assay of
Cascara Tablets
Hydroxyanthracene glycosides other than cascarosides
DEFINITION
Transfer the organic layer to a round-bottomed flask and
remove the solvent by distillation, evaporating almost to Cascara Tablets contain Standardised Cascara Dry Extract.
dryness. Dissolve the residue in 0.5 mL of methanol R, add They are coated.
10 mL of water R at 40 °C and transfer to a 50 mL The tablets comply with the requirements stated under Tablets and
volumetric flask, rinsing the round-bottomed flask with with the following requirements.
water R at 40 °C and adding the rinsings to the Content of total hydroxyanthracene derivatives
hydromethanolic solution. Allow to cool and dilute to 17.0 to 23.0 mg, of which not less than 60% consists of
50.0 mL with water R. Transfer 20.0 mL of the solution to a cascarosides, both expressed as cascaroside A.
100 mL round-bottomed flask with a ground-glass neck
IDENTIFICATION
containing 2 g of feme chloride R and 12 mL of hydrochloric
Carry out the method for thin-layer chromatography,
acid R. Attach a reflux condenser and place the flask in a
water-bath so that the level of the water is above that of the
liquid in the flask and heat for 4 h. Allow to cool, transfer (1) Boil a quantity of the powdered tablets containing the
the solution to a separating funnel and rinse the flask equivalent of 32 mg of total hydroxyanthracene derivatives
successively with 4 mL of 1 M sodium hydroxide and 4 mL of with 5 mL of 70% v/v of ethanol, cool and centrifuge. Decant
water R, adding the rinsings to the separating funnel. Shake the supernatant liquid immediately and use within
the contents of the separating funnel with 3 quantities, each 30 minutes.
of 30 mL, of a mixture of 1 volume of ether R and 3 volumes (2) Dissolve 20 mg of barbaloin and 2 mg of emodin in
of hexane R. Wash the combined organic layers with 70% v/v of ethanol and dilute to 10 mL with the same
2 quantities, each of 10 mL, of water R and discard the solvent.
rinsings. Dilute the organic layer to 100.0 mL with a mixture CHROMATOGRAPHIC CONDITIONS
of 1 volume of ether R and 3 volumes of hexane R. Take
(a) Use silica gel F254 precoated plates or high-performance
20.0 mL of the solution, evaporate carefully to dryness on a
silica gel F2S4 (Merck silica gel F254 HPTLC plates are
water-bath and dissolve the residue in 10.0 mL of a 5 g/L
suitable).
solution of magnesium acetate R in methanol R. Measure the
absorbance (2.2.25) at 440 nm and 515 nm, using (b) Use the mobile phase as described below.
methanol R as the compensation liquid. If the ratio of the (c) Apply 10 pL [or 2 |1L] of each solution, as bands.
absorbance at 515 nm to that at 440 nm is less than 2.4, the (d) Develop the plate to 10 cm [or 6 cm].
assay is invalid. (e) After removal of the plate, dry in air, spray with a 5% w/v
Calculate the percentage content of hydroxyanthracene solution of potassium hydroxide in 50% v/v ethanol, heat at
glycosides other than cascarosides, expressed as 100 to 105° for 15 minutes and examine under ultraviolet
cascaroside A, using the following expression: light (365 nm).
MOBILE PHASE
A X 6.95 13 volumes of water, 17 volumes of methanol and
m 100 volumes of ethyl acetate.
CONFIRMATION
i.e. taking the specific absorbance to be 180. The chromatogram obtained with solution (1) show
A ะะะ absorbance at 515 nm; yellowish-brown fluorescent bands with Rf values of between
m = mass of the substance to be examined, in grams. 0.2 and 0.25, an intense yellowish-brown fluorescent band
Cascarosides with an Rf value of about 0.3, a blue fluorescent band with
Dilute the aqueous layer to 50.0 mL with water R. Treat an Rf value of about 0.6, a yellowish-brown fluorescent band
20.0 mL of this solution as described above in the assay of with an Rf value of about 0.7 corresponding in colour and
hydroxyanthracene glycosides other than cascarosides. position to the band obtained with barbaloin in solution (2)
Measure the absorbance (2.2.25) at 440 nm and 515 nm. and a faint reddish fluorescent band with an Rf value of
IV-138 Cassia Oil 2016
about 0.9 corresponding in position to emodin in the combined organic layers with 2-quantities, each of
solution (2). 10 mL, of water and discard the rinsings. Dilute the organic
layer to 100.0 mL with a mixture of 1 volume of ether and
Top of the plate 3 volumes of hexane. Take 20.0 mL of the solution,
evaporate carefully to dryness on a water-bath and dissolve
A faint red fluorescent band Emodin a red fluorescent band the residue in 10.0 mL of a 0.5% w/v solution of magnesium
acetate in methanol. Measure the absorbance of the resulting
solution at 440 nm and at 515 nm, Appendix II B, using
A yellow-brown fluorescent band Barbaloin: a yellow-brown methanol in the reference cell. The assay is not valid if the
fluorescent band ratio of the absorbance at 515 nm to that at 440 nm is less
A blue fluorescent band than 2.4.
Calculate the percentage content of hydroxyanthracene
glycosides other than cascarosides, expressed as cascaroside
A, using the following expression:
An intense yellow-brown
fluorescent band A X 6.95
3 yellow-brown fluorescent bands m
i.e. taking the specific absorbance to be 180.

Solution (1) Solution (2) /4 = absorbance at 515 nm;
m = weight of the substance being examined, in grams.
Cascarosides
TESTS To the aqueous solution reserved from the preliminary
Disintegration extraction add sufficient water to produce 50.0 mL. Carry
Comply with the requirements stated under Tablets but for out the Assay for hydroxyanthracene gycosides other than
sugar-coated tablets the maximum time is 120 minutes. cascarosides, beginning at the words, ‘Transfer 20 mL ... ’.
ASSAY Measure the absorbance of the resulting solution at 440 nm
Carry out the assay within 24 hours, protectedfrom bright light. and at 515 nm, Appendix II B, using methanol in the
Add 80 mL of 70% v/v ethanol to a quantity of the powdered reference cell. The assay is not valid if the ratio of the
tablets containing 75 mg of total hydroxyanthracene absorbance at 515 nm to that at 440 nm is less than 2.7.
derivatives. Shake and allow to stand in the dark for at least Calculate the percentage content of cascarosides, expressed
8 hours. Dilute to 100.0 mL with 70% v/v of ethanol. Shake as cascaroside A, using the following expression:
and filter, discarding the first 20 mL of filtrate. Transfer
10.0 mL of the filtrate to a separating funnel, add 0.1 mL of A X 6.95
1M hydrochloric acid and shake with 2-quantities, each of m
20 mL, of a mixture of 1 volume of ether and 3 volumes of
hexane. Wash the combined organic extracts with 5 mL of i.e. taking the specific absorbance to be 180.
water. Discard the organic layer and return the rinsings to the A = absorbance at 515 nm;
hydroalcoholic layer. Shake with 4-quantities, each of 30 mL, m ะ= weight of the substance being examined, in grams.
of ethyl acetate freshly saturated with water prepared by
shaking 150 mL of ethyl acetate with 15 mL of water for
LABELLING
3 minutes and allowing to stand until the layers have The label states the nominal content of hydroxyanthracene
separated and the organic layer is clear. Combine the ethyl glycosides, expressed as cascarosides A.
acetate extracts and use the aqueous layer for the assay of
cascarosides and the organic layer for the assay of
Hydroxyanthracene glycosides other than cascarosides Cassia Oil *******
Transfer the organic layer to a round-bottomed flask and
remove the solvent by distillation, evaporating almost to
dryness. Dissolve the residue in 0.5 mL of methanol, add 8007-80-5
10 mL of water at 40° and transfer to a 50 mL volumetric Ph Eur__________________________________________________________ _____
flask, rinsing the round-bottomed flask with water at 40° and
adding the rinsings to the hydromethanolic solution. Allow to DEFINITION
cool and dilute to 50.0 mL with water. Transfer 20.0 mL of Essential oil obtained by steam distillation of the leaves and
the solution to a 100 mL round-bottomed flask with a young branches of Cinnamomum cassia Blume (C. aromaticuni
ground-glass neck containing 2 g of iron (ill) chloride Nees).
hexahydrate and 12 mL of 7m hydrochloric acid. Attach a CHARACTERS
reflux condenser and place the flask in a water-bath so that Appearance
the level of the water IS above that of the liquid in the flask Clear, mobile, yellow or reddish-brown liquid.
and heat for 4 hours. Allow to cool, transfer the solution to a
Characteristic odour reminiscent of cinnamic aldehyde.
separating funnel and rinse the flask successively with 4 mL
of Im sodium hydroxide and 4 mL of water, adding the IDENTIFICATION
rinsings to the separating funnel. Shake the contents of the First identification B
separating funnel with 3-quantities, each of 30 mL, of a Second identification A
mixture of 1 volume of ether and 3 volumes of hexane. Wash A. Thin-layer chromatography (2.2.27).
2016 Greater Celandine IV-139
Test solution Dissolve 0.5 mL of the essential essential oil to Temperature:

be examined in acetone R and dilute to 10 mL with the same
solvent.
Time Temperature
Reference solution Dissolve 50 pL of trans-cinnamic aldehyde Ry (min) (°C)
10 pL of eugenol R and 50 mg of coumarin R in acetone R and Column 0 - 10 60
dilute to 10 mL with the same solvent.
10 - 75 60 -> 190
75 - 160 190
Mobile phase methanol Ry toluene R (10:90 VIV).
Injection port 200
Development Over a path of 15 cm. Detector 240
Drying In air.
Detection A Examine in ultraviolet light at 365 nm. Detection Flame ionisation.
Results A The zone of blue fluorescence in the chromatogram Injection 0.2 pL.
obtained with the test solution is similar in position and Elution order Order indicated in the composition of the
colour to the zone in the chromatogram obtained with the reference solution, depending on the operating conditions
reference solution (coumarin). and the state of the column, coumarin may elute before or
Detection B Spray with anisaldehyde solution Ry examine in after trans-2-methoxycinnamaldehyde; record the
daylight while heating at 100-105 °C for 5-10 min. retention times of these substances.
Results B The chromatogram obtained with the reference System suitability: reference solution:
solution shows in its upper part a violet zone (eugenol) and — resolution: minimum 1.5 between the peaks due to trans-2-
above this zone a greenish-blue zone (trans-cinnamic methoxycinnamaldehyde and coumarin.
aldehyde). The chromatogram obtained with the test solution Identification of components Using the retention times
shows a zone similar in position and colour to the zone due determined from the chromatogram obtained with the
to trans-cinnamic aldehyde in the chromatogram obtained reference solution, locate the components of the reference
with the reference solution and may show a very faint zone solution in the chromatogram obtained with the test solution.
due to eugenol. Other faint zones are present.
Determine the percentage content of each of these
B. Examine the chromatograms obtained in the test for components. The percentages are within the following
chromatographic profile. ranges:
Results The principal peaks in the chromatogram obtained — trans-cinnamic aldehyde: 70 per cent to 90 per cent;
with the test solution are similar in retention time to those in — cinnamyl acetate: 1.0 per cent to 6.0 per cent;
the chromatogram obtained with the reference solution. — eugenol: maximum 0.5 per cent;
Eugenol may be absent from the chromatogram obtained — trans-2-methoxycinnamaldehyde: 3.0 per cent to
with the test solution. 15 per cent;
TESTS — coumarin: 1.5 per cent to 4.0 per cent.
Relative density (2.2.5) STORAGE
1.052 to 1.070. Protected from heat.
Refractive index (2.2.6) ______________________________________________________________ PhEur
1.600 to 1.614.
-1° to + 1°.
Chromatographic profile Greater Celandine * \
procedure. (Ph. Eur. monograph 1861) **
Test solution The essential oil to be examined. PhEur__________________________________________ ____________________
Reference solution Dissolve 100 pL of trans-cinnamic DEFINITION

aldehyde Ry 10 pL of cinnamyl acetate Ry 10 pL of eugenol Ry Dried, whole or cut aerial parts of Chelidonium majus L.
10 pL of trans-2-methoxycinnamaldehyde R and 20 mg of collected during flowering.
coumarin R in 1 mL of acetone R. Content
Column'. Minimum 0.6 per cent of total alkaloids, expressed as
— material', fused silica; chelidonine (C20H19NO5; Mr 353.4) (dried drug).
— size'. I = 60 m, 0 = about 0.25 mm;
— stationary phase: bonded macrogol 20 000 R.
IDENTIFICATION
A. The stems are rounded, ribbed, yellowish or greenish-
Carrier gas helium for chromatography R. brown, somewhat pubescent, about 3-7 mm in diameter,
Flow rate 1.5 mL/min. hollow and mostly collapsed. The leaves are thin, irregularly
Split ratio 1:100. pinnate, the leaflets ovate to oblong with coarsely dentate
margins, the terminal leaflet often 3-lobed; the adaxial
surface is bluish-green and glabrous, the abaxial surface paler
and pubescent, especially on the veins. The flowers have
2 deeply concavo-convex sepals, readily removed, and
4 yellow, broadly ovate, spreading petals about 8-10 mm
long; the stamens are numerous, yelloWj and a short style
IV-140 Greater Celandine 2016
arises from a superior ovary; long, capsular, immature fruits Plate TLC silica gel plate R.
are rarely present. Mobile phase anhydrous formic acid R, water R, propanol R
B. Microscopic examination (2.8.23). The powder is dark (1:9:90 FZP7F).
greyish-green or brownish-green. Examine under a Application 10 pL as bands.
numerous fragments of upper epidermis, composed of cells Drying In air.
with sinuous walls in surface view [B], accompanied by Detection Spray with potassium iodobismuthate solution R and
underlying palisade parenchyma [Ba]; numerous fragments of dry in air; spray with sodium nitrite solution R and allow to dry
lower epidermis in surface view [A, E] bearing anomocytic in air; examine in daylight.
stomata (2.8.3) [Aa] and bases of covering trichomes [Ab], Results See below the sequence of zones present in the
sometimes accompanied by underlying spongy parenchyma chromatograms obtained with the reference solution and the
[Ea]; long, uniseriate, multicellular covering trichomes, test solution. Furthermore, other weaker zones may be
usually fragmented, with thin-walled cells, sometimes present in the chromatogram obtained with the test solution.
collapsed [G]; vascular tissue from ±e leaves and stems
consisting of pitted and spirally thickened vessels [D]; groups
Top of the plate
of fibres [C]; articulated latex tubes with yellowish-brown
contents [F]; occasional fragments of the corolla [H]
consisting of thin-walled cells containing numerous pale
Methyl red: a red zone A brown zone
yellow droplets of oil [Ha]; spherical pollen grains about
30-40 pm in diameter with 3 pores and a finely pitted A brown zone
exine [J]. Papaverine: a greyish-brown zone A greyish-brown zone
2 brown zones
TESTS
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
(2.9.12) add 200 mL of dilute acetic acid R and heat on a
water-bath for 30 min, shaking frequently. Cool and dilute to
250.0 mL with dilute acetic acid R. Filter. Discard the first
20 mL of the filtrate. To 30.0 mL of the filtrate add 6.0 mL
of concentrated ammonia R and 100.0 mL of methylene
chloride R. Shake for 30 min. Separate the organic layer,
place 50.0 mL in a 100 mL round-bottomed flask and
evaporate to dryness in vacuo at a temperature not exceeding
40 °C. Dissolve the residue in about 2-3 mL of ethanol
(96 per cent) R, warming slightly. Transfer the solution to a
25 mL volumetric flask by rinsing the round-bottomed flask
Figure 1861.-1. - Illustration for identification test B ofpowdered with dilute sulfuric acid R and dilute to 25.0 mL with the
herbal drug of greater celandine same solvent. To 5.0 mL of the solution add 5.0 mL of a
C. Thin-layer chromatography (2.2.27). 10 g/L solution of chromotropic acid, sodium salt R in sulfuric
acid R in a 25 mL volumetric flask, stopper the flask and mix
carefully. Dilute to 25.0 mL with sulfuric acid R and stopper
(2.9.12) add 50 mL of dilute acetic acid R. Boil in a water
the flask.
bath under a reflux condenser for 30 min. Cool and filter.
To the filtrate add concentrated ammonia R until a strong Compensation liquid Prepare at the same time and in the same
alkaline reaction is produced. Shake with 30 mL of methylene manner as for the test solution: place in a 25 mL volumetric
chloride R. Dry the organic layer over anhydrous sodium flask 5.0 mL of dilute sulfuric acid R and 5.0 mL of a 10 g/L
sulfate R, filter and evaporate in vacuo to dryness. Dissolve solution of chromotropic acid, sodium salt R in sulfuric acid R,
the residue in 1.0 mL of methanol R. stopper the flask and mix carefully. Dilute to 25.0 mL with
sulfuric acid R and stopper the flask.
Reference solution Dissolve 2 mg of methyl red R and 2 mg of
papaverine hydrochloride R in 10 mL of ethanol (96 per cent) R.
2016 Centaury IV-141
Place both solutions on a water-bath for 10 min. Cool to epidermis of the testa showing large, brown reticulations and
about 20 °C and dilute if necessary to 25.0 mL with sulfuric a pitted surface.
acid R. Measure the absorbance (2.2.25) of the test solution c. Thin-layer chromatography (2.2.27).
at 570 nm by comparison with the compensation liquid.
Calculate the percentage content of total alkaloids, expressed (2.9.12) add 25 mL of methanol R, shake for 15 min and
as chelidonine, using the following expression: filter. Evaporate the filtrate to dryness under reduced
pressure and at a temperature not exceeding 50 °C. Take up
A X 2.23 the residue with small quantities of methanol R so as to
obtain 5 mL of solution, which may contain a sediment.
Reference solution Dissolve 1 mg of rutin R and 1 mg of
i.e. taking the specific absorbance of chelidonine to be 933.
swertiamarin R in methanol R and dilute to 1 mL with the
same solvent.
-—— --------------------------------------------------------------------------------------- Ph Eur F254 plate R (2-10 pm)].
Mobile phase water R, anhydrous formic acid R3 ethyl formate R
(4:8:88 VIVIV).
Centaury ★* ** Development In an unsaturated tank over a path of 12 cm [or
6 cm].
Drying In air.
Ph Elf_________________
DEFINITION Results A See below the sequence of the zones present in the
Whole or fragmented dried flowering aerial parts of chromatograms obtained with the reference solution and the
Centaurium erythraea Rafri ร. 1. including c. majus (H. et L.) test solution. Furthermore, other less intense quenching
Zeltner and c. suffruticosum (Griseb.) Ronn. (syn.: Erythraea zones may be present in the chromatogram obtained with the
centaurium Persoon; c. umbellatum Gilibert; c. minus Gars.). test solution.
CHARACTERS
Bitter taste. Top of the plate
IDENTIFICATION
A. The hollow cylindrical, light green to dark brown stem has
longitudinal ridges, and is branched only in its upper part.
The sessile leaves are entire, decussately arranged, and have Swertiamarin: a quenching zone A prominent quenching zone
an ovate to lanceolate lamina, up to about 3 cm long. Both (swertiamarin)
surfaces are glabrous and green to brownish-green. Rutin: a quenching zone
The inflorescence is diaxially branched. The tubular calyx is
green and has 5 lanceolate, acuminate teeth. The corolla
consists of a whitish tube divided into 5 elongated lanceolate
pink to reddish lobes, about 5-8 mm long. 5 stamens are
present attached to the top of the corolla tube. The ovary is
superior and has a short style, a broad bifid stigma and
100-105 °C for 5-10 min. Examine in daylight.
numerous ovules. Cylindrical capsules, about 7-10 mm long,
with small brown markedly rough seeds are frequently Results B See below the sequence of the zones present in the
present. chromatograms obtained with the reference solution and the
test solution. Furthermore, other less intense coloured zones
may be present in the chromatogram obtained with the test
greenish-yellow or brownish. Examine under a microscope,
solution.
following diagnostic characters: fragments from the stem with
lignified groups of fibres associated with narrow vessels, Top of the plate
tracheidal vessels occasional vessels with spiral thickening;
pined parenchyma of the pith and medullary rays; fragments
of leaf lamina with sinuous epidermal cells and striated
cuticle, especially over the margins and surrounding the Swertiamarin ะ a brown zone A brown zone (swertiamarin)
stomata; numerous stomata, mainly anisocytic (2.5.3);
Rutin: a yellow zone
fragments of the palisade mesophyll, each cell containing a
single prism crystal or, less frequently, a cluster crystal of A brownish-grey zone
calcium oxalate; fragments of calyx and corolla, those of the
calyx with straight-walled epidermal cells, those of the inner
epidermis of the corolla with obtuse papillae and radially
striated cuticle; parts of the endothecium with reticulate or A grey zone
ridge-shaped wall thickenings; triangularly rounded or Test solution
Reference solution
elliptical, yellow pollen grains, about 30 pm in diameter, with
a distinctly pitted exine and 3 germinal pores; fragments of
the wall of die fruit capsule composed of crossed layers of
fusiform cells; oil droplets from the seeds, fragments of the
-142 Centella 2016
TESTS Plate TLC silica gel G plate R.

Foreign matter (2.8.2) Mobile phase acetic acid R, formic acid Ry water R, ethyl
Maximum 3 per cent. acetate R (11:11:27:100 VIVIVIV).
Bitterness value (2.8.15) Application 10 pL, as bands.
Minimum 2000. Development Over a path of 15 cm.
Loss on drying (2.2.32) Drying In air.
100-105 °C; examine in daylight.
105 °C for 2 h.
Results The chromatograms obtained with the reference
Total ash (2.4.16) solution and the test solution show in the lower third a
Maximum 6.0 per cent. greenish-blue zone (asiaticoside). The chromatogram
----------------- ---------------------------------------------------------------------------------------- Ph Eur obtained with the test solution shows also below this zone a
violet zone (madecassoside); near the solvent front it shows a
light blue zone (asiatic acid) and just below a pinkish-violet
zone (madecassic acid); in the lower half it shows brown,
grey and brownish-green zones between the point of
Centella * * application and the zone due to madecassoside, and other
(Ph. Eur. monograph 1498) *** brownish-yellow or light yellow zones above the zone due to
asiaticoside.
PhEir______________________________________________________________
TESTS
DEFINITION Foreign matter (2.8.2)
Dried, fragmented aerial parts of Centella asiatica (L.) Urban. Maximum 7 per cent, of which maximum 5 per cent of
Content underground organs and maximum 2 per cent of other
Minimum 6.0 per cent of total triterpenoid derivatives, foreign matter.
expressed as asiaticoside (C48H78O19; Mr 959.15) (dried Loss on drying (2.2.32)
drug). Maximum 10.0 per cent, determined on 1.000 g of the
CHARACTERS powdered herbal drug (355) (2.9.12) by drying in an oven at
The leaves are very variable in size; the petiole is usually 105 °C for 2 h.
5-10, sometimes 15, times longer ±an the lamina, which is Total ash (2.4.16)
10-40 mm long and 20-40 mm, sometimes up to 70 mm, Maximum 12.0 per cent.
wide.
ASSAY
IDENTIFICATION Liquid chromatography (2.2.29).
A. The leaves are alternate, sometimes grouped together at Test solution Place 5.0 g of the powdered herbal drug (355)
the nodes, reniform or orbicular or oblong-elliptic and have (2.9.12) in a cellulose fingerstall in a continuous extraction
palmate nervation, usually with 7 veins, and a crenate apparatus (Soxhlet type). Add 100 mL of methanol R and
margin. Young leaves show a few trichomes on the lower heat for 8 h. Cool and dilute the extract to 100.0 mL with
surface while adult leaves are glabrous. The inflorescence, if methanol R. Filter through a 0.45 pm filter. Dilute 2.0 mL of
present, is a single umbel which usually consists of 3 flowers, the filtrate to 20.0 mL with methanol R.
rarely 2 or 4; the flowers are very small (about 2 mm)
Reference solution Dissolve 20.0 mg of asiaticoside R in
pentamerous and have an inferior ovary; the fruit, a
methanol R, if necessary using sonication, and dilute to
brownish-grey, orbicular cremocarp, up to 5 mm long, is very
20.0 mL with the same solvent. Dilute 2.0 mL of this
flattened laterally and has 7-9 prominent curved ridges.
solution to 100.0 mL with methanol R.
B. Reduce the drug to a powder (355) (2.9.12). The powder
Column'.
is greenish-grey. Examine under a microscope using chloral
— size'. I = 0.25 m, 0 = 4 mm;
characters: numerous fragments of leaf epidermis with
(5 pm).
polygonal cells having an irregularly striated cuticle, and
paracytic stomata (2.8.3) that are more numerous in the Phase mobile:
lower epidermis; fragments of petiole epidermis with — mobile phase A: acetonitrile for chromatography R'y
elongated cells; uniseriate, long, flexuous unicellular covering — mobile phase B: dilute 3 mL of phosphoric acid R to
trichomes, occasionally multicellular; young leaves; spiral 1000 mL with water R'y
vessels; resiniferous canals; calcium oxalate prisms and
macles up to 40 pm in diameter; bundles of narrow septate Time Mobile phase A Mobile phase B
fibres from the stem; fragments of the fruit: layers of wide ________ (min) (per cent V/V)_________ (per cent V/V)
cells in a parquetry arrangement, annular vessels, 0-65_________ 22___________________ 78
parenchyma cells containing simple or compound starch 65 - 66 55 45
granules.
66 - 76 95 5
76 - 85 22 78
(2.9.12) add 50 mL of ethanol (30 per cent VIV) R'y heat to
boiling under a reflux condenser and centrifuge. Flow rate 1.0 mL/min.
Reference solution Dissolve 5 mg of asiaticoside R in methanol R Detection Spectrophotometer at 200 nm.
2id dilute to 10 mL with the same solvent.
Injection 20 pL.
2016 Chamomile Flowers IV-143
Relative retention with reference to the solvent CHARACTERS

Madecassoside = about 5.8; asiaticoside = about 8.1; The flower-heads are white or yellowish-grey, composed of
madecassic acid = about 17.6; asiatic acid = about 21.7. solitary hemispherical capitula, made up of a solid conical
Calculate the response factor Rp of asiaticoside using the receptacle bearing the florets, each subtended by a
following expression: transparent small palea.
Strong and characteristic odour.
Al X Vi X 100 IDENTIFICATION
7ท1 X HPLCp
A. The capitula have a diameter of 8-20 mm; the receptacle
is solid; the base of the receptacle is surrounded by an
■^1 = area of the peak due to asiaticoside in the involucre consisting of 2-3 rows of compact and imbricated
chromatogram obtained with the reference bracts with scarious margins. Most florets are ligulate, but a
solution; few pale yellow tubular florets occur in the central region.
Fl = volume of the reference solution, in millilitres; Ligulate florets are white, dull, lanceolate and reflexed with a
m\ = mass of asiaticoside in the reference solution, dark brown, inferior ovary, a filiform style and a bifid stigma;
in milligrams; tubular florets have a five-toothed corolla tube,
HPLCp ะะะ purity determined for asiaticoside. 5 syngenesious, epipetalous stamens and a gynoecium similar
to that of the ligulate florets.
Calculate the mean response factor (rp) for asiaticoside
using the following expression: B. Separate the capitulum into its different parts. Examine
under a microscope using chloral hydrate solution R. All parts
of the flower-heads are covered with numerous small yellow
ERF. glistening glandular trichomes. The involucral bracts and
=N paleae have epidermal cells in longitudinal rows, sclerified at
the base and they are covered with conical trichomes, about
500 pm long, each composed of 3-4 very short base cells and
N a long, bent, terminal cell about 20 pm wide. The corolla of
£2 RF: = SUTn °f response factors of asiaticoside for the the ligulate flowers consists of papillary cells with cuticular
t=l chromatograms obtained with the reference striations. The ovaries of both kinds of florets have at then-
solution; base a sclerous ring consisting of a single row of cells.
N = number of injections of reference solution (N The receptacle and the ovaries contain small clusters of
= 4, at least). calcium oxalate. The pollen grains have a diameter of about
Calculate the percentage content of total triterpenoid 35 pm and are rounded or triangular with 3 germinal pores
derivatives, expressed as asiaticoside, using the following and a spiny exine.
expression: c. Thin-layer chromatography (2.2.27).
V โ A 4- (B X 1.017) + (C X 0.526) 4- (B X 0.509) (2.9.72) add 10 mL of methanol R and heat with shaking in a
water-bath at 60 °C for 5 min. Allow to cool and filter.
Reference solution Dissolve 2.5 mg of apigenin R and 2.5 mg of
apigenin 7-glucoside R in 10 mL of methanol R.
V = volume of the test solution, in millilitres;
tn = mass of the substance to be examined in the test Plate TLC silica gel plate R.
solution, in milligrams; Mobile phase glacial acetic acid R, water R, butanol R
A = area of the peak due to asiaticoside in the (17:17:66 VIVIV).
chromatogram obtained with the test solution; Application 10 pL, as bands.
B = area of the peak due to madecassoside in the Development Over a path of 10 cm.
Dying At 100-105 °C for 5 min.
c = area of the peak due to madecassic acid in the
chromatogram obtained with the test solution; Detection Spray the warm plate with a 10 g/L solution of
D = area of the peak due to asiatic acid in the diphenylboric acid aminoethyl ester R in methanol R, using about
chromatogram obtained with the test solution; 10 mL for a plate 200 mm square; subsequently spray with
RF = mean response factor of asiaticoside. the same volume of a 50 g/L solution of macrogol 400 R in
methanol R; allow to stand for about 30 min and examine in
---------------------------------------------------------------------------------------------------------- PhEur ultraviolet light at 365 nm.
solution shows in the upper third a yellowish-green
fluorescent zone (apigenin) and in the middle third a
Chamomile Flowers ***** yellowish fluorescent zone (apigenin 7-glucoside).
The chromatogram obtained with the test solution shows a
(Roman Chamomile Flower^ Ph Eur monograph 0380) *** yellowish-green fluorescent zone and a yellowish fluorescent
PhEur______________________________________________________________ zone similar in position and fluorescence to the zones due to
apigenin and apigenin 7-glucoside in the chromatogram
DEFINITION obtained with the reference solution; above the apigenin
Dried flower-heads of the cultivated double variety of 7-glucoside zone there is a brownish fluorescent zone
Chamaemelum nobile (L.) All. (Anthemis nobilis L.). (luteolin); immediately below the apigenin 7-glucoside zone
Content there is a light brownish fluorescent zone (apiin);
Minimum 7 ml/kg of essential oil (dried drug). immediately below the apiin zone there is a bright blue
IV-144 Cinchona Bark 2016
fluorescent zone and below this zone a bright blue thick-walled with an uneven lumen and with conspicuous,
fluorescent zone; other faint zones may be present. funnel-shaped pits, whole [A] or fragmented [F, J];
TESTS parenchymatous idioblasts filled with microprisms of calcium
Diameter of the flower-heads oxalate [E, G]; clusters of thin-walled phloem parenchyma
Maximum 3 per cent of flower-heads have a diameter smaller cells [L] accompanied by medullary rays in tangential section
than 8 mm. (DJ. Examine under a microscope using a 50 per cent VIV
solution of glycerol R. The powder shows a few starch
Deteriorated flower-heads granules 6-10 pm in diameter, mostly simple but occasionally
Brown or darkened flower-heads are absent. with 2 or 3 components, free [B] or included in
Loss on drying (2.2.32) parenchymatous cells [C].
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Use 20.0 g of whole drug, a 500 mL round-bottomed flask,
250 mL of water R as the distillation liquid and 0.50 mL of
xylene R in the graduated tube. Distil at a rate of
3-3.5 mL/min for 3 h.
---------------------------------------------------------------------------------------------------------- Ph Eur
Cinchona Bark * *
Cinchona; Red Cinchona Bark ***
Preparation
Cinchona Liquid Extract, Standardised
When Powdered Cinchona is prescribed or demanded,
material complying with the requirements below with the
exception of Identification test A and the test for Foreign
matter shall be dispensed or supplied.
Ph Eur___________________________________________________________ ___
DEFINITION
Whole or cut, dried bark of Cinchona pubescens Vahl
(Cinchona succirubra Pav.), of Cinchona calisaya Wedd., of
Cinchona ledgeriana Moens ex Trimen, or of their varieties or
hybrids.
herbal drug of cinchona bark
Content c. Thin-layer chromatography (2.2.27).
Minimum 6.5 per cent of total alkaloids, of which
30 per cent to 60 per cent consists of quinine-type alkaloids Test solution To 0.10 g of the powdered herbal drug (180)
(2.9.12) in a test-tube add 0.1 mL of concentrated ammonia R
(dried drug).
and 5 mL of methylene chloride R. Shake vigorously
CHARACTERS occasionally during 30 min and filter. Evaporate the filtrate
Intense bitter, somewhat astringent taste. to dryness on a water-bath and dissolve the residue in 1 mL
IDENTIFICATION of anhydrous ethanol R.
A. The stem and branch bark is supplied in quilled or curved Reference solution Dissolve 17.5 mg of quinine R, 2.5 mg of
pieces 2-6 mm thick. The outer surface is dull brownish-grey quinidine R3 10 mg of cinchonine R and 10 mg of
or grey and frequently bears lichens; it is usually rough, cinchonidine R in 5 mL of anhydrous ethanol R.
marked with transverse fissures and longitudinally furrowed Plate TLC silica gel plate R.
or wrinkled; exfoliation of the outer surface occurs in some Mobile phase diethylaniine R, ethyl acetate R, toluene R
varieties. The inner surface is striated and deep reddish- (10:20:70 VIVIP).
brown; the fracture is short in the outer part and fibrous in
the inner part.
Development Twice over a path of 15 cm.
reddish-brown. Examine under a microscope using chloral Drying At 100-105 °C, then allow to cool.
hydrate solution R. The powder shows the following diagnostic Detection A Spray with anhydrous formic acid R and allow to
characters (Figure 0174.-1): thin-walled cork cells filled with dry in air; examine in ultraviolet light at 365 nm.
reddish-brown contents, in surface view [K] and transverse Results A See below the sequence of zones present in the
section [H]; yellow, spindle-shaped striated phloem fibres up chromatograms obtained with the reference solution and the
to 90 pm in diameter and up to 1300 pm in length, very
2016 Cinchona Preparations IV-145
test solution. Furthermore, other fluorescent zones are x _ [4316 X 4348c] — [4316c X 434s] 100 2
present in the chromatogram obtained with the test solution. [4316g X 4348c] — [4316c X 4348g] m 1000
Top of the plate

_ [4ร16 X 4348g] — [4316g X 434ร] 100 2
[4316c X 4348g] — [4si6g X 4348c] 771 1000
Quinidine: a distinct blue A distinct blue fluorescent zone
fluorescent zone (quinidine) m - mass of the herbal drug used, in grams;
X = percentage content of quinine-type alkaloids;
Quinine: a distinct blue fluorescent A distinct blue fluorescent zone y — percentage content of cinchonine-type alkaloids;
zone (quinine) 4316 = absorbance of the test solution at 316 nm;
Reference solution Test solution y4348 = absorbance of the test solution at 348 nm;
4316c = absorbance of the reference solution containing
cinchonine at 316 nm, corrected to a
Detection B Spray with iodoplatviate reagent R.
concentration of 1 mg/1000 mL;
Results B See below the sequence of zones present in the 4316q = absorbance of the reference solution containing
chromatograms obtained with the reference solution and the quinine at 316 nm, corrected to a concentration
test solution. Furthermore, other zones are present in the of 1 mg/1000 mL;
chromatogram obtained with the test solution. 4348c = absorbance of the reference solution containing
cinchonine at 348 nm, corrected to a
Top of the plate concentration of 1 mg/1000 mL;
4348q = absorbance of the reference solution containing
quinine at 348 nm, corrected to a concentration
Cinchonine: a violet zone that A violet zone that becomes of 1 mg/1000 mL.
becomes violet-grey violet-grey (cinchonine)
Quinidine: a violet zone that
Calculate the content of total alkaloids (x + y), and calculate
A violet zone that becomes
becomes violet-grey violet-grey (quinidine) the relative content of quinine-type alkaloids using the
Cinchonidine: an intense dark An intense dark blue zone following expression:
blue zone (cinchonidine)
IQOz
Quinine: a violet zone that A violet zone that becomes x+y
becomes violet-grey violet-grey (quinine)
TESTS
Total ash (2.4.16)
^Maximum 6.0 per cent.
Standardised Cinchona Liquid ★ *
Loss on drying (2.2.32) Extract *****
Maximum 10 per cent, determined on 1.000 g of the (Ph. Eur. monograph 1818)
powdered herbal drug (355) (2.9.12) by drying in an oven at Ph Eur______________________________________________________________
105 °C for 2 h.
DEFINITION
ASSAY Liquid extract produced from Cinchona bark (0174).
Test solution In a 250 mL conical flask mix 1.000 g of the Content
powdered herbal drug (180) (2.9.12) with 10 mL of water R Minimum 4.0 per cent and maximum 5.0 per cent of total
and 7 mL of dilute hydrochloric acid R. Heat in a water-bath alkaloids, of which 30 per cent to 60 per cent are alkaloids of
for 30 min, allow to cool and add 25 mL of methylene the quinine type (C20H24N2O2; Afr 324.4).
chloride R, 50 mL of ether R and 5 mL of a 200 g/L solution
of sodium hydroxide R. Shake the mixture repeatedly for PRODUCTION
30 min, add 3 g of powdered tragacanth R and shake until Standardised cinchona liquid extract is produced from the
the mixture becomes clear. Filter through a plug of absorbent herbal drug by an appropriate procedure using:
cotton and rinse the flask and the cotton with 5 quantities, — ethanol (30 per cent VIV to 90 per cent P7P), or;
each of 20 mL, of a mixture of 1 volume of methylene — a mixture of diluted hydrochloric acid, ethanol
chloride R and 2 volumes of ether R. Combine the filtrate and (96 per cent VIV)y glycerol, water (1:2:5:20 VIV).
washings, evaporate to dryness and dissolve the residue in CHARACTERS
10.0 mL of anhydrous ethanol R. Evaporate 5.0 mL of this Appearance
solution to dryness, dissolve the residue in 0.1 M hydrochloric Brownish-red liquid.
acid and dilute to 1000.0 mL with the same acid. It has a bitter, astringent taste.
Reference solutions Dissolve separately 30.0 mg of quinine R
IDENTIFICATION
and 30.0 mg of cinchonine R in 0.1 M hydrochloric acid and
dilute each solution to 1000.0 mL with the same acid.
Test solution Dilute 1 mL of the extract to be examined in
Measure the absorbances (2.2.25) of the 3 solutions at
1 mL of anhydrous ethanol R.
316 nm and 348 nm using 0.1 M hydrochloric acid as the
compensation liquid. Reference solution Dissolve 2.5 mg of quinidine R> 10 mg of
cinchonidine R, 10 mg of cinchonine R and 17.5 mg of
Calculate the percentage content of alkaloids using the
quinine R in 5 mL of anhydrous ethanol R.
following equations:
IV-146 Cinnamon 2016
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel 20 mL, of a mixture of 1 volume of methylene chloride R and
plate R (2-10 gm)]. 2 volumes of ether R. Combine the filtrate and washings,
Mobile phase diethylamine R, ethyl acetate R, toluene R evaporate to dryness and dissolve the residue in 10.0 mL of
(10:20:70 VIVIV). ethanol (96 per cent) R. Evaporate 5.0 mL of this solution to
Application 10 pL [or 2 pL] as bands. dryness, dissolve the residue in 0.1 M hydrochloric acid and
dilute to 1000.0 mL with the same acid.
Development Twice over a path of 15 cm [or 6 cm].
Reference solution (a) Dissolve 30.0 mg of cinchonine R in
Drying At 100-105 cc then allow to cool.
0.1 M hydrochloric acid and dilute to 1000.0 mL with the
Detection A Spray with a 50 g/L solution of anhydrous formic same acid.
acid R and allow to dry in air; examine in ultraviolet light at
Reference solution (b) Dissolve 30.0 mg of quinine Rin 0.1 M
365 nm.
hydrochloric acid and dilute to 1000.0 mL with the same acid.
Results A See below the sequence of ±e zones present in the
Measure the absorbances (2.2.25) of the 3 solutions at
316 nm and 348 nm, using 0.1 M hydrochloric acid as the
compensation liquid.
Calculate the percentage content of alkaloids from the
Top of Lhe plate following equations:
_ [Al X A2a] - [Ala X A2] 100 2

Quinidine: a distinct blue A distinct blue fluorescent zone
fluorescent zone (quinidine) [Alb X A2a] — [Ala X A2b] m 1000
Quinine: a distinct blue fluorescent A distinct blue fluorescent zone [Al X A2b] - [Alb X A2] 100 2
zone (quinine) 2 [Ala X A2b] — [Alb X A2a]m x 1000
m ะ= mass of the liquid extract to be examined in

Detection B Spray with iodoplatinate reagent R.
grams;
Results B See below the sequence of the zones present in the ท1 = percentage content of quinine-type alkaloids;
chromatograms obtained with the reference solution and the พ2 = percentage content of cinchonine-type alkaloids;
test solution. Furthermore, other zones may be present in the Al = absorbance of the test solution at 316 nm;
chromatogram obtained with the test solution. A2 = absorbance of the test solution at 348 nm;
Ala = absorbance of reference solution (a) at 316 nm,
Top of the plate
corrected to a concentration of 1 mg/1000 mL;
Alb = absorbance of reference solution (b) at 316 nm,
Cinchonine: a violet-grey zone A violet-grey zone (cinchonine) corrected to a concentration of 1 mg/1000 mL;
A2a = absorbance of reference solution (a) at 348 nm,
Quinidine: a violet-grey zone A violet-grey zone (quinidine)
corrected to a concentration of 1 mg/1000 mL;
Cinchonidine: an intense dark blue An intense dark blue zone A2b = absorbance of reference solution (b) at 348 nm,
(cinchonidine)
corrected to a concentration of 1 mg/1000 mL.
Calculate the content of total alkaloids (ท1 + H2), anfl the
Quinine: a violet-grey zone A violet-grey zone (quinine)
relative content of quinine-type alkaloids, from the following
Reference solution Test solution expression:
TESTS ท1 X 100
Ethanol (2.9.10) Til + ท2
95 per cent to 105 per cent of the content stated on the
label.
LABELLING
Methanol and 2-propanol (2.9.11) The label states the solvent composition used for the
Maximum 0.05 per cent v/v of methanol and maximum production.
0.05 per cent v/v of 2-propanol.
_____________________________________________________________ __ Ph Eur
Minimum 12.0 per cent for glycerol-free standardised
cinchona liquid extract and minimum 30.0 per cent for
glycerol-containing standardised cinchona extract, determined
on 2.0 g. Cinnamon
ASSAY Cinnamon Bark; Ceylon Cinnamon *
Test solution In a 250 mL conical flask, mix about 1.000 g of (Ph. Eur. monograph 0387)
the extract to be examined with 10 mL of water R and 7 mL
of dilute hydrochloric acid R. Heat in a water-bath for 30 min, Preparation
allow to cool and add 25 mL of methylene chloride R, 50 mL Cinnamon Tincture
of ether R and 5 mL of a 200 g/L solution of sodium When Powdered Cinnamon is prescribed or demanded,
hydroxide R. Shake the mixture frequently for 30 min, add material complying with the requirements below with the
3 g of powdered tragacanth R and shake until the mixture exception of Identification test A and containing not less than
becomes clear. Filter through a plug of absorbent cotton, 1.0% v/w (10 mL/kg) of essential oil shall be dispensed or
rinse the flask and the cotton with 5 quantities, each of supplied.
2016 Ceylon Cinnamon Bark Oil IV-147
Pn Eur__________ _________________ ____ _____________________________

DEFINITION Test solution Shake 0.1 g of the powdered herbal drug (500)
Dried bark, freed from the outer cork and the underlying (2.9.12) with 2 mL of methylene chloride R for 15 min. Filter
parenchyma, of the shoots grown on cut stock of and evaporate the filtrate carefully almost to dryness on a
Cinnamomum verum J.Presl. water-bath. Dissolve the residue in 0.4 mL of toluene R.
Content Reference solution Dissolve 50 pL of cinnamic aldehyde R and
Minimum 12 mL/kg of essential oil. 10 pL of eugenol R in toluene R and dilute to 10 mL with the
same solvent.
CHARACTERS
Plate TLC silica gel GF254 plate R.
Characteristic, aromatic odour.
Mobile phase methylene chloride R.
IDENTIFICATION
Application 10 |1L as bands of 20 mm by 3 mm.
A. The bark is about 0.2-0.8 mm thick and occurs in closely
packed compound quills made up of single or double quills. Development Over a path of 10 cm.
The outer surface is smooth, yellowish-brown with faint scars Drying In air.
marking the position of leaves and axillary buds and has fine, Detection A Examine in ultraviolet light at 254 nm and mark
whitish and wavy longitudinal striations. The inner surface is the quenching zones, then examine in ultraviolet light at
slightly darker and longitudinally striated. The fracture is 365 nm and mark the fluorescent zones.
short and fibrous. Results A Examined in ultraviolet light at 254 nm, the
B. Microscopic examination (2.8.23). The powder is chromatograms obtained with the test solution and the
yellowish or reddish-brown. Examine under a microscope reference solution show a quenching zone due to
using chloral hydrate solution R. The powder shows the cinnamaldehyde in the median part and, just above it, a
following diagnostic characters (Figure 0387.-1): rounded weaker quenching zone due to eugenol; examined in
sclereids with pitted, channelled and moderately thickened ultraviolet light at 365 nm, the chromatogram obtained with
walls, single [E, F] or in groups [C]; numerous colourless, the test solution shows a fluorescent light blue zone due to
single fibres, often whole [A], or fragmented [D], with a o-methoxycinnamaldehyde just below the zone due to
narrow lumen, thickened, lignified walls and few pits; small cinnamaldehyde.
acicular crystals of calcium oxalate in parenchymatous Detection B Spray with phloroglucinol solution R.
cells [J]; very numerous oil droplets [B]. Cork fragments [G] Results B The zone due to cinnamaldehyde is yellowish-
are absent or very rare. Examine under a microscope using a brown and the zone due to o-methoxycinnamaldehyde is
50 per cent VIV solution of glycerol R. The powder shows violet.
abundant starch granules [H].
TESTS
Total ash (2.4.16)
ASSAY
Use 20.0 g of drug reduced to a powder (710) (2.9.12)
immediately before the determination, a 500 mL flask,
200 mL of 0.1 M hydrochloric acid as the distillation liquid,
rate of 2.5-3.5 mL/min for 3 h.
_____________________________________________________________ Ph Elf
Ceylon Cinnamon Bark Oil * :

Cinnamon Oil ***
Preparation
Concentrated Cinnamon Water
PhEtr__ _________________________________________________________
DEFINITION
Essential oil obtained by steam distillation of the bark of the
shoots of Cinnamomum verum J.Presl.
CHARACTERS
Appearance
Clear, mobile, light yellow liquid becoming reddish over
time.
Characteristic odour reminiscent of cinnamic aldehyde.
IDENTIFICATION
Figure 0387.-1. - Illustration for identification test B ofpowdered
herbal drug of cinnamon Second identification A
IV-148 Cinnamon Preparations 2016
A. Thin-layer chromatography (2.2.27). lit]ection 0.2 pL.

Test solution Dissolve 1 mL of the essential oil to be examined Elution order Order indicated in the composition of the
in acetone R and dilute to 10 mL with the same solvent. reference solution; depending on the operating conditions
Reference solution Dissolve 50 pL of trans-cinnamic aldehyde R, and the state of the column, coumarin may elute before or
10 pL of eugenol R, 10 pL of linalol R and 10 pL of after rrans-2-methoxycinnamaldchyde; record the
p-caryophyllene R in ethanol (96 per cent) R and dilute to retention times of these substances.
10 mL with the same solvent. System suitability: reference solution:
Plate TLC silica gel plate R. — resolution: minimum 1.5 between the peaks due to linalol
Mobile phase methanol R, toluene R (10:90 VIV). and p-caryophyllene.
Application 10 pL as bands. Identification of components Using the retention times
determined from the chromatogram obtained with the
Development Over a path of 15 cm. reference solution, locate the components of the reference
Drying In air. solution in the chromatogram obtained with the test solution.
Detection Spray with anisaldehyde solution R’> heat at Determine the percentage content of each of these
100-105 °C for 5-10 min and examine in daylight. components. The percentages are within the following
Residts The zones in the chromatogram obtained with the test ranges:
solution are similar in position and colour to those in the — cineole: maximum 3.0 per cent;
chromatogram obtained with the reference solution. — linalol: 1.0 per cent to 6.0 per cent;
B. Examine the chromatograms obtained in the test for — p-caryophyllene: 1.0 per cent to 4.0 per cent;
chromatographic profile. — safrole: maximum 0.5 per cent;
— trans-cinnamic aldehyde: 55 per cent to 75 per cent;
Results The principal peaks in the chromatogram obtained
— eugenol: maximum 7.5 per cent;
with the test solution are similar in retention time to those in
— coumarin: maximum 0.5 per cent;
the chromatogram obtained with the reference solution.
— trans-2-methoxycinnamaldehyde: 0.1 per cent to
Safrole, coumarin and cineole may be absent from the
1.0 per cent;
— benzyl benzoate: maximum 1.0 per cent.
TESTS
STORAGE
Protected from heat.
1.000 to 1.030.
______________________________________________________________ _ Ph Eur
Refractive index (2.2.6)
1.572 to 1.591.
-2° to + 1°.
Chromatographic profile Cinnamon Tincture * ไ
Gas chromatography (2.2.25): use ±e normalisation (Ph. Eur. monograph 1819) ***
procedure.
Ph Eur__________________________________________________________ _____
Test solution The essential oil to be examined.
Reference solution Dissolve 10 pL of cineole R, 10 pL of DEFINITION
linalol R, 10 pL of P-caryophyllene R, 10 pL of safrole R, Tincture produced from Cinnamon (0387).
100 pL of trans-cinnamic aldehyde R, 10 pL of eugenol R, PRODUCTION
20 mg of coumarin R, 10 pL of trans-2- The tincture is produced from 1 part of the drug and 5 parts
methoxycinnamaldehyde R and 10 pL of benzyl benzoate R in of ethanol (70 per cent VIV) by an appropriate procedure.
1 mL of acetone R.
CHARACTERS
Column’.
Appearance
— material: fused silica;
Clear, brownish-red liquid, with a characteristic odour.
— size: I — 60 m, 0 = 0.25 mm;
— stationary phase: bonded macrogol 20 000 R. IDENTIFICATION
Carrier gas helium for chromatography R. Thin-layer chromatography (2.2.27).
Flow rate 1.5 mL/min. Test solution Place 10 mL of the tincture to be examined,
10 mL of saturated sodium chloride solution R and 5 mL of
Split ratio 1 ะ 100.
toluene R in a ground glass-stoppered tube. Shake for 2 min
Temperature: and centrifuge for 10 min. Use the organic layer.
Reference solution Dissolve 5 pL of eugenol R} 25 pL of trans-
Time Temperature cinnamic aldehyde R and 5 pL of trans-2-
(min) _____ ea_____ methoxycinnamaldehyde R in toluene R and dilute to 10 mL
Column 0-10 60 with the same solvent.
10-75 60 -> 190 Plate TLC silica gel G plate R.
75 - 200 190 Mobile phase methylene chloride R.
Injection port 200 Application 20 pL, as bands.
240
Detector
Drying In air.
2016 Ceylon Cinnamon Leaf Oil IV-149
Results A See below the sequence of the zones present in the The water complies with the requirements stated under Aromatic
chromatograms obtained with the reference solution and the Waters and with the following requirements.
test solution.
TESTS
Ethanol content
Top of the plate 52 to 56% v/v, Appendix VUI F.
Weight per mL
Trans-2-methoxycinnamaIdehyde ะ A light blue fluorescent zone
0.914 to 0.922 g, Appendix V G.
a light blue fluorescent zone (.trans-2- methoxycinnamaldehyde)
A greenish fluorescent zone (above

the line of application)
Ceylon Cinnamon Leaf Oil * *
Reference solution Test solution (Ph. Eur. monograph 1608) *★*
PhEur_____________________________________________________________
Detection B Spray with a 200 g/L solution of phosphomolybdic DEFINITION

acid R in ethanol R. Examine in daylight while heating at Oil obtained by steam distillation of the leaves of
100-105 °C for 5-10 min. Cinnamomum verum J.s. Presl.
Results B See below the sequence of the zones present in the CHARACTERS
chromatograms obtained with the reference solution and the Appearance
test solution. Furthermore, other zones may be present in the Clear, mobile, reddish-brown or dark brown liquid.
Characteristic odour reminiscent of eugenol.
IDENTIFICATION
Top of the plate
1 blue zone (terpenhydrocarbons)
Eugenol: a blue zone A blue zone (eugenol) Test solution Dilute 1 mL of the substance to be examined in
Trnns-cinnamic aldehyde: a blue acetone R and dilute to 10 mL with the same solvent.
aldehyde) Reference solution Dilute about 50 pL of trans-cinnamic
Trflns-2-methoxycinnamaldehyde ะ A weak orange-brown zone aldehyde R} 10 pL of eugenol R, 10 pL of linalol R and 10 pL
an orange-brown zone (the colour (trflns-2-methoxycinnamaldehyde)
fades away) of p-caryophyllene R in alcohol R and dilute to 10 mL with the
same solvent.
2 or 3 blue zones above the line of
application Mobile phase methanol R) toluene R (10:90 VIV).
Reference solution Test solution Application 10 pL, as bands.
TESTS Drying In air.
Ethanol (2.9.10) Detection Spray with anisaldehyde solution R. Examine in day
64 per cent VIV to 70 per cent VIV. light while heating at 100-105 °C for 5-10 min.
Methanol and 2-propanol (2.9.11) Results The zones in the chromatogram obtained with the test
Maximum 0.05 per cent VIV of methanol and maximum solution are similar in position and colour to those in the
0.05 per cent VIV of 2-propanol. chromatogram obtained with the reference solution.
The zone due to rrans-cinnamic aldehyde may be very faint
Dry residue (2.8.16) or absent.
Minimum 1.5 per cent mlm, determined on 5.0 g.
B. Examine the chromatogram obtained in the test for
______________________________________________________________ PhEur
Concentrated Cinnamon Water solution. The peaks corresponding to cineole, safrole,
nuns-cinnamic aldehyde, cinnamyl acetate and coumarin may
DEFINITION be absent in the chromatogram obtained with the test
Cinnamon Oil 20 mL solution.
Ethanol (90 per cent) 600 mL
Water Sufficient to produce 1000 mL TESTS
Extemporaneous preparation 1.030 to 1.059.
Dissolve the Cinnamon Oil in the Ethanol (90 per cent) and 1.527 to 1.540.
add gradually, with vigorous shaking after each addition,
sufficient Water to produce 1000 mL. Add 50 g of previously Optical rotation (2.2.7)
sterilised Purified Talc, or other suitable filtering aid, allow to -2.5° to + 2.0°.
stand for a few hours, shaking occasionally, and filter.
IV-150 Citronella Oil 2016
Chromatographic profile CHARACTERS

Gas chromatography (2.2.28): use ±e normalisation Appearance
procedure. Pale yellow or brown-yellow liquid.
Test solution The substance to be examined. Very strong odour of citronellal.
Reference solution Dissolve 10 pL of cineole R, 10 pL of IDENTIFICATION
linalol R, 10 pL of p-caryophyllene R, 10 pL of safrole R,
First identification B.
10 pL of trans-cinnamic aldehyde R} 10 pL of cinnamyl
acetate R, 100 pL of eugenol R and 10 mg of coumarin R in Second identification A
1 mL of acetone R. A. Thin-layer chromatography (2.2.27).
Column: Test solution Dilute 0.1 g of citronella oil in 10.0 mL of
— material: fused silica, alcohol R.
— size: I = 60 m, 0 = 0.25 mm, Reference solution Dilute 20 |1L of citronellal R in 10.0 mL of
— stationary phase: macrogol 20 000 R. alcohol R.
Carrier gas helium for chromatography R. Plate TLC silica gel plate R.
Flow rate 1.5 mL/min. Mobile phase ethyl acetate R, toluene R (10:90 VIV).
Split ratio 1/100. Applicatiott 5 pL, as bands.
Temperature: Development Over a path of 15 cm.
Drying In air.
Time Temperature Detection Spray with anisaldehyde solution R and heat at
(min) (°C) 100-105 °C for 10 min. Examine in ultraviolet light at
Column 0 - 10 45 365 nm.
10 - 78 45 -> 180
78 - 88 180
Result See below the sequence of the zones present in the
Injection port 200
solutions. Furthermore, other zones are present in the
Detector 240
Top of the plate

Injection 0.2 pL. Citronellal: a violet zone A zone similar in colour to the
citronellal zone
Elution order The order indicated in the composition of the An orange zone (citronellol
reference solution. Record the retention times of these geraniol)
substances. Reference solution Test solution
— resolution: minimum of 1.5 between the peaks due to B. Examine the chromatograms obtained in the test for
linalol and p-caryophyllene. chromatographic profile.
Using the retention times determined from the Results The characteristic peaks in the chromatogram
chromatogram obtained with the reference solution, locate obtained with the test solution are similar in retention time to
the components of the reference solution in the those in ±e chromatogram obtained with the reference
chromatogram obtained with the test solution. solution. Neral and geranial may be absent in the
Determine the percentage content of these components. chromatogram obtained with the test solution.
The percentages are within the following ranges: TESTS
— cineole: maximum 1.0 per cent, Relative density (2.2.5)
— linalol: 1.5 per cent to 3.5 per cent, 0.881 to 0.895.
— p-caryophyllene: 1.5 per cent to 7.0 per cent,
— safrole: maximum 3.0 per cent,
1.463 to 1.475.
— trans-cinnamic aldehyde: maximum 3.0 per cent,
— cinnamyl acetate: maximum 2.0 per cent, Optical rotation (2.2.7)
— eugenol: 70 per cent to 85 per cent, -4° to + 1.5°.
— coumarin: maximum 1.0 per cent. Chromatographic profile
STORAGE Gas chromatography (2.2.28): use the normalisation
Protected from heat. procedure.
Test solution The substance to be examined.
.______ __ ______________ _________________________________________ PhEur
Reference solution Dilute 25 pL of limonene R, 100 pL of
citronellal R} 25 pL of citronellyl acetate R} 25 pL of citral R>
25 pL of geranyl acetate R, 25 |1L of citronellol R and 100 pL
of geraniol R in 5 mL of hexane R.
Citronella Oil Column:
(Ph. Eur. monograph 1609) *
— size: I = 60 m, 0 = 0.25 mm,
Ph Fir -------------- -------------------------------------------------------------------------------- — stationary phase: macrogol 20 000 R (0.2 pm).
DEFINITION Carrier gas helium for chromatography R.
Oil obtained by steam distillation from the fresh or partially Flow rate 1.0 mL/min.
dried aerial parts of Cymbopogon winterianus Jowitt. Split ratio 1:100.
2016 Clematis Armandii Stem IV-151
Temperature: corresponding to the medullary rays; the vessels are clearly

visible in transverse section. The pale yellow or whitish pith,
Time Temperature sometimes replaced by a hollow, is reduced.
(min) (°C)
Column 0-2 80
2-26 80 -> 150 hydrate solution R. The powder shows the following diagnostic
26 - 42 150 -> 185 characters: very numerous fragments of vessels, up to
42 - 49
250 pm in diameter, with pitted walls, isolated or associated
185 -> 250
with elongated tracheids about 15-25 Jim in diameter with
Injection port 260 lignified, thickened and pitted walls; fibres 25-30 pm in
Detector 260 diameter, with narrow lumen and thick and partly, slightly
pitted walls; parenchymatous cells of the secondary phloem
Detection Flame ionisation. and outer parts of the medullary rays, thin-walled, from the
secondary xylem, inner parts of the medullary rays and pith,
Injection 1 pL of the reference solution, 0.2 pL of the test
solution. with slightly thickened, pitted and lignified cell walls; sub-
rectangular or fusiform sclereids, about 100 pm long and
Elution order The order indicated in the composition of the 35 pm wide, with thick and pitted walls; rare orange-brown
reference solution. Record the retention times of these cork fragments. Examine under a microscope using a
substances. 50 per cent VIV solution of glycerol R. The powder shows
System suitability: reference solution: rare starch granules, simple or 2-3 compound, spherical or
— resolution: minimum of 1.2 between the peaks due to ovate, individual granules up to 17 pm in diameter, with a
geranyl acetate and citronellol. punctiform or slit-shaped hilum.
Using the retention times determined from the c. Thin-layer chromatography (2.2.27).
chromatogram obtained with the reference solution, locate Test solution To 1 g of the powdered herbal drug (1400)
the components of the reference solution in the (2.9.12) add 5 mL of methanol R and heat on a water-bath at
chromatogram obtained with the test solution. 60 °C for 5 min. Filter.
Determine the percentage content of each of these Reference solution Dissolve 4 mg of hederagenin R and 4 mg of
components. oleanolic acid R in 10 mL of methanol R.
The percentages are within the following values: Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel
— limonene: 1.0 per cent to 5.0 per cent, F254 plate R (2-10 pm)].
— citronellal: 30.0 per cent to 45.0 per cent,
Mobile phase acetic acid R3 acetone R3 toluene R
— citronellyl acetate: 2.0 per cent to 4.0 per cent,
(2:8:32 VIVIV).
— neral: maximum 2.0 per cent,
— geranial: maximum 2.0 per cent, Application 40 pL [or 10 pL] as bands of 10 mm [or 8 mm].
— geranyl acetate: 3.0 per cent to 8.0 per cent, Development Over a path of 13 cm [or 6 cm].
— citronellol: 9.0 per cent to 15.0 per cent, Diying In air.
— geraniol: 20.0 per cent to 25.0 per cent. Detection Treat with vanillin reagent R3 heat at 100 °C for
---------------------------------------------------------------------------------------------------------- Ph Eur 5 min and examine in daylight.
test solution. Furthermore, other mainly grey zones may be
Clematis Armandii stem * * present in the chromatogram obtained with the test solution.
(Ph Eur monograph 2463) *** Top of the plate

Ph Eur______________________________________________________________
DEFINITION
Whole or fragmented, dried stem of Clematis armandii
A bluish-violet zone
French., with cork removed, collected in spring or autumn.
Content Oleanolic acid: a reddish-violet A reddish-violet zone
Minimum 0.30 per cent of oleanolic acid (C3oH4803;
A weak light blue or grey zone
IDENTIFICATION
A. The whole stem is long and cylindrical, slightly twisted on Hederagenin: a greenish-brown
zone
itself, about 1-6.5 cm in diameter. It shows nodes, usually
An orange zone
swollen, with leaf and branch scars. The outer surface is
brownish-yellow or dull brownish-yellow, showing Reference solution Test solution
longitudinal grooves and striations corresponding to the ends
of the medullary rays. Rare cork remnants are easily removed
as longitudinal strips. The texture is hard. The fracture is TESTS
difficult. Avistolochia manshuriensis Kom. and other species of
Aristolochia
The fragmented stem occurs in thick slices, about 2-5 mm Examine the powdered herbal drug (355) (2.9.12) under a
thick, with uneven margins; most of the transverse section microscope using chloral hydrate solution R'i no cluster crystals
consists of the pale yellow or slightly brownish-yellow wood
are visible.
and shows numerous radial striations and cracks
IV-152 Clove 2016
Aristolochic acids (2.8.213 Method A)

It complies with the test. Clove * t
Loss on drying (2.2.32) (Ph. Eur. monograph 0376) ***
Maximum 12.0 per cent, determined on 1.000 g of the When Powdered Clove is prescribed or demanded, material
powdered herbal drug (1400) (2.9.12) by drying in an oven complying with the requirements below with the exception of
at 105 °C for 2 h. Identification test A and the test for Foreign matter and
Total ash (2.4.16) containing not less than 12.0% v/w (120 mUkg) of essential
Maximum 3.0 per cent. oil shall be dispensed or supplied.
Ph Fur ____________________________________________________ _______
ASSAY
Liquid chromatography (2.2.29). DEFINITION
Test solution Disperse 1.00 g of the powdered herbal drug Whole flower buds of Syzygium aromaticum (L.) Merr.
(355) (2.9.12) in methanol R3 add 3 mL of 6 M hydrochloric et L.M.Perry (syn. Eugenia caryophyllus (Spreng.) Bullock et
acid R and dilute to 30.0 mL with methanol R. Shake for 2 h. S.G.Harrison) dried until they become reddish-brown.
Filter, add to the filtrate 10 mL of water R by rinsing the Content
flask and the filter, and extract with 3 quantities, each of Minimum 150 mUkg of essential oil.
30 mL, of methylene chloride R. Combine the methylene
CHARACTERS
chloride extracts and evaporate to dryness. Dissolve the
residue in 10.0 mL of methanol R3 shake and filter through a Characteristic, aromatic odour.
membrane filter (nominal pore size 0.45 pm). IDENTIFICATION
Reference solution (a) Dissolve 10.0 mg of oleanolic acid CRS A. The flower bud is reddish-brown and consists of a
in methanol R and dilute to 20.0 mL with the same solvent. quadrangular stalked portion, the hypanthium, 10-12 mm
Reference solution (b) Dissolve 5.0 mg of ursolic acid R in long and 2-3 mm in diameter, surmounted by 4 divergent
reference solution (a) and dilute to 10.0 mL with the same lobes of sepals which surround a globular head 4-6 mm in
solution. diameter. A bilocular ovary containing numerous ovules is
situated in the upper part of the hypanthium. The head is
Column:
globular and dome-shaped, composed of 4 imbricated petals
— size: I = 0.25 m, 0 = 4.6 mm;
that enclose numerous incurved stamens and a short, erect
style with a nectary disc at the base. The hypanthium exudes
chromatography R (5 pm);
essential oil when indented with the finger-nail.
B. Reduce to a powder (355) (2.9.12). The powder is dark
Mobile phase 0.4 per cent VIV solution of acetic acid R3
brown and has the odour and taste of the unground drug.
methanol R (15:85 VIV).
Examine under a microscope using chloral hydrate solution R.
Flow rate 1.0 mUmin. The powder shows the following diagnostic characters:
Detection Spectrophotometer at 210 nm. fragments of the hypanthium showing the epidermis and
Injection 20 pL. underlying parenchyma containing large oil glands; short
Run time 1.2 times the retention time of ursolic acid. fibres occurring singly or in small groups, with thickened,
lignified walls and few pits; abundant fragments of
Retention time Oleanolic acid = about 21 min; ursolic
parenchyma containing cluster crystals of calcium oxalate;
acid ะ= about 22 min.
numerous triangular pollen grains about 15 pm in diameter
System suitability: reference solution (b): with 3 pores in the angles. Starch granules are absent.
oleanolic acid and ursolic acid. c. Thin-layer chromatography (2.2.27).
Calculate the percentage content of oleanolic acid using the Test solution Shake 0.1 g of the powdered herbal drug (500)
(2.9.12) with 2 mL of methylene chloride R for 15 min. Filter
and carefully evaporate the filtrate to dryness on a water
bath. Dissolve the residue in 2 mL of toluene R.
Al X 7712 X p
Reference solution Dissolve 20 pL of eugenol R in 2 mL of
A2 X 7721 X 2
toluene R.
A1 = area of the peak due to oleanolic acid in the
Mobile phase toluene R.
A2 = area of the peak due to oleanolic acid in the Application 10 pL of the reference solution and 20 pL of the
chromatogram obtained with reference solution test solution, as bands of 20 mm by 3 mm.
(a); Development Twice, in an unsaturated tank over a path of
mx = mass of the herbal drug to be examined used to 10 cm; allow the plate to stand for 5 min between the
prepare the test solution, in grams; 2 developments.
m2 = mass of oleanolic acid CRS used to prepare Drying In air.
reference solution (a), in grams; Detection A Examine in ultraviolet light at 254 nm and mark
p = percentage content of oleanolic acid in oleanolic the quenching zones.
acid CRS.
Results A In the chromatogram obtained with the test
_________________________________ Ph Eur solution there is in the median part a quenching zone due to
eugenol similar in position to the quenching zone in the
chromatogram obtained with the reference solution and there
may be a weak quenching zone due to acetyleugenol just
below the zone due to eugenol.
2016 Clove Oil IV-153
Detection B Spray with anisaldehyde solution R using 10 mL Detection A Examine in ultraviolet light at 254 nm and mark
for a plate 200 mm square and heat at 100-105 °C for the quenching zones.
5-10 min. Examine in daylight. Results A The chromatogram obtained with the test solution
Results B The zones due to eugenol in the chromatograms shows in the middle part a quenching zone (eugenol) that is
obtained with the test and reference solutions are strong similar in position to the quenching zone in the
brownish-violet and the zone due to acetyleugenol in the chromatogram obtained with the reference solution; just
chromatogram obtained with the test solution is faint violet below, there is a weak quenching zone (acetyleugenol) that is
blue. In the chromatogram obtained with the test solution similar in position to the zone of acetyleugenol in the
there are other coloured zones, particularly a faint red zone chromatogram obtained with ±e reference solution.
in the lower part and a reddish-violet zone due to Detection B Spray with anisaldehyde solution R and examine in
caryophyllene in the upper part. daylight while heating at 100-105 °C for 5-10 min.
TESTS Results B The zone due to eugenol in the chromatograms
Foreign matter (2. ร. 2) obtained with the test and reference solutions is strong
Maximum 6 per cent of peduncles, petioles and fruits, brownish-violet and the zone due to acetyleugenol in the
maximum 2 per cent of deteriorated cloves and maximum chromatogram obtained with the test solution is faint violet
0.5 per cent of other foreign matter. blue; in the chromatogram obtained with the test solution
Total ash {2.4.16) there are other coloured zones, particularly a faint red zone
Maximum 7.0 per cent. in the lower part and a reddish-violet zone (P-caryophyllene)
in the upper part.
ASSAY
Essential oil {2.8.12) chromatographic profile.
Use a 250 mL flask, 100 mL of water R as the distillation
Results The 3 principal peaks in chromatogram obtained with
liquid and 0.50 mL of xylene R in the graduated tube. Grind
the test solution are similar in retention time to the
5.0 g of the drug with 5.0 g of diatomaceous earth R to form a
3 principal peaks in the chromatogram obtained with the
fine, homogeneous powder and proceed immediately with the
reference solution.
determination using 4.0 g of the mixture. Distil at a rate of
2.5-3.5 mUmin for 2 h. TESTS
_________ ____________________________________________________ Ph Eur Relative density (2.2.5)
1.030 to 1.063.
1.528 to 1.537.
Clove Oil ★ ★ Optical rotation (2.2.7)

-2° to 0°.
(Ph. Eur. monograph 1091) *** Fatty oils and resinified essential oils (2.8.7)
Ph Elf______________________________________________________________ It complies with the test.
DEFINITION Solubility in alcohol {2.8.10)
Essential oil obtained by steam distillation from the dried 1.0 mL is soluble in 2.0 mL and more of ethanol
flower buds of Syzygium aromaticum (L.) Merr. et L.M.Perry (70 per cent V/V) R.
(syn. Eugenia caryophyllus (Spreng.) Bullock et S.G.Harrison). Chromatographic profile
Gas chromatography {2.2.28): use the normalisation
CHARACTERS
procedure.
Appearance
Clear, yellow liquid, which becomes brown when exposed to Test solution Dissolve 0.2 g of the substance to be examined
air. in 10 g of hexane R.
Reference solution Dissolve 7 mg of ^-caryophyllene R) 80 mg
Solubility
of eugenol R and 4 mg of acetyleugenol R in 10 g of hexane R.
Miscible with methylene chloride, with toluene and with fatty
oils. Column:
IDENTIFICATION — size: z = 60 m, 0 = about 0.25 mm;
First identification B. — stationary phase: macrogol 20 000 R.
Second identification A. Carrier gas helium for chromatography R.
A. Thin-layer chromatography (2.2.27). Flow rate 1.5 mL/min.
Test solution Dissolve 20 |1L of the substance to be examined Split ratio 1:100.
in 2.0 mL of toluene R. Temperature:
Reference solution Dissolve 15 |1L of eugenol R and 15 |1L of
acetyleugenol R in 2.0 mL of toluene R. Temperature
Time
Plate TLC silica gel F254 plate R. (min) _______ CS)_________
Mobile phase toluene R. Column 0-8 60
Application 20 pL of the test solution and 15 |1L of the 8-48 60 -> 180
reference solution, as bands. 180
48 - 53
Development Twice in an unsaturated tank over a path of 270
10 cm; allow to stand for 5 min between the 2 developments. Injection port
Detector 270
Drying In air.
IV-154 Coix Seed 2016
Detection Flame ionisation. Reference solution Dissolve 2 mg of oleic acid R and 2 mg of

Injection 1.0 |1T. triolein R in methanol R and dilute to 1 mL with the same
Elution order Order indicated in the composition of the solvent.
reference solution. Record the retention times of these Plate TLC octadecylsilyl silica gel plate R (5-40 pm) [or
substances. TLC octadecylsilyl silica gel plate R (2-10 pm)].
System suitability: reference solution: Mobile phase methylene chloride Ry glacial acetic acid Ry
— resolution: minimum 1.5 between the peaks due to eugenol acetone R (20:40:50 VIVIV).
and acetyleugenol; Application 10 pL [or 2 pL] as bands of 10 mm [or 8 mm].
— number of theoretical plates: minimum 30 000, calculated Development Over a path of 7 cm.
for the peak due to P-caryophyllene at 110 °C.
Drying In air.
Identification of components Using the retention times
Detection Treat with a 100 g/L solution of phosphomolybdic
acid R in ethanol (96 per cent) R, heat at 120 °C for about
reference solution, locate the components of the reference
3 min and examine in daylight.
solution on the chromatogram obtained with the test
solution. Results See below the sequence of zones present in the
components. The limits are within the following ranges:
— p-caryophyllene: 5.0 per cent to 14.0 per cent;
— eugenol: 75.0 per cent to 88.0 per cent;
— acetyleugenol: 4.0 per cent to 15.0 per cent. Top of the plate
STORAGE
Protected from heat.
______________________________________________________________ PhEur
A purple zone
Oleic acid: a purple zone A purple zone (oleic acid)
Coix Seed * * A faint purple zone
(Ph. Eur. monograph 2454) *** A purple zone
PhEir______________________________________________________________ A purple zone
DEFINITION
Dried, ripe, caryopsis, freed from the shell, of Coix lacryma- Triolein: a purple zone A purple zone (triolein)
jobi L. subsp. ma-yuen (Rom. Caill.) T.Koyama.
Content
Minimum 0.50 per cent of triolein (C57H104O6; Mr 885) Reference solution Test solution
(dried drug).
IDENTIFICATION
TESTS
A. The white or pale yellow caryopsis freed from the shell is
roughly ovoid or elongated-elliptical, about 4-8 mm long and
3-6 mm wide. The dorsal surface is rounded, milky white
and smooth; the ventral surface shows a deep longitudinal
105 °C for 2 h.
furrow; yellowish-brown remnants of the membranous floral
parts may be present. One end is obtusely rounded, the other Total ash (2.4.16)
end is relatively flat and slightly dented with an indistinct, Maximum 3.0 per cent.
pale brown hilum. ASSAY
B. Microscopic examination (2.8.23). The powder is light Liquid chromatography (2.2.29).
grey or light brown. Examine under a microscope using Test solution To 0.600 g of the powdered herbal drug (355)
chloral hydrate solution R. The powder shows the following (2.9.12) add 50 mL of the mobile phase and stir with a
diagnostic characters: fragments of endosperm with polygonal magnetic stirrer for 2 h. Sonicate for 30 min. Allow to cool,
cells arranged in a network; fragments of epicarp with dilute to 50.0 mL with the mobile phase and filter.
elongated, slightly sinuous cells; cells of the middle layer of
Reference solution (a) Dissolve 10.0 mg of triolein CRS in the
the pericarp are yellowish-brown, irregularly tube-like, slightly
curved and are iiregularly crossed. Examine under a mobile phase and dilute to 50.0 mL with the mobile phase.
microscope using a 50 per cent VIV solution of glycerol R. Reference solution (b) To 0.600 g of coix seed HRS add 50 mL
The powder shows very numerous starch granules, simple or of the mobile phase and stir with a magnetic stirrer for 2 h.
2-3 compound, spherical or slightly polyhedral, 3-20 pm in Sonicate for 30 min. Allow to cool, dilute to 50.0 mL with
diameter, with a stellate, Y-shaped, cleft-like or point-like the mobile phase and filter.
hilum. Reference solutions (c) 3 (d)3 (e)3 (f)3 (g)3 (h) Dilute reference
solution (a) to obtain 6 reference solutions of triolein, the
concentrations of which span the expected value in the test
Test solution To 1 g of the powdered herbal drug (710) solution.
(2.9.12) add 10 mi of light petroleum R1 and sonicate for
Column:
30 min. Filter and reduce in vacuo to 1 mL.
— size: I = 0.25 m, 0 = 4.6 mm;
2016 Cola IV-155
stationary phase', end-capped, octadecylsilyl silica gel for convex, corresponding to the cotyledons and usually
chromatography R (5 pm). occurring separated in the commercial drug; the cotyledons
Mobile phase methylene chloride R, acetonitrile R (35:65 VIV), are 3-4 cm long, 2-2.5 cm wide and 1-2 cm thick.
Flow rate 2.0 mUmin. In c. acuminata, the cotyledons are smaller and divided into
Detection Evaporative light-scattering detector; the following 4-6 irregular parts.
settings have been found to be suitable; if the detector has B. Reduce to a powder (355) (2.9.12). The powder is
different setting parameters, adjust the detector settings so as reddish-brown. Examine under a microscope using a
to comply with the system suitability criterion for signal-to- 50 per cent VIV solution of glycerol R. The powder shows the
noise ratio: following diagnostic characters: numerous ovoid or reniform
— carrier gas: nitrogen R; starch granules, 5-25 pm in size, with concentric striations
— flow rate: 0.8 mL7min; and a stellate, slighdy eccentric hilum; fragments of cotyledon
— evaporator temperature: 100 °C. tissue showing large, thick-walled, reddish polygonal cells
Injection 10 pL. filled with starch granules; occasional fragments of the
external epidermis of the cotyledons.
Run time 35 min.
Retention time Triolein ะ= about 18 min.
System suitability:
(2.9.12) add 5 mL of ethanol (60 per cent VIV) R. Shake
resolution: minimum 1.5 between the peak due to triolein mechanically at 40 °C for 30 min and filter.
and peak 2 in the chromatogram obtained with reference
Reference solution (a) Dissolve 25 mg of caffeine R in 10 mL
solution (b); use the chromatogram supplied with coix
of ethanol (60 per cent VIV) R.
seed HRS to identify peak 2;
— signal-to-noise ratio: minimum 30 for the peak due to Reference solution (b) Dissolve 50 mg of theobromine R in
triolein in the chromatogram obtained with reference 10 mL of the mobile phase. Filter.
solution (a). Plate TLC silica gel F254 plate R.
Establish a calibration curve with the logarithm of the mass Mobile phase water R, methanol R, ethyl acetate R
of triolein (in milligrams) per 50 mL of reference (10:13:77 VIVIV).
solutions (c), (d), (e), (f), (g) and (h) (corrected by the Application 20 pL, as bands.
assigned percentage content of triolein CRS) as the abscissa Development Over a path of 10 cm.
and the logarithm of the corresponding peak area as the
ordinate. Drying In air for 5 min.
Calculate the percentage content of triolein using the
following expression: Results A The chromatogram obtained with the test solution
shows 2 principal quenching zones which are similar in
position to the zones in the chromatograms obtained with
10A
reference solutions (a) and (b).
m X 10
Detection B Spray with a mixture of equal volumes of ethanol
(96 per cent) R and hydrochloric acid R and then with a
A = logarithm of the mass of triolein in the test
solution prepared immediately before use by dissolving 1 g of
solution, determined from the calibration curve and
iodine R and 1 g of potassium iodide R in 100 mL of ethanol
the area of the corresponding peak in ±e
(96 per cent) R.
m = mass of the herbal drug to be examined used to Results B The chromatogram obtained with the test solution
prepare the test solution, in grams. shows a reddish-brown principal zone similar in position and
colour to the zone in the chromatogram obtained with
______________________________________________________________ Ph Eur
reference solution (a).
TESTS
Cola ****** Maximum 12.0 per cent, determined on 2.00 g of the
(Ph. Eur. monograph 1504) *** 105 °C for 2 h.
Ph Eur______________________________________________________________ Total ash (2.4.16)
DEFINITION
Whole or fragmented dried seeds, freed from the testa, of ASSAY
Cola nitida (Vent.) Schott et Endl. (C. vera K. Schum.) and Liquid chromatography (2.2.29).
its varieties, as well as of Cola acuminata (P. Beauv.) Schott Test solution To 1.00 g (mf) of the powdered herbal drug
et Endl. (Sterculia acuminata p. Beauv.). (355) (2.9.12), add 50 mL of methanol R. Heat under a
Content reflux condenser on a water-bath for 30 min. Allow to cool
Minimum 1.5 per cent of caffeine (Mr 194.2) (dried drug). and filter. Rinse the filter with 10 mL of methanol R. Take up
the residue with 50 mL of methanol R. Proceed as before.
IDENTIFICATION Combine the filtrates and the washings in a 200.0 mL
A. The kernels have an oblong, somewhat obtuse, sub- volumetric flask and dilute to 200.0 mL with methanol R.
tetragonal shape, with deformations resulting from mutual Transfer 20.0 mL of this solution into a round-bottomed
pressure inside the fruit; they vary in size and mass, ranging flask and evaporate to dryness under reduced pressure. Take
from 5-15 g; the outside is hard, smooth and very dark up the residue with the mobile phase, transfer to a 50.0 mL
brown, the inside is more reddish-brown. In c. nitida and its
varieties, the kernels are divided in 2 parts, almost plano
FV-156 Colophony 2016
volumetric flask and dilute to 50.0 mL with the mobile Detection Spray with anisaldehyde solution R and heat at
phase. 100-105 °C for 10 min; examine in daylight.
Reference solution In a 100.0 mL volumetric flask, dissolve Results See below' the sequence of the zones present in the
30.0 mg (m2) of caffeine CRS and 15.0 mg of theobromine R chromatograms obtained with the reference solution and the
in the mobile phase and dilute to 100.0 mL with the mobile test solution. Furthermore, other coloured zones are present
phase. Transfer 10.0 mL of this solution to a 100.0 mL in the chromatogram obtained with the test solution.
volumetric flask and dilute to 100.0 mL with the mobile
phase.
Top of the plate
Column:
— size: I = 0.25 m, 0 = 4.6 mm; A purple band
A purple band
(5 pm).
Mobile phase methanol R} water R (25:75 P7P).
Flow rate 1 mL/min. 2 purple bands
Thymol ะ an orange band
Injection The chosen volume of each solution; loop injector.
— resolution: minimum 2.5 between the peaks due to caffeine
Linalol ะ a purple band Sequence of narrow purple bands
and theobromine. If necessary, adjust the volume of
พater R in the mobile phase. Purple extended baseline band
Calculate the caffeine content using the following expression:
m2 X Al X 50
mi X >42 TESTS
Acid value (2.5.1)
A1 = area of the peak due to caffeine in the 145 to 180, determined on 1.0 g.
chromatogram obtained with the test solution; Total ash (2.4.16)
A2 = area of the peak due to caffeine in the Maximum 0.2 per cent.
chromatogram obtained with the reference STORAGE
solution;
Do not reduce to a powder.
nil = mass of the herbal drug to be examined in the test
_____ ___________________________________________________ _ Ph Eur
solution, in grams;
m2 = mass of caffeine CRS in the reference solution, in
grams.
_______________________________________________________________ PhEur
Coriander
When Powdered Coriander is prescribed or demanded,
Colophony * * material complying with the appropriate requirements below
but containing not less than 0.2% v/w of essential oil shall be
dispensed or supplied.
Preparation Ph Eur_______________________________________________________ _ ______
Flexible Collodion
DEFINITION
PhEur________________________________________________________________
Dried cremocarp of Coriandruni sativum L.
DEFINITION
Content
Residue remaining after distillation of the volatile oil from the
Minimum 3 mITkg of essential oil (dried drug).
oleoresin obtained from various species of Pinus.
IDENTIFICATION
IDENTIFICATION
A. The fruit is brown or light brown, more or less spherical,
A. Translucent, pale yellow to brownish-yellow, angular, about 1.5-5 mm in diameter, or oval and 2-6 mm long.
irregularly-shaped, brittle, glassy pieces of different sizes the It consists of the entire cremocarp, with the mericarps usually
surfaces of which bear conchoidal markings. tightly connected. The fruit is glabrous and has 10 wavy,
B. Thin-layer chromatography (2.2.27). slightly raised primary ridges and 8 straight, more prominent
Test solution Dissolve 1 g in 10 mL of methanol R by gently secondary ridges. The mericarps are concave on the internal
warming. surface. The stylopod crowns the apex and a small fragment
Reference solution Dissolve 10 mg of thymol R and 10 mg of of the pedicel may be present.
linalol R in 10 mL of methanol R. B. Microscopic examination (2.8.23). The powder is brown.
Plate TLC silica gel plate R. Examine under a microscope using chloral hydrate solution R.
The powder shows the following diagnostic characters
(Figure 1304.-1): numerous oil droplets [B]; fragments of
Application 10 pL, as bands. endosperm [A] with small, thick-walled, regular cells
Development Over a path of 15 cm. containing microrosettes [Aa] and microcrystals of calcium
Drying In air. oxalate and oil droplets [Ab]; fragments of endocarp (surface
2016 Coriander Oil IV-157
view [C, J], transverse section [H]), with very narrow cells test solution. Furthermore, other faint zones may be present
having a parquetry arrangement [Ca, Ha] and usually in the chromatogram obtained with the test solution.
associated with a layer of thin-walled [Cb, Hb] or thicker-
walled [Ja] rectangular sclereids of the mesocarp; fragments
from the sclerenchymatous layer of the mesocarp [G] with Top of the plate
short, strongly thickened, pitted, fusiform cells occurring in A bluish-violet zone
layers with the cells of adjacent layers approximately at right
angles to one another; fragments of parenchyma of the
mesocarp (transverse section [E]) with small cells with Geranyl acetate: a violet-blue zone
slightly thickened walls [Ea], the remains of secretory canals
[Eb] and sclereids [Ec]; fragments of epicarp (surface view
[F]) with thin-walled polyhedral cells, some of which contain Linalol: an intense violet zone A violet zone (linalol)
small prisms of calcium oxalate [Fa]; rare fragments of A violet-blue zone
secretory canals with brown cells, (surface view [D]);
occasional fragments of vascular bundles [K].
TESTS
It complies with the test. None of the cremocarps show
perforations due to insects.
105 °C for 2 h.
Total ash {2.4.16)
ASSAY
Essential oil {2.8.12!)
Use a 500 mL round-bottomed flask, 200 mL of water R as
the distillation liquid and 0.5 mL of xylene R in the
graduated tube. Reduce the herbal drug to a coarse powder
and immediately use 30.0 g for the determination. Distil at a
rate of 2-3 mL/min for 2 h.
______________________________________________________________PhEur
Coriander Oil * *
Ph Elf______________________________________________________________
DEFINITION
Essential oil obtained by steam distillation from the fruits of
Figure 1304.-1. - Illustration for identification test B of powdered Coriandrum sativum L.
herbal drug of coriander CHARACTERS
c. Thin-layer chromatography (2.2.27). Appearance
Test solution To 0.5 g of the freshly powdered herbal drug Clear, colourless or pale yellow liquid.
(355) (2.9.72) add 5 mL of methanol R. Sonicate for 10 min IDENTIFICATION
and centrifuge or filter; use the supernatant or filtrate. First identification B
Reference solution Dissolve 10 pL of linalol R and 2 pL of Second identification A.
geranyl acetate R in 1.0 mL of toluene R. A. Examine by thin-layer chromatography (2.2.27).
Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel Test solution Dissolve 10 pL of the substance to be examined
F254 plate R (2-10 pm)]. in 1.0 mL of toluene R.
Mobile phase ethyl acetate R} toluene R (5:95 VIV). Reference solution Dissolve 10 pL of linalol R and 2 pL of
Application 10 pL [or 2 pL] as bands of 15 mm [or 8 mm]. geranyl acetate R in 1.0 mL of toluene R.
Development Over a path of 10 cm [or 6 cm]. Plate'. TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel
Drying In air for 5 min. F254 plate R (2-10 pm)].
Detection Treat with anisaldehyde solution R and heat at Mobile phase ethyl acetate R, toluene R (5:95 VIV).
100-105 °C for 5 min; examine in daylight. Application 10 pL [or 2 pL] as bands of 15 mm [or 8 mm].
Results See below the sequence of zones present in the Development Over a path of 10 cm [or 6 cm].
chromatograms obtained with the reference solution and the Drying In air for 5 min.
IV-158 Coriander Oil 2016
Detection Treat with anisaldehyde solution R and heat at Temperature:

100-105 °C for 5 min; examine in daylight.
Results See below the sequence of zones present in the Time Temperature
chromatograms obtained with the reference solution and the (°C)_____________
test solution. Furthermore, other faint zones may be present Column 0 - 10 60
in the chromatogram obtained with the test solution. 10 - 75 60 -> 190
75 - 120 190
Injection port 220
Top of he plate
Detector 240

Geranyl acetate: a violet-blue
Injection 0.2 pL.
Elution order Order indicated in the composition of reference
Linalol: an intense violet zone An intense violet zone (linalol)
A violet-blue zone — resolution: minimum 1.5 between the peaks due to linalol
Reference solution Test soludon and camphor.
Using ±e retention times determined from the
B. Examine the chromatograms obtained in the test for the components of reference solution (a) in the
chromatographic profile. chromatogram obtained with the test solution.
Results The characteristic peaks in the chromatogram Determine the percentage content of each of these
obtained with the test solution are similar in retention time to components. The percentages are within the following
those in the chromatogram obtained with the reference ranges:
solution. — a-pinene: 3.0 per cent to 7.0 per cent;
TESTS — limonene: 1.5 per cent to 5.0 per cent;
Relative density (2.2.5) — y-terpinene: 1.5 per cent to 8.0 per cent;
0.860 to 0.880. — p-cymene: 0.5 per cent to 4.0 per cent;
— camphor. 3.0 per cent to 6.0 per cent;
Refractive index (2.2.6) — linalol: 65.0 per cent to 78.0 per cent;
1.462 to 1.470. — a-terpineol: 0.1 per cent to 1.5 per cent;
Optical rotation (2.2.7) — geranyl acetate: 0.5 per cent to 4.0 per cent;
+ 7° to + 13°. — geraniol: 0.5 per cent to 3.0 per cent;
Acid value (2.5.7) — disregard limit: area of the peak in the chromatogram
Maximum 3.0, determined on 5.00 g of the substance to be obtained with reference solution (b) (0.05 per cent).
examined. Chiral purity
Chromatographic profile Gas chromatography (2.2.25).
Gas chromatography (2.2.25): use the normalisation Test solution Dissolve 0.02 g of the substance to be examined
procedure. in pentane R and dilute to 10 mL with the same solvent.
Test solution The substance to be examined. Reference solution Dissolve 10 pL of linalol R and 5 mg of
Reference solution (a) Dissolve 10 pL of tL-pinene Ry 10 pL of borneol R in pentane R and dilute to 10 mL with the same
limonene R, 10 pL of y-terpinene Ry 10 pL of p-cyniene Ry solvent.
10 mg of camphor Ry 20 pL of linalol Ry 10 pL of Column:
a-terpineol Ry 10 pL of geranyl acetate R and 10 pL of — material: fused silica;
geraniol R in 1 mL of heptane R. — size: z = 25 m, 0 = 0.25 mm;
Reference solution (b) Dissolve 5 pL of geraniol R in heptane R — stationary phase: modified P-cyclodextrin for chiral
and dilute to 10 mL with the same solvent. chromatography R (film thickness 0.25 pm).
Column'. Carrier gas helium for chromatography R.
— material: fused silica; Flow rate 1.3 mL/min.
— size: z = 60 m, 0 = 0.25 mm; Split ratio 1:30.
— stationary phase: macrogol 20 000 R (film thickness Temperature:
0.25 pm).
Carrier gas helium for chromatography R.
Time Temperature
Flow rate 1 mIJmin. (min) ___________m_____________
Split ratio 1:65. Column 0 - 65 50 -* 180
Injection port 230
Detector 230

Injection 1 pL.
2016 Dandelion Herb with Root IV-159
System suitability'. reference solution:

resolution', minimum 5.5 between the peaks due to
GR)-linalol (1st peak) and (ร)-linalol (2"d peak) and
minimum 2.9 between the peaks due to (ร)-linalol and
borneol (3rd peak).
Limit Calculate the percentage content of (J?)-linalol using
the expression:
A* X 100
As + Ar
As = area of the peak due to (ร)-linalol;

A/? = area of the peak due to (l?)-linalol.
— (R)-linalol'. maximum 14 per cent.
STORAGE
PhEur
Couch Grass Rhizome ★* **

(PA. Eur. monograph 1306) ***
Ph Elf _______________
DEFINITION Gb
Whole or cut, washed and dried rhizome of Agropyron
repens (L.) P.Beauv. (Elymus repens (L.) Gould); Figure 1306.-1. - Illustration for identification test B of powdered
the adventitious roots are removed. herbal drug of couch grass rhizome
IDENTIFICATION Water-soluble extractive
A. The shiny yellowish, light brown or yellowish-brown Minimum 25 per cent.
pieces of the rhizome are 2-3 mm thick and longitudinally To 5.0 g of the powdered herbal drug (355) (2.9.12) add
furrowed. At the nodes are the remains of very thin, more or 200 mL of boiling water R. Allow to stand for 10 min,
less branched roots and whitish or brownish scale-like leaves; shaking occasionally. Allow to cool, dilute to 200.0 mL with
the internodes, up to 6 cm long, are furrowed and hollow water R and filter. Evaporate 20.0 mL of the filtrate to
inside. The transverse section of the nodes shows a yellowish dryness on a water-bath. Dry the residue in an oven at
medulla. 100-105 °C. The residue weighs a minimum of 0.125 g.
B. Microscopic examination (2. ร. 22). The powder is whitish- Loss on drying (2.2.32)
yellow. Examine under a microscope using chloral hydrate Maximum 12.0 per cent, determined on 1.000 g of the
solution R. The powder shows the following diagnostic powdered herbal drug (355) (2.9.12) by drying in an oven at
characters (Figure 1306.-1): fragments of the epidermis in 105 °C for 2 h.
surface view [A] covered with a thick cuticle and composed Total ash (2.4.16)
of rectangular and elongated, thick-walled cells with pitted, Maximum 5.0 per cent.
slightly wavy walls, which usually alternate with small, thin Ash insoluble in hydrochloric acid (2.8.1)
walled, rounded or almost square twin cells; fragments in
transverse section [B] showing the epidermis [Ba] associated
___________ PhEur
with thick-walled cells of the hypodermis; fragments in
transverse section [F] consisting of endodermic cells with
U-shaped thickening of the walls [Fa] accompanied by
pericyclic fibres [Fb]; numerous fragments of moderately
thickened fibres [C]; groups of vessels [D, G] with slit Dandelion Herb with Root * **
shaped pits [Da] or with spiral and annular thickening [Ga],
accompanied by fibres [Db, Gb]; numerous fragments of the
cortical parenchyma and the pith with slightly thickened and Ph Elf---------------------------------------------------------------------- -- ----------------------------- ---
pitted cells [E]. DEFINITION

TESTS Mixture of whole or fragmented, dried aerial and
Cynodon dactylon, Imperata cylindrica underground parts of Taraxacum officinale F.H. Wigg.
Examine under a microscope using iodine solution Rl. CHARACTERS
No blue starch grains are visible. Bitter taste.
Foreign matter (2.8.2) IDENTIFICATION
Maximum 15 per cent of blackish-grey pieces of rhizome in A. The underground parts consist of dark brown or blackish
the cut herbal drug. fragments 2-3 cm long, deeply wrinkled longitudinally on the
outer surface. The thickened crown shows many scars left by
IV-160 Dandelion Herb \vi± Root 2016
the rosette of leaves. The fracture is short. A transverse Test solution To 2.0 g of the powdered herbal drug (355)
section show’s a greyish-white or brownish cortex containing (2.9.12) add 10 mL of methanol R. Heat in a water-bath at
concentric layers of brownish laticiferous vessels and a 60 °C or sonicate for 10 min. Cool and filter.
porous, pale yelloWj non-radiate wood. Leaf fragments are Reference solution Dissolve 2 mg of chlorogenic acid R and
green, glabrous or densely pilose. They are crumpled and 2 mg of rutin R in methanol R and dilute to 20 mL with the
usually show' a clearly visible midrib on the inner surface. same solvent.
The lamina, with deeply dentate margins, is crumpled.
The solitary flower heads, on hollow stems, consist of an
plate R (2-10 pm)].
involucre of green, foliaceous bracts surrounding the yellow
florets, all of which are ligulate; a few' achenes bearing a Mobile phase anhydrous formic acid R, water R, ethyl acetate R
W'hite, silk}', outspread pappus may be present. (10:10:80 VIVIV).
Drying In air.
Detection Heat at 100 °C for 5 min; spray with or dip briefly
into a 10 g/L solution of diphenylboric acid aminoethyl ester R
in methanol R and dry at 100 °C for 5 min; spray with or dip
briefly into a 50 g/L solution of macrogol 400 R in methanol R;
heat at 100 °C for 5 min and examine in ultraviolet light at
365 nm.
Results See below' the sequence of zones present in the
Top of the plate
A faint red zone
A faint yellow zone
Chlorogenic acid: a blue zone 2 light blue zones
Rutinะ a yellowish-brown zone
A light blue zone
TESTS
Figure 1851.-1.- Illustration for identification test B of powdered
herbal drug of dandelion herb with root
B. Microscopic examination (2.8.23). The powder is 105 °C for 2 h.
hydrate solution R. The powder shows the following diagnostic Total ash (2.4.16)
characters (Figure 1851.-1): fragments of cork [G] with Maximum 17.0 per cent.
flattened, thin-walled cells; reticulate lignified vessels [H] Ash insoluble in hydrochloric acid (2.8.1)
from the roots; fragments of parenchyma containing Maximum 5.0 per cent.
branched laticiferous vessels [F]; fragments of leaves, in Extractable matter
surface view, showing upper [E] and lower [C] epidermises Minimum 30.0 per cent.
consisting of interlocking lobed cells and anomocytic stomata To 2.000 g of the powdered herbal drug (250) (2.9.12) add
(2.8.3) [Ca, Ea]; elongated, multicellular covering trichomes 40 g of water R. Stir for 1 h and filter. Evaporate 10 g of the
with constrictions, which are more or less abundant filtrate to dryness on a water-bath and dry in an oven at
depending on the variety or sub-variety [B, D]; fragments of 100-105 °C for 2 h. The residue weighs a minimum of
the upper [E] epidermis usually accompanied by underlying 0.15 g.
palisade parenchyma [Eb] and fragments of the lower [C]
epidermis accompanied by underlying spongy parenchyma Bitterness value (2.8.15)
[Cb]; lignified, spirally or annularly thickened vessels; Minimum 100.
fragments of flower-stem epidermis with stomata and rigid ____________________________________ __________________________ _ Ph Eur
walled, elongated cells [A], pollen grains with a pitted exine

บ]. Examine under a microscope using glycerol R.
The powder shows angular, irregular inulin fragments, free or
included in the parenchyma cells.
2016 Dandelion Herb with Root IV-161
Dandelion Root f ** c. Thin-layer chromatography (2.2.27).

(Ph. Eur. monograph 1852) *** (2.9.12) add 10 ml of methanol R. Heat in a water-bath at
PhEtr___________________ 60 °C or sonicate for 10 min. Cool and filter.
DEFINITION Reference solution Dissolve 2 mg of chlorogenic acid R and
Whole or cut, dried underground parts of Taraxacum 2 mg of rutin R in methanol R and dilute to 20 mL with the
officinale F.H.Wigg. same solvent.
CHARACTERS
P254 plate R (2-10 pm)].
Bitter taste.
Mobile phase anhydrous formic acid R, water R} ethyl acetate R
IDENTIFICATION (10:10:80 VIVIV).
A. The dark brown or blackish taproot shows little branching Application 20 pL [or 5 pL] as bands of 10 mm [or 8 mm].
and is deeply wrinkled longitudinally on the outer surface.
The thickened crown shows many scars left by the rosette of
leaves. The fracture is short. A transverse section shows a Drying In air.
greyish-white or brownish cortex containing concentric layers Detection Heat at 100 cc for 5 min; spray with or dip briefly
of brownish laticiferous vessels and a porous, pale yellow, into a 10 g/L solution of diphenylboric acid aminoethyl ester R
non-radiate wood. in methanol R and dry at 100 °C for 5 min; spray with or dip
B. Reduce to a powder (355) (2.9.72). The powder is briefly into a 50 g/L solution of macrogol 400 R in methanol R;
yellowish-brown. Examine under a microscope using chloral heat at 100 °C for 5 min and examine in ultraviolet light at
hydrate solution R. The powder shows the following diagnostic 365 nm.
characters (Figure 1852.-1): fragments of brown or reddish- Results See below the sequence of zones present in the
brown cork, in surface view [G] and transverse section [C] chromatograms obtained with the reference solution and the
with flattened, thin-walled cells [Ca], sometimes test solution. Furthermore, other faint zones may be present
accompanied by parenchyma [Cb]; reticulate lignified vessels in the chromatogram obtained with the test solution.
[E, J, M]; fragments of parenchyma [A, D, K, L], some
containing branched laticiferous vessels, in longitudinal Top of tie plate
section [Ka] and transverse section [Da]; granular contents
of laticiferous vessels [B, H]. Examine under a microscope A light blue zone
using glycerol R. The powder shows numerous irregular,
angular inulin fragments, free [F] or included in the
Chlorogenic acid: a blue zone A blue zone (chlorogenic acid)
parenchyma cells [La],
Rutin; a yellowish-brown zone
TESTS
105 °C for 2 h.
Total ash (2.4.16)
Extractable matter
To 2.000 g of the powdered herbal drug (250) (2.9.72) add
40 g of water R. Stir for 1 h and filter. Evaporate 10 g of the
filtrate to dryness on a water-bath and dry in an oven at
100-105 °C for 2 h. The residue weighs a minimum of
0.10 g.
Minimum 100.
______________________________________ _______________________ Ph Eur
Figure 1852.-1. - Illustration for identification test B ofpowdered

herbal drug of dandelion root
IV-162 Devil's claw root 2016
Devil's Claw Root ** **

Harpagophytum * **
(Devil’s Claw Root3 Ph Eur monograph 1095)
Preparation
Devil’s Claw Dry Extract
Ph Eur_______________________________________________ _______________
DEFINITION
Cut and dried, tuberous secondary roots of Harpagophytum
procumbens DC. and/or Harpagophytum zeyheri Decne.
Content
Minimum 1.2 per cent of harpagoside (024แ3()0]1;
CHARACTERS
The root is greyish-brown or dark brown.
IDENTIFICATION
A. It consists of thick, fan-shaped or rounded slices or of
roughly crushed discs. The darker outer surface is traversed
by tortuous longitudinal wrinkles. The paler cut surface
shows a dark cambial zone and xylem bundles distinctly
aligned in radial rows. The central cylinder shows fine
concentric striations. Seen under a lens, the cut surface
presents yellow or brownish-red granules.
hydrate solution R. The powder shows the folloving diagnostic
characters (Figure 1095.-1): fragments of cork consisting of
yellowish-brown, thin-walled cells, in surface view [B] and in
Figure 1095.-1- Illustration for identification test B of
transverse section [C]; fragments of cortical parenchyma
powdered herbal drug of devil’s claw root
consisting of large, thin-walled cells [E, K, N, P], sometimes
containing reddish-brown granular inclusions and isolated
yellow droplets (P); fragments of reticulately thickened or
pitted vessels [D, F, G, M] and fragments of lignified Top of the plate
parenchyma [L], sometimes associated with vessels, from the
central cylinder; prism crystals [A] and rare small needles of
Harpagoside: a quenching zone A quenching zone: harpagoside
calcium oxalate in the parenchyma. The powder may also
show rectangular or polygonal sclereids with dark reddish-
brown contents [H, J]. With a solution of phloroglucinol in
hydrochloric acid, the parenchyma turns green.
c. Thin-layer chromatography (2.2.27). Detection B Spray with a 10 g/L solution of phloroglucinol R in
Test solution Heat 1.0 g of the powdered herbal drug (355) ethanol (96 per cent) R and then with hydrochloric acid R; heat
(2.9.72) with 10 mL of methanol R on a water-bath at 60 °C at 80 °C for 5-10 min and examine in daylight.
for 10 min. Filter and reduce the filtrate to about 2 mL Residts B See below the sequence of zones present in the
under reduced pressure at a temperature not exceeding chromatograms obtained with the reference solution and the
40 °C. test solution; the chromatogram obtained with the test
Reference solution Dissolve 1 mg of harpagoside R and 2.5 mg solution also shows several yellow or brown zones above the
offructose R in 1 mL of methanol R. zone due to harpagoside. Furthermore, other faint zones may
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel be present in the chromatogram obtained with the test
plate R (2-10 pm)]. solution.
Mobile phase water Ry methanol Ry ethyl acetate R
Top of the plate
(8:15:77 r/K/P).
Development Over a path of 10 cm [or 7.5 cm]. Harpagoside: a green zone A green zone (harpagoside)
chromatograms obtained with the reference solution and the A light green zone
test solution; the chromatogram obtained with the test Fructose: a yellowish-grey zone A yellowish-grey zone may be
solution shows other distinct zones, mainly above the zone present (fructose)
due to harpagoside. Furthermore, other faint zones may be A brown zone
present in the chromatogram obtained with the test solution. Reference solution Test solution
2016 Devil’s Claw Preparations IV-163
TESTS solvent that is at most equivalent in strength to ethanol

Starch (95 per cent V/V).
Examine the powdered herbal drug (355) (2.9.72) under a
CHARACTERS
microscope using water R. Add iodine solution Rl. No blue
colour develops. Appearance
Light brown powder.
Maximum 12.0 per cent, determined on 1.000 g of the IDENTIFICATION
powdered herbal drug (355) (2.9.72) by drying in an oven at Thin-layer chromatography (2.2.27).
105 °C. Test solution To 1.0 g of the extract to be examined add
Total ash (2.4.76) 10 mL of methanol R and heat in a water-bath at 60 °C for
Maximum 10.0 per cent. 10 min. Cool and filter.
Reference solution Dissolve 1.0 mg of harpagoside R and
ASSAY
2.5 mg of fructose R in 1.0 mL of methanol R.
Mobile phase water R, methanol R, ethyl acetate R
(2.9.72) add 100.0 mL of methanol R. Shake for 4 h and
(8:15:77 VIVIV).
filter through a membrane filter (nominal pore size 0.45 pm).
Reference solution Dissolve the contents of a vial of
harpagoside CRS in methanol R and dilute to 10.0 mL with Development Over a path of 10 cm.
the same solvent. Drying In a current of warm air.
Column". Detection Spray with a 10 g/L solution of phloroglucinol R in
size: I = 0.10 m, 0 ะ= 4.0 mm; ethanol (96 per cent) R and then with hydrochloric acid R", heat
stationary phase: octadecylsilyl silica gel for chromatography R at 80 °C for 5-10 min and examine in daylight.
(5 pm). Results See below the sequence of zones present in the
Mobile phase methanol R, water R (50:50 V/V). chromatograms obtained with the reference solution and the
Flow rate 1.5 mL/min. test solution. Furthermore, other faint zones may be present
Injection 10 pL.
Top of the plate
Run time 3 times the retention time of harpagoside.
Retention time Harpagoside = about 7 min.
Calculate the percentage content of harpagoside using the Harpagoside: a green zone A green zone (harpagoside)
7772 X Al X 1000
A light green zone
A2 X 7711
Fructose ะ a yellowish-grey zone A yellowish-grey zone may be
present (fructose)
A1 = area of the peak due to harpagoside in the A brown zone
A2 = area of the peak due to harpagoside in the Reference solution Test solution

solution;
7771 = mass of the herbal drug to be examined used to ASSAY
prepare the test solution, in grams; Liquid chromatography (2.2.29).
m2 = mass of harpagoside CRS in the reference solution, Test solution Introduce 0.350 g of the extract to be examined
in grams. into a 100 mL volumetric flask, add 90 mL of methanol R
and sonicate for 20 min. Cool to room temperature, dilute to
__ ____________________________________________________________ PhEur
100.0 mL with methanol R and filter through a membrane
filter (nominal pore size 0.2 pm).
Reference solution Dissolve the contents of 1 vial of
harpagoside CRS in methanol R and dilute to 10.0 mL with
Devil’s Claw Dry Extract * the same solvent.
(Ph. Eur. monograph 1871) *** Column:
— size: I = 0.10 m, 0 = 4.0 mm;
Ph Eur______________________________________________________________ — stationary phase: octadecylsilyl silica gel for chromatography R
DEFINITION (5 pm).
Dry extract obtained from Devil's claw root (1095). Mobile phase methanol R, water R (50:50 V!V).
Content Flow rate 1.5 mL/min.
Minimum 1.5 per cent of harpagoside (C24H30O11; Detection Spectrophotometer at 278 nm.
Mr 494.5) (dried extract). Injection 10 pL.
PRODUCTION Run time 3 times the retention time of harpagoside.
The extract is produced from the herbal drug by an Retention time Harpagoside = about 7 min.
appropriate procedure using either water or a hydroalcoholic
IV-164 Digitalis Leaf 2016
Calculate the percentage content of harpagoside using the b) glandular trichomes usually with a unicellular [C, D],
following expression: sometimes a multicellular, uniseriate [A, B, E] stalk and a
unicellular head [A, B, c, E] or bicellular head, in side view
Al X 7ท2 X 1000 [D] and in surface view [F] or exceptionally a tetracellular
>12 X 7711 head.
/41 = area of the peak due to harpagoside in the

A2 = area of the peak due to harpagoside in the
m1 = mass of the extract to be examined used to prepare
the test solution, in grams;
ไท2 = mass of harpagoside contained in 1 vial of
harpagoside CRS3 in grams.
_______________________________________________________________ PhEur
Digitalis Leaf * *
(Ph Eur monograph 0117) ***
When Powdered Digitalis is prescribed or demanded,
PhEir_______________________________________________________________
DEFINITION
Dried leaf of Digitalis purpurea L.
Content
Minimum 0.3 per cent of cardenolic glycosides, expressed as
digitoxin (Afr 765) (dried drug).
CHARACTERS
Faint but characteristic odour.
The whole leaf is about 10-40 cm long and 4-15 cm wide. Figure 0117.-1. - Illustration for identification test B of powdered
The lamina is ovate lanceolate or broadly ovate. The winged herbal drug of digitalis leaf
petiole is from 1/4 as long as to equal in length to the c. Thin-layer chromatography (2.2.27).
lamina. Test solution To 1.0 g of the powdered herbal drug (180)
IDENTIFICATION (2.9.12) add a mixture of 20 mL of ethanol
A. The leaf is brittle and often occurs broken. The upper (50 per cent VIV) R and 10 mL of lead acetate solution R. Boil
surface is green and the lower surface is greyish-green. for 2 min, allow to cool and centrifuge. Shake the
The apex is subacute and the margin is irregularly crenate, supernatant solution with 2 quantities, each of 15 mL, of
dentate or serrate. The base is decurrent. The venation is chloroform R3 separate the 2 layers by centrifugation if
pinnate, the lateral veins being prominent especially on the necessary. Dry the chloroform layers over anhydrous sodium
lower surface, leaving the midrib at about 45° and sulfate R and filter. Evaporate 10 mL of the solution to
anastomosing near the margin; a veinlet terminates in each dryness on a water-bath and dissolve the residue in 1 mL of
tooth of the margin and the lower veins run down the winged a mixture of equal volumes of chloroform R and methanol R.
petiole. The upper surface is rugose and pubescent; the lower Reference solution Dissolve 5 mg of purpureaglycoside A CRS,
surface shows a network of raised veinlets and is densely 2 mg of purpureaglycoside B CRS3 5 mg of digitoxin R and
pubescent. 2 mg of gitoxin R in a mixture of equal volumes of
B. Microscopic examination (2.8.23). Examine under a chloroform R and methanol R, then dilute to 10 mL with the
microscope using chloral hydrate solution R. The powder same mixture of solvents.
shows the following diagnostic characters (Figure 0117.-1): Plate TLC silica gel G plate R.
fragments of the upper epidermis, in surface view [K, L], Mobile phase water R3 methanol R, ethyl acetate R
with cells with a smooth cuticle and anticlinal walls that are (7.5:10:75 VIVIV).
slightly thickened, are straight or slightly sinuous, and may
Application 20 îL as bands of 2 cm by 0.3 cm.
show slight beading and pitting [La] and sometimes scars of
covering trichomes [Ka], accompanied by underlying palisade Development Over a path of 10 cm.
parenchyma [Lb]; fragments of the lower epidermis, in Drying Until the solvents have evaporated.
surface view [G], with markedly sinuous cells and Detection Treat with a mixture of 2 volumes of a 10 g/L
anomocytic stomata (2.8.3) [Ga]; trichomes are of 2 types: solution of chloramine R and 8 volumes of a 250 g/L solution
a) uniseriate covering trichomes with blunt apex, usually of trichloroacetic acid R in ethanol (96 per cent) Ri then heat at
consisting of 3-5 cells [H, J], often with 1 or more collapsed 100-105 °C for 10 min; examine in ultraviolet light at
cells [Ja], walls mostly finely warty or faintly striated; 365 nm.
2016 Dill Oil IV-165
Results The chromatogram obtained with the reference (50 per cent V/V) Ry 2 mL of dinitrobenzoic acid solution R and
solution shows a zone of light blue fluorescence in the lower 1 mL of 1 M sodium hydroxide.
part of the chromatogram, due to purpureaglycoside B, and, From the absorbances measured and the concentrations of
just above it, a zone of brownish-yellow fluorescence due to the solutions, calculate the content of cardenolic glycosides,
purpureaglycoside A; a zone of light blue fluorescence, due to expressed as digitoxin.
gitoxin, appears in the middle of the chromatogram and
above it a zone of brownish-yellow fluorescence, due to STORAGE
digitoxin; the zones in the chromatogram obtained with the Protected from moisture.
test solution are similar in position, colour and size to the ____________________________________________________________ Ph Eur
zones in the chromatogram obtained with the reference
solution. Other zones of fluorescence may also appear in the
D. Evaporate 5 mL of the chloroformic solution obtained in
identification test c to dryness on a water-bath. To the Dill Oil
residue add 2 mL of dinitrobenzoic acid solution R and 1 mL DEFINITION
of / M sodium hydroxide. A reddish-violet colour develops Dill Oil is obtained by distillation from the dried ripe fruits of
within 5 min. Anethum graveolens L.
E. Evaporate 5 mL of the chloroformic solution obtained in CHARACTERISTICS
identification test c to dryness on a water-bath. To the A clear, colourless or pale yellow liquid, visibly free from
residue add 3 mL of xanthydrol solution R and heat on a water; odour, characteristic of the crushed fruit.
water-bath for 3 min. A red colour develops.
TESTS
TESTS
Optical rotation
Digitalis lanata Ehrh. +70° to +80°, Appendix V F.
The presence of leaves with few or no trichomes and with
parallel venation or the presence of cells of the abaxial Refractive index
epidermis with beaded anticlinal walls and of cells of the 1.481 to 1.492, Appendix V E.
adaxial epidermis with numerous stomata indicates Solubility in ethanol
adulteration by Digitalis lanata Ehrh. Soluble, at 20°, in 1 volume or more of ethanol (90%) and in
Loss on drying (2.2.32) 10 volumes or more of ethanol (80%), Appendix X M.
Maximum 6.0 per cent, determined on 1.000 g of the Weight per mL
powdered herbal drug (355) (2.9.12) by drying in an oven at 0.895 to 0.910 g, Appendix V G.
105 °C. Content of carvone
Total ash (2.4.16) 43.0 to 63.0% พ/พ when determined by the following
iMaximum 12.0 per cent. method. To 1.5 g in a glass-stoppered tube (approximately
150 mm X 25 mm) add 10 mL of a solution prepared in the
following manner. Dissolve 7.0 g of hydroxylamine
hydrochloride in 90 mL of ethanol (90%) 3 warming gently if
ASSAY necessary, add 1.6 mL of dimethyl yellow solution and
Shake 0.250 g of the powdered herbal drug (180) (2.9.12) sufficient Im potassium hydroxide in ethanol (90%)) to produce
with 50.0 mL of water R for 1 h. Add 5.0 mL of a 150 g/L a pure yellow colour and dilute to 100 mL with ethanol
solution of lead acetate R, shake, and after a few minutes add (90%)). Titrate with Im potassium hydroxide in ethanol (90%))
7.5 mL of a 40 g/L solution of disodium hydrogen phosphate R. PS until the red colour changes to yellow. Place the tube in a
Filter through a pleated paper filter. Heat 50.0 mL of the water bath at 75° to 80° and, at 5-minute intervals, neutralise
filtrate with 5 mL of hydrochloric acid (150 g/L HC1) under with Im potassium hydroxide in ethanol (90%) PS; after
a reflux condenser on a water-bath for 1 h. Transfer to a 40 minutes complete the titration to the full yellow colour of
separating funnel, rinse the flask with 2 quantities, each of the indicator. This procedure gives an approximate value for
5 mL, of พater R and shake with 3 quantities, each of the carvone content of the oil. Repeat the procedure, using as
25 mL, of chloroform R. Dry the combined chloroform layers the colour standard for the end point of the titration the
over anhydrous sodium sulfate R and dilute to 100.0 mL with titrated liquid of the first determination with the addition of
chloroform R. Evaporate 40.0 mL of the chloroformic solution 0.5 mL of Im potassium hydroxide in ethanol (90%)) vs.
to dryness, dissolve the residue in 7 mL of ethanol Calculate the content of carvone from the second
(50 per cent V/V) R, add 2 mL of dinitrobenzoic acid solution R determination. Each mL of 1M potassium hydroxide in ethanol
and 1 mL of 1 M sodium hydroxide. At the same time prepare (90%)) VS is equivalent to 151.4 mg of carvone, CioH140.
a reference solution as follows. Dissolve 50.0 mg of STORAGE
digitoxin CRS in ethanol (96 per cent) R and dilute to 50.0 mL
Dill Oil should be kept in a well-filled container and
with the same solvent. Dilute 5.0 mL of the solution to protected from light. It darkens in colour on storage.
50.0 mL with ethanol (96 per cent) R. To 5.0 mL of the
resulting solution add 25 mL of water R and 3 mL of
hydrochloric acid (150 g/L HC1). Heat ±e solution under a
reflux condenser on a water-bath for 1 h and complete the
preparation as described above. Measure the absorbance
(2.2.25) of the 2 solutions at 540 nm several times during the
first 12 min until the maximum is reached, using as the
compensation liquid a mixture of 7 mL of ethanol
IV-166 Dioscorea Oppositifolia Rhizome 2016
Dioscorea Oppositifolia Rhizome ***** Application 15 pL as bands of 8 mm.

(Ph. Eur. monograph 2473) *** Drying In air.
Ph Eur_________________________________________________________ _____
Detection Treat with anisaldehyde solution R, heat at 100 °C
DEFINITION for 3 min, allow to cool for 10 min and examine in daylight.
Dried, whole or fragmented rhizome of Dioscorea Results The chromatogram obtained with the test solution
oppositifolia L. (syn. Dioscorea opposita Thunb.), collected in shows no brownish-green or orange-brown zones in the
winter when the stem and leaves are withered, with outer upper third of ±e chromatogram.
bark and fibrous roots removed. Loss on drying (2.2.32)
IDENTIFICATION Maximum 12.0 per cent, determined on 1.000 g of the
A. The rhizome occurs in sub cylindrical pieces, sometimes powdered herbal drug (355) (2.9.12) by drying in an oven at
flattened, about 15-30 cm long and 1.5-6 cm thick; the outer 105 °C for 2 h.
surface is yellowish-white or pale yellow, longitudinally Total ash (2.4.16)
furrowed and wrinkled, and bearing slit-shaped root scars Maximum 4.0 per cent.
with occasional patches of brownish cork. The fragmented Ash insoluble in hydrochloric acid (2.8.1)
rhizome occurs in whole or fragmented slices, about Maximum 0.5 per cent.
0.2-0.5 cm thick; some slices still bear slit-shaped root scars.
The texture is heavy, compact and tough; ±e fracture is Extractable matter
white and starchy. Minimum 7.0 per cent (dried drug).
B. Microscopic examination (2.8.23). The powder is whitish To 1.00 g of the powdered herbal drug (355) (2.9.12) add
or yellowish-white. Examine under a microscope using chloral 25 mL of water R, shake for 6 h and allow to macerate for
hydrate solution R. The powder shows the following diagnostic 18 h. Filter, evaporate the filtrate to dryness on a water-bath
characters: a few vessels with reticulate thickening not under reduced pressure and dry in an oven at 100-105 °C for
exceeding 50 pm in diameter; very thin-walled polyhedral or 2 h. The residue weighs a minimum of 70 mg.
ovoid parenchyma cells, some of which contain calcium _____________________________________________________________ _ Ph Euf
oxalate raphides up to 240 pm long; a few isolated calcium
oxalate needles from the raphides. Examine under a
microscope using a 50 per cent v/v solution of glycerol R.
The powder shows extremely abundant starch granules,
simple, ovoid or oblong, flattened with rounded extremities, Dog Rose ★ **
with a maximum dimension of up to 53 pm; the hilum is (Ph. Eur. monograph 1510) ***
usually eccentric, punctiform or V- or cross-shaped; rare,
Ph Eur_______________________________________________________________
2- to 4-compound starch granules may be present.
c. Examine the chromatograms obtained in the test for DEFINITION
Dioscorea bulbifera L. Rose hips made up by the receptacle and the remains of the
Results See below the sequence of zones present in the dried sepals of Rosa canina L., R. pendulina L. and other Rosa
chromatograms obtained with the reference solution and the species, with the achenes removed.
test solution. Furthermore, other faint zones may be present Content
in the chromatogram obtained with the test solution. Minimum 0.3 per cent of ascorbic acid (C6H8O6; Mf 176.1)
(dried drug).
Top of the plate IDENTIFICATION
3-5 violet zones A. It consists of fragments of the fleshy, hollow, urceolate
receptacle, bearing the remains of the reduced sepals, light
pink or orange-pink, the convex outer surface shiny and
Aescin ะ a violet zone A violet zone strongly wrinkled; bearing on its lighter inner surface
abundant bristle-like hairs.
Glucose: a yellow or green zone A yellow or brown zone
orange-yellow. Examine under a microscope using chloral
A violet zone hydrate solution R. The powder shows the following diagnostic
characters: numerous fragments of receptacle, the outer
Reference solution Test solution epidermis with orange-yellow contents and a thick cuticle,
the inner epidermis composed of thin-walled cells containing
cluster crystals and occasional prisms of calcium oxalate;
TESTS scattered lignified cells, isodiametric, with thickened and
Dioscorea bulbifera L pitted walls forming the trichome bases; abundant unicellar
trichomes, up to 2 mm long and 30-45 pm thick, tapering
Test solution To 0.5 g of the powdered herbal drug (355) towards each end, walls heavily thickened and with a waxy
(2.9.12) add 5 mL of methanol R. Sonicate for 10 min. cuticle which may show fissures in a spiral arrangement;
Centrifuge and use the supernatant. numerous oily orange-yellow globules.
Reference solution Dissolve 1 mg of aesciท R and 3 mg of c. Thin-layer chromatography (2.2.27).
glucose R in 7 mL of methanol R. Test solution To 5 g of the powdered herbal drug (355)
Plate TLC silica gel plate R (2-10 Jim). (2.9.12) add 25 mL of ethanol (96 per cent) R, shake for
Mobile phase water R, methanol R, methylene chloride R 30 min and filter.
(10:50:64 VIVIV).
2016 Drynana Rhizome IV-167
Reference solution Dissolve 10 mg of ascorbic acid R in 5.0 mL obtained during the preparation of the test solution. Measure
of ethanol (60 per cent VIV) R. the absorbance (2.2.25) at 520 nm using solution B as the
Plate TLC silica gel F254 plate R. compensation liquid.
Mobile phase acetone R, glacial acetic acid R, methanol R, Solution B Treat 2.0 mL of the reference solution as
toluene R (5:5:20:70 VIVIV/V). described above for solution A.
Application 20 pL of the test solution and 2 pL of the Calculate the percentage content of ascorbic acid from the
reference solution. following expression:
2.5 X Al X 7712
Drying In air.
Al X mi
Results A The chromatogram obtained with the test solution A1 = absorbance of the test solution;
shows a quenching zone similar in position to the principal 242 = absorbance of the reference solution;
zone in the chromatogram obtained with the reference พ1 = mass of the substance to be examined, in grams;
solution. m2 — mass of ascorbic acid used, in grams.
Detection B Spray with a 0.2 g/L solution of
_____________________________________________________________ Ph Eur
dichlorophenolindophenol, sodium salt R in ethanol
(96 per cent) R. Examine in daylight.
shows a white zone on a pink background (ascorbic acid)
similar in position and colour to the principal zone in the Drynaria Rhizome * *
(Ph Eur monograph 2563) * **
The chromatogram also shows an intense orange-yellow zone
near the solvent front and a yellow zone in the upper third PhEur______________________________________________________________
(carotenoids). DEFINITION
TESTS Dried rhizome of Drynaria fortune! (Kunze) J. Sm. The
Foreign matter (2.8.2) ramenta may be removed.
Maximum 1 per cent. Content
Loss on drying (2.2.32) Minimum 0.5 per cent of naringin (C27H32O14; Mr 580.5)
Maximum 10.0 per cent, determined on 1.000 g of the (dried drug).
powdered herbal drug (355) (2.9.12) by drying in an oven at IDENTIFICATION
105 °C. A. Long, flattened, slat-shaped rhizome, often curved and
Total ash (2.4.16) branched, 5-15 cm long and 1-1.5 cm thick. The surface is
Maximum 7.0 per cent. either completely covered in scaly, dark brown hairs (rhizome
ASSAY with ramenta) or glabrous with dark brown dots (rhizome
without ramenta). The upper surface and both sides show
Test solution In a round-bottomed flask, weigh 0.500 g of the
circular frond scars, rarely the frond bases. The lower surface
freshly powdered herbal drug (710) (2.9.12). Add a solution
shows scars or the remains of fibrous roots. The texture is
of 1.0 g of oxalic acid R in 50.0 mL of methanol R. Boil under
light, fragile, easily broken. The section is reddish-brown;
a reflux condenser for 10 min, and cool in iced water until
the steles form a ring of small yellow dots.
the temperature reaches 15-20 °C. Filter. Transfer 2.0 mL of
the filtrate to a 50 mL conical flask. Add successively, with B. Microscopic examination (2.8.23). The powder is reddish-
gentle shaking after each addition, 2.0 mL of brown. Examine under a microscope using chloral hydrate
dichlorophenolindophenol standard solution R and then, exactly solution R. The powder shows the following diagnostic
60 ร later, 0.5 mL of a 100 g/L solution of thiourea R in characters: numerous parenchyma fragments, consisting of
ethanol (50 per cent VIV) R and 0.7 mL of polyhedral cells with slighdy and regularly thickened and
dinitrophenylhydrazine-sulfuric acid solution R. Heat under a pitted walls; scalariform lignified vessels of variable diameter
reflux condenser at 50 °C for 75 min, and place immediately up to 60 pm; fragments of scaly hairs forming a tissue
in iced water for 5 min. Add dropwise 5.0 mL of a mixture consisting of many reddish-brown cells forming expansions
of 12 mL of water R and 50 mL of sulfuric acid R, taking care on the margins (rhizome with ramenta).
to carry out the addition over a period of minimum 90 ร and c. Thin-layer chromatography (2.2.27).
maximum 120 ร while maintaining vigorous stirring in iced Test solution To 0.5 g of the powdered herbal drug (355)
water. Allow to stand for 30 min at room temperature and (2.9.12) add 5 mL of methanol R and sonicate for 10 min.
measure the absorbance (2.2.25) at 520 nm using solution A Cool, centrifuge and use the supernatant.
as compensation liquid. Reference solution Dissolve 1 mg of naringin R and 1 mg of
Solution A Treat 2.0 ml. of the filtrate obtained during the hyperoside R in 2 mL of methanol R.
preparation of the test solution as described but adding the Plate TLC silica gel plate R (2-10 pm).
dinitrophenylhydrazine-sulfuric acid solution R just before the
Mobile phase acetic acid R, anhydrous formic acid R, water R,
absorbance is measured. ethyl acetate R (11:11:26:100 VIVIVIV).
Reference solution Dissolve 40.0 mg of ascorbic acid I? in a
freshly prepared 20 g/L solution of oxalic acid R in
methanol R and dilute to 100.0 mL with the same solvent. Development Over a path of 6 cm.
Dilute 5.0 mL of this solution to 100.0 mL with a freshly Drying In air.
prepared 20 g/L solution of oxalic acid R in methanol R. Treat Detection Treat with aluminium chloride reagent R; examine in
2.0 mL of the solution as described above for the filtrate ultraviolet light at 365 nm.
IV-168 Echinacea 2016
Results See below the sequence of zones present in the Al X m2 X p

chromatograms obtained with the reference solution and the A2 X mi X 20
in the chromatogram obtained with the test solution. Al = area of the peak due to naringin in the
Top of the plate A2 = area of the peak due to naringin in the
(c);
— พ] = mass of the herbal drug to be examined used to
Hyperoside: a yellow zone
m2 = mass of naringin CRS used to prepare reference
Naringin: a bluish-white zone A bluish-white zone (naringin) solution (a), in grams;
A bluish-white zone
p = percentage content of naringin in naringin CRS.
_____ _________________________________________________________ Ph Eur
Echinacea Angustifolia Root * *

TESTS (Narrow-leaved Coneflower Root, * **
Loss on drying (2.2.32) Ph Eur monograph 1821)
Maximum 13.0 per cent, determined on 1.000 g of the Ph Eur______________________________________________________________
105 °C for 2 h. DEFINITION
Dried, whole or cut underground parts of Echinacea
Total ash (2.4.16)
angustifolia (D.C.).
Content
Minimum 0.5 per cent of echinacoside (C35H.i6O20i
ASSAY
IDENTIFICATION
First identification A, B, c.
Second identification A, B, D.
(355) (2.9.12) in a 50 per cent v/v solution of methanol R
and dilute to 10.0 mL with the same solvent. Weigh, A. The root crown is up to about 30 mm in diameter and
sonicate for 45 min. Allow to cool, weigh and compensate shows only a few stem bases. The roots are not very
the loss of solvent with a 50 per cent P7F solution of numerous, up to about 15 mm in diameter, cylindrical or
methanol R, shake well. Filter through a membrane filter slightly tapering and sometimes spirally twisted, the outer
(nominal pore size 0.45 pm). surface is pale brown to yellowish-brown. The fracture is
short, dark brown with a radiate structure.
Reference solution (a) Dissolve 10.0 mg of naringin CRS in
methanol R and dilute to 20.0 mL with the same solvent. B. Reduce to a powder (355) (2.9.12). The powder is
greyish-brown. Examine under a microscope using chloral
Reference solution (b) Dissolve 5.0 mg of neohesperidin R in
reference solution (a) and dilute to 10.0 mL with reference
characters: narrow lignified fibres (up to about 800 pm in
solution (a).
length and 50 pm in diameter) joined together in long
Reference solution (c) Dilute 1.0 mL of reference solution (a) bundles surrounded by phytomelanin deposits; lignified
to 10.0 mL with methanol R. reticulately or scalariformly thickened vessels (up to about
Column'. 60 pm in diameter); abundant sclereids occuring singly or,
— size’. I = 0.15 m, 0 = 4.6 mm; more usually, in groups of 2 to 10, mostly elongated to
— stationary phase: octadecylsilyl silica gel for chromatography R rectangular, (up to about 150 pm in length and 40 pm wide),
(5 pm). with intercellular spaces filled with phytomelanin deposit;
Mobile phase acetonitrile R, 0.4 per cent vtv solution of acetic fragments of oleoresin canal (80-150 pm in diameter) with
add R (18:82 V/V). yellowish-orange to reddish-brown content; groups of
squarish to rectangular cells, about 30-45 pm from the outer
layers of the roots; abundant fine-walled pitted parenchyma
with sphaerocrystalline masses of inulin.
Injection 20 pL of the test solution and reference solutions (b)
and (c). Echinacea purpurea.
Run time Twice the retention time of naringin.
Retention time Naringin = about 9 min; chromatograms obtained with the reference solution and the
neohesperidin = about 12 min. test solution. Furthermore, other faint dark blue fluorescent
System suitability, reference solution (b): zones may be present between the zones of echinacoside and
— resolution: minimum 5.0 between the peaks due to cynarin in the chromatogram obtained with the test solution.
naringin and neohesperidin.
Calculate the percentage content of naringin using the
2016 Echinacea IV-169
1. caftaric acid 3. caffeic acid 5. echinacoside
2. chlorogenic acid 4. cynarin 6. cichoric acid
Figure 1821.-1. - Chromatogram for the assay of echinacoside in narrow-leaved coneflower root
Top of the plate Drying In a stream of cold air for about 10 min followed by
2 min at 100-105 °C.
Caffeic acid: a strong blue
fluorescent zone Detection Treat the hot plate using a 5 g/L solution of
diphenylboric acid aminoethyl ester R in ethyl acetate R} examine
in ultraviolet light at 365 nm after 30 min.
Cynarin: a strong greenish A greenish fluorescent zone
fluorescent zone (cynarin) Results The chromatogram obtained with the test solution
shows no greenish fluorescent zone just below the zone due
to caffeic acid in the chromatogram obtained with the
reference solution and no greenish fluorescent zone below the
Echinacoside: a strong greenish A strong greenish fluorescent zone zone due to cynarin in the chromatogram obtained with the
fluorescent zone (echinacoside) reference solution. In the chromatogram obtained with the
test solution no zones apart from faint dark blue fluorescent
Reference solution Test solution zones are visible between the zones due to echinacoside and
cynarin.
D. Examine the chromatograms obtained in the assay. Loss on drying (2.2.52)
Results The chromatogram obtained with the test solution Maximum 12.0 per cent, determined on 1.000 g of the
shows 1 major peak due to echinacoside and a minor peak powdered herbal drug (355) (2.9.12) by drying in an oven at
due to cynarin. Peaks due to caffeic acid, caftaric acid and 105 °C for 2 h.
chlorogenic acid are minor peaks or may be absent. Total ash (2.4.16)
TESTS Maximum 9.0 per cent.
Foreign matter (2. ร. 2) Ash insoluble in hydrochloric acid (2.8.1)
Maximum 3 per cent. Maximum 3.0 per cent.
Echinacea purpurea ASSAY
Thin layer chromatography (2.2.27). Liquid chromatography (2.2.29).
Test solution To 1.0 g of the powdered herbal drug (355) Test solution In a 100 mL volumetric flask place 0.500 g of
(2.9.12) add 10 mL of methanol Rs treat in an ultrasonic bath the powdered herbal drug (355) (2.9.12) and add 80 mLof
for 5 min. Centrifuge and use the supernatant solution. ethanol (70 per cent V/V) R. Treat in an ultrasonic bath for
Reference solution Dissolve 1 mg of echinacoside R, 1 mg of 15 min and dilute to 100.0 mL with ethanol
cynarin R and 0.5 mg of caffeic acid 7? in 5.0 mL of (70 per cent V/V) R. Mix the suspension and allow to stand
methanol R. for a few minutes so that visible solids settle. Filter a suitable
proportion of the solution through a membrane filter
(nominal pore size 0.45 pm) before injection.
F254 plate R (2-10 pm)].
Reference solution Dissolve 10.0 mg of chlorogenic acid CRS
Mobile phase anhydrous formic acid R) water R} methyl ethyl
and 10.0 mg of caffeic acid R in ethanol (70 per cent V/V) R>
ketone R, ethyl acetate R (3:3:9:15 V/V/V/V). sonicate for 15 min and dilute to 10.0 mL with the same
Application 25 pL [or 5 pL] of the test solution and 10 pL solvent. Dilute 4.0 mL of this solution to 100.0 mL with
[or 2 pL] of the reference solution as bands. ethanol (70 per cent V/V) R.
development Over a path of 15 cm [or 5 cm].
Column: IDENTIFICATION
— size: I = 0.25 m, 0 = 4.6 mm; A. The rhizome and roots arc 4-20 mm in diameter,
— stationary phase: octadecylsilyl silica gel for chromatography R cylindrical and sometimes spirally twisted, longitudinally
(5 gm); wrinkled or deeply furrowed; the outer surface is reddish-
— temperature: 35 °C. brown to greyish-brown.
Mobile phase: B. Reduce to a powder (355) (2.9.72). The powder is
— mobile phase A: phosphoric acid R3 water 1? (1:999 VIV), greyish-brown to light yellow. Examine under a microscope
— mobile phase B: acetonitrile Rj using chloral hydrate solution R. The powder shows the
Time
following diagnostic characters: short lignified fibres
Mobile phase A Mobile phase B
(100-300 pm in length, up to about 80 pm in diameter)
0 90 10 occurring singly or joined together in long bundles,
sometimes with phytomelanin deposits; lignified reticulately
0- 13 90 -» 78 10 -» 22
or scalariformly thickened vessels (up to about 70 pm in
13- 14 78-» 60 22 -» 40 diameter); abundant sclereids, occurring singly or in small
14 - 145 60 40
groups of less than 10, varying considerably in shape from
rounded to rectangular or irregular, sometimes much
Flow rate 1.5 mlVmin. elongated and fibre-like and measuring up to 400 pm in
length; all ±e sclereids have associated black, phytomelanin
deposits; fragments of oleoresin canals (up to 240 pm in
Injection 10 pL. diameter) with yellowish-orange content; groups of squarish
Relative retention With reference to chlorogenic acid: to rectangular cells of the outer layers (about 40 X 80 pm);
caftaric acid = about 0.8; caffeic acid = about 1.5; abundant thin-walled pitted parenchyma with
cynarin = about 1.6; echinacoside = about 1.7; sphaerocrystalline masses of inulin.
cichoric acid = about 2.3. c. Examine the chromatograms obtained in the test for other
System suitability: reference solution: Echinacea species and Parthenium integrifolium.
— resolution: minimum 10 between the peaks due to caffeic Results See below the sequence of zones present in the
acid and chlorogenic acid. chromatograms obtained with the reference solution and the
Locate the peaks due to caffeic acid and chlorogenic acid test solution. The chromatogram obtained with the test
using the chromatogram obtained with the reference solution. solution may also show a weak zone close to the solvent
Locate the peaks due to echinacoside and cynarin using the front.
chromatogram in Figure 1821.-1.
Calculate the percentage content of echinacoside from the Top of the plate
Ar X C2 X 100 X 2.221 A greenish-brown to brown zone

A2 X Cl A yellow zone
Al = area of the peak due to echinacoside in the A violet zone
chromatogram obtained wi± the test solution;

^Sitosterol: a violet to pink zone A violet to pink zone (^sitosterol)
solution; jV-Isobutyldodecatetraenamide: a
greyish-blue zone
Cl ะ= concentration of the test solution, in milligrams
per millilitre;
c2 = concentration of chlorogenic acid in the A dark grey-blue zone
reference solution, in milligrams per millilitre;
2.221 = peak correlation factor between chlorogenic
acid and echinacoside. Reference solution Test solution
STORAGE
Store uncomminuted. D. Examine the chromatograms obtained in the assay.
________________________________________________________________ Ph Eur Results The major peak in the chromatogram obtained with
the test solution is due to echinacoside. Peaks due to caftaric
acid, caffeic acid, cynarin, chlorogenic acid and cichoric acid
are minor peaks or may be absent.
Echinacea Pallida Root * TESTS
(Pale Conefiower Root, Ph Eur monograph 1822) *า Maximum 3 per cent.
Ph Elf______________ _______ -_______________________________________ Other Echinacea species and Parthenium integrifolium
DEFINITION Thin-layer chromatography (2.2.27).
Dried, whole or cut underground parts of Echinacea pallida Test solution To 1.0 g of the powdered herbal drug (355)
Nutt. (2.9.72) add 10 mL of methylene chloride R and sonicate for
5 min. Centrifuge and use the supernatant solution.
Content
Minimum 0.2 per cent of echinacoside (035แ46020> Reference solution Dissolve 1 mg of p-sitosterol R and a volume
Mt 786.5) (dried drug). of N-isobutyldodecatetraenamide solution R corresponding to
Figure 1822.-1. - Chromatogram for the assay of echinacoside in pale coneflower root
1 mg of N-isobutyldodecatetraenamide R in methanol R and Reference solution Dissolve 10.0 mg of chlorogenic acid CRS
dilute to 5.0 mL with the same solvent. and 10.0 mg of caffeic acid R in ethanol (70 per cent VIV) R,
Plate TLC silica gel 7*254 plate R (5-40 pm) [or TLC silica gel sonicate for 15 min and dilute to 10.0 mL with the same
7*254 plate R (2-10 pm)]. solvent. Dilute 4.0 mL of this solution to 100.0 mL with
Mobile phase anhydrous formic acid R, cyclohexane R, ethanol (70 per cent V/V) R.
ethyl acetate R, toluene R (0.9:3:6:24 VIVIVIV). Column-.
— size: I = 0.25 m, 0 = 4.6 mm;
Application 25 pL [or 5 pL] of the test solution and 10 pL
[or 4 pL] of the reference solution as bands.
(5 nm);
Development Over a path of 15 cm [or 5 cm]. — temperature: 35 °C.
Drying In a stream of cold air for about 10 min. Mobile phase:
Detection Treat the plate using anisaldehyde solution R and — mobile phase A: phosphoric acid R, water R (1:999 VIV);
heat at 105 °C for 3 min; examine in daylight. — mobile phase B: acetonitrile R;
shows no greyish-blue zone at tile position of Time Mobile phase B
Mobile phase A
N-isobutyldodecatetraenamide in the chromatogram obtained (min) (per cent V/V) (per cent v/n____
with the reference solution, and no blue zone at the position 0 90 10
of the violet zone due to /^-sitosterol in the chromatogram
0-13 90-» 78 10-» 22
13-14 78-» 60 22-» 40
Maximum 12.0 per cent, determined on 1.000 g of the 14-20 60 40
powdered herbal drug (355) (2.9. /2) by drying in an oven at
105 °C for 2 h.
Total ash {2.4.16)
Maximum 7.0 per cent. Detection spectrophotometer at 330 nm.
Ash insoluble in hydrochloric acid {2.8.1) Injection 10 pL.
Maximum 2.0 per cent. Relative retention With reference to chlorogenic add (retention
time = about 7 min): caftaric add ะ= about 0.8; caffeic
ASSAY
acid = about 1.5; cynarin = about 1.6;
Liquid chromatography (2.2.29). echinacoside = about 1.7; cichoric add = about 2.3.
Test solution In a 100 mL volumetric flask place 0.500 g of System suitability: reference solution:
the powdered herbal drug (355) {2.9.12) and add 80 mL of — resolution: minimum 10 between the peaks due to caffeic
ethanol (70 per cent VIV) R. Sonicate for 15 min and dilute to add and chlorogenic add.
100.0 mL with ethanol (70 per cent VIV) R. Mix the Locate the peaks due to caffeic add and chlorogenic add
suspension and allow to stand for a few minutes to allow using the chromatogram obtained with the reference solution.
visible solids to settle. Filter a suitable proportion of the Locate the peaks due to echinacoside, caftanc add and
solution through a membrane filter (nominal pore size cichoric acid using the chromatogram in Figure 1822.-1.
0.45 pm) before injection.
Calculate the percentage content of echinacoside using the seeds with oil droplets; fragments of the epidermis of ligulate
following expression: florets composed of red to violet papillous cells; spheroidal
pollen grains, 30-40 pm in diameter, with a spiny exine.
Al X c2 X 100 X 2.221
A? X Cl c. Thin-layer chromatography (2.2.27).
A1 = area of the peak due to echinacoside in the (2.9.12) add 10 mL of methanol R and sonicate for 5 min.
chromatogram obtained with the test solution; Centrifuge and use the supernatant solution.
A2 = area of ±e peak due to chlorogenic acid in the Reference solution Dissolve 0.5 mg of caffeic acid R and 0.5 mg
chromatogram obtained with the reference of chlorogenic acid R in 5.0 mL of methanol R.
solution;
Cj = concentration of the test solution, in milligrams plate R (2-10 pm)].
per millilitre;
C2 = concentration of chlorogenic acid in the Mobile phase anhydrous formic acid R} water R, methyl ethyl
reference solution, in milligrams per millilitre; ketone R3 ethyl acetate R (3:3:9:15 V/V/V/V).
2.221 = peak correlation factor between chlorogenic Application 25 pL [or 5 pL] of the test solution and 10 pL
acid and echinacoside. [or 2 pL] of the reference solution, as bands.
STORAGE
Uncomminuted. Drying In a stream of cold air for about 10 min, then at
100 °C for 2 min.
--------------------------------------------------------------------------------------------------------- Ph Eur
diphenylboric acid aminoethyl ester R in ethyl acetate R‘i after
30 min, examine in ultraviolet light at 365 nm.
Echinacea Purpurea Herb * ★ chromatograms obtained with the reference solution and the
test solution. Furthermore, other faint blue fluorescent zones
(Purple Coneflower Herb3 Ph Eur monograph 1823) *** may be present in the chromatogram obtained with the test
Ph Eur______________________________________________________________ solution.
DEFINITION
Dried, whole or cut flowering aerial parts of Echinacea Top of the plate
purpurea (L) Moench. An intense red fluorescent zone
Content Caffeic acid: a strong blue A blue fluorescent zone
Minimum 0.1 per cent for the sum of caftaric acid fluorescent zone
(C13H12O9; 312.2) and cichoric acid (C22H18O12; Mr 474.3)
(dried drug).
IDENTIFICATION
First identification A, B, c
Chlorogenic acid: a strong blue
Second identification A3 B, D fluorescent zone
A. The herbaceous perennial plant is 60-150 cm, rarely up to A faint yellow-orange fluorescent
180 cm high. The stem is green to red, upright and slightly zone
branched. The leaves are alternate, ovate to ovate-lanceolate,
irregularly serrate, rugose on both surfaces, dark green with
prominent light green veins; the lamina is thick and shiny.
The involucral bracts of the large capitulum are arranged in Reference solution Test solution
2 or 3 rows. The solid receptacle is slightly convex. Each of
the outer violet ligulate florets (4-6 cm) and of the inner D. Examine the chromatograms obtained in the assay.
violet-pink tubular florets is attached to a reddish acute and The principal peak in the chromatogram obtained with the
coriaceous bract, which overtops the tubular florets. test solution is due to cichoric acid and a smaller peak is due
The calyx is reduced to a very short crown, one of the sepals to caftaric acid. Peaks due to caffeic acid and chlorogenic
is up to 1 mm long. acid are minor or may be absent.
B. Reduce to a powder (355) {2.9.12). The powder is green.
TESTS
The powder shows ±e following diagnostic characters:
whitish-green groups of fibres, 150-200 pm in length,
10-15 pm in diameter, sometimes with black deposits;
105 °C for 2 h.
fragments of leaves in surface view showing anomocytic or
anisocytic stomata {2.8.3) (about 35-40 pm in length); Total ash {2.4.16)
uniseriate covering trichomes or fragments thereof consisting Maximum 12.0 per cent.
mainly of 3 or 4 thick-walled cells of which the apical cell is ASSAY
markedly longer than the others; fragments of leaves with Liquid chromatography (2.2.29).
rosette-like arranged epidermal cells around the base of the
Test solution In a 100 mL volumetric flask place 0.500 g of
covering trichomes; uniseriate glandular trichomes composed
the powdered herbal drug (355) {2.9.12) and add 80 mL of
of very thin-walled cells; pitted parenchymatous cells from
ethanol (70 per cent V/V) R. Sonicate for 15 min and dilute to
the pith of the stem as well as pitted elongated cells from the
100.0 mL with ethanol (70 per cent V/V) R. Mix the
mesocarp of the achenes; fragments of parenchyma from the
1. caftaric acid 2. chlorogenic acid 3. cichoric acid

Figure 1823.-1. - Chromatogram for the assay of caftaric acid and cichoric acid in purple coneflower herb
suspension and allow to stand for a few minutes to allow Calculate the percentage content of caftaric acid using the
visible solids to setde. following expression:
and 10.0 mg of caffeic acid R in ethanol (70 per cent V/V) R, Al X c2 X 100 X 0.881
sonicate for 15 min and dilute to 10.0 mL with the same A2 X Cl
solvent. Dilute 4.0 mL of this solution to 100.0 mL with
ethanol (70 per cent V/V) R.
Column: Calculate the percentage content of cichoric acid using the
— size: I = 0.25 m, 0 = 4.6 mm; following expression:
(5 pm); A3 X c2 X 100 X 0.695
— temperature: 35 °C. A2 X Cl
Mobile phase:
— mobile phase A: phosphoric acid R, water R (1:999 V/V); Al = area of the peak due to caftaric add in the
— mobile phase B: acetonitrile R; chromatogram obtained with the test solution;
Time Mobile phase A
Mobile phase B
(mln) (per cent V/V) (per cent V/V) solution;
0 90 10 A3 = area of the peak due to cichoric acid in the
0- ]3 90-» 78 10-» 22
Cl = concentration of the test solution, in milligrams
13-14 78-» 60 22-» 40 per millilitre;
14-20 60 40 c2 = concentration of chlorogenic acid in the
reference solution, in milligrams per millilitre;
0.695 = peak correlation factor based upon the liquid
Flow rate 1.5 mL/min. chromatography response observed;
Detection Spectrophotometer at 330 nm. 0.881 = peak correlation factor between caftaric acid
and chlorogenic acid.
Injection 10 |1L.
Relative retention With reference to chlorogenic acid (retention STORAGE
time = about 7 min): caftaric acid = about 0.8; caffeic Uncomminuted.
acid = about 1.5; cynarin = about 1.6; _____________________________________________________________ PhEur
echinacoside = about 1.7; cichoric acid = about 2.3.
— resolution: minimum 5 between the peaks due to caffeic
acid and chlorogenic acid.
Locate the peaks due to caffeic acid and chlorogenic acid
using the chromatogram obtained with the reference solution.
Locate the peaks due to caftaric acid and cichoric acid using
the chromatogram in Figure 1823.-1.
Echinacea Purpurea Root Test solution To 1.0 g of the powdered herbal drug (355)
(2.9.12) add 10 mL of methylene chloride R and sonicate for
(Purple Coneflower Root, Ph Eur monograph 1824) 5 min. Centrifuge and use the supernatant solution.
PhEur________________________________________________ Reference solution Dissolve 1 mg of p-sitosterol R and a volume
of N-isobutyldodecatetraenamide solution R corresponding to
DEFINITION
1 mg of N-isobutyldodecatetraenamide R in 5.0 mL of
Dried, whole or cut underground parts of Echinacea
methanol R.
purpurea (L.) Moench.
Content plate R (2-10 pm)].
Minimum 0.5 per cent for the sum of caftaric acid
Mobile phase anhydrous formic acid R, cyclohexane R, ethyl
(C13H12O9; 312.2) and cichoric acid (C22HI8O12; MT 474.3)
acetate R, toluene R (0 9:3:6:24 VIVIVIV).
(dried drug).
Application 25 pL [or 5 pL], as bands.
IDENTIFICATION
Development Over a path of about 15 cm [or 5 cm].
First identification A, B, c, E
Drying In a stream of cold air for about 10 min.
Second identification AJ B, D, E
Detection Dip the plate into anisaldehyde solution R for 1 ร and
A. . The rhizome is up to 15 cm long, branched, reddish-
heat at 100-105 °C for 3 min; examine in daylight.
brown to dark brown on the surface and carries many stem
bases; the inside is fibrous and white. The numerous roots Results See below the sequence of zones present in the
are spirally twisted, light to dark brown and show a fine cross chromatograms obtained with the reference solution and the
structuring on the surface. test solution. Furthermore, other faint zones may be present
yellow to pinkish-beige. Examine under a microscope using
chloral hydrate solution R. The powder shows the following Top of the plate
diagnostic characters: numerous light-brown spindle-shaped
fibres ±at are joined together in long bundles without black
deposits; rare sclereids from the rhizomes and roots, usually
occuring singly, those from the rhizomes being isodiametric, A bluish-violet zone
about 60 pm in diameter, with black deposits, those from the
roots being 50-120 pm in length with no black deposits; [^Sitosterol ะ a violet or pink zone A violet or pink zone (P-sitosterol)
secretory cavities up to 180 pm in diameter with yellow oil A^-lsobutyldodecatetraenamide: a A greyish-blue zone
droplets; squarish to rectangular cells of the outer layers, greyish-blue zone (Msobutyldodecatetraenamide)
some with reddish walls; bordered-pitted vessels from the
rhizome, 30-40 pm in diameter. A dark greyish-blue zone
c. Examine the chromatogram obtained in the test for other
Echinacea species and Parthenium integrifolium.
chromatograms obtained with the reference solution and the TESTS
test solution. Furthermore, faint greenish fluorescent zones
Other Echinacea species and Parthenium integrifolium
may be present just below the zone situated in the middle of
the chromatogram obtained with the test solution.
(2.9.12) add 10 mL of methanol R and sonicate for 5 min.
Top of the plate Centrifuge and use ±e supernatant solution.
Caffeic acid: a strong blue A strong blue fluorescent zone Reference solution Dissolve 1 mg of echinacoside R, 1 mg of
fluorescent zone cynarin R and 0.5 mg of caffeic acid R in 5.0 mL of
methanol R.
plate R (2-10 pm)].
Cynarin: a strong greenish
fluorescent zone Mobile phase anhydrous formic acid R, water R, methyl ethyl
A blue fluorescent zone ketone R, ethyl acetate R (3:3:9:15 VIVIVIV).
Application 10 pL [or 5 pL] of the test solution and 5 pL [or
2 pL] of the reference solution, as bands.
Echinacoside: a strong greenish
fluorescent zone
Drying In a stream of cold air for about 10 min, then at
105 °C for 2 min.
diphenylboric acid aminoethyl ester R in ethyl acetate R; after
D. Examine the chromatograms obtained in the assay. 30 min, examine in ultraviolet light at 365 nm.
The principal peak in the chromatogram obtained with the Results The chromatogram obtained with the test solution
test solution is due to cichoric acid and a smaller peak is due shows no greenish fluorescent zone corresponding to the
to caftaric acid. Peaks due to caffeic acid and chlorogenic zone due to echinacoside in the chromatogram obtained with
acid are minor or may be absent. the reference solution, and no greenish fluorescent zone
E. Thin-layer chromatography (2.2.27). corresponding to the zone due to cynarin in the
chromatogram obtained with the reference solution. No other
1 A 2 . . .lkJL
2 4 6 8 10 12 14 16 18 min
1. caftaric acid 2. chlorogenic acid 3. cichoric acid
Figure 1824.-1. - Chromatogram for the assay of caftaric acid and cichoric acid in purple coneflower root
zones apart from very faint dark blue fluorescent zones are Time Mobile phase A Mobile phase B
seen in the lower half of the chromatogram of the test (min) (per cent F/n (per cent V/V)
solution. 0 90 10
Foreign matter (2.5.2) 0-13 90 —> 78 10 —> 22

Maximum 3 per cent. 13-14 78 60 22 —> 40
Loss on drying (2.2.52) 14-20 60 40
powdered herbal drug (355) (2.9. 12) by drying in an oven at
105 °C for 2 h. Flow rate 1.5 mL/min.
Total ash (2.4.16) Detection Spectrophotometer at 330 nm.
Maximum 9.0 per cent. Injection 10 |1L.
Ash insoluble in hydrochloric acid (2.8.1) Relative retention With reference to chlorogenic acid (retention
Maximum 2.0 per cent. time ะะะ about 7 min): caftaric acid — about 0.8; caffeic
ASSAY acid = about 1.5; cynarin =ะ about 1.6;
Liquid chromatography (2.2.29). echinacoside = about 1.7; cichoric acid = about 2.3.
Test solution In a 100 mL volumetric flask place 0.500 g of System suitability: reference solution:
— resolution: minimum 5 between the peaks due to caffeic
the powdered herbal drug (355) (2.9.12) and add 80 mL of
ethanol (70 per cent VIV) R. Sonicate for 15 min and dilute to acid and chlorogenic acid.
100.0 mL with ethanol (70 per cent VIV) R. Mix the Locate the peaks due to caffeic acid and chlorogenic acid
suspension and allow to stand for a few minutes to allow using the chromatogram obtained with the reference solution.
visible solids to settle. Locate the peaks due to caftaric acid and cichoric acid using
the chromatogram in Figure 1824.-1.
and 10.0 mg of caffeic acid R in ethanol (70 per cent VIV) R, Calculate the percentage content of caftaric acid using the
sonicate for 15 min and dilute to 10.0 mL with the same following expression:
solvent. Dilute 4.0 mL of this solution to 100.0 mL with
ethanol (70 per cent VIV) R. Al X c2 X 100 X 0.881
Column: Al X Cl
— size: I = 0.25 m, 0 = 4.6 mm;
— stationary phase: octadecylsilyl silica gel for chromatography R Calculate the percentage content of cichoric add using the
(5 pm);
— temperature: 35-°C.
Mobile phase:
A3 X c2 X 100 X 0.695
— mobile phase A: phosphoric acid R3 water R (1:999 VIV)'3
— mobile phase B: acetonitrile R; Al X Cl
IV-176 Eclipta Herb 2016
■^1 = area of the peak due to caftaric acid in the Mobile phase anhydrous formic acid R, acetone R, toluene R
chromatogram obtained with the test solution; (1:6:11 J//P7F).
A2 = area of the peak due to chlorogenic acid in the Application 10 pL as bands of 8 mm.
solution;
Aj = area of the peak due to cichoric acid in the Drying In air.
chromatogram obtained with the test solution; Detection Heat at 100 °C for 5 min, treat die plate whilst still
Cl = concentration of the dried drug in the test hot with a 0.5 per cent v/v solution of diphenylboric acid
solution, in milligrams per millilitre; aminoethyl ester R in ethyl acetate R. Examine in ultraviolet
c2 = concentration of chlorogenic acid in the light at 365 nm.
reference solution, in milligrams per millilitre; Results See below the sequence of zones present in the
0.695 = peak correlation factor based upon the liquid chromatograms obtained with the reference solution and the
chromatography response observed; test solution. Furthermore, other faint fluorescent zones may
0.881 = peak correlation factor between caftaric acid be present in the chromatogram obtained with the test
and chlorogenic acid. solution.
STORAGE
บทcomminuted. Top of the plate
---------------------------------------------------------------------------------------------------------- Ph Eur A red fluorescent zone
A diffuse pale blue fluorescent

zone
A greenish-white fluorescent zone
Eclipta Herb * **
(Ph. Eur. monograph 2564) * ** A pale blue fluorescent zone
Ph Eur_______________________________________________________ ______
DEFINITION
Dried, whole or fragmented, flowering aerial parts of Eclipta Wedelolactone: a bluish-white A bluish-white fluorescent zone
prostrata L. fluorescent zone (wedelolactone)
Content A bluish-white fluorescent zone
Minimum 0.04 per cent of wedelolactone (C16H10O7;
Mt 314.3) (dried drug).
IDENTIFICATION
A. The cylindrical stems are striated longitudinally and are
2-5 mm in diameter. The external surface is brownish-green Rosmarinic acid: a bluish-white
or dark green and bears covering trichomes, flattened against fluorescent zone
the stem and all pointing upwards. The hairy, dark green A bluish-white fluorescent zone
leaves are opposite, always sessile, elongate, lanceolate, with Reference solution Test solution
entire or slightly dentate margins. The capimla are 2-6 mm
in diameter, with whitish flowers that do not extend beyond
the bracts of the involucre. The fruits are elliptical, flattened Loss on drying (2.2.32)
achenes, brown or pale brown, 2-3 mm long. Maximum 11.0 per cent, determined on 1.000 g of the
B. Microscopic examination (2.8.23). The powder is powdered herbal drug (355) (2.9.12) by drying in an oven at
greenish-brown. Examine under a microscope using chloral 105 °C for 2 h.
hydrate solution R. The powder shows the following diagnostic Total ash (2.4.16)
characters: numerous free, whole or broken covering Maximum 13.0 per cent.
trichomes, usually tricellular, up to 700 pm long, with a
broad basal cell, a relatively long median cell with thick and
warty walls, and a very short, pointed, sub-triangular distal
cell; fragments of lamina, some of which have trichomes, ASSAY
with sinuous epidermis cells and anomocytic stomata (2.8.3) Liquid chromatography (2.2.29).
with 3-4 subsidiary cells, often accompanied by palisade Test solution Disperse 0.300 g of the powdered herbal drug
parenchyma; covering trichomes similar to those previously (355) (2.9.12) in 10 mL of ethanol (70 per cent V/V) R in a
described; fragments of stems with different types of vascular conical flask and weigh. Heat under a reflux condenser for
bundles, sometimes accompanied by secretory canals; 1 h, cool and weigh again. Compensate the loss of solvent
bundles of fibres with thickened walls; pollen grains about with ethanol (70 per cent V/V) R, mix well and allow to stand.
20 pm in diameter, with 3 pores and a spiny exine. Filter through a membrane filter (nominal pore size
c. Thin-layer chromatography (2.2.27). 0.22 pm).
Test solution To 0.5 g of the powdered herbal drug (355) Reference solution (a) Dissolve 4.0 mg of wedelolactone CRS in
(2.9.12) add 5 mL of methanol R and sonicate at 60 °C for methanol R and dilute to 100.0 mL with the same solvent.
10 min. Allow to cool, centrifuge and use the supernatant. Reference solution (b) Dissolve 2 mg of ethyl
Reference solution Dissolve 1 mg of wedelolactone R and 1 mg parahydroxybenzoate R in reference solution (a) and dilute to
ofrosmarinic acid R in 1 mL of methanol R. 50 mL with reference solution (a).
2016 Eclipta Prostrata IV-177
Column: main root up to about 7 mm in diameter, with secondary

size: I = 0.15 m, 0 = 4.6 mm; branching.
stationary phase: end-capped octadecylsilyl silica gel for B. Reduce to a powder (355). The powder is greenish
chromatography R (5 pm). brown. Examine under a microscope using chloral hydrate
Mobile phase acetonitrile R, 0.2 per cent VIV solution of solution. The main diagnostic characters include numerous
phosphoric acid R (24:76 VIV). free, whole or broken, large covering trichomes, with warty or
Flow rate 1.0 mL/min. spiny walls, up to 700 pm long, uniseriate, usually tricellular,
Detection Spectrophotometer at 249 nm. with a broad basal cell, a long median cell and a short,
Injection 20 pL. pointed sub-triangular apical cell; less frequently, smaller,
unicellular, pointed covering trichomes from the midrib and
Run time 1.5 times the retention time of wedelolactone. stem. Fragments of leaf show sinuous walled epidermal cells,
Relative retention With reference to wedelolactone (retention underlying palisade, anomocytic stomata, cuticular striations
time = about 17 min): ethyl parahydroxybenzoate ะ- about and covering trichomes on both surfaces. Stem fragments
with unicellular and multicellular trichomes, epidermis of
System suitability: reference solution (b): elongated cells, or in mature stem, poorly developed
resolution: minimum 2.0 between the peaks due to rectangular cork cells; secondary cortex of parenchyma with
wedelolactone and ethyl parahydroxybenzoate. numerous air-spaces, pericyclic fibres thick-walled, lignified,
Calculate the percentage content of wedelolactone using the simple pined; secretory canals may be visible; xylem vessels
following expression: usually simple pitted or spirally thickened, xylem parenchyma
lignified and pitted. Fragments of root, if present, show
poorly developed cork, consisting of 3-5 rows of thin-walled
Al X 7712 X p
elongated cells, a secondary cortex of parenchyma, with
A2 X 7721 X 10 scattered stone cells and fibres either singly or in groups,
xylem vessels and tracheids, and fibres with peg-like
Al = area of the peak due to wedelolactone in the projections. Pollen grains with spiny exine and 3 pores.
chromatogram obtained with the test solution; c. Carry out the method for thin-layer chromatography,
A2 = area of the peak due to wedelolactone in the Appendix in A, using the following solutions.
chromatogram obtained with reference solution (1) Reduce to a powder (355). To 2.0 g of powdered sample
;
(a) add 40 mL of methanol. Mix with the aid of ultrasound for
1,11 = mass of the herbal drug to be examined used to 2 hours at 50° with occasional shaking and allow to cool.
prepare the test solution, in grams; Dilute to 50 mL with methanol and filter.
ทใ2 = mass of wedelolactone CRS used to prepare
(2) 0.05% w/v each of wedelolactone EPCRS and rosmarinic
acid in methanol.
p = percentage content of wedelolactone in
wedelolactone CRS. CHROMATOGRAPHIC CONDITIONS
---------------------------------------------------------------------------------------------------------- Ph Eur
(a) Use as the coating silica gel F254 (Merck silica gel
HPTLC plates are suitable).
(c) Apply 10 pL of each solution as 8 mm bands.
Eclipta Prostrata Whole Plant
(e) After removal of the plate, dry in air, heat at 100° for
DEFINITION 5 minutes, treat the plate whilst still hot with a 0.5% v/v
Eclipta Prostrata Whole Plant is the dried whole plant, either solution of diphenylboric acid aminoethyl ester in ethyl acetate
entire or fragmented, of Eclipta prostrata (L.) L. and examine under ultraviolet light (365 nm).
It contains not less than 0.04% of wedelolactone MOBILE PHASE
(C16H10O7), calculated with reference to the dried material.
I volume of anhydrous formic acid, 6 volumes of acetone and
IDENTIFICATION II volumes of toluene.
A. Stems cylindrical, four sided or flattened, 2 to 5 mm in SYSTEM SUITABILITY
diameter, greyish, with appressed, whitish hairs pointing
towards the tip, longitudinally striated, occasionally
solution (2) shows two clearly separated spots.
branching and nodes distinct. Leaves dark green, sessile or
subsessile, opposite, lanceolate, 2 to 8.5 cm long and 1 to
2.5 cm wide with an entire or slightly dentate margin and Top of the plate
appressed trichomes on both surfaces. Flowerheads 2 to A red zone
6 mm in diameter, greenish-brown, solitary or in pairs on A diffuse pale blue zone
unequal axillary peduncles, up to 8 involucral bracts, ovate, A greenish-white zone
with appressed hairs; ray florets spreading, no longer than the A pale blue zone
bracts, not toothed; disc florets with 4-toothed corolla,
pappus usually absent or reduced to minute teeth; 5 stamens,
A blue-white zone (wedelolactone) A blue-white zone (wedelolactone)
filaments epipetalous and anthers united into a tube; pistil
A blue-white zone
bicarpellary, ovary inferior, unilocular with one basal ovule.
Fruits 2 to 3 mm long, pappi persistent and coroniform;
unfertilised achenes pale yellow, flattened and smooth; Rosmarinic acid: A blue-white zone
A blue-white zone
fertilised achenes pale to dark brown, 3 to 4 angled, _______Solution (2)_______
Solution (1)
tuberculate and bulbous. Root, if present, cylindrical, greyish,
IV-178 Elder Flower 2016
CONFIRMATION p = percentage content of wedelolactone

The chromatogram obtained with solution (1) shows a (C16Hjo07) in wedelolactone EPCRS;
fluorescent band corresponding to wedelolactone and several d = percentage loss on drying of the herbal drug being
other fluorescent bands as shown in the table. Other examined.
fluorescent bands may be present.
TESTS
Loss on drying
When dried at 100° to 105c for 2 hours, loses not more than Elder Flower * **
11.0% of its weight. Use 1 g.
Total Ash
Not more than 22.0%, Appendix XI J, Method n. PhEur.______________________________________________________________
Acid-insoluble Ash DEFINITION

Not more than 11.0%, Appendix XI K. Dried flowers of Sambucus nigra L.
ASSAY Content
Carry out the method for liquid chromatography3 Minimum 0.80 per cent of flavonoids, expressed as
Appendix JU D, using the following solutions. isoquercitroside (C21H20012; Mr 464.4) (dried drug).
(1) Reduce to a powder (355). To 2.0 g of powdered sample IDENTIFICATION
add 40 mL of methanol. Mix with the aid of ultrasound for A. The flower, about 5 mm in diameter, has 3 small bracts,
2 hours at 50° with occasional shaking and allow to cool. visible under a lens, and may have a peduncle.
Dilute to 50 mL with methanol and filter. The 5-toothed calyx is small; the corolla is light yellow, with
(2) 0.005% w/v each of wedelolactone EPCRS and coumestrol 5 broadly oval petals fused at their bases into a tube.
in methanol. The filaments of the 5 yellow stamens alternate with the
petalร. The corolla is often isolated or attached to the
stamens, to which it is fused at the base. The ovary is inferior
(a) Use a stainless steel column (15 cm X 4.6 mm) packed and it bears a short style with 3 obtuse stigmata.
with octadecylsilyl silica gel for chromatography (5 pm) (Waters
Symmetry Cl8 is suitable).
greenish-yellow. Examine under a microscope using chloral
(b) Use isocratic elution and the mobile phase described hydrate solution R. The powder shows the following diagnostic
below. characters (Figure 1217.-1): numerous spherical, sometimes
(c) Use a flow rate of 1.0 mL per minute. ellipsoidal, pollen grains about 30 pm in diameter, with
(d) Use an ambient column temperature. 3 germinal pores and very finely pitted exine [G]; cells of the
(e) Use a detection wavelength of 249 nm. lower epidermis of the sepals often containing oil globules
and covered by a striated cuticle in surface view [A]; rare
fragments of the rim of the sepals showing unicellular
MOBILE PHASE marginal teeth, in transverse section [E]; petal fragments with
24 volumes of acetonitrile and 76 volumes of a 0.2% v/v numerous small globules of essential oil [H]; fragments of
solution of orthophosphoric acid. upper epidermis of the sepals [B] or petals [F], in surface
SYSTEM SUITABILITY
view, with slightly and irregularly thickened walls [Ba, Fa],
anomocytic stomata (2.8.3) [Bb, Fb] and a striated cuticle;
The assay is not valid unless, in the chromatogram obtained
mesophyll cells of petals and sepals with idioblasts containing
with solution (2):
numerous microsphenoid crystals of calcium oxalate [Be];
the symmetry factor of the peak due to wedelolactone is less fragments of anthers in transverse section [C] and in surface
than 1.2; view [D], showing the outer layer [Ca] and the cells of the
the symmetry factor of the peak due to coumestrol is less than fibrous layer [Cb, Cc, D].
DETERMINATION OF CONTENT Test solution To 0.5 g of the powdered herbal drug (355)
Calculate the content of wedelolactone in the sample using (2.9.12) add 5 mL of methanol R and sonicate for 10 min.
the declared content of wedelolactone (C16H10O7) in Centrifuge for 5 min.
wedelolactone EPCRS and the following expression: Reference solution Dissolve 1 mg of caffeic acid R3 1 mg of
chlorogenic acid R, 2.5 mg of hyperoside R and 2.5 mg of
rutin R in 10 mL of methanol R.
Al m2 Vi 100 Plate TLC silica gel plate R (2-10 pm).
Az Vz mi p 100-d Mobile phase anhydrous formic acid R, water R3 methyl ethyl
ketone R3 ethyl acetate R (10:10:30:50 VIVIVIV).
A1 = area of the peak due to wedelolactone in the
chromatogram obtained with solution (1); Drying In air.
A2 = area of the peak due to wedelolactone in the Detection Heat the plate for 5 min at 100 °C and treat with a
chromatogram obtained with solution (2); 1 g/L solution of diphenylboric acid aminoethyl ester R in ethyl
m1 = weight of the herbal drug being examined in mg; acetate R3 then treat with a 5 g/L solution of macrogol 400 R
m2 = weight of wedelolactone EPCRS in mg; in methylene chloride R'3 allow to dry in air for 30 min.
V1 ะ= dilution volume of solution (1) in mL; Examine in daylight (results A) and in ultraviolet light at
v2 = dilution volume of solution (2) in mL; 365 nm (results B).
2016 Elder Flower IV-17 9
Top of the plate
Caffeic acid: a blue fluorescent

zone
An intense, light blue fluorescent
2 light blue fluorescent zones
Hyperoside ะ an orange fluorescent

zone
Chlorogenic acid: a light blue An intense, light blue fluorescent
fluorescent zone zone
Rutin: an orange fluorescent zone An orange fluorescent zone
TESTS
Maximum 8 per cent of fragments of coarse pedicels and
other foreign matter and maximum 15 per cent of
discoloured, brown flowers. Carry out the determination on
10 g.
Sambucus ebulus L
Figure 1217.-1. - Illustration for identification B of powdered Examine the chromatograms obtained in identification c.
herbal drug of elder flower
Results A See below the sequence of zones present in the does not show a greenish-white zone above the zone due to
chromatograms obtained with the reference solution and the caffeic acid in the chromatogram obtained with the reference
test solution. Fur±ermore, other faint zones may be present solution; in the chromatogram obtained with the test
in the chromatogram obtained with the test solution. solution, no green fluorescent zone is seen just below the
orange fluorescent zone due to rutin in the chromatogram
Top of the plate obtained with the reference solution.
An orange zone 105 °C for 2 h.
Total ash (2.4.16)
Hyperoside: a dark yellow zone
ASSAY
Stock solution In a 100 mL round-bottomed flask, introduce
0.600 g of the powdered herbal drug (355) (2.9.72), add
1 mL of a 5 g/L solution of hexamethylenetetramine Rs 20 mL
Rutin: a dark yellow zone A dark yellow zone of acetone R and 2 mL of hydrochloric acid Rl. Boil the
mixture under a reflux condenser for 30 min. Filter the
mixture through a plug of absorbent cotton into a flask.
Add the absorbent cotton to the residue in the round-
bottomed flask and extract with 2 quantities, each of 20 mL,
Results B See below the sequence of zones present in the of acetone R} each time boiling under a reflux condenser for
10 min. Allow to cool, filter each extract through the plug of
test solution. Furthermore, other faint zones may be present absorbent cotton into the flask. After cooling, filter the
combined acetone extracts through a filter paper into a
volumetric flask and dilute to 100.0 mL with acetone R by
rinsing the flask and the filter paper. Introduce 20.0 mL of
this solution into a separating funnel, add 20 mL of water R
and shake the mixture with 1 quantity of 15 mL and then
3 quantities, each of 10 mL, of ethyl acetate R. Combine the
ethyl acetate extracts in a separating funnel, wash with
2 quantities, each of 50 mL, of water R) and filter the
IV-180 Eleutherococcus 2016
extracts over 10 g of anhydrous sodium sulfate R into a c. Thin-layer chromatography (2.2.27).

volumetric flask and dilute to 50.0 mL with ethyl acetate R. Test solution To 1.0 g of the powdered herbal drug (355)
Test solution To 10.0 mL of the stock solution add 1 mL of (2.9.72) add 10 mL of alcohol (50 per cent V/V) R and boil
aluminium chloride reagent R and dilute to 25.0 mL with a under reflux for 1 h. Cool and filter. Evaporate the filtrate to
5 per cent v/v solution of glacial acetic acid R in methanol R. dryness on a water-bath. Dissolve the residue in 2.5 mL of a
Compensation liquid Dilute 10.0 mL of the stock solution to mixture of 5 volumes of water R and 20 volumes of alcohol
25.0 mL with a 5 per cent v/v solution of glacial acetic (50 per cent V/V) R and filter.
acid R in methanol R. Reference solution Dissolve 2.0 mg of esculin R and 2.0 mg of
After 30 min, measure the absorbance (2.2.25) of the test catalpol R in 20 mL of a mixture of 2 volumes of water R and
solution at 425 nm, by comparison with the compensation 8 volumes of alcohol (50 per cent V/V) R.
liquid. Plate TLC silica gel plate R.
Calculate the percentage content of flavonoids, expressed as Mobile phase water Ry methanol Ry methylene chloride R
isoquercitroside, using ±e following expression: (4:30:70 V/V/V).
A X 1.25 Development Over a path of 10 cm.
m Drying In air.
i.e. taking the specific absorbance of isoquercitroside to
be 500. Results A The chromatogram obtained with the reference
A = absorbance at 425 nm; solution shows in the upper half a blue fluorescent zone
m = mass of the herbal drug to be examined, in grams. (esculin).
Detection B Spray with anisaldehyde solution R and examine in
______________________________________________________________ Ph Eur
daylight while heating at 100-105 °C for 5-10 min.
Results B See below the sequence of the zones present in the
test solution. Furthermore, other faint zones are present in
Eleutherococcus * * the chromatogram obtained with the test solution.
Siberian Ginseng ***
Top of the plate
A brown zone (eleutheroside B)
Ph Ell______________________________________________________________
Esculin: a blue fluorescent zone
DEFINITION (marked at 365 nm)
Dried, whole or cut underground organs of Eleutherococcus A reddish-brown zone
senticosus (Rupr. et Maxim.) Maxim. (eleutheroside E)
Content
Minimum 0.08 per cent for the sum of eleutheroside B (Mr Catalpol: a violet-brown zone
372.4) and eleutheroside E (Mr 742.7).
IDENTIFICATION
2 brown zones
A. The rhizome is knotty, of irregular cylindrical shape,
1.5 cm to 4.0 cm in diameter; the surface is rugged, Reference solution Test solution
longitudinally wrinkled and greyish-brown to blackish-brown;
the bark, about 2 mm thick, closely adheres to the xylem;
the heartwood is light brown and the sapwood is pale yellow; TESTS
the fracture shows short thin fibres in the bark and is coarsely Foreign matter (2.5.2)
fibrous, especially in the internal part of the xylem. Maximum 3 per cent.
The lower surface bears numerous cylindrical and knotty Loss on drying (2.2.22)
roots, 3.5 cm to 15 cm long and 0.3 cm to 1.5 cm in Maximum 10.0 per cent, determined on 1.000 g of the
diameter; with a smooth, greyish-brown to blackish-brown powdered herbal drug (355) (2.9.72) by drying in an oven at
surface; the bark is about 0.5 mm thick, closely adhering to 105 °C for 2 h.
the pale yellow xylem; the fracture is slightly fibrous;
Total ash {2.4.16)
in places where the outer layer has been removed, the outer
surface is yellowish-brown.
B. Reduce to a powder (355) (2.9.72). The powder is ASSAY
yellowish-brown. Examine under a microscope, using chloral Liquid chromatography (2.2.29).
hydrate solution R. The powder shows numerous groups of Test solution To 0.500 g of the powdered herbal drug (355)
thick-walled, lignified fibres; fragments of reticulate and (2.9.72) in a 100 mL round-bottomed flask, add 30 mL of a
bordered pitted vessels with a wide lumen; groups of mixture of equal volumes of alcohol R and water R. Heat in a
secretory canals, up to 20 pm in diameter with brown water-bath at 60 °C for 30 min. Allow to cool and filter
contents; parenchjonatous cells containing cluster crystals of through a sintered-glass filter (2.7.2). Collect the liquid in a
calcium oxalate 10 pm to 50 pm in diameter. Examine under 250 mL round-bottomed flask. Repeat this operation twice,
a microscope, using a 50 per cent v/v solution of glycerol R. using the residue obtained in the filtration step instead of the
The powder shows small starch granules, rounded to slightly powdered herbal drug. Add both fractions of supernatant to
angular in outline, single compounds or with 2 or the 250 mL round-bottomed flask. Evaporate under reduced
3 components. pressure until about 10 mL of supernatant is left in the flask.
2016 Eleutherococcus IV-181
Figure 1419.-1. - JJV spectrum of eleutheroside B for the assay

of eleutherococcus
Transfer the supernatant quantitatively to a

20.0 mL volumetric flask and dilute to 20.0 mL with a
mixture of equal volumes of alcohol R and water R. Filter
through a nylon filter (pore size 0.45 pm).
Reference solution (a) Dissolve 10 mg of ferulic acid R in a
mixture of equal volumes of methanol R and water R and Figure 1419.-2. - uv spectrum of eleutheroside E far the assay
dilute to 20.0 mL with the same mixture of solvents. of eleutherococcus
Reference solution (b) Dissolve 10 mg of caffeic acid R in a
mixture of equal volumes of methanol R and water R and — stationary phase: octadecylsUyl silica gel for chromatography R
dilute to 20.0 mL with the same mixture of solvents. (5 pm).
Reference solution (c) Transfer 1 mL of reference solution (a) Column:
to a 25 mL volumetric flask and dilute to 25.0 mL with a — size: I = 0.25 m, 0 = 4.6 mm,
mixture of equal volumes of methanol R and water R. Filter — stationary phase: octadecylsilyl silica gel for chromatography R
through a nylon filter (pore size 0.45 pm). (5 pm).
Reference solution (d) Transfer 1 mL of reference solution (a) Mobile phase:
and 1 mL of reference solution (b) in a mixture of equal — mobile phase A: phosphoric acid R> water R (0.5:99.5 P7F),
volumes of methanol R and water R and dilute to 25.0 mL — mobile phase B: acetonitrile for chromatography R3
with the same mixture of solvents. Filter through a nylon
filter (pore size 0.45 pm).
Precolumn:
— size: l ะะะ 4 mm, 0 = 4.6 mm,
IV-182 Ephedra Herb 2016
Time Mobile phase A Mobile phase B hydrate solution R. The powder shows the following diagnostic
(min) (per cent V/V) (per cent V/V) characters: fragments of the epidermis, in surface view,
0-5 90 10 composed of rectangular cells and numerous stomata with a
5 - 27 90 -> 80 10 -> 20 small depression at each end, the guard cells large and
27 - 30
broadly elliptical; epidermal fragments, in transverse section,
80 -» 50 20 -> 50
showing a thick cuticle and some of the cells extended to
30 - 35 50 50 form projections; fibres in groups or single, with thick,
usually lignified walls; fragments of lignified tissue composed
of small, bordered-pitted tracheids, vessels with spiral
thickening and groups of sclereids; groups of parenchyma,
Flow rale 1.0 mUmin. some with thickened and pitted walls; scattered prism crystals
Detection Spectrophotometer at 220 nm. of calcium oxalate.
Injection 20 pL of the test solution and reference solutions (c) c. Thin-layer chromatography (2.2.27).
and (d). Test solution To 0.2 g of the powdered herbal drug (355)
Retention time Eleutheroside B = about 10 min; (2.9.12) add 0.5 mL of concentrated ammonia R and 10 mL of
eleutheroside E = about 22 min. methylene chloride R. Boil in a water-bath under a reflux
Locate the peaks due to eleutheroside B and eleutheroside E condenser for 1 h. Allow to cool, filter and evaporate the
using the uv spectra shown in Figures 1419.-1 and 1419.-2. filtrate to dryness; dissolve the residue in 2 mL of
System suitability Reference solution (d): methanol R.
— resolution: minimum 15 between the peaks due to caffeic Reference solution Dissolve 1 mg of ephedrine hydrochloride CRS
acid and ferulic acid. and 1 mg of 2-indanamine hydrochloride R in 2 mL of
Calculate the total percentage content of eleutheroside B and methanol R.
eleutheroside E from the expression: Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
plate R (2-10 pm)].
Mb X c X 0.73 X 2) (Ê X c X 1,90 X 2) Mobile phase concentrated ammonia Ry methanol R, methylene
(j4r X m) (j4r X m) chloride R (0.5:5:20 P7P7F).
Application 10 pL [or 1 pL] as spots with a diameter of 5 mm
Ab = area of the peak due to eleutheroside B in the [or 2 mm].
chromatogram obtained with the test solution, Development Over a path of 10 cm [or 6 cm].
/4e = area of the peak due to eleutheroside E in the Drying In air.
chromatogram obtained with the test solution,
Detection Spray with a 2 g/L solution of ninhydrin R in ethanol
Ar = area of the peak due to ferulic acid in the
(96 per cent) Ry heat at 110 °C for 10 min and examine
immediately in daylight.
(c),
c = concentration of ferulic acid in reference Results See below the sequence of spots present in the
solution (c), in micrograms per millilitre, chromatograms obtained with the reference solution and the
m = mass of the herbal drug to be examined, in test solution. Furthermore, other faint spots may be present
milligrams. in the chromatogram obtained with the test solution.
_______________________________________________________________ Ph Eur
Top of the plate
2-Indanamine: a purple spot
Ephedra Herb * * A purple spot may be present

PhEur_______________________________________________________________ Ephedrine: a purple spot at the A purple spot (ephedrine) at the
border between the middle and border between the middle and
DEFINITION lower thirds lower thirds
Dried herbaceous stem of Ephedra sinica Stapf, Ephedra Reference solution Test solution
intermedia Schrenk et C.A.Mey. or Ephedra equisetina Bunge.
Content
Minimum 1.0 per cent of ephedrine (C10H15NO; Mr 165.2) TESTS
(dried drug). Loss on drying (2.2.32)
IDENTIFICATION powdered herbal drug (355) (2.9.12) by drying in an oven at
A. Thin cylindrical pale green or yellowish-green stems up to 105 °C for 2h.
30 cm long and 1-3 mm in diameter; longitudinally striated
Total ash (2.4.16)
and slightly rough; internodes varying in length between
1 cm and 6 cm; opposite and decussate leaves reduced to
sheaths surrounding the stem, carrying diminutive laminae ASSAY
1.5-4 mm long with 2 lobes (rarely 3), acutely triangular, Liquid chromatography (2.2.29).
apex greyish-white, base tubular and reddish-brown or Test solution To 0.200 g of the powdered herbal drug (355)
blackish-brown. Fracture slightly fibrous. (2.9.12) add 25.0 mL of methanol Ry weigh and sonicate for
B. Reduce to a powder (355) (2.9.12). The powder is 45 min. Allow to cool, weigh and adjust to the original mass
greenish-yellow. Examine under a microscope using chloral with methanol Ry shake well and filter. Transfer 1.0 mL of the
2016 Eucalyptus Leaf IV-183
filtrate to a small column (1 cm in diameter) packed with IDENTIFICATION

1.50 g of neutral aluminium oxide R (60-210 pm). Elute with A. The leaves, which are mainly greyish-green and relatively
a mixture of equal volumes of methanol R and water R. thick, are elongated, elliptical and slightly sickle-shaped and
Collect about 9 mL of the eluate, add 0.5 mL of phosphoric usually up to 25 cm in length and up to 5 cm in width.
acid R and dilute to 10.0 mL with a mixture of equal The petiole is twisted, strongly wrinkled and is 2-3 cm, rarely
volumes of methanol R and water R. 5 cm, in length. The coriaceous, stiff leaves are entire and
Reference solution (a) Dissolve 10.0 mg of ephedrine glabrous and have a yellowish-green midrib. Lateral veins
hydrochloride CRS in methanol R and dilute to 100.0 mL with anastomose near the margin to a continuous line.
the same solvent. Dilute 2.0 mL of the solution to 25.0 mL The margin is even and somewhat thickened. On both
with the mobile phase. surfaces there are minute, irregularly distributed, warty, dark
Reference solution (b) Dissolve 1 mg of ephedrine brown spots. Small oil glands may be seen in transmitted
hydrochloride CRS and 1 mg of terbutaline sulfate CRS in light.
methanol R and dilute to 10 mL with the same solvent. B. Microscopic examination (2.8.23). The powder is greyish-
Dilute 2 mL of the solution to 25 mL with the mobile phase. green. Examine under a microscope using chloral hydrate
Column-. solution R. The powder shows the following diagnostic
— size’. I = 0.25 m, 0 = 4.6 mm; characters (Figure 1320.-1): fragments of glabrous lamina
stationary phase-, octadecylsilyl silica gel for chromatography R (surface view [A, L], transverse section [F, H]), with small,
(5 pm). thick-walled epidermal cells bearing a thick cuticle [Fa, Ha],
numerous anomocytic stomata (2.8.3) greater than 80 pm in
Mobile phase acetonitrile Rly 0.1 per cent FZF solution of
diameter [Aa, La] with occasional groups of brown cork cells,
phosphoric acid R (15:85 V/V).
300 pm in diameter and brownish-black in their centre, and
Flow rate 2.0 mLAnin.
underlying palisade parenchyma [Ab, Fb]; fragments of
Detection Spectrophotometer at 207 nm. bilateral mesophyll (side view [G]), with 2-3 layers of
Injection 10 pL. palisade parenchyma [Ga] on each side and in the centre
Run time 3 times the retention time of ephedrine. several layers of spongy mesophyll [Gb] with elongated cells
System suitability-, reference solution (b): having the same orientation as the palisade cells and
containing prisms [B, Gd] and cluster crystals of calcium
— resolution-, minimum 3.5 between the peaks due to
terbutaline and ephedrine. oxalate [Gc, K]; large schizogenous oil glands, whole [E] or
usually broken, accompanied by palisade parenchyma [Ea];
Calculate the percentage content of ephedrine using the fragments of vessels [J] and thick-walled and slightly
following expression: channelled fibres [C] accompanied by crystal sheaths [Ca,
Ja]; crystal sheaths containing prisms of calcium oxalate [D].
Al X 7712 X p X 165.2
A2 X 7721 X 5 X 201.7
/41 = area of the peak due to ephedrine in the

A2 = area of the peak due to ephedrine in the
(a);
m2 = mass of ephedrine hydrochloride CRS used to
prepare reference solution (a), in grams;
p = percentage content of ephedrine hydrochloride in
ephedrine hydrochloride CRS.
_______________________________________________________________ Ph Eur
Eucalyptus Leaf * t
Ph Eur______________________________________________________________ _
DEFINITION
Whole or cut, dried leaves of older branches of Eucalyptus
globulus Labill.
Essential oil content.
— for the whole drug, minimum 20 mL/kg (anhydrous
drug);
— for the cut drug, minimum 15 mI7kg (anhydrous drug). Figure 1320.-1. - Illustration for identification test B of powdered
CHARACTERS herbal drug of eucalyptus leaf
Aromatic odour of cineole.
IV-184 Eucalyptus Oil 2016
Test solution Shake 0.5 g of the freshly powdered herbal drug Second identification A
(355) (2.9.72) with 5 mL of toluene R for 2-3 min and filter A. Thin-layer chromatography (2.2.27).
over about 2 g of anhydrous sodium sulfate R.
Test solution Dissolve 0.1 g of the essential oil to be examined
Reference solution Dissolve 50 pL of cineole R in toluene R and in toluene R and dilute to 10 mL with the same solvent.
Reference solution Dissolve 20 pL of a-terpineol R and 50 pL of
Plate TLC silica gel plate R. cineole R in toluene R and dilute to 5 mL with the same
Mobile phase ethyl acetate R, toluene R (10:90 V!V). solvent.
Application 10 pL as bands. Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
Development Over a path of 15 cm. plate R (2-10 pm)].
Drying In air. Mobile phase ethyl acetate R, toluene R (10:90 V/V).
Detection'. Treat with anisaldehyde solution R and heat at Application 10 pL [or 2 pL] as bands of 10 mm [or 6 mm].
100-105 °C for 10-15 min; examine in daylight. Development Over a path of 15 cm [or 6 cm].
Residts The chromatogram obtained with the reference Drying In air.
solution shows in the middle a zone due to cineole. Detection Spray with anisaldehyde solution R and heat at
The chromatogram obtained with the test solution shows a 100-105 °C for 5-10 min; examine in daylight.
principal zone similar in position and colour to ±e zone due
to cineole in the chromatogram obtained with the reference
solution, it also shows an intense violet zone (hydrocarbons)
near the solvent front and there may also be other fainter
in the chromatogram obtained with the test solution, near the
zones.
solvent front and at the level of a-terpineol.
TESTS
Top of he plate
Maximum 3 per cent of dark and brown leaves, maximum
5 per cent of stems and maximum 2 per cent of other foreign — —
matter. Cordate or ovate sessile leaves of young branches, 1,8-Cineole: a violet-brown zone An intense violet-brown zone
with numerous glands on both sides, visible as points in (1,8-cineole)
transmitted light, are not present. Carry out the
determination using 30 g of the herbal drug to be examined.
a-Terpineol: a violet-brown zone
Water {2.2.13)
Maximum 100 mL/kg, determined on 20.0 g of the Reference solution Test solution
powdered herbal drug (355) {2.9.12).
Total ash {2.4.16) B. Examine the chromatograms obtained in the test for
Maximum 6.0 per cent. chromatographic profile.
ASSAY Results The characteristic peaks due to a-pinene, P-pinene,
Essential oil {2.8.12) a-phellandrene, limonene and 1,8-cineole in the
Use 10.0 g of the herbal drug, cut immediately before chromatogram obtained with the test solution are similar in
determination, a 500 mL round-bottomed flask, 200 mL of retention time to those in the chromatogram obtained with
water R and 100 mL of glycerol R as the distillation liquid and reference solution (a). Sabinene and camphor may be present
0.5 mL of xylene R in the graduated tube. Distil at a rate of in the chromatogram obtained with the test solution.
2-3 mLAnin for 2 h. TESTS
________________________________________________________________Ph Eur Relative density (2.2.5)
0.906 to 0.927.
1.458 to 1.470.
Eucalyptus Oil * * Optical rotation (2.2.7)
0° to + 10°.
Solubility in alcohol {2.8.10)
Ph Eur _----------------------------------------------------------------------------------------------------------- It is soluble in 5 volumes of ethanol (70 per cent V/V) R.
DEFINITION Aldehydes
Essential oil obtained by steam distillation and rectification To 10 mL in a ground-glass-stoppered tube 25 mm in
from the fresh leaves or the fresh terminal branchlets of diameter and 150 mm long, add 5 mL of toluene R and 4 mL
various species of Eucalyptus rich in 1,8-cineole. The species of alcoholic hydroxylamine solution R. Shake vigorously and
mainly used are Eucalyptus globulus Labill., Eucalyptus titrate immediately with 0.5 M potassium hydroxide in alcohol
polybractea R.T.Baker and Eucalyptus smithii R.T.Baker. (60 per cent V/V) until the red colour changes to yellow.
CHARACTERS Continue the titration with shaking; the end-point is reached
when the pure yellow colour of the indicator is permanent in
Appearance
the lower layer after shaking vigorously for 2 min and
Colourless or pale yellow liquid.
allowing separation to take place. The reaction is complete in
Odour about 15 min. Repeat the titration using a further 10 mL of
Reminiscent of 1,8-cineole. the substance to be examined and, as a reference solution for
IDENTIFICATION the end-point, the titrated liquid from the 1st determination
First identification B to which has been added 0.5 mL of 0.5 M potassium
2016 Eucommia Bark IV-185
hydroxide in alcohol (60 per cent VIV). Not more than 2.0 mL
Qi 0.5 M potassium hydroxide in alcohol (60 per cent VIV) is Eucommia Bark
required in the 2nd titration.
Chromatographic profile PhEur______________________
procedure. DEFINITION
Test solution Dissolve 200 jiL of the essential oil to be Whole or fragmented, scraped, dried bark of the stem of
Eucommia ulmoides Oliv.
examined in heptane R and dilute to 10.0 mL with the same
solvent. Content
Reference solution (a) Dissolve 10 pL of ซ.-pinene R, 5 pL of fl- Minimum 0.10 per cent of pinoresinol diglucoside
pinene R, 5 pL of sabinene R, 5 pL of ซ.-phellandrene R, 10 pL (C32H42016; Afr 683) (dried drug).
Qi limonene R, 50 pL of cineole R and 5 mg of camphor R in IDENTIFICATION
heptane R and dilute to 10 mL with the same solvent. A. Pieces are flat, curved or channelled, varying in size, about
Reference solution (b) Dissolve 5 pL of limonene R in heptane R 3-7 mm thick. The outer surface is pale brown or greenish-
and dilute to 50.0 mL with the same solvent. Dilute 0.5 mL brown, markedly wrinkled or fissured, sometimes with
of the solution to 5.0 mL with heptane R. intentional scarring in a rhombus shape; some barks show
Column'. lenticels. The inner surface is dark reddish-brown or dark
— material', fused silica; purplish-brown, smooth to the touch. The texture is fragile,
— size-. / = 60 m, 0 = about 0.25 mm; easily broken, with the edges of the fracture connected by
— stationary phase', macrogol 20 000 R (film thickness fine, dense, silvery and elastic rubber threads.
0.25 pm). B. Microscopic examination (2.5.25). The powder is
Carrier gas helium for chromatography R. brownish. Examine under a microscope using chloral hydrate
characters: many ribbon-shaped latex fragments with a
Split ratio 1:50. granular surface, twisted or folded back on themselves;
Temperature'. numerous sclereids up to 180 pm long and 20-80 pm in
diameter, isolated or mostly in groups, with very thick and
Time Temperature markedly channelled walls, some sclereids have masses of
(min) (°C) latex in their lumen; fibres with very narrow lumens, usually
Column 0-5 60 associated in groups with sclereids; fragments of hard cork
with cells that are polygonal and about 15-40 pm in diameter
5 - 33 60 -> 200
in surface view and rectangular in transverse section, showing
33 - 38 200 walls that are irregularly thickened and with fine pits on 3
Injection port 220 sides and thin on the outer side; ovoid parenchyma cells.
Detector
c. To 1.0 g of the powdered herbal drug (355) (2.9.12) add
220
10 mL of methylene chloride R and allow to stand for 2 h.
Filter and evaporate the filtrate to dryness. Take up the
Detection Flame ionisation. residue with 1.0 mL of anhydrous ethanol R'i an elastic film is
Injection 1 pL. formed.
Elution order Order indicated in the composition of reference D. Thin-layer chromatography (2.2.27).
solution (a). Record the retention times of these substances. Test solution To 1 g of the powdered herbal drug (355)
System suitability: reference solution (a): (2.9.12) add 10 mL of methylene chloride R. Sonicate for
— resolution: minimum 1.5 between the peaks due to 10 min. Discard the liquid phase and repeat the extraction
limonene and cineole. with another 10 mL of methylene chloride R. Discard the
liquid phase again. Dry the residue in air. Add 7 mL of
methanol R. Sonicate in a centrifuge tube at 60 °C for
determined from the chromatogram obtained with reference
20 min. Centrifuge; use the supernatant.
solution (a), locate the components of reference solution (a)
in the chromatogram obtained with the test solution. Reference solution Dissolve 2 mg of 5,7-dihydroxy-4-
methylcoumarin R and 20 mg of p-sitosterol R in 10 mL of
methanol R.
components. The percentages are within the following
ranges: Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
— ซ-pinene: 0.05 per cent to 10.0 per cent; plate R (2-10 pm)].
— f-pinene: 0.05 per cent to 1.5 per cent; Mobile phase anhydrous formic acid R, ethyl acetate R, toluene R
— sabinene: maximum 0.3 per cent; (1:35:65 VIVIV).
— ซ-phellandrene: 0.05 per cent to 1.5 per cent; Application 20 pL [or 10 pL] as bands of 10 mm [or 8 mm].
— limonene: 0.05 per cent to 15.0 per cent; Development Over a path of 10 cm [or 6 cm].
— 1,8-cineole: minimum 70.0 per cent;
Drying In air.
— camphor, maximum 0.1 per cent;
Detection Treat with anisaldehyde solution R and heat at
— disregard limit: the area of the principal peak in the
100-105 °C for 5 min; examine in daylight.
chromatogram obtained with reference solution (b)
(0.05 per cent). Results See below the sequence of zones present in the
storage test solution. Furthermore, other faint zones may be present
At a temperature not exceeding 25 °C. in the chromatogram obtained with the test solution.
--------- ------------------------------------------------------------------------------------------------- PhEur
IV-186 Bitter Fennel 2016
Top of the plate Identification of peaks Use the chromatogram supplied with
A violet zone
eucommia bark HRS and the chromatogram obtained with
reference solution (a) to identify the peak due to pinoresinol
diglucoside and peak 2.
A violet zone Retention time Caffeine ะ= about 8 min; pinoresinol
P-Sitosterol: a blue zone A violet zone
diglucoside = about 10 min.
— resolution: minimum 2.0 between the peak due to
A violet zone pinoresinol diglucoside and peak 2.
5,7-Dihydroxy-4-
Calculate the percentage content of pinoresinol diglucoside
methylcoumarin ะ an orange using the following expression:
Several zones Al X 7712 X 5.6 X p

Several zones A2 X mi X 50
A1 = area of the peak due to pinoresinol diglucoside in

Loss on drying (2.2.32) the chromatogram obtained with the test solution;
Maximum 12.0 per cent, determined on 1.000 g of the A2 = area of the peak due to caffeine in the
powdered herbal drug (355) (2.9.12) by drying in an oven at chromatogram obtained with reference solution
105 °C. (b);
Total ash (2.4.16) prepare the test solution, in grams;
Maximum 10.0 per cent. m2 = mass of caffeine CRS used to prepare reference
ASSAY solution (b), in grams;
Liquid chromatography (2.2.29). p = percentage content of caffeine in caffeine CRS;
Test solution Treat 2.00 g of ±e freshly powdered herbal drug 5.6 = correction factor for caffeine with respect to
(355) (2.9.12) with 75 mL of methylene chloride R in a pinoresinol diglucoside.
continuous extraction apparatus (Soxhlet type) for 1 h. Cool. _______________________________________________________________ Ph Eur
Discard the organic solution and replace with 75 mL of
methanol R. Extract for 6 h in the same apparatus. Filter and
evaporate the filtrate to dryness. Take up the residue with
10.0 mL of a mixture of methanol R and water R
(30:70 V/V). Centrifuge. Filter through a membrane filter Bitter Fennel
(nominal pore size 0.45 pm).
Reference solution (a) Treat 2.00 g of eucommia bark HRS
Ph Eur_______________________________________________________________
with 75 mL of methylene chloride R in a continuous extraction
apparatus (Soxhlet type) for 1 h. Cool. Discard the organic DEFINITION
solution and replace with 75 mL of methanol R. Extract for Dry cremocarps and mericarps of Foeniculuni vulgare
6 h in the same apparatus. Filter and evaporate the filtrate to Mill. ssp. vulgare var. vulgare.
dryness. Take up the residue with 10.0 mL of a mixture of Content
methanol R and water R (30:70 VIV). Centrifuge. Filter — essential oil: minimum 40 mL/kg (anhydrous drug);
through a membrane filter (nominal pore size 0.45 pm). — anethole: minimum 60.0 per cent in the essential oil;
Reference solution (b) Dissolve 20.0 mg of caffeine CRS in — fenchone: minimum 15.0 per cent in the essential oil.
mobile phase A and dilute to 20.0 mL with mobile phase A. CHARACTERS
Dilute 1.0 mL of the solution to 25.0 mL with mobile
Bitter fennel is greenish-brown, brown or green.
phase A.
Column’. IDENTIFICATION
— size". I = 0.25 m, 0 = 4.0 mm; A. The fruit of bitter fennel is a cremocarp, of almost
— stationary phase: end-capped octadecylsilyl silica gel for cylindrical shape with a rounded base and a narrower summit
chromatography R. crowned with a large stylopod. It is generally 3-12 mm long
and 3-4 mm wide. The mericarps, usually free, are glabrous.
Mobile phase:
— mobile phase A: 1.0 g/L solution of phosphoric acid R; Each bears 5 prominent, slightly carenated ridges. When cut
— mobile phase B: acetonitrile R’, transversely, 4 vittae on the dorsal surface and 2 on the
commissural surface may be seen with a lens.
B. Microscopic examination (2.8.23). The powder is greyish-
brown or greyish-yellow. Examine under a microscope using
chloral hydrate solution R. The powder shows the following
0 - 35 87 -> 75 13 -> 25
diagnostic characters (Figure 0824.-1): yellow fragments of
35 - 40 75 ->0 25 -> 100 wide secretory canals, often made up of yellowish-brown
walled polygonal secretory cells [D, H]; reticulate
parenchyma of the mesocarp [B]; numerous fibre bundles
Flow rate 1.0 mL/min. [G] from the ridges [Ga], often accompanied by narrow
Detection Spectrophotometer at 278 nm. spiral vessels [Gb]; very numerous endosperm fragments [F]
Injection 20 pL. containing aleurone grains [Fb] and very small cluster
crystals of calcium oxalate [Fa]; some fibre bundles from the
2016 Bitter Fennel IV-187
carpophore [E]; fragments of the endocarp, in surface view Test solution Dilute the mixture of essential oil and xylene R
[A, K], consisting of thin-walled, transversely elongated cells, obtained in the determination of essential oil to 5.0 mL with
2-9 pm wide, having a parquetry arrangement, sometimes xylene R) by rinsing the apparatus.
accompanied by the inner layer of the mesocarp [Aa]; Reference solution Dissolve 5 mg of estragole 7? in 0.5 mL of
fragments of the epicarp with stomata accompanied by oil xylene R.
droplets [C]; very numerous oil droplets [J].
Column:
— size: I = 30-60 m, 0 = 0.3 mm;
— stationary phase: macrogol 20 000 R.
Carrier gas nitrogen for chromatography R.
Split ratio 1:200.
Temperature:
Time Temperature
(°C)
Column 0-4 60
4 - 26 60 -> 170
26 - 41 170
Injection port 220
Detector 270

Injection 1 pL.
Limit".
— estragole: maximum 5.0 per cent in the essential oil
obtained in the assay.
Maximum 1.5 per cent of peduncles and maximum
1.5 per cent of other foreign matter.
Figure 0824.-1. - Illustration for identification test B of powdered Water (2.2.18)
herbal drug of bitter fennel Maximum 100 mL/kg, determined on 20.0 g of the
Test solution Shake 0.3 g of the freshly powdered herbal drug Total ash {2.4.16)
(1400) (2.9.72) with 5.0 mL of methylene chloride R for Maximum 10.0 per cent.
15 min. Filter and carefully evaporate the filtrate to dryness ASSAY
on a water-bath at 60 °C. Dissolve the residue in 0.5 mL of Essential oil (2.5.72)
toluene R. Use a 500 mL round-bottomed flask and 200 mL of water R
Reference solution Dissolve 50 pL of anethole R and 10 pL of as the distillation liquid. Reduce the herbal drug to a coarse
fenchone R in 5.0 mL of hexane R. powder (1400) (2.9.72) and immediately use 5.0 g for the
Plate TLC silica gel GFวู^ plate R. determination. Introduce 0.50 mL of xylene R in the
graduated tube. Distil at a rate of 2-3 mL/min for 2 h.
Mobile phase hexane R, toluene R (20:80 VIV).
Anethole and fenchone
Application 10 pL as bands of 20 mm by 3 mm.
Gas chromatography (2.2.25) as described in the test for
Development Over a path of 10 cm. estragole with the following modifications.
Drying In air. Reference solution Dissolve 5 mg offenchone R and 5 mg of
Detection A Examine in ultraviolet tight at 254 nm. anethole R in 0.5 mL of xylene R.
Results A The chromatograms show in the central part a Elution order The order indicated in the composition of the
quenching zone due to anethole. reference solution; record the retention times of these
Detection B treat with sulfuric acid R and heat at 140 °C for substances.
5-10 min until a yellow zone due to fenchone appears in the STORAGE
lower third of the chromatograms. Protected from moisture.
Results B Anethole appears as a violet band in the central
part; the chromatogram obtained with the test solution also
shows a reddish-brown zone in its upper third (terpenes).
TESTS
Estragole
Gas chromatography {2.2.28): use the normalisation
procedure.
IV-188 Bitter-Fennel Fruit Oil 2016
Detection Spray with a freshly prepared 200 g/L solution of

Bitter-Fennel Fruit Oil * phosphomolybdic acid R in ethanol (96 per cent) R and heat at
(Ph. Eur. monograph 1826) *** 150 °C for 15 min; examine in daylight.
Ph Elf______________________________________________________________ Results See below the sequence of the zones present in the
DEFINITION
Essential oil obtained by steam distillation from the ripe fruits chromatogram obtained with the test solution.
of Foeniculum vulgare Miller, ssp. vulgare var. vulgare.
Content
— fenchone: 12.0 per cent to 25.0 per cent, Top of the plate
— mnis-anethole: 55.0 per cent to 75.0 per cent.
Anethole: a dark blue to dark A dark blue to dark violet zone
CHARACTERS violet zone (anethole)
Appearance
Characteristic odour. Fenchone: a blue or bluish-grey A blue or bluish-grey zone
(fenchone)
IDENTIFICATION
Second identification A. Reference solution Test solution
Test solution Dissolve 0.1 mL of the essential oil to be
examined in 5 mL of toluene R.
Reference solution Dissolve 10 pL offenchone R and 80 pL of
anethole R in 5 mL of toluene R. Results The characteristic peaks in the chromatogram
Mobile phase ethyl acetate R, toluene R (5:95 VIV).
solution.
Development Over a path of 15 cm. TESTS
Drying In air. Relative density (2.2.5)
0.961 to 0.975.
1. a-pinene 3. fenchone 5. cis-anethole 7. anisaldehyde
2. limonene 4. estragole 6. /rans-anethole
Figure 1826.-1. - Chromatogram for the test for chromatographic profile of bitter-fennel fruit oil
2016 Bitter-Fennel Herb Oil IV-189
Refiractive index (2.2.6)

1.528 to 1.539. Bitter-Fennel Herb Oil ★ *
Optical rotation (2.2.7) (Ph. Eur. monograph 2380) * **
+ 10.0° to + 24.0°. PhEur____________________________________________________________
Chromatographic profile DEFINITION
Essential oil obtained by steam distillation of the aerial parts
procedure.
of Foeniculum vulgare Mill. ssp. vulgare, var. vulgare collected
Test solution Dissolve 0.20 mL of the essential oil to be during fruiting.
solvent. CHARACTERS
Appearance
Reference solution Dissolve 20 pL of 1-pinene R, 20 pL of
Clear, pale or intense yellow liquid.
limonene R, 50 pL of fenchone R, 20 pL of estragole R> 100 pL
of anethole R and 20 pL of anisaldehyde R in heptane R and Anise-like odour.
dilute to 10.0 mL with the same solvent. IDENTIFICATION
Column: First identification B
— material: fused silica, Second identification A
size: I = 60 m, 0 = 0.25 mm,
stationary phase: macrogol 20 000 R (film thickness
0.25 pm). Test solution Dissolve 0.1 mL of the oil to be examined in
5 mL of toluene R.
Reference solution Dissolve 10 pL of fenchone R and 40 pL of
Flow rate 1 mUmin.
anethole R in 5 mL of toluene R.
Split ratio 1:200.
Temperature: plate R (2-10 pm)].
Time Temperature Application 10 pL [or 3 pL] as bands of 10 mm [or 8 mm].
(°C)
Column 0-4 60
Drying In air.
4-26 60 -> 170
Detection Spray with a freshly prepared 200 g/L solution of
26 - 41 170
phosphomolybdic acid R in ethanol (96 per cent) R and heat at
Injection port 220 150 °C for 15 min; examine in daylight.
Detector 270 Results See below the sequence of zones present in the
Detection Flame ionisation. in the chromatogram obtained with the test solution.
Injection 1.0 pL.
Elution order Order indicated in the composition of the Top of the plate
reference solution. Record the retention times of these
substances. Anethole: a dark blue or dark A dark blue or dark violet zone
violet zone (anethole)
estragole and rrans-anethole. Fenchone: a blue or bluish-grey A sometimes faint blue or
bluish-grey zone (fenchone)
the components of the reference solution on the Reference solution Test solution
chromatogram obtained with the test solution and locate
cis-anethole using Figure 1826.-1. (Disregard the peak due to
heptane). B. Examine the chromatograms obtained in the test for
Results:
components. The percentages are within the following
— Spanish type: the characteristic peaks due to a-pinene,
ranges:
P-pinene, p-myrcene, a-phellandrene, limonene,
— a-pinene: 1.0 per cent to 10.0 per cent,
fenchone, estragole and trans-anethole in the
chromatogram obtained with the test solution are
— fenchone: 12.0 per cent to 25.0 per cent,
similar in retention time to those in the chromatogram
— estragole: maximum 6.0 per cent,
— cis-anethole: maximum 0.5 per cent,
— Tasmanian type: the characteristic peaks due to
— trans-anethole: 55.0 per cent to 75.0 per cent,
a-pinene, a-phellandrene, limonene, fenchone,
— anisaldehyde: maximum 2.0 per cent.
estragole and rrans-anethole in the chromatogram
The ratio of a-pinene content to limonene content is greater obtained with the test solution are similar in retention
than 1.0. time to those in the chromatogram obtained with
storage reference solution (a).
PhEur
IV-190 Bitter-Fennel Herb Oil 2016
6 5 10 15 20 2'5 30 ' 35 ’ 40 กnin
1. a-pinene 4. fenchone 7. /rans-anethole
2. a-phellandrene 5. estragole 8. anisaldehyde
3. limonene 6. cis-anethole 9. anise ketone
Figure 2380.-2. - Chromatogram for the test for chromatographic profile of Tasmanian-type
bitter-fennel herb oil
TESTS Carrier gas helium for chromatography R.

Relative density (2.2.5) Flow rate 1 mUmin.
— Spanish type: 0.877 to 0.921; Split ratio 1:50.
— Tasmanian type: 0.940 to 0.973.
ไ'emperature:
— Spanish type: 1.487 to 1.501;
Time Temperature
— Tasmanian type: 1.512 to 1.538.
(°C)
Optical rotation (2.2.7) Column 0 - 35 70 -» 210
— Spanish type: + 42° to + 68°;
— Tasmanian type: -r 11° to + 35°. 35 - 42 210
Solubility in alcohol (2.8.10) Injection port 250

— Spanish type: 1 volume is soluble in 2 volumes and more Detector 270
of ethanol (90 per cent V/V) R;
— Tasmanian type: 1 volume is soluble in 10 volumes and
more of ethanol (85 per cent V!V) R. Detection Flame ionisation.
Chromatographic profile Injection 1 pL.
Gas chromatography (2.2.28): use the normalisation Elution order Order indicated in the composition of the
procedure. reference solution; record the retention times of these
Test solution Dissolve 0.20 mL of the oil to be examined in substances.
acetone R and dilute to 10.0 mL with the same solvent. System suitability: reference solution (a):
Reference solution (a) Dissolve 20 pL of ซ-pinene R3 10 pL of
p-myrcene and a-phellandrene.
P-pinene R3 20 pL of p-myrcene R3 20 pL of ซ-phellandrene R3
20 pL of limonene R, 40 pL of fenchone R, 10 pL of Using the chromatogram obtained with the reference
estragole R3 40 pL of anethole R3 10 pL of anisaldehyde R and solution, locate the relevant components for the type of the
10 pL of anise ketone R in acetone R and dilute to 10.0 mL essential oil to be examined in the chromatogram obtained
with the same solvent. with the test solution, and locate cis-anethole using
Figures 2380.-1 and 2380.-2.
Reference solution (b) Dissolve 5 pL of anethole R in 25.0 mL
of acetone R. Dilute 0.5 mL of this solution to 20.0 mL with Determine the percentage content of each of these
components.
acetone R.
Column: For Spanish-type bitter-fennel herb oil, ±e percentages are
— material: fused silica; within the following ranges:
— size: z = 60 m, 0 = 0.25 mm; — ซ-pinene: 2.0 to 8.0 per cent;
— stationary phase: macrogol 20 000 R (film thickness — p-pinene: 1.0 to 4.0 per cent;
— P-myrcene: 1.0 to 12.0 per cent;
0.25 pm).
— ซ-phellandrene: 1.0 to 25.0 per cent;
2016 Sweet Fennel IV-191
— limonene: 8.0 to 30.0 per cent; calcium oxalate cluster crystals [Fa]; some fibre bundles from
fenchone: 7.0 to 16.0 per cent; the carpophore [E]; fragments of the endocarp, in surface
estragole: 2.0 to 7.0 per cent; view [K, A], consisting of thin-walled, transversely elongated
cis-anethole: maximum 0.5 per cent; cells 2-9 |im wide, having a parquetry arrangement,
trans-anethole: 15.0 to 40.0 per cent; sometimes accompanied by the inner layer of the
anisaldehyde: maximum 1.0 per cent; mesocarp [Aa]; fragments of the epicarp with stomata
anise ketone: maximum 0.05 per cent; accompanied by oil droplets [C]; very numerous oil
disregard limit: the area of the principal peak in the droplets [ฎ.
(0.025 per cent).
For Tasmanian-type bitter-fennel herb oil, the percentages
are within the following ranges:
— ct-pinene: 2.0 to 11.0 per cent;
— ซ.-phellandrene: 1.0 to 8.5 per cent;
— limonene: 1.0 to 6.0 per cent;
— fenchone: 10.0 to 25.0 per cent;
— estragole: 1.5 to 6.0 per cent;
— cis-anethole: maximum 0.5 per cent;
trans-anethole: 45.0 to 78.0 per cent;
— anisaldehyde: maximum 1.0 per cent;
— anise ketone: maximum 0.05 per cent;
disregard limit: the area of the principal peak in the
(0.025 per cent).
STORAGE
LABELLING
The label states that the content is Spanish-type or
Tasmanian-type.
—— — ----- —________________________________________ PhEur
Sweet Fennel ★ ** Figure 0825.-1. - Illustration for identification test B of powdered

(Ph Eur monograph 0825) *** herbal drug of sweet fennel
PhEur—_____________________________________________________ c. Thin-layer chromatography (2.2.27).
Test solution Shake 0.3 g of the freshly powdered herbal drug
DEFINITION
(1400) (2.9.12) with 5.0 mL of methylene chloride R for
Dry cremocarps and mericarps of Foeniculum vulgare Mill,
15 min. Filter and carefully evaporate the filtrate to dryness
subsp. vulgare var. dulce (Mill.) Batt. & Trab.
on a water-bath at 60 °C. Dissolve the residue in 0.5 mL of
Content toluene R.
— essential oil: minimum 20 mUkg (anhydrous drug); Reference solution Dissolve 60 pL of anethole R in 5.0 mL of
— anethole: minimum 80.0 per cent in the essential oil. hexane R.
CHARACTERS Plate TLC silica gel GF254 plate R.
Sweet fennel is pale green or pale yellowish-brown. Mobile phase hexane R3 toluene R (20:80 VIV).
IDENTIFICATION Application 10 pL as bands of 20 mm by 3 mm.
A. The fruit of sweet fennel is a cremocarp of almost Development Over a path of 10 cm.
cylindrical shape with a rounded base and a narrowed
Drying In air.
summit crowned with a large stylopod. It is generally
3-12 mm long and 3-4 mm wide. The mericarps, usually
free, are glabrous. Each bears 5 prominent, slightly carenated Results A The chromatograms show in the central part a
ridges. When cut transversely, 4 vittae on the dorsal surface quenching zone due to anethole.
and 2 on the commissural surface may be seen with a lens. Detection B Spray with sulfuric acid R and heat at 140 °C for
B. Microscopic examination (2.8.23). The powder is greyish- 5 min; examine in daylight.
brown or greyish-yellow. Examine under a microscope using Results B The chromatograms show in the central part a
chloral hydrate solution R. The powder shows the following violet band due to anethole; the chromatogram obtained with
diagnostic characters (Figure 0825.-1.)ะ yellow fragments of the test solution also shows a reddish-brown zone in the
wide secretory canals, often made up of yellowish-brown upper third (terpenes).
walled polygonal secretory cells [D, H]; reticulate TESTS
parenchyma of the mesocarp [B]; numerous fibre Estragole and fenchone
bundles [G] from the ridges [Ga], often accompanied by Gas chromatography (2.2.28): use the normalisation
narrow spiral vessels [Gb]; very numerous endosperm procedure.
fragments [F] containing aleurone grains [Fb] and very small
IV-192 Fenugreek 2016
Test solution Dilute the mixture of essential oil and xylene R IDENTIFICATION
obtained in the assay of essential oil to 5.0 mL with xylene R, A. The seed is hard, flattened, brown or reddish-brown and
by rinsing the apparatus. more or less rhomboidal with rounded edges. It is 3-5 mm
Reference solution Dissolve 5 mg of estragole R and 5 mg of long, 2-3 mm wide and 1.5-2 mm thick. The widest surfaces
fenchone J? in 0.5 mL of xylene R. are marked by a groove that divides the seed into 2 unequal
Column: parts. The smaller part contains the radicle; the larger part
— size: I = 30-60 m, 0 = 0.3 mm; contains the cotyledons.
— stationary phase: macrogol 20 000 R. B. Reduce to a powder (355) (2.9.12). The powder is
Carrier gas nitrogen for chromatography R. yellowish-brown. Examine under a microscope using chloral
Flow rate 0.40 mUmin. hydrate solution R. The powder shows the following diagnostic
characters: fragments of the testa in sectional view with thick
Split ratio 1:200. cuticle covering lageniform epidermal cells, with an
underlying hypodermis of large cells, narrower at the upper
Time Temperature end and constricted in the middle, with bar-like thickenings
(min) __________ (°C) of the radial walls; yellowish-brown fragments of the
Column 0 -4 60 epidermis in surface view, composed of small, polygonal cells
4 - 26 60 -> 170 with thickened and pitted walls, frequently associated with
the hypodermal cells, circular in outline with thickened and
26 - 41 170
closely beaded walls; fragments of the hypodermis viewed
Injection port 220 from below, composed of polygonal cells whose bar-like
Detector 270
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
Detection Flame ionisation. irregularly thickened, sometimes elongated cells, containing
Injection 1 j-iL. mucilage.
Limits: c. Thin-layer chromatography (2.2.27).
— estragole: maximum 10.0 per cent in the essential oil; Test solution Place 1.0 g of the powdered herbal drug (710)
— fenchone: maximum 7.5 per cent in the essential oil. (2.9.12) in a 25 mL conical flask and add 5.0 mL of
Foreign matter (2.8.2) methanol R. Heat in a water-bath at 65 °C for 5 min. Cool
Maximum 1.5 per cent of peduncles and maximum and filter.
1.5 per cent of other foreign matter. Reference solution Dissolve 3.0 mg of trigonelline hydrochloride R
in 1.0 mL of methanol R.
Water (2.2.13)
Maximum 80 mJLTkg, determined on 20.0 g of the powdered Plate TLC silica gel 2๖54 plate R.
herbal drug (710) (2.9.12). Mobile phase water R, methanol R (30:70 V/V).
Total ash (2.4.16) Application 20 pL of the test solution and 10 pL of the
Maximum 10.0 per cent. reference solution, as bands.
ASSAY Development Over a path of 10 cm.
Essential oil (2.8.12) Drying In air.
Use 10.0 g of the herbal drug reduced to a coarse powder Detection A Examine in ultraviolet light at 254 nm.
(1400) (2.9.12) immediately before the assay, a 500 mL Results A The chromatogram obtained with the test solution
round-bottomed flask, 200 mL of water R as the distillation shows in its lower half a quenching zone similar in position
liquid, and 0.50 mL of xylene R in ±e graduated tube. Distil and fluorescence to the zone in the chromatogram obtained
at a rate of 2-3 mlVmin for 2 h. with the reference solution.
Anethole Detection B Spray with potassium iodobismuthate solution R2.
Gas chromatography (2.2.28) as described in the test for Results B The chromatogram obtained with the test solution
estragole and fenchone with the following modification. shows an intense orange-red zone similar in position and
Reference solution Dissolve 5 mg of anethole 7? in 0.5 mL of colour to the zone in the chromatogram obtained with the
xylene R. reference solution. It also shows in its upper half, a broad
STORAGE light brownish-yellow zone (triglycerides).
Protected from moisture. TESTS
Ph Eur Loss on drying (2.2.32)
powdered herbal drug by drying in an oven at 105 °C for
2 h.
Total ash (2.4.16)
Fenugreek Maximum 5.0 per cent.
(Ph. Eur. monograph 1323) Swelling index (2.8.4)
Minimum 6, determined on the powdered herbal drug (710)
Ph Eur _______.----------
(2.9.12) .
DEFINITION Ph
Dried, ripe seeds of Trigonella foenum-graecum L.
CHARACTERS
Strong characteristic aromatic odour.
2016 Feverfew IV-193
Feverfew ** ** Application 20 pL, as bands.

(Ph. Eur. monograph 1516) Drying In air.
Ph Eur________ ___________ _________________________________________
Detection Spray with a 5 g/L solution of vanillin R in a
DEFINITION mixture of 20 volumes of anhydrous ethanol R and
Dried, whole or fragmented aerial parts of Tanacetum 80 volumes of sulfuric acid R. Examine in daylight after
parthenium (L.) Schultz Bip. 5 min.
Content Results The chromatogram obtained with the test solution
Minimum 0.20 per cent of parthenolide (C15H20O3; Mr shows in its central part a blue principal zone that is similar
248.3) (dried drug). in position, colour and size to the principal zone in the
chromatogram obtained with the reference solution, and
CHARACTERS somewhat below the principal zone a 2nd blue zone may be
Camphoraceous odour. present; 1 or 2 blue zones are also present in its lower third;
IDENTIFICATION other violet zones may be present.
A. The leafy, more or less branched stem has a diameter of TESTS
up to 5 mm; it is almost quadrangular, channelled Foreign matter (2.5.2)
longitudinally and slightly pubescent. The leaves are ovate, Maximum 10.0 per cent of stem with a diameter greater than
2-5 cm long, sometimes up to 10 cm, yellowish-green, 5 mm and maximum 2.0 per cent of other foreign matter.
petiolate and alternate. They are pinnate or bipinnate, deeply
divided into 5-9 segments, each with a coarsely crenate
margin and an obtuse apex. Both surfaces are somewhat
pubescent and the midrib is prominent on the lower surface.
105 °C for 2 h.
When present, die flowering heads are 12-22 mm in
diameter with long pedicels; they are clustered into broad Total ash {2.4.16)
corymbs consisting of 5-30 flower-heads. The hemispherical Maximum 12.0 per cent.
involucre is 6-8 mm wide and consists of many overlapping ASSAY
bracts, which are rather narrow, obtuse and scarious and Liquid chromatography (2.2.29).
have membranous margins. The central flowers are yellow, Test solution Completely reduce about 50 g of the drug to be
hermaphrodite, tube-shaped with 5 teeth and have 5 stamens examined to a powder (355) (2.9.72). After homogenisation,
inserted in the corolla; the filaments of tile stamens are introduce 1.00 g of the powdered herbal drug into a flask
separate from each other but the anthers are fused into a and add 40 mL of methanol R. Heat in a water-bath at 60 °C
tube through which passes the style, bearing 2 stigmatic for 10 min. Allow to cool and filter. Rinse the filter with
branches. The peripheral flowers are female and have a 15 mL of methanol R. Take up the residue with 40 mL of
white, three-toothed ligule, 2-7 mm long. The fruit is an methanol R. Repeat the operation. Collect the filtrates and
achene, 1.2-1.5 mm long, brown when ripe, with 5-10 white rinsings and evaporate to dryness under reduced pressure.
longitudinal ribs. It is glandular and bears a short, crenate, Take up the residue with methanol R and dilute to 20.0 mL
membranous crown. with the same solvent. Dilute 10.0 mL of this solution to
B. Reduce to a powder (355) (2.9.72). The powder is 50.0 mL with ±e mobile phase. Filter (0.45 pm).
yellowish-green. Examine under a microscope using chloral Reference solution Dissolve 5.0 mg of parthenolide R in
hydrate solution R. The powder shows the following diagnostic methanol R and dilute to 10.0 mL with the same solvent.
characters: numerous large, multicellular, uniseriate covering Dilute 2.0 mL of this solution to 50.0 mL with the mobile
trichomes consisting of a rhomboidal basal cell, 3-5 smaller, phase.
thick-walled rectangular cells and a very long, flat, slender
Column:
terminal cell, often curved at a right angle to the axis of the
— size: I = 0.25 m, 0 = 4.6 mm;
basal cell; glandular trichomes with a short, biseriate,
2-4 celled stalk and a biseriate head of 4 cells around which
(5 pm).
the cuticle forms a bladder-like covering; epidermal cells with
very sinuous, anticlinal walls, a striated cuticle and Mobile phase acetonitrile R} water R (40:60 VIV).
anomocytic stomata (2.5.5); numerous spirally and annularly Flow rate 1 mL/min.
thickened vessels; stratified parenchyma and collenchyma. Detection Spectrophotometer at 220 nm.
Fragments of disc florets containing pale yellow amorphous Injection 20 pL.
masses and small rosette crystals of calcium oxalate may be
Retention time Parthenolide = about 11.5 min.
present; spherical pollen grains about 25 pm in diameter,
Calculate the percentage content of parthenolide using the
with 3 pores and a spiny exine may be present.
Test solution To 1 g of the powdered herbal drug (355) Al X 7712 X 40
{2.9.12) add 20 mL of methanol R. Heat in a water-bath at
A2 X mi
60 °C for 15 min. Allow to cool and filter. Evaporate to
dryness under reduced pressure and dissolve the residue in
2 mL of methanol R. Al = area of the peak due to parthenolide in the
Reference solution Dissolve 5 mg of parthenolide R in
methanol R and dilute to 5 mL with the same solvent. Ao = area of the peak due to parthenolide in the
solution;
Mobile phase acetone R, toluene R (15:85 VIV).
IV-194 Fig 2016
ผ1 = mass of the herbal drug to be examined in the test The external surface of the root is reddish-brown with
solution, in grams; irregular wrinkles, resembling transversely elongated lenticels,
ใท2 = mass of parthenolide in the reference solution, in and with fine rootlet scars. The texture is dense, compact
grams. and granular. The fracture is pale yellowish-brown or
------------------------------------------------------------------------ PAfw reddish-brown. The drug is powdery when it is fractured.
In the cortex there are 4-11 bundles giving rise to a cloud
like appearance. The central xylem is large, sometimes
distinguishable as a central lignified part.
Fig B. Microscopic examination (2.8.23). The powder is
DEFINITION yellowish-brown. Examine under a microscope using chloral
The sun-dried succulent fruit of Ficus carica L. hydrate solution R. The powder shows the following diagnostic
characters: cluster crystals of calcium oxalate 10-80 pm,
CHARACTERISTICS sometimes up to 160 pm, in diameter, with obtuse angles;
Odour, pleasantly fruity; taste, sweet. rare, relatively large, isolated tetragonal prism crystals;
Macroscopical Fruit compound: soft, fleshy, brown or fragments of parenchyma consisting of thin-walled, sub
yellowish brown, sometimes covered with a saccharine rounded or rectangular cells, sometimes containing brown,
efflorescence; at the summit a small opening surrounded by yellowish-brown or reddish-brown inclusions; rare fragments
scales and at the base a short, stalk-like prolongation; fruit up of cork consisting of several layers of regular cells filled with
to about 5 cm in length and breadth, consisting of a hollow brown contents; fragments of pitted vessels 15-180 pm in
receptacle bearing on the inner surface numerous drupelets, diameter; few groups of xylem fibres; scattered brown
each containing a stone about 1.5 to 2.0 mm long; seed masses, varying in shape, size and colour. Examine under a
containing endosperm and a curved embryo. microscope using a 50 per cent VIV solution of glycerol R.
Microscopical Receptacle: epidermal cells polyhedral, The powder shows simple or 2-9 compound starch granules,
stomata raised, trichomes unicellular, thick walled, of varying the simple granules are sub-rounded, 4-50 pm in diameter,
length up to about 300 pm; hypodermis composed of with a V-shaped, stellate or Y-shaped hilum, the large
rounded polyhedral cells, some containing small rosette granules show clearly visible layers.
crystals of calcium oxalate; parenchyma made up of large, c. Thin-layer chromatography (2.2.27).
irregular cells, forming the greater pan of the receptacle, Test solution To 0.500 g of the powdered herbal drug (355)
containing large rosette crystals of calcium oxalate and (2.9.12) add 5 mL of methanol R. Heat in a water-bath at
interspersed with numerous latex tubes, about 30 to 50 pm 60 °C for 15 min and filter.
wide, and slender vascular bundles. Pericarp: epicarp Reference solution Dissolve 1 mg of emodin R and 1 mg of
consisting of radially elongated cells with mucilaginous outer resveratrol R in 2 mL of methanol R.
walls; mesocarp of delicate, often disorganised cells; endocarp
of radially elongated sclereids with pitted walls. Endosperm
F254 plate R (2-10 pm)].
and embryo: small cells containing aleurone grains and fixed
oil; starch absent. Mobile phase glacial acetic acid R, anhydrous ethanol R,
toluene R (1:4:16 VIVIV).
Water-soluble extractive
Not less than 60.0% when determined by the following Application 20 pL [or 5 pL] as bands of 10 mm [or 8 mm].
method. To 25 g, minced, add 500 mL of water, boil under Development Over a path of 10 cm [or 6 cm].
a reflux condenser for 1 hour, cool and filter. To 20 mL of Dtying In air.
the filtrate add 20 g of washed and ignited sand, evaporate to Detection Examine in ultraviolet light at 365 nm.
dryness in a tared, flat bottomed shallow dish and dry the
residue to constant weight at 100°. Calculate the water
soluble extractive by subtracting the weight of sand from the
weight of the residue obtained.
STORAGE
Figs should be stored in a dry place.
Top of the plate
Fleeceflower Root * * Emodin ะ a yellow fluorescent

zone
(emodin)
Ph Eur________________________________________________________________ Resveratrol ะ a light blue
fluorescent zone
DEFINITION
Whole or fragmented dried tuberous root of Fallopia
multiflora (Thunb.) Haraldson (syn. Polygonum multiflorum
Thunb.). Reference solution Test solution
Content
Minimum 1.0 per cent of 2,3,5,4'-tetrahydroxystilbene-2-O-
P-D-glucoside (C20H22O9; Mr 406.4) (dried drug). TESTS
IDENTIFICATION
A The whole drug consists of an irregular, fusiform,
powdered herbal drug (355) (2.9.12) by drying m an oven at
tuberous, root 6-15 cm long and 4-12 cm in diameter;
105 °C for 2 h.
the fragmented drug consists of slices or irregular pieces.
2016 Frangula Bark IV-195
Total ash (2.4.16) A2 — area of the peak due to resveratrol in the

Maximum 5.0 per cent. chromatogram obtained with reference solution
Ash insoluble in hydrochloric acid (2.8.1) (a);
Maximum 2.0 per cent. พ! = mass of the herbal drug to be examined used to
ASSAY prepare the test solution, in grams;
พ2 = mass of resveratrol CRS used to prepare reference
Liquid chromatography (2.2.29). solution (a), in grams;
Test solution Weigh 0.250 g of the powdered herbal drug p = percentage content of resveratrol in resveratrol
(355) (2.9.12) in a 100 mL glass vial with a screw cap. CRS.
Add 50.0 mL of a 50 per cent VIV solution of methanol Ry 0.5 = correction factor for resveratrol with respect to
close and extract for 1 h using ultrasound. Filter the solution 2,3,5,4'-tetrahydroxystilbene-2-O-P-D-glucoside.
through a membrane filter (nominal pore size 0.45 pm).
____________________________________________________________ PhEur
Reference solution (a) Dissolve 10.0 mg of resveratrol CRS in
Reference solution (b) Weigh 0.250 g of fleeceflower root HRS in
a 100 mL glass vial with a screw cap. Add 50.0 mL of a Frangula Bark ★ *
50 per cent VIV solution of methanol Ry close and extract for
1 h using ultrasound. Filter the solution through a membrane
filter (nominal pore size 0.45 pm). Preparation
Column’. Standardised Frangula Bark Dry Extract
— size: I = 0.125 m, 0 = 4.6 mm; When Powdered Frangula Bark is prescribed or demanded,
— stationary phase: octadecylsilyl silica gel for chromatography R material complying with the requirements below, with the
(5 pm); exception of Identification test A and the test for Foreign
— temperature: 30 °C. matter, shall be dispensed or supplied.
Mobile phase: PhEis_____________________________________________________________
— mobile phase A: 0.1 per cent VIV solution of anhydrous DEFINITION
formic acid R’y
Dried, whole or fragmented bark of the stems and branches
— mobile phase B: acetonitrile R’y
of Rhamnus frangula L. (Frangula alnus Miller).
Content
Time Mobile phase A Mobile phase B Minimum 7.0 per cent of glucofrangulins, expressed as
(rain) (per cent V/V) (per cent V/V) glucofrangulin A (C27H30014; Afr 578.5) (dried drug).
0 - 15 90 -> 70 10 -> 30
IDENTIFICATION
15 - 16 70 20 30 -> 80
A. The bark occurs in curved, almost flat or rolled fragments
16 - 21 20 80 or in single or double quilled pieces usually 0.5-2 mm thick
and variable in length and width. The greyish-brown or dark
brown outer surface is wrinkled longitudinally and covered
Flow rate 1.0 mUmin. with numerous greyish, transversely elongated lenticels; when
Detection Spectrophotometer at 320 nm. the outer layers are removed, a dark red layer is exposed.
Injection 10 pL. The orange-brown or reddish-brown inner surface is smooth
Identification of peaks Use the chromatogram supplied with and bears fine longitudinal striations; it becomes red when
fleeceflower root HRS and the chromatogram obtained with treated with alkali. The fracture is short, fibrous in the inner
reference solution (b) to identify the peak due to 2,3,5,4'- part.
tetrahydroxystilbene-2-O"P-D-glucoside and peak 2 B. Microscopic examination (2.8.23). The powder is
(unknown). yellowish or reddish-brown. Examine under a microscope
Retention time 2,3,5,4/-tetrahydroxystilbene-2-O-P-D- using chloral hydrate solution R. The powder shows the
glucoside = about 12 min; peak 2 = about 13 min; following diagnostic characters (Figure 0025.-1): numerous
resveratrol ะ= about 17 min. phloem fibres, in tangential section [D] or in longitudinal
section [K], partially lignified, in groups [Da, Ka] with
crystal sheaths containing calcium oxalate prisms [Db, Kb],
— resolution: minimum 2.0 between the peak due to
sometimes including medullary rays [De]; reddish-brown
2,3,5,4'-tetrahydroxystilbene-2-O-p-D-glucoside and
fragments of cork [H]; fragments of phloem parenchyma, in
peak 2.
longitudinal section [G] containing calcium oxalate cluster
Calculate the percentage content of 2,3,5,4'- crystals [A, E] or in tangential section [C] including
tetrahydroxystilbene-2-O-P-D-glucoside using the following medullary rays [Ca] and cells containing calcium oxalate
expression: cluster crystals [Cb]; a few fragments of collenchyma [F];
isolated calcium oxalate cluster crystals [B] and prisms □โ).
Al X 7712 X p c. Examine the chromatogram obtained in test A for other
A2 X 7711 X 4 X 0.5 species of Rhamnus’i anthrones in ultraviolet light at 365 nm.
A[ = area of the peak due to 2,3,5,4'- shows 2 orange-brown zones (glucofrangulins) in the lower
tetrahydroxystilbene-2-O-P-D-glucoside in the third and 2-4 red zones (frangulinSi not always clearly
chromatogram obtained with the test solution; separated, and above them frangula-emodin) in the upper
third.
IV-196 Frangula Bark 2016
B. Application-. 10 pL of the test solution as a band.

Drying In air for maximum 5 min.
Detection Spray immediately with a 5 g/L solution of
nitrotetrazoliuni blue R in methanol R; examine immediately.
Results No violet or greyish-blue zones appear.
Maximum 1 per cent.
Loss on drying {2.2.32)
powdered herbal drug (355) {2.9.12) by drying in an oven at
105 °C for 2 h.
Total ash {2.4.16)
/Maximum 6.0 per cent.
ASSAY
Cany out the assay protected from blight light.
In a tared, round-bottomed flask with a ground-glass neck,
weigh 0.250 g of the powdered herbal drug (180) {2.9.12).
Add 25.0 mL of a 70 per cent v/v solution of methanol R;
mix and weigh. Heat in a water-bath under a reflux
condenser for 15 min. Allow to cool, weigh and adjust to the
original mass with a 70 per cent v/v solution of methanol R.
Filter and transfer 5.0 mL of the filtrate to a separating
funnel. Add 50 mL of water R and 0.1 mL of hydrochloric
acid R. Shake with 5 quantities, each of 20 mL, of light
petroleum R. Allow the layers to separate and transfer the
Figure 0025.-1. - Illustration for identification test B of powdered aqueous layer to a 100 mL volumetric flask. Combine the
herbal drug offrangula bark light petroleum layers and wash with 2 quantities, each of
15 mL, of water R. Use this water for washing the separating
D. To about 50 mg of the powdered herbal drug (180) funnel and add it to the aqueous solution in the volumetric
(2.9.12) add 25 mL of dilute hydrochloric acid R and heat the flask. Add 5 mL of a 50 g/L solution of sodium carbonate R
mixture on a water-bath for 15 min. Allow to cool, shake and dilute to 100.0 mL with water R. Discard the light
with 20 mL of ether R and discard the aqueous layer. Shake petroleum layer. Transfer 40.0 mL of the aqueous solution to
the ether layer with 10 mL of dilute ammonia Rl. a 200 mL round-bottomed flask with a ground-glass neck.
The aqueous layer becomes reddish-violet. Add 20 mL of a 200 g/L solution of ferric chloride R and heat
TESTS under a reflux condenser for 20 min in a water-bath with the
Other species of Rhamnus} anthrones water level above that of the liquid in the flask. Add 2 mL of
Thin-layer chromatography {2.2.27). hydrochloric acid R and continue heating for 20 min, shaking
frequently, until the precipitate is dissolved. Allow to cool,
transfer the mixture to a separating funnel and shake with
(2.9.12) add 5 mL of ethanol (70 per cent VIV) R and heat to
3 quantities, each of 25 mL, of ether R, previously used to
boiling. Cool and centrifuge. Decant the supernatant
rinse the flask. Combine the ether extracts and wash with
immediately and use within 30 min.
2 quantities, each of 15 mL, of water R. Transfer the ether
Reference solution Dissolve 20 mg of barbaloin R in ethanol layer to a volumetric flask and dilute to 100.0 mL with
(70 per cent VIV) R and dilute to 10 mL with the same ether R. Evaporate 20.0 mL carefully to dryness and dissolve
solvent. the residue in 10.0 mL of a 5 g/L solution of magnesium
Plates TLC silica gel plate R (2 plates). acetate R in methanol R. Measure the absorbance (2.2.2S) at
Mobile phase water R, methanol R} ethyl acetate R 515 nm using methanol R as the compensation liquid.
(13:17:100 VIVIV). Calculate the percentage content of glucofrangulins,
A. Application-. 10 pL as bands. expressed as glucofrangulin A, using the following expression:
A X 3.06
m
hydroxide R in ethanol (50 per cent VIV) R} and heat at
100-105 °C for 15 min; examine in ultraviolet light at i.e. taking the specific absorbance of glucofrangulin A to be
204.
365 nm.
solution shows a brownish-yellow zone due to barbaloin in
the central part; the chromatogram obtained with the test ________ ______________________________________________________ Ph Elf
solution shows no zones of intense yellow fluorescence and

no zone of orange or reddish fluorescence similar in position
to the zone due to barbaloin in the chromatogram obtained
with the reference solution.
2016 Indian Frankincense TV-197
Standardised Frangula Bark Dry ***** 5 quantities, each of 20 mL, of light petroleum Rl. Allow the
layers to separate and transfer the aqueous layer to a 100 mL
Extract ***** volumetric flask. Combine the light petroleum layers and
(Ph. Eur. monograph 1214) wash with 2 quantities, each of 15 mL, of water R. Use this
PhEir_________ ________ ______________________ water for washing the separating funnel and add it to the
aqueous solution in the volumetric flask. Add 5 mL of a
DEFINITION 50 g/L solution of sodium carbonate R and dilute to 100.0 mL
Standardised dry extract obtained from Frangula bark (0025). with water R. Discard the light petroleum layer. Transfer
Content 40.0 mL of the aqueous solution to a 200 mL round-
15.0 per cent to 30.0 per cent of glucofrangulins, expressed bottomed flask with a ground-glass neck. Add 20 mL of a
as glucofrangulin A (C27H30014; Mr 578.5) (dried extract). 200 g/L solution of ferric chloride R and heat under a reflux
The measured content does not deviate from that stated on condenser for 20 min in a water-bath with the water level
the label by more than ± 10 per cent. above that of the liquid in the flask. Add 2 mL of hydrochloric
acid R and continue heating for 20 min, shaking frequently,
PRODUCTION
until the precipitate is dissolved. Allow to cool, transfer the
The extract is produced from the herbal drug by a suitable mixture to a separating funnel and shake with 3 quantities,
procedure using ethanol (50-90 per cent P7I0- each of 25 mL, of ether R, previously used to rinse the flask.
CHARACTERS Combine the ether extracts and wash with 2 quantities, each
Appearance of 15 mL, of water R. Transfer the ether layer to a volumetric
Yellowish-brown, fine powder. flask and dilute to 100.0 mL with ether R. Evaporate
20.0 mL carefully to dryness and dissolve the residue in
IDENTIFICATION
10.0 mL of a 5 g/L solution of magnesium acetate R in
A. Thin-layer chromatography (2.2.27). methanol R. Measure the absorbance (2.2.25) at 515 nm
Test solution To 0.05 g of the extract to be examined add using methanol R as the compensation liquid.
5 mL of ethanol (70 per cent VIV) R and heat to boiling. Cool Calculate the percentage content of glucofrangulins,
and centrifuge. Decant the supernatant immediately and use expressed as glucofrangulin A, using the following expression:
within 30 min.
Reference solution Dissolve 20 mg of barbaloin R in ethanol A X 3.06
(70 per cent V/V) R and dilute to 10 mL with the same
solvent.
Plate TLC silica gel plate R. i.e. taking the specific absorbance of glucofrangulin A to be
Mobile phase water R, methanol R, ethyl acetate R 204, calculated on the basis of the specific absorbance of
(13:17:100 VIVIV). barbaloin.
m = mass of the extract to be examined, in grams.
Drying In air for 5 min. LABELLING
The label states the content of glucofrangulins.
Detection treat with a 50 g/L solution of potassium hydroxide R
in ethanol (50 per cent VIV) R and heat at 100-105 °C for _____________________________________________________________ PhEur
15 min; examine immediately after heating.

solution shows in the middle third a reddish-brown zone due
to barbaloin. The chromatogram obtained with the test Indian Frankincense *****
solution shows 2 orange-brown zones (glucofrangulins) in the (Ph. Eur. monograph 2310) ***
lower third and 2-4 red zones (frangulins, not always clearly
separated, and above them frangula-emodin) in the upper PhEur______________________________________________________________
third. DEFINITION
B. To about 25 mg add 25 mL of dilute hydrochloric acid R Air-dried gum-resin exudate, obtained by incision in the stem
and heat the mixture on a water-bath for 15 min. Allow to or branches of Boswellia serrata Roxb. ex Colebr.
cool, shake with 20 mL of ether R and discard the aqueous Content
layer. Shake the ether layer with 10 mL of dilute ammonia Rl. — 11-keto-fl-bosweUic acid (บ30พ4604; Mr 470.7): minimum
The aqueous layer becomes reddish-violet. 1.0 per cent (dried drug);
TESTS — acetyl-11-keto-fl-boswellic acid (C32H48O5; Mr 512.7):
Loss on drying {2.8.17) minimum 1.0 per cent (dried drug).
Maximum 5.0 per cent. IDENTIFICATION
ASSAY A. Indian frankincense consists of translucent, roundish or
Cany out the assay protected from bright light. irregularly shaped, variable size pieces of up to 3 cm. They
are yellowish or reddish-brown. Their surface is covered with
Into a tared round-bottomed flask with a ground-glass neck,
grey dust. The fracture is dull or slightly glossy.
weigh 0.100 g. Add 25.0 mL of a 70 per cent V/V solution
of methanol R} mix and weigh again. Heat the flask in a B. Thin-layer chromatography (2.2.27).
water-bath under a reflux condenser at 70 °C for 15 min. Test solution To 1.0 g of the powdered herbal drug (355)
Allow to cool, weigh and adjust to the original mass with a (2.9.12) add 90 mL of methanol R and sonicate for 10 min.
70 per cent V/V solution of methanol R. Filter and transfer Shake the mixture vigorously 3 or 4 times during this
5.0 mL of the filtrate to a separating funnel. Add 50 mL of procedure. Dilute to 100 mL with methanol R. Centrifuge
water R and 0.1 mL of hydrochloric acid R. Shake with and use the clear supernatant solution.
IV-198 Fraxinus rhynchophylla bark 2016
Reference solution Dissolve 2 mg of 11-keto-p-boswellic acid R Time Mobile phase A Mobile phase B
and 2 mg of acetyl-ไ 1-keto-p-boswellic acid R in 20 mL of (min) (per cent V/V) (per cent V/V)
methanol R. 0 - 12.5 16 6 84 -> 94
Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel 12.5 - 13.5 6 -> 0 94 -> 100
7*254 plate R (2-10 pm)].
13.5 - 28 0 100
Mobile phase anhydrous formic acid R, heptane R> ethyl
acetate R3 toluene R (3:10:20:80 VIVIVIV).
Injection 20 pL.
Drying In air.
Retention time 11-keto-P-boswellic acid = about 8 min; acetyl-
11-keto-P-boswellic acid = about 12 min.
System suitability", reference solution:
— resolution", minimum 6.0 between the peaks due to
test solution. The zones due to 11-keto-P-boswellic acid and
11-keto-P-boswellic acid and acetyl-11-keto-P-boswellic
acetyl-11-keto-p-boswellic acid in the test solution are of
acid.
approximately equivalent intensity. Furthermore, other weak
quenching zones may be present in the chromatogram Calculate the percentage content of 11-keto-P-boswellic acid
obtained with the test solution. using the following expression:
Al X mi X 5 X Pl
Top of the plate A2 X m
A1 = area of the peak due to 11-keto-P-boswellic acid

in the chromatogram obtained with the test
Acetyl-ll-keto-P-boswellic acid: A quenching zone solution;
a quenching zone (acetyl-11-keto-p-boswellic A2 = area of the peak due to 11-keto-P-boswellic acid
acid)
in the chromatogram obtained with the reference
1 l-Keto-0-boswellic acid: a A quenching zone
quenching zone (11-keto-P-boswellic acid) solution;
Reference solation
m = mass of the substance to be examined, in grams;
Test solution
nil = mass of 11-keto-p-boswellic acid R in the reference
solution, in grams;
TESTS p1 = percentage content of 11-keto-P-boswellic acid in
11-keto-p-boswellic acid R.
Maximum 8.0 per cent, determined on 1.000 g of the Calculate the percentage content of acetyl-11-keto-P"
powdered herbal drug (355) (2.9.12) by drying in an oven at boswellic acid using the following expression:
105 °C for 3 h.
A3 X 7ท2 X 5 X p2
Total ash (2.4.16)
Maximum 10.0 per cent. A4 X โท
ASSAY A3 = area of the peak due to acetyl-11-keto-p-boswellic

Liquid chromatography (2.2.29). acid in the chromatogram obtained with the test
Test solution To 1.0 g of the powdered herbal drug (355) solution;
(2.9.72) add 90 mL of methanol R and sonicate for 10 min. A4 = area of the peak due to acetyl-11-keto-P-boswellic
Shake the mixture vigorously 3 or 4 times during this acid in the chromatogram obtained with the
procedure. Dilute to 100.0 mL with methanol R. Centrifuge reference solution;
for 5 min. Dilute 1.0 mL of the clear solution to 10.0 mL m = mass of the substance to be examined, in grams;
with a mixture of 16 volumes of mobile phase A and m2 — mass of acetyl-11-keto-p-boswellic acid R in the
84 volumes of mobile phase B. reference solution, in grams;
p2 = percentage content of acetyl-11-keto-P-boswellic
Reference solution Dissolve 1.0 mg of 1 l-keto-P-bosweUic acid R
and 1.0 mg of acetyl-1 l-keto-fi-boswellic acid R in 20.0 mL of acid in acetyl-11-keto-p-boswellic acid R.
methanol R. Dilute 1.0 mL of this solution to 10.0 mL with a ______________________________________________________ _ Ph Elf
mixture of 16 volumes of mobile phase A and 84 volumes of
mobile phase B.
Column".
— size". I = 0.25 m, 0 = 4.6 mm; Fraxinus Rhynchophylla Bark ** **
— stationary phase", octadecylsilyl silica gel for chromatography R
(5 pm).
Mobile phase". Ph Pur ________________________________________ ______________
— mobile phase A", phosphoric acid R> water R (0.1:99.9 VIV); DEFINITION
— mobile phase B". phosphoric acid R, acetonitrile R
Whole or fragmented, dried branch or trunk bark of Fraxinus
(0.1:99.9 V/Vy, rhynchophylla Hance, collected in spring or autumn.
Content
Minimum 1.0 per cent for the sum of esculin (C15H16O9;
Mt 340.3) and esculetin (C9H6O4; Mr 178.1) (dried drug).
2016 Fraxinus rhynchophylla bark IV-199
IDENTIFICATION TESTS
A. The branch bark occurs as flexible, curved or channelled, Loss on drying (2.2.32)
rolled or folded pieces up to 60 cm long and 3 mm thick; Maximum 12.0 per cent, determined on 1.000 g of the
the outer surface is whitish-grey to dark brownish-grey, powdered herbal drug (355) (2.9.72) by drying in an oven at
sometimes in patches, and is smooth or slightly rough, dotted 105 °C.
With whitish-grey, rounded lenticels; the inner surface is
Total ash {2.4.16)
smooth, soft to the touch, yellowish-white or brown.
The fracture is fibrous.
The trunk bark occurs as compact, rigid, slat-shaped pieces,
Ash insoluble in hydrochloric acid {2.8.1)
up to 6 mm thick; the outer surface is brownish-grey, with
fine longitudinal furrows and many reddish-brown lenticels, ASSAY
rounded or slightly split transversally; the inner surface is Liquid chromatography (2.2.29).
smooth, orange-brown. The fracture is fibrous. Test solution To 0.500 g of the powdered herbal drug (355)
B. Microscopic examination (2.5.23). The powder is ,
(2.9.72) add 50.0 mL of methanol R and weigh. Heat on a
brownish. Examine under a microscope using chloral hydrate water-bath under a reflux condenser for 1 h. Cool and weigh
solution R. The powder shows the following diagnostic again. Compensate for the loss of solvent with methanol R
characters: large sclereids up to 300 pm in diameter, single or and mix. Filter through a membrane filter (nominal pore size
in groups, with a very narrow lumen; fragments of brownish 0.45 pm).
cork; ovoid parenchymatous cells. Reference solution (a) Dissolve 10.0 mg of esculin CRS in the
c. To 0.1 g of the powdered herbal drug (355) (2.9.72), add mobile phase and dilute to 50.0 mL with the mobile phase.
10 mL of water R previously heated to 60 °C. Allow to stand Reference solution (b) Dissolve 10.0 mg of esculetin CRS in
for 2 min and filter. Examined in ultraviolet light at 365 nm, 10 mL of acetonitrile R and dilute to 50.0 mL with the
the solution shows an intense blue fluorescence that fades mobile phase.
considerably after the addition of 2 mL of hydrochloric acid R. Reference solution (c) Mix 5.0 mL of reference solution (a)
D. Thin-layer chromatography (2.2.27). with 3.0 mL of reference solution (๖) and dilute to 10.0 mL
Test solution To 0.25 g of the powdered herbal drug (355) with the mobile phase.
(2.9.72) add 5 mL of methanol R. Heat in a water-bath at Column'.
60 °C for 1 min. Centrifuge and use the supernatant; filter, if — size: I = 0.15 m, 0 = 4.0 mm;
necessary. — stationary phase: end-capped octadecylsilyl silica gel for
Reference solution Dissolve 1 mg of esculin R and 1 mg of chromatography R (5 pm).
esculetin R in 2 mL of methanol R. Mobile phase acetonitrile R3 0.1 per cent V/V solution of
Plate TLC silica gel 7*254 plate R (5-40 pm) [or TLC silica gel phosphoric acid R (12:88 VIV).
F254 plate R (2-10 pm)]. Flow rate 0.75 mUmin.
Mobile phase anhydrous formic acid R, water R, ethyl acetate R Detection Spectrophotometer at 334 nm.
(10:10:80 VIVIV). Injection 10 pL.
Application 10 pL as bands of 15 mm [or 8 mm]. Run time 1.5 times the retention time of esculetin.
Development Over a path of 10 cm [or 6 cm]. Retention time Esculin = about 4.5 min; esculetin = about
Drying In air. 8.5 min.
Detection Treat with a solution containing 10 g/L of Identification of peaks Use the chromatogram obtained with
diphenylboric acid aminoethyl ester R and 50 g/L of macrogol reference solution (a) to identify the peak due to esculin and
400 R in methanol R. Examine in ultraviolet light at 365 nm. the chromatogram obtained with reference solution (b) to
Results See below the sequence of zones present in the identify the peak due to esculetin.
chromatograms obtained with the reference solution and the System suitability: reference solution (c):
test solution. Furthermore, other fluorescent zones may be — resolution: minimum 5.0 between the peaks due to esculin
present in the chromatogram obtained with the test solution. and esculetin.
Calcdate the percentage content of the sum of esculetin and
esculin using ±e following expression:
Top of the plate
Escdetin: a greenish-yellow A greenish-yellow fluorescent Al X 7712 X Pl X 0.5 A3 X 7713 X P2 X 0.3

fluorescent zone zone (esculetin)
A2 X 7711 A4 X 7711
A green fluorescent zone may be

present A1 = area of the peak due to esculin in the chromatogram
A blue fluorescent zone may be obtained with the test solution;
present A2 = area of the peak due to esculin in the chromatogram
obtained with reference solution (c);
Esculin: an intense blue An intense blue fluorescent zone Al = area of the peak due to esculetin in the
fluorescent zone (esculin) chromatogram obtained with the test solution;
A whitish-blue fluorescent zone A4 = area of the peak due to esculetin in the
chromatogram obtained with reference solution (c);
m2 = mass of esculin CRS used to prepare reference
IV-200 Fumitory 2016
m3 = mass of esculetin CRS used to prepare reference

Pl = percentage content of esculin in esculin CRS;
p2 = percentage content of esculetin in esculetin CRS.
--------------------------------------------------------------------------------------------- ------------- Ph Eur
Fumitory
Ph Eur_______________________________________________________ _
DEFINITION
Whole or fragmented, dried aerial parts of Fumaria
officinalis L. harvested in full bloom.
Content
protopine (C2oH19N05; Afr 353.4) (dried drug).
IDENTIFICATION
A. The hollow, angular stem is light green or greenish-
brown. The leaves are alternate, bipinnatisect with 2 or 3 leaf
segments, the ultimate lobes lanceolate or obovate; they are
greenish-blue and glabrous on both surfaces. The flowers are
small and occur in loose racemes; each has a short pedicel
and is subtended by a leafy bract; they are pink or purplish-
red, dark purple or brown at the apex; ±e calyx is short,
composed of 2 petalloid sepals and the corolla is tubular with
4 petals, the upper petal slightly spurred; there are 6 stamens Figure 1869.-1. - Illustration for identification test B of powdered
united by their filaments into 2 groups of 3. The greenish- herbal drug offumitory
brown, indehiscent fruitร are globular or keel-shaped,
truncated or slightly emarginate at the apex, and each Reference solution Dissolve 5 mg of protopine hydrochloride R
contains a small brown seed. and 5 mg of quinine R in 10 mL of methanol R.
B. Microscopic examination (2.8.23). The powder is green. Plate TLC silica gel plate R.
Examine under a microscope using chloral hydrate solution R. Mobile phase concentrated ammonia R} ethanol (96 per cent) R,
The powder shows the following diagnostic characters acetone R} toluene R (2:6:40:52 VIVIVIV).
(Figure 1869.-1): fragments of the leaf lamina (surface view
[D]) with the upper epidermis composed of irregularly
polygonal cells [Da], some of which contain microcrystals of Development Over a path of 15 cm.
calcium oxalate [Db], and underlying palisade parenchyma Drying In air.
[De]; marginal cells at the apex of the lamina elongated to Detection A Examine in ultraviolet light at 365 nm.
form blunt papillae [Dd], and with the lower epidermis [A] Results A See below the sequence of zones present in the
composed of cells having wavier walls [Aa] and underlying chromatograms obtained with the reference solution and the
spongy parenchyma [Ac]; anomocytic stomata (2.8.3) [Ab, test solution. Furthermore, other blue fluorescent zones are
De] on both surfaces; groups [G] of lignified fibres [Ga] and present in the chromatogram obtained with the test solution.
spiral [Gb], reticulate or bordered-pitted [B] vessels from the
stem; fragments of the epidermis of the petals [F] composed
of polygonal cells with sinuous or wavy anticlinal walls and Top of the plate
no papulae; spherical pollen grains [E], about 30 pm in
diameter, with a pitted exine and 6 large pores; fragments of
the fruit with polygonal cells with a thick, warty cuticle, from
the epicarp [H], and sinuous sclereids with thick and
channelled walls, from the endocarp [C].
Quinine: a blue fluorescent zone
A greenish-blue fluorescent zone
(2.9.12) add 15 mL of 0.05 M sulfuric acid and stir for
15 min. Filter. Dilute the filtrate to 20 mL with 0.05 M Reference solution Test solution
sulfuric acid. Add 1 mL of concentrated ammonia R and 10 mL
of ethyl acetate R. Stir and centrifuge. Collect the upper
organic layer. Repeat the extraction in the same manner. Detection B Treat with a mixture of potassium iodobismuthate
Collect the organic layers and dry over anhydrous sodium solution R2} acetic acid R and water R (1:2:10 VIVIV) until
sulfate k? Evaporate to dryness under reduced pressure. Take orange zones appear against a yellow background.
up the residue with 0.5 mL of methanol R. Results B See below the sequence of zones present in the
test solution. Furthermore, other less intense orange zones
2016 Garlic Powder FV-201
are present in the chromatogram obtained with the test CHARACTERS

solution. Appearance
Light yellowish powder.
Top of the plate
IDENTIFICATION
Protopine: an orange zone An orange zone (protopine) A. Examine under a microscope using chloral hydrate
2 orange zones solution R. The powder shows the following diagnostic
characters: numerous fragments of parenchyma and groups
of spiral or annular vessels accompanied by thin-walled
Quinine: an orange zone A faint orange zone
parenchyma.
Reference solution
Test solution To 1.0 g of garlic powder add 5.0 mL of
Test solution
methanol R, shake for 60 ร and filter.
Reference solution Dissolve 5 mg of alanine R in 10 mL of
TESTS water R and dilute to 20 mL with methanol R.
Cadmium (2.4.27) Plate TLC silica gel plate R.
Maximum 1.5 ppm.
Mobile phase glacial acetic acid R, propanol R, water R,
Loss on drying (2.2.22) anhydrous ethanol R (20:20:20:40 VIVIVIV).
Maximum 12.0 per cent, determined on 1.000 g of the Application 20 |1L of the test solution and 10 pL of ±e
powdered herbal drug (355) (2.9.72) by drying in an oven at reference solution, as bands.
105 °C for 2 h.
Total ash {2.4.16) Drying In air.
Detection Spray with a 2 g/L solution of ninhydrin R in a
ASSAY mixture of 5 volumes of glacial acetic acid R and 95 volumes
To 5.000 g of the powdered herbal drug (355) (2.9.72) add of butanol R and heat at 105-110 °C for 5-10 min; examine
5 mL of dilute ammonia R1 and 50 mL of ethyl acetate R. in daylight.
Shake for 15 min. Filter. Repeat the procedure in the same Results The chromatogram obtained with the reference
manner and combine the filtrates. Evaporate the filtrates to solution shows a violet zone (alanine) in its central third.
dryness under reduced pressure. Dissolve the residue by The chromatogram obtained with the test solution shows a
sonication for 10 min in 50 mL of 0.05 M sulfuric acid. Filter. violet or brownish-red zone similar in position to that in the
Dilute the filtrate to 100 mL with 0.05 M sulfuric acid. Adjust chromatogram obtained with the reference solution and
to pH 9-10 with concentrated ammonia R and then add corresponding to alliin; above and below this zone are other,
50 mL of ethyl acetate R. Shake gently. Collect the upper generally fainter, violet zones.
organic layer, after centrifugation if necessary. Repeat the
procedure in the same manner. Combine the organic layers TESTS
and dry over anhydrous sodium sulfate R. Evaporate to dryness Starch
under reduced pressure. Take up the residue with 100 mL of Examine the powdered herbal drug under a microscope using
anhydrous acetic acid R. Titrate with 0.02 M perchloric acid, water R. Add iodine solution Rl. No blue colour develops.
determining the end-point potentiometrically (2.2.20). Loss on drying (2.2.22)
1 mL of 0.02 M perchloric acid is equivalent to 7.068 mg of Maximum 7.0 per cent, determined on 1.000 g of the
protopine. powdered herbal drug by drying in an oven at 105 °C.
Calculate the percentage content of total alkaloids, expressed Total ash {2.4.16)
as protopine, using the following expression: Maximum 5.0 per cent.
ASSAY
n X 706.8
Liquid chromatography (2.2.29). Carty out the assay as quickly
m as possible.
ท = volume of 0.02 M perchloric acid used, in millilitres; Internal standard solution Dissolve 20.0 mg of butyl
m ะ= mass of the herbal drug to be examined, in parahydroxybenzoate CRS in 100.0 mL of a mixture of
milligrams. equal volumes of methanol R and water R.
_______________________________________________________________ Ph Eur
Test solution To 0.800 g of garlic powder add 20.0 mL of
water R and homogenise the mixture in an ultrasonic bath at
4 °C for 5 min. Allow to stand at room temperature for
30 min. Then centrifuge for 30 min. Dilute 10.0 mL of the
supernatant to 25.0 mL with a mixture of 40 volumes of a
Garlic Powder * ; 1 per cent VIV solution of anhydrous formic acid R and
60 volumes of methanol R (stock solution). Shake and
centrifuge for 5 min. Place 0.50 mL of the internal standard
Ph Eur_________________________________________ solution in a volumetric flask and dilute to 10.0 mL with the
DEFINITION stock solution.
Bulbs of Allium sativum L., cut, freeze-dried or dried at a Precolumn'.
temperature not exceeding 65 °C and powdered. — size: I = 20 mm, 0 = 4 mm,
— stationary phase: silanised octadecylsilyl silica gel for
Content
Minimum 0.45 per cent of allicin (C6H10OS2; Mr 162.3)
(dried drug).
IV-202 Gentian 2016
Column’. using chloral hydrate solution R. The powder shows the

— size-. I = 0.25 m, 0 = 4 mm, following diagnostic characters (Figure 0392.-1): fragments of
— stationary phase’, silanised octadecylsilyl silica gel for cork with polyhedral, thin-walled, yellowish-brown cells
chromatography R (5 pm). (surface view [E]); fragments of dermal tissue (transverse
Mobile phase Mix 40 volumes of a 1 per cent VIV solution of section [C]) consisting of thin-walled, yellowish-brown cork
anhydrous formic acid R and 60 volumes of methanol R. cells [Ca] and thick-walled collenchymatous cells
Flow rate 0.8 mL/min. (phelloderm) [Cb]; fragments of parenchyma (longitudinal
section [B], transverse section [D]) with moderately thick
walled cells containing droplets of oil [Ba, Da], small
Injection Loop injector, 1 pL of the internal standard solution prisms [Bb, Db] and minute needles of calcium
and 10 pL of the test solution. oxalate [Be, De]; isolated fragments of lignified vessels with
Calculate the percentage of allicin using the following spiral [H] or reticulate [G] thickening and up to 80 pm in
expression: diameter; fragments of xylem (longitudinal section [A],
transverse section [F]) consisting of vessels [Aa, Fa] and of
Si X 7722 X 22.75 moderately thick-walled parenchymatous cells containing
ร2 X 7721 droplets of oil [Ab, Fb].
ร1 = area of the peak due to allicin (principal peak) in

the chromatogram obtained with the test solution,
ร’2 = area of the peak due to butyl parahydroxybenzoate
solution,
พ1 = mass of the herbal drug to be examined, in grams,
m2 = mass of butyl parahydroxybenzoate in 100.0 mL
of the internal standard solution, in grams. 1 mg
of butylparahydroxybenzoate corresponds to 8.65
mg of allicin.
---------------------------------------------------------------------------------------------------------- Ph Eur
Gentian ** *
(Gentian Root, Ph. Eur. monograph 0392) ***
Preparations
Compound Gentian Infusion
Gentian Tincture
When Powdered Gentian is prescribed or demanded,
exception of Identification test A shall be dispensed or
supplied.
Ph Eur_______________________________________________________________
DEFINITION
Dried, fragmented underground organs of Gentiana lutea L.
CHARACTERS Figure 0392.-1. - Illustration for identification test B of powdered
Strong and persistent bitter taste. herbal drug of gentian root
IDENTIFICATION c. Thin-layer chromatography (2.2.27).
A. Gentian root occurs as single or branched subeylindrieal Test solution To 1.0 g of the powdered herbal drug (355)
pieces of various lengths (typically 5-15 cm) and usually (2.9.12) add 25 mL of methanol R, shake for 15 min and
5-40 mm in diameter. The surface is yellowish-brown or filter. Evaporate the filtrate to dryness under reduced
greyish-brown, and the colour of a transverse section is pressure, at a temperature not exceeding 50 °C. Take up the
yellowish or reddish-yellow, but not reddish-brown. The root residue with small quantities of methanol R so as to obtain
IS longitudinally wrinkled and bears occasional rootlet scars. 5 mL of a solution, which may contain a sediment.
The branches of the rhizome frequently bear a terminal bud Reference solution Dissolve 5 mg of hyperoside R and 5 mg of
and are always encircled by closely arranged leaf scars. phenazone R in 10 mL of methanol R.
The rhizome and root are brittle when dry and break with a Plate TLC silica gel F254 plate R.
short fracture but they absorb moisture readily to become Mobile phase water R, anhydrous formic acid R, ethyl formate R
flexible. The smoothed, transversely cut surface shows a (4:8:88 VIVIV).
bark, occupying about one-quarter of the radius, separated by
the well-marked cambium from an indistinctly radiate and
mainly parenchymatous xylem. Development In an unsaturated tank, over a path of 8 cm.
B. Microscopic examination (2.8.23). The powder is light Drying In air.
brown or yellowish-brown. Examine under a microscope Detection A Examine in น]traviolet light at 254 nm.
2016 Gentian Preparations IV-203

chromatograms obtained with the reference solution and the Compound Gentian Infusion
test solution. Furthermore, other zones may be present in the DEFINITION
chromatogram obtained with the test solution. Concentrated Compound Gentian 100 ml
Infusion
Water Sufficient to produce
Top of the plate
1000 ml
A prominent quenching zone The infusion complies with the requirements stated under Infusions.
Phenazone: a quenching zone
A weak quenching zone CONCENTRATED COMPOUND GENTIAN

(amarogentin) INFUSION
DEFINITION
Gentian, cut small and bruised 125 g
Hyperoside: a quenching zone A prominent quenching zone Dried Bitter-orange Peel, cut small 125 g
(gentiopicroside) Dried Lemon Peel, cut small 125 g
Reference solution Test solution Ethanol (25 per cent) 1200 ml
Detection B treat with a 100 g/L solution of potassium
hydroxide R in methanol R and then with a freshly prepared Macerate the Gentian, the Dried Bitter-orange Peel and the
2 g/L solution of fast blue B salt J? in a mixture of 50 volumes Dried Lemon Peel in a covered vessel for 48 hours with
of anhydrous ethanol R and 50 volumes of water R'} examine in 1000 ml of the Ethanol (25 per cent); express the liquid.
daylight. To the pressed marc add 200 ml of the Ethanol
(25 per cent), macerate for 24 hours, press and add the
liquid to the product of the first pressing. Allow to stand for
not less than 14 days; filter.
chromatogram obtained with the test solution. TESTS
Ethanol content
20 to 24% v/v, Appendix vni F.
Top of the plate
Total solids
A prominent dark violet zone Not less than 9.5% w/v, Appendix XI A.
A violet-red zone (amarogentin)
Gentian Tincture * **
Hyperoside: a brownish-red zone A weak light brown zone
(gentiopicroside) (Ph. Eur. monograph 1870) * **
Reference solution Test solution PhEis----------------------------------------------------------------------------------------------- ---------
DEFINITION
Tincture produced from Gentian root (0392).
TESTS
Other species of Gentiana PRODUCTION
Examine the chromatograms obtained in identification The tincture is produced from 1 part of the comminuted
test c, detection B. drug and 5 parts of ethanol (70 per cent V7P) by a suitable
Results The chromatogram obtained with the test solution procedure.
does not show violet zones immediately above the zone due CHARACTERS
to amarogentin. Appearance
Total ash (2.4.16) Yellowish-brown or reddish-brown liquid.
Maximum 6.0 per cent. It has a strong bitter taste.
Bitterness value (2.8.15) IDENTIFICATION
Minimum 10 000. Thin-layer chromatography (2.2.27).
Water-soluble extractive Test solution The tincture to be examined.
Minimum 33 per cent. Reference solution Dissolve 5 mg of phenazone R and 5 mg of
To 5.0 g of the powdered herbal drug (710) (2.9.12) add hyperoside R in 10 mL of methanol R.
200 mL of boiling water R. Allow to stand for 10 min,
Plate TLC silica gel F254 plate K-
shaking occasionally. Allow to cool, dilute to 200.0 mL with
Mobile phase water R, anhydrous formic acid Ry ethyl formate R
water R and filter. Evaporate 20.0 mL of the filtrate to
dryness on a water-bath. Dry the residue in an oven at (4:8:88 VIVIV).
100-105 °C. The residue weighs a minimum of 0.165 g. Application 20 pL, as bands.
_______________________ _______________________________________ PhEur Application
Development Over a path of 8 cm, in an unsaturated tank.
Drying In air.
IV-204 Gentian Preparations 2016
Results A See below the sequence of the zones present in the The mixture complies with the requirements stated under Oral
chromatograms obtained with the reference solution and the Liquids and with the following requirements.
test solution. Furthermore, other zones may be present in the Content of hydrochloric acid, HC1
chromatogram obtained with the test solution. 0.48 to 0.56% w/v.
ASSAY
Top of the plate
To 10 mL add 10 mL of water, adjust the pH to between 5.0
A prominent quenching zone and 6.0 with 2m sodium hydroxide and dilute to 25 mL with
Phenazone ะ a quenching zone water. Add 7 5 mL of acetate buffer pH 5.0 and titrate with
0.1m silver nitrate zs* determining the end point
A weak quenching zone
(amarogentin)
potentiometrically using a silver indicator electrode and a
glass reference electrode and stirring throughout the titration.
Each mL of 0.1m silver nitrate vs is equivalent to 3.646 mg
ofHCl.
Hyperoside: a quenching zone A prominent quenching zone
(gentiopicroside)
Detection B Spray with a 10 per cent v/v solution of

Alkaline Gentian Mixture
Alkaline Gentian Oral Solution
potassium hydroxide R in methanol R and then with a freshly
prepared 2 g/L solution of fast blue B salt R in a mixture of DEFINITION
ethanol R and water R (50:50 V/V). Examine in daylight. Alkaline Gentian Mixture is an oral solution containing
Residts B See below the sequence of the zones present in the 10% v/v of Concentrated Compound Gentian Infusion and
chromatograms obtained with the reference solution and the 5% w/v of Sodium Bicarbonate in a suitable vehicle.
test solution. Furthermore, other zones may be present in the Extemporaneous preparation
chromatogram obtained with the test solution. It is recently prepared according to the following formula.
Concentrated Compound Gentian 100 mL
Top of the plate Infusion
Sodium Bicarbonate 50 g
A prominent dark violet zone
Double-strength Chloroform Water 500 mL
A violet-red zone (amarogentin) Water Sufficient to produce
1000 mL
The mixture complies with the requirements stated under Oral
Hyperoside: a brownish-red zone A weak light brown zone
(gentiopicroside) Content of sodium bicarbonate, NaHCO3
Reference solution Test solution 4.75 to 5.25% w/v.
ASSAY
To 10 mL of the mixture add 100 mL of water and 25 mL
TESTS
of 0.5m hydrochloric acid zs, boil for 10 minutes and titrate
Ethanol content (2.9.10) the excess of hydrochloric acid with 0.5m sodium hydroxide
62 per cent V!V to 67 per cent v/v.
zs using 0.5 mL of methyl red solution as indicator. Each mL
Bitterness value (2.8. IS) of 0.5m hydrochloric acid zs is equivalent to 42.00 mg of
Minimum 1000. NaHCO3.
Minimum 5.0 per cent m/m, determined on 3.00 g.
_______________________________________________________________ Ph Eur
Ginger * $
Preparation
Acid Gentian Mixture Strong Ginger Tincture
Acid Gentian Oral Solution Ginger may be known in commerce as unbleached ginger.
DEFINITION When Powdered Ginger is prescribed or demanded, material
Acid Gentian Mixture is an oral solution containing 10% v/v complying with the appropriate requirements below shall be
of Concentrated Compound Gentian Infusion and 5% v/v of dispensed or supplied.
Dilute Hydrochloric Acid in a suitable vehicle. Ph Eur___________ _ _________________________________________________
Extemporaneous preparation DEFINITION
It is recently prepared according to the following formula. Dried, whole or cut rhizome of Zingiber officinale Roscoe,
Concentrated Compound Gentian 100 mL with the cork removed, either completely or from the wide,
Infusion flat surfaces only.
Dilute Hydrochloric Acid 50 mL
Double-strength Chloroform Water 500 mL Content
Water Sufficient to produce Minimum 15 mUkg of essential oil (anhydrous drug).
1000 mL
2016 Ginger Preparations IV-205
CHARACTERS
Characteristic aromatic odour.
Spicy and burning taste.
IDENTIFICATION
A. The rhizome is laterally compressed, bearing short,
flattened, obovate oblique branches on the upper side, each
sometimes having a depressed scar at the apex; the whole
rhizomes are about 5-10 cm long, 1.5-3 cm or 4 cm wide
and 1-1.5 cm thick, sometimes split longitudinally.
The scraped rhizome with a light-brown external surface
shows longitudinal striations and occasional loose fibres;
the outer surface of the unscraped rhizome varies from pale
to dark brown and is more or less covered with cork that
shows conspicuous, narrow, longitudinal and transverse
ridges; the cork readily exfoliates from the lateral surfaces but
persists between the branches. The fracture is short and
starchy with projecting fibres. The smoothed transversely cut
surface exhibits a narrow cortex separated by an endodermis
from a much wider stele; it shows numerous, scattered,
fibrovascular bundles and abundant scattered oleoresin cells
with yellow contents. The unscraped rhizome shows, in
addition, an outer layer of dark brown cork.
B. Reduce to a powder (355) (2.9.72). The powder is pale
yellow or brownish. Examine under a microscope using
diagnostic characters (Figure 1522.-1): groups of large, thin
walled, septate fibres, with one wall frequently dentate [C, D,
G]; fragments [K] containing vessels with reticulate
thickening [Ka] often accompanied by narrow, thin-walled
cells containing brown pigment [Kb] and amyliferous Figure 1522.-1. - Illustration for identification test B of powdered
parenchyma [Kc]; abundant reticulate vessels, fairly large, herbal drug of ginger
isolated [H, L]; abundant thin-walled parenchyma of the
resorcinol and citral in the chromatogram obtained with the
ground tissue [J, M], some cells containing brown
reference solution, 2 other less intense violet zones
oleoresin [Ja]; fragments of brown cork, usually seen in
(shogaols); other zones may be present.
surface view [F] but sometimes in transverse section [E].
Examine under a microscope using a 50 per cent VIV TESTS
solution of glycerol R. The powder shows abundant starch Water (2.2.13)
granules, simple, flattened, oblong or oval or irregular, up to Maximum 100 ml/kg, determined by distillation on 20.0 g
about 50 pm long and 25 pm wide, with a small point hilum of the powdered herbal drug (710) (2.9.72).
situated at the narrower end; sometimes, granules show faint, Total ash (2.4.16)
transverse striations, and may be free [A], agglomerated [B] Maximum 6.0 per cent.
or included in parenchymatous cells (Kc).
ASSAY
c. Thin-layer chromatography (2.2.27). Essential oil (2.8.12)
Test solution To 1.0 g of the powdered herbal drug (710) Use 20.0 g of the freshly, coarsely powdered herbal drug, a
(2.9.72) add 5 mL of methanol R. Shake for 15 min and 1000 mL round-bottomed flask, 10 drops of liquid paraffin R
filter. or other antifoam, 500 mL of water R as distillation liquid
Reference solution Dissolve 10 pL of citral R and 10 mg of and 0.5 mL of xylene R in the graduated tube. Distil at a rate
resorcinol R in 10 mL of methanol R. Prepare the solution of 2-3 mL/min for 4 h.
immediately before use. __________________________________________________ _ _______ Ph Eur
Mobile phase hexane R, ether R (40:60 VIV).
Development In an unsaturated tank, over a path of 15 cm. strong Ginger Tincture
Drying In air. Ginger Essence
Detection Spray with a 10 g/L solution of vanillin R in sulfuric
DEFINITION
acid R and examine in daylight while heating at 100-105 °C Ginger, in moderately coarse powder 500 g
for 10 min. Ethanol (90 per cent) Sufficient to produce
Results The chromatogram obtained with the reference 1000 ml
solution shows in the lower half an intense red zone
(resorcinol) and in the upper half 2 violet zones (citral);
the chromatogram obtained with the test solution shows The following directions apply.
below the zone due to resorcinol in the chromatogram Prepare by percolation. Appendix XI F.
obtained with the reference solution 2 intense violet zones
(gingerols) and in the middle, between the zones due to
IV-206 Ginger Preparations 2016
The tincture complies with the requirements for Tinctures stated and stomata [Ab] about 60 pm, wide, deeply sunken with
under Extracts and with the following requirements. 6-8 subsidiary cells; fragments of vascular tissue from the
TESTS petiole and veins [C] with xylem [Ca] and parenchyma,
Ethanol content some cells containing abundant cluster crystals of calcium
80 to 88% v/v, Appendix vm F, Method HL oxalate of various sizes [Cb].
Dry residue
2.0 to 3.0% w/v.
Relative density
Weak Ginger Tincture

DEFINITION
Strong Ginger Tincture 200 ml
Ethanol (90 per cent) Sufficient to produce 1000 ml
The tincture complies with the requirements for Tinctures stated
under Extracts and with the following requirements.
TESTS
Ethanol content
86 to 90% v/v, Appendix vm F, Method in.
Dry residue
Not less than 0.4% w/v. Use 10 ml.
Relative density
Ginkgo Leaf f \

Preparation
Ginkgo Leaf Dry Extract, Refined and Quantified
Ph Eur_______________________________________________________________
herbal drug of ginkgo leaf
DEFINITION c. Thin-layer chromatography (2.2.27).
Whole or fragmented, dried leaf of Ginkgo biloba L. Test solutioti To 2.0 g of the powdered herbal drug (710)
Content (2.9.12) add 10 mL of methanol R. Heat in a water-bath at
Not less than 0.5 per cent of flavonoids, expressed as flavone 65 °C for 10 min. Shake frequently. Allow to cool to room
glycosides (Mr 757) (dried drug). temperature and filter.
IDENTIFICATION Reference solution Dissolve 1.0 mg of chlorogenic acid R and
A. The leaf is greyish or yellowish-green or yellowish-brown. 3.0 mg of rutin R in 20 mL of methanol R.
The upper surface is slightly darker than the lower surface. Plate TLC silica gel plate R.
The petioles are about 4-9 cm long. The lamina is about Mobile phase anhydrous formic acid R) glacial acetic acid R)
4-10 cm wide, fan-shaped, usually bilobate or sometimes water R, ethyl acetate R (7.5:7.5:17.5:67.5 VIVIVIV).
undivided. Both surfaces are smooth, and the venation Application 20 |1L as bands.
dichotomous, the veins appearing to radiate from the base;
they are equally prominent on both surfaces. The distal
margin is incised, irregularly and to different degrees, and Drying Kt 100-105 °C.
irregularly lobate or emarginate. The lateral margins are Detection Spray the warm plate with a 10 g/L solution of
entire and taper towards the base. diphenylboric acid aminoethyl ester R in methanol R, then with
B. Reduce to a powder (355) (2.9.12). The powder is greyish the same volume of a 50 g/L solution of niacrogol 400 R in
or yellowish-green or yellowish-brown. Examine under a methanol R‘} allow to dry in air for about 30 min and examine
microscope using chloral hydrate solution R. The powder in ultraviolet light at 365 nm.
shows the followmg diagnostic characters (Figure 1828.-1): Results See below the sequence of zones present in the
irregularly-shaped fragments of the lamina [A, B, D, E], with chromatograms obtained with the reference solution and the
the upper epidermis, in surface view [D] and transverse test solution. Furthermore, other weak fluorescent zones may
section [E], consisting of elongated cells with irregularly be present in the chromatogram obtained with the test
sinuous walls [Da], often accompanied by palisade solution.
parenchyma [Db], and the lower epidermis, in surface view
[A] and transverse section [B], consisting of small cells, with
a finely striated cuticle and each cell shortly papillose [Aa],
2016 Ginkgo Preparations FV-207
Top of the plate Time Mobile phase A Mobile phase B

A yellowish-brown fluorescent
zone 0- 1 60 40
A green fluorescent zone 1 - 20 60 -> 45 40 -> 55
2 yellowish-brown fluorescent 20 - 21 45 ■> 0 55 -> 100

zones
21 - 25 0 100
An intense light blue fluorescent
zone sometimes overlapped by a
greenish-brown fluorescent zone
Chlorogenic acid: a light blue Flow rate 1.0 mL/min.
fluorescent zone
Detection spectrophotometer at 370 nm.
Injection 10 pL.
Rutin: a yellowish-brown 2 yellowish-brown fluorescent Relative retention With reference to quercetin (retention
fluorescent zone zones
time = about 12.5 min): kaempferol = about 1.4;
isorhamnetin = about 1.5.
A yellowish-brown fluorescent System suitability:
zone
Reference solution Test solution kaempferol and isorhamnetin.
Do not take into account peaks eluting before the quercetin
TESTS peak or after the isorhamnetin peak in the chromatogram
Maximum 5 per cent of stems and 2 per cent of other Calculate the percentage content of flavonoids, expressed as
foreign matter. flavone glycosides, using the following expression:
Maximum 11.0 per cent, determined on 1.000 g of the 2 x Fl X 7711 X 2.514 X p
powdered herbal drug (355) (2.9.12) by drying in an oven at Fl X 7712
105 °C for 2 h.
Total ash (2.4.16) F\ = sum of the areas of all the considered peaks in the
Maximum 11.0 per cent. chromatogram obtained with the test solution;
F2 = area of the peak due to quercetin in the
ASSAY chromatogram obtained with the reference
Flavonoids solution;
Liquid chromatography (2.2.29). m1 ะ= mass of quercetin used to prepare the reference
Test solution Heat 2.500 g of the powdered herbal drug (710) solution, in grams;
(2.9.12) in 50 mL of a 60 per cent VIV solution of acetone R m2 = mass of the herbal drug to be examined used to
under a reflux condenser for 30 min. Filter and collect the prepare the test solution, in grams;
filtrate. Extract the drug residue a 2nd time in the same p = percentage content of anhydrous quercetin in
manner, using 40 mL of a 60 per cent VIV solution of quercetin dihydrate R.
acetone R and filter. Collect the filtrates and dilute to _____________________________________________________________ PhEur
100.0 mL with a 60 per cent VIV solution of acetone R.
Evaporate 50.0 mL of the solution to eliminate the acetone
and transfer to a 50.0 mL vial, rinsing with 30 mL of
methanol R. Add 4.4 mL of hydrochloric acid Rl, dilute to
50.0 mL with water R and centrifuge. Place 10 mL of the Refined and Quantified Ginkgo * *
supernatant in a 10 mL brown-glass vial. Close with a rubber
seal and an aluminium cap and heat on a water-bath for Dry Extract *****
25 min. Allow to cool to room temperature. (Ph. Eur. monograph 1827)
Reference solution Dissolve 10.0 mg of quercetin dihydrate R in PhEur______________________________________________________________
20 mL of methanol R. Add 15.0 mL of dilute hydrochloric DEFINITION
acid R and 5 mL of water R and dilute to 50.0 mL with Refined and quantified dry extract produced from Ginkgo
methanol R.
leaf (1828)
Column'.
Content
— size: z = 0.125 m, 0 = 4 mm;
— flavonoids, expressed as flavone glycosides (MT 756.7):
22.0 per cent to 27.0 per cent (dried extract);
(5 pm);
— bilobalide: 2.6 per cent to 3.2 per cent (dried extract);
— ginkgolides A} B and C: 2.8 per cent to 3.4 per cent (dried
Mobile phase: extract);
— mobile phase A: 0.3 g/L solution of phosphoric acid R — ginkgolic acids: maximum 5 ppm (dried extract).
adjusted to pH 2.0;
— mobile phase B: methanol Rj PRODUCTION
The extract is produced from the herbal drug by an
appropriate procedure using organic solvents and their
mixtures with water, physical separation steps as well as other
suitable processes.
IV-208 Ginkgo Preparations 2016
CHARACTERS acid R and 5 mL of water R and dilute to 50.0 mL with

Appearance methanol R.
Bright yellow-brown, powder or friable mass. Column'.
IDENTIFICATION — size: I = 0.125 m, 0 ะ= 4 mm;
Thin-layer chromatography (2.2.27). — stationary phase: octadecylsilyl silica gel for chromatography R
(5 pm);
Test solution Dissolve 20.0 mg of the extract to be examined
— temperature: 25 cc.
in 10 mL of a mixture of 2 volumes of water R and
8 volumes of methanol R. Mobile phase:
— mobile phase A: 0.3 g/L solution of phosphoric acid R
adjusted to pH 2.0;
3.0 mg of rutin R in 20 mL of methanol R.
— mobile phase B: methanol Rj
Plate TLC silica gel plate R (5-40 pm) or [TLC silica gel
plate R (2-10 pm)].
Mobile phase anhydrous formic acid R, glacial acetic acid R, (min) (per cent V/V) (per cent V/V)
water R, ethyl acetate R (7.5:7.5:17 5:67 5 VIV/V/V). 0- 1 60 40
Application 20 pL [or 5 pL], as bands. 1 - 20 60 -> 45 40 -> 55
20 - 21 45 -> 0 55 -> 100
Drying 100-105 °C.
21 - 25 0 100
Detection Spray the plate whilst still hot with a 10 g/L
solution of diphenylboric acid aminoethyl ester R in methanol R}
then spray with a 50 g/L solution of macrogol 400 R in Flow rate 1.0 mL/min.
methanol Rj allow to dry in air for about 30 min and examine Detector Spectrophotometer at 370 nm.
in ultraviolet light at 365 nm. Injection 10 pL.
Results See below the sequence of zones present in the Relative retention With reference to quercetin (retention
chromatograms obtained with the reference solution and the time = about 12.5 min): kaempferol = about 1.4;
test solution. Furthermore, other, weaker fluorescent zones isorhamnetin = about 1.5.
System suitability Test solution:
solution.
kaempferol and isorhamnetin.
Top of the plate Determine the sum of the areas including all the peaks from
A blue fluorescent zone the peak due to quercetin to the peak due to isorhamnetin in
the chromatogram obtained with the test solution (see Figure
Several faint coloured zones
1827.-1).
A brown fluorescent zone flavone glycosides, using the following expression:
Fl X 7721 X 2.514 X p
An intense light blue fluorescent
F2 X 7722
zone sometimes overlapped by a
greenish-brown fluorescent zone
Chlorogenic acid ะ a light blue F] = sum of the areas of all the peaks from the peak
fluorescent zone due to quercetin to the peak due to isorhamnetin
One or two green fluorescent zones in the chromatogram obtained with the test
Rutin ะ a yellowish-brown One or two yellowish-brown solution;
fluorescent zone fluorescent zones F2 = area of the peak due to quercetin in the
Several green and yellowish-brown solution;
fluorescent zones m1 = mass of quercetin dihydrate CRS in the reference
solution, in grams;
Reference solution Test solution m2 = mass of the extract to be examined used to
p = percentage content of anhydrous quercetin in
ASSAY quercetin dihydrate CRS.
Flavonoids Terpene lactones
Liquid chromatography (2.2.29). Liquid chromatography (2.2.29).
Test solution Dissolve 0.200 g of the extract to be examined in Test solution Place 0.120 g of the extract to be examined in a
20 mL of methanol R. Add 15.0 mL of dilute hydrochloric 25 mL beaker and dissolve it in 10 mL of phosphate buffer
acid R and 5 mL of water R and dilute to 50.0 mL with solution pH 5.8 R by stirring. Transfer the solution into a
methanol R. Transfer 10.0 mL of this solution into a 10 mL chromatography column, about 0.15 m long and about
brown-glass vial. Close the vial with a tight rubber membrane 30 mm in internal diameter, containing 15 g of kieselguhr for
stopper and secure with an aluminium crimped cap. Heat on chromatography R. Wash the beaker with 2 quantities, each of
a water-bath for 25 min. Allow to cool to 20 °C. 5 mL, of phosphate buffer solution pH 5.8 R and transfer the
Reference solution Dissolve 10.0 mg of quercetin dihydrate CRS washings to the chromatography column. Allow to stand for
in 20 mL ofmethanol R. Add 15.0 mL of dilute hydrochloric 15 min. Elute with 100 mL of ethyl acetate R. Evaporate the
eluate to dryness at a pressure not exceeding 4 kPa in a
2016 Ginkgo Preparations LV-209
1. quercetin 2. kaempferol 3. isorhamnetin

Figure 1827.-1. - Chromatogram for the assay offlavonoids in refined and quantified ginkgo dry extract
water-bath at 50 °C. The residue of solvent is eliminated by System suitability:

an air-current. Take up the residue in 2.5 mL of the mobile — the chromatogram obtained with reference solution (b) is
phase. similar to the chromatogram supplied with ginkgo dry
Reference solution (a) Dissolve 30.0 mg of benzyl alcohol CRS extract for peak identification CRS.
in the mobile phase and dilute to 100.0 mL with the mobile Calculate ±e percentage content of bilobalide, using the
phase. following expression:
Reference solution (b) Place 0.120 g of the ginkgo dry extract for
peak identification CRS in a 25 mL beaker and dissolve it in Fl X mi X p X 0.025 X 1.20
10 mL of phosphate buffer solution pH 5.8 R by stirring, then Fs X 7712
proceed as described for the test solution.

Column'. Calcdate the percentage content of ginkgolide A, using the
— size:. I = 0.25 m, 0 = 4 mm; following expression:
— stationary phase', octylsilyl silica gel for chromatography R
(5 nm); F2 X mi X p X 0.025 X 1.22
— temperature: 25 °C. F5 X m2
Mobile phase tetrahydrofuran R, methanol R> water R
(10:20:75 VIVIV).
F3 X mi X p X 0.025 X 1.19
F5 X m2
Detection Refractometer maintained at 35 °C.
Injection 100 ||T- Calculate the percentage content of ginkgolide B, using the
Identification of peaks Use the chromatogram supplied with following expression:
ginkgo dry extract for peak identification CRS and the
chromatogram obtained with the reference solution (b) to F4 X 7ท1 X p X 0.025 X 1.27
identify the peaks due to bilobalide and ginkgolides A, B F5 X m2
and c.
Calculate the percentage content of ginkgolide c, using the
IV-210 Ginseng 2016
F\ = area of the peak due to bilobalide in the — symmeny factor. 0.8 to 2.0 for the peaks due to ginkgolic
chromatogram obtained with the test solution; acids C13, C15 and C17.
F2 = area of the peak due to ginkgolide A in the Calculate the content in parts per million of ginkgolic acids
chromatogram obtained with the test solution; expressed as ginkgolic acid Cl7, using the following
F3 = area of the peak due to ginkgolide B in the expression:
F4 = area of the peak due to ginkgolide c in the
Al X 7712 X p X 2000
A2 X 7711
F5 ะ= area of the peak due to benzyl alcohol in the
(a); A1 ะ= sum of the areas of the peaks due to the ginkgolic
พ1 = mass of benzyl alcohol CRS in reference solution acids Cl3, Cl5 and C17 in the chromatogram
(a), in grams; obtained with the test solution;
m2 = mass of the extract to be examined used to A2 = area of the peak due to ginkgolic acid C17 in the
prepare the test solution, in grams; chromatogram obtained with the reference
p = percentage content of benzyl alcohol in benzyl solution;
alcohol CRS. nil = mass of the extract to be examined used to
Calculate the percentage content of the sum of ginkgolides prepare the test solution, in grams;
A, B and c, using the following expression: m2 = mass of ginkgolic acids CRS used to prepare the
p = percentage content of ginkgolic acid c 17 in
+ (7b + Gc
ginkgolic acids CRS.
Ga = percentage content of ginkgolide A; __________ PhEif
(?B = percentage content of ginkgolide B;
Gc = percentage content of ginkgolide c.
Ginkgolic acids
Ginseng * โ
Test solution Dissolve 0.500 g of the powdered extract to be
examined in 8 mL of methanol R} sonicating if necessary, and (Ph. Eur. monograph 1523) *
dilute to 10.0 mL with the same solvent. Centrifuge if Ph Eur_______________________________________________ _ _____________
necessary.
DEFINITION
Reference solution Dissolve 10.0 mg of ginkgolic acids CRS in Whole or cut dried root, designated white ginseng; treated
8 mL of methanol R3 sonicating if necessary, and dilute to with steam and then dried, designated red ginseng, of Panax
10.0 mL with the same solvent. Dilute 2.0 mL of this ginseng c. A. Meyer.
solution to 10.0 mL with methanol R.
Content
Column:
Minimum 0.40 per cent for the sum of ginsenosides Rgl
— size: I = 0.25 m, 0 = 4.6 mm;
(C42H72014,2H2O; Mr 837) and Rbl (C54H92O23,3H2O;
— stationary phase: octylsilyl silica gel for chromatography R
Mr 1163) (dried drug).
(5 gm);
— temperature: 35 °C. IDENTIFICATION
Mobile phase: A. The principal root is fusiform or cylindrical, sometimes
— mobile phase A: dilute 0.1 mL of trifluoroacetic acid R to branched, up to about 20 cm long and 2.5 cm in diameter,
1000 mL with water R', and may be curved or markedly re-curved. The surface is
— mobile phase B: dilute 0.1 mL of trifluoroacetic acid R to pale yellow to cream in white ginseng, brownish-red in red
1000 mL with acetonitrile R', ginseng and shows longitudinal ridges. Stem scars may be
seen at the crown. The fracture is short. The transversely-cut
surface shows a wide outer zone with scattered orange-red
resin canals and a finely radiate inner region. The rootlets,
(min)
25 10 75-» 90
numerous in the lower part of white ginseng, are normally
0 - 30
absent in red ginseng.
30 - 35 10 90
35 - 36 10 ■> 25 90-> 75 yellow. Examine under a microscope using chloral hydrate
36-45 25 75 solution R. The powder shows the following diagnostic
characters: abundant fragments of thin-walled
parenchymatous cells and fragments of large secretory canals
Flow rate 1.0 mL/min. containing yellowish-brown resin, non-lignified tracheids and
Detection Spectrophotometer at 210 nm. partially-lignified vessels with spiral or reticulate thickening,
isolated or in groups; scattered cluster crystals of calcium
Injection 50 ]1L.
oxalate. Examine under a microscope using a mixture of
Identification of components Use the chromatogram supplied
with ginkgolic acids CRS and the chromatogram obtained with equal volumes of glycerol R and water R. The starch granules
are very abundant, simple or 2 or 3 compound, and range
the test solution to identify the peaks due to ginkgolic acids
from 1-10 pm in diameter. In red ginseng the starch granules
C13, C15 and Cl7. are often deformed and destroyed by treating with steam, or
System suitability, reference solution: may be absent.
— resolution-, minimum 2.0 between the peaks due to
ginkgolic acids Cl3 and Cl5;
2016 Ginseng IV-21
1. ginsenoside Rgl 3. ginsenoside Rf 5. ginsenoside Rc 7. ginsenoside Rd
2. ginsenoside Re 4. ginsenoside Rbl 6. ginsenoside Rb2
Figure 1523.-1. - Chromatogram for the assay of ginseng: test solution
Test solution Boil 1.0 g of the powdered herbal drug (355) TESTS
(2.9.72) under a reflux condenser with 10 mL of a Panax quinquefolium
70 per cent v/v solution of methanol 7? for 15 min. Filter Examine the chromatograms obtained in the assay.
after cooling and dilute to 10.0 mL with methanol R. The chromatogram obtained with the test solution shows a
Reference solution Dissolve 5.0 mg of aescin R and 5.0 mg of peak due to ginsenoside Rf (see Figure 1523.-1). In the case
arbutin R in 1 mL of methanol R. of a substitution by Panax quinquefolium no peak due to
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel ginsenoside Rf is present.
plate R (2-10 pm)]. Loss on drying (2.2.52)
Mobile phase ethyl acetate R, water R> butanol R Maximum 10.0 per cent, determined on 1.000 g of the
(25:50:100 V/V/V)) allow the mixture to separate for 10 min. powdered herbal drug (355) (2.9.72) by drying in an oven at
Use the upper layer. 105 °C.
Application 20 pL [or 4 pL] as bands. Total ash (2.4.16)
Development Over 10 cm [or 5 cm] in an unsaturated tank. Maximum 7.0 per cent.
Drying In air. Ash insoluble in hydrochloric acid (2.8.1)
Detection spray with anisaldehyde solution R and heat at
105-110 °C for 5-10 min. Examine in daylight. ASSAY
Results See below the sequence of the zones present in the Liquid chromatography (2.2.29).
chromatograms obtained with the reference solution and the Test solution Reduce about 50 g to a powder (355) (2.9.72).
test solution. Place 1.00 g of the powdered herbal drug and 70 mL of a
50 per cent v/v solution of methanol R in a 250 mL round-
bottomed flask. After adding a few grains of pumice, boil on
Top of the plate a water-bath under a reflux condenser for 1 h. After cooling,
Arbutin: a brown zone centrifuge and collect the supernatant. Treat the residue as
described above. Mix the collected liquids and evaporate to
dryness under reduced pressure at a temperature not
A violet zone (ginsenosides Rgl exceeding 60 °C. Take up the residue with 20.0 mL of a
+ Rg2) mixture of 20 volumes of acetonitrile R and 80 volumes of
A faint violet zone (ginsenoside Rf) water R. Dilute 2.0 mL of the solution to 10.0 mL with a
A violet zone (ginsenoside Re) mixture of 20 volumes of acetonitrile R and 80 volumes of
water R. Filter through a suitable membrane filter (nominal
pore size 0.45 pm) before injection.
A violet zone (ginsenoside Rd) Reference solution Dissolve 3.0 mg of ginsenoside Rgl R,
A faint violet zone 3.0 mg of ginsenoside Re R} 3.0 mg of ginsenoside Rf R and
3.0 mg of ginsenoside Rbl R in methanol R and dilute to
10.0 mL with the same solvent.
A violet zone (ginsenoside Rc) Column'.
Aescin: a grey zone A violet zone (ginsenosides Rbl
— size: I = 0.125 m, 0 = 4.6 mm;
+ Rb2) — stationary phase: octadecylsilyl silica gel for chromatography R
Reference solution Test solution (5 pm);
IV-212 Ginseng Dry Extract 2016
Mobile phase'.
— mobile phase A: water R adjusted to pH 2 with phosphoric Ginseng Dry Extract * *
acid R’j (Ph. Eur. monograph 2356) *■**
— mobile phase B: acetonitrile R-3
Ph Eur _________________________________________ ____________
DEFINITION
(min)
Dry extract produced from Ginseng (1523).
(per cent V/V) (per cent r/V)
0-8 80 20 Content
Minimum 4.0 per cent of the sum of ginsenosides Rbl, Rb2,
8-40 80 -> 60 20 -» 40
Rc, Rd, Re, Rf, Rgl and Rg2, expressed as ginsenoside Rbl
40-45 60-» 40 40 -» 60 (C54H02O23; Mr 1109) (dried extract).
45-47 40 —> 0 60 -> 100 PRODUCTION
47-52 0 100 The extract is produced from the herbal drug by a suitable
52-55 0 —> 80
procedure using a hydroalcoholic solvent equivalent in
100 ->20
strength to ethanol (35-90 per cent VIV).
CHARACTERS
Flow rate 1.0 mUmin. Appearance
Detection Spectrophotometer at 203 nm. Light brownish-yellow, hygroscopic powder or brittle mass.
Equilibration 20 min. IDENTIFICATION
Injection 20 pL. Thin-layer chromatography (2.2.27).
Elution order Order indicated in the composition of the Test solution Dissolve 0.15 g of the extract to be examined in
reference solution; record the retention times of these 10 mL of a 70 per cent VIV solution of methanol R.
substances. Reference solution Dissolve 0.15 g of ginseng dry extract HRS in
System suitability-, reference solution: 10 mL of a 70 per cent VIV solution of methanol R.
— resolution-, minimum 1.0 between the peaks due to Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
ginsenoside Rgl and ginsenoside Re. plate R (2-10 pm)].
Locate the peaks due to ginsenoside Rbl and Mobile phase ethyl acetate R3 water R, butanol R
ginsenoside Rgl in the chromatogram obtained with the test (25:50:100 VIVIV)-3 allow the phases to separate for 10 min
solution. and use the upper layer.
Calculate the percentage content of ginsenosides Rbl and Application 20 pL [or 4 pL] as bands of 10 mm [or 8 mm].
Rgl using the following expression:
Development Over a path of 10 cm [or 5 cm] in an
unsaturated tank.
Al X 7712 X Pl A2 X m3 X P2
Drying In air.
>เ3 X โท1 X 100 A4 X 7ท1 X 100
105-110 °C for 5-10 min; examine in daylight.
ฬ] = area of the peak due to ginsenoside Rbl in the Results See below the sequence of zones present in the
chromatogram obtained with the test solution, chromatograms obtained with the reference solution and the
A2 = area of the peak due to ginsenoside Rgl in the test solution. Furthermore, other faint zones may be present
chromatogram obtained with the test solution, in the chromatograms obtained with the test solution and the
A^ = area of the peak due to ginsenoside Rbl in the reference solution.
solution,
A4 = area of the peak due to ginsenoside Rgl in the Top of the plate
solution,
m1 = mass of the herbal drug to be examined, in grams,
m2 = mass of ginsenoside Rbl in the reference solution, A violet zone (ginsenosides Rgl A violet zone (ginsenosides
+ Rg2) Rgl + Rg2)
in milligrams,
A faint violet zone (ginsenoside Rf) A faint violet zone (ginsenoside Rf)
m3 = mass of ginsenoside Rgl in the reference solution,
in milligrams, A violet zone (ginsenoside Re) A violet zone (ginsenoside Re)
p1 = percentage content of ginsenoside Rbl in the
reagent,
A violet zone (ginsenoside Rd) A violet zone (ginsenoside Rd)
p2 = percentage content of ginsenoside Rgl in the
reagent. A faint violet zone A faint violet zone
__________________________________________________ Ph Eur A violet zone (ginsenoside Rc) A violet zone (ginsenoside Rc)
A faint violet zone A faint violet zone
A violet zone (ginsenosides Rbl A violet zone (ginsenosides

+ Rb2) Rbl + Rb2)
Several unresolved violet and Several unresolved violet and
greenish zones greenish zones
2016 Goldenrod LV-213
TESTS Flow rate 1.0 mL/min.

Loss on drying (2.8.17) Detection Spectrophotometer at 203 nm.
Injection 20 pL.
ASSAY Elution order Ginsenoside Rgl, ginsenoside Re,
Liquid chromatography (2.2.29). ginsenoside Rf, ginsenoside Rbl, ginsenoside Rg2,
Buffer solution Dissolve 3.5 g of disodium hydrogen phosphate ginsenoside Rc, ginsenoside Rb2, ginsenoside Rd; depending
dihydrate R and 7.2 g of potassium dihydrogen phosphate R in on the operating conditions and the state of the column,
water R and dilute to 1000 mL with the same solvent. ginsenoside Rbl may elute before or after ginsenoside Rg2.
Test solution Dissolve 0.100 g of the extract to be examined in Identification of peaks Use the chromatogram supplied with
the buffer solution and dilute to 10.0 mL with the buffer ginseng dry extract HRS and the chromatogram obtained with
solution. Prepare a ready-to-use sample-preparation cartridge reference solution (a) to identify the peaks due to
containing 0.50 g of octadecylsilyl silica gel (45 |im), using ginsenosides Rgl, Re, Rf, Rc, Rb2 and Rd; use the
5 mL of methanol R followed by 20 mL of water R. Apply chromatogram obtained with reference solution (b) to
5.0 mL of the solution to be analysed to the top of the identify the peak due to ginsenoside Rbl; use the
cartridge. Wash the cartridge with 20 mL of water R followed chromatogram obtained with reference solution (c) to identify
by 15 mL of a 30 per cent VIV solution of methanol R. the peak due to ginsenoside Rg2.
Discard the eluates after confirming that no ginsenosides are Relative retention With reference to ginsenoside Rb 1
present, otherwise repeat the preparation of the solution with (retention time = about 33 min): ginsenoside Rgl = about
another brand of cartridge where no ginsenosides are eluted 0.53; ginsenoside Re = about 0.54; ginsenoside
with a 30 per cent V/V solution of methanol R. Elute the Rf = about 0.88; ginsenoside Rg2 = about 0.98;
cartridge with 20 mL of methanol R'y collect the eluate. Under ginsenoside Rc = about 1.04; ginsenoside Rb2 = about 1.08;
reduced pressure, evaporate the eluate to dryness. Dissolve ginsenoside Rd = about 1.17.
the residue in 2.0 mL of methanol R. Filter through a suitable System suitability Reference solution (d):
membrane filter (nominal pore size 0.45 pm). — resolution: minimum 1.5 between the peaks due to
Reference solution (a) Dissolve 0.100 g of ginseng dry ginsenosides Rg2 and Rbl.
extract HRS in the buffer solution and dilute to 10.0 mL with Calculate the percentage content of the sum of
the buffer solution. Prepare a ready-to-use sample ginsenosides Rbl, Rb2, Rc, Rd, Re, Rf, Rgl and Rg2,
preparation cartridge containing 0.50 g of octadecylsilyl silica expressed as ginsenoside Rbl, using the following expression:
gel (45 pm), using 5 mL of methanol R followed by 20 mL of
water R. Apply 5.0 mL of the solution to be analysed to the Al X 7712 X p X 0.8
top of the cartridge. Wash the cartridge with 20 mL of A2 X 7711
water R followed by 15 mL of a 30 per cent VIV solution of
methanol R. Discard the eluates after confirming that no A1 = sum of the areas of the peaks due to ginsenosides
ginsenosides are present, otherwise repeat the preparation of Rbl, Rb2, Rc, Rd, Re, Rf, Rgl and Rg2 in the
the solution with another brand of cartridge where no chromatogram obtained with the test solution;
ginsenosides are eluted with a 30 per cent VIV solution of A2 = area of the peak due to ginsenoside Rbl in the
methanol R. Elute the cartridge with 20 mL of methanol R’i chromatogram obtained with reference solution
collect the eluate. Under reduced pressure, evaporate the
(b);
eluate to dryness. Dissolve the residue in 2.0 mL of พ1 = mass of the extract to be examined used to
methanol R. Filter through a suitable membrane filter prepare the test solution, in grams;
(nominal pore size 0.45 pm). m2 = mass of ginsenoside Rbl CRS used to prepare
Reference solution (b) Dissolve 3.0 mg of ginsenoside Rbl CRS reference solution (b), in grams;
in methanol R and dilute to 5.0 mL with the same solvent. p = percentage content of ginsenoside Rbl in
Reference solution (c) Dissolve 3.0 mg of ginsenoside Rg2 R in ginsenoside Rbl CRS.
methanol R and dilute to 5.0 mL with the same solvent. _______________________________________ _ _____________________ Ph Eur
Reference solution (d) Dilute 1.0 mL of reference solution (b)
to 2.0 mL with reference solution (c).
Column'.
— size: I = 0.125 m, 0 = 4.6 mm;
— stationary phase: octadecylsilyl silica gel for chromatography R Goldenrod ** *
(5 pm); (Ph. Eur. monograph 1892) ***
Ph Eur---------------------------------------------------- - ---------------------------------------------------
Mobile phase:
— mobile phase A: water R adjusted to pH 2 with phosphoric DEFINITION
acid Ry Whole or cut, dried, flowering aerial parts of Solidago gigantea
— mobile phase B: acetonitrile Rl'y Ait. or Solidago canadensis L.J their varieties or hybrids and/or
mixtures of these.
Time Mobile phase A Mobile phase B Content
(min) (per cent V/V) (per cent V/V) Minimum 2.5 per cent of flavonoids, expressed as hyperoside
0-8 80 20 (C21H20O12; 464.4) (dried drug).
8 - 40 80 -> 60 20-» 40 IDENTIFICATION
40 - 45 60-» 40 40-» 60 A. The stems are greenish-yellow or greenish-brown, partly
tinted reddish, roundish, more or less conspicuously grooved,
45-47 40 -» 0 60 -» 100
glabrous and smooth in the lower part, slightly or densely
IV-214 Goldenrod 2016
pubescent in the upper part. They are solid with a whitish a few rare fragments of the ovary with twin covering
pith. trichomes [D].
The leaves are green, sessile, lanceolate, with a serrate c. Thin-layer chromatography (2.2.27).
margin, 8-12 cm long and about 1-3 cm wide, the upper Test solution To 0.75 g of the powdered herbal drug (355)
surface is green and more or less glabrous, the lower surface (2.9.12) add 5 mL of methanol R and boil in a water-bath
is greyish-green and pubescent, especially on the veins. under a reflux condenser for 10 min. Cool and filter.
The inflorescence consists of a number of unilateral, curved
Reference solution Dissolve 1.0 mg of chlorogenic acid Ry
racemes which together form a pyramidal panicle at the end
2.5 mg of quercitrin R and 2.5 mg of rutin R in 10 mL of
of the stems.
methanol R.
Mobile phase anhydrous formic acid Ry water R) methyl ethyl
ketone Ry ethyl acetate 7? (6:6:18:30 VIVIVIV).
Application 20 |1L of the test solution and 10 |1L of the
Detection treat with a 10 g/L solution of diphenylboric acid
solution of macrogol 400 R in methanol R. Allow to stand for
30 min. Examine in ultraviolet light at 365 nm.
Top of the plate
A bluish-green fluorescent zone
Quercitrin: a yellowish-brown A faint to intense yellowish-brown

fluorescent zone fluorescent zone (quercitrin)
A more or less intense

yellowish-brown zone
Figure 1892.-1 - Illustration for identification test B of fluorescent zone (chlorogenic acid) and/or a
powdered herbal drug ofgoldenrod yellow fluorescent zone
Rutin: an orange fluorescent zone A faint to intense yellowish-brown
fluorescent zone (rutin)
Each capitulum has an involucre composed of linear-
lanceolate, imbricated yellowish-green bracts, surrounding a
single row of yellow ligulate florets about the same length as Reference solution Test solution
the involucre; yellow, radially arranged tubular florets, as
long as, or longer, than the ligulate florets; a brownish
inferior ovary surmounted by a white pappus of silky hairs. TESTS
B. Microscopic examination (2.8.23). The powder is green or Foreign matter (2.8.2)
greyish-green. Examine under a microscope using chloral Maximum 5 per cent of brownish parts and maximum
hydrate solution R. The powder shows the following diagnostic 2 per cent of other foreign matter.
characters (Figure 1892.-1): pappus bristles, usually broken, Loss on drying (2.2.32)
consisting of multiseriate trichomes composed of elongated Maximum 10 per cent, determined on 0.500 g of the
cells with the tips free from the surface and forming pointed powdered herbal drug (355) (2.9.12) by drying in an oven at
projections over the entire length [J]; fragments of the 105 °C for 2 h.
mesophyll [F] with vascular bundles [Fa], oil glands [Fb] Total ash (2.4.16)
and palisade parenchyma [Fc]; fragments of the leaf Maximum 7.0 per cent.
epidermises [E, H] with sinuous or wavy-walled cells
accompanied by palisade parenchyma [Ea] (upper
epidermis), with whip-like, multicellular covering trichomes
[Eb] and anomocytic stomata (mainly on the lower ASSAY
epidermis) (2.8.3) [Ha]; multicellular uniseriate covering Stock solution In a 100 mL round-bottomed flask, introduce
trichomes from the stems and the margins of the leaves with 0.200 g of the powdered herbal drug (250) (2.9.72), add
up to 5 or 6 thick-walled cells [C]; in Solidago canadensis L., 1 mL of a 5 g/L solution of hexamethylenetetramine Ry 20 mL
covering trichomes with a terminal cell that may be bent at a of acetone R and 2 mL of hydrochloric acid Rl. Boil the
right angle [G]; fragments of the style [A] with short papillae mixture under a reflux condenser for 30 min. Filter the
[Aa] and long, slender papillae with wrinkled walls [Ab]; liquid through a small plug of absorbent cotton into a
pollen grains with 3 germinal pores and a spiny exine [B]; 100 mL flask. Add the absorbent cotton to the residue in the
2016 European Goldenrod IV-215
round-bottomed flask, extract with 2 quantities, each of margin. Each capitulum contains 6-12 widely separated
20 mL of acetone R, each time boiling under a reflux female ray florets, about twice as long as the bracts, and
condenser for 10 min. Allow to cool. Filter the combined about 10-30 hermaphrodite, tubular florets. All florets are
acetone extracts through a filter paper into a volumetric flask. yellow. The brown, inferior ovary tapers towards the base
Rinse the flask and the filter paper and dilute to 100.0 mL and has a ribbed surface, covered with scattered hairs; it is
with acetone R. Introduce 20.0 mL of the solution into a surmounted by a whitish pappus composed of smooth or
separating funnel, add 20 mL of water R and shake the rough, bristly hairs.
mixture with 1 quantity of 15 mL and then with 3 quantities,
each of 10 mL, of ethyl acetate R. Combine the ethyl acetate
extracts in a separating funnel, wash twice with 50 mL of
tuater R and filter the extracts over 10 g of anhydrous sodium
sulfate R into a volumetric flask. Dilute to 50.0 mL with ethyl
acetate Rj rinsing the separating funnel and the sodium
sulfate.
Test solution To 10.0 mL of the stock solution add 1.0 mL of
5 per cent VIV solution of glacial acetic acid R in methanol R.
Compensation solution Dilute 10.0 mL of the stock solution to
25.0 mL with a 5 per cent VIV solution of glacial acetic
Measure the absorbance of tile test solution (2.2.25) at
425 nm after 30 min by comparison with the compensation
solution.
A X 1.25
m
i.e. taking the value of the specific absorbance of hyperoside

to be 500.
A = absorbance measured at 425 nm;
m ะะะ mass of the herbal drug to be examined, in grams.
----------------------------------- --------------------------------------------------------------------- Ph Eur
European Goldenrod * * Figure 1893.-1. - Illustration for identification test B of powdered

herbal drug of European goldenrod
(Ph. Eur. monograph 1893) *** B. Microscopic examination (2.8.23). The powder is light
Ph fir __________________________________________________________ green. Examine under a microscope using chloral hydrate
DEFINITION solution R. The powder shows the following diagnostic
characters (Figures 1893.-1 and 1893.-2): fragments of the
Whole or fragmented, dried, flowering aerial parts of Solidago
upper epidermis of the leaf in surface view [B, H, M],
virgaurea L.
covered by a distinctly striated cuticle, composed of
Content polygonal cells with straight, beaded, thickened walls [Ba,
Minimum 0.5 per cent and maximum 1.5 per cent of Ma], uniseriate, multicellular covering trichomes [Ha] or
flavonoids, expressed as hyperoside (C2iH2o012; Mr 464.4) rounded, thick-walled, covering trichome scars with a pitted
(dried drug). lumen [Mb], and a few anomocytic stomata (2.8.3) [Bb]
IDENTIFICATION sometimes accompanied by underlying palisade
A. The stem is cylindrical, striated, the lower part often parenchyma [Be]; fragments of the lower epidermis of the
reddish-violet, sometimes entirely glabrous or pubescent with leaf in surface view [A, K, N] covered by a slightly striated
short, bent, apically directed hairs. The basal leaves are cuticle composed of cells with sinuous walls in the area of the
obovate or oblanceolate, with a serrate margin, and taper at lamina [Aa] or wi± more rigid walls near the veins [N],
the base into a long, winged petiole; the cauline leaves are numerous anomocytic stomata (2.8.3) [Ab], occasional
alternate, smaller than the basal leaves and more elliptical in glandular trichomes with a unicellular stalk and a unicellular
outline, with an entire or slightly toothed margin; they are head [Ka, Na], covering trichomes some of which are
sessile or with only a short petiole. Both surfaces of the leaves pennant-like [Ac, F], uniseriate, multicellular, with 1-3 thin
are glabrous or only slightly pubescent with a prominent walled basal cells [Fa], a flagella-like distal cell [Fb], and an
reticulate venation on the lower surface. The capitula form a enlarged, more or less rounded cell [Fc] between them,
tightly packed panicle. At the base of the pedicels there are others are uniseriate, multicellular (up to about 10 cells),
2 small, linear bracts with scarious margins. The involucre with thick, finely wrinkled walls and a rigid conical distal cell
consists of 2-4 rows of loosely arranged, imbricate bracts, in side view [E]; rare fragments from the ovary [G] bearing
each bract greenish-yellow with a smooth and shiny inner paired, covering trichomes with a distinctly pitted central wall
and a bifid apex in surface view [Ga] or in side view [Gb];
surface, the outer surface hairy or glabrous, with a scarious
IV-216 European Goldenrod 2016
vascular tissue from the stems [L] composed of vessels [La] TESTS
and groups of fibres [Lb]; fragments of the epidermis of the Foreign matter (2.8.2)
petals with a striated cuticle, through which run fine spiral Maximum 5 per cent of brown coloured matter and
vessels [ร], and bearing biseriate glandular trichomes in side maximum 5 per cent of other foreign matter.
view [P]; spherical pollen grains, with 3 germinal pores and a
Solidago gigantea Ait. and Solidago canadensis L
spiny exine [J]; abundant pappus hairs and their
fragments [C, D], multiseriate with the marginal cells
overlapping outwards; fragments of parenchyma [Q], some Test solution To 0.75 g of the powdered herbal drug (355)
showing cells containing small, isolated cluster crystals of (2.9.12) add 5 mL of methanol R and heat on a water-bath
calcium oxalate [Qa]; fragments of bracts [R] with a finely under a reflux condenser for 10 min. Cool and filter.
striated cuticle, polygonal cells [Ra], bearing pennant-like Reference solution Dissolve 1.0 mg of chlorogenic acid Ry
covering trichomes [Rb] and whose margin bears uniseriate, 2.5 mg of quercitrin R and 2.5 mg of rutin R in 10 mL of
multicellular covering trichomes [Rc]. methanol R.
Mobile phase anhydrous formic acid Ry water Ry methyl ethyl
ketone Ry ethyl acetate R (6:6:18:30 VIVI VIV).
Drying In air.
Detection Treat the plate with a 10 g/L solution of
with a 50 g/L solution of macrogol 400 R in methanol R.
Examine in ultraviolet light at 365 nm after 30 min.
shows no strong orange fluorescent zone similar in position
to the zone of quercitrin in the chromatogram obtained with
the reference solution.
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Stock solution Ina 100 mL round-bottomed flask, place
0.200 g of the powdered herbal drug (355) (2.9.12)y add
1 mL of a 5 g/L solution of hexamethylenetetramine Ry 20 mL
of acetone R and 2 mL of hydrochloric acid Rl. Boil the
mixture in a water-bath under a reflux condenser for 30 min.
Filter the liquid through a small plug of absorbent cotton
into a 100 mL flask. Add the absorbent cotton to the residue
Figure 1893.-2. - Illustration for identification test B of powdered in the round-bottomed flask and extract with 2 quantities,
herbal drug of European goldenrod each of 20 mL, of acetone Ry each time boiling under a reflux
condenser for 10 min. Allow to cool. Filter the combined
c. Thin-layer chromatography (2.2.27) as described in the
acetone extracts through filter paper, dilute to 100.0 mL with
test for Solidago gigantea Ait. and Solidago canadensis L.
acetone Ry rinsing the volumetric flask and the filter paper
Results See below the sequence of the zones present in the with acetone. Introduce 20.0 mL of the solution into a
chromatograms obtained with the reference solution and the suitable separating funnel, add 20 mL of water R and shake
test solution. Furthermore, other fluorescent zones may be the mixture with 1 quantity of 15 mL and then with
present in the chromatogram obtained with the test solution. 3 quantities, each of 10 mL, of ethyl acetate R. Combine the
ethyl acetate extracts in a separating funnel, wash twice with
Top of the plate 50 mL of water R and filter the extracts over 10 g of
anhydrous sodium sulfate R into a volumetric flask. Dilute to
50.0 mL with ethyl acetate Ry rinsing the separating funnel
Quercitrin: an orange fluorescent and the sodium sulfate.
zone Test solution To 10.0 mL of the stock solution add 1.0 mL of
Chlorogenic acid: a light blue A light blue fluorescent zone 5 per cent VIV solution of glacial acetic acid R in methanol R.
Compensation liquid Dilute 10.0 mL of the stock solution to
Rutin ะ an orange fluorescent zone An orange fluorescent zone 25.0 mL with a 5 per cent VIV solution of glacial acetic
(rutin)
After 30 min, measure the absorbance (2.2.25) of the test
Reference solution Test solution solution at 425 nm by comparison with the compensation
liquid.
2016 Goldenseal Root IV-217

A X 1,25
m
i.e. taking the specific absorbance of hyperoside to be 500.

A = measured absorbance at 425 nm;
พ = mass of the herbal drug to be examined, in grams.
———— ------------------------- ------------------------------------------------------Ph Eur
Goldenseal Root ** **
(Goldenseal Rhizome, Ph Eur monograph 1831) * **
Ph Eur______________
DEFINITION
Whole or cut, dried rhizome and root of Hydrastis
canadensis L.
Content
— hydrastine (C21H21NO6; Afr 383.4): minimum
2.5 per cent (dried drug);
— berberine (C20H18NO4; Mr 336.4): minimum
3.0 per cent (dried drug).
IDENTIFICATION
A. The rhizome is tortuous and knotty, about 5 cm long and Figure 1831.-1. - Illustration for identification test B of powdered
5-10 mm thick. The surface is yellowish or brownish-grey, herbal drug of goldenseal rhizome
irregularly wrinkled, and bears the remains of numerous Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
slender, wiry roots; stem bases and scale leaves occur on the plate R (2-10 pm)].
upper surface. The fracture is short and resinous. Mobile phase anhydrous formic acid R, water R, ethyl acetate R
The transversely cut surface is yellowish-brown and shows a (10:10:80 VIVIV).
fairly wide bark, a ring of 12-20 widely separated xylem
bundles and a large, central pith.
Development Over a pa± of 15 cm [or 6 cm].
greenish-yellow. Examine under a microscope using chloral Drying In air.
hydrate solution R. The powder shows the following diagnostic Detection Examine in ultraviolet light at 365 nm.
characters (Figure 1831.-1): abundant thin-walled fragments Results See below the sequence of zones present in the
of parenchyma [A, G, K]; occasional fragments of yellowish- chromatograms obtained with the reference solution and the
brown cork from the rhizome and roots, in surface view J] test solution. Furthermore, other fluorescent zones may be
or in transverse section [F]; groups of small vessels with present in the chromatogram obtained with the test solution.
conspicuous perforations in the oblique end walls [L] and
with simple or bordered, slit-shaped pits [B, D, E];
Top of the plate
infrequent groups of thin-walled, pined fibres [H], usually
found associated with the vessels; numerous ovoid or
spherical, orange-brown granular masses. Examine under a Berberine: a bright yellow A bright yellow fluorescent zone
microscope using a 50 per cent v/v solution of glycerol R. fluorescent zone (berberine)
The powder shows abundant starch granules [C], mostly Hydrastine: a deep blue A deep blue fluorescent zone
simple but sometimes compound with up to 4 components; fluorescent zone (hydrastine)
the granules are small, spherical or ovoid, up to about 10 pm
in diameter, occasionally with a small, rounded or slit-shaped A bright light blue fluorescent
hilum. zone (hydrastinine)
c. Thin-layer chromatography (2.2.27). A deep blue fluorescent zone
Test solution To 250 mg of the powdered herbal drug (180) Reference solution Test solution
(2.9.12) add 4 mL of a mixture of 20 volumes of water R and
80 volumes of methanol R. Sonicate for 10 min and filter.
Wash the residue with 2 quantities, each of 2 mL, of TESTS
methanol R. Combine the solutions and dilute to 20 mL with Loss on drying (2.2.52)
methanol R. Maximum 10.0 per cent, determined on 1.000 g of the
Reference solution Immediately before use, dissolve 5 mg of powdered herbal drug (180) (2.9.12') by drying in an oven at
hydrastine hydrochloride R and 5 mg of berberine chloride R in 105 °C for 2 h.
20 mL of methanol R. Total ash (2.4.16)
IV-218 Hamamelis Leaf 2016
Ash insoluble in hydrochloric acid (2.8.7) p = percentage content of hydrastine in hydrastine

Maximum 4.0 per cent. hydrochloride CRS or berberine in berberine chloride
ASSAY CRS.
Liquid chromatography (2.2.29). ___ ___________________________________________________________PhE'J-

(2.9J2) in a 100 mL round-bottomed flask, add 50 mL of a
1 per cent VIV solution of concentrated ammonia R in ethanol
(96 per cent) R and boil the mixture under a reflux condenser
for 30 min. Allow to cool to room temperature and filter the Hamamelis Leaf * *
liquid through a plug of absorbent cotton into a flask. (Ph. Eur. monograph 0909) **
Add the plug of absorbent cotton to the residue in the
Ph Eur_______________________________________________ _______________
round-bottomed flask and repeat the extraction with a further
2 quantities, each of 30 mL, of a 1 per cent v/v solution of DEFINITION
concentrated ammonia R in ethanol (96 per cent) R, each time Whole or cut, dried leaf of Hamamelis virginiana L.
boiling under a reflux condenser for 10 min and filtering Content
through a plug of absorbent cotton in the same flask as Minimum 3 per cent of tannins, expressed as pyrogallol
previously. Filter the combined filtrates through a filter paper (C6H6O3; Mr 126.1) (dried drug).
into a 250 mL round-bottomed flask, and rinse the flask and
the filter with 20 mL of a 1 per cent VIV solution of IDENTIFICATION
concentrated ammonia R in ethanol (96 per cent) R. Evaporate A. The leaf is green or greenish-brown, often broken,
the filtrate to dryness in vacuo in a water-bath at 55 °C. crumpled and compressed into more or less compact masses.
Dissolve the residue in 50.0 mL of the mobile phase. Dilute The lamina is broadly ovate or obovate; tire base is oblique
10.0 mL of this solution to 50.0 mL with the mobile phase. and asymmetric and the apex is acute or, rarely, obtuse.
The margins of the lamina are roughly crenate or dentate.
Reference solution Immediately before use, dissolve 10.0 mg of
The venation is pinnate and prominent on the abaxial
hydrastine hydrochloride CRS and 10.0 mg of berberine
surface. Usually, 4-6 pairs of secondary veins are attached to
chloride CRS in methanol R and dilute to 100.0 mL with the
the main vein, emerging at an acute angle and curving gently
same solvent.
to the marginal points where there are fine veins often at
Column'. right angles to the secondary veins.
— size'. I = 0.125 m, 0 = 4 mm;
— stationary' phase: end-capped octadecylsilyl silica gel for
brownish-green. Examine under a microscope using chloral
Mobile phase Dissolve 9.93 g of potassium dihydrogen characters (Figure 0909.-1): fragments of adaxial epidermis
phosphate R in 730 mL of water R, add 270 mL of with wavy anticlinal walls, in surface view [C, J], often
acetonitrile R and mix. accompanied by small, cylindrical cells of the palisade
Flow rate 1.2 mL/min. parenchyma, in surface view [Ja], or elongated, in transverse
Detection Spectrophotometer at 235 nm. section [F]; fragments of abaxial epidermis with stomata
Injection 10 pL. mainly paracytic (2.8.3), in surface view [B], which may be
accompanied by irregular-shaped cells of spongy7 mesophyll
[K, L]; star-shaped covering trichomes, either entire or
— elution order, order indicated in the composition of the
broken [A, D, M], composed of 4-12 unicellular branches
reference solution; record the retention times of these
that are united by their bases, elongated, conical and curved,
substances;
usually up to 250 pm long, thick-walled and with a clearly
visible lumen whose contents are often brown; fibres are
hydrastine and berberine.
lignified and thick-walled, isolated or in groups, and
Using the retention times determined from the accompanied by a sheath of prismatic calcium oxalate crystals
chromatogram obtained with the reference solution, locate in [N, P]; sclereids, frequently enlarged at 1 or both ends,
the chromatogram obtained with the test solution the 150-180 pm long, whole or fragmented [H]; fragments of
components of the reference solution. annular or spiral vessels [E]; isolated prisms of calcium
Calculate the percentage content of each alkaloid (hydrastine oxalate [G].
and berberine) using the following expression: c. Thin-layer chromatography (2.2.27).
™2*p. x 2.5 (2.9.72) add 10 mL of ethanol (60 per cent VIV) R, shake for
A2 X 7ท1 15 min and filter.
Reference solution (a) Dissolve 30 mg of tannic acid R in 5 mL
A1 = area of the peak due to hydrastine or berberine in of ethanol (60 per cent VIV) R.
the chromatogram obtained with the test solution; Reference solution (b) Dissolve 5 mg of gallic acid R in 5 mL
A2 = area of the peak due to hydrastine or berberine in of ethanol (60 per cent VIV) R.
the chromatogram obtained with the reference Plate TLC silica gel G plate R.
solution;
Mobile phase anhydrous formic acid R, water R, ethyl formate R
prepare the test solution, in grams; (10:10:80 VIVIV).
m2 = mass of hydrastine hydrochloride CRS or berberine Application 10 pL, as bands.
chloride CRS used to prepare the reference Development Over a path of 10 cm.
solution, in grams; Drying At 100-105 °C for 10 min, then allow to cool.
2016 Hawthorn Berries IV-219
Hawthorn Berries
PhEur___________________________________________________________
DEFINITION
Dried false fruits of Crataegus monogyna Jacq. (Lindm.) or
c. laevigata (Poir.) DC. (syn. c. oxyacantha L.) or their
hybrids or a mixture of these false fruits.
Content
Minimum 0.06 per cent of procyanidins, expressed as
cyanidin chloride (CisHijClOe; Mr 322.7) (dried drug).
IDENTIFICATION
A. The false fruit of c. monogyna is obovate or globular,
generally 6-10 mm long and 4-8 mm wide, reddish-brown or
dark red. The surface is pitted or, more rarely, reticulated.
The upper end of the fruit is crowned by the remains of
5 reflexed sepals surrounding a small, sunken disc with a
shallow, raised rim. The remains of the style occur in the
centre of the disc with tufts of stiff, colourless hairs at the
base. At the lower end of the fruit is a short length of pedicel
or, more frequently, a small, pale, circular scar where the
pedicel was attached. The receptacle is fleshy and encloses a
yellowish-brown, ovoid fruit with a hard, thick wall
containing a single, elongated, pale brown, smooth and shiny
seed.
The false fruit of c. laevigata is up to 13 mm long.
It contains 2-3 stony fruits, ventrally flattened, with short
hairs at the top. Frequently, in the centre of the disc of the
false fruit occur the remains of the 2 styles.
Figure 0909.-1. - Illustration for identification test B of powdered red. Examine under a microscope using chloral hydrate
herbal drug of hamamelis leaf solution R. The powder shows the following diagnostic
Detection Spray with ferric chloride solution R2 until bluish-grey characters (Figure 1220.-1): covering trichomes [F] from
zones (phenolic compounds) appear. inside the disc that are long, unicellular, frequently bent,
Results The chromatogram obtained with the test solution tapering to a point, with much thickened and lignified walls;
fragments of the red outer layer of the receptacle, in surface
shows in its lower third a principal zone similar in position to
the principal zone in the chromatogram obtained with view [G]; fragments of the inner layers of the receptacle [A],
some cells containing cluster crystals [Aa] or prisms [Ab] of
reference solution (a) and, in its upper part, a narrow zone
calcium oxalate; occasional fragments [J, K] including groups
similar in position to the principal zone in the chromatogram
of sclereids [Ka] and vascular bundles [Ja, Kb] associated
obtained with reference solution (b); the chromatogram
obtained with the test solution shows, in addition, several with rows of cells containing prisms of calcium oxalate [Jb,
slightly coloured zones in the central part. Kc]; fragments of the pericarp [B] consisting of parenchyma
including some cells containing cluster crystals of calcium
TESTS oxalate [Ba] and groups of sclereids of various sizes with
Foreign matter (2.8.2) numerous pits [Bb]; thick-walled sclereids [E, H], some
Maximum 7 per cent of stems and maximum 2 per cent of channelled (E), some with conspicuously branched
other foreign matter, determined on 50 g. channels (H); a few fragments of the testa [C] having an
Loss on drying (2.2.52) outer layer composed of hexagonal, mucilaginous cells [Ca]
iMaximum 10.0 per cent, determined on 2.000 g of the beneath which is a yellowish-brown pigment layer containing
powdered herbal drug (355) (2.9.12) by drying in an oven at numerous prisms of calcium oxalate [Cb]; parenchyma of the
105 °C for 4 h. endosperm and cotyledons consisting of cells containing
aleurone grains and globules of fixed oil [D].
Total ash (2.4.16)
Maximum 7.0 per cent. c. Thin-layer chromatography (2.2.27).
(2.9.12) add 10 mL of methanol R and heat on a water bath
at 65 °C for 5 min, shaking frequently. Allow to cool to
ASSAY room temperature and filter. Dilute the filtrate to 10 mL
Tannins (2.8.14) with methanol R.
Use 0.750 g of the powdered herbal drug (180) (2.9.12). Reference solution Dissolve 2 mg of chlorogenic acid R> 2 mg of
____________________ • __________________________________ Ph Eur caffeic acid Rj 5 mg of hyperoside R and 5 mg of rutin R in
20 mL of methanol R.
IV-220 Hawthorn Leaf and Flower 2016
and c. azarolus L.), which are characterised by the presence

of more than 3 hard stones.
105 cc for 2 h.
Total ash (2.4.16)
ASSAY
30 mL of ethanol (70 per cent V/V) R. Heat under a reflux
condenser for 30 min and filter. Wash the residue with
10.0 mL of ethanol (70 per cent V/V) R. Add to the filtrate
15.0 mL of hydrochloric acid R1 and 10.0 mL of water R.
Heat under a reflux condenser for 80 min. Allow to cool,
filter and wash the residue with ethanol (70 per cent VIV) R
until the filtrate is colourless. Dilute the filtrate to 250.0 mL
with ethanol (70 per cent V/V) R. Evaporate 50.0 mL of this
solution in a round-bottomed flask to about 3 mL and
transfer to a separating funnel. Rinse the round-bottomed
flask sequentially with 10 mL and 5 mL of water R and
transfer to the separating funnel. Shake the combined
solution with 3 quantities, each of 15 mL, of butanol R.
Combine the organic layers and dilute to 100.0 mL with
butanol R.
Measure the absorbance (2.2.25) of the solution at 555 nm.
Calculate the percentage content of procyanidins, expressed
as cyanidin chloride, using the following expression:
Figure 1220.-1. - Illustration for identification test B of powdered A X 500
herbal drug of hawthorn berries 1200 X m

Mobile phase anhydrous formic acid Ry water Ry methyl ethyl
ketone Ry ethyl acetate R (10:10:30:50 VIVIVIV). i.e. taking the specific absorbance of cyanidin chloride to be
1200.
Application 30 |1L of the test solution and 10 pL of the
reference solution, as bands. m = mass of the substance to be examined, in grams.
______________________________________________________________ Ph Eur
Detection spray whilst hot with a 10 g/L solution of
diphenylboric acid aminoethyl ester R in methanol Ry
subsequently spray with a 50 g/L solution of macrogol 400 R
in methanol R’y allow to dry in air for 30 min and examine in Hawthorn Leaf and Flower ** *
ultraviolet light at 365 nm. (Ph. Eur. monograph 1432) *
Preparations
solution shows in the lower half, in order of increasing Rp
Hawthorn Leaf and Flower Dry Extract
values, a yellowish-brown fluorescent zone (rutin), a light
blue fluorescent zone (chlorogenic acid) and a yellowish- Quantified Hawthorn Leaf and Flower Liquid Extract
brown fluorescent zone (hyperoside); in the upper third Ph Eur_________________________________________ ____________
appears a light blue fluorescent zone (caffeic acid). DEFINITION
The chromatogram obtained with the test solution shows Whole or cut, dried flower-bearing branches of Crataegus
3 zones similar in position and fluorescence to the zones due monogyna Jacq. (Lindm.), c. laevigata (Poir.) DC.
to chlorogenic acid, hyperoside and caffeic acid in the (syn. c. oxyacanthoides Thuill.; c. oxyacantha auct.) or their
chromatogram obtained with the reference solution, and hybrids or, more rarely, other European Crataegus species
3 weak reddish fluorescent zones, one corresponding to the including c. pentagyna Waldst. et Kit. ex Willd., c. nigra
zone due to rutin in the chromatogram obtained with the Waldst. et Kit. and c. azarolus L.
reference solution and both of the others located above the
zone due to hyperoside; below and above the zone due to Content
caffeic acid some light blue zones appear. Minimum 1.5 per cent of total flavonoids, expressed as
hyperoside (C21H20O12; Mr 464.4) (dried drug).
TESTS
IDENTIFICATION
A. The stems are dark brown, woody, 1-2.5 mm in diameter,
Maximum 5 per cent of deteriorated false fruit and
bearing alternate, petiolate leaves with small, often deciduous
maximum 2 per cent of other foreign matter. It does not
stipules and corymbs of numerous small white flowers.
contain false fruits of other Crataegus species (C. nigra
The leaves are more or less deeply lobed with slightly serrate
Waldst. et Kit., c. pentagyna Waldst. et Kit. ex Willd.
or almost entire margins; those of c. laevigata are pinnately
2016 Hawthorn Leaf and Flower TV-221
lobed or pinnatifid with 3, 5 or 7 obtuse lobes, those of Top of the plate

c. monogyna pinnatisect with 3 or 5 acute lobes; the adaxial
surface is dark green or brownish-green, the abaxial surface is
lighter greyish-green and shows a prominent, dense, A yellowish-green fluorescent
reticulate venation. The leaves of c. laevigata, c. monogyna zone (vitexin)
and c. pentagyna are glabrous or bear only isolated Hyperoside: a yellowish-orange A yellowish-orange fluorescent
fluorescent zone zone (hyperoside)
trichomes, those of c. azarolus and c. nigra are densely
pubescent. The flowers have a brownish-green tubular calyx fluorescent zone (chlorogenic acid)
composed of 5 free, reflexed sepals, a corolla composed of A yellowish-green fluorescent
5 free, yellowish-white or brownish, rounded or broadly ovate zone (vitexin-2"-rhamnoside)
and shortly unguiculate petals and numerous stamens.
The ovary is fused to the calyx and consists of 1-5 carpels,
each with a long style and containing a single ovule;
in c. monogyna there is 1 carpel, in c. laevigata 2 or 3, in
c. azarolus 2 or 3, or sometimes only 1, in c. pentagyna 5 or,
rarely, 4. TESTS
B. Reduce to a powder (355) (2.9.12). The powder is Maximum 8 per cent of lignified branches with a diameter
yellowish-green. Examine under a microscope using chloral greater than 2.5 mm and maximum 2 per cent of other
hydrate solution R. The powder shows the following diagnostic foreign matter.
characters: unicellular covering trichomes, usually with a
thick wall and wide lumen, almost straight or slightly curved,
pitted at the base; fragments of leaf epidermis with cells
which have sinuous or polygonal anticlinal walls and with
large anomocytic stomata (2.8.3) surrounded by 4-7 105 °C for 2 h.
subsidiary cells; parenchymatous cells of the mesophyll Total ash (2.4.16)
containing calcium oxalate clusters, usually measuring Maximum 10.0 per cent.
10-20 pm, those associated with the veins containing groups ASSAY
of small prism crystals; fragments of petals showing rounded Stock solution Into a 200 mL flask introduce 0.400 g of the
polygonal epidermal cells, strongly papillose, with thick walls, powdered herbal drug (250) (2.9.12) and 40 mL of ethanol
the cuticle of which clearly shows wavy striations; fragments (60 per cent VIV) R. Heat in a water-bath at 60 °C for
of anthers showing endothecium with an arched and 10 min, shaking frequently. Allow to cool and filter through a
regularly thickened margin; fragments of stems containing plug of absorbent cotton into a 100 mL volumetric flask.
collenchymatous cells, bordered pitted vessels and groups of Transfer the absorbent cotton with the drug residue back to
lignified sclerenchymatous fibres with narrow lumina; the 200 mL flask, add 40 mL of ethanol (60 per cent VIV) R
numerous spherical to elliptical or triangular pollen grains up and heat again in a water-bath at 60 °C for 10 min, shaking
to 45 pm in diameter, with 3 germinal pores and a faintly frequently. Allow to cool and filter into the same 100 mL
granular exine. volumetric flask. Rinse the 200 mL flask with a further
c. Thin-layer chromatography (2.2.27). quantity of ethanol (60 per cent V/V) R, filter and transfer to
Test solution To 1.0 g of the powdered herbal drug (355) the same 100 mL volumetric flask. Dilute to 100.0 mL with
(2.9.12) add 10 mL of methanol R and heat in a water-bath ethanol (60 per cent VIV) R and filter.
at 65 cc under a reflux condenser for 5 min. Cool and filter. Test solution Introduce 5.0 mL of the stock solution into a
Reference solution Dissolve 1.0 mg of chlorogenic acid R and round-bottomed flask and evaporate to dryness under
2.5 mg of hyperoside J? in 10 mL of methanol R. reduced pressure. Take up the residue with 8 mL of a
Plate TLC silica gel plate R. mixture of 10 volumes of methanol R and 100 volumes of
anhydrous acetic acid R and transfer to a 25 mL volumetric
Mobile phase anhydrous formic acid R, water R, methyl ethyl flask. Rinse the round-bottomed flask with 3 mL of a
ketone R) ethyl acetate R (10:10:30:50 VIVIVIV). mixture of 10 volumes of methanol R and 100 volumes of
Application 20 pL as bands. anhydrous acetic acid R and transfer to the same 25 mL
Development Over a path of 15 cm. volumetric flask. Add 10.0 mL of a solution containing
Drying Pit 100-105 °C. 25.0 g/L of boric acid R and 20.0 g/L of oxalic acid R in
anhydrous formic acid R and dilute to 25.0 mL with anhydrous
diphenylboric acid aminoethyl ester R in methanol R, then spray acetic acid R.
with a 50 g/L solution of macrogol 400 R in methanol R', allow Compensation liquid Introduce 5.0 mL of the stock solution
to dry in air for about 30 min and examine in ultraviolet light into a round-bottomed flask and evaporate to dryness under
at 365 nm. reduced pressure. Take up the residue with 8 mL of a
mixture of 10 volumes of methanol R and 100 volumes of
anhydrous acetic acid R and transfer to a 25 mL volumetric
flask. Rinse the round-bottomed flask with 3 mL of a
anhydrous acetic acid R and transfer to the same 25 mL
volumetric flask. Add 10.0 mL of anhydrous formic acid R and
dilute to 25.0 mL with anhydrous acetic acid R.
solution at 410 nm, by comparison with the compensation
liquid.
IV-222 Hawthorn Preparations 2016

Top of the plate
expressed as hyperoside, using the following expression:
A light yellow fluorescent zone
A X 1.235 Hyperoside: a yellowish-orange A yellowish-orange fluorescent


i.e. taking the specific absorbance of hyperoside to be 405. fluorescent zone (chlorogenic acid)
A yellowish-green fluorescent zone
ไท = mass of the herbal drug to be examined, in grams. (vitexin 2z,-rhamnoside)
_________________________________________________ ___ __________ Ph Eur

Rutin: a yellowish-orange A yellowish-orange fluorescent
fluorescent zone
Hawthorn Leaf and Flower Dry * *

TESTS
Extract ***** Loss on drying (2.2.32)
(Ph. Eur. monograph ใ865) Maximum 6.0 per cent, determined on 0.500 g of the extract
PnEir__________________________________________________ to be examined by drying in an oven at 105 °C for 2 h.
DEFINITION ASSAY
Dry extract produced from Hawthorn leaf and flower (1432). Stock solution Dissolve 0.100 g of the extract to be examined
Content in ethanol (60 per cent VIV) R and dilute to 100.0 mL with
— for aqueous extracts: minimum 2.5 per cent of total the same solvent.
flavonoids, expressed as hyperoside (C21H20012; Test solution Introduce 5.0 mL of the stock solution into a
Afr 464.4) (dried extract); round-bottomed flask and evaporate to dryness under
— for hydroalcoholic extracts: minimum 6.0 per cent of total reduced pressure. Take up the residue in 8 mL of a mixture
flavonoids, expressed as hyperoside (C2JH20012; of 10 volumes of methanol R and 100 volumes of anhydrous
Mr 464.4) (dried extract). acetic acid R and transfer to a 25 mL volumetric flask. Rinse
the round-bottomed flask with 3 mL of a mixture of
PRODUCTION 10 volumes of methanol R and 100 volumes of anhydrous
The extract is produced from the herbal drug by a suitable acetic acid R and transfer to the same 25 mL volumetric flask.
procedure using either water or a hydroalcoholic solvent at Add 10.0 mL of a solution containing 25.0 g/L of bone
least equivalent in strength to ethanol (45 per cent VIV). acid R and 20.0 g/L of oxalic acid R in anhydrous formic acid R
CHARACTERS and dilute to 25.0 mL with anhydrous acetic acid R.
Appearance Compensation liquid Introduce 5.0 mL of the stock solution
Light brown or greenish-brown powder. into a round-bottomed flask and evaporate to dryness under
IDENTIFICATION reduced pressure. Take up the residue in 8 mL of a mixture
of 10 volumes of methanol R and 100 volumes of anhydrous
acetic acid R and transfer to a 25 mL volumetric flask. Rinse
Test solution Suspend 0.2 g of the extract to be examined in the round-bottomed flask with 3 mL of a mixture of
20 mL of ethanol (70 per cent VIV) R and filter. 10 volumes of methanol R and 100 volumes of anhydrous
Reference solution Dissolve 1 mg of chlorogenic acid R} 2.5 mg acetic acid R and transfer to the same 25 mL volumetric flask.
of hyperoside R and 2.5 mg of rutin J? in 10 mL of Add 10.0 mL of anhydrous formic acid R and dilute to
methanol R. 25.0 mL with anhydrous acetic acid R.
Plate TLC silica gel plate R. After 30 min, measure the absorbance (2.2.25) of the test
Mobile phase anhydrous formic acid R, water R, methyl ethyl solution at 410 nm, by comparison with the compensation
ketone R, ethyl acetate R (10:10:30:50 VIVIVIV). liquid.
Application 20 pL of the test solution and 10 pL of the Calculate the percentage content of total flavonoids,
reference solution, as bands. expressed as hyperoside, using the following expression:
Drying Ax 100-105 °C. A X 1.235
Detection spray the still-warm plate with a 10 g/L solution of m
diphenylboric acid aminoethyl ester R in methanol R} then spray
with a 50 g/L solution of macrogol 400 R in methanol R-, allow A = absorbance at 410 nm;
ไท ะะะ mass of the extract to be examined, in grams.
to dry in air for 30 min and examine in ultraviolet light at
365 nm. __________________________________________________ _________ . PhEtr
Results See below sequence of zones present in the

2016 Hop Strobile LV-223
Quantified Hawthorn Leaf and ****** reduced pressure. Take up the residue with 8 mL of a
Flower Liquid Extract V glacial acetic acid R and transfer into a 25 mL volumetric
(Ph. Eur. monograph 1864) flask. Rinse the round-bottomed flask with 3 mL of a
Pa Eur__________ _________ mixture of 10 volumes of methanol R and 100 volumes of
glacial acetic acid R and transfer into the 25 mL volumetric
DEFINITION flask. Add 10.0 mL of a solution containing 25.0 g/L of boric
Quantified liquid extract produced from Hawthorn leaf with acid R and 20.0 g/L of oxalic acid R in anhydrous formic acid R
flower (1432). and dilute to 25.0 mL with anhydrous acetic acid R.
Content Compensation liquid Introduce 5.0 mL of the stock solution
0.8 per cent to 3.0 per cent of flavonoids, expressed as into a round-bottomed flask and evaporate to dryness under
hyperoside (C21H20O12; Mr 464.4). reduced pressure. Take up the residue with 8 mL of a
PRODUCTION mixture of 10 volumes of methanol R and 100 volumes of
The extract is produced from the herbal drug and ethanol glacial acetic acid R and transfer into a 25 mL volumetric
(30 per cent VIV to 70 per cent V/V) by an appropriate flask. Rinse the round-bottomed flask with 3 mL of a
procedure. mixture of 10 volumes of methanol R and 100 volumes of
IDENTIFICATION flask. Add 10.0 mL of anhydrous formic acid R and dilute to
Thin-layer chromatography (2.2.27). 25.0 mL with anhydrous acetic acid R.
Test solution Dilute 1.0 g in methanol R and dilute to 5 mL After 30 min measure the absorbance (2.2.25) of the test
with the same solvent. Shake and filter. solution at 410 nm.
Reference solution Dissolve 1.0 mg of chlorogenic acid R and Calculate the percentage content of total flavonoids,
2.5 mg of hyperoside R in methanol R and dilute to 10 mL expressed as hyperoside, from the following expression:
with the same solvent.
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel A X 1.235
plate R (2-10 pm)].
Mobile phase anhydrous formic acid R, water R3 methyl ethyl
ketone R3 ethyl acetate R (10:10:30:50 V/V/V/V). i.e. taking the value of the specific absorbance of hyperoside
to be 405.
A = absorbance at 410 nm,
Development Over a path of 15 cm [or 6 cm]. m = mass of the extract to be examined, in grams.
____________________________________________________________ Ph Eur
aminoethyl ester R in methanol R. Subsequently spray with a
50 g/L solution of macrogol 400 R in methanol R. Allow the
plate to dry in air for about 30 min. Examine in ultraviolet
light at 365 nm. Hop Strobile * **
Results See below the sequence of zones present in the (Ph. Eur. monograph 1222) * **
chromatograms obtained with the reference solution and the Ph Eur_____________________________________________________________
present in the chromatogram obtained with the test solution. DEFINITION
Dried, generally whole, female inflorescence of Humulus
lupulus L.
Top of the plate
CHARACTERS
IDENTIFICATION
Hyperoside: a yellowish-orange A yellowish-orange fluorescent A. Hop strobiles are generally isolated and 2-5 cm long,
petiolate, ovoid, made up of many oval, greenish-yellow,
sessile, membranous, overlapping bracts. The external bracts
A yellowish-green fluorescent zone are flattened and symmetrical. The internal bracts are longer
and asymmetrical at the base because of a fold generally
encircling an induviate fruit (achene). The ovary or rarely the
fruit, the base of the bracts and especially the induvial fold,
are covered with small orange-yellow glands.
TESTS B. Reduce to a powder (355) (2.9.12). The powder is
Ethanol (2.9.10)
95 per cent v/v to 105 per cent v/v of the quantity stated
characters (Figure 1222.-1): fragments of bracts and
on the label.
bracteoles covered by polygonal, irregular or wavy-walled
assay epidermal cells [D, L, M]; unicellular, conical, straight or
Stock solution Dilute about 0.400 g, accurately weighed, in curved covering trichomes with thin, smooth walls,
ethanol (60 per cent V/V) R and dilute to 100.0 mL with the fragmented [E, G] or attached to an epidermis [A]; rare
same solvent. anomocytic stomata (2.8.3) [K]; glandular trichomes, usually
Test solution Introduce 5.0 mL of the stock solution into a free, with bicelidar biseriate stalks and heads consisting of
round-bottomed flask and evaporate to dryness under 8 small cells [H, N], rarely attached to an epidermis [La];
IV-224 White Horehound 2016
fragments of mesophyll containing small calcium oxalate in position to the zones in the chromatogram obtained with
cluster crystals [J]; many characteristic orange-yellow the reference solution: at about the level of the zone due to
glandular trichomes with short, bicellular bisenate stalks, curcumin is a faint zone due to xanthohumol, near the level
bearing a part widening into a cup, 150-250 pm in diameter, of the zone due to dimethylaminobenzaldehydc arc zones due
made up of a hemispherical layer of secretory’ cells with a to humulones, and near the level of the zone due to Sudan
cuticle that has been detached and distended by the orange are zones due to lupulones.
accumulation of oleoresinous secretions, in surface new [B] Detection B Examine in ultraviolet light at 365 nm.
or in side view [C]; fragments of elongated sclerenchymatous
Results B In the chromatogram obtained with the test
cells of the testa with thick walls showing striations and
solution the zones due to lupulones show blue fluorescence,
numerous pits [F].
the zones due to humulones show brown fluorescence and
the zone due to xanthohumol shows dark brown
fluorescence.
Detection c spray with dilute phosphomolybdotungstic reagent Ry
expose to ammonia vapour and examine in daylight.
Results c In the chromatogram obtained with the test
solution the zones due to humulones and to lupulones arc
bluish-grey and the zone due to xanthohumol is greenish-
grey; in the chromatogram obtained with the reference
solution the zones are bluish-grey or brownish-grey.
TESTS
Matter extractable by ethanol (70 per cent V/V)
300 mL of ethanol (70 per cent vrv) R and heat for 10 min
on a water-bath under a reflux condenser. Allow to cool,
filter, and discard the first 10 mL of the filtrate. Evaporate
30.0 mL of the filtrate to dryness on a water-bath and dry m
an oven at 100-105 °C for 2 h. The residue weighs a
minimum of 0.250 g.
105 °C for 2 h.
Total ash (2.4.76)
____________________________________________________________ __ Ph Eur

herbal drug of hop strobile
White Horehound * โ
c. Thin-layer chromatography (2.2.27). (Ph. Eur. monograph 1835) *
Test solution To 1.0 g of the freshly powdered herbal drug Ph Eur——----------------- --
(355) (2.9.12) add 10 mL of a mixture of 3 volumes of
DEFINITION
พater R and 7 volumes of methanol R’, shake for 15 min and
Whole or fragmented dried flowering aerial parts of
filter.
Marrubium vulgare L.
Reference solution Dissolve 1.0 mg of Sudan orange R} 2.0 mg
of curcumin R and 2.0 mg of dimethylaminobenzaldehyde R in
Content
20 mL of methanol R. Minimum 0.7 per cent of maiTubiin (C2oH280‘b Mr 332.4)
(dried drug).
Plate TLC silica gel F254 plate R.
Mobile phase anhydrous acetic acid R} ethyl acetate R3
CHARACTERS
cyclohexane R (2:38:60 VIVIV). Bitter taste.
Application 20 pL as bands. IDENTIFICATION
Development Over a path of 15 cm. A. The stems are up to 50 cm long, quadrangular, up to
7 mm wide, young stems are densely covered with whitish
Drying In air.
downy hairs, older stems are greenish-grey and less hairy.
Detection A Examine in ultraviolet light at 254 nm. The lower leaves are broadly ovate to almost orbicular, upper
Results A The chromatogram obtained with the reference leaves less broadly ovate, both petiolate; lamina 1.5-4 cm
solution shows 3 quenching zones; in the lower quarter is the long, 1-3.5 cm wide, apex sub-acute, base tapering or
faint zone due to curcumin, somewhat below the middle is somewhat cordate, margin dentate to crenate, petiole up to
the zone due to dimethylaminobenzaldehyde, and above, the 3 cm long; venation pinnate, prominent on the lower surface,
zone due to Sudan orange. The chromatogram obtained with distinctly depressed on the upper surface. Both leaf surfaces
the test solution shows a number of quenching zones similar are densely covered with fine, white, woolly hairs, older
2016 White Horehound IV-225
leaves having fewer hairs on die dark greyish-green upper Top of the plate
surface. The flowers are small, sessile in dense axillary
Guaiazulene: a A bluish-violet zone A bluish-violet zone
clusters. The calyx is 5 mm long, persistent, with 5 long and reddish-violet zone
5 short, alternating, hooked, recurved fringing spines; throat
of calyx with an internal ring of long silky hairs; corolla
/ mm long, dull white, 4-lobed, upper lobe 2-lipped, lower- A bluish-violet zone A bluish-violet zone
lobe 3-lipped; 4 short stamens; style with bifid stigma.
B. Reduce to a powder (710) (2.9.72). The powder is An intense bluish-violet A bluish-violet zone
Cholesterol: a
greyish-green. Examine under a microscope under chloral bluish-violet zone zone (marrubiin) (marrubiin)
hydrate solution R. The powder shows the following diagnostic A bluish-violet zone A bluish-violet zone
characters: fragments of leaves with sinuous, polygonal
A bluish-violet zone A bluish-violet zone
epidermal cells, diacytic stomata (2.8.3), more numerous on
the lower surface and cells of the mesophyll with small Reference solution Test solution (a) Test solution (b)
needles and cluster crystals of calcium oxalate; covering
trichomes very numerous, twisted or coiled, 100-200 pm
long, unicellular or multicellular and unscriate with 2-6 cells, TESTS
enlarged at the joints; stellate trichomes of 2 types, one with Loss on drying (2.2.32)
15-20 branches arising from a short unicellular stalk and the Maximum 10.0 per cent, determined on 1.000 g of the
other with fewer branches arising from a sessile base; 8-celled powdered herbal drug (710) (2.9.72) by drying in an oven at
secretory trichomes of lamiaceous type; glandular trichomes 105 °C for 2 h.
with 1 or 2 celled stalk and 1 to 4 celled head; the covering Total ash (2.4.16)
trichomes on the inner surface of the calyx are up to Maximum 15.0 per cent.
1000 pm long with 2 to 3 cells, strongly thickened at the Ash insoluble in hydrochloric acid (2.8.1)
swollen joint and with the upper cell elongated; pollen grains Maximum 3.0 per cent.
spherical, about 25 pm in diameter with smooth exine;
fragments of vascular tissue from the stems and veins. ASSAY
Test solution Reduce 50 g of the drug to a powder (250)
Test solution (a) To 1.0 g of the powdered herbal drug (710)
(2.9.72) and homogenise. To 1.00 g of the powdered herbal
(2.9.72) add 2 mL of dilute hydrochloric acid R and 8 mL of
drug in a 50 mL round-bottomed flask add 15 mL of a
methanol R. Heat under a reflux condenser for 30 min, cool
mixture of 2 volumes of dilute hydrochloric acid R and
and filter.
8 volumes of methanol R. Heat in a water bath at 80 °C
Test solution (b) To 1.0 g of the powdered herbal drug (710) under a reflux condenser for 30 min. Allow to cool at room
(2.9.72) add 10 mL of methanol R. Heat under a reflux temperature and filter through a plug of adsorbent cotton
condenser for 30 min, cool and filter. into a 25 mL volumetric flask. Dilute to 25.0 mL with
Reference solution Dissolve 10 mg of cholesterol R and 10 mg of methanol R by rinsing the round-bottomed flask and the filter.
guaiazulene R in 10 mL of methanol R. Reference solution Dissolve 2.0 mg of marrubiin R in 10.0 mL
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel of methanol R.
plate R (2-10 pm)]. Column:
Mobile phase methanol R, toluene R (5:95 VIV). — size: z = 0.25 m, 0 = 4 mm,
Application 20 pL [or 5 pL] of test solutions (a) and (b) and — stationary phase: end-capped octadecylsilyl silica gel for
10 pL [or 2 pL] of the reference solution, as bands. chromatography R (5 pm).
Development Over a path of 10 cm [or 6 cm]. Mobile phase:
— mobile phase A: acetonitrile Ry
Drying In air.
— mobile phase B: dilute 0.5 mL of phosphoric acid R to
Detection Spray with a 5 g/L solution of vanillin R in a 1000 mL with water R,
mixture of 20 volumes of ethanol (96 per cent) R and
80 volumes of sulfuric acid R and examine in daylight
immediately after heating at 130 °C for 5-10 min. (min) (per cent V/V) (per cent V/V)
Results See below the sequence of the zones present in the 0-> 15 40->90 60 -> 10
chromatograms obtained with the reference solution and test
15->20 90-> 40 10 -> 60
solutions (a) and (b). Further zones in the chromatograms
obtained with test solutions (a) and (b) may be present. 20->25 40 60
The zone due to marrubiin in the chromatogram obtained

with test solution (a) is more intense than that in the
chromatogram obtained with test solution (b). During
extraction with hydrochloric acid and methanol, conversion Detection Spectrophotometer at 217 nm.
of pre-marrubiin to marrubiin takes place which leads to an Injection 20 pL.
increase in intensity of the zone. Locate the peak due to marrubiin by comparison with the
Calculate the percentage content of marrubiin from the
41 X Ttt2 X p X 2.5
A2 X mi
FV-226 Horsetail 2016
^] = area of the peak due to marrubiin in the

chromatogram obtained with the test solution,
A2 = area of the peak due to marrubiin in the
solution,
nil = mass of the herbal drug to be examined, in
milligrams,
m2 = mass of marrubiin R) in milligrams,
p = percentage content of marrubiin in marrubiin R.
Ph Eur
Horsetail * *♦
(Equisetum Siem, Ph Eur monograph 1825) ***
Ph Eur______________________________________________________ _________
DEFINITION
Whole or cut, dried sterile aerial pans of Equisetum arvense L.
Content
Minimum 0.3 per cent of total flavonoids, expressed as
isoquercitroside (C2iH2o012; 464.4) (dried drug).
IDENTIFICATION
A. It consists of fragments of grooved main stems, branches
with longitudinal sharp ridges and leaves in whorls, united at Figure 1825.-1. - Illustration for identification test B of powdered
the base into a sheath, light green or greenish-grey. herbal drug of equisetum stem
The fragments are rough to the touch, brittle and crunchy c. Examine the chromatograms obtained in the test for
when crushed. The main stems are about 1-4.5 mm in Equisetum palustre.
diameter, hollow, jointed at the nodes, which occur at Results See below the sequence of zones present in the
intervals of about 1.5-4.5 cm; distinct vertical grooves are chromatograms obtained with reference solution (b) and the
present on the internodes, ranging in number from 4 to 14 test solution. Furthermore, other weak fluorescent zones may
or more. The central hollow is less than 50 per cent but be present in the chromatogram obtained with the test
more than 25 per cent of the diameter of the main stem. solution.
Verticils of widely spaced and erect branches, usually simple,
each about 1 mm thick with 3-5 longitudinal, sharp ridges, Top of the plate
occur at the nodes; at the end of each ridge is a protruding,
2 red fluorescent zones
distinct collenchymatic bundle under the epidermis.
The branches are not hollow. The leaves are small, linear, Caffeic acid: a greenish-blue
verticillate at each node, concrescent at the base; they form a fluorescent zone
toothed sheath around the stem with the number of teeth 2 greenish-blue fluorescent zones
corresponding to the number of grooves on the stem. Each
tooth, often brown, is lanceolate-triangular. The lowest
internode of each branch is longer than the sheath of the
stem to which it belongs. Hyperoside: an orange fluorescent
zone
2 greenish-blue fluorescent zones
greenish-grey. Examine under a microscope using chloral
characters (Figure 1825.-1): fragments of the epidermis in
Rutin ะ an orange fluorescent zone
surface view [B, C] composed of rectangular cells with wavy
walls and paracytic stomata (2.8.3) in 2-4 rows, the Reference solution (b) Test solution
2 subsidiary cells are in the same plane as the epidermis,
cover the guard cells and show radial ridges; small silica
TESTS
pilulae are scattered on the surface of the subsidiary cells and
appear more frequent at the margin forming a distinct ring
Maximum 5 per cent.
surrounding the subsidiary cells (C); 2-celled papillae on the
ridges, less distinct on the main stem [A] but large and Equisetum palustre
rectangular on the branches, oriented longitudinally [F]; Thin-layer chromatography (2.2.27).
in surface view, the epidermis of the main stems consists of Test solution To 1.0 g of the powdered herbal drug (355)
elongated cells [G], the epidermis of the secondary branches (2.9.12) add 10 mL of methanol R. Heat in a water-bath at
shows the 2-celled papillae which resemble pairs of small 60 °C for 10 min with occasional shaking. Allow to cool.
cells separated by a larger cell [D]; fragments of large-celled Filter.
parenchyma [H] and groups of long unlignified fibres with Reference solution (a) To 100.0 mg of Equisetum palustre HRS
narrow lumens; small vessels with spiral or annular add 10 mL of methanol R. Heat in a water-bath at 60 °C for
thickening [E], 10 min with occasional shaking. Allow to cool. Filter.
2016 Iceland Moss IV-227
Reference solution (b) Dissolve 1.0 mg of caffeic acid R, 2.5 mg A X 1.25

of hyperoside R and 2.5 mg of rutin R in 20 mL of m
methanol R.
Plate TLC silica gel plate R (2-10 pm). i.e. taking the specific absorbance of isoquercitroside to be
Mobile phase anhydrous formic acid R3 glacial acetic acid R, 500.
water R, ethyl acetate R (7.5:7.5:18:67 VIVIVIV). A = absorbance at 425 nm;
Application 5 pL as bands of 8 mm. m = mass of the substance to be examined, in grams.
Development Over a path of 6 cm. ___________________________________________________ _______ Ph Eur
Drying In a current of cold air for 5 min.

Detection Heat at 100 °C for 3 min and treat the still-warm
plate with a 10 g/L solution of diphenylboric acid aminoethyl
ester R in methanol R, then treat with a 50 g/L solution of
macrogol 400 R in methanol R3 allow to dry in a current of
cold air and examine after 10 min in ultraviolet light at
Iceland Moss ** **
365 nm. (Ph. Eur. monograph 1439) ** *
System suitability The chromatogram obtained with reference Ph Eur____________________________________________________ ___ ____
solution (a) shows 2 greenish fluorescent zones just above the
DEFINITION
line of application.
Whole or cut, dried thallus of Cetraria islandica (L.)
Results In the chromatogram obtained with the test solution, Acharius s.l.
any greenish fluorescent zones just above the line of
application are not more intense than the corresponding IDENTIFICATION
zones (characteristic of E. palustre L.) in the chromatogram A. The thallus, up to 15 cm long, is irregularly dichotomous
obtained with reference solution (a). and consists of glabrous, groove-shaped or almost flat, stiff,
brittle bands, 0.3-1.5 cm wide and about 0.5 mm thick,
sometimes serrated with the margin appearing ciliated
(pycnidia). The upper surface is greenish or greenish-brown,
the lower surface is greyish-white or light brownish and
105 °C for 2 h.
shows whitish, depressed spots (so-called respiratory cavities).
Ash insoluble in hydrochloric acid (2.8.1) On the apices of the terminal lobes, very rarely, there are
Minimum 3.0 per cent and maximum 15.0 per cent. brown, discoid apothecia.
Total ash (2.4.16) B. Reduce to a powder (355) (2.9.12). The powder is
Minimum 12.0 per cent and maximum 27.0 per cent. greyish-brown. Examine under a microscope, using chloral
ASSAY hydrate solution R. The powder shows the following diagnostic
characters: numerous fragments of the pseudoparenchyma
consisting of narrow-lumened, thick-walled hyphae from the
0.800 g of the powdered herbal drug (355) (2.9.12) and add
marginal layer and wide-lumened hyphae from the adjacent
1 mL of a 5 g/L solution of hexamethylenetetramine R) 20 mL
layer consisting of loosely entwined hyphae, in which, in the
of acetone R and 2 mL of hydrochloric acid Rl. Boil the
medullary zone, greenish or brownish algae cells up to 15 pm
mixture under a reflux condenser for 30 min. Filter the
in diameter, are embedded; occasionally marginal fragments
liquid through a plug of absorbent cotton into a flask.
of the thallus with tube-like or cylindrical spermogonia, up to
Add the absorbent cotton to the residue in the round-
about 160 pm wide and up to about 400 pm long.
bottomed flask and extract with 2 quantities, each of 20 mL,
of acetone R, each time boiling under a reflux condenser for c. To 1.0 g of the powdered herbal drug (355) (2.9.12) add
10 min. Allow to cool and filter each extract through a plug 10 mL of water R and boil for 2-3 min. The greyish-brown
of absorbent cotton into the flask. After cooling, filter the solution forms a gel after cooling which gives a blue colour
combined acetone extracts through a filter paper into a with iodine solution R.
volumetric flask and dilute to 100.0 mL with acetone R by D. Thin-layer chromatography (2.2.27).
rinsing the flask and the filter paper. Introduce 20.0 mL of Test solution To 1.0 g of the powdered herbal drug (355)
the solution into a separating funnel, add 20 mL of water R (2.9.12) add 5 mL of acetone R and heat in a water-bath
and shake the mixture with 1 quantity of 15 mL and then under a reflux condenser for 2-3 min. Cool and filter.
3 quantities, each of 10 mL, of ethyl acetate R. Combine the Reference solution Dissolve 5 mg of anethole R and 5 mg of
ethyl acetate extracts in a separating funnel, wash with caffeic acid Rin 2 mL of acetone R.
2 quantities, each of 50 mL, of water R, and filter the
extracts over 10 g of anhydrous sodium sulfate R into a
plate R (2-10 pm)].
volumetric flask. Dilute to 50.0 mL with ethyl acetate R.
Mobile phase acetone R, methanol R, anhydrous formic acid R,
Test solution To 10.0 mL of the stock solution add 1 mL of
toluene R (5:5:10:80 VIVIVIV).
5 per cent VIV solution of glacial acetic acid R in methanol R. Application 20 pL [or 4 pL] of the test solution and 10 pL
[or 2 pL] of the reference solution, as bands.
Compensation solution Dilute 10.0 mL of the stock solution to
25.0 mL with a 5 per cent VIV solution of glacial acetic Development Over a path of 10 cm [or 6 cm].
acid R in methanol-R. Drying In air.
Measure the absorbance (2.2.25) of the test solution after Detection spray with anisaldehyde solution R. Heat at
30 min, by comparison with the compensation solution at 100-105 °C for 5-10 min and examine in daylight.
425 nm. Calculate the percentage content of flavonoids, Results See below the sequence of zones present in the
expressed as isoquercitroside, using the following expression: chromatograms obtained with the reference solution and the
IV-228 Ipecacuanha 2016
test solution. Furthermore, other faint zones may be present having rounded ridges completely encircling the root;
in the chromatogram obtained with the test solution. the fracture is short in the bark and splintery in the wood.
The transversely cut surface shows a wide greyish bark and a
Top of the plate small uniformly dense wood. The rhizome occurs as short
lengths usually attached to roots, cylindrical, up to 2 mm in
A greyish-blue zone
diameter, finely wrinkled longitudinally and with pith
Anethole: a blue or bluish-violet occupying approximately one-sixth of the whole diameter.
zone
c acuminata The root in general resembles the root of
c. ipecacuanha, but differs in the following particulars: it is
2 weak greyish-blue zones often up to 9 mm thick; the external surface is greyish-brown
or reddish-brown with transverse ridges at intervals of usually
A weak greyish-brown or grey zone
1-3 mm, the ridges being about 0.5-1 mm wide, extending
about half-way round the circumference and fading at the
A greyish-violet zone
extremities into the general surface level.
Caffeic acid: a greyish-blue zone
grey or yellowish-brown. Examine under a microscope, using
Reference solution
diagnostic characters: parenchymatous cells, raphides of
Test solution
calcium oxalate up to 80 pm in length either in bundles or
scattered throughout the powder; fragments of tracheids and
TESTS vessels usually 10-20 pm in diameter, with bordered pits;
Foreign matter (2.8.2) larger vessels and sclereids from the rhizome. Examine under
Maximum 5 per cent. a microscope using a 50 per cent VIV solution of glycerol R.
The powder shows simple or two- to eight-compound starch
Lead (2.4.27)
granules contained in parenchymatous cells, the simple
Maximum 10.0 ppm.
granules being up to 15 pm in diameter in c. ipecacuanha
Loss on drying (2.2.32) and up to 22 pm in diameter in c. acuminata.
105 °C for 2 h. Test solution To 0.1 g of the powdered herbal drug (180)
(2.9.12) in a test-tube add 0.05 mL of concentrated
Total ash (2.4.16) ammonia R and 5 mL of ether R and stir the mixture
Maximum 3.0 per cent. vigorously with a glass rod. Allow to stand for 30 min and
Swelling index (2.8.4) filter.
Minimum 4.5, determined on the powdered herbal drug Reference solution Dissolve 2.5 mg of emetine hydrochloride CRS
(355) (2.9.12). and 3 mg of cephaeline hydrochloride CRS in methanol R and
--------------------------------------------------------------------------------------------------- --------- Ph Eur dilute to 20 mL with the same solvent.
Mobile phase concentrated ammonia R, methanol R, ethyl
acetate R, toluene R (2:15:18:65 VIVIVIV).
Ipecacuanha * * Application 10 pL, as bands.
(Ipecacuanha Rootj Ph Eur monograph 0094) *** Development Over a path of 10 cm.
Preparations Drying In air.
Prepared Ipecacuanha Detection A Spray with a 5 g/L solution of iodine R in ethanol
Ipecacuanha Liquid Extract (96 per cent) R and heat at 60 °C for 10 min. Examine in
daylight.
Standardised Ipecacuanha Liquid Extract
Results A The chromatograms obtained with the test solution
Standardised Ipecacuanha Tincture
and with the reference solution show in the lower part a
Ph Elf_______________________________________________________________ yellow zone due to emetine and below a light brown zone
DEFINITION due to cephaeline.
Fragmented and dried underground organs of Cephaelis Detection B Examine in ultraviolet light at 365 nm.
ipecacuanha (Brot.) A. Rich., known as Matto Grosso Results B The zone due to emetine shows an intense yellow
ipecacuanha, or of Cephaelis acuminata Karsten, known as fluorescence and that due to cephaeline a light blue
Costa Rica ipecacuanha, or of a mixture of both species. fluorescence. The chromatogram obtained with the test
The principal alkaloids are emetine and cephaeline. solution shows also faint fluorescent zones.
Content With c. acuminata the principal zones in the chromatogram
Minimum 2.0 per cent of total alkaloids, expressed as obtained with the test solution are similar in position,
emetine (C29H4oN204; Alr 480.7) (dried drug). fluorescence and size to the zones in the chromatogram
CHARACTERS obtained with the reference solution.
Slight odour. With c. ipecacuanha the only difference is that the zone due
to cephaeline in the chromatogram obtained with the test
IDENTIFICATION solution is much smaller than the corresponding zone in the
A. c. ipecacuanha. The root occurs as somewhat tortuous chromatogram obtained with the reference solution.
pieces, dark reddish-brown or very dark brown, seldom more
than 15 cm long or 6 mm thick, closely annulated externally,
2016 Ipecacuanha IV-229
TESTS Test solution To 0.1 g of the drug to be examined in a test

Loss on drying (2.2.52) tube add 0.05 mL of concentrated ammonia R and 5 mL of
Maximum 10.0 per cent, determined on 1.000 g of the ether R and stir the mixture vigorously with a glass rod. Allow
d herbal drug (180) (2.9.72) by drying in an oven at to stand for 30 min and filter.
105 °C.
Reference solution Dissolve 2.5 mg of emetine hydrochloride CRS
Total ash (2.4.16) and 3 mg of cephaeline hydrochloride CRS in methanol R and
Maximum 5.0 per cent. dilute to 20 mL with the same solvent.
Ash insoluble in hydrochloric acid (2.8.1) Plate TLC silica gel plate R.
Maximum 3.0 per cent. Mobile phase concentrated ammonia R, methanol R, ethyl
ASSAY acetate R, toluene R (2:15:18:65 VIVIV/V).
To 7.5 g of the powdered herbal drug (180) (2.9.12) in a dry Application 10 pL, as bands.
flask, add 100 mL of ether R and shake for 5 min. Add 5 mL Development Over a path of 10 cm.
of dilute ammonia R1, shake for 1 h. Add 5 mL of water R Drying In air.
and shake vigorously. Decant the ether layer into a flask
Detection A spray with a 5 g/L solution of iodine R in ethanol
through a plug of cotton. Wash the residue in the flask with
(96 per cent) R; heat at 60 °C for 10 min and examine in
2 quantities, each of 25 mL, of ether R, decanting each
daylight.
portion through the same plug of cotton. Combine the ether
solutions and eliminate the ether by distillation. Dissolve the Results A The chromatograms obtained with the test solution
residue in 2 mL of ethanol (90 per cent VIV) R, evaporate to and the reference solution show in the lower part a yellow
dryness and heat at 100 °C for 5 min. Dissolve the residue in zone due to emetine and below it a light brown zone due to
5 mL of previously neutralised ethanol (90 per cent VIV) R, cephaeline.
warming on a water-bath. Add 15.0 mL of 0.1 M hydrochloric Detection B Examine in ultraviolet light at 365 nm.
acid and titrate the excess acid with 0.1 M sodium hydroxide Results B The zone due to emetine shows an intense yellow
using 0.5 mL of methyl red mixed solution R as indicator. fluorescence and that due to cephaeline a light blue
1 mL of 0.1 M hydrochloric acid is equivalent to 24.03 mg fluorescence. The chromatogram obtained with the test
of total alkaloids, expressed as emetine. solution also shows faint fluorescent zones.
STORAGE With prepared c. acuminata, the principal zones in the
chromatogram obtained with the test solution are similar in
Protected from moisture.
position, fluorescence and size to the zones in the
---------------- ----------------------------------------------------------------------------------------- Ph Eur chromatogram obtained with the reference solution.
With prepared c. ipecacuanha, the only difference is that the
zone due to cephaeline in the chromatogram obtained with
the test solution is much smaller than the corresponding zone
Prepared Ipecacuanha ** \ in the chromatogram obtained with the reference solution.
TESTS
PhEir_____________ Maximum 5.0 per cent, determined on 1.000 g by drying in
DEFINITION an oven at 105 °C.
Ipecacuanha root powder (180) (2.9.12) adjusted, if Total ash (2.4.16)
necessary, by the addition of powdered lactose or Maximum 5.0 per cent.
ipecacuanha root powder with a lower alkaloidal content. Ash insoluble in hydrochloric acid (2.8.1)
Content Maximum 3.0 per cent.
1.9 per cent to 2.1 per cent of total alkaloids, expressed as ASSAY
emetine (C29H4oN204; Mx 480.7) (dried drug).
To7.5ginadry flask, add 100 mL of ether R and shake for
CHARACTERS 5 min. Add 5 mL of dilute ammonia Rl, shake for 1 h, add
Appearance 5 mL of water R and shake vigorously. Decant the ether layer
Light grey or yellowish-brown powder. into a flask through a plug of cotton. Wash the residue in the
Slight odour. flask with 2 quantities, each of 25 mL, of ether R, decanting
each portion through the same plug of cotton. Combine the
IDENTIFICATION ether solutions and eliminate the ether by distillation.
A. Examine under a microscope, using chloral hydrate Dissolve the residue in 2 mL of ethanol (90 per cent VIV) R,
solution R. The powder shows the following diagnostic evaporate the ethanol to dryness and heat at 100 °C for
characters: parenchymatous cells, raphides of calcium oxalate 5 min. Dissolve the residue in 5 mL of previously neutralised
up to 80 pm in length either in bundles or scattered ethanol (90 per cent VIV) R, warming on a water-bath, add
throughout the powder; fragments of tracheids and vessels 15.0 mL of 0.1 M hydrochloric acid and titrate the excess add
usually 10-20 pm in diameter, with bordered pits; larger with 0.1 M sodium hydroxide using 0.5 mL of methyl red mixed
vessels and sclereids from the rhizome. Examine under a solution R as indicator.
microscope using a 50 per cent VIV solution of glycerol R. 1 mL of 0.1 M hydrochloric acid is equivalent to 24.03 mg of
The powder shows simple or 2-8-compound starch granules total alkaloids, expressed as emetine.
contained in parenchymatous cells, the simple granules being
up to 15 pm in diameter in Cephaelis ipecacuanha and up to STORAGE
22 pm in diameter in c. acuminata. Examined in glycerol In an airtight container.
(85 per cent) R3 it may be seen to contain lactose crystals.
IV-230 Ipecacuanha Preparations 2016
Ipecacuanha Liquid Extract Standardised Ipecacuanha Liquid *******

DEFINITION Extract *****
Ipecacuanha Liquid Extract is prepared from Ipecacuanha by (Ph. Eur. monograph 1875)
a method stated under the general monograph for Extracts.
Ph Eur______________________________________________________________
It contains not less than 1.90% and not more than 2.10% of
total alkaloids, calculated as emetine, C29H40N2O4. DEFINITION
Standardised liquid extract produced from Ipecacuanha root
EXTEMPORANEOUS PREPARATION (0094).
Content
Prepare by extracting Ipecacuanha with Ethanol
1.80 per cent to 2.20 per cent of total alkaloids, calculated as
(80 per cent) according to the following formula and
emetine (C29H40N2O4; Mr 480.7).
directions.
Ipecacuanha in fine powder 1000 g PRODUCTION
Ethanol (80 per cent) A sufficient quantity The extract is produced from the herbal drug and ethanol
Exhaust the Ipecacuanha by percolation. Appendix XI F, with (60 to 80 per cent V/V) by an appropriate procedure.
Ethanol (80 per cent), reserving the first 750 mL of the CHARACTERS
percolate. Remove the effianol from the remainder of the Appearance
percolate by evaporation under reduced pressure at a Dark brown liquid.
temperature not exceeding 60° and dissolve the residual
IDENTIFICATION
extract in the reserved portion. Determine the proportion of
alkaloids in the liquid thus obtained by the Assay described
below. To the remainder of the liquid add sufficient Ethanol Test solution Dilute 5.0 mL of the extract to be examined to
(80%) to produce an Ipecacuanha Liquid Extract containing 50 mL with ethanol (70 per cent V/V) R. To 2.0 mL of this
2% w/v of total alkaloids calculated as emetine. Allow to solution add 2 mL of water R and 0.1 mL of concentrated
stand for not less than 24 hours; filter. ammonia R. Add 10 mL of ether R and shake. Separate the
upper layer, dry it over about 2 g of anhydrous sodium
The extract complies with the requirements stated under Extracts
sulfate R and filter.
and with the following requirements.
Reference solution Dissolve 2.5 mg of emetine hydrochloride CRS
TESTS and 3 mg of cephaeline hydrochloride CRS in methanol R and
Ethanol content dilute to 10 mL with the same solvent.
63 to 69% v/v, Appendix vm F, Method LU.
Relative density Mobile phase concentrated ammonia R, methanol R, ethyl
0.910 to 0.960, Appendix V G. acetate R, toluene R (2:15:18:65 V/V/V/V).
Dry residue Application 10 pL as bands.
The requirement for Dry residue does not apply to
Ipecacuanha Liquid Extract.
Drying In air.
ASSAY
Detection A Spray with a 5 g/L solution of iodine R in ethanol
To 5 mL in a separating funnel add 20 mL of water, 5 mL of (96 per cent) R. Heat at 60 °C for 10 min and allow to cool
Im sulfuric acid and 10 mL of chloroform and shake well. for 30 min. Examine in daylight.
Transfer the chloroform extract to a second separating funnel
Results A See below the sequence of the zones present in the
containing a mixture of 4 mL of ethanol (96%) and 20 mL of
0.05m sulfuric acid, shake, allow to separate and discard the chromatograms obtained with the reference solution and the
chloroform layer. Continue the extraction of the liquid in the
first separating funnel with two further 10 mL quantities of
chloroform, transferring the chloroform solution each time to
the second separating funnel and washing as before. Transfer Top of the plate
the acidic liquid from the second separating funnel to the
first separating funnel, make distinctly alkaline with
5m ammonia and shake with successive quantities of
chloroform until complete extraction of the alkaloids is effected, Emetine: a yellow zone A yellow zone (emetine)
Appendix XI G, washing each chloroform solution with the
Cephaeline: a light brown zone A light brown zone (cephaeline)
same 10 mL of water contained in a third separating funnel.
Remove the chloroform, add to the residue 2 mL of ethanol Reference solution Test solution
(96%), evaporate to dryness and dry for 5 minutes at 80° in
a current of air. Dissolve the residue in 2 mL of ethanol
Detection B Examine the plate in ultraviolet light at 365 nm.
(96%), previously neutralised to methyl red solution, add
10 mL of 0.05m sulfuric acid PS and titrate with 0.1m sodium Results B See below the sequence of the zones present in the
hydroxide vs using methyl red mixed solution as indicator. Each chromatograms obtained with the reference solution and the
mL of 0.05m sulfuric acid vs is equivalent to 24.03 mg of test solution. Furthermore, other faint fluorescent zones are
total alkaloids, calculated as emetine, 029แ40พ204. present in the chromatogram obtained with the test solution.
2016 Ipecacuanha Preparations IV-231
Top of the plate IDENTIFICATION

Test solution To 2.0 mL of the tincture to be examined add
2 mL of water R and 0.1 mL of concentrated ammonia R.
Emetine: an intense yellow An intense yellow fluorescent zone
fluorescent zone (emetine)
Add 10 mL of ether R and shake. Separate the ether layer,
dry it over about 2 g of anhydrous sodium sulfate R and filter.
(cephaeline) Reference solution Dissolve 2.5 mg of emetine hydrochloride CRS
Reference solution Test solution and 3 mg of cephaeline hydrochloride CRS in methanol R and
With a liquid extract from Cephaelis acuminata root, the zones Plate TLC silica gel plate R.
of emetine and cephaeline in the chromatogram obtained Mobile phase concentrated ammonia Ry methanol Ry ethyl
with the test solution are of similar size. acetate Ry toluene R (2:15:18:65 VIVIVIV).
With a liquid extract from Cephaelis ipecacuanha root, the Application 10 |1L as bands.
zone of emetine is much larger than the zone of cephaeline in Development Over a path of 10 cm.
the chromatogram obtained with the test solution. Drying In air.
TESTS Detection A Spray with a 5 g/L solution of iodine R in ethanol
Ethanol (2.9.10) (96 per cent) R and heat at 60 °C for 10 min. Examine in
95 per cent to 105 per cent of the quantity stated on the daylight.
label. Results A See below the sequence of zones present in the
ASSAY chromatograms obtained with the reference solution and the
Dilute 1.00 g of the extract to be examined to 10 mL with test solution.
ethanol (70 per cent VIV) R and transfer to a chromatography
column about 0.2 m long and about 15 mm in internal Top of the plate
diameter, containing 8 g of basic aluminium oxide Ry using a
glass rod. After infiltration into the aluminium oxide layer,
rinse the flask, glass rod and internal wall of the column with
3 quantities, each of 2 mL, of ethanol (70 per cent VIV) R. Emetine: a yellow zone A yellow zone (emetine)
Elute in portions with 40 mL of ethanol (70 per cent VIV) R. Cephaeline: a light brown zone A light brown zone (cephaeline)
Avoid disturbance or drying of the surface of the aluminium
oxide layer. Collect the whole of the eluate. Evaporate the
eluate on a water-bath to about 10 mL. Allow to cool.
Add 10.0 mL of 0.02 M hydrochloric acid and 20 mL of Detection B Examine the plate in ultraviolet light at 365 nm.
carbon dioxide-free water R. Titrate the excess acid with Results B See below the sequence of zones present in the
0.02 M sodium hydroxide using 0.15 mL of methyl red mixed chromatograms obtained with the reference solution and the
solution R as indicator. test solution. Furthermore, other faint fluorescent zones are
Perform a blank assay by replacing the extract to be present in the chromatogram obtained with the test solution.
examined with 10.0 mL of alcohol of the strength stated on
the label.
Top of the plate
1 mL of 0.02 M hydrochloric acid is equivalent to 4.807 mg of
total alkaloids, calculated as emetine.
---------------------- ------ ----------------------------------------------------------------------------- Ph Eur
Emetine: an intense yellow An intense yellow fluorescent zone
fluorescent zone (emetine)
(cephaeline)
Standardised Ipecacuanha *
Tincture ***< With a tincture from Cephaelis acuminata root, the zones of
(Ph. Eur. monograph 1530) emetine and cephaeline in the chromatogram obtained with
FhEir__________________________________________________________ the test solution are similar in size.
DEFINITION With a tincture from Cephaelis ipecacuanha root, the zone of
Tincture produced from Ipecacuanha root (0094). emetine is much larger than the zone of cephaeline in the
Content
0.18 per cent mhn to 0.22 per cent mlm of total alkaloids, TESTS
calculated as emetine (C29H40N2O4; Mr 480.7). Ethanol (2.9.10}
95 per cent to 105 per cent of the quantity stated on the
PRODUCTION label.
The tincture is produced from the herbal drug and ethanol
(70 per cent VIV) by an appropriate procedure. ASSAY
Transfer 10.00 g of the tincture to be examined to a
CHARACTERS chromatography column about 0.2 m long and about 15 mm
Appearance in internal diameter, filled with 8 g of basic aluminium
Yellowish-brown liquid. oxide R. After infiltration into the aluminium oxide layer rinse
the internal wall of the column with 3 quantities, each of
IV-232 Ipecacuanha Preparations 2016
2 mL, of ethanol (70 per cent VIV) R. Elute in portions, with ASSAY
40 mL of ethanol (70 per cent V/V) R. Avoid whirling or To 25 mL in a separating funnel add 20 mL of water and
drying of the surface of the aluminium oxide layer. Collect 5 mL of Im sulfuric acid, shake with three 10 mL quantities
the whole of the eluate. Evaporate the eluate on a water-bath of chloroform and wash each chloroform extract with a
to about 10 mL. Allow to cool. Add 10.0 mL of mixture of 20 mL of 0.05m sulfuric acid and 4 mL of ethanol
0.02 M hydrochloric acid and 20 mL of carbon dioxide-free (96%) contained in a second separating funnel. Transfer the
water R. Titrate the excess acid with 0.02 M sodium hydroxide acid-ethanol mixture from the second separating funnel to
using 0.15 mL of methyl red mixed solution R as indicator. the first, make the combined liquids distinctly alkaline to
Perform a blank assay replacing the tincture to be examined litmus paper with 5m ammonia and extract with successive
with 10.0 mL of alcohol of the strength stated on the label. quantities of chloroform until complete extraction of the
1 mL of 0.02 M hydrochloric acid is equivalent to 4.807 mg of alkaloids is effected, Appendix XI G. Wash each chloroform
total alkaloids, calculated as emetine. extract with the same 10 mL of "water, combine the
chloroform extracts, evaporate the chloroform, add 2 mL of
----------------------------------------------------------------------------------------------------------- Ph Eur
ethanol (96%) to the residue, evaporate to dryness and dry
the residue at 80° in a current of air for 5 minutes. Dissolve
the residue in 2 mL of ethanol (96%) previously neutralised
to methyl red solution, add 10 mL of 0.01m sulfuric acid zs
Paediatric Ipecacuanha Emetic Mixture and titrate the excess of acid with 0.02m sodium hydroxide Izs
using methyl red solution as indicator. Each mL of
Paediatric Ipecacuanha Emetic; Paediatric Ipecacuanha
0.01m sulfuric acid vs is equivalent to 4.806 mg of
Emetic Oral Solution
C29H40N2O4.
DEFINITION
Paediatric Ipecacuanha Emetic Mixture is an oral solution.
Ipecacuanha Liquid Extract 70 mL
Hydrochloric Acid 2.5 mL Isatis Root * *
Glycerol 100 mL
Syrup Sufficient to produce 1000 mL (Ph. Eur. monograph 2566) *
The mixture complies with the requirements stated under Oral Ph Eur______________________________________________________________
Liquids and with the following requirements. DEFINITION

Content of total alkaloids Dried root of Isatis tinctoria L. (/. indigotica Fortune)
0.12 to 0.16% w/v, calculated as emetine, C29H40N2O4. collected in autumn.
IDENTIFICATION Content
Carry out the method for thin-layer chromatography, Minimum 1.0 per cent of arginine (C6H14N4O2; Mr 174.2)
Appendix ni A, using the following solutions. (dried drug).
(1) Mix 5 mL with 10 mL of Im sulfuric acid, shake with two IDENTIFICATION
10 mL quantities of chloroform and discard the chloroform. A. The root is cylindrical, slightly tortuous, 10-20 cm long,
Add sufficient 5m ammonia to make the aqueous solution 0.5-1 cm in diameter, externally greyish-yellow or brownish-
distinctly alkaline to litmus paper, extract with four 10 mL yellow, wrinkled longitudinally and lenticellate transversally,
quantities of chloroform, evaporate the combined extracts to with rootlets or rootlet scars. Root stock slightly expanded,
dryness, cool the residue and dissolve it in 0.5 mL of ethanol exhibiting dark green or dark brown petiole bases arranged in
(96%). whorls, and dense tubercles. The fracture is yellowish-white,
(2) 0.1% w/v of cephaeline hydrochloride EPCRS in ethanol brown or dark brown in bark and yellow or brown in wood.
(96%). B. Microscopic examination (2.8.23). The powder is whitish-
(3) 0.1% w/v of emetine hydrochloride EPCRS in ethanol yellow or yellow. Examine under a microscope using chloral
(96%). hydrate solution R. The powder shows the following diagnostic
characters: fragments of cork consisting of 5-8 thin-walled
layers; fragments of xylem with reticulate structure; thin
(a) Use as the coating silica gel G. walled, rounded parenchyma cells. Examine under a
(b) Use the mobile phase as described below. microscope using a 50 per cent v/v solution of glycerol R.
(c) Apply 2 J1L of each solution. The powder shows abundant, single or compound (2, 3 or 4)
(d) Develop the plate to 15 cm. starch grains. The starch grains, 1.5-3.4 pm in diameter, with
spot, cleft or V-shaped hilum.
(e) After removal of the plate, dry it at 105° to 110° for
30 minutes, allow to cool and spray with dilute potassium c. Thin-layer chromatography (2.2.27).
iodobismuthate solution. Test solution To 0.5 g of the powdered herbal drug (355)
(2.9.12) add 5 mL of ethanol (70 per cent V/V) R and
MOBILE PHASE
sonicate for 10 min. Centrifuge and use the supernatant.
10 volumes of diethylamine and 90 volumes of chloroform.
Reference solution Dissolve 4 mg of arginine R and 4 mg of
CONFIRMATION cysteine hydrochloride R in 1 mL of ethanol
The principal spots in the chromatogram obtained with (70 per cent V/V) R.
solution (1) correspond in colour and position to the spots in Plate TLC silica gel F254 plate R (5-40 nm) [or TLC silica gel
the chromatograms obtained with solutions (2) and (3). F254 plate R (2-10 pm)].
Disregard any secondary spots. Mobile phase anhydrous formic acid R, water R, acetonitrile R
(2:8:30 V/V/V).
Application 4 pL as bands of 10 mm [or 8 mm].
2016 Ispaghula Husk IV-233
Development Over a path of 8.5 cm [or 6 cm]. to comply with the system suitability criterion for the signal-
Drying In air. to-noise ratio:
Detection Expose to concentrated ammonia R vapour for 5 min, — carrier gas: nitrogen R’i
treat with ninhydrin solution R4, then heat at 120 °C for — pressure: 330 kPa;
3 min. — evaporator temperature: 80 °C.
Results See below the sequence of zones present in the Injection 10 nL.
chromatograms obtained with the reference solution and the Run time 25 min.
test solution. Furthermore, other faint coloured zones may be System suitability.
present in the chromatogram obtained with the test solution. — resolution: minimum 1.5 between the peaks due to
cysteine and arginine in the chromatogram obtained with
reference solution (b);
Top of the plate
— signal-to-noise ratio: minimum 50 for the peak due to
arginine in the chromatogram obtained with reference
solution (a).
Establish a calibration curve with the logarithm of the
A prominent brown zone
concentration (in milligrams per 10 mL) of reference
Cysteine: a brown zone solutions (c), (d), (e), (f), (g) and (h) (corrected by the
assigned percentage content of arginine CRS) as the abscissa
A brown zone
and the logarithm of the corresponding peak areas as the
ordinate.
Arginine: a brown zone A brown zone (arginine) Calculate the percentage content of arginine using the
A faint brown zone
Reference solution Test solution 10A

m X 10
TESTS
Loss on drying (2.2.32) A ะ= logarithm of the concentration of arginine in ±e
Maximum 9.0 per cent, determined on 1.000 g of the test solution, determined from the calibration
powdered herbal drug (355) (2.9.72) by drying in an oven at curve;
105 °C for 2 h. m = mass of the herbal drug to be examined used to
prepare the test solution, in grams.
Total ash (2.4.16)
Maximum 5.0 per cent. ____________________________________________________________ Ph Eur

ASSAY
Liquid chromatography (2.2.29). Ispaghula Husk * *
Test solution To 0.100 g of the powdered herbal drug (355) (Ph. Eur. monograph 1334) ***
(2.9.12) add 20 mL of ethanol (70 per cent VIV) R, sonicate
Preparations
for 20 min, filter, and evaporate the filtrate to dryness.
Ispaghula Husk Oral Powder
Dissolve the residue in ethanol (70 per cent VIV) R and dilute
to 10.0 mL with the same solvent. Ispaghula Husk Grandes
Ispaghula Husk Effervescent Granules
Reference solution (a) Dissolve 25.0 mg of arginine CRS in
ethanol (70 per cent VIV) R and dilute to 50.0 mL with the Ph Eur _____________________________________________________________
same solvent. DEFINITION
Reference solution (b) Dissolve 3.0 mg of cysteine Episperm and collapsed adjacent layers removed from the
hydrochloride 1? in 6.0 mL of reference solution (a) and dilute seeds of Plantago ovata Forssk. (P. ispaghula Roxb.).
to 10.0 mL with ethanol (70 per cent V/V) R.
IDENTIFICATION
Reference solutions (c)3 (d)3 (e)3 (f)3 (g) 3 (h) Dilute reference A. The husk consists of pinkish-beige fragments or flakes up
solution (a) to obtain 6 reference solutions of arginine, the to about 2 mm long and 1 mm wide, some showing a light
concentrations of which span the expected value in the test brown spot corresponding to the location of the embryo
solution. before it was removed from the seed.
Column-.
— size: 1 = 0.15 m, 0 = 4.6 mm; yellow. Examine under a microscope using lactic reagent R.
— stationary phase', end-capped octadecylsilyl silica gel for The powder shows the following diagnostic characters:
chromatography R (3 pm); mainly fragments of the episperm with polygonal cells filled
— temperature: 30 °C. with mucilage; fragments of the inner layers of the testa with
Mobile phase trifluoroacetic acid R, water R (0.2:99.8 VIV). brownish thin-walled cells often associated with the outer
Plow rate 0.2 mL/min. layers of the endosperm. Examine under a microscope using
Detection Evaporative light-scattering detector; the following a 50 per cent VIV solution of glycerol R. The powder shows
settings have been found to be suitable; if the detector has occasional starch granules, single or in groups of 2-4,
different setting parameters, adjust the detector settings so as measuring 3-25 pm in diameter.
IV-234 Ispaghula Preparations 2016
Test solution To 10 mg of the powdered herbal drug (355) Loss on drying

(2.9.72) in a thick-walled centrifuge tube, add 2 mL of a When dried at 100° to 105°, loses not more than 12.0% of
230 g/L solution of trifluoroacetic acid R and shake vigorously. its weight. Use 1 g.
Stopper the test tube and heat at 120 cc for 1 h. Centrifuge Ash
the hydrolysate, transfer the clear supernatant into a 50 mL Not more than 5.0%, Appendix XI J, Method n.
flask, add 10 mL of water R and evaporate to dryness under
reduced pressure. Take up the residue in 10 mL of water R STORAGE
and evaporate again to dryness under reduced pressure. Take Ispaghula Husk Granules should be protected from moisture.
up the residue with 2 mL of methanol R.
Reference solution (a) Dissolve 10 mg of arabinose R in a small
quantity of water R and dilute to 10 mL with methanol R.
Reference solution (b) Dissolve 10 mg of xylose R in a small Ispaghula Husk Effervescent Granules
DEFINITION
Reference solution (c) Dissolve 10 mg of galactose R in a small
Ispaghula Husk Effervescent Granules contain Ispaghula
Husk in a suitable, effervescent basis.
The granules comply with the requirements stated under Granules
Mobile phase water R, acetonitrile 7? (15:85 VIV). and with the following requirements.
Application 10 J1L, as bands.
TESTS
Development Over a path of 15 cm. Disintegration
Detection Spray with aminohippuric acid reagent R and heat at Carry out the test stated under Effervescent Granules with
120 °C for 5 min; examine in daylight. the following modifications. Stir the contents of the beaker
Results The chromatogram obtained with the test solution occasionally to disperse the mucilage formed; evolution of gas
shows 2 orange-pink zones (arabinose and xylose) and a is complete after 5 minutes.
yellow zone (galactose) similar in position and colour to ±e Swelling index
zones in the chromatograms obtained with the reference Not less than 40, Appendix XI c. Use a quantity of the
solutions. powdered granules containing 1.0 g of Ispaghula Husk and a
TESTS 100 mL ground-glass-stoppered cylinder graduated in 1 mL
Foreign matter (2.5.2) divisions.
Carry out the determination using 5.0 g.
powdered herbal drug (355) (2.9.72) by drying in an oven at Ispaghula Husk Oral Powder
105 °C for 2 h.
DEFINITION
Total ash (2.4.16) Ispaghula Husk Oral Powder contains Ispaghula Husk with
Maximum 4.0 per cent. or without suitable excipients.
Swelling index (2.8.4) The powder complies with the requirements stated under Oral
Minimum 40, determined on 0.1 g of the powdered herbal Powders and with the following requirements.
drug (355) (2.9.72).
IDENTIFICATION
________________________________________________________________ Ph Eur
A. Carry out the method for thin-layer chromatography,
Appendix III A, using the following solutions.
(1) Add 2 mL of a 23% w/v solution of trifluoroacetic acid to
a quantity of the powder containing 10 mg of Ispaghula
Ispaghula Husk Granules Husk in a thick-walled centrifuge tube, shake vigorously,
close the tube and heat at 120° for 1 hour. Centrifuge the
DEFINITION
hydrolysate, transfer the clear supernatant liquid into a
Ispaghula Husk Granules contain Ispaghula Husk with or
50 mL flask, add 10 mL of water and evaporate the solution
without suitable excipients.
to dryness under reduced pressure. Take up the residue in
The granules comply with the requirements stated under Granules 10 mL of water, again evaporate to dryness under reduced
and with the following requirements. pressure and take up the residue in 2 mL of methanol.
IDENTIFICATION (2) Dissolve 10 mg of arabinose in a small quantity of water
A. Powder the granules and examine under a microscope and dilute to 10 mL with methanol.
using lactic reagent. Fragments of the episperm with polygonal (3) Dissolve 10 mg of xylose in a small quantity of water and
cells filled with mucilage and fragments of the inner layers of dilute to 10 mL with methanol.
the testa with brownish thin-walled cells often associated with (4) Dissolve 10 mg of galactose in a small quantity of water
the outer layers of the endosperm are seen.
and dilute to 10 mL with methanol.
B. When mounted in ruthenium red solution, the particles of
the powder are stained red.
(a) Use as the coating silica gel.
TESTS
Swelling index
Not less than 40, Appendix XI c. Use a quantity of the (c) Apply 10 pL of each solution.
granules containing 1.0 g of Ispaghula Husk and a 100-mL (d) Develop the plate to 15 cm.
ground-glass-stoppered cylinder graduated in 1 mL divisions.
2016 Ivy Leaf IV-235
(e) After removal of the plate, dry in air, spray with to dryness under reduced pressure. Take up the residue in
aminohippuric acid reagent, heat at 120° for 5 minutes and 2 mL of methanol R.
Reference solution (a) Dissolve 10 mg of arabinose R in a small
MOBILE PHASE quantity of water R and dilute to 10 mL with methanol R.
15 volumes of water and 85 volumes of acetonitrile. Reference solution (b) Dissolve 10 mg of xylose R in a small
CONFIRMATION quantity of water R and dilute to 10 mL with methanol R.
The chromatogram obtained with solution (1) shows two Reference solution (c) Dissolve 10 mg of galactose R in a small
orange-pink zones (arabinose and xylose) and a yellow zone quantity of water R and dilute to 10 mL with methanol R.
(galactose) similar in position and colour to the zones in the Plate TLC silica gel plate R
chromatograms obtained with solutions (2), (3) and (4). Mobile phase water R, acetonitrile R (15:85 VIV).
B. When mounted in ruthenium red solution, the particles of Application 10 J1L, as bands.
the powder are stained red. Development Over a path of 15 cm.
TESTS Detection Spray with aminohippuric acid reagent R and heat at
Swelling index 120 °C for 5 min. Examine in daylight.
Not less than 40, Appendix XI c. Use a quantity of the oral Results See below the sequence of the zones present in the
powder containing 1.0 g of Ispaghula Husk and a 100 mL chromatograms obtained with the reference and the test
ground-glass-stoppered cylinder graduated in 1 mL divisions. solutions.
Ash
Not more than 4.0%, Appendix XI J, Method II. Use a Top of the plate
quantity of the powder containing 1 g of Ispaghula Husk.
Xylose: an orange-pink zone An orange-pink zone (xylose)
STORAGE
Arabinose: an orange-pink zone An orange-pink zone (arabinose)
Ispaghula Husk Oral Powder should be protected from
moisture. Galactose: a yellow zone A yellow zone (galactose)
Ispaghula Seed / T TESTS

(Ph. Eur. monograph 1333) *** Carry out the determination using 10.0 g.
Ph Eir_____________ Swelling index (2.8.4)
DEFINITION Minimum 9.
Dried ripe seeds of Plantago ovata Forssk. Loss on drying (2.2.32)
(P. ispaghula Roxb.). Maximum 10.0 per cent, determined on 1.000 g of the
IDENTIFICATION 105 °C for 2 h.
A. Ispaghula seed is pinkish-beige, smooth, boat-shaped and
curved. It is 1.5 mm to 3.5 mm long, 1.5 mm to 2 mm wide
Total ash (2.4.16)
and 1 mm to 1.5 mm thick. The concave surface shows in
the centre a light coloured spot corresponding to the hilum. _________________________________________ ■ _____________ Ph Elf
The convex surface shows a light brown spot corresponding

to the location of the embryo and takes up about one quarter
of the length of the seed.
brown. Examine under a microscope using lactic reagent R.
Ivy Leaf * *
The powder shows mainly fragments of the episperm with (Ph. Eur. monograph 2148) ***
polygonal cells filled with mucilage; fragments of the inner Ph Eur_________________ _ _________________________________________
layers of the testa with brownish thin-walled cells often
associated with the outer layers of the endosperm; fragments DEFINITION
of the endosperm with cells with thick cellulose walls Whole or cut, dried leaves of Hedera helix L., collected in
containing aleurone grains and oil droplets; a few fragments spring and summer.
of embryo with thin-walled cells. Examine under a Content
microscope using a 50 per cent VIV solution of glycerol R. Minimum 3.0 per cent of hederacoside c (C59H96O26J
The powder shows starch granules, single or in groups of Mr 1221) (dried drug).
2 to 4 and measuring 3 Jim to 25 Jim in diameter. IDENTIFICATION
c. Thin-layer chromatography (2.2.27). A. Whole leaves are coriaceous, 4-10 cm in length and width,
Test solution To 50 mg of the powdered herbal drug (355) cordate at the base. The lamina is palmately 3-5 lobed, the
(2.9.72) in a thick-walled centrifuge tube add 2 mL of a lobes more or less triangular with entire margins. The upper
230 g/L solution of trifluoroacetic acid R, and shake surface is dark green with a paler, radiate venation, the lower
vigorously. Stopper the test tube and heat the mixture at surface more greyish-green and the venation is distinctly
120 °C for 1 h? Centrifuge the hydrolysate, transfer the clear raised. The petioles are long, cylindrical, about 2 mmin
supernatant into a 50 mL flask, add 10 mL of water R and diameter and grooved longitudinally. Scattered white hairs
evaporate the solution to dryness under reduced pressure. occur on the petioles and on the surfaces of younger leaves,
Take up the residue in 10 mL of water R and evaporate again the older leaves are glabrous. Occasional entire, ovate-
IV-236 Ivy Leaf 2016
rhombic to lanceolate leaves 3-8 cm long from the flowering Application 20 pL as bands of 15 mm.
stems may be present. Development Over a path of 12 cm.
B. Microscopic examination (2.8.23). The powder is green.
Drying Kl 100-105 °C.
Detection Treat with alcoholic solution of sulfuric acid Ry heat at
110 °C for 10 min and examine in daylight.
(Figure 2148.-1): fragments of the upper epidermis (surface
view [F]), showing cells with thickened, rather sinuous, finely Results See below the sequence of zones present in the
pitted anticlinal walls [Fa] usually accompanied by chromatograms obtained with the reference solution and the
underlying palisade parenchyma [Fb] including some cells test solution. Furthermore, other zones may be present in the
containing cluster crystals of calcium oxalate [Fc]; fragments chromatogram obtained with the test solution.
of the lower epidermis (surface view [E]), showing cells with
sinuous, irregularly thickened and pitted walls [Ea], stomata Top of the plate
that are mostly anomocytic [Eb] but occasionally anisocytic
(2.8.3)3 surrounded by cells including some that show faint
cuticular striations; the lower epidermis is accompanied by
underlying spongy’ parenchyma [Ec] including some cells
a-Hederin: a purple zone A very faint purple zone
containing cluster crystals of calcium oxalate [Ed]; scattered (a-hederin)
stellate covering trichomes may be present, composed of A broad yellow zone
4-8 branches joined at the base on a multicellular, biseriate 2-3 purple or green zones
stalk (surface view [B], side view [A]); cluster crystals of
calcium oxalate, about 40 pm in diameter, scattered [C] or
occurring throughout the parenchyma [Ed, Fc]; groups of Hederacoside C: a purple zone A purple zone (hederacoside C)
lignified fibro-vascular tissue from the veins [D]. Reference solution Test solution
TESTS
Maximum 10 per cent of discoloured leaves, maximum
10 per cent of stems, and maximum 2 per cent of other
foreign matter.
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Solvent mixture water Ry methanol R (20:80 VIV).
(2.9.12) in a 250 mL round-bottomed flask add 50 mL of
±e solvent mixture and heat under a reflux condenser in a
water-bath at 80 °C for 1 h. Cool and filter through a plug of
absorbent cotton into a 100 mL volumetric flask. The plug
of absorbent cotton together with the residue is again
extracted with 30 mL of the solvent mixture under reflux for
30 min. Filter and combine the filtrates. Rinse the round-
bottomed flask and the plug of absorbent cotton with the
solvent mixture and use the solvent mixture to dilute the
contents of the volumetric flask to exactly 100.0 mL. Filter
through a suitable membrane before use.
Reference solution To 20.0 mg of ivy leaf dry extract HRS add
5.0 mL of methanol R and sonicate for 10 min. Filter through
Figure 2148.-1. - Illustration for identification test B ofpowdered a membrane filter (nominal pore size 0.45 pm).
herbal drug of ivy leaf
Column-.
c. Thin-layer chromatography (2.2.27). — size-. I = 0.125 m, 0 = 4 mm;
Test solution Extract 0.50 g of the powdered herbal drug — stationary phase-, end-capped octadecylsilyl silica gel for
(355) (2.9.12) under a reflux condenser in a water-bath at chromatography R (5 pm).
60 °C with 5 mL of methanol R for 30 min. Cool and filter. Mobile phase-.
Reference solution Dissolve 1.0 mg of hederacoside c R and — mobile phase A-. mix 14 volumes of acetonitrile R with
1.0 mg of a-hederin R in 1.0 mL of methanol R. 88 volumes of water R and adjust to pH 2.0 with
Plate TLC silica gel plate R. phosphoric acid Ry
— mobile phase B: phosphoric acid Ry acetonitrile R
Mobile phase anhydrous formic add Ry acetone Ry methanol Ry
ethyl acetate R (4:20^0:30 VIVIVIV). (0.2:99.8 V!V)y
2016 Java Tea IV-237
Time Mobile phase A Mobile phase B characters: fragments of epidermis, with cells with sinuous
___ (min) (per cent V/V) (per cent V/V) outlines, bearing unicellular or bicellular conical covering
0-5 100 0 trichomes and articulated uniseriate trichomes up to 450 pm
5-6 100 -> 94 0 -> 6 long, consisting of 3-8 cells with thick pitted walls; capitate
6 - 40
trichomes with unicellular or bicellular heads; secretory
94 -> 60 6 -> 40
trichomes with unicellular stalks and usually tetracellular
40-41 60 -> 0 40 -> 100 heads; diacytic stomata (2.8.3), which are more numerous on
41 - 55 0 100
the lower epidermis.
Flow rate 1.5 mUmin. Test solution Shake 1 g of the powdered herbal drug (710)
(2.9.12) with 10 mL of methanol R in a water-bath at 60 cc
for 5 min and filter the cooled solution.
Injection 20 pL.
Reference solution Dissolve 1 mg of sinensetin R in methanol R
System suitability: reference solution: and dilute to 20 mL with the same solvent.
retention time: hederacoside c = about 20 min;
if necessary3 adjust the time intervals of the gradient.
Mobile phase methanol R, ethyl acetate R, toluene R
Calculate the percentage content of hederacoside c with
(5:40:55 V/V/V).
reference to the dried drug using the following expression:
>11 X 7712 X 20 X p Development Over a path of 10 cm.
>12 X 7711 Drying In air.
>11 = area of the peak due to hederacoside c in the
chromatogram obtained with the test solution; chromatogram obtained with the reference solution and the
A2 = area of the peak due to hederacoside c in the
test solution. Furthermore, red fluorescent zones are present
chromatogram obtained with the reference solution; in the lower third and near the solvent front of the
nh = mass of the herbal drug to be examined used to chromatogram obtained with the test solution.
m2 = mass of ivy leaf dry extract HRS used to prepare the
reference solution, in grams; Top of the plate
p = percentage content of hederacoside c in ivy leaf dry 1 or 2 more or less intense blue or
extract HRS. violet-blue fluorescent zones
-------------------------------------- ----- -------------------------------------------------------------- Ph Eur

Sinensetin: an intense light blue A major blue fluorescent zone
fluorescent zone (sinensetin)
2 bluish fluorescent zones

Java Tea ** **
Reference solation Test solation
Ph Elf________ ______________________________________________________
TESTS
DEFINITION
Fragmented, dried leaves and tops of stems of Orthosiphon
stamineus Benth. (O. aristatus Miq.; o. spicatus Bak.).
1 mm and maximum 2 per cent of other foreign matter.
Content
Minimum 0.05 per cent of sinensetin (C20H20O7; Mr 372.4)
(dried drug).
IDENTIFICATION 105 °C for 2 h.
A. The leaves are friable, up to 7.5 cm in length and 2.5 cm Total ash (2.4.16)
in width. The petiole is short. The lamina is oval or Maximum 12.5 per cent.
lanceolate, the apex acuminate and the base cuneate.
The abaxial surface of the leaves is light greyish-green and ASSAY
the adaxial surface is dark green or brownish-green. Liquid chromatography (2.2.29).
The venation is pinnate with few secondary veins. Examined Test solution Heat 2.5 g of the powdered herbal drug (355)
under a lens (X 10), the secondary veins, after running (2.9.12) and 100 mL of methylene chloride R on a water-bath
parallel to the midrib, diverge at an acute angle. The margin for 30 min with stirring. Filter. Collect the filtrate and repeat
is irregularly and roughly dentate, sometimes crenate and the the operation twice, in the same manner, on the filtration
abaxial surface is slightly curved. The petioles are thin, residue. Combine the filtrates. Evaporate the solvent under
quadrangular, 4-8 mm long and, like the primary venation, reduced pressure. Dissolve the residue in 25.0 mL of die
usually violet-coloured. Occasionally, inflorescences in mobile phase, using an ultrasonic bath if necessary. Filter the
clusters of bluish-white or violet flowers, not yet opened, are solution through a nitrocellulose filter with a pore size of
found. 0.45 pm.
B. Reduce to a powder (355) (2.9.12). The powder is dark Reference solution Dissolve 5 mg (m2) R in 80 mL
green. Examine under a microscope using chloral hydrate of the mobile phase using an ฟtrasonic bath if necessary and
solution R. The powder shows the following diagnostic dilute to 100.0 mL with the mobile phase.
IV-238 Juniper 2016
7712 X Fl X 25
7711 X F2
Fl = area of the peak due to sinensetin in the

chromatogram obtained with tile test solution;
F2 = area of the peak due to sinensetin in the
solution;
nil = mass of the herbal drug to be examined, in grams;
>ท2 = mass of sinensetin in the reference solution, in
grams.
Ph
Juniper * :
(Ph Eur monograph 1532) * **
Ph Eur_______ _________________________________________ _____________
DEFINITION
Dried ripe cone berry of Juniperus communis L.
Content
Minimum 10 mI7kg of essential oil (anhydrous drug).
CHARACTERS
Strongly aromatic odour, especially if crushed.
IDENTIFICATION
A. The berry-shaped cone is globular, up to 10 mm in
diameter, and violet-brown or blackish-brown, frequendy
A. Lamina, in transverse F. Margin of the lamina with a bluish bloom. It consists of 3 fleshy scales. The apex
section, showing a secretory G. Secretory trichome has a 3-rayed closed cleft and 3 not very clearly defined
trichome with a tetracellular H. Lower epidermis, in surface projections. A remnant of peduncle is frequendy attached at
head (Aa) and a capitate view, with diacytic stomata the base. The fleshy part is crumbly and brownish.
trichome with a unicellular (Ha), capitate trichome with a It contains 3 or, more rarely, 2 small, elongated, extremely
head (Ab) unicellular head (Hb) and hard seeds that have 3 sharp edges and are slightly rounded
B. Upper epidermis, in multicellular covering trichome at the back, acuminate at the apex. The seeds are fused with
surface view, showing a (He) the fleshy part of the cone berry in the lower part on the
capitate trichome with a J. Covering trichomes on the outside of their bases. Very large, oval oil glands containing
bicellular head (Ba) and margin of the lamina sticky resin lie at the outer surface of the seeds.
underlying palisade B. Microscopic examination (2.8.23). The powder is brown.
parenchyma (Bb) Examine under a microscope using chloral hydrate solution R.
c. and E. Articulated The powder shows the following diagnostic characters:
covering trichomes (usually fragments of epidermis of the cone berry wall containing cells
only fragments observed) with thick, pitted, colourless walls and brown glandular
D. Lower epidermis, in content, occasiormally with anomocytic stomata (2.5.3);
surface view, with diacytic fragments of the 3-rayed apical cleft of the cone berry with
stomata (Da) and secretory spaces and epidermal cells interlocked by papillous
trichome with a tetracellular outgrowths; fragments of the hypodermis with
head (Db) collenchymatous thickened cells; fragments of the mesocarp
Figure 1229.-1. - Illustration of powdered herbal drug ofJava consisting of large thin-walled parenchymatous cells, usually
tea (see Identification B) rounded, with large intercellular spaces and irregular, large,
usually scarcely pitted, yellow idioblasts (barrel cells);
Column’. fragments of schizogenous oil cells; fragments of the testa
— size: I = 0.25 m, 0 = 4.6 mm; with thick-walled, pitted, colourless sclereids containing 1 or
— stationary phase: octadecylsilyl silica gel for chromatography R several prism crystals of calcium oxalate; fragments of the
(5 pm). endosperm and embryonic tissue with thin-walled cells
containing fatty oil and aleurone grains.
Mobile phase tetrahydrofuran R, acetic acid R, water R,
methanol R (5:8:42:45 VIVIVIV). c. Thin-layer chromatography (2.2.27).
Flow rate 0.5 mL/min. Test solution Dilute the oil-xylene mixture obtained in the
assay to 5.0 mL with hexane R.
Reference solution Dissolve 4.0 mg of guaiazulene R and 50 pL
Injection 20 pL.
of cineole R in 10 mL of hexane R.
Calculate the percentage content of sinensetin using the
2016 Juniper Oil IV-239
Application 20 pL of the test solution and 10 pL of the Mobile phase ethyl acetate Ry toluene R (5:95 VIV).
Drying In air.
Drying In air.
Detection Treat with anisaldehyde solution Ry heat at
100-105 c for 5-10 min and examine in daylight.
100-105 °C until the zones appear; examine immediately in
Results The chromatogram obtained with the reference daylight.
solution shows a red zone (guaiazulene) in the upper half and Results See below the sequence of zones present in the
a brownish-violet or greyish-violet zone (cineole) in the lower
half; the chromatogram obtained with the test solution shows
test solution.
a strong violet zone (mono- and sesquiterpenes) similar in
position to the zone due to guaiazulene in the chromatogram
obtained with the reference solution, a reddish-violet zone a Top of the plate 1
little above the zone due to cineole in the chromatogram An intense brownish-violet zone
obtained with the reference solution, a greyish-violet zone
(terpinen-4-ol) a little below the zone due to cineole in the A brown zone
chromatogram obtained with the reference solution, and just A violet-pink zone
below that a blue zone; a faint violet zone may be present in
Terpinen-4-ol: a brownish-violet A brownish-violet zone
a similar position to the zone due to cineole; further zones (terpinen-4-ol)
are present.
TESTS a-Terpineol: a violet or A violet or brownish-violet zone
brownish-violet zone (a-terpineol)
Maximum 5 per cent of unripe or discoloured cone berries Reference solution Test solution 1
and maximum 2 per cent of other foreign matter.

Water (2.2.13) B. Examine the chromatograms obtained in the test for
Maximum 120 mL/kg, determined on 20.0 g of the crushed chromatographic profile.
drug.
Total ash (2.4.16) obtained with the test solution are similar in retention time to
Maximum 4.0 per cent. those in the chromatogram obtained with the reference
ASSAY solution.
Essential oil (2.8.12) TESTS
Use 20.0 g of the herbal drug reduced to a coarse powder Relative density (2.2.5)
using a suitable mill immediately before the assay, a 500 mL 0.857 to 0.876.
round-bottomed flask, 200 mL of water R as the distillation Refractive index (2.2.6)
liquid and 0.5 mL of xylene R in the graduated tube. Distil at 1.471 to 1.483.
a rate of 3-4 mL/min for 90 min.
---------------------------------- - ----------------------------------------------------------------------- Ph Eur
-15° to -0.5°.
Peroxide value (2.5.5)
Maximum 20.
Juniper Oil * * It complies with the test for fatty oils and resinified essential
(Ph. Eur. monograph 1832) *** oils.
PhEir_________________________________________________________
DEFINITION procedure.
Essential oil obtained by steam distillation from the ripe, Test solution Dissolve 60 mg of the substance to be examined
non-fermented berry cones of Juniperus communis L. in trimethylpentane R and dilute to 5.0 mL with the same
A suitable antioxidant may be added. solvent.
CHARACTERS Reference solution Mix 25 pL each of ซ-pinene Ry sabinene Ry
Appearance fi-pinene Ry p-myrcene Ry ซ-phellandrene Ry limonene Ry
Mobile, colourless or yellowish liquid. terpinen-4-ol Ry bornyl acetate R and P~caryophyllene R and
Characteristic odour. dilute to 25.0 mL with trimethylpentane R.
Column:
IDENTIFICATION
material: fused silica;
size: I = 30 m (a film thickness of 1 pm may be used) to
Second identification A.
60 m (a film thickness of 0.2 pm may be used),
A. Thin-layer chromatography (2.2.27). 0 = 0.25-0.53 mm;
Test solution Dissolve 0.2 mL of the substance to be stationary phase: poly(dimethyl) (diphenyl) siloxane R.
examined in 5 mL of heptane R.
Reference solution Dissolve 20 mg of ซ.-terpineol R and 20 i-iL
of terpinen-4-ol R in 25 mL of heptane R.
Split ratio 1:50.
IV-240 Kelp 2016
Temperature: central ribs (pseudoveins). F. vesiculosus typically shows a

foliose blade with smooth edges and bears occasional ovoid,
Time Temperature single or paired, air vesicles. The ends of certain branches are
(min) (°C) of ovoid shape and a little widened. They bear numerous
Column 0- 1 60 reproductive organs (conceptacles). F. serratus has a foliose
1 - 58 60*230 blade with a serrate margin and no vesicles, the branches
bearing conceptacles are less swollen. The thallus of
Injection port 250
A. nodosum is irregularly branched, without pseudo-midrib.
Detector 250 It shows single ovoid air vesicles; the falciform conceptacles
are located at the end of small branches.
Detection Flame ionisation. B. Reduce to a powder (355) (2.9. 72). The powder is
greenish-brown. Examine under a microscope using chloral
Injection 0.5 J1L.
hydrate solution R. The powder shows fragments of surface
Elution order Order indicated in the composition of the tissue with regular isodiametric cells with brown contents,
reference solution. Record the retention times of these and fragments of deep tissue with colourless, elongated cells
substances. arranged in long filaments with large mucilaginous spaces
System suitability, reference solution: between them. Thick-walled cells in files and in closely
resolution', minimum 1.5 between the peaks due to sabinene packed groups, from the pseudovcin, are sometimes visible,
and [Lpinene. c. To 1 g of the powdered herbal drug (355) (2.9.72) add
Using the retention times determined from the 20 mL of a 2 per cent VIV solution of hydrochloric acid R.
chromatogram obtained with the reference solution, locate Shake vigorously and filter. Wash the residue with 10 mL of
the components of the reference solution in the water R and filter. To the residue add 10 mL of a 200 g/L
chromatogram obtained with the test solution. solution of sodium carbonate R. Shake and centrifuge. Collect
Determine the percentage content of the components. the supernatant. Adjust to pH 1.5 using sulfuric acid R.
Disregard the peak due to trimethylpentane and peaks K white, flocculent precipitate is slowly formed.
comprising less than 0.01 per cent of the total surface area. TESTS
The percentages are within the following ranges: Arsenic (2.4.27)
- ช.-pinene: 20 per cent to 50 per cent; maximum 90 ppm.
- sabinene'. maximum 20 per cent; Cadmium (2.4.27)
- p-pinene: 1.0 per cent to 12 per cent; maximum 4 ppm.
- p~myrcene: 1.0 per cent to 35 per cent; Lead (2.4.27)
- a.-phellandrene: maximum 1.0 per cent; maximum วิ ppm.
- limonene'. 2.0 per cent to 12 per cent; Mercury (2.4.27)
- terpinen-4-ol: 0.5 per cent to 10 per cent; maximum 0.1 ppm.
- bornyl acetate: maximum 2.0 per cent; Swelling index (2.5.4)
Minimum 6.
- p-caryophyllene: maximum 7.0 per cent.
STORAGE Maximum 15.0 per cent, determined on 1.000 g by drying in
At a temperature not exceeding 25 °C. an oven at 105 °C, for 2 h.
------------------------------------------------------------------------------------------------------------ Ph Eur
Total ash (2.4.16)
Maximum 24 per cent.
Kelp ASSAY
Bladderwrack; Fucus Total iodine
(Ph. Eur. monograph 1426) To 1.000 g of the powdered herbal drug, in a tall silica
crucible, add 5 mL of water R and 5 g of potassium
Ph Eur.________________________
hydroxide R. Stir with a magnesium rod. Heat on a water
DEFINITION bath. Add 1 g of potassium carbonate R. Mix, add the tip of
Fragmented dried thallus of Fucus vesiculosus L. the magnesium rod with the residues of the drug and dry,
or F. serratus L. or AscophyUum nodosum Le Jolis. first on a water-bath then over an open flame. Incinerate
Content raising the temperature progressively to not more than
Minimum 0.03 per cent and maximum 0.2 per cent of total 600 °C. Allow to cool. Add 20 mL of water R and heat
iodine (Ar 126.9) (dried drug). gently to boiling, stirring with a glass rod. Filter the hot
mixture through an unpleated filter, into a conical flask.
CHARACTERS Rinse the residue with 4 quantities, each of 20 mL, of hot
Salty and mucilaginous taste. water R. Rinse the filter and the crucible with 50 mL of hot
Unpleasant marine odour. water R. Combine the solutions. Allow to cool. Neutralise
with dilute sulfuric acid R in the presence of methyl orange
IDENTIFICATION
solution R. Add 3 mL of dilute sulfuric acid R and 1 mL of
A. The drug consists of fragments with a corneous
bromine water R. The solution is yellow. After 5 min add
consistency, blackish-brown to greenish-brown, sometimes
0.6 mL of a 50 g/L solution of phenol R. The solution is
covered with whitish efflorescence. The thallus consists of a clear. Acidify with 5 mL of phosphoric acid R and add 0.2 g of
ribbon-like blade, branching dichotomously with prominent
2016 Knotgrass IV-241
potassium iodide R. Allow to stand for 5 min protected from

light. Add 1 mL of starch solution R and titrate with 0.01 M
sodium thiosulfate.
1 mL of 0.01 M sodium thiosulfate is equivalent to 0.2115 mg
of iodine.
LABELLING
The label states the species of kelp present.
---------------- ---------------------------------------------------------------------------------------- Ph Eur
Knotgrass * **
PhEtr_______ ______________________________________________________
DEFINITION
Whole or fragmented, dried flowering aerial parts of
Polygonum aviculare L. ร. I.
Content
Minimum 0.30 per cent of flavonoids, expressed as
hyperoside (C2iH2o012; Mr 464.4) (dried drug).
IDENTIFICATION
A. The stem is 0.5-2 mm thick, branched, with nodes,
cylindrical or slightly angular, and longitudinally striated.
It bears sessile or shortly petiolate, glabrous, entire leaves,
which differ widely in shape and size. The sheath-like stipules
(ochrea) are lacerate and silvery. The small, axillary flowers
have 5 greenish-white perianth segments, the tips of which
are often red. The dry, indehiscent fruits are 2-4 mm, brown Figure 1885.-1. - Illustration for identification test B of powdered
or black, triangular, usually punctate or striate. herbal drug of knotgrass
B. Microscopic examination (2.&23). The powder is
greenish-brown. Examine under a microscope using chloral Application 20 gL as bands.
hydrate solution R. The powder shows the following diagnostic Development Over a path of 10 cm.
characters (Figure 1885.-1): fragments of lower [A] and Drying At 100-105 °C.
upper [D] leaf epidermises with a striated cuticle and Detection treat with a 10 g/L solution of diphenylboric acid
anisocytic stomata (2.8.3) [Aa, Da]; polygonal cells of the aminoethyl ester R in methanol R', subsequently treat with a
upper epidermis [D] with slightly thickened beaded walls, 50 g/L solution of macrogol 400 R in methanol R. Allow to dry
often associated with palisade parenchyma [Db]; cells of the in air for about 30 min. Examine in ultraviolet light at
lower epidermis [A], with thin, sinuous walls; fragments of 365 nm.
the margin of the lamina of the leaf with irregular cells [J];
fragments of parenchyma [G] with numerous cells containing
in the chromatograms obtained with the reference solution
cluster crystals of calcium oxalate, some of which are very
and the test solution. Furthermore, other fluorescent zones
large [Ga], often associated with vessels [Gb]; groups of
are present in the chromatogram obtained with the test
fibres [B, C] with thick walls [Ba, Cb] from the hypodermis
solution.
of the stem associated either with the epidermis [Ca] or with
parenchyma consisting of cells containing cluster crystals of
calcium oxalate [Bb]; fragments of the ochrea [E] with Top of the plate
elongated, thin-walled cells [Ea], along which run very Caffeic acid: a light blue 1 or 2 blue fluorescent zones
elongated fibres [Eb]; globular pollen grains with a smooth fluorescent zone (caffeic acid)
exine and 3 germinal pores [H]; occasional brown fragments
of the exocarp composed of cells with thick, sinuous 1 or 2 yellowish-green fluorescent
walls [F]. zones
c. Thin-layer chromatography (2.2.27). A yellow fluorescent zone
Test solution To 1.0 g of the powdered herbal drug (355) Hyperoside: a yellowish-brown
(2.9.72) add 10 mL of methanol R. Heat the mixture in a fluorescent zone
water-bath under a reflux condenser for 10 min. Cool and A yellowish-brown fluorescent
zone
filter.
Reference solution Dissolve 1 mg of caffeic acid R, 1 mg of fluorescent zone (chlorogenic acid)
chlorogenic acid R and 2.5 mg of hyperoside R in 10 mL of
methanol R.
A yellowish-brown fluorescent
Plate TLC silica gel plate R. zone
Mobile phase anhydrous formic acid R) glacial acetic acid R> Reference solution Test solution
water R3 ethyl acetate R VIVIVIV).
IV-242 Kudzuvine Root 2016
TESTS IDENTIFICATION
Foreign matter (2.8.2) A. Small, square pieces or thick, rectangular slices, 5-35 cm
Maximum 2 per cent of roots and maximum 2 per cent of long and 0.5-1 cm thick. The outer bark is pale brown, with
other foreign matter. longitudinal wrinkles and rough; the section is yellowish-
Loss on drying (2.2.32) white and shows indistinct striations. The texture is strongly
Maximum 10.0 per cent, determined on 1.000 g of the fibrous.
powdered herbal drug (710) (2.9.12) by drying in an oven at B. Microscopic examination (2.8.23). The powder is pale
105 °C for 2 h. brown. Examine under a microscope using chloral hydrate
Total ash (2.4.16) solution R. The powder shows the following diagnostic
Maximum 10.0 per cent. characters: thick-walled lignified fibres, which occur in
groups, surrounded by a calcium oxalate prism sheath; crystal
ASSAY cells with thickened walls; rare sclereids, subrounded or
Stock solution In a 100 mL round-bottomed flask, place elliptical, about 50 pm in diameter; relatively large bordered-
0.800 g of the powdered herbal drug (355) (2.9.72), and add pitted vessels with hexagonal or elliptical pits arranged very
1 mL of a 5 g/L solution of hexamethylenetetramine Ry 20 mL densely. Examine under a microscope using a
of acetone R and 2 mL of hydrochloric acid Rl. Boil the 50 per cent VIV solution of glycerol R. The powder shows
mixture under a reflux condenser for 30 min. Filter the numerous starch granules, simple or 2-20 compound;
liquid through a plug of absorbent cotton into a flask. the starch granules are spheroidal, semi-rounded or polygonal
Add the absorbent cotton to the residue in the round- with a pointed, cleft or stellate hilum, about 15 pm in
bottomed flask and extract with 2 quantities, each of 20 mL, diameter.
of acetone R, each time boiling under a reflux condenser for
10 min. Allow to cool, filter each extract through the plug of
absorbent cotton into the flask. Filter the combined acetone Test solution Sonicate 0.5 g of die powdered herbal drug
extracts through a filter paper into a volumetric flask and (355) (2.9.12) with 5 mL of methanol Ry then centrifuge;
dilute to 100.0 mL with acetone Ry rinsing the flask and the use the supernatant.
filter paper. Introduce 20.0 mL of the solution into a Reference solution Dissolve 5 mg of puerarin R and 5 mg of
separating funnel, add 20 mL of water R and shake the daidzin R in 5 mL of methanol R.
mixture with 1 quantity of 15 mL and then 3 quantities, each Plate TLC silica gel F254 plate R (2-10 pm).
of 10 mL, of ethyl acetate R. Combine the e±yl acetate Mobile phase water Ry methylene chloride Ry methanol Ry ethyl
extracts in a separating funnel and wash with 2 quantities, acetate R (10:20:22:40 VIVIVIV) y use the lower layer.
each of 50 mL, of water R. Dry ±e extracts over 10 g of
anhydrous sodium sulfate Ry filter into a 50 mL volumetric
flask and dilute to volume with ethyl acetate R. Development Over a path of 6 cm.
Test solution To 10.0 mL of the stock solution add 1 mL of Drying In air.
aluminium chloride reagent R and dilute to 25.0 mL with a Detection Examine in ultraviolet light at 254 nm.
5 per cent VIV solution of glacial acetic acid R in methanol R. Results See below the sequence of zones present in the
Compensation liquid Dilute 10.0 mL of the stock solution to chromatograms obtained with the reference solution and the
25.0 mL with a 5 per cent VIV solution of glacial acetic test solution. Furthermore, other zones may be present in the
acid R in methanol R. chromatogram obtained with the test solution.
Measure the absorbance (2.2.25) of the test solution after
30 min by comparison with the compensation liquid at Top of the plate
425 nm. Calculate the percentage content of flavonoids,
calculated as hyperoside, using the following expression:
A X 1.25 A quenching zone
Daidzin: a quenching zone A quenching zone
i.e. taking the specific absorbance of hyperoside to be 500. Puerarin: a quenching zone A quenching zone
At least 5 quenching zones
-------------------------------------------------------- —-------------------------------- --------------Ph Eur
TESTS
Kudzuvine Root ; * Foreign matter (2.8.2)
Maximum 5 per cent.
Ph Eur_________ __ ___ _________________________ ___________ _ ________ Maximum 10.0 per cent, determined on 1.000 g of the
DEFINITION powdered herbal drug (355) (2.9.12) by drying in an oven at
Fragmented, dried root of Pueraria lobata (Willd.) Ohwi. 105 °C.
Content Total ash (2.4.16)
Minimum 6.5 per cent of total isoflavonoids, expressed as Maximum 7.0 per cent.
puerarin (C12H2009; Mf 416.4) (dried drug), of which Ash insoluble in hydrochloric acid (2.8.1)
minimum 45 per cent consists of puerarin. Maximum 1.0 per cent.
2016 Thomson Kudzuvine IV-243
ASSAY Calculate the percentage content of total isoflavonoids

Liquid chromatography (2.2.29). (3-hydroxypuerarin, puerarin, 3-methoxypuerarin, 6-O"-D-
Test solution Introduce 0.100 g of the powdered herbal drug xylosylpuerarin and daidzin) using the following expression:
(355) (2.9.72) into a 250 mL conical flask, add 50.0 mL of
ethanol (30 per cent VIV) R and weigh. Heat under a reflux Al X 7ท2 X p
condenser for 30 min. Allow to cool and weigh again. Adjust A2 X 7ท1
to the initial mass with ethanol (30 per cent VIV) R, mix well
and filter. Al = sum of the areas of the peaks due to the
isoflavonoids (3-hydroxypuerarin, puerarin,
Reference solution Introduce an amount of kudzuvine root dry
3-methoxypuerarin, 6-O"-D-xylosylpuerarin and
extract HRS corresponding to 3.0 mg of puerarin into a
daidzin) in the chromatogram obtained with the
250 mL conical flask, add 50.0 mL of ethanol
test solution;
(30 per cent VIV) R and weigh. Heat under a reflux
A2 = area of the peak due to puerarin in the
condenser for 30 min. Allow to cool and weigh again. Adjust
and filter. solution;
nil — mass of the herbal drug to be examined used to
Column 2 columns coupled in series: prepare the test solution, in grams;
size: I — 0.10 m, 0 = 4.6 mm; m2 =■ mass of kudzuvine root dry extract HRS used to
stationary phase: monolithic octadecylsilyl silica gel for prepare the reference solution, in grams;
chromatography R. p = percentage content of puerarin in kudzuvine root
Mobile phase: dry extract HRS.
— mobile phase A: glacial acetic acid Ry water R
____________________________________________________ PhEur
(0.1:99.9 VIV);
Time
(min)
Mobile phase A
(per cent V/V)
Mobile phase B
(per cent V/V) Thomson Kudzuvine Root ★ *
0 - 16.5 90 -> 71 10 -» 29 (Ph. Eur. monograph 2483) *'**
PhEur.____________________________________________________________
Flow rate 3.0 โทบmin. DEFINITION
Detection Spectrophotometer at 260 nm. Whole or fragmented, dried root of Pueraria thomsonii Benth.,
Injection 10 pL. with the outer bark removed.
Identification of peaks Use the chromatogram supplied with Content
kudzuvine root dry extract HRS and the chromatogram Minimum 0.4 per cent of total isoflavonoids, expressed as
obtained with the reference solution to identify the peaks due puerarin (C12H20O9; Mr 416.4) (dried drug), of which
to the isoflavonoids (3-hydroxypuerarin, puerarin, minimum 55 per cent consists of puerarin.
3-methoxypuerarin, 6-O"-D-xylosylpuerarin and daidzin).
IDENTIFICATION
Relative retention With reference to puerarin (retention A. Cylindrical, subfusiform or semi-cylindrical, 12-15 cm
time = about 3.4 min): 3-hydroxypuerarin = about 0.7; long and 4-8 cm in diameter, sometimes in longitudinally or
3-methoxypuerarin = about 1.09; 6-O"-D- obliquely cut thick slices, varying in size. Externally
xylosylpuerarin = about 1.15; daidzin = about 1.4. yellowish-white or pale brown. The root is heavy, texture
System suitability: reference solution: hard and starchy, a transverse section shows pale brown
— peak-to-valley ratio: minimum 10, where Hp = height concentric rings formed by fibres, a longitudinal section
above the baseline of the peak due to 3-methoxypuerarin shows several longitudinal striations formed by fibres.
and Hv ะ= height above the baseline of the lowest point of B. Microscopic examination {2.8.23). The powder is
the curve separating this peak from the peak due to yellowish-white. Examine under a microscope using chloral
puerarin. hydrate solution R. The powder shows the following diagnostic
Calculate the percentage content of puerarin using the characters: thick-walled lignified fibres, which occur in
following expression: groups, surrounded by a calcium oxalate prism sheath; crystal
cells with thickened walls; rare sclereids, subrounded or
Al X m2 Xp elliptical, about 50 pm in diameter; relatively large bordered-
A.2 X mi
pitted vessels with hexagonal or elliptical pits, arranged very
densely. Examine under a microscope using a
50 per cent VIV solution of glycerol R. The powder shows
Al = area of the peak due to puerarin in the numerous starch granules, simple or 2-20 compound;
chromatogram obtained with the test solution; the starch granules are spheroidal, semi-rounded or polygonal
A2 = area of the peak due to puerarin in the with a pointed, cleft or stellate hilum, about 15 pm in
chromatogram obtained with the reference diameter.
solution;
พ1 = mass of the herbal drug to be examined used to
prepare the test solution, in grams; Test solution Sonicate 0.5 g of the powdered herbal drug
(355) {2.9.12) with 5 mL of methanol R) then centrifuge;
nt2 = mass of kudzuvine root dry extract HRS used to
prepare the reference solution, in grams; use the supernatant.
p = percentage content of puerarin in kudzuvine root Reference solution Dissolve 5 mg of daidzin R and 5 mg of
dry extract HRS. puerarin R in 5 mL of methanol R.
IV-244 Thomson Kudzuvine 2016
Plate TLC silica gel F254 plate R (2-10 pm). Time Mobile phase A Mobile phase B
Mobile phase water Ry methylene chloride Ry methanol Ry ethyl
0 - 16.5 90 -> 71 10 -> 29
acetate R (10:20:22:40 VlVIVIVfy use the lower layer.
Application 7 |1L as bands of 8 mm.
Development Over a path of 6 cm. Flow rate 3.0 mUmin.
Drying In air. Detection Spectrophotometer at 260 nm.
Detection Examine in ultraviolet light at 254 nm. Injection 10 pL.
Results See below the sequence of zones present in the Identification of peaks Use the chromatogram supplied with
chromatograms obtained with the reference solution and the kudzuvine root dry extract HRS and the chromatogram
test solution. Furthermore, other zones may be present in the obtained with the reference solution to identify the peaks due
chromatogram obtained with the test solution. to the isoflavonoids (puerarin, 3-mcthoxypuerarin,
6-O"-D-xylosylpuerarin and daidzin).
Top of the plate Relative retention With reference to puerarin
(retention time = about 3.4 min):
A weak quenching zone 6-O"-D-xylosylpuerarin = about 1.15; daidzin = about 1.4.
— peak-to-valley ratio: minimum 10, where Hp = height
Daidzin: a quenching zone A weak quenching zone
above the baseline of the peak due to 3-methoxypuerarin
Puerarin: a quenching zone A weak quenching zone and Hv = height above the baseline of the lowest point of
the curve separating this peak from the peak due to
puerarin.
Several quenching zones
Calculate the percentage content of puerarin using the
Reference solution Test solution following expression:
Al X ไท2 X p
TESTS A2 X mi
Maximum ว per cent.
Loss on drying (2.2.32) chromatogram obtained with the test solution;
Maximum 10.0 per cent, determined on 1.000 g of the A2 — area of the peak due to puerarin in the
powdered herbal drug (355) (2.9.12) by drying in an oven at chromatogram obtained with the reference
105 °C. solution;
Total ash (2.4.16) m1 = mass of the herbal drug to be examined used to
Maximum 7.0 per cent. prepare the test solution, in grams;
Ash insoluble in hydrochloric acid (2.8.1) m2 = mass of kudzuvine root dty extract HRS used to
Maximum 1.0 per cent. prepare the reference solution, in grams;
p = percentage content of puerarin in kudzuvine root
ASSAY dry extract HRS.
Calcdate the percentage content of total isoflavonoids
Test solution Introduce 1.00 g of the powdered herbal drug (puerarin, 6-O"-D-xylosylpuerarin and daidzin) using the
(355) (2.9.12) into a 250 mL conical flask, add 50.0 mL of following expression:
ethanol (30 per cent VIV) R and weigh. Heat under a reflux
condenser for 30 min. Allow to cool and weigh again. Adjust
to the initial mass with ethanol (30 per cent VIV) Ry mix well Al X m2 X p
and filter. A2 X mi
Reference solution Introduce an amount of kudzuvine root dry

Al = sum of the areas of the peaks due to the
extract HRS corresponding to 3.0 mg of puerarin into a
isoflavonoids (puerarin, 6-O"-D-xylosylpuerarin
250 mL conical flask, add 50.0 mL of ethanol
and daidzin) in the chromatogram obtained with
(30 per cent VIV) R and weigh. Heat under a reflux
condenser for 30 min. Allow to cool and weigh again. Adjust the test solution;
and filter.
solution;
Column 2 columns coupled in series: m1 = mass of the herbal drug to be examined used to
— size: z = 0.10 m, 0 = 4.6 mm; prepare the test solution, in grams;
— stationary phase: monolithic octadecylsilyl silica gel for m2 = mass of kudzuvine root dry extract HRS used to
chromatography R. prepare the reference solution, in grams;
Mobile phase: p =ะ percentage content of puerarin in kudzuvine root
— mobile phase A: glacial acetic add Ry water R dry extract HRS.
(0.1:99.9 VIV);
— mobile phase B: acetonitrile R’y
2016 Lavender Flower IV-245
Lavender Flower
Pn Etr___________ ______________ ____________________________________
DEFINITION
Dried flower of Lavandula angustifolia Mill. (L. officinalis
Chaix).
Content
Minimum 13 mL/kg of essential oil (anhydrous drug).
CHARACTERS
Strongly aromatic odour.
IDENTIFICATION
First identification A, B, D
Second identification A, B, c
A. The flower has a short peduncle and consists of a bluish-
grey tubular calyx divided distally into 4 very short teeth and
a small rounded lobe, a blue bilabial corolla with the upper
lip bifid and the lower lip trilobate and 4 didynamous
stamens with ovoid anthers.
B. Microscopic examination (2.8.23). The powder is bluish-
grey. Examine under a microscope using chloral hydrate
characters (Figure 1534.-1): covering trichomes bifurcating at
one or more levels [C, L]; secretory trichomes with short
stalks and 8-celled heads of the Laniiaceae type in side
view [H], in surface view [M]; glandular trichomes with
unicellular [O] or multicellular [K] stalks and unicellular
heads; glandular trichomes with long uneven stalks and Figure 1534.-1. - Illustration for identification test B of powdered
unicellular heads, separated from the stalk by an intermediary
herbal drug of lavender flower
cell with a smooth cuticle, certain trichomes show a crown of
small spheroid protuberances just below the insertion point
D. Examine the chromatograms obtained in the test for other
of the intermediary cell on the stalk [G]; fragments of
papillose epidermis from the inner surface of the petals, in species and varieties of lavender.
surface view [J] 5 in side view [P]; fragments of calyx Results The 5 principal peaks in the chromatogram obtained
epidermis with sinuous-walled cells and containing prismatic with the reference solution are similar in retention time to the
crystals of calcium oxalate [Q]; spherical pollen grains which corresponding peaks in the chromatogram obtained with the
have a diameter of about 45 pm and an exine with 6 slit-like test solution. Among them are mainly linalol and linalyl
germinal pores and 6 ribbon-like groins radiating from the acetate peaks.
poles [A, D, E, F]; rare fragments of leaf epidermis with TESTS
stomata, mostly of the diacytic type (2.8.3) [B]; fragments of Foreign matter (2.8.2)
vascular tissue with spiral vessels included in parenchyma Maximum 3 per cent of stems and maximum 2 per cent of
with some cells containing small calcium oxalate cluster other foreign matter.
crystals [N].
Other species and varieties of lavender
c. Thin-layer chromatography (2.2.27). Gas chromatography (2.2.28): use the normalisation
Test solution To 0.5 g of the powdered herbal drug (355) procedure.
(2.9.12) add 5 mL of hexane R, shake for 5 min and filter. Test solution Dilute 0.2 mL of the essential oil-xylene mixture
Reference solution Dissolve 10 pL of linalol R and 10 pL of obtained in the assay to 5 mL with hexane R, add 1 g of
linalyl acetate R in 5 mL of hexane R. anhydrous sodium sulfate R, shake and use the supernatant.
Plate TLC silica gel plate R. Reference solution Dissolve 0.1 g of limonene Rj 0.2 g of
Mobile phase ethyl acetate R, toluene R (5:95 VIV). cineole R, 0.05 g of camphor R, 0.4 g of linalol R3 0.6 g of
Application 10 pL as bands. linalyl acetate R and 0.2 g of (L-terpineol R in 100 mL of
hexane R.
Column:
Drying In air. — material: fused silica;
Detection Spray with anisaldehyde solution R. Heat at — size: / = 60 m, 0 = 0.25 mm;
100-105 °C for 5-10 min and examine in daylight. — stationary phase: macrogol 20 000 R.
Results The chromatogram obtained with the reference Carrier gas helium for chromatography R.
solution shows in the lower third a greyish-blue zone (linalol) Flow rate 1.5 mL/min.
and in the middle third a greyish-blue zone (linalyl acetate).
Split ratio 1:100.
The chromatogram obtained with the test solution shows the
zones due to linalol and linalyl acetate and in the middle,
between these zones, a redish-violet zone
(epoxydihydrocaryophyllene). Further zones are also present.
IV-246 Lavender Oil 2016
Temperature: Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
plate R (2-10 pm)].
Time Temperature Mobile phase ethyl acetate Ry toluene R (5:95 VIV).
(°C) Application 10 pL [or 2 pL] as bands of 10 mm [or 6 mm].
Column 0 - 15 70
15 - 70 70 -> 180
Drying In air.
Injection port 220
Detector 220 100-105 °C for 5-10 min; examine immediately in daylight.
Detection Flame ionisation. chromatograms obtained with the reference solution and the
test solution. Furthermore, other violet-red or greenish-brown
Injection The same volume of each solution.
zones are present in the chromatogram obtained with the test
Elution order Order indicated in the composition of the solution above the zone of linalyl acetate up to the solvent
reference solution. Record the retention times of these front.
substances.
System suitability’, reference solution: Top of the plate
— resolution’, minimum 1.5 between the peaks due to
limonene and cineole; A violet-red or greenish-brown
zone
— number of theoretical plates’, minimum 30 000, calculated
for the peak due to limonene at 110 °C.
Using the retention times determined from the Linalyl acetate: a violet or brown A violet or brown zone (linalyl
acetate)
A violet-red zone
the 6 components of the reference solution in the
chromatogram obtained with the test solution. Disregard the
peaks due to hexane and xylene.
Limit".
1,8-Cineole: a violet-brown zone Possibly a weak violet-brown
— camphor, maximum 1 per cent. zone (1,8-cineole)
Water (2.2.13) Linalol: a violet or brown zone A violet or brown zone (linalol)
Maximum 100 mI7kg, determined on 20.0 g.
A weak yellowish-brown zone
Total ash (2.4.16)
Several unresolved zones
ASSAY
Use 20.0 g of the herbal drug, a 1000 mL round-bottomed B. Examine the chromatograms obtained in the test for
flask, 500 mL of water R as the distillation liquid and 0.5 mL chromatographic profile.
of xylene R in the graduated tube. Distil at a rate of Results The characteristic peaks in the chromatogram
2-3 mI7min for 2 h. obtained with the test solution are similar in retention time to
________________________________________________________________ Ph Eur those in the chromatogram obtained with reference
solution (a).
TESTS
Lavender Oil * * 0.878 to 0.892.
(Ph. Eur. monograph 1338) *** Refractive index (2.2.6)

1.455 to 1.466.
PhEtr_______________________________________________________________
DEFINITION -12.5° to - 6.0°.
Essential oil obtained by steam distillation from the flowering Acid value (2.5.1)
tops of Lavandula angustifolia Mill. (Lavandula officinalis Maximum 1.0, determined on 5.0 g of the substance to be
Chaix). examined dissolved in 50 mL of the prescribed mixture of
CHARACTERS solvents.
Appearance Chromatographic profile
Colourless or pale yellow, clear liquid. Gas chromatography (2.2.28): use the normalisation
Odour Complex, reminiscent of linalyl acetate. procedure.
IDENTIFICATION Test solution Dissolve 200 pL of the essential oil to be
solvent.
Reference solution (a) Dissolve 5 pL of limonene Ry 5 pL of
A. Thin-layer chromatography (2.2.27). cineole Ry 5 pL of 3-octanone Ry 5 mg of camphor Ry 40 pL of
Test solution Dissolve 20 pL of the essential oil to be linalol R, 50 pL of linalyl acetate Ry 10 pL of terpinen-4-ol Ri
examined in 1 mL of toluene R. 5 pL of lavandulyl acetate Ry 5 pL of lavandulol R and 5 mg
Reference solution Dissolve 10 pL of linalol Ry 10 pL of of a-terpineol R in heptane R and dilute to 10 mL with the
cineole R and 10 pL of linalyl acetate R in 1 mL of toluene R. same solvent.
2016 Lavender Oil IV-247
Reference solution (b) Dissolve 5 pL of limonene R in heptane R Carrier gas helium for chromatography R.
and dilute to 50.0 mL with the same solvent. Dilute 0.5 mL Flow rate 1.3 mL/min.
of the solution to 5.0 mL with heptane R.
split ratio 1:30.
Column'.
Temperature:
— size: I = 60 m, 0 = 0.25 mm;
stationary phase: macrogol 20 000 R (film thickness Time Temperature
0.25 pm). (min) __________ (°C)________
Column 0 - 65 50 -> 180
Flow rate 1.5 mUmin. Injection port 230
split ratio 1:50. Detector 230

Temperature:
Time Temperature Injection 1 pL.
(min) (°C)
Column 0 - 15
Elution order (R)-linalol, (ร)-linalol, borneol, (R)-linalyl
70
acetate, (ร)-!inalyl acetate; depending on the operating
15 - 70 70 -> 180 conditions and the state of the column, borneol may elute
Injection port 220 before or after (ร)-linalol.
Detector 220
(R)-linalol and (ร)-!inalol; minimum 2.9 between the
Detection Flame ionisation. peaks due to (ร)-linalol and borneol; minimum 2.0
Injection 1 pL. between the peaks due to (R)-linalyl acetate and
(ร)-linalyl acetate.
solution (a). Record the retention times of these substances. Calculate the percentage content of the specified
(ร)-enantiomers using the following expression:
terpinen-4-ol and lavandulyl acetate. . As . ■ X 100
Using the retention times determined from the As 4- Ar
the components of reference solution (a) in the As = area of the peak due to the corresponding
chromatogram obtained with the test solution. (ร)-enantiomer;
Determine the percentage content of each of these Ar = area of the peak due to the corresponding
components. The percentages are within the following (R)-enantiomer.
ranges: Limits:
— limonene: maximum 1.0 per cent; — (ร)-linalol: maximum 12 per cent;
— 1,8-cineole: maximum 2.5 per cent; — (ร)-linalyl acetate: maximum 1 per cent.
— 3-octanone: 0.1 per cent to 5.0 per cent;
STORAGE
— camphor, maximum 1.2 per cent;
— linalol: 20.0 per cent to 45.0 per cent;
_________________________________ ____ _______________________ Ph Eur
— linalyl acetate: 25.0 per cent to 47.0 per cent;
— terpinen-4-ol: 0.1 per cent to 8.0 per cent;
— lavandulyl acetate: minimum 0.2 per cent;
— lavandulol: minimum 0.1 per cent;
— ซ.-terpineol: maximum 2.0 per cent;
Spike Lavender Oil ; t
chromatogram obtained with reference solution (b) (Ph. Eur. monograph 2419) ***
(0.05 per cent).
PhEir-------------------------------------------------------------- -- ---------------------------------------
Chiral purity
DEFINITION
Gas chromatography (2.2.25).
Essential oil obtained by steam distillation of the flowering
Test solution Dissolve 0.02 g of the essential oil to be tops of Lavandula latifolia Medik.
examined in pentane R and dilute to 10 mL with the same
solvent. CHARACTERS
Reference solution Dissolve 10 pL of linalol R (mixture of
Appearance
Clear, mobile, light yellow or greenish-yellow liquid.
(R)-linalol and (ร)-linalol), add 5 mg of borneol R and 10 pL
of linalyl acetate R (mixture of (R)-linalyl acetate and Odour reminiscent of cineole and camphor.
(ร)-linalyl acetate) in pentane R and dilute to 10 mL with the IDENTIFICATION
same solvent. First identification B.
Column: Second identification A.
— material: fused silica; A. Thin-layer chromatography (2.2.27).
— size-, z = 25 m, 0 = 0.25 mm;
Test solution Dissolve 20 pL of the substance to be examined
— stationary phase: modified ft-cyclodextrin for chiral
chromatography R (film thickness 0.25 pm). in 1 mL of toluene R.
IV-248 Lemon Balm 2016
Reference solution Dissolve 10 pL of cineole R, 10 pL of Reference solution (b) Dissolve 5 pL of limonene R in 50.0 mL
linalol R and 10 pL of linalyl acetate R in 1 mL of toluene R. of heptane R. Dilute 0.5 mL of this solution to 5.0 mL with
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel heptane R.
plate R (2-10 pm)]. Column’.
Mobile phase ethyl acetate R, toluene 7? (5:95 VIV). — material’, fused silica;
Application 10 pL [or 2 pL], as bands of 10 mm [or 6 mm]. — size’. / = 60 m, 0 = 0.25 mm;
— stationary phase’, macrogol 20 000 R (film thickness
0.25 pm).
Drying In air.
Canier gas helium for chromatography R.
Detection spray with anisaldehyde solution R and heat at
Flow rate 1.5 mDmin.
100-105 °C for 5-10 min; examine immediately in daylight.
split ratio 1:50.
chromatograms obtained with the reference solution and the Temperature
in the chromatogram obtained with the test solution. Time Temperature
(°C)_____________
Column 0 - 15 70
Top of the plate 15 - 70 70 - 180
Injection port 220
A pink zone
Detector 220
Linalyl acetate: a violet or brown A faint violet or brown zone may

be present (linalyl acetate) Detection Flame ionisation.
Injection 1 pL.
spike lavender oil CRS and the chromatogram obtained with
Cineole: a violet-brown zone An intense violet-brown zone reference solution (a) to identify the peaks due to limonene,
(cineole)
cineole, camphor, linalol, linalyl acetate, a-terpineol and
Linalol: a violet or brown zone An intense violet or brown zone
(linalol) traHs-a-bisabolene.
A greyish or brownish zone System suitability: reference solution (a):
A faint violet zone
chromatogram supplied with spike lavender oil CRS;
Reference solution Test solution — resolution: minimum 1.5 between the peaks due to
limonene and cineole.
components. The percentages arc within the following
ranges:
Results The characteristic peaks in the chromatogram — limonene: 0.5 per cent to 3.0 per cent;
obtained with the test solution are similar in retention time to — cineole: 16.0 per cent to 39.0 per cent;
the peaks due to limonene, cineole, camphor, linalol, linalyl — camphor. 8.0 per cent to 16.0 per cent;
acetate, a-terpineol and mnzs-a-bisabolene in the — linalol: 34.0 per cent to 50.0 per cent;
chromatogram obtained with reference solution (a). — linalyl acetate: maximum 1.6 per cent;
TESTS — a-terpineol: 0.2 per cent to 2.0 per cent;
Relative density (2.2.5) — trans-a-bisabolene: 0.4 per cent to 2.5 per cent;
0.894 to 0.907. — disregard limit: the area of the principal peak in the
(0.05 per cent).
1.461 to 1.468.
Optical rotation (2.2.7) STORAGE
—7 to 4- 2°. At a temperature not exceeding 25 °C.
____________________________________________ __________________ PnEj
Acid value (2.5.7)
Maximum 1.5, determined on 5.00 g of the substance to be
examined.
Solubility in alcohol (2.5.10)
1.0 mL of the substance to be examined is soluble, Lemon Balm ★ ไ
sometimes with opalescence, in 3.0 mL of ethanol
(70 per cent VIV) R. (Melissa Leaf Ph Eur monograph 1447)
Chromatographic profile Preparation
Gas chromatography (2.2.25): use the normalisation Lemon Balm Dry Extract
procedure. Ph Eur------------------------------------------------------------------------------------------------- --------
Test solution Dissolve 200 pL of the substance to be examined DEFINITION

in heptane R and dilute to 10.0 mL with the same solvent. Dried leaf of Melissa officinalis L.
Reference solution (a) Dissolve 200 pL of spike lavender Content
oU CRS in heptane R and dilute to 10.0 mL with the same Minimum 1.0 per cent of rosmarinic acid (CigHifiOgj
solvent. Mr 360.3) (dried drug).
2016 Lemon Balm IV-249
CHARACTERS the graduated tube of the apparatus with the aid of a small
Odour reminiscent of lemon. portion of xylene Ry and dilute to 1.0 mL with the same
IDENTIFICATION solvent.
A. The leaves have a petiole of varying length; the lamina is Reference solution Dissolve 1.0 pL of citronellol R and 10.0 pL
broadly ovate, up to about 8 cm long and 5 cm wide, acute of citral R (composed of neral and geranial) in 25 mL of
at the apex and rounded to cordate at the base; the margins xylene R.
are crenate to dentate. The upper surface is intense green, Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
the lower surface is paler green and shows a conspicuous plate R (2-10 pm)].
midrib and a raised, reticulate venation; scattered hairs occur Mobile phase ethyl acetate R, hexane R (10:90 VIV).
on the upper surface and along the veins on the lower Application 20 pL [or 4 pL] as bands.
surface, which is also finely punctuate.
Development In an unsaturated tank over a path of 15 cm
[or 6 cm].
greenish. Examine under a microscope using chloral hydrate
solution R. The powder shows the following diagnostic Dtying In air.
characters (Figure 1447.-1): fragments of the upper Detection Spray with anisaldehyde solution R and heat at
epidermis, in surface view, with sinuous walls [A, B, G], 100-105 °C for 10-15 min; examine in daylight.
sometimes accompanied by palisade parenchyma [Aa]; Results See below the sequence of zones present in the
fragments of the lower epidermis [D] with diacytic stomata chromatograms obtained with the reference solution and the
(2.5.2) [Db]; short, straight, unicellular, conical covering test solution. Furthermore, other zones may be present in the
trichomes with a finely striated cuticle, free [E] or attached to chromatogram obtained with the test solution.
an epidermis [Da]; multicellular, uniseriate covering
trichomes with pointed ends and thick, warty cuticles [C];
Top of the plate
eight-celled secretory trichomes of lamiaceous type, in surface
view [Ga]; secretory trichomes with unicellular to tricellular
stalks and unicellular or, more rarely, bicellular heads, in Citronella!: a grey or A grey or greyish-violet zone
surface view [Ba] or in transverse section [F]. greyish-violet zone at the border (citronella!) at the border between
between the upper and middle the upper and middle thirds
thirds
A reddish-violet zone
Citral: 2 greyish-violet or 2 greyish-violet or bluish-violet

bluish-violet zones at the border zones (citral) at the border
between the middle and lower between the middle and lower
thirds thirds
TESTS
1 mm and maximum 2 per cent of other foreign matter,
determined on 20 g.
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Test solution Use brown-glass flasks. Disperse 0.100 g of the
powdered herbal drug (355) (2.9.12) in 90 mL of ethanol
(50 per cent VIV) R. Boil in a water-bath under a reflux
condenser for 30 min, cool, and filter into a 100 mL
volumetric flask. Rinse the flask and the filter with 10 mL of
ethanol (50 per cent V/V) R and dilute to 100.0 mL with the
Figure 1447.-1.- Illustration for identification test B of powdered same solvent. Filter through a 0.45 pm filter.
herbal drug of melissa leaf
Reference solution (a) Dissolve 20.0 mg of rosmarinic acid CRS
c. Thin-layer chromatography (2.2.27). in ethanol (50 per cent V/V) R and dilute to 100.0 mL with
Test solution Place 2.0 g of the powdered herbal drug (355) the same solvent. Dilute 20.0 mL of this solution to
{2.9.12) in a 250 mL round-bottomed flask and add 100 mL 100.0 mL with ethanol (50 per cent VIV) R.
of water R. Distil for 1 h using the apparatus for the Reference solution (b) Dissolve 5.0 mg offerulic acid R in
determination of essential oils in herbal drugs (2.8.12) and reference solution (a) and dilute to 50.0 mL with the same
0.5 mL of xylene R in the graduated tube. After distillation solution.
transfer the organic phase to a 1 mL volumetric flask, rinsing
IV-250 Lemon Balm Preparations 2016
Colimin'. IDENTIFICATION
— stationary phase’, octadecylsilyl silica gel for chromatography R Test solution To 0.2 g of the extract to be examined add
(5 pm). 5 mL of methanol R. Sonicate for 5 min and filter.
Mobile phase'.
Reference solution Dissolve 1.0 mg of hyperoside Ry 1.0 mg of
— mobile phase A: phosphoric acid Ry acetonitrile R, water R
rutin R and 5.0 mg of rosmarinic acid R in 10 mL of
(1:19:80 V!VIV)',
methanol R.
— mobile phase B: phosphoric acid Ry methanol Ry acetonitrile R
(1:40:59 VIVIV)', Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
plate R (2-10 pm)].
Mobile phase anhydrous formic acid Ry water Ry ethyl acetate R
(6:6:90 V/V/V).
(min) (percent V/V) (per cent V/V)
0 - 20 100 -> 55 0 -> 45
20 - 25 55 -> 0
45 -» 100
Drying In air.
25 - 30 0 -> 100 100 ->0
Detection Heat at 100 °C for 5 min, spray the plate whilst still
hot with a 5 g/L solution of diphenylboric acid aminoethyl
Flow rate 1.2 mUmin. ester R in ethyl acetate Ry and examine in ultraviolet light at
Detection Spectrophotometer at 330 nm. 365 nm.
Injection 20 pL. Results See below the sequence of fluorescent zones present
Relative retention With reference to rosmarinic acid (retention
and the test solution. Furthermore, other weaker fluorescent
time = about 11 min): ferulic acid ะะ- about 0.8.
test solution.
— resolution: minimum 4.0 between the peaks due to ferulic
acid and rosmarinic acid.
Calculate the percentage content of rosmarinic acid using the Top of the plate
Rosmarinic acid: a light blue An intense light blue fluorescent
Al X m2 X p X 0.2 fluorescent zone zone (rosmarinic acid)
A2 X 7711 A blue fluorescent zone
AI = area of the peak due to rosmarinic acid in the A blue fluorescent zone
A2 = area of the peak due to rosmarinic acid in the
chromatogram obtained with reference solution (a); Hyperoside: an orange or
nil = mass of the herbal drug to be examined used to greenish-yellow fluorescent
zone
m2 = mass of rosmarinic acid CRS used to prepare
reference solution (a), in grams; Rutin: an orange or greenish-yellow
fluorescent zone
p = percentage content of rosmarinic acid in rosmarinic
acid CRS.
________________________________________________________________ Ph Eur
TESTS
Lemon Balm Dry Extract ASSAY
(Melissa Leaf Dry Extract, Ph Eur monograph 2524) ***
Test solution Use brown glass flasks. To 0.200 g of the extract
Ph Eur_____________________________________________________________ to be examined add 50 mL of ethanol (50 per cent VIV) R.
DEFINITION Sonicate for 10 min and dilute to 100.0 mL with ethanol
Dry extract produced from Melissa leaf (1447). (50 per cent V/V) R. Filter through a membrane filter
Content
Reference solution (a) Dissolve 20.0 mg of rosmarinic acid CRS
Minimum 2.0 per cent of rosmarinic acid (C18H16O8;
in ethanol (50 per cent V/V) R and dilute to 100.0 mL with
Mr 360.3) (dried extract).
the same solvent. Dilute 20.0 mL of this solution to
PRODUCTION 100.0 mL with ethanol (50 per cent V/V) R.
The extract is produced from the herbal drug by a suitable Reference solution (b) Dissolve 5 mg offerulic acid R in
procedure using either hot water (not less than 70 °C) or a reference solution (a) and dilute to 50 mL with reference
hydroalcoholic solvent that is at most equivalent in strength solution (a).
to ethanol (70 per cent VIV).
Column:
CHARACTERS — size: I = 0.25 m, 0 = 4.6 mm;
Appearance — stationary phase: octadecylsilyl silica gel for chromatography R
Brown or greenish-brown, amorphous powder. (5 pm).
2016 Lemon Oil IV-251
Mobile phase'. c. Carry out the method for thin-layer chromatography,

mobile phase A: phosphoric acid R, acetonitrile R, water R Appendix in A, using the following solutions.
(1:19:80 VlVIVy,
(1) Add 1 g of freshly cut peel to 10 mL of methanol and
mobile phase B: phosphoric acid R, methanol R, acetonitrile R
heat in a water-bath at 65° for 5 minutes, shaking frequently.
(1:40:59 VIVIVy,
Allow to cool and filter.
(2) 0.01% w/v each of caffeic acid and naringin and
Time Mobile phase A Mobile phase B 0.025% w/v of rutin in methanol.
0 - 20 100 -> 55 CHROMATOGRAPHIC CONDITIONS
0 -» 45
20 - 25 (a) Using a TLC silica gel plate.
55-> 0 45 -> 100
Flow rate 1.2 mUmin. (c) Apply 20 pL of each solution as bands.
Injection 20 pL. (e) After removal of the plate, dry ±e plate at 105° and spray
the warm plate with a 1 % w/v solution of diphenylboric acid
Relative retention With reference to rosmarinic acid (retention aminoethyl ester in methanol and then with a 5% w/v solution
time = about 11 min): ferulic acid = about 0.8. of polyethylene glycol 400 in methanol. Allow the plate to stand
System suitability'. reference solution (b): for 30 minutes and examine under ultraviolet light (365 nm).
resolution', minimum 4.0 between the peaks due to ferulic
MOBILE PHASE
acid and rosmarinic acid.
A mixture of 10 volumes of anhydrous formic acid, 10 volumes
Calculate the percentage content of rosmarinic acid using the
of water, 30 volumes of butan-2-one and 50 volumes of ethyl
acetate.
Al X 7712 X p X 0.2 CONFIRMATION
A2 X 7711 The chromatogram obtained with solution (1):

exhibits a yellowish-brown fluorescent zone corresponding in
Al ะ= area of the peak due to rosmarinic acid in the colour and position to the zone for rutin in the
chromatogram obtained with the test solution; chromatogram obtained with solution (2);
A2 = area of the peak due to rosmarinic acid in the above it an intense red fluorescent zone and further above a
chromatogram obtained with reference solution very weak greenish fluorescent zone corresponding in colour
;
(a) and position to the zone for naringin in the chromatogram
III ไ = mass of the extract to be examined used to obtained with solution (2);
prepare the test solution, in grams; other coloured zones are present in the chromatogram.
1แ2 = mass of rosmarinic acid CRS used to prepare
reference solution (a), in grams; TESTS
p = percentage content of rosmarinic acid in rosmarinic Volatile oil
acid CRS. Not less than 2.0% v/w (1.7% พ/พ), Appendix XI E, using
300 mL of water as the distillation liquid and no xylene in
---------------------------------------------------------------------------------------------------------- Ph Eur
the graduated tube. Use 20 g, soaked in water and macerated
in a suitable blender, and distil for 3 hours.
Dried Lemon Peel

DEFINITION
Lemon Oil
Dried Lemon Peel is the dried outer part of the pericarp of (Ph. Eur. monograph 0620) *
the ripe, or nearly ripe, fruit of Citrus Union (L.) Burm. f. Preparation
CHARACTERISTICS Terpeneless Lemon Oil
It has the macroscopical and microscopical characters Ph Eur__ _____ ____________________________________________________
described tinder Identification tests A and B.
DEFINITION
IDENTIFICATION Essential oil obtained by suitable mechanical means, without
A. Dried Lemon Peel consists of strips or pieces, showing a the aid of heat, from the fresh peel of Citrus Union (L.)
marked thickening of the epicarp around the calyx. The outer Burman fil.
surface is yellow and somewhat rough from the presence of
CHARACTERS
numerous minute pits, each corresponding to an oil gland;
Appearance
the inner surface with only a small remnant of white, spongy
Clear, mobile, pale yellow or greenish-yellow liquid. It may
pericarp. Fracture, short.
become cloudy at low temperatures.
B. Prepare thin cross sections from material softened by
Characteristic odour.
soaking in water and examine under a microscope using
chloral hydrate solution. The sections show a yellow epidermis IDENTIFICATION
composed of small, thin-walled cells and an underlying First identification B
parenchyma with numerous large oil glands and scattered Second identification A
small strands of vascular tissue; prismatic crystals of calcium
oxalate occur throughout the parenchyma and are
particularly abundant in the layers adjacent to the epidermis.
IV-252 Lemon Oil 2016
A. Thin-layer chromatography (2.2.27). Optical rotation (2.2.7)

Test solution Mix 1 mL of the substance to be examined in + 57° to + 70°.
1 mL of toluene R. Absorbance (2.2.25)
Reference solution Dissolve 10 mg of citropten R and 50 pL of Dissolve 0.250 g of the substance to be examined in
citral R in toluene R and dilute to 10 mL with the same alcohol R) mix and dilute to 100.0 mL with the same solvent.
solvent. Measure the absorbance over the range 260 nm to 400 nm.
Plate TLC silica gel GF254 plate R. If a manual instrument is used, measure the absorbance at
5 nm intervals from 260 nm to about 12 nm before the
Mobile phase ethyl acetate R, toluene R (15:85 ViV).
expected absorption maximum, then at 3 nm intervals for
Application 10 pL, as bands. 3 readings and at 1 nm intervals to about 5 nm beyond the
Development Over a path of 15 cm. maximum and finally at 10 nm intervals to 400 nm. Plot a
Drying In air. curve representing the absorption spectrum with the
Detection A Examine in ultraviolet light at 254 nm. absorbances as ordinates and the wavelengths as abscissae.
Draw as a baseline the tangent between A and B
(Figure 0620.-1). The absorption maximum c is situated at
315 + 3 nm. From c draw a line perpendicular to the axis
test solution.
of abscissae and intersecting AB at D. Deduct the
absorbance corresponding to point D from that
Top of the plate corresponding to point c. The value c - D is 0.20 to 0.96
and for Italian-type lemon oil it is not less than 0.45.
A quenching zone (bergamotin)
Citral: a quenching zone A quenching zone (citral)
A dark blue zone (5-geranyloxy-

7-methoxycoumarin)
Citropien: a light blue fluorescent A light blue fluorescent zone
(citropten)
A quenching zone (psoralen
derivative)
A quenching zone (biakangelicin)

Results B See below the sequence of the zones present in ±e
test solution.
Top of the plate

(bergamotin)
Citral: a quenching zone A quenching zone (citral)
A bright blue fluorescent

zone (5-geranyloxy-7-
methoxycoumarin)
Citropten: a bright blue A bright violet-blue fluorescent
fluorescent zone zone (citropten)
(psoralen derivative)
Wavelength (nm)
An orange zone (biakangelicin)

Figure 0620.-1. - Typical spectrum of lemon oil for the test for
absorbance
B. Examine the chromatograms obtained in the test for Fatty oils and resinified essential oils (2.8.7)
chromatographic profile. It complies with the test for fatty oils and resinified essential
Results The characteristic peaks in the chromatogram oils.
obtained with the test solution are similar in retention time to Chromatographic profile
those in the chromatogram obtained with the reference Gas chromatography (2.2.28) ะ use the normalisation
solution. procedure.
TESTS Test solution The substance to be examined.
Relative density (2.2.5) Reference solution Dissolve 20 pL of p-pinene R> 10 pL of
0.850 to 0.858. sabinene R, 100 pL of limonene R, 10 pL of y-terpinene R,
Refractive index (2.2.6) 5 pL of P-caryophyUene R, 20 pL of citral R> 5 pL of
1.473 to 1.476. a.-terpineol R> 5 pL of neryl acetate R and 5 pL of geranyl
acetate R in 1 mL of acetone R.
2016 Lemon Preparations IV-253
Column:
— material: fused silica, Terpeneless Lemon Oil
size: l = 30 ทา (a film thickness of 1 pm may be used) to Preparations
60 m (a film thickness of 0.2 pm may be used), Lemon Spirit
0 = 0.25-0.53 mm, Compound Orange Spirit
DEFINITION
Terpeneless Lemon Oil may be prepared by concentrating
Flow rate 1.0 mUmin. Lemon Oil under reduced pressure until most of the terpenes
split ratio 1:100. have been removed or by solvent partition. It contains not
Temperature: less than 40% พ/พ of aldehydes calculated as citral,
C10H16อ.
Time Temperature CHARACTERISTICS
co A clear, colourless or pale yellow liquid, visibly free from
Column 0 -6 water; odour and taste, those of lemon.
6 - 21 45 -> 90
21 - 39
TESTS
90 -> 180
39 - 55 180
Optical rotation
Injection port 220 -5° to +2°, Appendix V F.
Detector 220
Refiractive index
1.475 to 1.485, Appendix V E.
Solubility in ethanol
Detection Flame ionisation. Soluble, at 20°, in 1 volume of ethanol (80%)),
Injection 0.5 |1L of the reference solution and 0.2 pL of the Appendix X M.
test solution. Weight per mL
Elution order Order indicated in the composition of the 0.880 to 0.895 g, Appendix V G.
reference solution. Record the retention times of these
ASSAY
substances.
Carry out the method for determination of aldehydes,
System suitability: reference solution: Appendix X K, using 1 g, omitting the toluene and using a
— resolution: minimum 1.5 between the peaks due to volume, not less than 7 mL, of alcoholic hydroxylamine solution
P-pinene and sabinene and minimum 1.5 between the that exceeds by 1 to 2 mL the volume of 0.5m potassium
peaks due to geranial and geranyl acetate. hydroxide in ethanol (60%) vs required. Each mL of
Using the retention times determined from the 0.5m potassium hydroxide in ethanol (60%)) PS is equivalent to
chromatogram obtained with the reference solution, locate 76.73 mg of C]0H16o.
the components of the reference solution in the
STORAGE
Terpeneless Lemon Oil should be kept in a well-filled
Determine the percentage content of these components.
container and protected from light.
— P-pinene: 7.0 per cent to 17.0 per cent,
— sabinene: 1.0 per cent to 3.0 per cent,
— y-terpinene: 6.0 per cent to 12.0 per cent, Lemon Spirit
— p-caryophyllene: maximum 0.5 per cent,
DEFINITION
— neral: 0.3 per cent to 1.5 per cent,
Terpeneless Lemon Oil 100 mL
— a-terpineol: maximum 0.6 per cent,
— neryl acetate: 0.2 per cent to 0.9 per cent,
— geranial: 0.5 per cent to 2.3 per cent, The spirit complies with the requirements stated under Spirits and
— geranyl acetate: 0.1 per cent to 0.8 per cent. with the following requirements.
Residue on evaporation (2.5.9) Content of aldehydes
1.8 per cent to 3.6 per cent after heating on the water-bath 3.45 to 4.60% w/v, calculated as citral, CioH160.
for 4 h. TESTS
STORAGE Ethanol content
At a temperature not exceeding 25 °C. 84 to 88% v/v, Appendix vni F.
LABELLING Weight per mL
The label states, where applicable, that the contents are 0.814 to 0.823 g, Appendix V G.
Italian-type lemon oil. ASSAY
_______________________________________________________________ Ph Eur Carry out the method for the determination of aldehydes,
Appendix X K, using 10 mL, omitting the toluene and using
a volume, not less than 7 mL, of alcoholic hydroxylamine
solution that exceeds by 1 to 2 mL the volume of
0.5m potassium hydroxide in ethanol (60%)) PS required. Each
mL of 0.5m potassium hydroxide in ethanol (60%) vs is
equivalent to 76.73 mg of CloH16O.
IV-254 Lemon Preparations 2016
Lemon Syrup transverse section [D, F]; these fragments are usually
accompanied by palisade parenchyma [Aa, Hb]; fragments of
DEFINITION the lower epidermis of the lamina in surface view [E],
Lemon Spirit 5 mL covered by a striated cuticle and composed of cells more
Citric Acid iMonohydrate 25 g irregular and somewhat sinuous in outline, with abundant
Invert Syrup 100 mL anomocytic stomata (2.8.3) [Ea] and numerous glandular
Syrup Sufficient to produce 1000 mL trichomes in surface view [Eb] and/or their scars [Ec];
Extemporaneous preparation fragments of the lamina in transverse section [G] with
The following directions apply. 2 layers of palisade parenchyma [Ga] and spongy
parenchyma [Gb]; lignified tissue from the veins [C].
Dissolve the Citric Acid Monohydrate in some of the Syrup,
add the Invert Syrup, the Lemon Spirit and sufficient Syrup
to produce 1000 mL and mix.
CHARACTERISTICS
Lemon Syrup has a weight per mL of about 1.33 g.
Content of citric acid monohydrate, C6H8O7,H2O
2.2 to 2.6% w/v.
ASSAY
Mix 8 g with 100 mL of water and titrate with 0.1m sodium
hydroxide using phenolphthalein solution R1 as indicator.
Each mL of 0.1m sodium hydroxide KS is equivalent to
7.005 mg of C6H8O7,H2O. Determine the weight per mL)
Appendix V G, and calculate the content of C6H8O7,H2O,
weight in volume.
Lemon Verbena Leaf *****

(Ph. Eur. monograph 1834) •***
Ph Eur_______________________________________________________________
DEFINITION
Whole or fragmented, dried leaves of Aloysia citrodora Palau
(syn. Aloysia triphylla (L'Her.) Kuntze; Verbena triphylla
L'Her.; Lippia citriodora Kunth).
Content
— acteoside (C09H36O15; Afr 625): minimum 2.5 per cent,
expressed as ferulic acid (dried drug);
— essential oil: minimum 3.0 mLkg for the whole drug and
minimum 2.0 mL/kg for the fragmented drug (dried herbal drug of lemon verbena leaf
drug). c. Examine the chromatograms obtained in die test for
Verbena officinalis.
CHARACTERS
After grinding, it has a characteristic odour reminiscent of
lemon.
IDENTIFICATION in the chromatogram obtained with the test solution. Zones
A. The leaves are simple with short petioles. They are may be present in the chromatogram obtained with the test
narrow, lanceolate, and about 4 times longer than they are solution below the zone due to rutin in the chromatogram
wide. The entire, slightly undulating margins are curled obtained with the reference solution.
towards the upper surface. The upper surface is dark green
and rough to the touch; the lower surface is paler green and
Top of the plate
shows a prominent midrib with secondary veins running to
the margins.
B. Microscopic examination (2.8.23). The powder is light An intense greyish-green zone
green. Examine under a microscope using chloral hydrate
Arbutin: a blue or brown zone
characters (Figure 1834.-1): fragments of the upper
epidermis of the lamina in surface view [A, B, H], composed
Rutin ะ a dark brownish-yellow A blue or violet zone
of polygonal cells with numerous short, unicellular, thick
walled cystolithic trichomes, each arising from a rosette of
cells at the base and containing calcium concretions (B),
glandular trichomes with a unicellular stalk and a unicellular, ______________ ______ __________ -1
globular head of variable size in surface view [Ha] and
2016 Lime Flower IV-255
TESTS Time Mobile phase A Mobile phase B

Verbena officinalis (min) (per cent V/V) (per cent V/V)
Thin-layer chromatography (2.2.27). 0 - 20 93 -» 83 7-» 17
Test solution To 0.50 g of the powdered herbal drug (710) 20 - 30 83 17
(2.9. /2) add 5 mL of methanol R. Heat in a water-bath at 83 -» 75 17-» 25
30 - 35
60 °C for 10 min. Cool and filter.
35 - 40 75 + 20 25-» 80
Reference solution Dissolve 10 mg of arbutin R and 10 mg of
nam R in methanol R and dilute to 10 mL with the same 40 - 45 20 -» 93 80-» 7
solvent.
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel Flow rate 1.0 mL/min.
plate R (2-10 pm)].
Mobile phase anhydrous formic acid R, glacial acetic acid R,
Injection 20 pL.
water R, ethyl acetate R (11:11:27:100 VIVIVIV).
Application 20 pL [or 5 pL] as bands. — resolution: minimum 3.5 between the peaks due to ferulic
Development Over a path of about 12 cm [or 6 cm]. acid and acteoside.
Drying In air. Calculate the percentage content of acteoside, expressed as
Detection Spray with anisaldehyde solution R and dry at ferulic acid, using the following expression:
100-105 °C for about 10 min; examine in daylight.
Results The chromatogram obtained with the test solution Al X 777-2 X p X 0.5 X 3.1
shows no brownish-grey zone at a position between that of A.2 X 7711
arbutin and rutin in the chromatogram obtained with the
reference solution. A1 = area of the peak due to acteoside in the
Loss on drying chromatogram obtained with the test solution;
Maximum 10.0 per cent, determined on 1.000 g of the /42 = area of the peak due to ferulic acid in the
powdered herbal drug (710) (2.9.12) by drying in an oven at chromatogram obtained with the reference
105 °C for 2 h. solution;
nil = mass of the herbal drug in the test solution, in
Total ash (2.4.16)
grams;
ni2 = mass of ferulic acid CRS in the reference solution,
Ash insoluble in hydrochloric acid (2.8.1) in grams;
Maximum 3.5 per cent. p ะ= percentage content of ferulic acid in ferulic acid
ASSAY CRS',
Acteoside 3.1 = correlation factor between acteoside and ferulic
Liquid chromatography (2.2.29). acid.
Test solution To 1.00 g of the powdered herbal drug (710) Essential oil (2.8.12)
(2.9.12) add 50.0 mL of the reference solution and stir for Introduce 25.0 g of the freshly crushed herbal drug into a
2 h with a magnetic stirrer. Centrifuge for 15 min and pass 1000 mL flask and add 500 mL of a 10 g/L solution of
the supernatant through a membrane filter (nominal pore sodium chloride R as the distillation liquid. Use 0.50 mL of
size 0.45 pm). xylene R in the graduated tube. Distil at a rate of
Reference solution Dissolve 10.0 mg of feridic acid CRS in 3.0-3.5 mUmin for 3 h.
ethanol (60 per cent VIV) R and dilute to 100.0 mL with the _____________________________________________________________ Ph Eur
same solvent.
Precolumn'.
— size: I = 0.01 m, 0 = 4.0 mm;
(5 pm).
Lime Flower * J
Column: (Ph. Eur. monograph 0957) **
— size: I = 0.25 m, 0 = 4.0 mm; Ph Eur---------------------------------------------------------------- -—-----------------------------------
(5 pm); DEFINITION
— temperature: 20 °C. Whole, dried inflorescence of Tilia cordata Miller, of Tilia
platyphyUos Scop., of Tilia X vulgaris Heyne or a mixture of
Mobile phase:
— mobile phase A: 0.3 per cent VIV solution of phosphoric these.
acid Ry CHARACTERS
— mobile phase B: acetonitrile R; Faint aromatic odour.
Faint, sweet and mucilaginous taste.
IDENTIFICATION
A. The inflorescence is yellowish-green. The main axis of the
inflorescence bears a linguiform bract, membranous,
yellowish-green, practically glabrous, the central vein of
which is joined for up to about half of its length with the
peduncle. The inflorescence usually consists of 2-7 flowers,
occasionally up to 16. The sepals are detached easily from
IV-256 Linseed 2016
the perianth; they are up to 6 mm long, their abaxial surface TESTS

is usually glabrous, their adaxial surface and their borders are Foreign matter (2.8.2)
strongly pubescent. The 5 spatulate, thin petals are yellowish- Maximum 2 per cent, determined on 30 g. There are no
white, up to 8 mm long. They show fine venation and their inflorescences with a bract bearing at the abaxial face stellate,
borders only are sometimes covered with isolated trichomes. five- to eight-rayed trichomes and flowers having an apparent
The numerous stamens are free and usually constitute double corolla by transformation of five stamens into petal
5 groups. The superior ovary has a pistil with a somewhat like staminoids and having a pistil which is not lobular nor
5-lobate stigma. indented. Hexamerous flowers occur only occasionally (Tilia
B. Separate the inflorescence into its different pans. Examine americana L., Tilia tomentosa Moench).
under a microscope using chloral hydrate solution R. Loss on drying (2.2.32)
The adaxial epidermis of the bract shows cells with straight Maximum 12.0 per cent, determined on 1.000 g of the
or slightly sinuous anticlinal walls; the abaxial epidermis powdered herbal drug (355) (2.9.12) by drying in an oven at
shows cells with wavy-sinuous anticlinal walls and 105 °C for 2 h.
anomocytic stomata (2.8.ร). Isolated cells in ±e mesophyll
contain small calcium oxalate cluster crystals. Total ash (2.4.16)
The parenchyma of the sepals shows, particularly near the Maximum 8.0 per cent.
veins, numerous mucilaginous cells and cells containing small ______________________________________________________________ Ph Elf
calcium oxalate clusters. The adaxial epidermis of sepals

bears bent, thick-walled covering trichomes, unicellular or
stellate with up to 5 cells. The epidermal cells of the petals
show straight anticlinal walls with a striated cuticle without
stomata. The parenchyma of the petals shows small calcium Linseed ; **
oxalate clusters and especially in its acuminate pan (Ph. Eur. monograph 0095) ***
mucilaginous cells. The pollen grains have a diameter of
When Powdered Linseed is prescribed or demanded, material
about 30-40 pm and are oval or slightly triangular with
complying with the appropriate requirements below shall be
3 germinal pores and a finely granulated exine. The ovary is
glabrous or densely covered with trichomes, often very
Ph Elf —______________________________________________ __ __________
twisted, unicellular or stellate with 2-4 branches.
c. Thin-layer chromatography (2.2.27). DEFINITION
Test solution Shake 1.0 g of the powdered herbal drug (355) Dried, ripe seeds of Linum usitatissimum L.
(2.9.72) with 10 mL of methanol J? in a water-bath at 65 °C IDENTIFICATION
for 5 min. Allow to cool and filter. A. The seed has a flattened, elongated ovoid shape. The testa
Reference solution Dissolve 2.0 mg of caffeic acid R, 5 mg of is dark reddish-brown or yellow, smooth and shiny.
hyperoside R and 5 mg of rutin R in 10 mL of methanol R. The seeds are 4-6 mm long, 2-3 mm wide and 1.5-2 mm
Plate TLC silica gel plate R. thick; one end is rounded and the other end forms an
Mobile phase anhydrous formic acid R, water R, methyl ethyl oblique point near which the hilum appears as a slight
ketone R, ethyl acetate R (10:10:30:50 VIVIVIV). depression. When viewed with a lens, the surface of the seed
coat is seen to be minutely pitted. Inside the testa a narrow,
whitish endosperm and an embryo composed of 2 large,
Development Over a path of 15 cm. flattened, yellowish and oily cotyledons are present;
Drying At 100-105 °C. the radicle points towards the hilum.
Detection Spray the warm plate with a 10 g/L solution of B. Microscopic examination (2.8.23). The powder is greasy
diphenylboric acid aminoethyl ester R in methanol R. ITen spray to the touch. Examine under a microscope using chloral
with a 50 g/L solution of macrogol 400 R in methanol R. Allow hydrate solution R. The powder shows the following diagnostic
to dry for about 30 min and examine in ultraviolet light at characters (Figure 0095.-1): fragments of the outer testa
365 nm. [A, B] with cells that are polygonal in surface view [Aa] or
Results The chromatogram obtained with the reference narrow in transverse section [Ba], and filled with mucilage
solution shows in order of increasing RF value yellowish- [Bb]; fragments of the collenchymatously thickened sub-
orange or brownish-orange fluorescent zones due to rutin and epidermal layer, in transverse section [Be], or in surface view
hyperoside and a greenish-blue fluorescent zone due to [Ab] with rounded cells with triangular intercellular spaces
caffeic acid. In the chromatogram obtained with the test often attached to the sclerenchymatous layer composed of
solution, the main zone shows brownish-yellow or orange elongated cells, with thickened and pitted walls [Ca], some
fluorescence. This zone is situated just above the zone due to with strongly thickened and pitted walls [G]; fragments, in
hyperoside in the chromatogram obtained with the reference surface view [C], consisting of the hyaline layer with thin
solution. In daylight, this zone stands out from the other walled cells [Cb] often remaining attached to the layer of
zones as the main zone. At the Rj: level of rutin there is also elongated sclereids and crossing them at approximately right
a brownish-yellow fluorescent zone. Below this zone, 2 yellow angles [Ca]; fragments of the inner testa, in surface view [D],
fluorescent zones may be present. Between the zones due to composed of moderately thickened polygonal cells filled with
rutin and hyperoside, orange and yellow fluorescent zones are brown-orange pigment; small polyhedral masses of pigment
visible. Between the zones due to hyperoside and caffeic acid, [H]; numerous fragments of parenchyma from the testae,
up to 5 yellow or orange fluorescent zones are present. with large, slightly and regularly thickened cells, in surface
Immediately below the zone due to caffeic acid is a a blue view n, L]; parenchyma of the endosperm [K] and
fluorescent zone. cotyledons [E] containing aleurone grains and oil droplets;
very numerous isolated oil droplets [F].
2016
Liquorice IV-257
Content
Minimum 4.0 per cent of 18p-glycyrrhizic acid (C42H62O| 6;
Mr 823) (dried drug).
IDENTIFICATION
A. The root has few branches. Its bark is brown or brownish-
grey with longitudinal striations and bears traces of lateral
roots. The cylindrical stolons are 1-2 cm in diameter; their
external appearance is similar to that of the root but there are
occasional small buds. The fracture of the root and the
stolon is granular and fibrous. The cork layer is thin;
the secondary phloem region is thick and light yellow with
radial striations. The yellow xylem cylinder is compact, with
a radiate structure. The stolon has a central pith, which is
absent from the root. The external part of the bark is absent
from the peeled root.
B. Microscopic examination (2.8.23). The powder is light
yellow or faintly greyish. Examine under a microscope using
diagnostic characters: fragments of yellow thick-walled fibres,
700-1200 pm long and 10-20 pm wide with a punctiform
lumen, often accompanied by crystal sheaths containing
prisms of calcium oxalate 10-35 pm long and 2-5 pm wide.
The walls of the vessels are yellow, 5-10 pm thick, lignified
and have numerous bordered pits with a slit-shaped aperture;
fragments of cork consisting of thin-walled cells and isolated
prisms of calcium oxalate occur as well as fragments of
parenchymatous tissue. Fragments of cork are absent from
the peeled root. Examine under a microscope using a
mixture of equal volumes of glycerol R and water R.
Figure 0095.-1. - Illustration for identification test B of powdered The powder shows the following diagnostic characters:
herbal drug of linseed simple, round or oval starch granules, 2-20 pm in diameter,
TESTS c. Thin-layer chromatography (2.2.27).
Foreign matter (2.8.2) Test solution To 0.50 g of the powdered herbal drug (180)
Maximum 10 per cent of seeds with a dull coat and (2.9.12) in a 50 mL round-bottomed flask add 16.0 mL of
maximum 1.5 per cent of other foreign matter. water R and 4.0 mL of hydrochloric acid R1 and heat on a
Swelling index (2.8.4) water-bath under a reflux condenser for 30 min. Cool and
Minimum 4. filter. Dry the filter and the round-bottomed flask at 105 °C
for 60 min. Place the filter in the round-bottomed flask, add
Cadmium (2.4.27)
20.0 mL of ether R and heat in a water-bath at 40 °C under
Maximum 0.5 ppm.
a reflux condenser for 5 min. Cool and filter. Evaporate the
Loss on drying (2.2.32) filtrate to dryness. Dissolve the residue in 5.0 mL of ether R.
Maximum 8.0 per cent, determined on 1.000 g of the Reference solution Dissolve 5.0 mg of glycyrrhetic acid R and
powdered herbal drug (355) (2.9.12) by drying in an oven at 5.0 mg of thymol R in 5.0 mL of ether R.
105 °C for 2 h.
Total ash (2.4.16)
Mobile phase concentrated ammonia Ry water Ry ethanol
(96 per cent) Ry ethyl acetate R (1:9:25:65 VIVIVIV).
----------- ----------------------------------------------------------------------------------------------- Ph Eur
Application 10 pL.
Detection A Examine in ฟtraviolet light at 254 nm.
Liquorice * <
Results A The chromatograms obtained with the test solution
(Liquorice Rooty Ph Eur monograph 0277) *** and the reference solution show in the lower half a
Preparations quenching zone due to glycyrrhetic acid.
Liquorice Dry Extract for Flavouring Purposes Detection B Treat with anisaldehyde solution Ry and heat at
Liquorice Liquid Extract 100-105 °C for 5-10 min; examine in daylight.
When Powdered Liquorice is prescribed or demanded, Results B The chromatogram obtained with the reference
material complying with the appropriate requirements below solution shows in the lower half a violet zone due to
shall be dispensed or supplied. glycyrrhetic acid and in the upper third a red zone due to
thymol. The chromatogram obtained with the test solution
Ph Eur______________ :________________________________________________
shows in the lower half a violet zone corresponding to the
DEFINITION zone of glycyrrhetic acid in the chromatogram obtained with
Dried, unpeeled or peeled, whole or cut root and stolons of the reference solution and a yellow zone (isoliquindigenine)
Glycyrrhiza glabra L. and/or of Glycyrrhiza inflata Bat. and/or in the upper third under the zone of thymol in the
Glycyrrhiza uralensis Fisch.
IV-258 Liquorice Preparations 2016
chromatograin obtained with the reference solution. Further B = declared percentage content of monoammonium
zones may be present. glycyrrhizate CRS;
TESTS m = mass of the herbal drug to be examined used to
Loss on drying (2.2.32) prepare the test solution, in grams;
Maximum 10.0 per cent, determined on 1.000 g of the 823 = molecular mass of 18P-glycyrrhizic acid;
powdered herbal drug (355) (2.9.12) by drying in an oven at 840 = molecular mass of monoammonium glycyrrhizate
105 °C for 2 h. (without any water of crystallisation).
Total ash (2.4.16) LABELLING
Maximum 10.0 per cent for the unpeeled drug and The label states whether the drug is peeled or unpeeled.
maximum 6.0 per cent for the peeled drug. _______ _____ ___________________________________ _ _______ ■ Ph Eur

Maximum 2.0 per cent for the unpeeled drug and maximum
0.5 per cent for the peeled drug.
Maximum 20 pg per kilogram of herbal drug.
Liquorice Dry Extract for * *
ASSAY
Flavouring Purposes *****
Liquid chromatography (2.2.29). (Ph. Eur. monograph 2378)
Ph Eur_________________________________________________ ____________
Test solution Place 1.000 g of the powdered herbal drug (180)
(2.9.12) in a 150 mL ground-glass conical flask. DEFINITION
Add 100.0 mL of an 8 g/L solution of ammonia R and treat Dry extract produced from Liquorice root (0277).
in an ultrasonic bath for 30 min. Centrifuge a part of the
Content
solution and dilute 1.0 mL of the supernatant layer to
5.0 per cent to 7.0 per cent of 18p-glycyrrhizic acid
5.0 mL with an 8 g/L solution of ammonia R. Filter through
(C42H62016; Mr 823) (dried extract).
a membrane filter (nominal pore size 0.45 pm); use the
filtrate as the test solution. PRODUCTION
Solution A Dissolve 0.130 g of monoammonium The extract is produced from the cut herbal drug by a
glycyrrhizate CRS in an 8 g/L solution of ammonia R and suitable procedure using water.
dilute to 100.0 mL with the same solvent. CHARACTERS
Reference solution (a) Dilute 5.0 mL of solution A to Appearance
100.0 mL wi± an 8 g/L solution of ammonia R. Yellowish-brown or brown powder.
Reference solution (b) Dilute 10.0 mL of solution A to Very sweet taste.
100.0 mL with an 8 g/L solution of ammonia R.
IDENTIFICATION
Reference solution (c) Dilute 15.0 mL of solution A to Thin-layer chromatography (2.2.27).
100.0 mL with an 8 g/L solution of ammonia R.
Solvent mixture ethyl acetate R3 methanol R (50:50 L7P).
Column’.
— size’. /=0.10 m, 0 = 4 mm;
30 mL of hydrochloric acid R1 and boil on a water-bath under
a reflux condenser for 60 min. After cooling, extract the
(5 pm).
mixture with 2 quantities, each of 20 mL, of ethyl acetate R.
Mobile phase glacial acetic acid R, acetonitrile R) water R Combine the organic layers and filter through a filter covered
(6:30:64 VIVIV). with anhydrous sodium sulfate R. Evaporate the filtrate to
Flow rate 1.5 mUmin. dryness in vacuo and dissolve the residue in 2.0 mL of the
Detection spectrophotometer at 254 nm. solvent mixture.
Injection 10 pL. Reference solution Dissolve 5.0 mg of glycyrrhetic acid R and
Establish a calibration curve with the mass of 5.0 mg of thymol 7? in 5.0 mL of the solvent mixture.
monoammonium glycyrrhizate in the reference solutions, in Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica
grams, as the abscissa and the corresponding peak areas as gel F254 plate R (2-10 pm)].
the ordinate. Mobile phase concentrated ammonia R, water R} ethanol
Using the retention times and the peak areas determined (96 per cent) R, ethyl acetate R (1:9:25:65 VIVIVIV).
from the chromatograms obtained with the reference Application 20 pL [or 10 pL] as bands.
solutions, locate and integrate the peak due to Development Over a path of 15 cm [or 7 cm].
18p-glycyrrhizic acid in the chromatogram obtained with the
test solution.
Calculate the percentage content of 18P-glycyrrhizic acid
using the following expression:
A = mass equivalent of monoammonium glycyrrhizate

in the test solution, determined from the
calibration curve, in grams;
2016 Liquorice Preparations IV-259
___________ Top of the plate A1 ะะะ area of the peak due to 18p-glycyrrhizic acid in
the chromatogram obtained with the test
Thymol: a red zone solution;
A2 = area of the peak due to 18p~glycyrrhizic acid in
A yellow zone the chromatogram obtained with the reference
solution;
mx = mass of the extract to be examined used to
Glycyrrhetic acid: a violet zone A violet zone (glycyrrhetic acid) m2 = mass of monoammonium glycyrrhizate CRS used
to prepare the reference solution, in grams;
p = percentage content of 18p-glycyrrhizic acid in
Reference solution Test solution monoammonium glycyrrhizate CRS;
0.979 ะ= peak correlation factor between 18P"
glycyrrhizic acid and monoammonium
TESTS glycyrrhizate.
____________________________________________________________ PfiEur
Maximum 80 pg per kilogram of extract.
The maximum content applies to the pure undiluted extract. Liquorice Liquid Extract
Where excipients are added to reduce the strength of the
extract, the maximum content should be reduced DEFINITION
proportionally. Liquorice Liquid Extract is prepared by extracting Liquorice
with Purified Water and adding sufficient Ethanol
ASSAY
(90 per cent) to give an ethanol content of 18% v/v in the
Liquid chromatography (2.2.29). final extract.
Solvent mixture water R, methanol R (20:80 VtV).
Test solution Place 0.200 g of the extract to be examined in a The following formula and directions apply.
150 mL ground-glass conical flask. Add 100.0 mL of the Liquorice, unpeeled, in coarse powder 1000 g
solvent mixture and sonicate for 2 min. Filter through a Purified Water A sufficient quantity
membrane filter (nominal pore size 0.45 pm). Ethanol (90 per cent) A sufficient quantity
Reference solution Dissolve 50.0 mg of monoammonium Exhaust the Liquorice with Purified Water by percolation,
glycyrrhizate CRS in the solvent mixture and dilute to Appendix XI F. Boil the percolate for 5 minutes and set
50.0 mL with the solvent mixture. Dilute 1.0 mL of this aside for not less than 12 hours. Decant the clear liquid, filter
solution to 10.0 mL with the solvent mixture. the remainder, mix the two liquids and evaporate until the
Column'. weight per mL of the liqmd is 1.198 g, Appendix V G. Add to
— size'. I ะะะ 0.10 m, 0 = 4.0 mm; this liquid, when cold, one quarter of its volume of Ethanol
— stationary phase', octadecylsilyl silica gel for chromatography R (90 per cent). Allow to stand for not less than 4 weeks; filter.
(5 pm). The extract complies with the requirements stated under Extracts
Mobile phase glacial acetic acid R, acetonitrile R, water R and with the following requirements.
(6:30:64 VIVIV).
IDENTIFICATION
Detection Spectrophotometer at 254 nm. Appendix in A, using the following solutions.
Injection 10 pL. (1) Extract 5 mL of the liquid extract with 20 mL of
Run time 3 times the retention time of 18p-glycyrrhizic acid. chloroform. Heat 2.5 mL of the aqueous layer with 30 mL of
Retention time 18P-glycyrrhizic acid = about 9 min. 0.5m sulfuric acid under a reflux condenser for 1 hour, cool
and extract with two 20 mL quantities of chloroform. Dry the
combined chloroform extracts over anhydrous sodium sulfate,
monoammonium glycyrrhizate CRS and the chromatogram
filter and evaporate the filtrate to dryness; dissolve the
obtained wiffi the reference solution to identify the peaks due
residue in 4 mL of a mixture of equal volumes of chloroform
to 18p-glycyrrhizic acid and 18a-glycyrrhizic acid.
and methanol.
— the chromatogram obtained with the reference solution is (2) Dissolve 10 mg of glycyrrhetinic acid in 2 mL of a mixture
similar to the chromatogram supplied with of equal volumes of chloroform and methanol.
monoammonium glycyrrhizate CRS; CHROMATOGRAPHIC CONDITIONS
— resolution: minimum 2.0 between the peaks due to (a) Use as the coating silica gel F254 (Merck 10 X 20 cm
18p-glycyrrhizic acid and 18a-glycyrrhizic acid. plates are suitable).
Calculate the percentage content of 18p~glycyrrhizic acid, (b) Use the mobile phase as described below.
using the following expression: (c) Apply 10 pL of solution (1) and 5 pL of solution (2).
Al X 7712 X p X 0.979 (e) After removal of the plate, allow it to dry and examine
A2 X 7711 X 5
under ultraviolet light (254 nm).
MOBILE PHASE
5 volumes of methanol and 95 volumes of chloroform.
IV-260 Liquorice Root 2016
CONFIRMATION in water. The powder shows abundant starch granules,

The chromatogram obtained with solution (1) exhibits a dark mostly simple, spherical to ovoid, 3 pm to 20 pm in
spot corresponding to the principal spot in the chromatogram diameter.
obtained with solution (2). c. Carr}' out the method for thin-layer chromatography)
TESTS Appendix III A, using the following solutions.
Ethanol content (1) Add 16 mL of water and 4 mL of hydrochloric acid R1 to
16 to 20% v/v, Appendix VUI F, Method in. 0.5 g of the powdered drug, heat on a water-bath under
Ammonia reflux for 30 minutes, cool and filter. Dry the filter paper and
Place 5 mL in a distillation apparatus of about 500 mL residue in a flask at 105° for 60 minutes, add 20 mL of
capacity fitted with an efficient splash-head and a delivery ether, heat on a water-bath at 40°C under reflux for
tube that dips below the surface of a mixture of 10 mL of 5 minutes, cool and filter. Evaporate the filtrate to dryness
0.05m sulfuric acid PS and 50 mL of water contained in the and dissolve the residue in 5 mL of ether.
receiver. Add 200 mL of water) a little pumice powder and (2) 0.1% w/v each of glycyrrhetic acid and thymol in ether.
30 mL of a solution prepared by dissolving 1.43 g of CHROMATOGRAPHIC CONDITIONS
potassium dihydrogen orthophosphate and 9.1 g of dipotassium (a) Use as the coating substance silica gel F254 (Merck 10
hydrogen orthophosphate in 100 mL of water and distil until X 20 cm plates are suitable).
about 175 mL of distillate has been collected. Titrate the
excess of acid with 0.1m sodium hydroxide PS using methyl red
solution as indicator. Not less than 5 mL of 0.1m sodium (c) Apply 10 pL of each solution as a band.
hydroxide PS is required. (d) Develop the plate to 15 cm.
Dry residue (e) Remove the plate and allow it to dr}' in air for 5 minutes.
40 to 45% w/v, Appendix XIP Examine under ultraviolet light (254 nm). spray the plate with
anisaldehyde solution and heat at 100° to 105° for 10 minutes
Relative density
and examine in daylight.
MOBILE PHASE
Ochratoxin A
Not more than 40 ppb, Appendix XI S2. 1 volume of concentrated ammonia) 9 volumes of water)
25 volumes of ethanol (96°/o) and 65 volumes of ethyl acetate.
SYSTEM SUITABILITY
Under ultra-violet light, the chromatogram obtained with
Liquorice Root for use in TCM solution (2) shows in the lower half a quenching zone due to
glycyrrhetic acid. When sprayed, the chromatogram obtained
DEFINITION with solution (2) shows in the lower half the violet zone of
Liquorice Root for use in TCM is the dried impeded root glycyrrhetic acid and in the upper third the red zone of
and rhizome of Glycyrrhiza uralensis Fisch., and/or Glycyrrhiza thymol.
inflata Bat. and/or Glycyrrhiza glabra L.
CONFIRMATION
It is collected in spring and autumn, separated from the
The chromatogram obtained with solution (1) shows in the
rootlets and dried in the รนท.
lower half a violet zone corresponding to the zone of
It contains not less than 2.0% of glycyrrhizic acid glycyrrhetic acid in the chromatogram obtained with solution
(042แ62016), calculated with reference to the dried material. (2) and a yellow zone (isoliquiridigenine) in the upper third
IDENTIFICATION under the zone of thymol in the chromatogram obtained with
A. The root has few branches. Its bark is brownish-grey or solution (2). Further zones may be present.
brown with longitudinal striations and bears traces of lateral TESTS
roots. The cylindrical stolons are 1-2 cm in diameter; their Total Ash
external appearance is similar to that of the root but there are Not more than 7.0%, Appendix XI J, Method II.
occasional small buds. The fracture of the root and the
stolon is granular and fibrous. The cork layer is thin; Acid-insoluble ash
the secondary phloem region is thick and light yellow with Not more than 2.0%, Appendix XI K, Method II.
radial striations. The yellow xylem cylinder is compact, with Loss on drying
a radiate structure. The stolon has a central pith, which is When dried for 2 hours at 100° to 105°, loses not more than
absent from the root. 12.0%. Use 1 g.
B. Reduce to a powder. The powder is yellowish-brown. Ochratoxin A
Examine under a microscope using chloral hydrate solution. Not more than 20 ppb, Appendix XI S2.
The powder shows abundant fibres occurring in groups, each
ASSAY
group surrounded by a calcium oxalate prism crystal sheath,
Carry out the method for liquid chromatography)
the individual fibres with very thick, partially lignified walls
Appendix in D, using the following solutions.
and few small pits; lignified bordered-pitted vessels, singly or
(1) Mix 1 g of the powdered drug with 100 mL of 0.8% w/v
in small groups and sometimes accompanied by lignified
parenchymatous cells with moderately thickened and pitted of ammonia) place in an ultrasonic bath for 30 minutes,
centrifuge, dilute 1 mL of supernatant solution to 5 mL with
walls, some of the individual vessels are very large, with pit
apertures much elongated and the borders difficult to 0.8% w/v of ammonia and filter through a 0.45-pm filter.
discern; prism crystals of calcium oxalate up to about 35 gm (2) 0.0065% w/v of monoammonium glycyrrhizate EPCRS in
long occur scattered and in parenchymatous tissue; fragments 0.8% w/v of ammonia.
of cork composed of thin-walled, slightly lignified polygonal
cells. Examine under a microscope using 50% พ/พ glycerol
2016 Liquorice Root IV-261
(3) 0.013% w/v of monoammonium glycyrrhizate EPCRS in

0.8% w/v of ammonia. Processed Liquorice Root for use
(4) 0.0195% w/v of monoammonium glycyrrhizate EPCRS in in TCM
0.8% w/v of ammonia.
DEFINITION
CHROMATOGRAPHIC CONDITIONS Processed Liquorice Root for use in TCM is the processed
(a) Use a stainless steel column (12.5 cm X 4 mm) packed Liquorice Root for use in TCM.
with octadecylsilyl silica gel for chromatography (5 pm) (Hypersil It contains not less than 2.0% of glycyrrhizic acid
ODS ss is suitable). (C42H62O16) calculated with reference to the dried material.
(b) Use isocratic elution and the mobile phase described PRODUCTION
below.
Processed Liquorice Root for use in TCM is cleaned,
(c) Use a flow rate of 1.5 mL per minute. softened thoroughly, sliced transversely or longitudinally to
(d) Use ambient column temperature. form uniform pieces and dried.
(e) Use a detection wavelength of 254 nm. IDENTIFICATION
(f) Inject 10 pL of each solution. A. The transversely-cut pieces are 0.5 to 2.5 cm in diameter,
MOBILE PHASE irregularly circular to ovoid, up to about 3 mm thick.
The outer surface is dark reddish-brown and longitudinally
6 volumes of glacial acetic acid) 30 volumes of acetonitrile and
wrinkled. The transverse surface is cream to yellow and
64 volumes of water.
shows a thin layer of cork and a pale brown cambium line
SYSTEM SUITABILITY separating the radiate phloem region from the distinctly
The assay is not valid unless (a) the column efficiency) radiate xylem. In pieces cut from the rhizome there is a
determined on the peak due to glycyrrhizic acid in the central, whitish pith; in those cut from the roots the radiate
chromatogram obtained with solution (2), is at least 30,000 xylem continues to the centre.
theoretical plates per metre and (b) the symmetry factor of the The longitudinally-cut pieces have been sliced obliquely and
peak is not more than 1.3. usually include a small portion of the outer surface at the
DETERMINATION OF CONTENT tapering ends; they are about 6 to 8 cm long, 1 to 1.5 cm
wide and 3 mm thick. The outer surface is reddish-brown
Inject solution (4). Adjust the sensitivity of the system so that
and longitudinally wrinkled with scattered transverse ridges;
the height of the peaks is at least 50% of the full scale of the
the smooth cut surface is cream to yellow, faintly fibrous and
recorder. Inject solutions (2), (3) and (4) and determine the
shows a distinct cambium; on some of the pieces the vessel
peak areas. Prepare a calibration curve with the concentration
cavities are visible in the central region.
of the solutions (g per 100 mL) as the abscissa and the
corresponding areas as the ordinate. Inject solution (1). B. Reduce to a powder. The powder is yellowish-brown.
Using the retention time and the peak area from the Examine under a microscope using chloral hydrate solution.
chromatograms obtained with solutions (2), (3) and (4), The powder shows abundant fibres occurring in groups, each
locate and integrate the peak due to glycyrrhizic acid in the group surrounded by a calcium oxalate prism crystal sheath,
chromatogram obtained with solution (1). the individual fibres have very thick, partially lignified walls
and few small pits; lignified bordered-pitted vessels, singly or
Calculate the percentage content of glycyrrhizic acid from the
in small groups and sometimes accompanied by lignified
parenchymatous cells with moderately thickened and pitted
walls, some of the individual vessels are very large, with pit
A X 5 X B X 822 apertures much elongated and the borders difficult to
m 840 discern; prism crystals of calcium oxalate up to about 35 pm
long occur scattered and in parenchymatous tissue; fragments
A ะะะ concentration of monoammonium glycyrrhizate in of cork composed of thin-walled, slightly lignified polygonal
solution (1) determined from the calibration curve, cells. Examine under a microscope using 50% พ/พ glycerol
in g per 100 mL, in water. The powder shows abundant starch granules,
B = declared percentage content of monoammonium mostly simple, spherical to ovoid, 3 pm to 20 pm in
glycyrrhizate EPCRS) diameter.
m = weight of the substance being examined in grams, c. Carry out the method for thin-layer chromatography)
822 = molecular weight of glycyrrhizic acid, Appendix in A, using the following solutions.
840 = molecular weight of monoammonium glycyrrhizate (1) Add 16 mL of water and 4 mL of hydrochloric acid (25%)
(without any water of crystallisation). to 0.5 g of the powdered drug, heat on a water-bath under
STORAGE reflux for 30 minutes, cool and filter. Dry the filter paper and
residue in a flask at 105° for 60 minutes, add 20 mL of ether,
Liquorice Root for use in TCM should be protected from
heat on a water-bath at 40° under reflux for 5 minutes, cool
moisture.
and filter. Evaporate the filtrate to dryness and dissolve the
residue in 5 mL of ether.
(2) 0.1% w/v each of glycyrrhetic acid and thymol in ether.
(a) Use as the coating substance silica gel F2S4 (Merck 10 X
20 cm plates are suitable).
(c) Apply 10 pL of each solution as a band.
IV-262 Long Pepper 2016
(e) Remove the plate and allow it to dr}’ in air for 5 minutes. chromatogram obtained with solution (2), is at least 30,000
Examine under ultraviolet light (254 nm). Spray the plate with theoretical plates per metre and (b) the symmetry factor of the
anisaldehyde solution and heat at 100° to 105° for 10 minutes peak is not more than 1.3.
MOBILE PHASE
Inject solution (4). Adjust the sensitivity of the system so that
1 volume of concentrated ammonia, 9 volumes of water, the height of the peaks is at least 50% of the full scale of the
25 volumes of ethanol (96%) and 65 volumes of ethyl acetate. recorder. Inject solutions (2), (3) and (4) and determine the
SYSTEM SUITABILITY peak areas. Prepare a calibration curve with the concentration
Under ultra-violet light, the chromatogram obtained with of the solutions (g per 100 mL) as the abscissa and the
solution (2) shows in the lower half a quenching zone due to corresponding areas as the ordmate. Inject solution (1).
glycyrrhetic acid. When sprayed, the chromatogram obtained Using the retention time and the peak area from the
with solution (2) shows in the lower half the violet zone of chromatograms obtained with solutions (2), (3) and (4),
glycyrrhetic acid and in the upper third the red zone of locate and integrate the peak due to glycyrrhizic acid in the
thymol. chromatogram obtained with solution (1).
CONFIRMATION Calculate the percentage content of glycyrrhizic acid from the
The chromatogram obtained with solution (1) shows in the
lower half of violet zone corresponding to the zone of
glycyrrhetic acid in the chromatogram obtained with solution 5 „ 822
A X — X B X
(2) and a yellow zone (isoliquiridigenine) in the upper third m 840
under the zone of thymol in the chromatogram obtained with
solution (2). Further zones may be present. A = concentration of monoammonium glycyrrhizate in
TESTS the solution (1) determined from the calibration
Total Ash curve, in g per 100 mL,
B = declared percentage content of monoammonium
Not more than 5.0%, Appendix XI J, Method II.
glycyrrhizate EPCRS,
Acid-insoluble ash m = weight of the substance being examined in grams,
Not more than 1.0%, Appendix XI K, Method II. 822 = molecular weight of glycyrrhizic acid,
Loss on drying 840 = molecular weight of the monoammonium
When dried for 2 hours at 100° to 105°, loses not more than glycyrrhizate (without any water of crystallisation).
STORAGE
Ochratoxin A
Not more than 20 ppb, Appendix XI ร2. Processed Liquorice Root for use in TCM should be
protected from moisture.
ASSAY
Appendix III D, using the following solutions.
(1) Mix 1 g of the powdered drug with 100 mL of 0.8% w/v
of ammonia, place in an ultrasound bath for 30 minutes, Long Pepper ** *1
centrifuge, dilute 1 mL of the supernatant solution to (Ph. Eur. monograph 2453) *
5 mL with 0.8% w/v of ammonia and filter through a
Ph Eur__________________________________________________ ____________
0.45-pm filter.
(2) 0.0065% w/v of monoammonium glycyrrhizate EPCRS in DEFINITION
0.8% w/v of ammonia. Dried, ripe or nearly ripe fruiting spikes of Piper longum L.
(3) 0.013% w/v of monoammonium glycyrrhizate EPCRS in or Piper retrofractum Vahl (syn. p. chaba Hunter and
0.8% w/v of ammonia. p. officinarum (Miq.) c. DC.) or a mixture of both species.
(4) 0.0195% w/v of monoammonium glycyrrhizate EPCRS in Content
0.8% w/v of ammonia. — essential oil', minimum 6.0 mUkg (dried drug);
— piperine (C17H19NO3; Mr 285.3): minimum 3.0 per cent
(dried drug).
(a) Use a stainless steel column (12.5 cm X 4 mm) packed
with octadecylsUyl silica gel for chromatography (5 pm) (Hypersil IDENTIFICATION
ODS ss is suitable). A. p. longum. The fruiting spikes are cylindrical or irregularly
(b) Use isocratic elution and the mobile phase described cylindrical, 1-2.5 cm long (rarely longer than 2.5 cm),
3-5 mm in diameter, blackish-brown or almost black.
below.
The spikes are quite compact, tough, composed of small
(c) Use a flow rate of 1.5 mL per minute. fruits firmly fixed on the receptacle in regular or oblique
(d) Use ambient column temperature. rows. The berries are spherical, about 1 mm in diameter.
(e) Use a detection wavelength of 254 nm. The bracts are black, small, punctiform, confined to
(f) Injection 10 pL of each solution. depressions between adjacent berries. The remains of the
peduncle may be present at the base of the cylinder. Spikes
MOBILE PHASE
can be easily broken; the fracture is irregular and granular.
6 volumes of glacial acetic acid, 30 volumes of acetonitrile and
p retrofractum The fruiting spikes are similar to those of
64 volumes of water.
p. longum but clearly more robust, straight and cylindrical,
SYSTEM SUITABILITY 2.5-4 cm long (rarely smaller than 2.5 cm), 5-8 mm in
The assay is not valid unless (a) the column efficiency, diameter, brown or reddish-brown. The berries are also
determined on the peak due to glycyrrhizic acid in die firmly fixed on the receptacle but, in contrast to those of
2016 Long Pepper IV-263
p. longuni, arranged more obviously in spiral rows. c. Thin-layer chromatography (2.2.27).

The bracts are more prominent than those of p. longum.
B. Microscopic examination {2.8.23}. The powder is grey or (2.9.72) add 5 mL of methanol R. Sonicate for 10 min,
Light brown. Examine under a microscope using chloral centrifuge and use the supernatant.
Reference solution Dissolve 10 mg of borneol R and 15 mg of
characters (Figure 2453.-1): fragments of the endocarp
piperine R in 10 mL of methanol R.
(surface view [A]), consisting of sclereids, more or less
elongated, about 75 pm long, pitted, with irregularly Plate TLC silica gel F254 plate p (5-40 pm) [or TLC silica gel
thickened walls and wide channels [Aa], associated with the F254 plate R (2-10 pm)].
reddish-brown pigmented layer of the testa with indistinct Mobile phase ethyl acetate R} cyclohexane R (30:50 VIV}.
cells [Ab] and also associated with the inner testa with Application 10 pL [or 5 pL] as bands of 10 mm [or 8 mm].
rectangular, brown, thin-walled cells [Ac]; fragments of the Development Over a path of 15 cm [or 6 cm].
endocarp (transverse section [D]), showing sclereids with Drying In air.
irregularly thickened inner walls on the 3 lower sides [Da],
usually associated with the testa (namely the pigmented layer
with indistinct cells [Db] and the layer of the inner testa Results A See below the sequence of zones present in the
[De]); fragments of the parenchyma of the mesocarp [B, C] chromatograms obtained with the reference solution and the
containing more or less polygonal sclereids, isolated [Ba] or test solution. Furthermore, other faint quenching zones may
in groups [Bb], and oil cells about 50 pm in diameter [Ca]; be present in the chromatogram obtained with the test
numerous ovoid or polygonal cells of the parenchyma of the solution.
seed [Dd]; fragments of the epicarp [F] with thin-walled
cells; rare fragments from the centre of the spike [E], Top of the plate
consisting of parenchymatous cells [Eb] and elongated
sclereids up to 400 pm long [Ea]; a few fragments of vascular
tissue [H] with spiral or striated vessels [Ha] and fibres [Hb]. 2 strong quenching zones
Examine under a microscope using a 50 per cent VIV
A quenching zone
solution of glycerol R. Starch is visible as rounded, compound
granules, each about 20 pm in diameter [Ga], made up of
tiny individual granules, ovoid or polyhedral by compression, A quenching zone
free [Gb] or included in the parenchymatous cells of the seed
[๐]. Piperine: a quenching zone A strong quenching zone
(piperine)
Detection B Treat with anisaldehyde solution R and heat at

100 °C for 5 min; examine in daylight.
Top of the plate
A purple-grey zone
Borneol : a yellowish-brown zone A violet zone
A purple-grey zone
Piperine: a green or brownish A green or brownish zone

zone (piperine)
TESTS
Figure 2453.-1. - Illustration for identification test B ofpowdered Foreign matter {2.8.2}
herbal drug of long pepper Maximum 3 per cent.
IV-264 Loosestrife 2016
Loss on drying {2.2.32) /Il X พ2 X p

Maximum 11.0 per cent, determined on 1.000 g of the A2 X 7ท1 X 2
freshly powdered herbal drug (355) {2.9.12) by drying in an
oven at 105 cc for 2 h. A1 = area of the peak due to piperine in the
Total ash {2.4.16) chromatogram obtained with the test solution;
Maximum 5.0 per cent. A2 = area of the peak due to piperine in the
ASSAY (a);
Essential oil {2.8.12) nil ะ= mass of the herbal drug to be examined used to
Use 25.0 g of the freshly, coarsely powdered herbal drug prepare the test solution, in grams;
(1400) {2.9.12) 3 a 1000 mL round-bottomed flask, 400 mL m2 = mass of pipeline CRS used to prepare reference
of water R as the distillation liquid and 0.5 mL of xylene R in solution (a), in grams;
±e graduated tube. Distil at a rate of 2-3 mUmin for 3 h. p = percentage content of piperine in pipeline CRS.
Piperine Ph
Liquid chromatography {2.2.29). Cany out the assay protected
from light.
(355) (2.9.12) in 40 mL of ethanol (96 per cent) R. Sonicate
for 20 min and filter. Rinse the flask and the filter with 5 mL
Loosestrife
of ethanol (96 per cent) R3 combine the filtrate and washings (Ph. Eur. monograph 1537) *
and dilute to 50.0 mL with the same solvent. Filter through Ph Eur______________________________________________________________
a membrane filter (nominal pore size 0.45 pm).
DEFINITION
Reference solution (a) Dissolve 15.0 mg of piperine CRS in
ethanol (96 per cent) R and dilute to 100.0 mL with the same Dried flowering tops, whole or cut, of Lyihnnn salicaria L.
solvent. Content
Reference solution (b) Disperse 0.250 g of long pepper for system iMinimum 5.0 per cent of tannins, expressed as pyrogallol
suitability HRS (355) (2.9.12) in 40 mL of ethanol (C6H603; Mt 126.1) (dried drug).
(96 per cent) R. Sonicate for 20 min and filter. Rinse the flask IDENTIFICATION
and the filter with 5 mL of ethanol (96 per cent) R, combine A. The stems are rigid, 4-angled, branching at the top,
the filtrate and washings and dilute to 50.0 mL with the brownish-green, longitudinally wrinkled and pubescent.
same solvent. Filter through a membrane filter (nominal pore The leaves are opposite, decussate, rarely verticillate in threes
size 0.45 pm). and sometimes alternate at the inflorescence which forms a
Column: long terminal spike. The leaves are sessile, lanceolate and
— size: I = 0.15 m, 0 = 4.6 mm; cordate at the base, 5-15 cm long and 1-2.5 cm wide,
— stationary phase: end-capped octadecylsilyl silica gel for pubescent on the lower surface; the subsidiary veins form
chromatography R (5 pm). arcs that anastomose near the leaf margin. The flowers have
Mobile phase: a pubescent, tubular, persistent gamosepalous calyx, 4-8 mm
— mobile phase A: water R-3 long, consisting of 6 sepals bearing 6 small, triangular teeth
— mobile phase B: acetonitrile Rj alternating with 6 large, acute teeth at least half as long as
the tube; a polypetalous corolla consisting of 6 violet-pink
petals, each expanded at the top with a wavy outline and
Time Mobile phase A Mobile phase B narrowing at the base. The androecium consists of 2 verticils
of 6 stamens (1 verticil with short, barely emerging stamens,
0-5 50 50
the other with long stamens extending well out of the
5 - 20 50 4 5 50 4 95 corolla). The fruit, if formed, is a small capsule included in
20 - 22 540 95 4 100
the persistent calyx.
Flow rate 1.0 mL/min. hydrate solution R. The powder shows the following diagnostic
Detection Spectrophotometer at 343 nm. characters (Figure 1537.-1): unicellular [Ea] or
Injection 10 pL. bicellular [Aa], uniseriate, thick-walled, finely pitted covering
trichomes from the epidermis of the leaf [A] and stem [E];
numerous uniseriate, unicellular [Ga] or bicellular [Gb],
long pepper for system suitability HRS and the chromatogram
thin-walled, finely pitted, annularly striated covering
obtained with reference solution (b) to identify the peak due trichomes from the calyx, in side view [G]; transparent violet
to piperine and peak 2.
pink fragments from the petals [F] consisting of epidermal
Retention time Piperine = about 10 min. cells with sinuous walls and a grainy cuticle [Fa], covering
System suitability: reference solution (b): fine spiral vessels [Fb]; fragments of parenchyma from the
— peak-to-valley ratio: minimum 4, where Hp = height above leaf [D] with numerous cells containing cluster crystals of
the baseline of peak 2 and Hv = height above the baseline calcium oxalate [Da], associated with spiral vessels [Db];
of the lowest point of the curve separating the peak due pollen grains with 3 pores and a thin and slightly granular
to piperine from peak 2. exine [C]; fragments of the upper epidermis of the leaf [A]
Calculate the percentage content of piperine using the with large polygonal cells and sinuous walls, covered by a
following expression: finely striated cuticle [Ab]; fragments of the lower epidermis
of the leaf [B] with smaller polygonal cells [Ba] and
anomocytic stomata [Bb] {2.8.3) ■ 3 fragments of the stem [E]
2016
Lovage Root IV-265
consisting of polygonal cells with straight anticlinal walls and chlorogenic acid in the chromatogram obtained with the
a striated cuticle [Eb]. reference solution, a yellow fluorescent zone similar in
position to the zone due to hyperoside in the chromatogram
obtained with the reference solution, and a bright green
fluorescent zone corresponding to the zone due to vitexin in
TESTS
105 °C.
Total ash {2.4.16)
ASSAY
Tannins {2.8.14)
____________________________________________________________ Ph Eur
Lovage Root * *
Ph Elf_____________________________________________________________
DEFINITION
Whole or cut, dried rhizome and root of Levisticum officinale
Koch.
Content
Minimum 4.0 mL/kg of essential oil for the whole drug and
minimum 3.0 mL/kg of essential oil for the cut drug (dried
drug).
herbal drug of loosestrife IDENTIFICATION
A. The rhizome and the large roots are often split
longitudinally. The rhizome is short, up to 5 cm in diameter,
Test solution To 1.0 g of the powdered herbal drug (355) light greyish-brown or yellowish-brown, simple or with
(2.9.72) add 10 mL of methanol R and heat in a water-bath several protuberances; the roots, showing little ramification,
at 65 °C for 5 min with frequent shaking. Cool and filter. are the same colour as the rhizome; they are usually up to
Dilute the filtrate to 10 mL with methanol R. 1.5 cm thick and up to about 25 cm long; the fracture is
Reference solution Dissolve 0.5 mg of chlorogenic acid R, 1 mg usually smooth and shows a very wide yellowish-white bark
of hyperoside Ry 1 mg of rutin R and 1 mg of vitexin R in and a narrow brownish-yellow wood.
10 mL of methanol R. B. Microscopic examination {2.8.23). The powder is
Blate TLC silica gel plate R. brownish-yellow. Examine under a microscope using chloral
Mobile phase anhydrous acetic acid Ry anhydrous formic acid Ry hydrate solution R. The powder shows the following diagnostic
water Ry ethyl acetate R (7.5:7.5:18:67 VIVIVIV). characters: cork cells, polygonal or rounded in surface view,
with brown contents; abundant parenchyma, mostly thin
walled and rounded but some with thicker walls; groups of
Development Over a path of 15 cm. small, reticulately thickened vessels embedded in small-celled,
Drying Kt 100-105 °C. unlignified parenchyma; fragments of larger vessels with
Detection treat the still-warm plate with a 10 g/L solution of reticulate thickening, up to 125 pm in diameter; fragments of
diphenylboric acid aminoethyl ester R in methanol R. secretory canals up to 180 pm wide. Examine under a
Subsequently treat with a 50 g/L solution of macrogol 400 R microscope using a 50 per cent v/v solution of glycerol R.
in methanol R. Allow to dry in air for 30 min and examine in The powder shows starch granules, simple, rounded or
ultraviolet light at 365 nm. ovoid, up to about 12 pm, and numerous larger, compound
Results The chromatogram obtained with the reference granules, many with several components.
solution shows in the lower third a yellowish-brown c. Examine the chromatograms obtained in ±e test for
fluorescent zone due to rutin and in the middle third a light species of Angelica and Ligusticum described in the European
blue fluorescent zone due to chlorogenic acid, above it a Pharmacopoeia.
yellowish-brown.fluorescent zone due to hyperoside and a Results A See below the sequence of zones present in the
green fluorescent zone due to vitexin. The chromatogram chromatograms obtained with the reference solution and the
obtained with the test solution shows a bright green test solution. Furthermore, other weak fluorescent zones may
fluorescent zone slightly above the zone due to rutin in the be present in the chromatogram obtained with the test
chromatogram obtained with the reference solution, a yellow solution.
fluorescent zone similar in position to the zone due to
IV-266 Magnolia Officinalis Bark 2016
Top of the plate Application 4 pL, as bands of 8 mm.

(Z)-Ligustilide; a bluish-white A bluish-white fluorescent zone Development Over a path of 6 cm.
fluorescent zone Drying In air.
Osthole: a blue fluorescent zone
shows no blue fluorescent zone just below or above the zone
Imperatorin: a whitish fluorescent A weak whitish fluorescent zone due to imperatorin in the chromatogram obtained with the
zone
reference solution.
A weak whitish fluorescent zone Results B The chromatogram obtained with the test solution
shows no zone at or just below the zone due to imperatorin
Detection c Treat the plate with a solution of 20 mL of
Results B See below the sequence of zones present in the sulfuric acid R in 180 mL of ice-cooled methanol R; heat at
chromatograms obtained with the reference solution and the 100-105 °C for 5 min and examine in daylight.
test solution. Furthermore, other weak quenching zones may
Results c The chromatogram obtained with the test solution
shows no purple zone between the 2 reddish zones at the top
solution.
of the chromatogram and the zone due to (Z)-ligustilide in
the chromatogram obtained with the reference solution;
Top of the plate the chromatogram obtained with the test solution shows no
(Z)-Ligustilide: a bluish fluorescent A bluish fluorescent zone
purple zone between the zones due to (Z)-ligustilide and
osthole in the chromatogram obtained with the reference
solution.
Osthole: a quenching zone A weak quenching zone Foreign matter (2.8.2)
Maximum 3 per cent, determined on 50 g.
Imperatorin: a quenching zone
Reference solution Test solution powdered herbal drug (355) (2.9.12) by drying in an oven at
105 °C for 2 h
Total ash (2.4.16)
Results c See below the sequence of zones present in the Maximum 8.0 per cent.
test solution. Furthermore, other faint zones may be present Ash insoluble in hydrochloric acid (2.8.1)
in the chromatogram obtained with the test solution. Maximum 2.0 per cent.
ASSAY
Top of the plate Essential oil (2.8.12)
Use a 2 L flask, 10 drops of liquid paraffin R, 500 mL of
2 prominent reddish zones
พater R as the distillation liquid and 0.50 mL of xylene R in
(zyLigustilide: a grey zone A grey zone the graduated tube. Reduce the herbal drug to a
powder (500) (2.9.12) and immediately use 40.0 g for the
determination. Distil at a rate of 2-3 mUmin for 4 h.
Osthole: a violet zone
________________________________________________________ ■ PnEur
Imperatorin: a grey zone
2 purple zones
Magnolia Officinalis Bark * **
A distinct brown zone
Ph Eur____________________________________________ ___ ____________ —
DEFINITION
TESTS Dried bark from the stem and branch of Magnolia officinalis
Species of Angelica and Ligusticum described in the Rehder et E.H. Wilson.
European Pharmacopoeia Content
Thin-layer chromatography (2.2.27). Minimum 2.0 per cent for the sum of magnolol (C18H18O2S
Test solution To 1 g of the freshly powdered herbal drug Afr 266.3) and honokiol (C18H1802; Mr 266.3) (dried drug).
(355) (2.9.12) add 4 mL of heptane R and sonicate for 5 min.
IDENTIFICATION
Centrifuge the mixture and use the supernatant.
A. Fragments of stem and branch bark, quilled singly or
Reference solution Dissolve 1 mg of imperatorin R, 1 mg of double quilled, about 30 cm long and 2-7 mm thick.
(Z)-ligustilide R and 1 mg of osthole R in 10 mL of The outer surface is brownish-grey, rough, sometimes scaly,
methanol R. easily exfoliated, with distinct lenticels and longitudinal
Plate TLC silica gel F254 plate R (2-10 pm). striations. The inner surface is reddish-brown or dark brown,
Mobile phase glacial acetic acid R} ethyl acetate R) toluene R smooth, with numerous fine, longitudinal striations.
(1:10:90 P7P7P). The texture is hard and difficult to break. The fracture is
2016 Magnolia Officinalis Bark IV-267
granular, brownish-grey in the outer layers and reddish- Application 5 pL [or 2 pL] as bands of 15 mm [or 8 mm].
brown or dark brown in the inner layers.
B Microscopic examination (2.5.22). The powder is
Drying In air.
hydrate solution R. The powder shows the following diagnostic Detection A Examine in ultraviolet light at 254 nm.
characters (Figure 2567.-1): numerous sclereids [D] of Results A See below the sequence of zones present in the
various shapes and sizes, free or in groups, often branched, chromatograms obtained with the reference solution and the
up to 100 pm long, with very thick, striated walls and test solution. Furthermore, other faint zones may be present
conspicuous pit canals; oval [F] or rounded [G] oil cells, up in the chromatogram obtained with the test solution.
to 100 pm in diameter, with orange-yellow contents; narrow,
duck-walled fibres, often in bundles [C, H], accompanied by Top of the plate
fusiform medullary rays (tangential section [Ca]) or
consisting of files of rectangular cells (longitudinal section
[Ha]); brown cork fragments with regularly and finely Eugenol: a faint quenching zone
thickened walls [B]; fragments of phloem parenchyma [A]
consisting of cells with irregular walls [Aa], accompanied by
fusiform medullary rays (tangential section [Ab]). Examine Magnolol ะ a dark blue fluorescent A dark blue fluorescent zone
under a microscope using a 50 per cent VIV solution of zone (magnolol)
glycerol R. The powder shows rounded, ovoid or polyhedral Honokiol: a quenching zone A quenching zone (honokiol)
starch granules, about 2-12 pm in diameter, simple or 2-6
compound, free [J] or included in parenchyma cells [E].
Detection B Treat with vanillin reagent Ry heat at 100-105 °C

for 5-10 min and examine in daylight.
test solution. Furthermore, other faint zones of various
colours may be present in the chromatogram obtained with
the test solution.
Top of the plate
Eugenol: a brown zone
Magnolol: a pinkish-violet zone A pinkish-violet zone (magnolol)
Honokiol ะ a dark violet zone A dark violet zone (honokiol)
TESTS
Figure 2567.-1. - Illustration for identification test B of powdered 105 °C for 2 h.
herbal drug of Magnolia officinalis bark
Total ash {2.4.16)
c. Thin-layer chromatography (2.2.27). Maximum 5.0 per cent.
Test solution Reduce to a powder (355) (2.9.72), avoiding
heating. To 0.5 g of the powdered herbal drug add 5 mL of
methanol Ry sonicate for 5 min, centrifuge, and use the
supernatant. Filter through a membrane filter (nominal pore ASSAY
size 0.45 pm) if necessary. Liquid chromatography (2.2.29).
Reference solution Dissolve 1 mg of honokiol R, 1 mg of Test solution To 0.500 g of the powdered herbal drug (355)
magnolol R and 2 mg of eugenol R in 1 mL of methanol R. (2.9.12) add 80 mL of methanol R and heat in a water-bath
Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel under a reflux condenser for 30 min. Cool, then dilute to
100.0 mL with methanol R. Filter through a membrane filter
^254 plate R (2-10 pm)].
Mobile phase methanol Ry ethyl acetate Ry toluene R
(4:8:120 VIVIF)
IV-268 Magnolia Officinalis Flower 2016
Reference solution (a) Dissolve 4.0 mg of honokiol CRS and

4.0 mg of magnolol CRS in methanol R and dilute to 20.0 mL Magnolia Officinalis Flower
with the same solvent. (Ph. Eur. monograph 2568)
Reference solution (b) Dissolve 2.0 mg of honokiol CRS in Pn Eur_ _____________________________________
2.0 mL of acetonitrile R. To 1.0 mL of the solution add
15 pL of acetic anhydride R and mix. Heat at 50 °C for DEFINITION
60 min. Cool. Add successively, mixing after each addition, Steamed and dried, unopened flower of Magnolia officinalis
16 pL of concentrated ammonia R) 1.0 mL of acetonitrile R and Rehder et E.H. Wilson.
2.0 mL of water R. Filter through a membrane filter Content
(nominal pore size 0.45 pm). Minimum 0.20 per cent of the sum of magnolol (C18HJ8O2;
Column’. Mr 266.3) and honokiol (C18H18O2; Mr 266.3) (dried drug).
— size'. I ะ= 0.25 m, 0 = 4.6 mm;
IDENTIFICATION
A. The greyish-yellow pedicel is short (0.5-2 cm) and densely
chromatography R1 (5 pm);
tomentose. The brown or reddish-brown flower bud is
elongated, conical, 4-7 cm long and 1.5-2.5 cm in diameter
Mobile phase 0.5 per cent VIV solution of acetic acid R, at the base; it usually consists of 12 perianth segments in
acetonitrile for chromatography R (40:60 VIV). several whorls. The stamens are numerous with a fine, short
Flow rate 1.0 mL/min. filament and a linear, yellowish-brown anther. The carpels
Detection Spectrophotometer at 290 nm. are free and numerous, spirally arranged on a conical
Injection 10 pL. receptacle.
Run time Twice the retention time of honokiol for the test B. Microscopic examination (2.8.23). The powder is reddish-
solution and reference solution (a); 3 times the retention time brown. Examine under a microscope using chloral hydrate
of honokiol for reference solution (b). solution R. The powder shows the following diagnostic
characters: fragments of the perianth segments with
Relative retention With reference to honokiol (retention polyhedral or elliptical epidermal cells, with irregularly
time = about 8 min): magnolol = about 1.4; honokiol thickened walls and anomocytic stomata (4-6 subsidiary7 cells)
monoacetate isomer 1 = about 1.5; honokiol monoacetate (2.8.3)) accompanied by parenchyma that includes oval or
isomer 2 = about 1.6; honokiol diacetate = about 2.6.
rounded oil cells about 50 pm in diameter with orange
System suitability: reference solution (b): yellow contents; certain fragments contain epidermal cells
— resolution: minimum 1.8 between the peaks due to with rounded papillae; numerous, branched sclereids, with
honokiol monoacetate isomers 1 and 2. channelled walls and a large lumen, about 15 pm in
Calculate the sum of the percentage contents of honokiol and diameter; numerous elliptical pollen grains about 50 pm long
magnolol using the following expression: and 40 pm wide, with a smooth exine.
c. Examine the chromatograms obtained in the test for other
Al X 7712 X Pl X 5 A3 X m3 X p2 X 5 Magnolia species.
A2 X rm A4 X 7711 Detection A Examine in ultraviolet light at 254 nm.
A1 = area of the peak due to honokiol in the chromatograms obtained with the reference solution and the
chromatogram obtained with the test solution; test solution. Furthermore, other faint zones may be present
A2 = area of the peak due to honokiol in the in the chromatogram obtained with the test solution.
A3 = area of the peak due to magnolol in the
Top of the plate
A4 = area of the peak due to magnolol in the
chromatogram obtained with reference solution (a); Eugenol: a faint quenching zone
ทใ1 = mass of the herbal drug to be examined used to
m2 = mass of honokiol CRS used to prepare reference Magnolol: a dark blue fluorescent A dark blue fluorescent zone
solution (a), in grams; (magnolol)
m3 = mass of magnolol CRS used to prepare reference Honokiol: a quenching zone A quenching zone (honokiol)
Pl = percentage content of honokiol in honokiol CRS')
p2 = percentage content of magnolol in magnolol CRS.
-------------------------------------------------------------------------------------------- --------------Ph Eur
Detection B Treat with vanillin reagent R) heat at 100-105 °C
for 5-10 min and examine in daylight.
the test solution.
2016 Magnolia Officinalis Flower IV-269
________ Top of the plate supernatant to the same 20.0 mL flask. Cool, then dilute to
20.0 mL with methanol R. Filter through a membrane filter
Reference solution (a) Dissolve 5.0 mg of honokiol CRS in
Eugenol: a brown zone methanol R and dilute to 5.0 mL with the same solvent.
Reference solution (b) Dissolve 6.0 mg of magnolol CRS in
Magnolol: a pinkish-violet zone A pinkish-violet zone (magnolol)
Honokiol: a dark violet zone A dark violet zone (honokiol) Reference solution (c) Dissolve 2.0 mg of honokiol R in 2.0 mL
of acetonitrile R. Add 30 pL of acetic anhydride R and mix.
Heat at 50 °C for 60 min. Cool. Add successively, mixing
after each addition, 32 pL of concentrated ammonia Rj 2.0 mL
Reference solution Test solution of acetonitrile R and 4.0 mL of water R. Filter through a
Column'.
TESTS — size'. I = 0.15 m, 0 ะ= 4.6 mm;
Other Magnolia species — stationary phase: end-capped polar-embedded octadecylsilyl
Thin-layer chromatography (2.2.27). amorphous organosilica polymer 7? (3.5 pm);
Test solution Reduce the herbal drug to a powder (710) — temperature: 25 ± 2 °C.
(2.9.72)
, avoiding heating. To 0.5 g of the powdered herbal Mobile phase:
drug add 2.5 mL of methanol R. Sonicate for 15 min at a — mobile phase A: anhydrous formic acid R, water R
power of 80 พ and a frequency of 37 kHz (sonication time (0.1:99.9 P7F);
may be adapted according to the power and frequency used), — mobile phase B: acetonitrile for chromatography R-,
then centrifuge at 1500-2000 g for 10 min and transfer the
supernatant to a 5 mL flask. Add 2 mL of methanol R to the Time Mobile phase A Mobile phase B
residue, sonicate for 15 min and centrifuge. Transfer the (per cent V/V) (per cent V/V)
supernatant into the same 5 mL flask. Dilute to 5 mL with 0 - 20 47 53
methanol R. Filter through a membrane filter (nominal pore 20 - 22 47 -> 5 53 -> 95
size 0.45 pm) if necessary.
22 - 27 5 95
Reference solution Dissolve 1 mg of honokiol R, 1 mg of
magnolol R and 2 mg of eugenol R in 4 mL of methanol R.
Plate TLC silica gel F254 plate R (2-10 pm). Flow rate 1.0 mUmin.
Mobile phase methanol R, ethyl acetate R, toluene R Detection Spectrophotometer at 292 nm.
(1:5:30 VIVIV). Injection 20 pL.
Application 8 pL as bands of 8 mm. Relative retention With reference to honokiol (retention
Development Over a path of 7 cm. time ะ= about 10 min): magnolol = about 1.3; honokiol
Drying In air. monoacetate isomer 1 = about 1.4; honokiol monoacetate
isomer 2 ะ= about 1.5; honokiol diacetate = about 1.9.
System suitability: reference solution (c):
shows no blue fluorescent zone in the lower part of the plate
honokiol monoacetate isomers 1 and 2.
and no green fluorescent zone in the upper part, nor any
other fluorescent zone. If necessary, dilute the test solution to obtain peaks of
honokiol and magnolol that are similar in height to the
Loss on drying (2.2.52) corresponding peaks in reference solutions (a) and (b).
Calculate the sum of the percentage contents of honokiol and
magnolol using the following expression:
105 °C.
Total ash (2.4.16) Al X 1ท2 X 0.16 X Pl X d A3 X 7713 X P2 X d
Maximum 8.0 per cent. A2 X 7711 A4 X 7711
ASSAY
Liquid chromatography (2.2.29). A1 = area of the peak due to honokiol in the
Test solution Reduce the herbal drug to a powder (710) chromatogram obtained with the test solution;
(2.9.12) using a blade grinder equipped with a double-walled A2 = area of the peak due to honokiol in the
grinding chamber cooled to a temperature of about 10 °C. chromatogram obtained with reference solution
To 0.500 g of the powdered herbal drug add 10 mL of (a);
methanol R. Sonicate for 1 h at a power of 80 พ and a A3 = area of the peak due to magnolol in the
frequency of 37 kHz (sonication time may be adapted chromatogram obtained with the test solution;
according to the power and frequency used). Change the A4 = area of the peak due to magnolol in the
water of the ultrasonic bath after 30 min of sonication to chromatogram obtained with reference solution
prevent heating. Centrifuge at 1500-2000 g for 15 min.
Transfer the supernatant to a 20.0 mL flask. Add 9.5 mL of m1 = mass of the herbal drug to be examined used to
methanol R to the residue. Repeat the sonication for 1 h. prepare the test solution, in grams;
Change the water of the ultrasonic bath after 30 min of m2 = mass of honokiol CRS used to prepare reference
sonication to prevent heating. Centrifuge. Transfer the solution (a), in grams;
"270 Mallow Flower 2016
= mass of magnolol CRS used to prepare reference

p\ = percentage content of honokiol in honokiol CRS;
p2 = percentage content of magnolol in magnolol CRS;
d = dilution factor of the test solution.
-------- -- ----------------------------------------------------------------------------------------------- Ph Eur
Mallow Flower * **
Ph Elf_______________________________________________________________
DEFINITION
Whole or fragmented dried flower of Malva sylvestris L. or its
cultivated varieties.
IDENTIFICATION
A. The flower consists of an epicalyx with 3 oblong or
elliptical-lanceolate parts that are shorter than those of the
calyx and situated immediately below it; a calyx with
5 pubescent triangular lobes, gamosepalous at the base;
a corolla 3-4 times longer than the calyx with 5 wedge-
shaped, notched petals fused to the staminal tube at their
base; numerous stamens, the filaments of which fuse into a
staminal tube covered by small star-shaped trichomes and
occasional simple trichomes visible using a lens; numerous
wrinkled carpels, glabrous or sometimes pubescent, enclosed
in the staminal tube and arranged into a circle around a Figure 1541.-1. - Illustration for identification test B of powdered
central style ending with numerous filiform stigmas. herbal drug of mallow flower
In cultivated varieties, the epicalyx is 3-7 partite, the calyx
5-8 partite and the corolla 5-10 partite. Reference solution 0.5 g/L solution of quinaldine red R in
B. Reduce to a powder (355) (2.9.72). The powder is bluish- ethanol (96 per cent) R.
grey. Examine under a microscope using chloral hydrate Plate TLC silica gel plate R.
solution R. The powder shows the following diagnostic Mobile phase glacial acetic acid R} water R, butanol R
characters (Figure 1541.-1): unicellular, thick-walled, (15:30:60 VIVIV).
flexuous covering trichomes, from the calyx and the epicalyx,
Application 10 pL of the test solution and 5 pL of the
up to 2 mm in length, whole [L] or, most often, fragmented
[Q]; fragments of ±e epidermis of the sepals in surface view
[D, J] with anomocytic stomata (2.8.3) [De]; club-shaped Development Over a path of 10 cm.
glandular trichomes with multicellular heads [Db] and short Dtying In air.
unicellular covering trichomes, somewhat curved, either Detection Examine in daylight.
isolated [J] or in star-shaped groups of 2-6 [Da]; fragments Results The chromatogram obtained with the reference
of covering trichomes [N]; isolated glandular trichomes in solution shows an orange-red zone in the upper part of the
surface view, [F]; or in transverse section [G]; fragments of middle third ; the chromatogram obtained with the test
the mesophyll of the calyx and the epicalyx whose cells solution shows, below the zone in the chromatogram
contain small cluster crystals of calcium oxalate [K]; veins of obtained with the reference solution, 2 violet zones in the
the sepals [P] with vessels [Pa] accompanied by cells with middle third, with the principal zone (6 "-malonyl malvin)
cluster crystals of calcium oxalate [Pb]; fragments of petal situated just below the other violet zone (malvin).
epidermis, with elongated cells and sinuous margins, narrow
in the wild plant [A], shorter and broader in the cultivated TESTS
varieties [B], bearing sessile glandular trichomes with Loss on drying (2.2.22)
multicellular club-shaped heads [Ba, c, E]; fragments of Maximum 12.0 per cent, determined on 1.000 g of the
petal mesophyll [H] consisting of large mucilage cells [He], powdered herbal drug by drying in an oven at 105 °C.
sometimes cells with small cluster crystalร of calcium oxalate Total ash (2.4.16)
[Hb] and spiral vessels [Ha]; spherical pollen grains, about Maximum 14.0 per cent.
150 pm in diameter, with a roughly spiny exine [M]. Ash insoluble in hydrochloric acid (2.8.1)
Illustration for identification test B of powdered herbal drug Maximum 2.0 per cent.
of mallow flower Swelling index (2.8.4)
c. Thin-layer chromatography (2.2.27). Minimum 15, determined on 0.2 g of the powdered herbal
Test solution To 1 g of the powdered herbal drug (355) drug (710) (2.9.72) moistened with 0.5 mL of anhydrous
(2.9.72) add 10 mL of ethanol (60 per cent V/V) R. Stir for ethanol R.
15 min and filter. Ph
2016 Mallow Leaf IV-271
Mallow Leaf *****

RiEif_______________________________________________
DEFINITION
Whole or fragmented, dried leaf of Malva sylvestris L., Malva
negleaa Wallr. or a mixture of both species.
IDENTIFICATION
A. The leaves of M. sylvestris are up to 12 cm long and up to
15 cm wide with 3, 5 or 7 lobes and sinuate at the base;
the leaves of M. neglecta are up to 9 cm long and wide,
round or kidney-shaped with 5-7 indistinct lobes. The leaves
of both species have irregular dentate margins and are green
or brownish-green. The abaxial surface of the lamina bears
more hairs and shows a more prominent venation than the
adaxial surface. The major veins on the upper surface of the
leaves and the petioles may be violet. The petioles are as long
as the leaves, up to 2 mm wide, rounded and somewhat
flattened, longitudinally slightly grooved, green or brownish-
green or violet. The fragmented drug consists of occasionally
agglomerated, crumpled pieces of leaves showing prominent
veins.
B. Microscopic examination (2.8.23). The powder is green or
yellowish-green. Examine under a microscope using chloral
characters (Figure 2391.-1): fragments of the lamina, in
transverse section [F], consisting of the lower epidermis, in
surface view [C], and the upper epidermis, in surface view
[D] or in transverse section [Fb], with cells that show
straight, or more or less sinuous anticlinal walls; stomata
mostly anisocytic (2.8.3) on both surfaces [Ca, Da]; long
covering trichomes with thickened walls and tapering to a Figure 2391.-1. - Illustration for identification test B of powdered
point at the apex, usually unicellular, whole [A, Fa] or
herbal drug of mallow leaf
fragmented [Db], but in M. Sylvestris they may be stellate
with 2-8 components [H], each strongly pitted at the base;
club-shaped glandular trichomes composed of 2-6 cells [E]
occur in both species; fragments of the mesophyll consisting
of palisade parenchyma, in surface view [De] or in transverse
section [Fc], and spongy mesophyll cells containing mucilage, test solution.
cells containing cluster crystals of calcium oxalate, often
associated with vessels [B]; occasional spherical pollen grains, Top of the plate
110-170 pm in diameter, with a spiny exine [G].
Hyperoside: a yellow fluorescent
Test solution To 2.0 g of the powdered herbal drug (710) zone
(2.9.12) add 20 mL of an 80 per cent v/v solution of A yellow fluorescent zone
tetrahydrofuran Ry extract for 10 min using sonication and
filter.
Reference solution Dissolve 3 mg of hyperoside R and 3 mg of Rutin: a yellow fluorescent zone
rutin R in 20 mL of methanol R. A yellow fluorescent zone
plate R (2-10 pm)].
Mobile phase anhydrous formic acid Ry anhydrous acetic acid Ry An orange fluorescent zone
water Ry ethyl formate Ry 3-pentanone R An orange fluorescent zone
(4:11:14:20:50 VIVIVIVIV).
Development Over a path of 10-12 cm [or 6 cm].
Drying In air. TESTS
Detection Heat at 100 °C for 10 min; spray or dip the warm Foreign matter (2.8.2)
plate in a 10 g/L solution of diphenylboric acid aminoethyl Maximum 5 per cent of foreign organs, maximum 5 per cent
ester R in methanol R-y remove the solvent with cold air; spray of leaves with blisters of spores of Puccima malvacearum and
or dip the plate in a 50 g/L solution of macrogol 400 R in maximum 2 per cent of foreign elements.
methanol Ry dry in air and examine after 15 min in ultraviolet Foreign organs can be flowers, fruits and parts of the stem.
light at 365 nm. The blisters of spores on the leaves are mostly 1 mm wi e,
IV-272 Mandarin Epicarp and Mesocarp 2016
and red or brown. Examine under a microscope using chloral Mobile phase water R, anhydrous formic acid R> ethyl acetate R
hydrate solution R. The spores of Puccinia malvacearum are (10:15:75 VIVIV).
oblong or oval with brownish walls and a small appendage. Application 5 pL of the test solution and 10 pL of the
Loss on drying (2.2.32) reference solution as bands of 8 mm.
Maximum 12.0 per cent, determined on 1.000 g of the Development Over a path of 6 cm.
Drying In air, then heat at 110-120 °C for 5 min.
105 °C for 2 h.
Detection Treat with a 10 g/L solution of aluminium chloride R
Total ash (2.4.16) in ethanol (96 per cent) R and heat at 110-120 °C for 5 min;
Maximum 17.0 per cent. treat the warm plate with a 10 g/L solution of diphenylboric
Ash insoluble in hydrochloric acid (2.8.1) acid aminoethyl ester R in methanol R, and then with a 50 g/L
Maximum 3.0 per cent. solution of macrogol 400 R in methanol R. After 60 min
Swelling index (2.8.4) examine the chromatograms in น]traviolet light at 365 run.
Minimum 7, determined on 1.0 g of the powdered herbal Results See below the sequence of fluorescent zones present
drug (710) (2.9.12). in the chromatograms obtained with the reference solution
_______________________________________________________________ Ph Eur
test solution.
Top of the plate

Mandarin Epicarp and Mesocarp *****
Caffeic acid: a light blue
(Ph. Eur. monograph 2430) *** fluorescent zone
PhEir_______________________________________________________ _______
DEFINITION
Dried epicarp and mesocarp of the ripe fruit of Citrus
reticulata Blanco or its cultivars, partly freed from the white Hesperidin: a greenish-brown A greenish-brown fluorescent
spongy tissue of the mesocarp. fluorescent zone zone (hesperidin)
Content
Minimum 3.5 per cent of hesperidin (C28H34O15; Mr 611) A yellowish-green fluorescent
(dried drug). zone
IDENTIFICATION
A. The pericarp consists of irregular pieces, usually in strips, A greenish fluorescent zone
up to 4 cm long, up to 2 cm wide and 1-3 mm thick. Test solution
Reference solution
The outer surface is yellowish or reddish-brown with spots of
oil glands; the inner surface appears yellowish-white, rough,
bearing yellowish-white or yellowish-brown vascular bundles. TESTS
B. Microscopic examination (2.8.23). The powder is light Bitter-orange epicarp and mesocarp
orange-brown. Examine under a microscope using chloral Examine the chromatograms obtained in the assay.
hydrate solution R. The powder shows the following diagnostic Results The chromatogram obtained with the test solution
characters: fragments of epicarp, in surface view, consisting shows no peak at the retention time of naringin with an area
of small polygonal cells, subsquare or rectangular, about of more than 1 per cent of the area of the peak due to
18-30 pm long, with slightly thickened anticlinal walls and hesperidin.
occasional rounded anomocytic stomata (2.8.3) with
indistinct subsidiary cells; fragments of pericarp, in transverse
section, showing the epicarp covered by a thick cuticle, sub-
epicarpal layers with collenchymatous thickenings and cells of
105 °C for 2 h.
the mesocarp, some of which contain one or more prism
crystals of calcium oxalate about 30 pm wide and 50 pm Total ash (2.4.16)
long; rare fragments of schizolysigenous oil glands; very Maximum 7.0 per cent.
numerous groups of cells of various shapes and sizes from the ASSAY
mesocarp, in surface view or side view; free prism crystals of Liquid chromatography (2.2.29).
calcium oxalate; small droplets of orange-yellow essential oil. Test solution Place 0.125 g of the powdered herbal drug (355)
Examine under a microscope using a 20 g/L solution of (2.9.12) in a 100 mL round-bottomed flask. Add 50.0 mL of
potassium hydroxide R. The mounting medium becomes methanol R, stir for 2 h and filter through a membrane filter
yellow because of the presence of hesperidin in the drug. (nominal pore size 0.45 pm).
c. Thin-layer chromatography (2.2.27). Reference solution (a) Dissolve 10.0 mg of hesperidin CRS in
Test solution To 1 g of the powdered herbal drug (355) methanol R and dilute to 50.0 mL with the same solvent.
(2.9.12) add 10 mL of methanol R and heat in a water-bath Reference solution (b) Dissolve 5.0 mg of naringin R in
at 65 °C for 5 min, shaking frequently. Allow to cool and reference solution (a) and dilute to 25.0 mL with reference
filter.
solution (a).
Reference solution Dissolve 1 mg of caffeic acid R and 2 mg of
hesperidin R in 5 mL of methanol R.
Plate TLC silica gel plate R (2-10 pm).
2016 Mandarin Oil IV-273
Column: Development Over a path of 15 cm [or 6 cm].

size: I = 0.25 ทไ3 0 = 4.6 mm;
Drying In air.
stationary phase: end-capped, octadecylsilyl silica gel for
chromatography R (5 gin); Detection A Examine in ultraviolet light at 365 nm.
— temperature: 40 °C. Results A The intense blue fluorescent zone in the
Mobile phase acetonitrile R3 glacial acetic acid R, methanol R, chromatogram obtained with the test solution is similar in
position and fluorescence to the zone due to methyl
water R (2.7:3.7:22:71.6 VIVIVIV).
N-methylanthranilate in the chromatogram obtained with the
Flow rate 1 mL/min.
reference solution. Furthermore, other fluorescent zones may
Detection Spectrophotometer at 283 nm. be present in the chromatogram obtained with the test
Injection 10 gL. solution.
Run time Twice the retention time of hesperidin. Detection B Spray with a 200 g/L solution of phosphomolybdic
Retention time Naringin = about 15 min; hesperidin = about acid R in ethanol (96 per cent) R and heat at 100 °C for
20 min. 10 min; examine in daylight.
System suitability: reference solution (b): Results B See below the sequence of the zones present in the
resolution: minimum 2.0 between the peaks due to chromatograms obtained with the reference solution and the
naringin and hesperidin. test solution. Furthermore, other zones may be present in the
Calculate the percentage content of hesperidin using the chromatogram obtained with the test solution.
Top of t le plate
Al X 7712 X p
>12 X 7711 A blue zone
Guaiazulene: a blue zone A blue zone

si 1 = area of the peak due to hesperidin in the
chromatogram obtained with the test solution; A blue zone
^2 = area of the peak due to hesperidin in the
(a); A blue zone
W1 = mass of the herbal drug to be examined used to
a-Terpineol: a blue zone A blue zone (a-terpineol)
= mass of hesperidin CRS used to prepare reference
solution (a), in grams; Reference solution Test solution
p = percentage content of hesperidin in hesperidin
CRS.
----------------------------------------------------------------------------------------------------------- Ph Eur
Mandarin Oil * ** solution.
TESTS
DEFINITION 0.848 to 0.855.
Essential oil obtained without heating, by suitable mechanical Refractive index (2.2.6)
treatment, from the peel of the fresh fruit of Citrus reticulata 1.474 to 1.478.
Blanco. Optical rotation (2.2.7)
CHARACTERS + 64° to + 75°.
Appearance Fatty oils and resinified essential oils (2.8.7)
Greenish, yellow or reddish orange liquid showing blue It complies with the test.
fluorescence. Chromatographic profile
Characteristic odour. Gas chromatography (2.2.28): use the normalisation
IDENTIFICATION procedure.
First identification B. Test solution Dilute 0.20 g of the substance to be examined to
10.0 mL with heptane R.
Reference solution (a) Dilute 5 gL of a-pinene R, 5 gL of
K. Thin-layer chromatography (2.2.27).
sabinene R, 5 gL of fi-pinene R, 5 gL of fl-myrcene R3 5 gL of
Test solution Dilute 0.1 mb of the substance to be examined p-cymene R, 70 gL of limonene R3 20 gL of y-terpinene R and
to 1 mL with toluene R. 5 gL of methyl N-methylanthranUate R to 5.0 mL with
Reference solution Dissolve 2 gL of methyl N- heptane R.
methylanthranilate R, 4 mg of guaiazulene R and 10 mg of Reference solution (b) Dissolve 5 gL of limonene R in 50 mL of
2-terpineol R in 10 mL of toluene R. heptane R. Dilute 0.5 mL of this solution to 5.0 mL with
Plate TLC silica gel plate R (5-40 gm) [or TLC silica gel heptane R.
plate R (2-10 gm)]. Column:
Mobile phase ethyl acetate R3 toluene R (15:85 VIV). — material: fused silica;
Application 10 gL [or 2 gL] as bands. — size: I = 60 m, 0 = 0.25 mm;
FV-274 Marshmallow Leaf 2016
— stationary phase', poly (dimethyl) (diphenyl) siloxane R (film B. Microscopic examination (2.8.23). The powder is greyish-
thickness 0.25 pm). green. Examine under a microscope using chloral hydrate
Carrier gas helium for chromatography R. solution R. The powder shows the following diagnostic
Flow rate 1.4 mUmin. characters (Figure 1856.-1): numerous long, rigid, unicellular
covering trichomes with thick walls, pointed at the apex,
Split ratio 1:70.
often fragmented [C], angular and pitted at the base where
Temperature’. they are sometimes still united to form stellate structures with
up to 8 components, in surface view [B] or in transverse
Time Temperature section [EJ; few secretory trichomes, isolated, with unicellular
(min) _ (°C) stalks and globular, multicellular heads [F]; fragments of the
Column 0 - 90 50 -> 230 lower [A] and upper [D] leaf epidermises in surface view
Injection port 250 with anomocytic [Aa] or paracytic [Da] stomata (2.8.3),
glandular trichomes [Ab] and basal cells of covering
Detector 250
trichomes [Ac], often accompanied by palisade
parenchyma [Db]; cluster crystals of calcium oxalate,
Detection Flame ionisation. isolated [H] or included in the parenchyma of the
mesophyll [Gc, Kb]; fragments of veins [G] with small,
Injection 1 |1L.
spiral [Gb] or annular [Ga] vessels, often accompanied by
Elution order Order indicated in the composition of reference sheaths containing cluster crystals of calcium oxalate [Gc];
solution (a); record the retention times of these substances. fragments of the lamina, in transverse section [K], showing
System suitability: reference solution (a): the epidermises bearing broken covering trichomes [Ka], a
— resolution: minimum 1.5 between the peaks due to symmetrical, heterogeneous mesophyll with some cells
sabinene and P-pinene and minimum 1.5 between the containing cluster crystals of calcium oxalate [Kb]; occasional
peaks due to />-cymene and limonene. pollen grains, spherical, with a roughly spiny exine, about
Identification of components Using the retention times 150 pm in diameter [J]. Examine under a microscope using
determined from the chromatogram obtained with reference ruthenium red solution R. The powder shows groups of
solution (a), locate the components of reference solution (a) parenchyma containing mucilage, which stains orange-red.
Disregard the peak due to heptane.
— CL-pinene: 1.6 per cent to 3.0 per cent;
— sabinene: maximum 0.3 per cent;
— P-pinene: 1.2 per cent to 2.0 per cent;
— p-myrcene: 1.5 per cent to 2.0 per cent;
— p-cymene: maximum 1.0 per cent;
— limonene: 65.0 per cent to 75.0 per cent;
— y-terpinene: 16.0 per cent to 22.0 per cent;
— methyl hl-methylanthranilate: 0.30 per cent to
0.60 per cent;
— disregard limit: area of the principal peak in the
chromatogram obtained with reference solution (b).
Residue on evaporation (2.8.9)
1.6 per cent to 4.0 per cent, determined after heating on a
water-bath for 4 h.
STORAGE
------------------------------------------------------------------------------------------------------------ PhEur
Marshmallow Leaf * ★
PhEur, ■ ______________________________ ___________________________
DEFINITION
Whole or cut, dried leaf of Althaea officinalis L. Figure 1856.-1. - Illustration for identification test B of powdered
IDENTIFICATION herbal drug of marshmallow leaf
A. The leaves have long petioles and are about 7-10 cm long; c. Thin-layer chromatography (2.2.27).
the lamina is cordate or ovate with 3-5 shallow lobes and Test solution To 1 g of the powdered herbal drug (355)
crenate or dentate margins; the venation is palmate. (2.9.12) add 10 mL of methanol R. Heat in a water-bath
The petioles and both surfaces of the lamina are greyish- under a reflux condenser for 5 min. Allow to cool and filter.
green and densely pubescent. Rarely, fragments of the Distil the filtrate under reduced pressure until the
inflorescence or immature fruits may be present. total volume is about 2 mL.
2016 Marshmallow Root IV-275

2.5 mg of quercitrin R in 10 mL of methanol R. Marshmallow Root * **
Plate TLC silica gel plate R. (Ph. Eur. monograph 1126) ***
Mobile phase anhydrous formic acid R, glacial acetic acid R, Ph Eur____________________________________ __
water R> ethyl acetate R (11:11:27:100 VIVIVIV).
DEFINITION
Peeled or unpeeled, whole or cut, dried root of Althaea
Development Over a path of 15 cm. officinalis L.
Drying Kt 100-105 °C.
IDENTIFICATION
Detection Spray with a 10 g/L solution of diphenylboric acid A. The unpeeled, non-fragmented drug consists of
ammoethyl ester R in methanol R, then with a 50 g/L solution cylindrical, slightly twisted roots, up to 2 cm thick, with deep
of macrogol 400 R in methanol R; allow to dry in air for longitudinal furrows. The outer surface is greyish-brown and
30 min and examine in ultraviolet light at 365 nm. bears numerous rootlet scars. The fracture is fibrous
Results See below the sequence of zones present in the externally, rugged and granular internally. The section shows
chromatograms obtained with the reference solution and the a more or less thick, whitish bark with brownish periderm,
test solution. Furthermore, other fluorescent zones may be separated by the well-marked, brownish cambium from a
present in the chromatogram obtained with the test solution. white xylem. The stratified structure of the bark and the
radiate structure of xylem become more distinct when
moistened.
Top of the plate
The peeled drug has a greyish-white, finely fibrous outer
A blue fluorescent zone surface. Cork and external cortical parenchyma are absent.
A yellow fluorescent zone B. Microscopic examination (2.8.23). The powder is greyish-
Quercitrin: an orange zone
brown (unpeeled root) or whitish (peeled root). Examine
under a microscope using chloral hydrate solution R.
(Figure 1126.-1): fragments of colourless, mainly unlignified,
thick-walled fibres [C, D, M] with split or pointed ends [D],
An orange fluorescent zone sometimes accompanied by parenchymatous cells of the
medullary rays [M], or grouped [C]; fragments of vessels,
bordered-pitted or with reticulate or scalariform thickenings
Chlorogenic acid: a blue
fluorescent zone [G, H]; cluster crystals of calcium oxalate about 20-35 pm,
mostly 25-30 pm in size, isolated [K] or included in
parenchymatous cells [B]; fragments of parenchyma [E] with
An orange fluorescent zone cells containing mucilage [Ea, F]; fragments of cork with
An intense yellow fluorescent
TESTS
Maximum 4 per cent of leaves infected by Puccinia
malvacearum) showing red spots, and maximum 2 per cent of
other foreign matter.
105 °C for 2 h.
Total ash (2.4.16)
Swelling index (2.8.4)
Minimum 12, determined on 0.2 g of the powdered herbal
drug (355) (2.9.12).
__ ____________________________________________________________ Ph Eur
Figure 1126.-1. - Illustrationfor identification test B of powdered

herbal drug of marshmallow root
IV-276 Mastic 2016
thin-walled, tabular cells in surface view [A] and transverse Top of the plate
section [L] (unpeeled root). Examine under a microscope
A violet zone
using ruthenium red solution R. The powder show's groups of
parenchyma containing mucilage, which stains orange-red.
Examine under a microscope using water R. The powder A pale violet zone
show's numerous starch granules [J], about 3-25 pm in size,
occasionally with a longitudinal hilum. The starch granules A very pale violet zone
are mosdy simple Qa], a few' being 2-4 compound [Jb].
TESTS Eugenol: a brown zone A blue zone
Borneol: a greenish-blue zone A bluish-violet zone
Maximum 2 per cent of brown deteriorated drug.
Loss on drying (2.2.32) A dark violet zone
Maximum 12.0 per cent, determined on 1.000 g of the Reference solution Test solution
pow’dered herbal drug (710) (2.9.12) by drying in an oven at
105 CC for 2 h.
Total ash (2.4.16) TESTS
Maximum 6.0 per cent for the peeled root and maximum Acid value (2.5.1)
8.0 per cent for the unpeeled root. 50 to 70, determined on 1.0 g.
Swelling index (2.8.4) Water (2.2.13)
Minimum 10, determined on the powdered herbal drug Maximum 10 mL/kg, determined on 25.0 g of the drug
(710) (2.9.12). reduced to a coarse pow'der (1400) (2.9.12).
_______________________________________________________________ Ph Eur Total ash (2.4.16)
ASSAY
Mastic * ★ Use a 500 mL round-bottomed flask and 200 mL of water R
as the distillation liquid. Reduce the drug to a coarse pow'der
(Ph. Eur. monograph 1876) *** (1400) (2.9.12) and immediately use 20.0 g for the
PhEir_______________________________________________________________
determination. Introduce 0.50 mL of xylene R in the
graduated tube. Distil at a rate of 2-3 mUmin for 2 h.
DEFINITION
Dried resinous exudate obtained from stems and branches of STORAGE
Pistacia lentiscus L. var. latifolius Coss. Do not powder.
_______________________________________________ _______________ Pn El!
Content
Minimum 10 mITkg of essential oil (anhydrous drug).
IDENTIFICATION
A. Small light yellow to greenish-yellow, non-uniform,
spherical or pyriform, clear or opaque, hard glassy fragments. Matricaria Flowers * *
B. Thin-layer chromatography (2.2.27). (Matricaria Flower, Ph Eur monograph 0404)
Test solution Dissolve 1 g of the substance to be examined in
Preparation
10 mL of methylene chloride R and filter after 1-2 min.
Matricaria Liquid Extract
Reference solution Dissolve 25 mg of eugenol R and 25 mg of
Ph Eur_____________________—
borneol R in 3 mL of methylene chloride R.
Plate TLC silica gel plate R. DEFINITION
Mobile phase light petroleum R, toluene R (5:95 VIV). Dried capitula of Matricaria recutita L. (Chamomilla
recutita (L.) Rauschert).
Content
— blue essential oil: minimum 4 mIJkg (dried drug);
Drying In air. — total apigenin 7-glucoside (C2]H2oOio)- minimum
Detection spray with vanillin reagent R and heat at 0.25 per cent (dried drug).
100-105 °C for 5 min.
IDENTIFICATION
Results See below the sequence of the zones present in the A. Capitula, when spread out, consisting of an involucre
chromatograms obtained with the reference solution and the made up of many bracts arranged in 1-3 rows; an elongated-
test solution. Furthermore, other zones of various colours conical receptacle, occasionally hemispherical (young
may be present in the chromatogram obtained with the test capitula); 12-20 marginal ligulate florets with a white ligule;
solution. several dozen yellow central tubular florets. The involucre
bracts are ovate or lanceolate, with a brownish-grey scarious
margin. The receptacle is hollow, without paleae. The corolla
of the ligulate florets has a brownish-yellow tube at the base
extending to form a white, elongated-oval ligule. The inferior
ovary is dark brown, ovoid or spherical, and has a long style
and bifid stigma. The tubular florets are yellow and have a
five-toothed corolla tube, 5 syngenesious, epipetalous
2016
Matricaria Flowers IV-277
stamens and a gynoecium similar to that of the ligulate Loss on drying (2.2.32)
florets.
B. Separate the capitulum into its different parts. Examine powdered herbal drug (355) (2.9.12) by drying in an oven at
under a microscope using chloral hydrate solution R. 105 °C for 2 h.
The bracts have a margin composed of thin-walled cells and Total ash (2.4.16)
a central region composed of elongated sclereids with Maximum 13.0 per cent.
occasional stomata (2.8.3). The inner epidermis of the
corolla of the ligulate florets, in surface view, consisting of ASSAY
thin-walled, polygonal cells, slightly papillose, those of the Essential oil (2.8.12)
outer epidermis markedly sinuous and strongly striated; Use 30 g of whole drug, a 1000 mL flask, 300 mL of water R
corolla of the tubular florets with longitudinally elongated as distillation liquid and 0.50 mL of xylene R in the
epidermal cells, and with small groups of papillae near the graduated tube. Distil at a rate of 3-4 mVmin for 4 h.
apex of the lobes. Glandular trichomes each consisting of a Towards the end of this period, stop the flow of water to the
short stalk and a head of 2-3 tiers of 2 cells each occur on condenser assembly but continue distilling until the blue,
the outer surfaces of the bracts and on the corollas of both steam-volatile components have reached the lower end of the
types of florets. The ovaries have a sclerous ring at the base condenser. Immediately re-start the flow of water to the
and the wall is composed of vertical bands of thin-walled, condenser assembly to avoid warming the separation space.
longitudinally elongated cells with numerous glandular Stop the distillation after a further 10 min.
trichomes, alternating with fusiform groups of small, radially Total apigenin 7-glucoside
elongated cells containing mucilage. The cells at the apex of Liquid chromatography (2.2.29).
the stigmas are extended to form rounded papillae. Test solution Reduce 40 g of the drug to a powder (500)
Numerous small, cluster crystals of calcium oxalate occur in (2.9.12). Place 2.00 g of the powdered herbal drug in a
the inner tissues of the ovanes and the anther lobes. Pollen 500 mL round-bottomed flask. Add 200 mL of ethanol
grains spherical to triangular, about 30 pm in diameter with (96 per cent) R. Heat the mixture under a reflux condenser
3 pores and a spiny exine. on a water-bath for 15 min. Cool and filter. Rinse the filter
c. Thin-layer chromatography (2.2.27). and the residue with a few millilitres of ethanol
Test solution Dilute 50 pL of essential oil obtained in the (96 per cent) R. To the filtrate add 10 mL of freshly prepared
assay of essential oil in 1 mL of xylene R. dilute sodium hydroxide solution R and heat the mixture under
Reference solution Dissolve 2 pL of chamazulene Ri 5 pL of a reflux condenser on a water-bath for about 1 h. Cool.
Dilute to 250.0 mL with ethanol (96 per cent) R. To 50.0 mL
(-)-v-bisabolol R and 10 mg of bornyl acetate R in 5 mL of
toluene R. of the solution add 0.5 g of citric acid R. Shake for 5 min and
filter. Dilute 5.0 mL of this solution to 10.0 mL with the
mobile phase (initial mixture).
Mobile phase ethyl acetate Ri toluene R (5:95 VIV). Reference solution (a) Dissolve 10.0 mg of apigenin
Application 10 pL, as bands. 7-glucoside R in 100.0 mL of methanol R. Dilute 25.0 mL of
Development Over a path of 10 cm. this solution to 200.0 mL with the mobile phase (initial
Drying In air. mixture).
Detection Spray with anisaldehyde solution R and heat at Reference solution (b) Dissolve 10.0 mg of 5,7-dihydroxy-
100-105 °C for 5-10 min. Examine immediately in daylight. 4-methylcoumarin R in 100.0 mL of methanol R. Dilute
25.0 mL of this solution to 100.0 mL of the mobile phase
(initial mixture). To 4.0 mL of this solution add 4.0 mL of
reference solution (a) and dilute to 10.0 mL with the mobile
test solution. Furthermore, other zones are present in the
phase (initial mixture).
Precolumn'.
— size'. I = 8 mm, 0 = 4.6 mm;
Top of the plate — stationary phase', octadecylsilyl silica gel for chromatography R
1 or 2 blue or bluish-violet zones
(5 pm).
Column'.
Chamazulene: a red or A red or reddish-violet zone
— size’. I = 0.25 m, 0 -= 4.6 mm;
reddish-violet zone (chamazulene)
(5 pm).
Bornyl acetateะ a yellowish-brown Mobile phase:
zone
— mobile phase A: phosphoric acid Ri water R (0.5:99.5 V/V);
A brown zone (en-yne-
dicycloether)
— mobile phase B: phosphoric acid Ri acetonitrile R
(0.5:99.5 VIV);
(-)-a-Bisabolol: a reddish-violet A reddish-violet or bluish-violet

or bluish-violet zone zone ((-)-a-bisabolol)
0-9 25
9- 19 75 -> 25 25 -> 75
TESTS 19-24 25 75
Broken drug
Maximum 25 per cent, determined on 20.0 g, passes through
a sieve (710) (2.9.12). Flow rate 1 mL/min.
IV-278 Matricaria Oil 2016
Injection 20 pL, Top of the plate

System suitability', reference solution (b): A blue zone (chamazulene)
Guaiazulene: a blue zone
— resolution', minimum 1.8 between the peaks due to
apigenin 7-glucoside and 5,7-dihydroxy-
4-methylcoumarin.
Calculate the percentage content of total apigenin 7-glucoside

xPx 0.625 100-105 °C for 5-10 min. Examine immediately in daylight.
A2 X 7711
Results B See below for the sequence of the zones present in
/31 = area of the peak due to apigenin 7-glucoside in the chromatograms obtained with the reference solution and
the chromatogram obtained with the test solution; the test solution. Furthermore, yellowish-brown to greenish-
A2 = area of the peak due to apigenin 7-glucoside in yellow zones (lower third), violet zones (lower third) and
the chromatogram obtained with the reference further weak zones may be present in the chromatogram
solution; obtained with the test solution.
พ] = mass of the herbal drug in the test solution, in
grams; Top of the plate
m2 = mass of apigenin 7-glucoside R in reference solution
(a), in grams; 1 or 2 blue to bluish-violet zones
p = percentage content of apigenin 7-glucoside in the Guaiazuleneะ a red to reddish-violet A red to reddish-violet zone
reagent. zone (chamazulene)
----------------------------------------------------------------------------------------------------------- Ph Eur
Bornyl acetate: a yellowish-brown A brown zone (en-yne-dicydoether)
to greyish-green zone
(—Fct-Bisabolol: a reddish-violet to A reddish-violet to bluish-violet

Matricaria Oil * * bluish-violet zone zone ((-)-<x-bisabolol)
A brownish zone
PhEir_______________________________________________________________
DEFINITION
Blue essential oil obtained by steam distillation from the fresh B. Examine the chromatograms obtained in the test for
or dried flower-heads or flowering tops of Matricaria chromatographic profile.
recutita L. (Chamomdla recutita L. Rauschert). There are Results The characteristic peaks due to (-)-a-bisabolol and
2 types of matricaria oil which are characterised as rich in chamazulene in the chromatogram obtained with the test
bisabolol oxides, or rich in (—)-a-bisabolol. solution are similar in retention time to those in the
CHARACTERS chromatogram obtained with the reference solution.
Appearance TESTS
Clear, intensely blue, viscous liquid. Chromatographic profile
Intense characteristic odour. Gas chromatography (2.2.25): use the normalisation
IDENTIFICATION procedure.
First identification B Test solution Dissolve 20 pL of the essential oil to be
examined in cyclohexane R and dilute to 5.0 mL with the
same solvent.
Reference solution Dissolve 20 pL of (-)-a-bisabolol R> 5 mg of
Test solution Dissolve 20 pL of the substance to be examined chamazulene R and 6 mg of guaiazulene R in cyclohexane R
in 1.0 mL of toluene R. and dilute to 5.0 mL with the same solvent.
Reference solution Dissolve 2 mg of guaiazulene R, 5 pL of Column'.
(-)-a-bisabolol R and 10 mg of bornyl acetate R in 5.0 mL of — material', fused silica,
toluene R. — size', z = 30 m (a film thickness of 1 pm may be used) to
Plate TLC silica gel plate R. 60 m (a film thickness of 0.2 pm may be used),
Mobile phase ethyl acetate R3 toluene R (5:95 V/V). 0 = 0.25-0.53 mm, when using a column longer than
30 m, an adjustment of the temperature programme may
be necessary,
Development Over a path of 10 cm. — stationary phase', macrogol 20 000 R.
Drying In air. Carrier gas helium for chromatography R.
Detection A Examine in daylight.
Flow rate 1-2 mUmin.
Results A See below for the sequence of the zones present in
Split ratio 1:100.
the chromatograms obtained with the reference solution and
the test solution.
2016
Matricaria Oil IV-279
1. {S-farnesene 3. bisabolone 5. chamazulene
2. bisabolo! oxide B 4. (—>-ct-bisabolol 6. bisabolol oxide A
Figure 1836.-1. - Chromatogram of matricaria oil rich in bisabolol oxides
1. p-farnesene 3. bisabolone 5. chamazulene
2. bisabolol oxide B 4. (->a-bisabolol 6. bisabolol oxide A
Figure 1836.-2. - Chromatogram of matricaria oil rich in (-/-cMsabolol

IV-280 Matricaria Preparations 2016
Temperature'. 47.5 volumes of water and 50 volumes of ethanol

(96 per cent).
Time Temperature CHARACTERS
___________ (°C) Appearance
Column 0-40 70 —> 230 Brownish, clear liquid.
40-50 230 Intense characteristic odour and characteristic bitter taste.
injection port 250 Solubility
Detector Miscible with water and with ethanol (96 per cent) with
250
development of turbidity, soluble in ethanol
(50 per cent P7F).
Detection Flame ionisation. IDENTIFICATION
Injection 1.0 pL. A. Thin-layer chromatography (2.2.27).
Elution order Order indicated in the composition of the Test solution Place 10 mL of the extract to be examined in a
reference solution. Record the retention times of these separating funnel and shake with 2 quantities, each of
substances. 10 mL, of pentane R. Combine the pentane layers, dry over
Relative retention With reference to chamazulene (retention 2 g of anhydrous sodium sulfate R and filter. Evaporate the
time = about 34.4 min): P-famesene = about 0.5; bisabolol filtrate to dryness on a water-bath and dissolve the residue in
oxide B = about 0.8; bisabolone = about 0.87; (-)-a- 0.5 mL of toluene R.
bisabolol = about 0.9; bisabolol oxide A =ะ about 1.02. Reference solution Dissolve 4 mg of guaiazulene R, 20 mg of
System suitability: reference solution: (-)-^t-bisabolol R and 20 mg of bornyl acetate R in 10 mL of
— resolution: minimum 1.5 between the peaks due to toluene R.
chamazulene and guaiazulene. Plate TLC silica gel 7*254 plate R-
Using the retention times determined from the Mobile phase ethyl acetate R, toluene R (5:95 P7P).
(-)-a-bisabolol and chamazulene in the chromatogram
obtained with the test solution; locate bisabolol oxides Development Over a path of 10 cm.
(bisabolol oxide B, bisabolone and bisabolol oxide A) using Drying In air.
Figures 1836.-1 and 1836.-2 (disregard the peak due to Detection A Examine in ultraviolet light at 254 nm.
cyclohexane). The chromatogram obtained with the test Results A The chromatogram obtained with the test solution
solution does not show a peak with the retention time of shows several quenching zones, of which 2 main zones are in
guaiazulene. the middle third (en-yne-dicycloether).
Determine the percentage content of the components. Detection B Examine in ultraviolet light at 365 nm.
The limits are within the following ranges. Results B The chromatogram obtained with the test solution
shows in the middle part an intense blue fluorescent zone
Matricaria oil rich in Matricaria oil rich in (hemiarin).
bisabolol oxides (-)-a-bisabolol
Detection c Spray with anisaldehyde solution R and examine in
(per cent) (per cent)
daylight while heating at 100-105 °C for 5-10 min.
Bisabolol oxides 29-81
Results c The chromatogram obtained with the reference
- (-)-a-Bisabolol 10-65
solution shows in the lower third a reddish-violet or bluish-
Chamazdene 2> 1.0 i 1.0 violet zone ((-)-a-bisabolol), in the middle third a yellowish-
Total of bisabolol oxides ะ> 20 brown or greyish-green zone (bornyl acetate) and in the
and (-Hx-Bisabolol upper third a red or reddish-violet zone (guaiazulene).
The chromatogram obtained with the test solution shows in
the lower third yellowish-brown or greenish-yellow and violet
STORAGE zones and a reddish-violet or bluish-violet zone due to
At a temperature not exceeding 25 °C. (-)-a-bisabolol in the chromatogram obtained with the
______________________________________________________________________ 1 PhEur reference solution; a brownish zone (en-yne-dicycloether)
similar in position to the zone due to bornyl acetate in the
chromatogram obtained with the reference solution; a red or
reddish-violet zone (chamazulene) corresponding to
guaiazulene in the chromatogram obtained with the reference
Matricaria Liquid Extract solution and immediately above it 1 or 2 blue or bluish-violet
(Ph. Eur. monograph 1544) *** zones; further weak zones may be present in the
Ph Elf______________________________________________________________
DEFINITION Test solution The extract to be examined.
Liquid extract produced from Matricaria flower (0404).
Reference solution Dissolve 1.0 mg of chlorogenic acid R>
Content 2.5 mg of hyperoside R and 2.5 mg of rutin R in 10 mL of
Minimum 0.30 per cent of blue residual oil. methanol R.
PRODUCTION Plate TLC silica gel plate R.
The extract is produced from the herbal drug by a suitable Mobile phase anhydrous formic acid R, glacial acetic acid R,
procedure for liquid extracts using a mixture of 2.5 volumes water R, ethyl acetate R (7.5:7.5:18:67 VIVIVIV).
of a 10 per cent mhn solution of ammonia (NH3),
2016 Meadowsweet IV-281
Application 10 pL as bands. IDENTIFICATION

Development Over a path of 15 cm. A. The stem, up to 5 mm in diameter, is greenish-brown,
Drying At 100-105 °C. stiff, angular, hollow except at the apex, and has regular,
Detection spray the warm plate with a 10 g/L solution of straight, longitudinal furrows. The petiolate leaf, compound
diphenylboric acid aminoethyl ester R in methanol R) imparipinnate, has 2 reddish-brown angular stipules.
subsequently spray with a 50 g/L solution of macrogol 400 R It consists of 3-9 pairs of leaflets, unevenly dentate, some of
in methanol Rj allow to dry in air for about 30 min and which are small and fan-shaped. The leaflets are dark green
examine in ultraviolet light at 365 nm. and glabrous on the upper surface, tomentose and lighter,
sometimes silvery on the lower surface. The terminal leaflet,
the largest, is divided into 3 segments. The veins are
solution shows in the middle part a light blue fluorescent
prominent and brown on the lower surface.
zone (chlorogenic acid), below it a yellowish-brown The inflorescence is complex and composed of very
fluorescent zone (rutin) and above it a yellowish-brown
numerous flowers arranged in irregular cymose panicles.
fluorescent zone (hyperoside). The chromatogram obtained
The flowers are creamish-white and about 3-6 mm in
With the test solution shows a yellowish-brown fluorescent diameter; the calyx consists of 5 dark green, reflexed and
zone corresponding to the zone of rutin in the chromatogram hairy sepals fused at the base to a concave receptacle;
obtained with the reference solution, a light blue fluorescent the 5 free petals, which are readily detached, are pale yellow,
zone corresponding to the zone of chlorogenic acid in the obovate and distinctly narrowed at the base; the stamens are
chromatogram obtained with the reference solution, a numerous with rounded anthers and they extend beyond the
yellowish-brown fluorescent zone similar in position to the petals; the gynoecium consists of about 4-6 carpels, each with
zone of hyperoside in the chromatogram obtained with the a short style and a globular stigma; the carpels become
reference solution; it also shows above the yellowish-brown twisted together spirally to form yellowish-brown fruits with a
fluorescent zone a green fluorescent zone, then several bluish helicoidal twist. Unopened flower buds are frequently
or greenish fluorescent zones and near the solvent front a present. If the fruit is present, it has a helicoidal twist and
yellowish fluorescent zone. contains brownish seeds.
TESTS B. Microscopic examination (2.8.23). The powder is green or
Ethanol (2.9.10) yellowish-green. Examine under a microscope using chloral
38 per cent VIV to 53 per cent VIV. hydrate solution R. The powder shows ±e following diagnostic
Dry residue (2.8.16) characters (Figure 1868.-1): fragments of the epidermises of
Minimum 12.0 per cent. the leaves and sepals [C, E, F] with sinuous or wavy
cells [Ca, Ea, Fa], short, thick-walled, conical covering
ASSAY trichomes thickened at the base, in surface view [Eb] and in
Place 20.0 g in a 1000 mL round-bottomed flask, add side view บ], unicellular covering trichomes, thin-walled, very
300 mL of water R and distil until 200 mL has been long and flexuous, with pointed ends in surface view [Fc]
collected in a flask. Transfer the distillate into a separating and in side view [A], or their scars (flexuous trichome [Fd],
funnel. Dissolve 65 g of sodium chloride R in the distillate and conical trichome [Fe]) and occasional clavate glandular
shake with 3 quantities, each of 30 mL, of pentane R trichomes with a 1- to 3-celled ([Ed] and [G], respectively),
previously used to rinse the reflux condenser and the flask. uniseriate stalk, a multicellular head and dense brown
Combine the pentane layers, dry over 2 g of anhydrous sodium contents; fragments of the upper epidermis often
sulfate R and filter into a tared 100 mL round-bottomed flask accompanied by palisade parenchyma [Cb] including some
which has been dried in a desiccator for 3 h. Rinse the hypertrophied cells containing a cluster crystal of calcium
anhydrous sodium sulfate and the filter with 2 quantities, oxalate [Cc]; fragments of ±e lower epidermis with
each of 20 mL, of pentane R. Evaporate the pentane in a anomocytic stomata (2.8.3) [Ec, Fb], sometimes
water-bath at 45 °C. The residue of pentane is eliminated in accompanied by spongy parenchyma [Ff] with some cells
a current of air for 3 min. Dry the flask in a desiccator for containing cluster crystals of calcium oxalate [Fg]; fragments
3 h and weigh. The residual oil is blue (chamazulene). of the petals [H] with thin-walled epidermal cells, some
----------- —--------------------------------------------------------------------------------------------- Ph Eur showing rounded papillae [Ha]; numerous spherical pollen
grains with 3 pores and a faintly pitted exine [Bb]; fragments
of the anther [B, D] whose fibrous layer shows specific
thickenings, in surface view [D] and in side view [Ba];
Meadowsweet ***** fragments of the ovary [K] with an epidermis bearing
stomata [Ka] and with parenchyma containing prism crystals
(Ph. Eur. monograph 1868) *** of calcium oxalate [Kb]; fragments of vascular tissue [L] with
annular, spiral or pitted vessels from the leaves and stems.
DEFINITION
Whole or cut, dried flowering tops of Filipendula ulmaria (L.)
Maxim, (syn. spiraea ulmaria L.).
Content
Minimum 1 mL/kg of essential oil (dried drug).
CHARACTERS
Aromatic odour of methyl salicylate, after crushing.
TV-282 Melilot 2016
TESTS
Maximum 5.0 per cent of stems with a diameter greater than
5 mm and maximum 2.0 per cent of other foreign matter.
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Use 50.0 g of the cut herbal drug, a 1000 mL flask, 300 mL
of dilute hydrochloric acid R as ±e distillation liquid, and
0.5 mL of xylene R in the graduated tube. Distil at a rate of
2-3 mUmin for 2 h.
__________________________________________________________ ____ PnEif
Melilot * *
Ph Eur _ __________________________________________________ __________
DEFINITION
Whole or cut, dried aerial parts of Melilotus officinalis (L.)
Lam.
Figure 1868.-1. - Illustration for identification test B of powdered Content
herbal drug of meadowsweet Minimum 0.3 per cent of coumarin (C9H6O2; Mr 146.1)
c. Thin-layer chromatography (2.2.27). (dried drug).
Test solution Xylene solution obtained in the assay. IDENTIFICATION
Reference solution Dissolve 0.1 mL of methyl salicylate R and A. The stem is green, cylindrical, glabrous and finely ridged.
0.1 mL of salicylaldehyde R in xylene R and dilute to 5 mL The leaves are alternate, petiolate and trifoliate with
with the same solvent. 2 lanceolate stipules; the leaflets are up to about 3 cm long
Plate TLC silica gel plate R. and 20 mm wide, elongated or ovate with a finely dentate
margin, acute at the apex and base; the upper surface is dark
Mobile phase hexane R, toluene R (50:50 V/V).
green and glabrous, the lower surface paler green with short,
Application 10 pL as bands. fine hairs, especially at the base. The inflorescence is
Development Over a path of 10 cm. racemose with numerous pale yellow flowers, about 7 mm
Drying In air. long, each having a hairy calyx with 5 deeply-divided,
Detection treat with 3 mL of ferric chloride solution R3 and unequal teeth, and a papilionate corolla. The fruit is an
examine in daylight. indehiscent pod, often persistent within the calyx, yellowish-
brown, short and tapering at the apex; the surface is glabrous
and transversely wrinkled.
test solution. Furthermore, other zones are present in the B. Microscopic examination (2.8.23). The powder is
chromatogram obtained with the test solution. yellowish-green. Examine under a microscope using chloral
characters (Figure 2120.-1): fragments of the leaf lamina in
Top of the plate surface view [D] showing unevenly thickened, slightly
sinuous epidermal cells; numerous stomata [Db], mostly
anomocytic (2.8.3) with 3-6 subsidiary cells [Da] and
Methyl salicylate ะ a violet-brown A violet-brown zone (methyl
frequently, underlying palisade parenchyma [De]; uniseriate
salicylate) covering trichomes with 2 short, smooth-walled basal cells
and a long terminal cell, bent at right angles, with a thick
Salicylaldehyde: a violet-brown A violet-brown zone wall and a warty cuticle [A, B]; occasional glandular
(salicylaldehyde)
trichomes with a short, 2- or 3- celled stalk and ovoid,
biseriate head with 4 indistinct cells [H]; fragments of the
petals composed of cells with wavy walls [M]; fragments of
vascular tissue from the stem [F, G], including large
vessels [G], sometimes associated with unlignified septate
fibres [Fa] and a sheath of parenchymatous cells containing
prisms of calcium oxalate [Fb]; fragments of mesophyll [J]
including some cells which may occasionally contain cluster
2016 Melilot IV-283
crystals of calcium oxalate Ja]; fragments of the stem Top of the plate
epidermis with elongated, straight-walled cells and
Coumarin: a greenish-yellow A greenish-yellow fluorescent
anomocytic (2.8.3) stomata [L]; fragments of the fibrous fluorescent zone zone (coumarin)
layer of the anthers in surface view [E] and in transverse
section [K]; spherical or ovoid pollen grains about 25 pm
long with 3 germinal pores and a smooth exine [C]- A blue fluorescent zone
o-Coumaric acid: a A greenish-yellow fluorescent

greenish-yellow fluorescent
zone present
TESTS
105 °C for 2 h.
Total ash (2.4.16)
ASSAY
Test solution Completely reduce about 50 g of the herbal drug
to a powder (500) (2.9.12). To 5.00 g of the powdered
herbal drug add 90 mL of methanol R and boil under a reflux
condenser for 30 min. Allow to cool. Filter under vacuum
through a fibre-glass filter. Take up the residue and the
fragmented filter with 90 mL of methanol R. Treat in the
same manner as before. Combine the filtrates and dilute to
Reference solution Dissolve 25.0 mg of coumarin CRS in
Figure 2120.-1. - Illustration for identification test B of powdered Column:
herbal drug of melilot — size: I = 0.25 m, 0 = 4 mm;
c. Thin-layer chromatography (2.2.27). — stationary phase: end-capped octadecylsilyl silica gel for
(2.9.12) add 3 mL of methanol R. Heat on a water-bath at Mobile phase acetonitrile R, 5 g/L solution of phosphoric acid R
100 °C for 1 min and filter. (22:78 V/V).
Reference solution Dissolve 50 mg of coumarin CRS and 20 mg Flow rate 1.7 mL/min.
of o-coumaric acid R in 50 mL of methanol R. Detection Spectrophotometer at 275 nm.
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel Injection 20 pL.
plate R (2-10 pm)]. System suitability:
Mobile phase dilute acetic acid R) ether R, toluene R — retention time: coumarin = about 7.8 min.
(10:50:50 V/V/V), use the upper layer. Calcdate the percentage content of coumarin using the
Application 25 pL [or 3 pL] as bands of 10 mm [or 8 mm]. following expression:
Al X 7ท2 X p
Drying In air.
Al X 7711
Detection Spray with 2 M alcoholic potassium hydroxide R and
A1 = area of the peak due to coumarin in the
A2 = area of the peak due to coumarin in the
solution;
the test solution. m1 = mass of the herbal drug to be examined used to
m2 = mass of coumarin CRS used to prepare the
p ะะะ percentage content of coumarin in coumarin CRS.
IV-284 Menthol Preparations 2016
Menthol and Benzoin Inhalation CHARACTERS

No rancid odour.
Menthol and Benzoin Inhalation Vapour
IDENTIFICATION
DEFINITION
A. The achene is strongly compressed, elongate-obovate,
Menthol and Benzoin Inhalation is an inhalation vapour,
about 6-8 mm long, 3 mm broad and 1.5 mm thick;
solution.
the outer surface is smooth and shiny with a grey or pale
Racementhol or 20 g
brown ground colour variably streaked dark brown
Levomenthol
longitudinally to give an overall pale greyish or brown colour,
Benzoin Inhalation Sufficient to produce 1000 mL
the fruit is tapering at the base and crowned at the apex with
Extemporaneous preparation a glistening, pale yellow extension forming a collar about
The following directions apply. 1 mm high surrounding the remains of the style.
Dissolve the Racementhol or the Levomenthol in a portion of Cut transversely, the fruit shows a narrow, brown outer area
the Benzoin Inh al anon, add sufficient Benzoin Inhalation to and 2 large, dense, white oily cotyledons.
produce 1000 mL and mix. B. Microscopic examination (2.5.25). The powder is
The inhalation complies with the requirements stated under brownish-yellow with darker specks. Examine under a
Preparations for Inhalation and with the following requirements. microscope using chloral hydrate solution R. The powder
shows the following diagnostic characters: fragments of the
epicarp composed of colourless cells, polygonal in surface
Not less than 2.8% w/v, calculated as cinnamic acid,
view, the lumen appearing fairly large or as a small slit,
C9H8๐2.
depending on the orientation; groups of parenchymatous cells
Total solids from the pigment layer, some of them containing colouring
9.0 to 12.0% w/v when determined by drying at 105° for matter which appears bright red; very abundant groups of
4 hours, Appendix XI A. Use 2 mL. large sclereids from the testa with bright yellow pitted walls
ASSAY and a narrow lumen; occasionally fragments of small-celled
Boil 10 mL with 25 mL of 0.5m ethanolic potassium hydroxide parenchyma with pitted and beaded walls; abundant thin
under a reflux condenser for 1 hour. Evaporate the ethanol, walled parenchymatous cells from the cotyledons containing
disperse the residue in 50 mL of hot water, cool, add 80 mL oil globules and scattered cluster crystals of calcium oxalate,
of water and 1.5 g of magnesium sulfate dissolved in 50 mL of a few larger, prismatic crystals of calcium oxalate.
water. Mix thoroughly and allow to stand for 10 minutes. c. Thin-layer chromatography (2.2.27).
Filter, wash the residue on the filter with 20 mL of water, Test solution To 1.0 g of the powdered herbal drug (500)
acidify the combined filtrate and washings with hydrochloric (2.9J2) add 10 mL of methanol R. Heat under reflux in a
acid and extract with four 40 mL quantities of ether. Discard water-bath at 70 ๐๐ for 5 min. Cool and filter. Evaporate the
the aqueous solution, combine the ether extracts and extract filtrate to dryness and dissolve the residue in 1.0 mL of
with successive quantities of 20, 20, 10, 10 and 10 mL of methanol R.
sodium hydrogen carbonate solution, washing each aqueous Reference solution Dissolve 2 mg of silibinin R and 5 mg of
extract with the same 20 mL of ether. Discard the ether taxifolin R in 10 mL of methanol R.
layers, carefully acidify the combined aqueous extracts with
hydrochloric acid and extract with successive quantities of 30,
20, 20 and 10 mL of chloroform, filtering each extract through Mobile phase anhydrous formic acid R, acetone R, methylene
anhydrous sodium sulfate supported on absorbent cotton. Distil chloride R (8.5:16.5:75 VIVIV).
the chloroform from the combined filtrates until 10 mL Application 30 pL of the test solution and 10 pL of the
remains and remove the remainder in a current of air. reference solution, as bands.
Dissolve the residue, with the aid of gentle heat, in 10 mL of Development Over a path of 10 cm.
ethanol (96%), previously neutralised to phenol red solution, Drying At 100-105 °C.
cool and titrate with 0.1m sodium hydroxide PS using phenol
Detection-. Treat the still-warm plate with a 10 g/L solution of
red solution as indicator. Each mL of 0.1m sodium hydroxide
diphenylboric acid aminoethyl ester R in methanol R and
Izs is equivalent to 14.82 mg of total balsamic acids,
subsequently treat with a 50 g/L solution of macrogol 400 R
calculated as cinnamic acid, C9H8๐2.
in methanol R. Allow to dry for 30 min and examine in
ultraviolet light at 365 nm.
Milk-thistle Fruit ***** test solution. Furthermore, other orange and yellowish-green
(Ph. Eur. monograph 1860) *** fluorescent zones are present between the zones due to
silibinin and taxifolin in the chromatogram obtained with the
Preparation
Refined and Standardised Milk Thistle Dry Extract test solution.
Ph Eur__________________________________________ ____________________
DEFINITION
Mature fruit, devoid of the pappus, of Silybum marianum L.
Gaertner.
Content
Minimum 1.5 per cent of silymarin, expressed as silibinin
(๐25แ22Oio; Afr 482.4) (dried drug).
2016 Milk Thistle Preparations IV-285
Top of the plate solution the peak due to silidianin may vary in size, be absent
Silibinin: a yellowish-green or be present as the principal peak.
A yellowish-green fluorescent
fluorescent zone zone (silibinin) Retention time Silibinin B = about 30 min; if necessary, adjust
the time periods of the gradient.
Taxifolin: an orange fluorescent System suitability, reference solution:
zone (taxifolin) — resolution: minimum 1.8 between the peaks due to
t\ yellowish-green fluorescent
silibinin A and silibinin B;
zone (silicristin) — the chromatogram obtained is similar to the
chromatogram supplied with milk thistle dry extract HRS.
Calculate the percentage content of total silymarin, expressed
A light blue fluorescent zone (line
of application) as silibinin, using the following expression:
(Al 4- Ao 4- A3 4- Aa + A5 4- A6) X m 1 X p X 5
(A74-A8) xm2
TESTS
Maximum 8.0 per cent, determined on 1.000 g of the A1 = area of the peak due to silicristin in the
powdered herbal drug (500) (2.9.12) by drying in an oven at chromatogram obtained with the test solution;
105 °C for 2 h. A2 = area of the peak due to silidianin in the
Total ash (2.4.16)
A3 = area of the peak due to silibinin A in the
ASSAY A4 = area of the peak due to silibinin B in the
Liquid chromatography (2.2.29). chromatogram obtained with the test solution;
Test solution Place 5.00 g of the powdered herbal drug (500) A5 = area of the peak due to isosilibinin A in the
(2.9.72) in a continuous-extraction apparatus. Add 100 mL chromatogram obtained with the test solution;
of light petroleum R and heat in a water-bath for 8 h. Allow Afy = area of the peak due to isosilibinin B in the
the defatted drug to dry at room temperature. In a chromatogram obtained with the test solution;
continuous-extraction apparatus, extract the latter with A7 = area of the peak due to silibinin A in the
100 mL of methanol R in a water-bath for 5 h. Evaporate the chromatogram obtained with the reference
methanolic extract in vacuo to a volume of about 30 mL. solution;
Filter into a 50 mL volumetric flask, rinsing the extraction A8 = area of the peak due to silibinin B in the
flask and the filter, and diluting to 50.0 mL with methanol R. chromatogram obtained with the reference
Dilute 5.0 mL of this solution to 50.0 mL with methanol R. solution;
tn 1 = mass of milk thistle dry extract HRS used to
Reference solution Dissolve a quantity of milk thistle dry
extract HRS corresponding to 10.0 mg of silibinin in
Column:
p = combined percentage content of silibinin A and
— size: I = 0.125 m, 0 = 4 mm;
silibinin B in milk thistle dry extract HRS.
_____________________________________________________________ Ph Eur
Mobile phase:
— mobile phase A: phosphoric acid Ry methanol R, water R
(0.5:35:65 VIVIV)y
— mobile phase B: phosphoric acid R’y methanol Ry water R
(0.5:50:50 0/P7F);
Refined and Standardised Milk
Time MobUe phase A Mobile phase B
Thistle Dry Extract ****
(min)(per cent V/V)(per cent V/V) (Ph. Eur. monograph 2071)
0 - 28 100 -» 0 0 -> 100 Ph Eur----------------------------------------------------------------------------------- -----------------
28 - 35 0 100 DEFINITION
35 - 36 0 -> 100 100 -> 0 Dry extract, refined and standardised, produced from Milk
thistle fruit (1860).
36 - 51 100 0
Content
90 per cent to 110 per cent of the nominal content of
Flow rate 0.8 mL/min. silymarin, expressed as silibinin (C25H22O10; Mr 482.4),
Detection Spectrophotometer at 288 nm. stated on the label. The nominal content of silymarin is
within the range 30 per cent mhn to 65 per cent m/m (dried
Injection 10 J1L. extract).
The content of silymarin corresponds to:
mdk thistle dry extract HRS and the chromatogram obtained — sum of the contents of silicristin and silidianin (both
with the reference solution to identify the peaks due to C25H22O10; Mr 482.4): 20 per cent to 45 per cent,
silicristin, silidianin, silibinin A, silibinin B, isosilibinin A and calculated with reference to total silymarin;
isosilibinin B. In the chromatogram obtained with the test
IV-286 Milk Thistle Preparations 2016
— Sinn of the contents of silibinin A and silibinin B (both Reference solution Dissolve a quantity of milk thistle dry
C25H22O10J Mr 482.4): 40 per cent to 65 per cent, extract HRS corresponding to 10.0 mg of silibinin in
calculated with reference to total silymarin; methanol R and dilute to 100.0 mL with the same solvent.
— Sinn of the contents of isosilibinin A and isosilibinin B (both Column’.
Q25H22O10; Mr 482.4): 10 per cent to 20 per cent, — size'. I = 0.125 m, 0 = 4 mm;
calculated with reference to total silymarin. — stationary phase: end-capped octadecylsilyl silica gel for
PRODUCTION chromatography R (5 pm).
The extract is produced from the herbal drug by an Mobile phase:
appropriate procedure, using one or more of the following — mobile phase A: phosphoric acid Ry methanol Ry water R
solvents: (0.5:35:65 VIVIV)’,
— ethyl acetate; — mobile phase B: phosphoric acid Ry methanol Ry water R
— acetone or mixture of acetone and water; (0.5:50:50 JZ/F/F);
— ethanol or mixture of ethanol and water;
— methanol or mixture of methanol and water. Time Mobile phase A Mobile phase B
CHARACTERS (min) (per cent V/V) (per cent V7V)
Appearance 0 - 28 100 -> 0 0 -> 100
Yellowish-brown, amorphous powder. 28 - 35 0 100
IDENTIFICATION 35 - 36 0 -> 100 100 -> 0

Thin-layer chromatography (2.2.27). 36 - 51 0
100
5 mL of methanol R.
Reference solution Dissolve 2 mg of silibinin R and 5 mg of Flow rate 0.8 mUmin.
taxifolin J? in 10 mL of methanol R. Detection Spectrophotometer at 288 nm.
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel Injection 10 pL.
plate R (2-10 pm)]. Identification of peaks Use the chromatogram supplied with
Mobile phase anhydrous formic acid Ry acetone Ry methylene milk thistle dry extract HRS and the chromatogram obtained
chloride R (8.5:16.5:75 VIVIV). with the reference solution to identify the peaks due to
Application 10 pL [or 8 pL] of the test solution and 10 pL silicristin, silidianin, silibinin A, silibinin B, isosilibinin A and
[or 2 pL] of the reference solution, as bands. isosilibinin B. In the chromatogram obtained with the test
Development Over a path of 10 cm [or 6 cm]. solution the peak due to silidianin may vary in size or be
absent.
Retention time Silibinin B = about 30 min; if necessary, adjust
Detection’. Treat the still-warm plate with a 10 g/L solution of the time periods of the gradient.
diphenylboric acid aminoethyl ester R in methanol R and
subsequently treat with a 50 g/L solution of macrogol 400 R
in methanol R. Allow to dry for 30 min and examine in
silibinin A and silibinin B;
ultraviolet light at 365 nm.
— the chromatogram is similar to the chromatogram
Results See below the sequence of zones present in the supplied with milk thistle dry extract HRS.
Calculate the percentage content of total silymarin, expressed
test solution. Furthermore, other yellowish-green fluorescent
as silibinin, using the following expression:
zones may be present between the zones due to silibinin and
taxifolin in the chromatogram obtained with the test solution.
(Al 4" A2 4" A3 4" A4 4" As 4- Ae) X m 1 X p
Top of the plate (A7 4- As) X m2
Silibinin: a yellowish-green A yellowish-green fluorescent zone
fluorescent zone (silibinin) Calculate the percentage content of the sum of silicristin and
silidianin, with reference to total silymarin, using the
Taxifolin: an orange fluorescent An orange fluorescent zone following expression:
(taxifolin)
(silicristin) (Al 4- A2) X 100
Al 4" A2 4“ A3 4” A4 4- As 4- Ae
A fluorescent zone (line of
application) Calculate the percentage content of the sum of silibinin A
Reference solution Test solution and silibinin B, with reference to total silymarin, using the
(A3 + A4) X 100

Maximum 5.0 per cent. Al 4- A2 4- A3 4- A4 4- As 4- Ae
ASSAY
Calculate the percentage content of the sum of isosilibinin A
and isosilibinin B, with reference to total silymarin, using the
Test solution Dissolve 60.0 mg of the extract to be examined following expression:
in methanol R and dilute to 100.0 mL with the same solvent.
2016 MintOil IV-287
(>*5 + A6) X 100

A + A2 + A3 4- A4 + A5 + A6 chromatograms obtained with the reference solution and the
test solution. Furthermore, a quenching zone may be present
-^1 = area of the peak due to silicristin in the in the upper third of the chromatogram obtained with the
chromatogram obtained with the test solution; test solution.
A2 = area of the peak due to silidianin in the
Top of the plate
Ai ะะะ area of the peak due to silibinin A in the
chromatogram obtained with the test solution; Carvone and pulegone: a A quenching zone
A4 ะ= area of the peak due to silibinin B in the quenching zone
chromatogram obtained with the test solution; A quenching zone

A5 ะะะ area of the peak due to isosilibinin A in the Reference solution Test solution
A$ ะ= area of the peak due to isosilibinin B in the
chromatogram obtained with the test solution; Detection B Spray with anisaldehyde solution R and heat at
A2 — area of the peak due to silibinin A in the 100-105 °C for 5-10 min. Examine immediately in daylight.
chromatogram obtained with the reference Results B See below the sequence of the zones present in the
solution; chromatograms obtained with the reference solution and the
As ะ= area of the peak due to silibinin B in the test solution. Furthermore, the zone due to cineole in the
chromatogram obtained with the reference reference solution is absent in the chromatogram obtained
solution; with the test solution. No yellowish-brown zone below the
,M1 = mass of milk thistle dry extract HRS used to intense reddish-violet zone is present in the chromatogram
prepare the reference solution, in grams; obtained with the test solution.
Top of the plate
p = combined percentage content of silibinin A and
silibinin B in milk thistle dry extract HRS. An intense reddish-violet zone
(near the solvent front)
-------------------------------------- - ------------------------------------------------------------------ Ph Eur
Menthyl acetate: a bluish-violet A bluish-violet zone (menthyl
acetate)
A strongly greenish zone
A greenish zone
Dementholised Mint Oil ★* ** Carvone and pulegone: a reddish A reddish zone

zone
(Partly Dementholised Mint Oil, ***
Cineole: a violet zone
Ph Eur monograph 1838)
Ph Eur__________
A distinctly violet zone
DEFINITION
Essential oil obtained by steam distillation from the fresh, Menthol: an intense blue zone A very intense blue zone
(menthol)
flowering aerial parts, recently gathered from Mentha
canadensis L. (syn. M. aruensis L. var. glabrata (Benth) Fem.,
Af. aruensis var. piperascens Malinv. ex Holmes), followed by
partial separation of menthol by crystallisation.
CHARACTERS chromatographic profile.
Appearance Results The characteristic peaks in the chromatogram
Colourless, pale yellow or greenish-yellow liquid. obtained with the test solution are approximately similar in
Characteristic odour. retention time to those in the chromatogram obtained with
the reference solution. Carvone may be absent from the
IDENTIFICATION
First identification: B.
Second identification A TESTS
0.888 to 0.910.
Test solution Dissolve 0.1 mL of the substance to be
examined in 1.0 mL of toluene R. Refiractive index (2.2.6)
1.456 to 1.470.
Reference solution Dissolve 4 pL of carvone R, 4 pL of
pulegone R, 10 pL of menthyl acetate R, 20 pL of cineole R and Optical rotation (2.2.7)
50 mg of menthol R in 5 mL of toluene R. -16.0° to -34.0°.
Plate TLC silica gel F254 plate R. Acid value (2.5J)
Maximum 1.0, determined on 5.00 g of the substance to be
Mobile phase ethyl acetate R, toluene R (5:95 V/V). examined dissolved in 50 mL of the prescribed mixture of
Application 10 pL, as bands. solvents.
Development Over a path of 15 cm. Chromatographic profile
Drying In air. Gas chromatography (2.2.25): use the normalisation
Detection A Examine in ultraviolet light at 254 nm. procedure.
FV-288 Motherwort 2016
Test solution Dissolve 0.20 g of the substance to be examined

in hexane R and dilute to 10.0 mL with the same solvent. Motherwort
Reference solution Dissolve 10 mg of limonene R, 20 mg of (Ph. Eur. monograph 1833) *
cineole R, 40 mg of menthone R3 10 mg of isomenthone R, Ph Eur____________________________________________ ___________
40 mg of menthyl acetate R, 20 mg of isopulegol R> 60 mg of
menthol R, 20 mg of pulegone R and 10 mg of carvone R in DEFINITION
hexane R and dilute to 10.0 mL with the same solvent. Whole or cut, dried flowering aerial parts of Leonurus
Column: cardiaca L.
— material: fused silica, Content
— size: z = 30 m (a film thickness of 1 gm may be used) to Minimum 0.2 per cent of flavonoids, expressed as hyperoside
60 m (a film thickness of 0.2 gm may be used), (C2iH2o012; Alj. 464.4) (dried drug).
0 = 0.25-0.53 mm, IDENTIFICATION
A. The stem pieces are hairy, longitudinally striated,
Carrier gas helium for chromatography R. quadrangular, hollow, and up to about 10 mm wide; they
Flow rate 1.5 mJL/min. bear opposite and decussate, petiolate leaves and, in the axils
Split ratio 1:100. of the upper leaves, about 6-12 small flowers, arranged in
Temperature: sessile whorls forming a long, leafy spike. The lower leaves
are ovate-orbicular, palmately 3- to 5-lobed, rarely 7-lobed,
the lobes irregularly dentate. The upper leaves are entire or
Time Temperature slightly trifid, lanceolate with a serrate margin and cuneate at
(min) (°C) the base. The upper surface of the leaves is green with
Column 0 - 10 60
scattered hairs, the lower surface is paler green, densely
10 - 70 60-» 180 pubescent and shows a prominent palmate and reticulate
70 - 75 180
venation. The flowers have a funnel-shaped calyx, 3 mm to
5 mm long with 5 stiff, recurved teeth; the corolla is
Injection port 200 2-lipped, the upper lip pink and pubescent on the outer
Detector 220 surface, the lower lip white with purplish spots; stamens 4,
densely pubescent.
B. Microscopic examination (2.8.23). The powder is green.
Detection Flame ionisation. Examine under a microscope using chloral hydrate solution R.
Injection 1.0 pL. The powder shows the following diagnostic characters
Elution order Order indicated in the composition of the (Figure 1833.-1): numerous covering trichomes [A],
reference solution. Record the retention times of these whole [Aa] or fragmented [Ab], uniseriate, with warty walls,
substances. composed of 2-8 cells with slight swellings at the junctions,
System suitability: reference solution: up to 1500 pm long; fragments of die upper epidermis, in
— resolution: minimum 1.5 between the peaks due to surface view [B], with cells with straight or sinuous anticlinal
limonene and cineole. walls [Ba], often accompanied by palisade parenchyma [Bb];
Using the retention times determined from the fragments of the lower epidermis, in surface view [C], with
chromatogram obtained with the reference solution, locate cells with sinuous anticlinal walls [Ca], diacytic stomata
the components of the reference solution in ±e (2.8.3) [Cb], bearing glandular trichomes with a short
chromatogram obtained with the test solution. unicellular stalk and a globular head composed of
8-16 cells [Cc],-glandular trichomes with a uni- or bicellular
Determine the percentage content of these components. stalk and a bi- or tetracellular head [Cd] and sometimes
The percentages are within the following ranges: covering trichomes; fragments of the lamina, in transverse
limonene: 1.5 per cent to 7.0 per cent, section [D], composed of epidermises bearing glandular
cineole: maximum 1.5 per cent, trichomes with a globular head consisting of 8-16 cells [Da]
menthone: 17.0 per cent to 35.0 per cent, or a bi- or tetracellular head [Db], a 1-layered palisade
mesophyll extending almost halfway across the section [De],
isomenthone: 5.0 per cent to 13.0 per cent,
and a loosely arranged spongy parenchyma [Dd]; fragments
menthyl acetate: 1.5 per cent to 7.0 per cent, of the calyx [G] with an epidermis consisting of polygonal
isopulegol: 1.0 per cent to 3.0 per cent, cells bearing uni- or bicellular conical covering trichomes,
menthol: 30.0 per cent to 50.0 per cent, with spiny walls [Ga], often associated with fusiform
pulegone: maximum 2.5 per cent, mesophyll cells with thick walls and containing small prism
crystals of calcium oxalate [Gb]; isolated glandular
carvone: maximum 2.0 per cent.
trichomes [H], either with a multicellular stalk and a
The ratio of cineole content to limonene content is less unicellular head from the anthers [Ha] or a uni- or
than 1. multicellular stalk and bi- to tetracellular head [Hb];
STORAGE spherical pollen grains, about 25-30 pm in diameter, with
At a temperature not exceeding 25 °C. 3 pores and 3 furrows and a smooth exine [E]; thick-walled,
lignified fibres [F]; fragments from the stem with spirally and
--------------------------------------------------------------------------------------- ------------- --------Ph Eur
annularly thickened vessels [K]; occasional fragments of
pericarp [J] consisting of lobed cells with thick, pitted walls,
each containing a single prism crystal of calcium oxalate [Ja].
2016 Mullein Flower IV-289

Foreign matter (2. ร. 2)
Maximum 2 per cent of brown or yellow leaves and
maximum 2 per cent of other foreign matter.
powdered herbal drug (355) (2.9.12} by drying in an oven at
105 °C for 2 h.
Total ash {2.4.16}
ASSAY
Stock solution In a 100 mL round-bottomed flask place 1.00 g
of the powdered herbal drug (355) (2.9.12}, add 1 mL of a
5 g/L solution of hexamethylenetetramine R, 20 mL of
acetone R and 2 mL of hydrochloric acid Rl. Boil the mixture
under a reflux condenser for 30 min. Filter the liquid
through a plug of absorbent cotton into a flask. Add the
absorbent cotton to the residue in the round-bottomed flask
and extract with 2 quantities, each of 20 mL, of acetone R,
each time boiling under a reflux condenser for 10 min. Allow
to cool and filter each extract through the plug of absorbent
cotton into the flask. After cooling, filter the combined
acetone extracts through a paper filter into a volumetric flask
and dilute to 100.0 mL with acetone R by rinsing the flask
and the paper filter. Introduce 20.0 mL of ±e solution into a
separating funnel, add 20 mL of water R and shake the
mixture with 1 quantity of 15 mL and then 3 quantities, each
of 10 mL, of ethyl acetate R. Combine the ethyl acetate
extracts in a separating funnel, wash with 2 quantities, each
of 50 mL, of พater R, filter the extracts over 10 g of
Figure 1833.-1. - Illustration for identification test B of powdered anhydrous sodium sulfate R into a volumetric flask and dilute
herbal drug of motherwort to 50.0 mL with ethyl acetate R.
Test solution To 0.5 g of the powdered herbal drug (355) Test solution To 10.0 mL of the stock solution add 1 mL of
(2.9.22) add 5 mL of methanol R. Heat on a water-bath at aluminium chloride reagent R and dilute to 25.0 mL with a
65 °C for 5 min with shaking. Cool and filter. 5 per cent vtv solution of glacial acetic acid R in methanol R.
Reference solution Dissolve 5 mg of naphthol yellow ร R and Compensation liquid Dilute 10.0 mL of the stock solution to
2.0 mg of catalpol R in 5.0 mL of methanol R. 25.0 mL with a 5 per cent vtv solution of glacial acetic
Plate TLC silica gel plate R. acid R in methanol R.
Mobile phase glacial acetic acid R, water R} ethyl acetate R Measure the absorbance (2.2.25) of the test solution after
(20:20:60 P7P7F). 30 min, by comparison with the compensation liquid at
Application 20 |1L as bands. 425 nm. Calculate the percentage content of flavonoids,
Development Over a path of 10 cm. calculated as hyperoside, using the following expression:
Drying In air. A X 1.25
Detection Treat with dimethylaminobenzaldehyde solution R2,
using about 5 mL for a plate 200 mm square; heat at
100-105 °C for 10 min until the spots appear; examine in i.e. taking the specific absorbance of hyperoside to be 500.
daylight. A = absorbance at 425 nm;
tn = mass of the substance to be examined, in grams.
chromatograms obtained with the reference solution and the ______________________________________________ _____ _________ PhEur
test solution. Furthermore, other weak greyish-blue zones

solution.
Mullein Flower * *
Top of the plate
A wide white zone
PhEur_____________________________________________________________
A greyish-blue zone (iridoid)
DEFINITION
Dried flower, reduced to the corolla and the androecium, of
Naphthol yellow ระ an intense 1 or 2 greyish-blue zones (iridoid) Verbascum thapsus L., V. densiflorutn Bertol. (F. thapsiforme
yellow zone Schrad), and V. phlomoides L.
Catalpol: a greyish-blue zone IDENTIFICATION
A. The corolla of V. thapsus is pale yellow, yellow or brown,
funnel-shaped, about 20 mm in diameter, with 5 slightly
unequal and spreading lobes. The corolla lobes are densely
IV-290 Mullein Flower 2016
hairy on the outer surface, glabrous on the inner surface, c. Thin-layer chromatography (2.2.27).
with a fine network of light brown veins. There are Test solution Heat 1.0 g of the powdered herbal drug (355)
5 stamens, alternating with the petal lobes; 2 of these are (2.9.72) in 10 mL of methanol R in a water-bath at 60 c for
long, with glabrous filaments, the other 3 shorter, with 5 min, with stirring. Cool and filter.
densely tomentose filaments. The anthers are attached
Reference solution Dissolve 1 mg of caffeic acid R, 2.5 mg of
transversely. In V. phlomoides the corolla is up to about hyperoside R and 2.5 mg of rutin R in methanol R and dilute
30 mm in diameter, bright yellow or orange, and the anthers
to 10 mL with the same solvent.
are obliquely attached to the filaments. The corolla of
V. densiflorum, about 30 mm in diameter, is almost flat and Plate TLC silica gel plate R.
deeply divided into 5 slightly unequal lobes, with rounded Mobile phase anhydrous formic acid R, water R} methyl ethyl
apices. ketone R, ethyl acetate R (10:10:30:50 VI VIVI V).
B. Reduce to a powder (355) (2.9.72). The powder is yellow Application 10 J1L of the reference solution and 30 pL of the
or yellowish-brown. Examine under a microscope using test solution, as bands.
chloral hydrate solution R. The powder shows ±e following Development Over a path of 15 cm.
diagnostic characters (Figure 1853.-1): many covering Drying At 100-105 °C.
trichomes from the corolla, whole and fragmented, Detection Spray the warm plate with a 10 gL solution of
pluricellular, of the candelabra type, with a central uniseriate diphenylboric acid aminoethyl ester R in methanol R) then with a
axis from which whorls of branch cells arise at the position of 50 g/L solution of macrogol 400 R in methanol Ri allow to dry
the cross walls and at the apex, in side view [A, B] or in in air for 30 min and examine in ultraviolet light at 365 nm.
surface view [F]; the covering trichomes from the stamen
filaments [G] are unicellular, long, thin-walled and tubular, Results See below the sequence of zones present in the
have a distinctly granular or striated surface with a sharp tip
[Ga] or sometimes with a club-shaped tip [Gb, Gc];
numerous pollen grains, ovoid with a finely granular exine in the chromatogram obtained with the test solution.
with 3 pores [D]; fragments of the fibrous layer of the anther Top of the plate
with thickened walls giving a characteristic star-shaped
A yellow or yellowish-green
appearance [C]; yellow fragments of the petals, in surface fluorescent zone
view [E], the epidermal cells polygonal and isodiametric [Ea]; Caffeic acid: a greenish-blue
fragments of the underlying mesophyll consisting of irregular fluorescent zone
parenchymatous cells [Eb] sometimes accompanied by spiral A bluish fluorescent zone
vessels [Ec]. A greenish fluorescent zone
A bluish fluorescent zone
Hyperoside: a yellowish-brown
fluorescent zone
A greenish fluorescent zone
Rutin: a yellowish-brown
fluorescent zone
D. Boil 1.0 g of the powdered herbal drug (355) (2.9.72)

with 15 mL of water R for 1 min. Filter. Add 1 mL of
hydrochloric acid R and boil for 1 min. A greenish-blue colour
develops and, after a few minutes, cloudiness appears and
then a blackish precipitate (iridoids).
TESTS
Maximum 5 per cent of brown petals and maximum
2 per cent of fragments of the calyx and other foreign matter,
determined on 20 g.
(2.9.72), moistened with 2 mL of ethanol (96 per cent) R.
105 °C for 2 h.
Total ash (2.4.16)
herbal drug of mullein flower
STORAGE
In an airtight container.
PnEif
2016 Myrrh Preparations IV-291
Myrrh ***** Results The chromatogram obtained with the test solution
shows no blue or violet fluorescent zones in the lower third
(Ph- Eur. monograph 1349) * ** of the chromatogram.
Preparation Matter insoluble in ethanol
Myrrh Tincture Maximum 70 per cent.
Ph Elf________________ ____________ Place 1.00 g of the powdered herbal drug (250) (2.9.12) in a
flask. Add 30 mL of ethanol (96 per cent) R and shake
DEFINITION
vigorously for 10 min. Filter the supernatant through a tared
Gum-resin, hardened in air, obtained by incision or produced sintered-glass filter (16) (2.1.2) avoiding the transfer of
by spontaneous exudation from the stem and branches of sediment from the flask. Repeat the extraction with
Commiphora molmol Engler and/or other species of 2 quantities, each of 20 mL, of ethanol (96 per cent) R.
Commiphora.
Quantitatively transfer the sediment to the filter by rinsing
CHARACTERS the flask with ethanol (96 per cent) R. Dry the filter and the
Bitter taste. residue in an oven at 100-105 °C and weigh.
IDENTIFICATION Loss on drying (2.2.22)
A. The light or dark orange-brown, irregular or roundish Maximum 15.0 per cent, determined on 1.000 g of the
grains or pieces of different size show components of various powdered herbal drug (355) (2.9.12) by drying in an oven at
colours. Their surface is mostly covered with grey or 105 °C for 2 h.
yellowish-brown dust. Total ash (2.4.16)
B. Reduce to a powder (355) (2.9.12). The powder is Maximum 7.0 per cent.
brownish-yellow or reddish-brown. Examine under a __________________________ ______ ___________________________ Ph Eur
microscope, using chloral hydrate solution R. The pow’der
shows the following diagnostic characters: a few tissue
fragments from the original plants including: reddish-brown
cork fragments; single or grouped polyhedral or elongated
stone cells with partly strongly thickened, pitted and lignified Myrrh Tincture ** X
walls with a brownish content; fragments of thin-walled (Ph. Eur. monograph 1877) ***
parenchyma and sclerenchymatous fibres; irregular prismatic
Ph Elf_________________ _ _________________________________________
or polyhedral crystals of calcium oxalate, about 10-25 pm in
size. DEFINITION
c. Examine the chromatograms obtained in the test for Tincture produced from Myrrh (1349).
Commiphora niukul. PRODUCTION
Detection Spray with anisaldehyde solution R, and examine in The tincture is produced from 1 part of the drug and 5 parts
daylight while heating at 100-105 °C for 10 min. of ethanol (90 per cent VIV) by a suitable procedure.
Results The chromatogram obtained with the reference CHARACTERS
solution shows in the lower third an orange-red zone
Clear yellowish-brown or orange-brown liquid.
(thymol) and in the middle third a violet zone (anethole).
The chromatogram obtained with the test solution shows an IDENTIFICATION
intense violet zone (furanoeudesma-l,3-diene), exceeding the Thin-layer chromatography (2.2.27).
other zones in size and intensity, above the zone of anethole Test solution Dilute 5 mL of the tincture to be examined to
in the chromatogram obtained with the reference solution; 10 mL with alcohol R.
a violet zone similar in position to the zone of anethole in the Reference solution Dissolve 10 mg of thymol R and 40 pL of
chromatogram obtained with the reference solution; 2 intense anethole R in 10 mL of ether R.
violet zones similar in position to the zone of thymol in the
chromatogram obtained with the reference solution, the
upper one due to curzerenone and the lower one to Mobile phase ethyl acetate R, toluene R (2:98 VIV).
2-methoxyfuranodiene. Further mostly violet zones are Application 10 pL, as bands.
present in the chromatogram obtained with the test solution. Development Over a path of 15 cm.
Commiphora mukul Detection spray with anisaldehyde solution R and examine in
Thin-layer chromatography (2.2.27). daylight whilst heating at 100-105 °C for 10 min.
Test solution To 0.5 g of the powdered herbal drug (355) Results See below the sequence of the zones present in the
(2.9.12) add 5.0 mL of ethanol (96 per cent) R and warm the chromatograms obtained with the reference solution and the
mixture on a water-bath for 2-3 min. Cool and filter. test solution. Furthermore, other zones mostly violet, are
Reference solution Dissolve 10 mg of thymol R and 40 |1L of present in the chromatogram obtained with the test solution.
anethole R in 10 mL of ethanol (96 per cent) R.
Mobile phase ethyl acetate R, toluene R (2:98 V/V).
Drying In air.
Detection Examine in น]traviolet light at 365 nm.
IV-292 Niaouli Oil, Cineole Type 2016
Top of the plate Top of the plate

An intense violet zone exceeding
the others in size and intensity
(furanoeudesma-l,3-diene) A faint grey zone
Anethole: a violet zone A violet zone
Thymol ะ an orange-red zone Two intense violet zones

(curzerenone and below A purple zone
2-methoxyfuranodiene)
1,8-Cineole: a violet-brown zone An intense violet-brown zone
(1,8-cineole)
TESTS trans-Nerolidol: a dark violet

zone
Ethanol content (2.9.10)
82 per cent V/V to 88 per cent PZE
An intense violet-brown zone
Methanol and 2-propanol (2.9.11)
Maximum 0.05 per cent VIV of methanol and maximum A violet-brown zone
0.05 per cent VIV of 2-propanol.
Minimum 4.0 per cent inIm.
STORAGE B. Examine the chromatograms obtained in die test for
Plastic containers are not recommended. chromatographic profile.
-----------------------------------------------------------------------------------------------------------Ph Eur
those in the chromatogram obtained with reference
solution (a).
TESTS
Cineole Type Niaouli Oil Relative density (2.2.5)
(Ph Eur monograph 2468) 0.904 to 0.925.
Ph Ecr___________________________________
1.463 to 1 472.
DEFINITION
Essential oil obtained by steam distillation from young leafy -4° to T 1°.
branches of Melaleuca quinquenervia (Cav.) S.T.Blake.
Methyleugenol and isomethyleugenol
CHARACTERS Gas chromatography (2.2.28) as described in the test for
Appearance chromatographic profile with the following modifications.
Colourless or pale yellow liquid. Reference solution Dissolve 5 pL of methyleugenol R and 5 pL
Aromatic odour of cineole. of isomethyleugenol R in heptane R and dilute to 50.0 mL with
IDENTIFICATION the same solvent. Dilute 0.5 mL of the solution to 5.0 mL
First identification'. B. with heptane R.
Second identification A. Elution order Order indicated in the composition of the
reference solution; record the retention times of
A.. Thin-layer chromatography (2.2.27).
methyleugenol and isomethyleugenol.
Test solution Dissolve 100 pL of the essential oil to be
Identification of peaks Using the retention times determined
examined in toluene R and dilute to 10.0 mL with the same
from the chromatogram obtained with the reference solution,
solvent.
locate the components of the reference solution in the
Reference solution Dissolve 25 pL of trans-nerolidol R and chromatogram obtained with the test solution.
50 pL of cineole R in toluene R and dilute to 5.0 mL with the
Limits:
same solvent.
— methyleugenol: maximum 0.05 per cent;
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel — isomethyleugenol: maximum 0.05 per cent.
plate R (2-10 pm)].
Mobile phase ethyl acetate R, toluene R (5:95 VIV). Gas chromatography (2.2.28): use the normalisation
Application 10 pL [or 2 pL] as bands of 10 mm [or 8 mm]. procedure.
Development Over a path of 15 cm [or 6 cm]. Test solution Dilute 0.2 mL of the essential oil to be examined
Drying In air. to 10.0 mL with heptane R.
Detection Treat with anisaldehyde solution R and heat at Reference solution (a) Dilute 10 pL of ช-pinene R} 5 pL of
100-105 °C for 3 min; examine in daylight. fi-pinene Ri 10 pL of limonene R, 50 pL of cineole Ri 5 pL of
Results See below the sequence of zones present in the p-cymene Ri 5 pL of benzaldehyde Ri 5 mg of ช-terpineol R and
chromatograms obtained with the reference solution and the 5 pL of trans-nerolidol R in heptane R and dilute to 10 mL
test solution. Furthermore, other faint zones may be present with the same solvent.
in the chromatogram obtained with the test solution. Reference solution (b) Dissolve 5 pL of limonene R in heptane R
and dilute to 50.0 mL with the same solvent. Dilute 0.5 mL
of the solution to 5.0 mL with heptane R.
2016 Neroli Oil IV-293
Column:
CHARACTERS
— material: fused silica; Appearance
size: I = 60 โท, 0 = 0.25 mm; Clear, pale-yellow or dark-yellow liquid.
stationary phase: niacrogol 20 000 R (film thickness
0.25 pm). Characteristic odour.
Carrier gas helium for chromatography R. IDENTIFICATION
Flow rate 1.3 mUmin. First identification B.
split ratio 1:50. Second identification A.
Temperature: A. Examine the chromatograms obtained in the test for
bergapten.
Time Temperature chromatograms obtained with the reference solution and the
(°C) test solution. Furthermore other zones may be present in the
Column 0-5 65
chromatograms obtained with the test solution.
5 - 65 65 -> 185
65 - 80 185 230 Top of the plate

Injection port 230
Detector 250 Methyl anthranilate: a blue A faint blue fluorescent zone
fluorescent zone (methyl anthranilate)

Injection 1 pL. Bergapten: a greenish-yellow
fluorescent zone
Elution order Order indicated in the composition of reference —
solution (a); record the retention times of these substances.
Identification of peaks Using the retention times determined
from the chromatogram obtained with reference solution (a),
locate the components of reference solution (a) in the Detection B Spray with anisaldehyde solution R-, heat at
chromatogram obtained with the test solution; the peak due 100-105 °C for 10 min; examine the chromatograms in
to vindiflorol elutes with a relative retention of about 1.02 ultraviolet light at 365 nm.
พํth reference to mnzs-nerolidol.
System suitability: reference solution (a): chromatograms obtained with the reference solution and the
— resolution: minimum 1.5 between the peaks due to test solution. In the chromatogram obtained with the test
limonene and 1,8-cineole. solution the zone due to linalol is more intense than the zone
Determine the percentage content of each of the following due to linalyl acetate.
— a-pinene: 5.0 per cent to 15.0 per cent;
Top of the plate
— fl-pinene: 1.0 per cent to 4.0 per cent;
— limonene: 5.0 per cent to 10.0 per cent; A brown fluorescent zone
— 1,8-cineole: 45.0 per cent to 65.0 per cent; Linalyl acetate: a brownish-red An intense brownish-red
— 0.05 per cent to 4.0 per cent; fluorescent zone fluorescent zone (linalyl acetate)
— benzaldehyde: 0.05 per cent to 0.5 per cent;
— a-terpineol: 3.0 per cent to 8.0 per cent;
Methyl anthranilate: a blue A faint blue fluorescent zone
— trans-nerolidol: 0.05 per cent to 1.5 per cent; fluorescent zone (methyl anthranilate)
— viridiflorol: 2.5 per cent to 9.0 per cent; A faint brownish-red fluorescent
chromatogram obtained with reference Linalol: a brownish-red A brownish-red fluorescent zone
solution (b) (0.05 per cent). fluorescent zone (linalol)
Bergapten: a greenish-yellow
STORAGE fluorescent zone
___ —_________________________________________________________ Ph Eur
Several blue and brownish-red
fluorescent zones
Neroli Oil ** * B. Examine the chromatograms obtained in the test for

(Ph. Eur. monograph 1175) *** chromatographic profile.
Results The principal peaks in the chromatogram obtained
PhEa _________________________________________________________ _____
with the test solution are similar in retention time to the
DEFINITION principal peaks in the chromatogram obtained with the
Neroli oil is obtained by steam distillation from the fresh reference solution.
flowers of Citrus aurantium L. subsp. aurantium L. TESTS
(C. aurantium L. subsp. amara Engl.). Relative density (2.2.5)
0.863 to 0.880.
IV-294 Neroli Oil 2016
Refractive index (2.2.6) System suitability: reference solution (a):

1.464 to 1.474. — resolution: minimum 1.5 between the peaks due to
Optical rotation (2.2.7) p-pinene and sabinene.
Acid value (2.5./) chromatogram obtained with reference solution (a), locate
Maximum 2.0. the components of reference solution (a) in the
Bergapten
Thin-layer chromatography (2.2.27). Limits:
Test solution Dissolve 0.1 g of the substance to be examined — limonene: 9.0 per cent to 18.0 per cent,
in ethanol (96 per cent) R and dilute to 5.0 mL with the same — linalol: 28.0 per cent to 44.0 per cent,
solvent. — linalyl acetate: 2.0 per cent to 15.0 per cent,
Reference solution Dissolve 2 pL of methyl anthranilate R, — tt-terpineol: 2.0 per cent to 5.5 per cent,
10 pL of linalyl acetate R3 20 pL of linalol R and 5 mg of — neryl acetate: maximum 2.5 per cent,
bergapten R in ethanol (96 per cent) R and dilute to 10.0 mL — geranyl acetate: 1.0 per cent to 5.0 per cent,
with the same solvent. — trans-nerolidol: 1.0 per cent to 5.0 per cent,
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel — methyl anthranilate: 0.1 per cent to 1.0 per cent,
plate R (2-10 pm)]. — (E3E)-farnesol: 0.8 per cent to 4.0 per cent,
Mobile phase ethyl acetate R3 toluene 1? (15:85 VIV). — disregard limit: area of the peak in the chromatogram
Application 10 pL [or 2 pL] as bands. obtained with reference solution (b).
Development Over a path of 15 cm [or 8 cm]. Chiral purity
Gas chromatography (2.2.25).
Drying In air.
Test solution Dissolve 20 mg of the substance to be examined
in pentane R and dilute to 10.0 mL with the same solvent.
Results The chromatogram obtained with the test solution Reference solution To 10 pL of linalol R add 10 pL of linalyl
does not show a zone corresponding to the zone due to
acetate R. Dilute to 10.0 mL with pentane R.
bergapten in ±e chromatogram obtained with the reference
Column:
solution.
Chromatographic profile — size: l ะ= 25 m, 0 — 0.25 mm,
Gas chromatography (2.2.25): use the normalisation — stationary phase: modified p-cyclodextrin for chiral
procedure. chromatography R (film thickness 0.25 pm).
Test solution The substance to be examined. Carrier gas helium for chromatography R.
Reference solution (a) Dissolve 20 pL of P-pinene R3 5 mg of Flow rate 1.3 mUmin.
sabinene R3 40 pL of limonene R3 40 pL of linalol R3 20 pL of
Split ratio 1:30.
linalyl acetate R3 5 mg of a-terpineol R3 5 pL of neryl acetate R3
Temperature:
5 pL of geranyl acetate R3 5 pL of trans-nerolidol R, 5 pL of
methyl anthranilate R and 5 pL (E3E)-farnesol R in 2 mL of Time Temperature
heptane R. (min) co_________
Reference solution (b) Dissolve 5 pL of methyl anthranilate R in Column 0 - 65 50 -> 180
heptane R and dilute to 10 mL with the same solvent. Injection port 230
Column’.
Detector 230
— size’. I = 60 โท3 0 = 0.25 mm,
— stationary phase: macrogol 20 000 R (film thickness
0.25 pm). Injection 1 pL.
รุys tern suitability: reference solution:
Flow rate 1.5 mL/min. (R)(-)-linalol (1st peak) and (ร)(+)-linalol (2nd peak);
Split ratio 1:100. minimum 2.7 between the peaks due to (R)(-)-linalyl
Temperature: acetate (3rd peak) and (ร)(+)-linalyl acetate 4th peak).
Calculate the percentage content of the specified
Time Temperature (ร)-enantiomers from the following expression:
(min) CC)
Column 0-4
4 - 42.8 75 -> 230
42.8 - 63 230
Injection port 270 A1 = area of the corresponding (ร)-enantiomer,
Detector 270 A2 = area of the corresponding (R)-enantiomer.
Limits:
— (ร)(+)-linalol: maximum 30 per cent,
— (ร) (+)-linalyl acetate: maximum 5 per cent.
Injection 0.2 pL.
STORAGE
PtiEir
2016 Nettle Leaf IV-295
Nettle Leaf ***** small glandular trichomes [F] (35-65 pm), with a uni- or
bicellular stalk and a bi- or quadricellular head, isolated [Fa],
(Ph. Eur. monograph 1897) **★* or on fragments of the epidermis [Fb]; fragments of the
upper epidermis of the leaves in surface view [G] or in
transverse section [D] showing slightly sinuous cells [Da,
definition Gc], unicellular, straight or slightly curved covering
Whole or cut dried leaves of บทica dioica L., Unica urens L., trichomes, enlarged at the base, up to 700 pm long [De, Ga]
or a mixture of the 2 species. and abundant large cystoliths [Db, Ea, Gb], empty or
Content containing dense, granular masses of calcium carbonate;
Minimum 0.3 per cent for the sum of caffeoylmalic acid and palisade parenchyma in surface view [E], with rounded cells
chlorogenic acid, expressed as chlorogenic acid (C]6H18O9; [Eb] surrounding cystoliths [Ea], or in transverse section
354.3) (dried drug). [Dd]; fragments of lower epidermis of leaves showing sinuous
IDENTIFICATION or wavy-walled cells [H], anomocytic [Ha] or anisocytic
stomata [Hb] (2.8.3) accompanied by spongy mesophyll in
A- The leaves are dark green, dark greyish-green or
surface view [He] and in transverse section [De] containing
brownish-green on the upper surface, paler on the lower
small cluster crystals of calcium oxalate in surface view [Hd]
surface; scattered stinging hairs occur on both surfaces, also
and in transverse section [Df]; occasional small groups of
small covering trichomes that are more numerous along the
vessels, accompanied by parenchyma containing cluster
margins and on the veins on the lower surface. The lamina is
crystals of calcium oxalate [J].
strongly shrunken, ovate or oblong, up to 100 mm long and
50 mm wide, with a coarsely serrate margin and a cordate or c. Thin-layer chromatography (2.2.27).
rounded base. The venation is reticulate and distinctly Test solution To 1 g of the powdered herbal drug (355)
prominent on the lower surface. The petiole is green or (2.9.12) add 10 mL of methanol R. Boil under a reflux
brownish-green, rounded or flattened, about 1 mm wide, condenser for 15 min. Cool and filter. Evaporate to dryness
longitudinally furrowed and twisted; it bears stinging hairs in vacuo at 40 °C. Dissolve the residue in 2 mL of
and covering trichomes. methanol R.
Reference solution Dissolve 1 mg of scopoletin R and 2 mg of
chlorogenic acid R in 20 mL of methanol R.
plate R (2-10 pm)].
Mobile phase anhydrous formic acid R, methanol R, water R)
ethyl acetate R (2.5:4:4:50 VIVIVIV).
Drying In air.
Detection Heat at 100 °C for 5 min; spray the sdll-warm plate
in methanol R-, examine in ultraviolet light at 365 nm.
test solution. Furthermore, other faint blue or yellow
fluorescent zones may be present in the lower half of the
Top of the plate
Scopoletin: an intense blue A blue fluorescent zone

fluorescent zone (scopoletin)
Chlorogenic acid: a blue A blue fluorescent zone

Figure 1897.-1. - Illustration for identification test B of powdered A brownish-yellow zone
herbal drug of nettle leaf
B. Reduce to a powder (355) (2.9.12). The powder is green
or greyish-green. Examine under a microscope using chloral
TESTS
characters (Figure 1897.-1): fragments of unicellular stinging
hairs [A, B, C], up to 2 mm long, composed of an elongated Maximum 5 per cent of stems and maximum 5 per cent of
tapering cell with a slightly swollen stinging tip that readily other foreign matter (including inflorescences).
breaks off, arising from a raised, multicellular base [Ca];
IV-296 Nettle Root 2016

Maximum 12.0 per cent, determined on 1.000 g of the Nettle Root t \
powdered herbal drug (355) (2.9.72) by drying in an oven at (Ph. Eur. monograph 2538) * **
105 °C for 2 h.
Ph Eur______________________________________________________________
Total ash {2.4.16)
Maximum 20.0 per cent. DEFINITION
Ash insoluble in hydrochloric acid {2.8.1) Dried, whole or fragmented underground parts of Urtica
Maximum 4.0 per cent. dioica L. or Urtica urens L., their hybrids or their mixtures.
ASSAY IDENTIFICATION
Liquid chromatography (2.2.29). A. Irregular cylindrical rhizome, greyish-brown, sporadically
purple or greenish, 3-10 mm in diameter, the internodes are
up to 2-3 cm long, with deep longitudinal furrows,
(2.9.72) add 25.0 mL of a 40 per cent VIV solution of
alternating with short, slightly swollen nodes; the nodes bear
methanol R. Extract for 30 min in an ultrasonic bath at 40 °C
numerous roots that are externally greyish-brown, 0.5-2 mm
and filter.
in diameter and up to about 10 cm long. The transverse
Reference solution Dissolve 10.0 mg of chlorogenic acid CRS in section shows a thin, dark bark, the inner part is creamish-
100.0 mL of a 40 per cent P7K solution of methanol R. white with a hollow centre. The fracture of the rhizome is
Dilute 5.0 mL of this solution to 25.0 mL with a fibrous and uneven, especially in the inner part.
40 per cent VIV solution of methanol R.
B. Microscopic examination {2.8.23). The powder is pale
Precolumn'.
characters: fragments of brownish cork with thin-walled cells;
fragments of vessels with bordered pits, 50-150 pm in
Column: diameter; fragments of fibres with thick lignified walls,
— size: 7 = 0.125 m, 0 = 4 mm; occurring singly or in groups; abundant fragments of
— stationary phase: end-capped octadecylsilyl silica gel for parenchyma with thin-walled cells, some containing large
chromatography R {5 pm);
cluster crystals of calcium oxalate; rare, scattered, simple
— temperature: 25 °C. crystals of calcium oxalate; fragments of the medullary rays
Mobile phase: with moderately thick-walled, pitted cells.
— mobile phase A: mix 15 volumes of methanol R and
85 volumes of water R and adjust to pH 2.0 with dilute
c. Thin-layer chromatography {2.2.27).
phosphoric add R'}
(2.9.72) add 10 mL of methanol R. Sonicate for 10 min and
— mobile phase B: methanol R’y
filter. Evaporate the filtrate to dryness and dissolve the
Time Mobile phase A Mobile phase B residue in 2 mL of methanol R.
(min) (per cent V/V) (per cent V/V) Reference solution Dissolve 1 mg of scopoletin R in 10 mL of
0 - 1 100 0 methanol R (solution A). Dissolve 10 mg of arbutin R and
1 - 25 100 -> 85 0 15 20 mg of P-sitosterol R in methanol R, add 1 mL of solution A
and dilute to 10 mL with methanol R.
25 - 35 85 15
35 - 36 85 -> 0 15 4 100 F254 plate R (2-10 pm)].
Mobile phase anhydrous formic add Rj methanol R, water R)
Flow rate 1 mUmin.
ethyl acetate R (2.5:4:4:50 VIVIVIV).
Injection 20 pL.
Relative retention With reference to chlorogenic acid (retention
time = about 13 min): caffeoylmalic acid = about 2.2. Drying In air for 5 min.
Calculate the percentage content of caffeoylmalic acid and Detection A Examine in ultraviolet light at 365 nm.
chlorogenic acid, expressed as chlorogenic acid, using the Results A See below the sequence of zones present in the
following expression: chromatograms obtained with the reference solution and the
test solution. Futhermore, other fluorescent zones may be
Al X 7712 X p
A-2 X 7711 X 20
Top of the plate
A1 = sum of the areas of the peaks due to caffeoylmalic
acid and chlorogenic acid in the chromatogram A blue or greenish-blue
fluorescent zone
chromatogram obtained with the reference Scopoletin: a blue fluorescent A blue fluorescent zone
solution; (scopoletin)

m2 = mass of chlorogenic add CRS used to prepare the
reference solution, in grams; Arbutin: a greenish-brown zone
p ะ= percentage content of chlorogenic acid in
chlorogenic acid CRS.
Ph Eur
2016 Notoginseng Root IV-297
B Treat with anisaldehyde solution R and heat at is surrounded by warty protuberances at the crown.
100-105 c for 5-10 min; examine in daylight. The texture of the root is compact. The fracture is smooth,
B See below the sequence of zones present in the shiny, brownish-grey and shows a yellowish-grey ring
chromatograms obtained with the reference solution and the (cambial zone) and many radial striations.
test solution. Furthermore, other zones may be present in the B. Reduce to a powder (355) (2.9.12). The powder is light
chromatogram obtained with the test solution. yellowish-grey. Examine under a microscope using chloral
characters: abundant fragments of thin-walled
__ _______ Top of the plate
parenchymatous cells; fragments of secretory canals
A purple zone containing yellowish-brown resin; rare lignified vessels about
30 pm in diameter, reticulate or pitted; rare cork fragments.
^-sitosterol: a purple zone A purple zone (0-sitosterol) solution of glycerol R. The starch granules, often deformed,
are very abundant, single or in groups of 2-3, and 1-10 pm
in diameter.
A faint purple zone
c. Examine the chromatogram obtained in the test for Panax
ginseng or Panax quinquefolium.
Reference solution Test solution test solution. Furthermore, other faint zones may be present
TESTS
Loss on drying (2.2.32) Top of the plate
Maximum 12.0 per cent, determined on 1.000 g of the A violet zone (at the solvent front)
105 °C for 2 h.
Lead (2.4.27) Arbutin: a brown zone
Maximum 7.0 ppm.
Total ash (2.4.16)
A violet zone (ginsenosides Rgl
Maximum 12.0 per cent. + Rg2)
Maximum 4.0 per cent. 2 faint violet zones
Extractable matter
Aescin ะ a grey zone
To 2.000 g of the powdered herbal drug (250) (2.9.12) add a
mixture of 6 g of water R and 14 g of ethanol (96 per cent) R Several violet and greenish zones
and extract for 2 h, shaking frequently. Filter, evaporate
Reference solution Test solation
5.000 g of the filtrate to dryness on a water-bath and dr}' in
an oven at 105 °C for 2 h. The residue weighs a minimum of
35 mg.
TESTS
--- ------------------------------------------------------------------------------------------------------- Ph Eur Panax ginseng or Panax quinquefoliunt
(2.9.12) add 10 mL of a 70 per cent VIV solution of
Notoginseng Root ** * methanol R and boil under a reflux condenser for 15 min.
Filter after cooling and dilute to 10.0 mL with methanol R.
(Ph. Eur. monograph 2383) *** Reference solution Dissolve 5.0 mg of aescin R and 5.0 mg of
arbutin R in 1 mL of methanol R.
DEFINITION Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
Whole or fragmented taproot, without secondary roots, of plate R (2-10 pm)].
Panax pseudoginseng Wall. var. notoginseng (Burk.) Hoo et Mobile phase ethyl acetate R, water Ry butanol R
Tseng [Panax notoginseng (Burk.) F.H. Chen ex C.Y. พน et (25:50:100 VIV/V)y allow to stand for 10 min and use the
K.M. Feng] treated with steam and dried. upper layer.
Content Application 20 pL, as bands of 15 mm [or 4 pL of the test
Minimum 3.8 per cent for the sum of ginsenosides Rgl solution and 2 pL of the reference solution, as bands of
(C42H72o 14,2H2O; 837) and Rbl (C54H92O23,3H2O; 8 mm].
Afr 1163) (dried drug). Development In an unsaturated tank, over a path of 10 cm [or
5 cm].
IDENTIFICATION
A. The primary root is conical, subconical or cylindrical, up Drying In air for 30 min.
to 6 cm long and 4 cm in diameter. The outer surface, Detection spray with anisaldehyde solution R and heat at
showing shallow transverse striations and secondary root 105-110 °C for 5-10 min; examine in daylight.
scars, is brownish-grey or yellowish-grey. The aerial stem scar
IV-298 Nutmeg Oil 2016
Results In the chromatogram obtained with the test solution, Calculate the sum of the percentage contents of
the absence of a violet zone immediately above the zone due ginsenosides Rbl and Rgl using the following expression:
to arbutin in the chromatogram obtained with the reference
solution suggests the presence of Panax ginsengy in the A\ X 7712 X 2 X Pl A2 X 7ท3 X 2 X P2
chromatogram obtained with the test solution, the presence mi X A3 mi X A4
of a brown zone immediately below’ the violet zone due to
the ginsenosides Rgl -r Rg2 suggests the presence of Panax A1 = area of the peak due to ginsenoside Rbl in the
quinquefolium. chromatogram obtained with the test solution;
Loss on drying (2.2.32) A2 = area of the peak due to ginsenoside Rgl in the
Maximum 12.0 per cent, determined on 1.000 g of the chromatogram obtained with the test solution;
powdered herbal drug (355) (2.9.12) by drying in an oven at A3 = area of the peak due to ginsenoside Rbl in the
105 °C for 2 h. • chromatogram obtained with the reference
Total ash (2.4.16) solution;
Maximum 6.0 per cent. A4 = area of the peak due to ginsenoside Rgl in the
Ash insoluble in hydrochloric acid (2.8.1) solution;
Maximum 1.0 per cent. ไท1 = mass of the dried drug to be examined, in grams;
ASSAY ไท2 = mass of ginsenoside Rbl R in the reference
Liquid chromatography (2.2.29). solution, in grams;
ไท3 = mass of ginsenoside Rgl R in the reference
Test solution Reduce about 50 g to a powder (355) (2.9.12).
Place 0.250 g of the powdered herbal drug and 70 mL of a solution, in grams;
50 per cent VIV solution of methanol R in a 250 mL round- Pl = percentage content of ginsenoside Rbl in
bottomed flask. After adding a few' grains of pumice, boil on ginsenoside Rbl R-y
a water-bath under a reflux condenser for 1 h. After cooling, p2 = percentage content of ginsenoside Rgl in
centrifuge and collect the supernatant. Treat ±e residue as ginsenoside Rgl R.
described above. Mix the collected liquids and evaporate to _______________________________________________________ _______ Ph Elf
dryness under reduced pressure at a temperature not
exceeding 60 °C. Take up the residue with 10.0 mL of a
buffer solution, adjusted to pH 4.5, containing 3.5 g of
sodium dihydrogen phosphate R and 7.2 g of potassium
dihydrogen phosphate R in 1000 mL of water R (solution A). Nutmeg Oil
Wash a cartridge containing about 0.36 g of octadecylsilyl (Ph. Eur. monograph 1552)
silica gel for chromatography R with 5 mL of methanol R
Ph Eur________________________
followed by 20 mL of water for chromatography R. Apply
5.0 mL of solution A to ±e cartridge. Elute with 20 mL of DEFINITION
water for chromatography Ry followed by 15 mL of a Essential oil obtained by steam distillation of the dried and
30 per cent VIV solution of methanol R. Discard the eluates crushed kernels of Myristica fragrans Houtt.
after confirming that no ginsenosides are present, otherwise
CHARACTERS
repeat the assay with another type of cartridge. Elute the
Appearance
cartridge with 20 mL of methanol R and evaporate the eluate
Colourless or pale yellow liquid.
to dryness. Take up the residue with 5.0 mL of methanol R.
Spicy odour.
Reference solution Dissolve 3.0 mg of ginsenoside Rbl Ry
3.0 mg of ginsenoside Rgl R and 3.0 mg of ginsenoside Rf R in IDENTIFICATION
methanol R and dilute to 5.0 mL with the same solvent. First identification B
Column: Second identification A
— size: I = 0.10 m, 0 = 4.6 mm; A. Thin-layer chromatography (2.2.27).
— stationary phase: aminopropylsilyl silica gel for
Test solution Dissolve 1 mL of the substance to be examined
in toluene R and dilute to 10 mL with the same solvent.
Mobile phase:
Reference solution Dissolve 20 pL of myristicine R in 10 mL of
— mobile phase A: acetonitrile Ry
toluene R.
— mobile phase B: water for chromatography Ry
Mobile phase ethyl acetate Ry toluene R (5:95 VIV).
(min) (per cent V/V) (per cent V/V) Application 10 |1L as bands.
0 - 14 90 10 Development Over a path of 15 cm.
14 - 18 90-» 80 10->20 Drying In air.
18-55 80 20 Detection Spray with vanillin reagent Ry heat at 100-105 °C for
10 min and examine in daylight.
Flow rate 2 mL/min.
solution shows in the upper third a pink or reddish-brown
Detection Spectrophotometer at 203 nm. zone (myristicine); the chromatogram obtained with the test
Injection 20 pL. solution shows a series of zones of which 1 is similar in
System suitability: reference solution: position and colour to the zone in the chromatogram
— resolution: minimum 3.0 between the peaks due to obtained with the reference solution; above this zone a
ginsenosides Rf and Rgl. brownish zone (safrole) and a violet zone (hydrocarbons) are
2016 Oak Bark IV-299
present; below the myristicine zone, 5 blue zones of variable — y-terpinene: 2.0 per cent to 6.0 per cent;
intensity are present. — terpinen-4-ol: 2.0 per cent to 6.0 per cent;
B. Examine the chromatograms obtained in the test for — safrole: maximum 2.5 per cent;
chromatographic profile. — myristicine: 5.0 per cent to 12.0 per cent.
The principal peaks in the chromatogram obtained STORAGE
test s°lut*on are similar in retention time to those in Protected from heat.
___________________________________________________________ Ph Eur
TESTS
0.885 to 0.905.
475
1. to 1.485.
Oak Bark * *
Optical rotation (2.2.7) (Ph. Eur. monograph 1887) ***
+ 8° to + 18°. Ph Eur____________________________________________________________
Chromatographic profile DEFINITION

Gas chromatography (2.2.25): use the normalisation Cut and dried bark from the fresh young branches of Quercus
procedure. robur L., Q. petraea (Matt.) Liebl. and Q. pubescens Wind.
Test solution The substance to be examined. Content
Reference solution Dissolve 15 pL of d-pinene R, 15 pL of Minimum 3.0 per cent of tannins, expressed as pyrogallol
fi-pinene Ry 15 pL of sabinene Ry 5 pL of car-3-ene Ry 5 pL of (C6H6O3; Afr 126.1) (dried drug).
limonene Ry 5 pL of y-terpinene Ry 5 pL of terpinen-4-ol Ry
IDENTIFICATION
5 pL of safrole R and 10 pL of myristicine R in 1 mL of
hexane R. A. The bark occurs in channelled or quilled pieces, not more
than 3 mm thick. The outer surface is light grey or greenish-
Column:
grey, rather smooth, with occasional lenticels. The inner
surface is dull brown or reddish-brown and has slightly raised
size: I = 25-60 m, 0 = about 0.3 mm;
longitudinal striations about 0.5-1 mm wide. The fracture is
— stationary phase: bonded macrogol 20 000 R.
splintery and fibrous.
Carrier gas helium for chromatography R. B. Reduce to a powder (355) (2.9.72). The powder is light
Elotu rate 1.5 mUmin. brown or reddish-brown and fibrous. Examine under a
Split ratio 1:100. microscope using chloral hydrate solution R. The powder
Temperature: shows the following diagnostic characters: groups of thick
walled fibres surrounded by a moderately thickened
parenchymatous sheath containing prism crystals of calcium
Time Temperature
oxalate; fragments of cork composed of thin-walled tabular
(°C)
cells filled with brownish or reddish contents; abundant
Column 0 - 10 50
sclereids, isolated and in groups, some large with thick,
10 - 75 50 -> 180 stratified walls and branching pits, others smaller and
75 - 130 180 thinner-walled with simple pits, often with dense brown
contents; fragments of parenchyma containing cluster crystals
Injection port 200 - 220
of calcium oxalate; occasional fragments of sieve tissue, thin
Detector 240 - 250 walled, some showing sieve areas on the oblique end-walls,
c. To 1 g of the powdered herbal drug (710) (2.9.72) add
10 mL of ethanol (30 per cent V/V) R and heat the mixture
under a reflux condenser on a water-bath for 30 min. Cool
Injection 0.2 pL. and filter. To 1 mL of this solution add 2 mL of a 10 g/L
Elution order Order indicated in the composition of the solution of vanillin R in hydrochloric acid R. A red colour
reference solution; record the retention times of these develops.
substances.
TESTS
System suitability: reference solution: Loss on drying (2.2.32)
— resolution: minimum 1.5 between the peaks due to Maximum 10.0 per cent, determined on 1.000 g of the
(Lpinene and sabinene. powdered herbal drug (710) (2.9.72) by drying in an oven at
Identification of components Using the retention times 105 °C for 2 h.
Total ash (2.4.16)
reference solution, locate the components of the reference
solution in the chromatogram obtained with the test solution.
Determine the percentage content of each of these ASSAY
components. The percentages are within the following Tannins (2.8.14)
ranges: Use 0.700 g of the powdered herbal drug (710) (2.9.72).
— (L-pinene: 15 per cent to 28 per cent; _ __________________________________ _________ ________________ Ph Eur
— fi-pinene: 13 per cent to 18 per cent;

— sabinene: 14 per cent to 29 per cent;
— car-3-ene: 0.5 per cent to 2.0 per cent;
— limonene: 2.0 per cent to 7.0 per cent;
IV-300 Olive Leaf 2016
Olive Leaf * Results See below the sequence of zones present in the
(Ph. Eur. monograph 1878) *** test solution. Furthermore, other faint zones may be present
Preparation in the chromatogram obtained with the test solution.
Olive Leaf Dry Extract
PhEir_____________________________________________________ _ _______
DEFINITION
Dried leaf of Olea europaea L.
Content
Minimum 5.0 per cent of oleuropein (C25H32O13; Mr 540.5)
(dried drug).
IDENTIFICATION
A. The leaf is simple, thick and coriaceous, lanceolate to
obovate, 30-50 mm long and 10-15 mm wide, with a
mucronate apex and tapering at the base to a short petiole;
the margins are entire and reflexed abaxially. The upper
surface is greyish-green, smooth and shiny, the lower surface
paler and pubescent, particularly along the midrib and main
lateral veins.
characters: fragments of the epidermis in surface view with
small, thick-walled polygonal cells and, in the lower
epidermis only, small anomocytic stomata (2.8.ร); fragments
of the lamina in sectional view showing a thick cuticle, a
palisade composed of 3 layers of cells and a small-celled
spongy parenchyma; numerous sclereids, very thick-walled
and mostly fibre-like with blunt or, occasionally, forked ends,
isolated or associated with the parenchyma of the mesophyll;
abundant, very large peltate trichomes, with a central
unicellular stalk from which radiate some 10-30 thin-walled
cells that become free from the adjoining cells at the margin
of the shield, giving an uneven, jagged appearance.
c. Thin-layer chromatography (2.2.27). A. Peltate trichome, seen F. Fragment of the lamina, in
from above transverse section, showing a
B. Peltate trichome, seen thick cuticle (Fa), palisade
(2.9.12) add 10 mL of methanol R. Boil under a reflux
from below parenchyma composed of 3
condenser for 15 min. Cool and filter.
c. Palisade parenchyma layers of cells (Fb), and spongy
Reference solution Dissolve 10 mg of oleuropein R and 1 mg of D, G, H and L. Fibre-like parenchyma (Fc)
rutin R in 1 mL of methanol R. sclereids, some J. Fragment of lower epidermis
Plate TLC silica gel plate R. accompanied by with anomocytic stomata (Ja)
Mobile phase water R3 methanol R, methylene chloride R parenchymatous fragments and cicatrix of peltate trichome
(1.5:15:85 VIVIV). of the spongy mesophyll CJb)
Application 10 pL, as bands. E. Spongy parenchyma K. Fragment of upper epidermis,
in surface view, with underlying
palisade parenchyma (Ka) and
Drying In air. sclereids of the spongy mesophyll
Detection spray with vanillin reagent R and heat at (Kb)
100-105 °C for 5 min; examine in daylight. Figure 1878.-1. - Illustration of powdered herbal drug of olive
leaf (see Identification B)
TESTS
Top of the plate
A dark violet-blue zone (solvent powdered herbal drug (355) (2.9.12) by drying in an oven at
front)
105 °C for 2 h.
A dark violet-blue zone
Oleuropein ะ a brownish-green A brownish-green zone

(oleuropein)
Rutin: a brownish-yellow zone

2016 Olive Leaf Dry Extract IV-301
Total ash (2.4.16) PRODUCTION

Maximum 9.0 per cent. The extract is produced from the herbal drug by a suitable
ASSAY procedure using ethanol (65-96 per cent VIV).
Liquid chromatography (2.2.29). CHARACTERS
Test solution In a flask, place 1.000 g of the powdered herbal Appearance
druร (355) (2.9.12) and add 50 mL of methanol R. Heat in a Greenish-brown or brown, amorphous powder.
water-bath at 60 °C for 30 min with shaking. Allow to cool
IDENTIFICATION
and filter into a 100 mL volumetric flask. Rinse the flask and
the filter with methanol R and dilute to 100.0 mL with the
same solvent. Dilute 2.5 mL of this solution to 25.0 mL with Test solution To 0.25 g of the extract to be examined add
water R. 10 mL of methanol R. Sonicate for 15 min and filter.
Reference solution Dissolve 5.0 mg of oleuropein CRS in Reference solution Dissolve 5 mg of oleuropein R and 1 mg of
5.0 mL of methanol R. Dilute 1.0 mL of this solution to rutin R in 1 mL of methanol R.
25.0 mL with water R. Plate TLC silica gel plate R (5-40 gm) [or TLC silica gel
Column: plate R (2-10 gm)].
size: l ะ= 0.15 m, 0 ะ= 3.9 mm; Mobile phase water R, anhydrous formic acid R, ethyl acetate R
stationary phase: octadecylsilyl silica gel for chromatography R (7:13:80 VIVIV).
(5 gm); Application 10 gL [or 2 gL] as bands of 10 mm [or 8 mm].
— temperature: 25 °C. Development Over a path of 10 cm [or 6 cm].
Mobile phase:
Drying In air.
mobile phase A: dilute 1.0 mL of glacial acetic acid R to
100 mL with water R) Detection Spray with anisaldehyde solution R and heat at
— mobile phase B: methanol R-, 100-105 °C for 5 min; examine in daylight.
Time Mobile phase A Mobile phase B chromatograms obtained with the reference solution and the
(min) (per cent V7V) (per cent V/V) test solution. Furthermore, other faint zones may be present
0-5 85 -> 40 15 -> 60 in the chromatogram obtained with the test solution.
5 - 12 40 -> 20 60 -> 80
12 - 15 20 85 Top of the plate
80 -> 15
A dark violet-blue zone
Flow rate 1 mL/min.
Oleuropein: a brownish-green zone A brownish-green zone
Injection 20 gL. (oleuropein)
Retention time Oleuropein = about 9 min.
Calculate the percentage content of oleuropein using the Rutin: a yellow zone
following expression: Reference solution Test solution
Al X 7ท2 X p X 8
/เ2 X 7711
TESTS
A1 ะ= area of the peak due to oleuropein in the Loss on drying (2.8.17)
chromatogram obtained with the test solution; Maximum 8.0 per cent.
/I2 = area of the peak due to oleuropein in the ASSAY
Liquid chromatography (2.2.29). Prepare the solutions
solution;
immediately before use.
Ml = mass of the herbal drug to be examined in the test
solution, in grams; Test solution To 0.250 g of the extract to be examined add
m2 = mass of oleuropein CRS in the reference solution, 50 mL of methanol R. Sonicate for 15 min and filter into a
in grams; 100 mL volumetric flask. Rinse the flask and the filter with
p = percentage content of oleuropein in oleuropein 2 mL of methanol R and dilute to 100.0 mL with water R.
CRS. Reference solution (a) Dissolve 10.0 mg of oleuropein CRS in
10.0 mL of methanol R and dilute to 25.0 mL with water R.
---------------------------------------------------------------------------------------------------------- Ph Eur
Reference solution (b) Dissolve 4 mg of rutin R in 10 mL of
Column:
Olive Leaf Dry Extract —- size: l ะ= 0.15 m, 0 = 4.6 mm;
— stationary phase: end-capped octadecylsUyl silica gel for
(Ph. Eur. monograph 2313) chromatography R (5 gm);
Ph Elf-___________ __ _________________________________ — temperature: 25 °C.
Mobile phase trifluoroacetic acid R, methanol R, water R
definition
(1:400:600 VIVIV).
Dry extract produced from Olive leaf (1878).
Flow rate 1 mL/min.
Content
Minimum 16.0 per cent of oleuropein (C25H32013;
Mr 540.5) (dried extract). Injection 20 gL.
IV-302 Opium 2016
Run time Twice the retention time of oleuropein. grains with 3 pores and a very finely pitted exine [E] and
Relative retention With reference to oleuropein (retention fragments of elongated fibres [D]. Fragments of epicarp
time = about 11 min): rutin = about 0.7. (surface view [B, c, G], transverse section [H]), consisting of
รุ),stem suitability reference solution (b): polygonal cells with thick walls defining a stellate lumen, and
— resolution', minimum 3.0 between the peaks due to rutin sometimes anomocytic stomata (2.8.8) [Ba] may also be
and oleuropein. present. Some elements of various origin introduced during
handling of the latex may also be present in small quantities
Calculate the percentage content of oleuropein using the
(fragments of covering trichomes and starch granules).
Al X 7712 X p X 4
Al = area of the peak due to oleuropein in the

A2 = area of the peak due to oleuropein in the
(a);
ทใ1 = mass of the extract to be examined used to
m2 = mass of oleuropein CRS used to prepare reference
p = percentage content of oleuropein in oleuropein
CRS.
Ph Eur
Opium
(Raw Opium3 Ph. Eur. monograph 0777) Figure 0777.-1. - Illustration for identification test A of powdered
Preparations raw opium
Opium Tincture B. Thin-layer chromatography (2.2.27).
Prepared Opium Test solution Triturate 0.10 g of the powdered substance to be
Standardised Opium Dry Extract examined (500) (2.9.12) with 5 mL of ethanol
Standardised Opium Tincture (70 per cent VIV) R. Transfer to a 25 mL conical flask. Rinse
with 3 mL of ethanol (70 per cent VIV) R and transfer the
Ph Eur_______________________________________________________________
rinsings to the same 25 mL conical flask. Heat in a water
Raw opium is intended only as starting material for the bath at 50-60 °C, while stirring, for 30 min. Cool, filter,
manufacture of galenical preparations. It is not dispensed as such. wash the filter with ethanol (70 per cent V/V) R and dilute the
DEFINITION combined filtrates to 10 mL with the same solvent.
Air-dried latex obtained by incision from the unripe capsules Reference solution Dissolve 5 mg of morphine hydrochloride R in
of Papaver somniferum L. a solution prepared as follows and dilute to 5 mL with the
Content same solution: dissolve 2 mg of papaverine hydrochloride R>
— morphine (C]7H19NO3; Mr 285.3): minimum 12 mg of codeine phosphate R and 12 mg of noscapine
10.0 per cent (dried drug); hydrochloride R in ethanol (70 per cent VIV) R and dilute to
— codeine (C18H21NO3; Afr 299.4): minimum 2.0 per cent 25 mL with the same solvent.
(dried drug). Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
plate R (2-10 pm)].
CHARACTERS
Appearance Mobile phase concentrated ammonia R, ethanol (96 per cent) Ri
Blackish-brown masses of various sizes, which tend to be soft acetone R3 toluene R (2:6:40:40 VIVIVIV), use a freshly
and shiny and, after drying, become hard and brittle. prepared mixture.
IDENTIFICATION
Strip off any covering3 cut the substance to be examined into thin
slices3 dry at about 60 cc for 48 h, if necessary3 and reduce to a Drying At 100-105 °C for 15 min.
powder (500) (2.9.12). Detection Allow to cool and treat with potassium iodobismuthate
A. Microscopic examination (2.8.28). The powder (500) solution R2 and then with a 4 g/L solution of sulfuric acid R;
(2.9.12) is light brown. Examine under a microscope using examine in daylight.
chloral hydrate solution R. Before heating, the powder shows Results See below the sequence of zones present in the
the following diagnostic characters (Figure 0777.-1): granules chromatograms obtained with the reference solution and the
of latex agglomerated in irregular masses [A] and light brown test solution. A dark red zone (thebaine) situated between
elongated filaments. After heating, some fragments of the zone due to codeine and the zone due to papaverine may
vessels n, K] and rather elongated, refringent crystals [F] are be present in the chromatogram obtained with the test
also visible, as well as a smaller number of round pollen
2016 Opium IV-303
solution. Furthermore, other faint zones may be present in Run time Twice the retention time of thebaine.
the chromatogram obtained with the test solution. System suitability: reference solution (c):
Top of the plate
morphine and codeine.
Noscapine: an orange-red or red An orange-red or red zone Calculate the percentage content of the relevant alkaloid
(noscapine)
Papaverine: an orange-red or red An orange-red or red zone Al X 7712 X F X p

(papaverine)
A2 X 7721
Codeine: an orange-red or red An orange-red or red zone A1 ะะะ area of the peak due to the relevant alkaloid in the
(codeine)
Morphine: an orange-red or red An orange-red or red zone
zone (morphine)
A2 = area of the peak due to the relevant alkaloid in the
Reference solution
chromatogram obtained with reference solution (a)
Test solution
for thebaine or reference solution (c) for morphine
and codeine;
c. To 1.0 g of the powdered substance (500) (2.9.72) add
nil = mass of the substance to be examined used to
5 mL of water R3 shake for 5 min and filter. To the filtrate
add 0.25 mL of ferric chloride solution R2. A red colour
m2 = mass of the relevant alkaloid used to prepare
develops which does not disappear upon the addition of
reference solution (a) for thebaine, reference solution
0.5 mL of dilute hydrochloric acid R.
(b) for morphine or reference solution (c) for
TESTS codeine, in grams;
Thebaine F = 6.250 for the determination of thebaine;
Ijquid chromatography (2.2.29). p = percentage content of the relevant alkaloid in the
Test solution Suspend 1.000 g of the substance to be corresponding CRS.
examined, cut into thin slices, in 50 mL of ethanol Limit:
(50 per cent V/V) R, mix using sonication for 1 h, allow to — thebaine: maximum 3.0 per cent (dried drug).
cool and dilute to 100.0 mL with the same solvent. Allow to Loss on drying (2.2.32)
stand. To 10.0 mL of the supernatant add 5 mL of Maximum 15.0 per cent, determined on 1.000 g of the
ammonium chloride buffer solution pH 9.5 R, dilute to 25.0 mL substance to be examined, cut into thin slices, by drying in
with water R and mix. Transfer 20.0 mL of this solution to a an oven at 105 °C for 4 h.
chromatography column about 0.15 m long and about
30 mm in internal diameter containing 15 g of kieselguhr for
Total ash (2.4.16)
chromatography R. Allow to stand for 15 min. Elute with
2 quantities, each of 40 mL, of a mixture of 15 volumes of ASSAY
2-propanol R and 85 volumes of methylene chloride R. Evaporate Liquid chromatography (2.2.29) as described in the test for
the combined eluates to dryness in vacuo at 40 °C. Transfer thebaine with the following modifications.
the residue to a volumetric flask using the mobile phase and Injection Test solution and reference solution (c).
dilute to 25.0 mL with the mobile phase. System suitability: reference solution (c):
Reference solution (a). Dissolve 5.0 mg of thebaine CRS in the — repeatability: maximum relative standard deviation of
mobile phase and dilute to 50.0 mL with the mobile phase. 1.0 per cent for the area of the peak due to morphine
Reference solution (b) Dissolve 12.0 mg of morphine after 6 injections.
hydrochloride trihydrate CRS in the mobile phase and dilute to Calcdate the percentage content of morphine and the
15.0 mL with the mobile phase. percentage content of codeine using the expression given in
Reference solution (c) Dissolve 10.0 mg of codeine CRS in the the test for thebaine, with F = 10.417 for morphine and
mobile phase and dilute to 50.0 mL with the mobile phase. F = 3.125 for codeine.
To 10.0 mL of the solution add 10.0 mL of reference To obtain the p value to be used for the calculation of the
solution (b). morphine content, multiply the percentage content of
Precolumn: morphine hydrochloride in morphine hydrochloride
— size: l ะะะ 4 mm, 0 = 4.0 mm; trihydrate CRS by 0.887.
— stationary phase: octylsilyl silica gel for chromatography R ________________________________ PhEur
(5 pm).
Column:
— size: l ะ= 0.25 m, 0 = 4.0 mm;
— stationary phase: end-capped octylsilyl silica gel for
chromatography R (5 |im). Prepared Opium *
Mobile phase Dissolve 1.0 g of sodium heptanesulfonate
monohydrate R in 420 mL of water R, adjust to pH 3.2 with a
4.9 g/L solution of phosphoric acid R and add 180 mL of Ph Elf---------------------------------------------- -------------- ------------------- - ---------- -------
acetonitrile R. DEFINITION
Flow rate 1.5 mL/min. Raw opium (0777) powdered (180) (2.9.12) and dried at a
temperature not exceeding 70 °C, with a morphine content
adjusted, if necessary, by adding a suitable excipient or raw
Injection 20 pL of the test solution and reference solutions (a)
opium powder with a lower alkaloidal content.
and (c).
IV-304 Opium 2016
Content Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
— morphine (C17H19NO3; Afr 285.3): 9.5 per cent to plate R (2-10 pm)].
10.5 per cent (dried preparation); Mobile phase concentrated ammonia R, ethanol (96 per cent) R,
— codeine (Ci8H2iNO3; Mr 299.4): minimum 1.0 per cent acetone R, toluene R (2:6:40:40 V/V/V/V), use a freshly
(dried preparation). prepared mixture.
CHARACTERS Application 20 pL [or 6 pL] as bands of 10 mm [or 8 mm].
Appearance Development Over a path of 15 cm [or 8 cm].
Yellowish-brown or dark brown powder.
Drying At 100-105 °C for 15 min.
IDENTIFICATION Detection Allow to cool and treat with potassium iodobismuthate
A. Microscopic examination (2.8.23). The powder (500) solution R2 and then with a 4 g/L solution of sulfuric acid R'j
(2.9.12) is light brown. Examine under a microscope using examine in daylight.
chloral hydrate solution R. Before heating, the powder shows Results See below the sequence of zones present in the
the following diagnostic characters (Figure 1840.-1): granules chromatograms obtained with the reference solution and the
of latex agglomerated in irregular masses [A] and light brown test solution. A dark red zone (thebaine) situated between
elongated filaments. After heating, some fragments of vessels the zone due to codeine and the zone due to papaverine may
[J, K] and rather elongated, refringent crystals [F] are also be present in the chromatogram obtained with the test
visible, as well as a smaller number of round pollen grains solution. Furthermore, other faint zones may be present in
with 3 pores and a very finely pitted exine [E] and fragments the chromatogram obtained with the test solution.
of elongated fibres [D]. Fragments of epicarp (surface view
[B, c, G], transverse section [H]), consisting of polygonal
cells with thick walls defining a stellate lumen, and Top of the plate
sometimes anomocytic stomata (2.8.3) [Ba] may also be Noscapine: an orange-red or red An orange-red or red zone
present. Some elements of various origin introduced during zone (noscapine)
handling of the latex may also be present in small quantities
(fragments of covering trichomes and starch granules).
Papaverine: an orange-red or red An orange-red or red zone
zone (papaverine)
Codeine: an orange-red or red An orange-red or red zone

(codeine)
Morphine: an orange-red or red An orange-red or red zone
zone (morphine)
c. To 1.0 g of the preparation to be examined add 5 mL of

water R, shake for 5 min and filter. To the filtrate add
0.25 mL of ferric chloride solution R2. A red colour develops
which does not disappear upon the addition of 0.5 mL of
dilute hydrochloric acid R.
TESTS
Thebaine
Test solution Suspend 1.000 g of the preparation to be
examined in 50 mL of ethanol (50 per cent V/V) R, mix using
sonication for 1 h, allow to cool and dilute to 100.0 mL with
the same solvent. Allow to stand. To 10.0 mL of the
supernatant add 5 mL of ammonium chloride buffer solution
Figure 1840.-1. - Illustration for identification test A of prepared pH 9.5 R> dilute to 25.0 mL with water R and mix. Transfer
opium 20.0 mL of this solution to a chromatography column about
B. Thin-layer chromatography (2.2.27). 0.15 m long and about 30 mm in internal diameter
containing 15 g of kieselguhr for chromatography R. Allow to
Test solution Triturate 0.10 g of the preparation to be
stand for 15 min. Elute with 2 quantities, each of 40 mL, of
examined with 5 mL of ethanol (70 per cent V/V) R. Transfer
a mixture of 15 volumes of 2-propanol R and 85 volumes of
to a 25 mL conical flask. Rinse with 3 mL of ethanol
methylene chloride R. Evaporate the combined eluates to
(70 per cent V/V) R and transfer the rinsings to the same
dryness in vacuo at 40 °C. Transfer the residue to a
25 mL conical flask. Heat in a water-bath at 50-60 °C, while
volumetric flask using the mobile phase and dilute to
stirring, for 30 min. Cool, filter, wash the filter with ethanol
25.0 mL with the mobile phase.
(70 per cent V/V) R and dilute the combined filtrates to
10 mL with the same solvent. Reference solution (a) Dissolve 5.0 mg of thebaine CRS in the
mobile phase and dilute to 50.0 mL with the mobile phase.
Reference solution Dissolve 5 mg of morphine hydrochloride R in
a solution prepared as follows and dilute to 5 mL with the Reference solution (b) Dissolve 12.0 mg of morphine
same solution: dissolve 2 mg of papaverine hydrochloride R3 hydrochloride trihydrate CRS in the mobile phase and dilute to
12 mg of codeine phosphate R and 12 mg of noscapine 15.0 mL with the mobile phase.
hydrochloride R in ethanol (70 per cent V/V) R and dilute to Reference solution (c) Dissolve 10.0 mg of codeine CRS in the
25 mL with the same solvent. mobile phase and dilute to 50.0 mL with the mobile phase.
2016 Opium Preparations IV-305
To 10.0 mL of the solution add 10.0 mL of reference To obtain the p value to be used for the calculation of the
solution (๖). morphine content, multiply the percentage content of
Pncolumn: morphine hydrochloride in morphine hydrochloride
— size: l ะ= 4 mm, 0 ะ= 4.0 mm; trihydrate CRS by 0.887.
stanonary phase: octylsilyl silica gel for chromatography R ______________________________________________________ ____ Ph Eur
(5 pm).
Column:
size: I = 0.25 m, 0 = 4.0 mm;
stationary phase: end-capped octylsilyl silica gel for
chromatography R (5 pm). Standardised Opium Dry Extract *****
Mobile phase Dissolve 1.0 g of sodium heptanesulfonate
monohydrate R in 420 mL of water R, adjust to pH 3.2 with a
4.9 g/L solution of phosphoric acid R and add 180 mL of Ph Eur____________________________________________________________
acetonitrile R. DEFINITION
Flow rate 1.5 mUmin. Standardised dry extract produced from Raw opium (0777).
Detection Spectrophotometer at 280 nm. Content
Injection 20 pL of the test solution and reference solutions (a) — morphine (C17H19NO3; Mr 285.3): 19.6 per cent to
and (c). 20.4 per cent (dried extract);
Run time Twice the retention time of thebaine. — codeine (C18H21NO3; Mr 299.4): minimum 2.0 per cent
รุ)’stem suitability: reference solution (c):
(dried extract).
resolution: minimum 2.5 between the peaks due to Content adjusted if necessary by adding a suitable excipient
morphine and codeine. (e.g. lactose, dextrin).
Calculate the percentage content of the relevant alkaloid PRODUCTION
using the following expression: The extract is produced from the drug by a suitable
procedure using water.
Al X m2 X F X p CHARACTERS
A2 X mi Appearance
Brown, amorphous powder.
•<41 = area of the peak due to the relevant alkaloid in the
IDENTIFICATION
chromatogram obtained with reference solution (a) Test solution Triturate 0.05 g of the extract to be examined
for thebaine or reference solution (c) for morphine with 5 mL of ethanol (70 per cent VIV) R. Transfer to a
and codeine; 25 mL conical flask. Rinse with 3 mL of ethanol
nil = mass of the preparation to be examined used to (70 per cent V/V) R and transfer the rinsings to the same
prepare the test solution, in grams; 25 mL conical flask. Heat in a water-bath at 50-60 °C, while
พ2 = mass of the relevant alkaloid used to prepare stirring, for 30 min. Cool, filter, wash the filter with ethanol
reference solution (a) for thebaine, reference solution (70 per cent V/V) R and dilute the combined filtrates to
(b) for morphine or reference solution (c) for 10 mL with the same solvent.
codeine, in grams; Reference solution Dissolve 5 mg of morphine hydrochloride R in
F = 6.250 for the determination of thebaine; a solution prepared as follows and dilute to 5 mL with the
p = percentage content of the relevant alkaloid in the same solution: dissolve 2 mg of papaverine hydrochloride R>
corresponding CRS. 12 mg of codeine phosphate R and 12 mg of noscapine
Limit: hydrochloride R in ethanol (70 per cent V/V) R and dilute to
25 mL with the same solvent.
— thebaine: maximum 3.0 per cent (dried preparation).
plate R (2-10 pm)].
preparation to be examined by drying in an oven at 105 °C Mobile phase concentrated ammonia Rf ethanol (96 per cent) R,
for 4 h. acetone R) toluene R (2:6:40:40 VIVIVIV)\ use a freshly
prepared mixture.
Total ash {2.4.16)
ASSAY
thebaine with the following modifications. Detection Allow to cool and treat with potassium iodobismuthate
solution R2 and then with a 4 g/L solution of sulfuric acid R'i
Injection Test solution and reference solution (c).
— repeatability: maximum relative standard deviation of
1.0 per cent for the area of the peak due to morphine
test solution. A dark red zone (thebaine) situated between
after 6 injection’s. the zone due to codeine and the zone due to papaverine may
Calculate the percentage content of morphine and the be present in the chromatogram obtained with the test
percentage content of codeine using the expression given in solution. Furthermore, other faint zones may be present in
the test for thebaine, with F = 10.417 for morphine and the chromatogram obtained with the test solution.
F - 3.125 for codeine.
IV-306 Opium Preparations 2016
Top of the plate System suitability: reference solution (c):

Noscapine: an orange-red or red
An orange-red or red zone
(noscapine) morphine and codeine.
Calculate the percentage content of the relevant alkaloid
(papaverine)
Al X ๓2 X F X p
A2 X mi
Codeine: an orange-red or red An orange-red or red zone
(codeine)
Morphine: an orange-red or red
(morphine) chromatogram obtained with the test solution;
B. To 0.5 g of the extract to be examined add 5 mL of and codeine;
water R) shake for 5 min and filter. To the filtrate add nil ะ= mass of the extract to be examined used to prepare
0.25 mL offerric chloride solution R2. A red colour develops the test solution, in grams;
which does not disappear upon the addition of 0.5 mL of m2 = mass of the relevant alkaloid used to prepare
dilute hydrochloric acid R. reference solution (a) for thebaine, reference solution
TESTS
codeine, in grams;
Thebaine F = 6.250 for the determination of thebaine;
Liquid chromatography (2.2.29). p = percentage content of the relevant alkaloid in the
Test solution Suspend 0.500 g of the extract to be examined corresponding CRS.
in 50 mL of ethanol (50 per cent V/V) Rj mix using sonication
Limit:
for 1 h, allow to cool and dilute to 100.0 mL with the same
— thebaine: maximum 6.0 per cent (dried extract).
solvent. Allow to stand. To 10.0 mL of the supernatant add
5 mL of ammonium chloride buffer solution pH 9.5 R, dilute to Loss on drying (2.2.22)
25.0 mL with water R and mix. Transfer 20.0 mL of this Maximum 5.0 per cent, determined on 1.000 g of the extract
solution to a chromatography column about 0.15 m long and to be examined by drying in an oven at 105 °C for 4 h.
about 30 mm in internal diameter containing 15 g of ASSAY
kieselguhr for chromatography R. Allow to stand for 15 min. Liquid chromatography (2.2.29) as described in the test for
Elute with 2 quantities, each of 40 mL, of a mixture of thebaine with the following modifications.
15 volumes of 2-propanol R and 85 volumes of methylene Injection Test solution and reference solution (c).
chloride R. Evaporate the combined eluates to dryness
in vacuo at 40 °C. Transfer the residue to a volumetric flask
— repeatability: maximum relative standard deviation of
using the mobile phase and dilute to 25.0 mL with the
1.0 per cent for the area of the peak due to morphine
mobile phase.
after 6 injections.
Reference solution (a) Dissolve 5.0 mg of thebaine CRS in the
Calculate the percentage content of morphine and the
mobile phase and dilute to 50.0 mL with the mobile phase.
percentage content of codeine using the expression given in
Reference solution (b) Dissolve 12.0 mg of morphine the test for thebaine, with F = 10.417 for morphine and
hydrochloride trihydrate CRS in the mobile phase and dilute to F = 3.125 for codeine.
To obtain the p value to be used for the calculation of the
Reference solution (c) Dissolve 10.0 mg of codeine CRS in the morphine content, multiply the percentage content of
mobile phase and dilute to 50.0 mL with the mobile phase. morphine hydrochloride in morphine hydrochloride
To 10.0 mL of the solution add 10.0 mL of reference trihydrate CRS by 0.887.
solution (b). ___________________________________________________________ ___ PnELT
Precolumn-.
— size'. 1=4 mm, 0 = 4.0 mm;
(5 pm).
Column: Standardised Opium Tincture
— size: I = 0.25 m, 0 = 4.0 mm; (Ph. Eur. monograph 1841) *
chromatography R (5 pm). PhEur------------------------------------------------------------------------ —------------------ ----------
Mobile phase Dissolve 1.0 g of sodium heptanesulfonate DEFINITION

monohydrate R in 420 mL of water R} adjust to pH 3.2 with a Standardised tincture produced from Raw opium (0777).
4.9 g/L solution of phosphoric acid R and add 180 mL of Content
acetonitrile R. — morphine (C17H19NO3; Mr 285.3): 0.95 per cent to
Flow rate 1.5 mL/min. 1.05 per cent;
Detection Spectrophotometer at 280 DID. — codeine (CI8H21NO3; Alr 299.4): minimum 0.1 per cent.
Injection 20 pL of the test solution and reference solutions (a) PRODUCTION
and (c). The tincture is produced from the drug by an appropriate
Run time Twice the retention time of thebaine. procedure using equal volumes of ethanol (70 per cent F/J7)
and water.
2016 Opium Preparations IV-307
CHARACTERS Reference solution (a) Dissolve 5.0 mg of thebaine CRS in the

Appearance mobile phase and dilute to 50.0 mL with the mobile phase.
Reddish-brown liquid.
Reference solution (b) Dissolve 12.0 mg of morphine
identification hydrochloride trihydrate CRS in the mobile phase and dilute to
Thin-layer chromatography (2.2.27). 15.0 mL with the mobile phase.
Test solution Dilute 1.0 mL of the tincture to be examined to Reference solution (c) Dissolve 10.0 mg of codeine CRS in the
10 mL with ethanol (70 per cent VIV) R. mobile phase and dilute to 50.0 mL with the mobile phase.
Reference solution Dissolve 5 mg of morphine hydrochloride R in To 10.0 mL of the solution add 10.0 mL of reference
a solution prepared as follows and dilute to 5 mL with the solution (b).
same solution: dissolve 2 mg of papaverine hydrochloride R, Precolumn'.
12 mg of codeine phosphate R and 12 mg of noscapine — size'. I = 4 mm, 0 ะ= 4.0 mm;
hydrochloride R in ethanol (70 per cent V/V) R and dilute to — stationary phase: octylsilyl silica gel for chromatography R
25 mL with the same solvent. (5 pm).
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel Column:
plate 2? (2-10 pm)]. — size: I = 0.25 m, 0 = 4.0 mm;
Mobile phase concentrated ammonia R, ethanol (96 per cent) R,
acetone R, toluene R (2:6:40:40 V/V/V/V); use a freshly
prepared mixture. Mobile phase Dissolve 1.0 g of sodium heptanesulfonate
monohydrate R in 420 mL of water R3 adjust to pH 3.2 with a
4.9 g/L solution of phosphoric acid R and add 180 mL of
Development Over a path of 15 cm [or 8 cm]. acetonitrile R.
Drying Kt 100-105 °C for 15 min. Flow rate 1.5 mL/min.
Detection Allow to cool and treat with potassium iodobismuthate Detection Spectrophotometer at 280 nm.
solution R2 and then with a 4 g/L solution of sulfuric acid R; Injection 20 pL of the test solution and reference solutions (a)
and (c).
Results See below the sequence of zones present in the Run time Twice the retention time of thebaine.
test solution. A dark red zone (thebaine) situated between
the zone due to codeine and the zone due to papaverine may
morphine and codeine.
solution. Furthermore, other faint zones may be present in Calcdate the percentage content of the relevant alkaloid
the chromatogram obtained with the test solution. using the following expression:
Top of the plate

Al X m2 X F X p
A.2 X 7ท1
Noscapine: an orange-red or red An orange-red or red zone
(noscapine)
chromatogram obtained with ±e test solution;
(papaverine) A2 = area of the peak due to the relevant alkaloid in the
Codeine: an orange-red or red zone An orange-red or red zone
(codeine) and codeine;
Morphine; an orange-red or red An orange-red or red zone m 1 = mass of the tincture to be examined used to prepare
(morphine) the test solution, in grams;
Reference solution Test solution m2 = mass of the relevant alkaloid used to prepare
reference solution (a) for thebaine, reference solution
TESTS codeine, in grams;
Ethanol (2.9.10) F = 1.563 for the determination of thebaine;
31 per cent V/V to 34 per cent V/V. p = percentage content of the relevant alkaloid in the
Thebaine corresponding CRS.
Liquid chromatography (2.2.29). Limit:.
Test solution Dilute 2.000 g of the tincture to be examined to — thebaine: maximum 0.3 per cent.
25.0 mL with ethanol (50 per cent V/V) R. To 10.0 mL of the Dry residue (2.8.16)
solution add 5 mL of ammonium chloride buffer solution Minimum 4.0 per cent nilnij determined on 3.00 g.
pH 9.5 R, dilute to 25.0 mL with water R and mix. Transfer
20.0 mL of this solution to a chromatography column about ASSAY
0.15 m long and about 30 mm in internal diameter
containing 15 g of kieselguhr for chromatography R. Allow to thebaine with the following modifications.
stand for 15 min. Elute with 2 quantities, each of 40 mL, of Injection Test solution and reference solution (c).
a mixture of 15 volumes of 2-propanol R and 85 volumes of System suitability: reference solution (c):
methylene chloride R. Evaporate the combinated eluates to — repeatability: maximum relative standard deviation of
diyness in vacuo at 40 °C. Transfer the residue to a 1.0 per cent for the area of the peak due to morphine
volumetric flask using the mobile phase and dilute to after 6 injections.
IV-308 Opium Preparations 2016
Calculate the percentage content of morphine and the Appendix II B, using in the reference cell a solution prepared
percentage content of codeine using the expression given in at the same time and in the same manner but using 8 mL of
the test for thebaine, with F = 2.604 for morphine and water in place of the solution of sodium nitrite. Calculate the
F = 0.781 for codeine. content of C17H19NO3 taking 124 as the value of
To obtain the p value to be used for the calculation of the A(l%, 1 cm) at the maximum at 442 nm.
morphine content, multiply the percentage content of
morphine hydrochloride in morphine hydrochloride
trihydrate CRS by 0.887.
----------------------------------------------------------------------------------------------------------- Ph Eur Concentrated Camphorated Opium
Tincture
DEFINITION
Opium Tincture 400 mL
Opium Tincture Benzoic Acid 40 g
DEFINITION Racemic Camphor 24 g
Opium, sliced 200 g Anise Oil or Star Anise Oil 24 mL
Ethanol (90 per cent) A sufficient quantity Ethanol (96 per cent) 400 mL
Purified Water A sufficient quantity Water Sufficient to produce 1000 mL
Extemporaneous preparation Extemporaneous preparation
The following directions apply. The following directions apply.
Pour 500 mL of boiling Purified Water on to the Opium and Dissolve the Benzoic Acid, the Racemic Camphor and the
allow to stand for 6 hours; add 500 mL of Ethanol Anise Oil or Star Anise Oil in the Ethanol (96 per cent), add
(90 per cent), mix thoroughly and allow to stand in a the Opium Tincture and sufficient Water to produce
covered vessel for 24 hours; strain, press the marc, mix the 1000 mL, mix and filter if necessary.
liquids and allow to stand for not less than 24 hours; filter. The tincture complies with the requirements for Tinctures stated
Determine the concentration of morphine, calculated as under Extracts and with the following requirements.
anhydrous morphine, in the tincture so prepared by the Content of anhydrous morphine, C17H19NO3
Assay. To the remainder of the liquid add sufficient of a 0.36 to 0.44% w/v.
mixture of equal volumes of Ethanol (90 per cent) and
TESTS
Purified Water to produce an Opium Tincture containing
1 % w/v of anhydrous morphine. Ethanol content
54 to 59% v/v, Appendix VIII F, Method in.
under Extracts and with the following requirements. Relative density
Content of anhydrous morphine, C17H19NO3
0.925 to 1.075% w/v. ASSAY
Dilute 10 mL to 100 mL with ethanol (50%) and carry out
TESTS
the Assay described under Camphorated opium Tincture
Ethanol content using 10 mL of the diluted solution.
41 to 46% v/v, Appendix VIII F, Method in.
Relative density
ASSAY Camphorated Opium Tincture
Dilute 5 mL to 100 mL with ethanol (45%). To 10 mL of DEFINITION
the resulting solution add 5 mL of water and 1 mL of Opium Tincture 50 mL
5m ammonia and extract with 30 mL of a mixture of equal Benzoic Acid 5g
volumes of ethanol (96%) and chloroform and then with two Racemic Camphor 3g
22.5 mL quantities of a mixture of 2 volumes of chloroform Anise Oil or Star Anise Oil 3 mL
and 1 volume of ethanol (96%), washing each extract with the Ethanol (60 per cent) Sufficient to produce 1000 mL
same 20 mL of a mixture of equal volumes of ethanol (96%)
and water. Evaporate the combined extracts just to dryness, Extemporaneous preparation
extract the residue with two 5 mL quantities of calcium The following directions apply.
hydroxide solution, filter and wash the filter with 10 mL of Dissolve the Benzoic Acid, the Racemic Camphor and the
calcium hydroxide solution. To the combined filtrate and Anise Oil or Star Anise Oil in 900 mL of Ethanol
washings add 0.1 g of ammonium sulfate, extract wi± two (60 per cent), add the Opium Tincture and sufficient
10 mL quantities of ethanol-free chloroform, wash the Ethanol (60 per cent) to produce 1000 mL and mix. Filter, if
combined extracts with 10 mL of water and discard the necessary.
chloroform solution. To the combined alkaline liquid and The tincture complies with the requirements for Tinctures stated
aqueous washings add 10 mL of Im hydrochloric acid, heat on under Extracts and with the following requirements.
a water bath to remove any chloroform, cool and dilute to Content of anhydrous morphine, C17H19NO3
100 mL with water. To 10 mL of this solution add 10 mL of
0.045 to 0.055% w/v.
0.1m hydrochloric acid and 8 mL of a freshly prepared
1.0% w/v solution of sodium nitrite, allow to stand for
15 minutes, add 12 mL of 5m ammonia, dilute to 50 mL
with water and measure the absorbance of a 4-cm layer of the
resulting solution at the maximum at 442 nm,
2016
Bitter-Orange Flower IV-309
tests
K]; fragments of the epidermis of the sepals in surface view
Ethanol content [D] and in transverse section [A, C], accompanied by
56 to 60% v/v, Appendix vni F, Method in. underlying mesophyll [B], some cells of which contain prisms
Relative density of calcium oxalate [Aa, Ba, Db], unicellular covering
0.90 to 0.92, Appendix V G. trichomes [Ca] and numerous anomocytic stomata (2.8.3)
assay [Da]; fragments of the epidermis of the petals in surface view
[F, G,J], with a distinctly striated cuticle; fragments of large
To 10 mL add 5 mL of water and 1 mL of 5m ammonia and
schizolysigenous oil glands in transverse section [E], which
w30 rrL^ a mixture eclual volumes of ethanol measure up to 100 pm in diameter. Examine under a
(96/a) and chloroform and then with two 22.5 mL quantities
microscope using a 20 g/L solution of potassium hydroxide R.
of a mixture of 2 volumes of chloroform and 1 volume of
The mounting medium becomes yellow because of the
ethanol (96%), washing each extract with the same 20 mL of
presence of hesperidin in the drug.
a mixture of equal volumes of ethanol (96%) and water.
Evaporate the combined extracts almost to dryness, extract
the residue with 10 mL of calcium hydroxide solution, filter
and wash the filter with 10 mL of calcium hydroxide solution.
To the combined filtrate and washings add 0.1 g of
ammonium sulfate, extract with two 10 mL quantities of
ethanol-free chloroform, wash the combined extracts with
10 mL of water and discard the chloroform solution. To the
combmed alkaline liquid and aqueous washings add 10 mL
of Im hydrochloric acid, heat on a water bath to remove any
chloroform, cool and dilute to 100 mL with water. To 10 mL
of this solution add 10 mL of 0.1m hydrochloric acid and
8 mL of a freshly prepared 1.0% w/v solution of sodium
nitrite, allow to stand for 15 minutes, add 12 mL of
5m ammonia, dilute to 50 mL with water and measure the
absorbance of a 4-cm layer of the resulting solution at the
maximum at 442 nm, Appendix II B, using in the reference
cell a solution prepared at the same time and in the same
manner but using 8 mL of water in place of the solution of
sodium nitrite. Calculate the content of C17H19NO3 taking
124 as the value of A(1 %, 1 cm) at the maximum at
442 nm.
Bitter-Orange Flower f **
DEFINITION
Whole, dried, unopened flower of Citrus aurantium L.
ssp. aurantium (C. aurantium L. ssp. amara Engl.).
Content
Minimum 8.0 per cent of total flavonoids, expressed as Figure 1810.-1. - Illustration for identification test B of powdered
naringin (C27H32O14; Mr 580.5) (dried drug). herbal drug of bitter-orange flower
IDENTIFICATION
sweet-orange flower.
A. The flower buds are white or yellowish-white and may
reach up to 25 mm in length. The dialypetalous corolla is Results See below the sequence of zones present in the
composed of 5 thick, oblong and concave petals dotted with chromatograms obtained with the reference solution and the
oil glands visible under a hand lens; the short, yellowish- test solution.
green persistent gamosepalous calyx has 5 spreading sepals,
connate at the base and forming a star-shaped structure Top of the plate
attached to the yellowish-green peduncle, which is about
A weak yellow fluorescent zone
5-10 mm long. The flower buds contain at least 20 stamens
with yellow anthers and with filaments fused at the base into A weak yellow fluorescent zone
groups of 4 or 5; the ovary is superior, brownish-black and Hesperidia: a greenish-yellow A greenish-yellow fluorescent
spherical, consists of 8-10 multi-ovular loculi and is fluorescent zone zone (hesperidin)
surrounded at the base by an annular granular hypogynous Naringin: a yellow fluorescent A yellow fluorescent zone
disc; the thick, cylindrical style ends in a capitate stigma. zone (naringin)
A red fluorescent zone
(neoeriocitrin)
brownish-yellow. Examine under a microscope using chloral A yellow fluorescent zone
hydrate solution R. The powder shows the following diagnostic (diosmin and neodiosmin)
characters (Figure 1810.-1): very numerous spherical pollen Reference solution Test solution
grains, with a finely pitted exine and 3-5 germinal pores [H,
rV-310 Orange Oil 2016
TESTS
Sweet-orange flower Orange Oil
Thin-layer chromatography (2.2.27). DEFINITION
Test solution To 0.5 g of the powdered herbal drug (355) Orange Oil is obtained by mechanical means from the fresh
(2.9. 72) add 5 mL of methanol R. Heat with stirring at 40 °C peel of the sweet orange, Citrus sinensis (L.) Osbeck.
for 10 min. Filter. CHARACTERISTICS
Reference solution Dissolve 3.0 mg of naringin R and 3.0 mg of A yellow to yellowish brown liquid, visibly free from water;
hesperidin R in 10 mL of methanol R. odour, that of orange.
Plate TLC silica gel plate R. TESTS
Mobile phase water R, anhydrous formic acid R, ethyl acetate R Optical rotation
(10:15:75 VIVIV). -r94° to +99°, Appendix V F. On distillation, the first 10%
Application 10 pL as bands. of the distillate has an optical rotation the same as, or only
Development Over a path of 10 cm. slightly lower than, the original oil.
Drying In air; heat in an oven at 110-120 °C for 5 min. Refractive index
Detection Spray the hot plate with a 10 g/L solution of 1.472 to 1.476, Appendix V E.
diphenylboric acid aminoethyl ester R in methanol R and then Residue on evaporation
with a 50 g/L solution of macrogol 400 R in methanol R; after 1.0 to 5.0% when determined by the method for residue on
at least 1 h, examine in ultraviolet light at 365 nm. evaporation of volatile oils, Appendix X M. Use 2 g and heat
Results The chromatogram obtained with the test solution for 4 hours.
show’s a yellow' zone similar in position to the zone of Solubility in ethanol
naringin in the chromatogram obtained with the reference Soluble at 20°, in 7 pans of ethanol (90%), Appendix X M.
solution, and immediately below it a red zone (neoeriocitrin). A bright solution is rarely obtained due to the presence of
Loss on drying (2.2.22) waxy non-volatile substances.
Maximum 11.0 per cent, determined on 1.000 g of the Weight per mL
powdered herbal drug (355) (2.9.72) by drying in an oven at 0.842 to 0.848 g, Appendix V G.
105 °C. Content of aldehydes
Total ash {2.4.16) Not less than 1.0% พ/พ, calculated as decanal, C10H20O.
Maximum 10.0 per cent. Carry out the method for the determination of aldehydes,
Appendix X K, using 10 g, omitting the toluene and using a
ASSAY
volume, not less than 7 mL, of alcoholic hydroxylamine solution
Stock solution To 0.175 g of the powdered herbal drug (355)
that exceeds by 1 to 2 mL the volume of 0.5m potassium
(2.9.72) add 95 mL of ethanol (50 per cent VIV) R. Heat on a
hydroxide in ethanol (60%) KS required. Each mL of
water-bath under a reflux condenser for 30 min. Allow to
0.5m potassium hydroxide in ethanol (60%) KS is equivalent
cool and filter through a sintered-glass filter (2.7.2). Rinse
to 78.76 mg of C10H20O.
the filter with 5 mL of ethanol (50 per cent VIV) R. Combine
the filtrate and the rinsings in a volumetric flask and dilute to STORAGE
100.0 mL with ethanol (50 per cent VIV) R. Orange Oil should be kept in a well-filled container and
Test solution Into a test tube (10 mm X 180 mm) introduce protected from light.
0.150 g of powdered magnesium R (250) (2.9.72), a magnetic
stirring bar 25 mm long and 2.00 mL of the stock solution.
Maintain the test tube upright, centrifuge at 125 £ and
carefully add dropwise, especially at the beginning, 2.0 mL of
hydrochloric acid R, and then 6.0 mL of ethanol
Sweet Orange Oil * *
(50 per cent VIV) R. Stopper the tube and mix by inverting. (Ph. Eur. monograph 1811) ***
Compensation solution Into a 2nd test tube, introduce 2.00 mL PhEur_______________________________________________ —
of the stock solution and carefully add dropwise, especially at
the beginning, 2.0 mL of hydrochloric acid R and then 6.0 mL DEFINITION
of ethanol (50 per cent VIV) R. Essential oil obtained without heating, by suitable mechanical
treatment from the fresh peel of the fruit of Citrus
sinensis (L.) Osbeck {Citrus aurantium L. var. dulcis L.).
solution at 530 nm.
A suitable antioxidant may be added.
expressed as naringin, using the following expression: CHARACTERS
Appearance
Clear, pale yellow or orange, mobile liquid, which may
A X 9.62
become cloudy when chilled.
Characteristic odour of fresh orange peel.
i.e. taking the specific absorbance of the reaction product of IDENTIFICATION

naringin to be 52. First identification B
/4 = absorbance at 530 nm; Second identification A
m = mass of the substance to be examined, in grams. A. Thin-layer chromatography (2.2.27).
--------------------------------------------- ------ ------- ------------------------------------------------- Ph Eur Examine the chromatograms obtained in the test for
bergapten.
2016 Orange Oil IV-311
Results A See below the sequence of the zones present in the Drying In air.
chromatograms obtained with the reference solution and the Detection A Examine in ultraviolet light at 365 nm.
test solution.
Top 0 the plate shows no greenish-yellow fluorescent zone present in the
chromatogram obtained with the.reference solution.
Detection B spray with anisaldehyde solution R and heat at
Bergapten: a greenish-yellow 100-105 °C for 10 min; examine the plate in ultraviolet light
fluorescent zone at 365 nm.
Many blue fluorescent zones Gas chromatography (2.2.25): use the normalisation
procedure.
Test solution Dilute 300 |1L of the substance to be examined
Resuks B See below the sequence of the zones present in the to 1 mL with acetone R.
chromatograms obtained with the reference solution and the Reference solution (a) Dilute 10 pL of (L-pinene R3 10 pL of
test solution. p-pinene R, 10 pL of sabinene R3 20 pL of p-myrcene R3 800 pL
of limonene R> 10 pL of octanal R3 10 pL of decanal R3 10 pL
Top o the plate of linalol R3 10 pL of citral R (composed of neral and
geranial) and 10 pL of valencene R in 1 mL of acetone R.
A brown fluorescent zone
Reference solution (b) Dissolve 5 pL of P-pinene R in 10 mL of
Linalyl acetate: a A faint brownish-orange
brownish-orange fluorescent
acetone R. Dilute 0.5 mL to 10 mL with acetone R.
fluorescent zone (linalyl acetate)
Column:
— size: z = 30 m, 0 = 0.53 mm,
Many orange fluorescent zones
— stationary phase: macrogol 20 000 R (film thickness 1 pm).
Linalol: a brownish-orange A brownish-orange fluorescent
fluorescent zone
zone (linalol)
Bergapten ะ a faint greenish-yellow Flow rate 1 mLZmin.
fluorescent zone Split ratio 1:100.
Many brownish-orange Temperature:
fluorescent zones
Time Temperature
Many blue fluorescent zones (min) (°C)
Reference solution Column 0-6 50
Test solution
6 - 31 50-> 150
31 - 41 150-» 180
B. Examine the chromatograms obtained in the test for 41 - 55 180
chromatographic profile. Injection port 250
Results The characteristic peaks in the chromatogram Detector 250
solution.
Injection 0.5 pL.
TESTS
Relative density (2.2.5) solution (a). Record the retention times of these substances.
0.842 to 0.850.
System suitability Reference solution (a)
Refractive index (2.2.6) — resolution: minimum 3.9 between the peaks due to
1.470 to 1.476. p-pinene and sabinene and minimum 1.5 between the
Optical rotation (2.2.7) peaks due to valencene and geranial.
+ 94° to + 99°. Using the retention times determined from the
Peroxide value (2.5.5, Method B) chromatogram obtained with reference solution (a), locate
Maximum 20. the components of reference solution (a) in the
It complies with the test for fatty oils and resinified essential Determine the percentage content of these components.
oils. The limits are within the following ranges:
Bergapten — p-pinene: 0.02 per cent to 0.3 per cent,
Thin-layer chromatography (2.2.27). — sabinene: 0.2 per cent to 1.1 per cent,
Test solution Dilute 0.2 mL of the substance to be examined — p-myrcene: 1.7 per cent to 2.5 per cent,
in 1 mL of alcohol R. — limonene: 92.0 per cent to 97.0 per cent,
Reference solution Dissolve 2 mg of bergapten R3 10 |1L of — octanal: 0.1 per cent to 0.4 per cent,
linalol R and 20 |iL of linalyl acetate R in 10 mL of alcohol R. — decanal: 0.1 per cent to 0.4 per cent,
Plate TLC silica gel plate R. — linalol: 0.2 per cent to 0.7 per cent,
— neral: 0.02 per cent to 0.10 per cent,
Mobile phase ethyl acetate R3 toluene R (15:85 VIV).
— valenceneะ 0.02 per cent to 0.5 per cent,
Application 10 |1L, as bands. — geranial: 0.03 per cent to 0.20 per cent.
IV-312 Orange Oil 2016
Disregard limit Area of the peak in the chromatogram

obtained with reference solution (b) (0.01 per cent). Dried Bitter-Orange Peel **
Residue on evaporation (Bitter-Orange Epicarp and Mesocarp,
1.0 per cent to 5.0 per cent. Ph. Eur. monograph 1603)
Evaporate 5.0 g to dryness on a water-bath and dry at Preparations
100-105 °C for 4 h. Orange Peel Infusion
STORAGE Orange Tincture
At a temperature not exceeding 25 °C. Ph Eur _ ___________ —__
--------------------------------------------------------------------------------------------------------- - Ph Eur DEFINITION

Dried epicarp and mesocarp of the ripe fruit of Citrus
aurantium L. ssp. aurantium (C. aurantium L. ssp. amara
Engl.) partly freed from the white spongy tissue of the
Terpeneless Orange Oil mesocarp and endocarp.
Preparation Content
Compound Orange Spirit Minimum 20 mUkg of essential oil (anhydrous drug).
DEFINITION
Terpeneless Orange Oil may be prepared by concentrating IDENTIFICATION
orange oil under reduced pressure until most of the terpenes A. The herbal drug consists of elliptical or irregular pieces
have been removed or by solvent partition. 5-8 cm long, 3-5 cm broad and about 3 mm thick.
CHARACTERISTICS The outer surface is yellowish or reddish-brown and
distinctly punctate, the inner surface is yellowish or
A clear, yellow or orange-yellow liquid, visibly free from
brownish-white.
water.
TESTS
Optical rotation
Not more than 4-60c, Appendix V F.
Refractive index
Solubility in ethanol
Soluble, at 20°, in 1 part of ethanol (90%)), Appendix X M.
Weight per mL
Content of aldehydes
Not less than 18% พ/พ, calculated as decanal, C10H20O.
Carry out the method for the determination of aldehydes,
Appendix X K, using 1.5 g, omitting the toluene and using a
volume, not less than 7 mL, of alcoholic hydroxylamine solution
that exceeds by 1 to 2 mL the volume of 0.5m potassium
hydroxide in ethanol (60%) vs required. Each mL of
0.5m potassium hydroxide in ethanol (60%) vs is equivalent to
78.76 mg of CjoH2oO.
STORAGE
Terpeneless Orange Oil should be kept in a well-filled
container and protected from light.
Compound Orange Spirit

DEFINITION
Terpeneless Orange Oil 2.5 mL
Terpeneless Lemon Oil 25 pm
Anise Oil or Star Anise Oil 4.25 mL
Coriander Oil 6.25 mL Figure 1603.-1. - Illustration for identification test B of powdered
Ethanol (90 per cent) Sufficient to produce 1 ooo mL herbal drug of bitter-orange epicarp and mesocarp
The spirit complies with the requirements stated under Spirits and
with the following requirements. B. Microscopic examination (2.8.23). The powder is light
TESTS solution R. The powder shows the following diagnostic
Ethanol content characters (Figure 1603.-1): fragments of epicarp (surface
86 to 90% v/v, Appendix VUI F. view [B]) composed of small polygonal cells with slightly
Weight per mL thickened anticlinal walls, filled with chromatophores and
0.828 to 0.841 g, Appendix V G. some anomocytic stomata (2.8.3)‘, fragments of epicarp
2016 Orange Preparations IV-313
showing the thickened cuticle (side view [N]); fragments of to dryness on a water-bath and dry in an oven at 100-105 °C
pencarp (transverse section [A]) showing the cuticularised for 3 h. Allow to cool in a desiccator over diphosphorus
epicarp [Aa], the sub-epicarpal layers with cells with pentoxide R and weigh. The residue weighs a minimum of
collenchymatous thickening [Ab], some of which contain a 0.100 g
pnsm of calcium oxalate [Ac], and fragments of
ASSAY
schizolysigenous oil glands [Ad]; fragments of the
collenchymatous sub-epicarpal layers (surface view [G]);
Use a 500 mL round-bottomed flask, 200 mL of water R as
groups of parenchyma cells [L], some of which contain a
the distillation liquid and 0.50 mL of xylene R in the
prism of calcium oxalate [C]; numerous fragments of
graduated tube. Reduce the drug to a powder (710) (2.9.72)
mesocarp [D, E, F, H, K, M]; free prisms of calcium
and immediately use 15.0 g for the determination. Distil at a
oxalate [J]. Examine under a microscope using a 20 g/L
rate of 2-3 mUmin for 90 min.
solution of potassium hydroxide R. The mounting medium
becomes yellow due to the presence of hesperidin in the _______ ____________________________________________________ Ph Ear
herbal drug.
(2.9.72) add 10 mL of methanol R and heat in a water-bath
Orange Peel Infusion
at 65 °C for 5 min with shaking. Allow to cool and filter. DEFINITION
Reference solution Dissolve 1.0 mg of naringin R and 1.0 mg of Concentrated Orange Peel Infusion 100 mL
caffeic acid R in 1 mL of methanol R. Water Sufficient to produce 1000 mL
Plate TLC silica gel plate R. The infusion complies with the requirements stated under Infusions.
Mobile phase water R, anhydrous formic acid R, ethyl acetate R
(10:15:75 VIVIV). CONCENTRATED ORANGE PEEL
Application 20 pL as bands. INFUSION
Development Over a path of 10 cm. DEFINITION
Drying In air, then in an oven at 110-120 °C for 5 min. Dried Bitter-Orange Peel, cut small 500 g
Detection Spray the warm plate with a 10 g/L solution of Ethanol (25 per cent) 1350 mL
diphenylboric acid aminoethyl ester R in methanol R and then Extemporaneous preparation
with a 50 g/L solution of macrogol 400 R in methanol R. After The following directions apply.
at least 1 h, examine in ultraviolet light at 365 ท๓. Macerate the Dried Bitter-Orange Peel in a covered vessel for
Results See below the sequence of zones present in the 48 hours with 1000 mL of the Ethanol (25 per cent) and
chromatograms obtained with the reference solution and the press out the liquid. To the pressed marc add 350 mL of the
test solution. Furthermore, other fluorescent zones are Ethanol (25 per cent), macerate for 24 hours, press and add
present in the chromatogram obtained with the test solution. the liquid to the product of the first pressing. Allow to stand
for not less than 14 days and filter.
Top of the plate TESTS
A light blue fluorescent zone Ethanol content
18 to 23% v/v, Appendix VUI F.
Total solids
Caffeic acid: a light blue 10 to 15% w/v, Appendix XI A. Use 1 mL.
fluorescent zone
A light blue fluorescent zone Weight per mL
Naringin: a dark green A dark green fluorescent zone

fluorescent zone (naringm)
(neoeriocitrin)
Orange Tincture * I
An orange fluorescent zone (Bitter-Orange Epicarp and Mesocarp Tincture, *
Reference solution Test solution Ph Eur monograph 1604)
Ph Elf-------------------------------------------------------------------------------------------------------- -
DEFINITION
TESTS
Tincture produced from Bitter-orange epicarp and
Water (2.2. 13)
mesocarp (1603).
xMaximum 10.0 per cent, determined by distillation on 20.0 g
of the powdered herbal drug (355) (2.9.72). PRODUCTION
The tincture is produced from 1 part of the freshly powdered
Total ash (2.4.16)
herbal drug (2000) (2.9.72) and 5 parts of alcohol
(70 per cent VIV) by an appropriate procedure.
Extractable matter
Minimum 25.0 per cent. CHARACTERS
To 2.000 g of the powdered herbal drug (250) (2.9.72) add Liquid with a bitter taste.
10.0 mL of a mixture of 30 volumes of water R and
70 volumes of ethanol (96 per cent) R and extract for 2 h,
shaking frequently. Filter, evaporate 2.000 mL of the filtrate
IV-314 Orange Preparations 2016
IDENTIFICATION
Examine by thin-layer chromatography (2.2.27). Oregano * *
Test solution The tincture to be examined. (Ph. Eur. monograph 1880) ***
Reference solution Dissolve 1.0 mg of naringin R and 1.0 mg of PhEur________________________________________________ -____________
caffeic acid R in 1 mL of methanol R.
DEFINITION
Plate TLC silica gel plate R. Dried leaves and flowers separated from the stems of
Mobile phase water R, anhydrous formic acid R, ethyl acetate R Origanum onites L. or Origanum vulgare L. subsp. hirtum
(10:15:75 PZP7F). (Link) letsw.j or a mixture of both species.
Application 20 |1L, as bands. Content
Development Over a path of 10 cm. — essential oil: minimum 25 mL/kg (anhydrous drug);
Drying In air, and heat in an oven at 110-120 cc for 5 min. — sum of the contents of carvacrol and thymol (both C10H14O;
Mx 150.2): minimum 60 per cent in the essential oil.
Detection Spray the warm plate with a 10 g/L solution of
diphenylboric acid aminoethyl ester R in methanol R and then IDENTIFICATION
with a 50 g/L solution of macrogol 400 R in methanol R. After A. o. onites. The leaf is yellowish-green, usually 4-22 mm
1 h, examine in ultraviolet light at 365 nm. long and 3-14 mm wide. It has a long or short petiole or is
Results See below the sequence of the zones present in the sessile. The lamina is ovate, elliptic or ovate-lanceolate.
chromatograms obtained with the reference and test Margins are entire or serrate, the apex is acute or obtuse.
solutions. Furthermore, other zones are present in the The veins are yellowish and conspicuous on the adaxial
chromatogram obtained with the test solution. surface. Flowers are solitary or seen as broken parts of the
corymb. The calyx is bract-like and inconspicuous.
The corolla is white, on top of inflorescences or single
Top of the plate
flowers, or inconspicuous. The bracts arc imbricate and
A light blue fluorescent zone green like the leaves. The drug contains yellowish or
A light blue fluorescent zone yellowish-brown stem parts.
o vulgare (subsp. hirtum). The leaf is green and usually
Caffeic acid: a light blue fluorescent
3-28 mm long and 2.5-19 mm wide. It is petiolate or sessile.
A light blue fluorescent zone The lamina is ovate or ovate-eliptic. The margins are entire
or serrate, the apex is acute or obtuse. Flowers are rare,
found as broken parts of the corymbs. Bracts are greenish-
Naringin: a dark green fluorescent A dark green fluorescent zone
(naringin)
yellow and imbricate. The calyx is corolla-like and
A red fluorescent zone inconspicuous. The corolla is white, on top of inflorescences,
(neoeriocitrin) slightly conspicuous or inconspicuous.
B. Reduce to a powder (710) (2.9.12). The powder is green
Reference solution Test solution (O. vulgare) or yellowish-green (O. onites). Examine under a
microscope using chloral hydrate solution R (Figure 1880.-1).
o onites Powder shows fragments of leaf epidermis [A, D, G]
TESTS composed of cells with sinuous walls, diacytic stomata
Ethanol content (2.9.10) (2.8.3) [Ga], covering trichomes and glandular trichomes;
63 per cent to 67 per cent v/v. there are 2 types of glandular trichomes: some of lamiaceous
Methanol and 2-propanol (2.9.11) type with 8-16 cells, in surface view [Da], and a very
Maximum 0.05 per cent v/v of methanol and maximum common type with a unicellular head and uni- [Gc], bi- [II]
0.05 per cent VIV of 2-propanol. or tricellular stalk; the covering trichomes have smooth, thick
walls; some are multicellular [B, Gb], often broken [Aa], and
Dry residue
contain prisms of calcium oxalate, while others, which are
Minimum 6.0 per cent mlnI, determined on 2.00 g of
rare, are unicellular and conical [C]; scars from covering and
tincture to be examined.
glandular trichomes are visible on the epidermises [Gd, Ge];
________________________________________________________________ PhEur pollen grains, with smooth exine, are frequent [E, F].
o vulgare Subsp. hirtum powder shows fragments of the
upper epidermis with cells with sinuous, beaded walls,
accompanied by palisade parenchyma [J]; fragments of the
Orange Syrup lower epidermis [N] composed of cells with finely and
irregularly thickened walls, diacytic stomata (2.8.3) [Na],
DEFINITION covering trichomes and glandular trichomes; there are 2 types
Orange Tincture 60 mL of glandular trichomes: some of lamiaceous type with
Syrup Sufficient to produce 1000 mL 12 cells, in surface view [Nb], and a rare type with a
The syrup complies with the requirements stated under Oral unicellular head [Nc] and bi- or tricellular stalk; the covering
Liquids and with the following requirement. trichomes have thick, warty walls and contain fine needles of
calcium oxalate; some are conical, multicellular and serrate
TESTS [L, M], while others, which are rare, are unicellular [K];
Weight per mL there are occasional pollen grains, with smooth exine [E, F].
2016 Oregano IV-315
Top of the plate
A bluish-purple zone
A pale green zone
Thymol: a pink zone A pink zone (thymol)
Carvacrol ะ a pale violet zone A pale violet zone (carvacrol)
A pale purple zone
A grey zone
A pale green zone
A bluish-purple zone
An intense brown zone
TESTS
Water (2.2.13)
Total ash (2.4.16)
ASSAY
Figure 1880.-1. - Illustration for identification test B of powdered Essential oil (2.8.12)
herbal drug of oregano Use 30.0 g of the drug to be examined, a 1000 mL round-
c. Thin-layer chromatography (2.2.27). bottomed flask and 400 mL of water R as the distillation
liquid. Distil at a rate of 2-3 mUmin for 2 h without xylene R
in the graduated tube.
(2.9.72) add 5 ml of methylene chloride R and shake for
3 min, then filter through about 2 g of anhydrous sodium Carvacrol and thymol
sulfate R. Gas chromatography (2.2.28): use the normalisation
procedure.
Reference solution Dissolve 1 mg of thymol R and 10 pL of
carvacrol R in 10 mL of methylene chloride R. Test solution Filter the essential oil obtained in the assay of
essential oil over a small amount of anhydrous sodium sulfate R
Blate TLC silica gel plate R.
and dilute to 5.0 mL with heptane R by rinsing the apparatus
Mobile phase methylene chloride R. and the anhydrous sodium sulfate.
Application 20 pL as bands. Reference solution Dissolve 0.20 g of thymol R and 50 mg of
Development Over a path of 15 cm. carvacrol R in heptane R and dilute to 5.0 mL with the same
Drying In air. solvent.
Detection Spray with anisaldehyde solution R using 10 mL for a Column:
plate 200 mm square and heat at 100-105 °C for 10 min. — material: fused silica;
Results See below the sequence of zones present in the — size: / = 60 m, 0 = 0.25 mm;
chromatograms obtained with the reference solution and the — stationary phase: macrogol 20 000 R (film thickness
test solution. Furthermore, other zones are present in the 0.25 pm).
lower third and upper part of the chromatogram obtained Carrier gas nitrogen for chromatography R or helium for
with the test solution. chromatography R.
Split ratio 1:100.
Temperature:
Time Temperature
(min) ___________ cs__________
Column 0 - 45 40 -> 250
Injection port 190
Detector 210

IV-316 Orientvine Stem 2016
Injection 0.2 pL. thin-walled cells containing fine, needle-shaped crystals of

Elution order Order indicated in the composition of the calcium oxalate; fragments of xylem consisting of reticulate or
reference solution; record the retention times of these pitted vessels, up to 200 pm in diameter, accompanied by
substances. ligneous parenchyma with slightly and regularly thickened
System suitability, reference solution: and pitted cells. Examine under a microscope using a
— resolution: minimum 1.5 between the peaks due to thymol 50 per cent VIV solution of glycerol R. The powder shows
and carvacrol. simple, spherical starch granules about 10 pm in diameter,
Using the retention times determined from the free or contained in parenchymatous cells.
chromatogram obtained with the reference solution, locate c. Thin-layer chromatography (2.2.27).
the components of the reference solution in the Test solution To 2 g of the powdered herbal drug (355)
chromatogram obtained with the test solution. (2.9.12) add 25 mL of ethanol (96 per cent) R and heat under
Determine the percentage content of the sum of carvacrol reflux for 1 h. Filter and evaporate the filtrate to dryness.
and thymol. Dissolve the residue in 2 mL of ethanol (96 per cent) R.
----------------------------------------------------------------------------------------- _____ PhEur
Reference solution Dissolve 5 mg of sinomenine R and 5 mg of
papaverine hydrochloride R in 5 mL of ethanol (96 per cent) R.
Mobile phase concentrated ammonia R) water R, toluene R,
methanol R, ethyl acetate R (2:10:20:30:40 VIVIVIVIV).
Orientvine stem * * Application 8 pL as bands of 8 mm.
(Ph. Eur. monograph 2450) *** Development Over a path of 6 cm.
Ph Etr_______________________________________________________________ Drying In air.
DEFINITION Detection A Examine in ultraviolet light at 254 nm.
Dried, whole or fragmented stem of Sinomenium acutum Results A Sec below the sequence of zones present in the
(Thunb.) Rehder et E.H.Wilson, collected in late autumn chromatograms obtained with the reference solution and the
and early winter. test solution. Furthermore, other faint fluorescent zones may
Content
solution.
Minimum 0.5 per cent of sinomenine (C19H23NO4;
Mr 329.4) (driecLdrug). Top of the plate
IDENTIFICATION
Papaverine: a quenching zone
A. Whole drug. Long cylindrical stem, somewhat curved,
60 cm long or more, 0.5-2 cm in diameter. The outer bark is A quenching zone
greenish-brown or brown, sometimes greyish-brown, wi±

relatively wide longitudinal striations and prominent
verrucose lenticels; the nodes are slightly swollen and
branched. The texture is light, hard and difficult to break; Sinomenine: a quenching zone A quenching zone (sinomenine)
the fracture is uneven, greyish-yellow or pale greyish-brown;
the bark is thin (about 1/10 of the diameter); the medullary
rays are very conspicuous; the pith is yellowish-white or pale
yellowish-brown. A dark blue fluorescent zone
Fragmented drug Fragments of stems, in discs, about 1.5 cm
in diameter and 0.3 cm thick, with greenish-brown, brown or
greyish-brown outer surface; a transverse section shows a
narrow, pale yellow cortical zone, it is mainly occupied by Detectio?! B Treat with a 10 g/L solution of sodium nitrite R in
the vascular system (about 3/4 of the section) consisting of potassium iodobismuthate solution R5 and allow to dry in air.
very numerous vascular bundles (about 15-20) in a circle Examine in daylight.
around the yellowish-white or pale yellowish-brown, small, Results B See below the sequence of zones present in the
circular pith; each bundle is delimited on the outside by a chromatograms obtained with the reference solution and the
narrow and continuous, wavy, light brown zone and is test solution. Furthermore, other faint zones may be present
separated from the next bundle by a narrow, light brown in the chromatogram obtained with the test solution.
medullary ray; the xylem vessels with a relatively wide
interior lumen are clearly visible.
Top of the plate
yellowish-brown or greyish-brown. Examine under a An orange zone
Papaverine: an orange zone
shows the following diagnostic characters: rare fragments of
3 light orange zones
epidermis with polyhedral cells, in surface view, covered with
a thick, pale yellow cuticle about 50 pm in diameter; Sinomenine: an orange zone An orange zone (sinomenine)
sclereids isolated or in groups, of various sizes and shapes
(subsquare, fusiform, elliptical or irregular), with thickened,
pitted walls with conspicuous pit canals, free or included in 2 orange zones
fragments of parenchyma; pale yellow or yellow fibres, A light orange zone
30-70 pm in diameter with thick, distinctly channelled walls
and a very narrow lumen; fragments of parenchyma with Reference solution Test solution
2016 Wild Pansy IV-317
TESTS
Loss on drying (2.2.32) Wild Pansy ;* **
Maximum 10.0 per cent, determined on 1.000 g of the (Wild Pansy (Flowering Aerial Parts), ***
herbal drug (355) (2.9.72) by drying in an oven at Ph Eur monograph 1855)
105 °C for 3 h.
PhEur___________________________________________________________
Total ash (2.4.16)
Dried flowering aerial parts of Viola aruensis Murray and/or
Aristolochic acids (2.8.21, Method A)
Viola tricolor L.
It complies with the test.
Content
ASSAY Minimum 1.5 per cent of flavonoids, expressed as violanthin
Liquid chromatography (2.2.29). (C27H3o014; A4r 578.5) (dried drug).
IDENTIFICATION
(3วว) (2.9.12) in 20.0 mL of ethanol (70 per cent VIV) R in a
A. The stem is angular and hollow. The leaves are oval,
conical flask and weigh. Sonicate for 20 min. Cool and weigh
petiolate, with a cordate base or elongated and obtuse, with
again. Compensate the loss of solvent with ethanol
lyrate stipules, divided in the middle. The flowers, with a
(70 per cent VIV) R and stopper the flask. Shake thoroughly
long peduncle, are zygomorphic, with 5 oval, lanceolate
and filter through a membrane filter (nominal pore size
0.45 pm). sepals, an appendage pointed outwards and 5 petals of which
the lower one bears a spur; in Viola aruensis, the petals are
Reference solution (a) Dissolve 3.0 mg of sinomenine CRS in shorter than the calyx, the lower petal is cream coloured,
methanol R and dilute to 10.0 mL with the same solvent. with black lines, the 4 upper petals may be cream coloured
Reference solution (b) Disperse 0.250 g of orientvine stem HRS or violet blue; in Viola tricolor, the petals are longer than the
in 10.0 mL of ethanol (70 per cent VIV) R in a conical flask calyx and violet coloured, more or less tinged with yellow.
and weigh. Sonicate for 20 min. Cool and weigh again. The androecium consisting of 5 stamens bears at the apex a
Compensate the loss of solvent with ethanol membranous connective appendage with 2 spurs.
(70 per cent VIV) R and stopper the flask. Shake thoroughly The trilocular ovary shows a short style and globular
and filter through a membrane filter (nominal pore size stigmata. The fruit are navicular capsules, three-lobed,
0.45 pm). yellowish brown, 5 mm to 10 mm long. The pale yellow,
Column'. pyriform seeds are about 1 mm long, bearing a caruncle.
— size: I = 0.15 m, 0 = 4.6 mm; B. Reduce to a powder (355) (2.9.12). The powder is
stationary phase: end-capped octadecylsilyl silica gel for greenish. Examine under a microscope using chloral hydrate
chromatography R (5 pm). solution R. The powder shows the following diagnostic
Mobile phase Adjust a 1.8 g/L solution of disodium hydrogen characters: fragments of the epidermis of the leaves in surface
phosphate R to pH 8.0 with a 0.8 g/L solution of sodium view with wavy-walled cells and anomocytic stomata (2.8.3);
dihydrogen phosphate R, then adjust to pH 9.0 with a 10 g/L conical unicellular covering trichomes, widened at the base
solution of triethylamine R. Mix 60 volumes of this solution and sharply pointed at the apex, with a striated cuticle;
with 40 volumes of acetonitrile R. glandular trichomes with a multicellular head, and a short,
Flow rate 1.0 mL/min. multicellular stalk in the indentations of the leaf margins;
cluster crystals of calcium oxalate, sometimes included in
Detection Spectrophotometer at 262 nm. parenchyma; fragments of the corolla with wavy-walled
Injection 20 pL. epidermal cells, those from the mid-region papillose and with
Retention time Sinomenine = about 3 min. some extended to form flask or bottle-shaped projections,
System suitability: reference solution (b) ะ those from the base of the petals with covering trichomes up
— resolution: minimum 1.5 between peak 1 and the peak due to about 300 pm long with characteristic hump-like swellings
to sinomenine; identify peak 1 using the chromatogram along their length; spherical or polyhedral pollen grains,
supplied with orientvine stem HRS. 60 pm to 80 pm in diameter, with finely pitted exines and 5
Calculate the percentage content of sinomenine using the pores (Viola aruensis) or 4 pores (Viola tricolor)', occasional
following expression: fragments of spiral and reticulate vessels and groups of fibres
from the stem.
Al X 7712 X 2 X p
Test solution Heat in a water-bath at 65 °C for 5 min, with
A2 X mi
frequent stirring, 2.0 g of the powdered herbal drug (355)
(2.9.12) in 10 mL of alcohol (70 per cent VIV) R. Cool and
A1 = area of the peak due to sinomenine in the filter.
Reference solution Dissolve 2.5 mg of rutin R} 2.5 mg of
A2 = area of the peak due to sinomenine in the
hyperoside R and 1.0 mg of caffeic acid R in methanol R and
;
(a)
= mass of the herbal drug to be examined used to Plate TLC silica gel plate R.
prepare the test solution, in grams; Mobile phase anhydrous formic acid R3 acetic acid R, water R,
= mass of sinomenine CRS used to prepare reference ethyl acetate R (11:11:27:100 VIV/VIV).
solution (a)> in grams; Application 10 pL, as bands.
p = assigned percentage content of sinomenine in Development Over a path of 12 cm.
sinomenine CRS.
PhEur
พ-318 Passion Flower 2016
Detection Spray with a solution containing 10 g/L of reduced pressure. Take up the residue with 8 mL of a
diphenylboric acid aminoethyl ester R and 50 g/L of macrogol mixture of 10 volumes of methanol R and 100 volumes of
400 R in methanol R. Allow the plate to dry in air for 30 min. glacial acetic acid R and transfer into a 25 mL volumetric
Examine in ultraviolet light at 365 nm. flask. Rinse the round-bottomed flask with 3 mL of a
Results See below the sequence of the zones present in the mixture of 10 volumes of methanol R and 100 volumes of
chromatograms obtained with the reference solution and the glacial acetic acid R and transfer into the same 25 mL
test solution. Furthermore, other zones may be present in the volumetric flask as used previously. Add 10.0 mL of a
chromatogram obtained with the test solution. solution containing 25.0 g/L of boric acid R and 20.0 g/L of
oxalic acid R in anhydrous formic acid R and dilute to 25.0 mL
with anhydrous acetic acid R.
Top of the plate
Compensation liquid Introduce 5.0 mL of the stock solution
Caffeic acid: a greenish-blue to
light blue fluorescent zone
into a round-bottomed flask and evaporate to dryness under
reduced pressure. Take up the residue with 8 mL of a
glacial acetic acid R and transfer into a 25 mL volumetric
glacial acetic acid R and transfer into the same 25 mL
Hyperoside: a yellowish-brown volumetric flask as used previously. Add 10.0 mL of
fluorescent zone anhydrous formic acid R and dilute to 25.0 mL with anhydrous
acetic acid R.
Measure the absorbance (2.2.25) of the test solution at
405 nm after 30 min.
Rutin: a yellowish-brown An intense yellowish-brown
fluorescent zone fluorescent zone (rutin) expressed as violanthin from the expression:
A yellowish-green fluorescent A X 1.25

m
A yellowish-green fluorescent A = measured absorbance at 405 nm,

___________________________________________________________ ___ PnEur
TESTS
Foreign matter (2. ร. 2) Passion Flower * *
Maximum 3 per cent. Passiflora ***
Swelling index (2. ร. 4)
.
(2.9.72) Preparation
Passion Flower Dry Extract
Ph Eur_______________________________________________________________
powdered herbal drug (355) (2.9.72) by drying in an oven at DEFINITION
105 °C for 2 h. Fragmented or cut, dried aerial parts of Passiflora
Total ash (2.4.76) incamata L. It may also contain flowers and/or fruits.
Maximum 15.0 per cent. Content
ASSAY Minimum 1.5 per cent of total flavonoids, expressed as
Stock solution In a 200 mL flask, introduce 0.300 g of the vitexin (C2iH2o010; Mr 432.4) (dried drug).
powdered herbal drug (250) (2.9.72) and 40 mL of alcohol IDENTIFICATION
(60 per cent V/V) R. Heat in a water-bath at 60 °C for A. The green or greenish-grey or brownish stem is ligneous,
10 min, shaking frequently. Allow to cool and filter through a hollow, longitudinally striated, glabrous or very slightly
plug of absorbent cotton into a 100 mL volumetric flask. pubescent, with a diameter that is generally less than 8 mm.
Transfer the absorbent cotton with the drug residue back The green or greenish-brown leaves are alternate, finely
into the 200 mL flask, add 40 mL of alcohol dentate and pubescent, deeply divided into 3 acute lobes of
(60 per cent VIV) R and heat again in a water-ba± at 60 °C which the central lobe is the largest. The midrib is much
for 10 min, shaking frequently. Allow to cool and filter into more prominent on the lower surface. The petiole is
the same 100 mL volumetric flask as used previously. Rinse pubescent and bears 2 dark nectaries near the lamina.
the 200 mL flask with a further quantity of alcohol The tendrils are very numerous and grow from the axils of
(60 per cent VIV) R, filter and transfer to the same 100 mL the leaves; they are fine, smooth, round and terminated in
volumetric flask. Dilute to volume with alcohol cylindrical spirals. The radiate flowers, if present, have 3
(60 per cent VIV) R and filter. small bracts and a corolla consisting of 5 white, elongated
Test solution Introduce 5.0 mL of the stock solution into a petals with several rows of filiform, petaloid appendices.
round-bottomed flask and evaporate to dryness under If present, the greenish or brownish fruit is flattened and
2016
Passion Flower Preparations IV-319
o\nl; It contains several flattened, brownish-yellow, pitted ASSAY

seeds.
B. Reduce to a powder (355) (2.9.72). The powder is light 0.200 g of the powdered herbal drug (250) (2.9.72) and add
green. Examine under a microscope using chloral hydrate 40 mL of ethanol (60 per cent VIV) R. Heat in a water-bath at
solution R. The powder shows the following diagnostic 60 °C under a reflux condenser for 30 min while shaking
characters: fragments of the leaf epidermis with sinuous walls frequently. Allow to cool and filter the mixture through a
and anomocytic stomata (2.5.5); numerous cluster crystals of plug of absorbent cotton in a 100 mL flask. Transfer the
calcium oxalate isolated or aligned along the veins; many absorbent cotton with ±e drug residue into the round-
isolated or grouped fibres from the stems associated with bottomed flask. Add 40 mL of ethanol (60 per cent VIV) R
pitted vessels and tracheids; uniseriate trichomes with 1-3 and heat again in a water-bath at 60 °C under reflux for
thin-walled cells, straight or slightly curved, ending in a point 10 min. Allow to cool and filter the mixture and the first
or sometimes a hook. In addition, the powder shows, if filtrate from the 100 mL flask through a filter paper into the
flowers are present, papillose epidermises of the petals and 100 mL volumetric flask. Dilute to 100 mL with the same
appendages and pollen grains with a reticulate exine; and if solvent, while rinsing the flask, round-bottomed flask and
mature fruits are present, scattered brown tannin cells and filter.
brownish-yellow, pitted fragments of the testa. Test solution Introduce 5.0 mL of stock solution into a flask.
c. Examine the chromatograms obtained in the test for other Evaporate to dryness under reduced pressure and take up the
species of Passiflora. residue with 10 mL of a mixture of 10 volumes of methanol R
Results The chromatogram obtained with the test solution and 100 volumes of glacial acetic acid R. Add 10 mL of a
shows below the zone due to rutin in the chromatogram solution consisting of 25 g/L of boric acid R and 20 g/L of
obtained with the reference solution a zone of intense yellow oxalic acid in anhydrous formic acid R and dilute to 25.0 mL
fluorescence, above it a zone of green fluorescence with anhydrous acetic acid R.
(diglycosylflavone), below the zone due to hyperoside in the Compensation liquid Introduce 5.0 mL of the stock solution
chromatogram obtained with the reference solution a zone of into a second flask. Evaporate to dryness under reduced
yellow fluorescence (iso-orientin) and above a zone of green pressure and take up the residue with 10 mL of a mixture of
fluorescence (isovitexin), above the zone due to hyperoside in 10 volumes of methanol R and 100 volumes of glacial acetic
the chromatogram obtained with the reference solution a acid R. Add 10 mL of anhydrous formic acid R and dilute to
zone of brownish-yellow fluorescence (orientin) and above it 25.0 mL with anhydrous acetic acid R.
a zone of green fluorescence (vitexin). These latter 2 zones After 30 min, measure the absorbance (2.2.25) of the test
may be absent. Further zones may be present. solution at 401 nm, by comparison with the compensation
TESTS liquid.
Other species of Passiflora Calcdate the percentage content of total flavonoids,
Thin-layer chromatography (2.2.27). expressed as vitexin, using the following expression:
A X 0.8
(2.9.12) add 5 mL of methanol R. Heat to boiling under a
reflux condenser for 10 min. Cool and filter. m
Reference solution Dissolve with heating 2.0 mg of rutin R and i.e. taking the specific absorbance of vitexin to be 628.
2.0 mg of hyperoside R in 10 mL of methanol R.
A = absorbance at 401 nm,
Plate TLC silica gel plate R. m = mass of the herbal drug to be examined, in grams.
Mobile phase anhydrous formic acid R, water Rj methyl ethyl _____________________________________________________________ PhEur
Drying In air. Passion Flower Dry Extract * **
aminoethyl ester R in methanol R and then with a 50 g/L (Ph. Eur. monograph 1882) *★*
solution of macrogol 400 R in methanol R. Allow to dry in air PhEur__________________________________________________________ —
for 30 min. Examine in ultraviolet light at 365 nm.
DEFINITION
Results The chromatogram obtained with the reference Dry extract produced from Passion flower (1459).
solution shows in the lower third a zone of yellowish-brown
Content
fluorescence due to rutin and in the middle third a zone of
Minimum 2.0 per cent of flavonoids, expressed as vitexin
yellowish-brown fluorescence due to hyperoside.
(C21H20O10; Afr 432.4) (dried extract).
The chromatogram obtained with the test solution shows no
intense zones of greenish-yellow or orange-yellow PRODUCTION
fluorescence between the zone due to diglycosylflavones and The extract is produced from the herbal drug and ethanol
that due to iso-orientin (P. coerulea and p. edulis). (40 per cent VIV to 90 per cent VIV) 3 methanol
Total ash (2.4.16) (60 per cent VIV) or acetone (40 per cent VIV) by an
Maximum 13.0 per cent. appropriate procedure.
Loss on drying (2.2.52) CHARACTERS
Maximum 10.0 per cent, determined on 1.000 g of the Appearance
powdered herbal drug (355) (2.9.72) by drying in an oven at Greenish-brown amorphous powder.
105 °C for 2 h. IDENTIFICATION
IV-320 Pelargonium Root 2016
Tat solution To 0.25 g of the extract to be examined add flask. Rinse the round-bottomed flask with 3 mL of a
methanol R. Shake, filter and dilute to 5 mL with methanol R. mixture of 10 volumes of methanol R and 100 volumes of
Reference solution Dissolve 2.0 mg of hyperoside R and 2.0 mg glacial acetic acid R and transfer into the 25 mL volumetric
of rutin R in methanol R and dilute to 10 mL with the same flask. Add 10.0 mL of anhydrous formic acid R and dilute to
solvent. 25.0 mL with anhydrous acetic acid R.
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel After 30 min, measure the absorbance (2.2.25) of the test
plate R (2-10 pm)]. solution at 401 nm.
Mobile phase anhydrous formic acid R, water Ry methyl ethyl Calculate the percentage content of total flavonoids,
ketone Ry ethyl acetate R (10:10:30:50 VIVIVIV). expressed as vitexin, from the following expression:
A X 0.8
rn
Detection Spray with a 10 g/L solution of diphenylboric acid i.e. taking the specific absorbance of vitexin to be 628.
aminoethyl ester R in methanol R. Subsequently spray with a /4 = absorbance at 401 nm,
50 g/L solution of macrogol 400 R in methanol R. Allow the m = mass of the extract to be examined, in grams.
plate to dry in air for about 30 min. Examine in ultraviolet ______________________________________________________________ PhEuf
light at 365 nm.
test solution. Other fluorescent zones may be present in the
chromatogram obtained with the test solution. Pelargonium Root t **
Top of he plate
PhEur______________________________________________________________
DEFINITION
Dried, usually fragmented, underground organs of
Pelargonium sidoides DC and/or Pelargonium reniforme Curt.
Hyperoside ะ a yellowish-orange Content
fluorescent zone Minimum 2.0 per cent of tannins, expressed as pyrogallol
A green fluorescent zone (C6H603; Alr 126.1) (dried drug).
A yellow fluorescent zone IDENTIFICATION
Rutin: a yellowish-orange A. The root is covered with dark, partly reddish-brown,
fluorescent zone longitudinally fissured bark. The transverse section shows,
underneath the cork layer, yellow or white wood, which
A green fluorescent zone clearly shows partly brownish medullary rays.
B. Reduce to a powder (355 (2.9.72)). The powder is
brownish-red. Examine under a microscope using chloral
Reference solution Test solution hydrate solution R. The powder shows the following diagnostic
characters: multilayer cork cells consisting of almost uniform,
rectangular cells; fragments of parenchyma underneath the
TESTS cork containing sclereids with a wide lumen; numerous
Loss on drying (2.8.17) calcium oxalate cluster crystals. Examine under a microscope
Maximum 5.0 per cent, determined on 0.500 g. using a 50 per cent VIV solution of glycerol R. The powder
ASSAY shows simple starch granules without striations or cracks.
Stock solution To 50 mg of the extract to be examined add c. Thin-layer chromatography (2.2.27).
ethanol (60 per cent VIV) R. Shake, filter and dilute to Test solution To 0.5 g of the powdered herbal drug (355)
100.0 mL with ethanol (60 per cent VIV) R. (2.9.72) add 10 mL of methanol Ry shake for 15 min and
Tat solution Introduce 5.0 mL of the stock solution into a filter.
round-bottomed flask and evaporate to dryness under Reference solution Dissolve 1 mg of scopoletin R and 2 mg of
reduced pressure. Take up the residue with 8 mL of a esculin R in 20 mL of methanol R.
mixture of 10 volumes of methanol R and 100 volumes of Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel
glacial acetic acid R and transfer into a 25 mL volumetric F254 plate R (2-10 pm)].
Mobile phase water Ry methanol Ry ethyl acetate R
(10:14:76 VIVIV).
flask. Add 10.0 mL of a solution containing 25.0 g/L of boric Application 10 pL [or 5 pL] as bands.
acid R and 20.0 g/L of oxalic acid R in anhydrous formic acid R Development Over a path of 10 cm [or 6 cm].
and dilute to 25.0 mL with anhydrous acetic acid R. Drying In air.
Compensation liquid Introduce 5.0 mL of the-stock solution Detection Spray with alcoholic potassium hydroxide solution R.
into a round-bottomed flask and evaporate to dryness under Examine in ultraviolet light at 365 nm.
reduced pressure. Take up the residue with 8 mL of a Results See below the sequence of zones present in the
mixture of 10 volumes of methanol R and 100 volumes of chromatograms obtained with the reference solution and the
glacial acetic acid R and transfer into a 25 ml. volumetric test solution. Furthermore, other blue fluorescent zones may
2016 White Peony Root IV-321
be present in the chromatogram obtained with the test accompanying the vessel fragments, they are 15 to 40 pm in
solution. diameter, with thickened, slightly lignified and frequently
pitted walls. Examine under a microscope using a 50% v/v
Top of the plate solution of glycerol in water. Many of the parenchymatous
cells contain masses of starch grains which may also be found
scattered throughout the powder.
Scopoletin: a very bright blue A weak blue fluorescent zone c. In the Assay, the chromatogram obtained with solution
fluorescent zone (scopoletin)
(1) shows a peak with the same retention time as the
principal peak in the chromatogram obtained with
One or two bright blue fluorescent solution (2).
D. Carry out the method for thin-layer chromatography,
Esculin ะ a very bright blue
fluorescent zone
A blue fluorescent zone (1) Shake 0.5 g of the powdered root with 10 mL of ethanol
for 5 minutes, filter and evaporate the filtrate to dryness and
A weak blue fluorescent zone dissolve the residue in 1 mL of absolute ethanol.
(2) 0.1% w/v each of paeoniflorin BPCRS and 4'-
hydroxyacetophenone BPCRS in ethanol.
(a) Use as the coating silica gel (Merck silica gel 60 precoated
plates are suitable).
TESTS (b) Use the mobile phase described below.
Loss on drying (2.2.52) (c) Apply 10 pL for each solution, as a band.
105 °C. (e) Dry the plate in air. spray with a 5% w/v solution of
vanillin in sulfuric acid, heat at 105° for 5 minutes and
Total ash (2.4.16)
examine immediately.
MOBILE PHASE
iMaximum 3.0 per cent. 0.2 volumes of formic acid, 5 volumes of ethyl acetate,
10 volumes of methanol and 40 volumes of dichloromethane.
ASSAY
SYSTEM SUITABILITY
Tannins (2.8.14)
Use 0.750 g of the powdered herbal drug (180) (2.9.12). The test is not valid unless, in the chromatogram obtained
with solution (2), two clearly separated bands are observed.
---------------------------------------------------------------------------------------------------------- Ph Eur
CONFIRMATION
The bluish-purple band with an Rf value of approximately
0.4 in the chromatogram obtained with solution (1)
corresponds in colour and position to that in the
White Peony Root chromatogram obtained with solution (2). Other bands are
DEFINITION present in the chromatogram obtained with solution (1) as
White Peony Root is the dried whole root of Paeonia lactiflora shown below.
Pallas (Paeonia albiflora Pallas; Paeonia edulis Salisb.; Paeonia
officinalis Thunb.). It contains not less than 1.6% of Top of the plate
paeoniflorin (C23H28O11), calculated with reference to the
A dark pink band
dried material.
It is collected in summer and autumn, washed clean, with
the two ends and rootlet removed and dried. Several bluish bands 4‘-hydroxyacetophenone:
an orange band
IDENTIFICATION
A. Cylindrical, straight or slightly curved, two ends truncate,
5 to 20 cm long, 1 to 2.5 cm in diameter. Externally light
brown to reddish brown, with longitudinal wrinkles, rootlet A bluish-purple band Paeoniflorin:
a bluish-purple band
scars and with laterally elongated lenticels. Texture compact,
easily broken, fracture relatively even, internally whitish or
pale brownish-red. Cambium ring distinct and rays radial.
B. Reduce to a powder. The powder is pale yellow. Examine
under a microscope using chloral hydrate solution. The powder Solution (1)____ Solution (2)
shows abundant rounded, rectangular or elongated
parenchymatous cells which occur singly or in groups, some
with pitted or slightly beaded walls; single cluster crystals of
calcium oxalate, 11 to 35 pm in diameter, isolated or packed TESTS
in rows in parenchymatous cells, sometimes with several Tree Peony
crystals in each cell; fragments of lignified vessels, 20 to In the Assay, the chromatogram obtained with solution (1)
65 pm in diameter, usually reticulately thickened but shows no peak with a relative retention of approximately 1.2
occasionally bordered-pitted; fibres occur rarely, usually with reference to paeoniflorin.
IV-322 White Peony Root 2016
Loss on drying A2 = Area of the peak due to paeoniflorin in the

When dried for 3 hours at 100° to 105°, loses not more than chromatogram obtained with solution (2).
12.0% of its weight. Use 1 g. 1ท1 = Weight of the drug being examined in mg.
Acid-insoluble ash m2 = Weight of paeoniflorin BPCRS in mg.
Not more than 0.5%, Appendix XI K. V1 = Dilution volume of solution (1) in mL.
V2 = Dilution volume of solution (2) in mL.
Ash
p = Percentage content of C23H28o 11 in paeoniflorin
Not more than 6.5%, Appendix XI J.
BPCRS.
Cadmium d = Percentage loss on drying of the herbal drug being
Maximum 3 ppm, Appendix vn. examined.
Lead
STORAGE
Maximum 5 ppm, Appendix vn. White Peony Root should be protected from moisture.
ASSAY
Carr}7 out the method for liquid chromatography,
Appendix in D, using the following solutions prepared in
0.05m potassium dihydrogen orthophosphate (solution A).
(1) Finely powder not less than 5 g of the herbal drug being Processed White Peony Root
examined. Mix 0.15 g of the powdered drug with 3 mL of DEFINITION
methanol and place in an ultrasonic bath for 30 minutes. Processed White Peony Root is White Peony Root that has
Dilute to 10 mL with solution A, mix, filter through a 0.45- been the boiled, peeled and dried. It contains not less than
pm filter and use the filtrate. 1.6% of paeoniflorin (C23H28O11), calculated with reference
(2) Dissolve a quantity of paeoniflorin BPCRS in methanol and to the dried material.
dilute with solution A to produce a solution containing
PRODUCTION
0.05% w/v in 3 volumes of methanol and 7 volumes of
It is collected in summer and autumn, washed clean, with
solution A and mix.
the two ends and rootlets removed, either peeled after boiling
(3) Dissolve a quantity of 4'-hydroxyacetophenone BPCRS in in water or boiled in water after peeling and dried in the รนท.
solution (2) to produce a solution containing 0.05% w/v of The dried root is washed and soaked thoroughly prior to
4 '-hydroxyacetophenone. being sliced transversely or longitudinally and dried.
IDENTIFICATION
(a) Use a stainless steel column (15 cm X 4.6 mm) packed A. The root slices are greyish-white to pale brown, darker at
with octadecylsilyl silica gel for chromatography (5 pm) (Luna the edges, and up to about 2 mm thick. They are smooth
C18 is suitable). and compact with a short and even fracture. Those that have
(b) Use isocratic elution and the mobile phase described been cut transversely occur in oval pieces up to about 4.5 cm
below. in diameter and the cut surface shows a distinctly radiate
(c) Use a flow rate of 1 mL per minute. xylem with narrow lines of vascular tissue separated by wide
(d) Use a column temperature of 30°. medullary rays. The longitudinally cut slices arc up to 10 cm
long and 1.5 cm wide and the cut surface shows raised
(e) Use a detection wavelength of 230 nm.
strands of vascular tissue running longitudinally.
B. Reduce to a powder. The powder is pale yellow. Examine
MOBILE PHASE under a microscope using chloral hydrate solution. The powder
30 volumes of methanol and 70 volumes of solution A. shows abundant rounded, rectangular or elongated
SYSTEM SUITABILITY
parenchymatous cells which occur singly or in groups, some
with pitted or slightly beaded walls; many of the cells contain
The test is not valid unless, in the chromatogram obtained pale yellow or pinkish masses of gelatinised starch which are
with solution (3), the number of theoretical plates for the peak also found scattered throughout the powder; single cluster
due to paeoniflorin is at least 3000, the symmetry factor for crystals of calcium oxalate, 11 to 35 pm in diameter, isolated
the peak due to paeoniflorin is not more than 1.3 and the or packed in rows in parenchymatous cells, sometimes with
resolution factor between the two peaks is not less than 1.7. several crystals in each cell; fragments of lignified vessels,
DETERMINATION OF CONTENT 20 to 65 pm in diameter, usually reticulately thickened but
Using the retention time and the peak area from the occasionally bordered-pitted; fibres occur rarely, usually
chromatograms obtained with solution (2), locate and accompanying the vessel fragments, they are 15 to 40 pm in
integrate the peak due to paeoniflorin in the chromatogram diameter, with thickened, slightly lignified and frequently
obtained with solution (1). pitted walls.
Calculate the content of C23H28011 in the sample, using the c. In the Assay, the chromatogram obtained with solution
declared content of paeoniflorin (C23H28011) in (1) shows a peak with the same retention time as the
paeoniflorin BPCRS and the following expression: principal peak in the chromatogram obtained with
solution (2).
D. Carry out the method for thin-layer chromatography3
A^nr^v, 100 Appendix in A, using the following solutions.
A/ \4 Xm/PX100-d
(1) Shake 0.5 g of the powdered root with 10 mL of ethanol
for 5 minutes, filter, evaporate the filtrate to dryness and
dissolve the residue in 1 mL of ethanol.
Al = Area of the peak due to paeoniflorin in the
chromatogram obtained with solution (1). (2) 0.1% w/v each of paeoniflorin BPCRS and 4'-
hydroxyacetophenone BPCRS in ethanol.
2016 White Peony Root FV-323
CHROMATOGRAPHIC CONDITIONS (1) Finely powder not less than 5 g of the herbal drug being
(a) Use as the coating silica gel (Merck silica gel 60 precoated examined. Mix 0.15 g of the powdered drug with 3 mL of
plates are suitable). methanol and place in an ultrasonic bath for 30 minutes.
(b) Use the mobile phase described below. Dilute to 10 mL with solution A, mix, filter through a 0.45-
(c) Apply 10 pL of each solution, as a band. pm filter and use the filtrate.
(d) Develop the plate to 15 cm. (2) Dissolve a quantity of paeoniflorin BPCRS in methanol and
dilute with solution A to produce a solution containing
(e) Dry the plate in air. Spray with a 5% w/v solution of 0.05% w/v in 3 volumes of methanol and 7 volumes of
vanillin in sulfuric acid, heat at 105° for 5 minutes and solution A and mix.
examine immediately.
(3) Dissolve a quantity of 41-hydroxyacetophenone BPCRS in
MOBILE PHASE
solution (2) to produce a solution containing 0.05% w/v of
A mixture of 0.2 volumes of formic acid, 5 volumes of ethyl 4 '-hydroxyacetophenone.
acetate, 10 volumes of methanol and 40 volumes of CHROMATOGRAPHIC CONDITIONS
dichloromethane.
SYSTEM SUITABILITY with octadecylsilyl silica gel for chromatography (5 pm) (Luna
The test is not valid unless, in the chromatogram obtained C18 is suitable).
with solution (2), two clearly separated spots are observed. (b) Use isocratic elution and the mobile phase described
CONFIRMATION below.
The bluish-purple spot with an Rf value of approximately 0.4 (c) Use a flow rate of 1 mL per minute.
in the chromatogram obtained with solution (1) corresponds (d) Use a column temperature of 30°.
in colour and position to that in the chromatogram obtained (e) Use a detection wavelength of 230 nm.
with solution (2). Other spots are present in the
chromatogram obtained with solution (1) as shown below.
MOBILE PHASE
A mixture of 30 volumes of methanol and 70 volumes of

Top of the plate solution A.
A dark pink band SYSTEM SUITABILITY
with solution (3), the number of theoretical plates for each
Several bluish bands 4’-hydroxyacetophenone: peak due to paeoniflorin is at least 3000, the symmetry factor
an orange band for ±e peak due to paeoniflorin is not more than 1.3 and the
resolution factor between the two peaks is not less ±an 1.7.
A bluish-purple band Paeoniflorin:
a bluish-purple band Using the retention time and the peak area from the
chromatograms obtained with solution (2), locate and
integrate the peak due to paeoniflorin in the chromatogram
obtained with solution (1).
Calculate the content of บ23บ2ร011 in the sample, using the
Solution (1) Solution (2) declared content of paeoniflorin (023บ2ร011) in
paeoniflorin BPCRS and the following expression:
TESTS A, V1 100
xntxpxioo-d
Tree Peony
In the Assay, the chromatogram obtained with solution (1)
shows no peak with a relative retention of approximately 1.2 Ai = Area of the peak due to paeoniflorin in the
with reference to paeoniflorin. chromatogram obtained with solution (1).
Loss on drying A2 = Area of the peak due to paeoniflorin in the
When dried for 3 hours at 100° to 105°, loses not more than chromatogram obtained with solution (2).
12.0% of its weight. Use 1 g. m 1 = Weight of the drug being examined in mg.
Acid-insoluble ash m2 = Weight of paeoniflorin BPCRS in mg.
Not more than 0.5%, Appendix XI K. Vi = Dilution volume of solution (1) in mL.
Ash
v2 = Dilution volume of solution (2) in mL.
p = Percentage content of C23H28O11 in paeoniflorin
BPCRS.
Cadmium d = Percentage loss on drying of the herbal drug being
Maximum 3 ppm, Appendix vn. examined.
Lead
STORAGE
Maximum 5 ppm, Appendix vn. Processed White Peony Root should be protected from
ASSAY moisture.
Appendix in D, using the following solutions prepared in
0.05m potassium dihydrogen orthophosphate (solution A).
IV-324 Pepper 2016
Pepper * **
Ph Elf_______________________________________________________________
DEFINITION
Dried, ripe or nearly ripe fruit of Piper nigrum L. with an
unbroken pericarp (black pepper) or with the outer layers of
the pericarp removed (white pepper).
Content
— essential oil: minimum 25 mL/kg (anhydrous drug);
— piperine (C17H19NO3; Mr 285.3): minimum 3.0 per cent
(anhydrous drug).
IDENTIFICATION
A. Wllite pepper. Spheroid berries, 3-5 mm in diameter,
slightly flattened at one pole and with a small protuberance
at the other, with smooth, externally matt, brownish-grey,
greyish-white or pale yellowish-white surface, with numerous
pale, linear striations between apex and base.
Black pepper Spheroid berries, 3-6 mm in diameter, externally
blackish-brown, with raised reticular wrinkles, bearing fine
remains of the style at the apex and a scar of the peduncle at
the base. The texture is hard, the epicarp can be stripped,
the endocarp is greyish-white or pale yellow. The fracture is
greyish-white, starchy, possessing a small space at the centre.
B. Microscopic examination (2.8.23). Figure 2477.-1. - Illustration for identification test B of powdered
White pepper The powder is light grey. Examine under a herbal drug of pepper
microscope using chloral hydrate solution R. The powder Reference solution Dissolve 10 mg of borneol R and 15 mg of
shows the following diagnostic characters (Figure 2477.-1): piperine R in 10 mL of methanol R.
fragments of the endocarp in surface view, consisting of more
or less polygonal sclereids about 20-30 pm in diameter,
which have irregularly thickened walls [Ac, c, Fa] and which F254 plate R (2-10 pm)].
may or may not be associated with the testa [A, F], Mobile phase ethyl acetate R, cyclohexane R (30:50 ViV).
consisting of a layer of indistinct, reddish-brown pigmented Application 10 pL [or 5 pL] as bands of 10 mm [or 8 mm].
cells constituting the ‘pigmented layer’ [Ab, Fb] and a layer Development Over a path of 15 cm [or 6 cm].
of very thin-walled polygonal cells constituting the ‘hyaline Diying In air.
layer’ [Aa]; fragments of the endocarp, in transverse
section [G], showing sclereids with thickened inner walls on
the 3 lower sides [Ga], usually associated with the testa Results A See below the sequence of zones present in the
(pigmented layer [Gb] and hyaline layer [Gc]); fragments of chromatograms obtained with the reference solution and the
the parenchyma of the mesocarp [D] containing large oil test solution. Furthermore, other faint quenching zones may
cells 50-75 pm in diameter [Da]; numerous thin-walled, be present in the chromatogram obtained with the test
ovoid or polygonal cells of the parenchyma of the seed [E]; solution.
rare, elongated sclereids, with thickened walls, from the fruit
peduncle [B]; a few fragments of vascular tissue with narrow Top of the plate
spiral vessels 0โเ. Examine under a microscope using a
50 per cent v/v solution of glycerol R. Rounded, compound
starch granules [H], about 30 pm in diameter, made up of 3 quenching zones
tiny individual granules, ovoid or polyhedral by compression,
free [Hb] or included in the parenchymatous cells of the
seed [Ha]. A quenching zone
Black pepper The powder is grey. Examine under a Piperine ะ a quenching zone A strong quenching zone
microscope using chloral hydrate solution R. In addition to the (piperine)
diagnostic characters described for white pepper, the
powdered black pepper shows the following diagnostic
characters (Figure 2477.-1): fragments of the epicarp [K]
with extremely thin-walled, brownish-red pigmented, Reference solution Test solution
polygonal or ovoid cells, which contain small prisms of
calcium oxalate [Ka], and which are associated with the
outer layers of the mesocarp consisting of groups of sclereids Detection B Treat with anisaldehyde solution R and heat at
with strongly thickened walls [Kb]. 100 °C for 5 min; examine in daylight.
c. Thin-layer chromatography (2.2.27). Results B See below the sequence of zones present in the
Test solution To 0.5 g of the powdered herbal drug (355) test solution. Furthermore, other zones may be present in the
(2.9.12) add 5 mL of methanol R. Sonicate for 10 min,
centrifuge and use the supernatant.
2016 Peppermint Leaf IV-325

A strong purple zone
0-5 50 50
A purple zone
5 - 20 50-> 5 50-» 95
20 - 22 5+0 95 -> 100

Borneol: a yellowish-brown zone
A purple-grey zone Flow rate 1.0 mL/min.

Injection 10 pL.
A violet-grey zone
Retention time Piperine = about 10 min.
A grey zone
Piperine: a green or brownish A green or brownish zone long pepper for system suitability HRS and the chromatogram
(piperine)
obtained with reference solution (b) to identify the peak due
to piperine and peak 2.
A grey zone System suitability: reference solution (b):
Reference solution
— peak-to^valley ratio: minimum 4, where Hp = height above
Test solution
the baseline of peak 2 and Hv = height above the baseline
of the lowest point of the curve separating the peak due
TESTS to piperine from peak 2.
Foreign matter (2.8.2) Calculate the percentage content of piperine using the
Maximum 3 per cent. following expression:
Water (2.2.13) Al X 7712 X p
Maximum 120 mL7kg, determined on 20.0 g of the freshly,
A2 X 7ท1 X 2
coarsely powdered herbal drug (1400) (2.9.12) reduced using
a knife mill.
ฬ1 = area of the peak due to piperine in the
Total ash (2.4.16) chromatogram obtained with the test solution;
Maximum 6.0 per cent. i42 = area of the peak due to piperine in the
ASSAY chromatogram obtained with reference solution
Essential oil (2.8.12) ;
(a)
Use 10.0 g of the freshly, coarsely powdered herbal drug ni\ = mass of the herbal drug to be examined used to
(1400) (2.9.12) 3 a 1000 mL round-bottomed flask, 400 mL prepare the test solution, in grams;
of water R as the distillation liquid and 0.5 mL of xylene R in m2 = mass of piperine CRS used to prepare reference
the graduated tube. Distil at a rate of 2-3 mL/min for 3 h. solution (a), in grams;
p = percentage content of piperine in piperine CRS.
Piperine
Liquid chromatography (2.2.29). Cany out the assay protected _____________________________________________________________ PhEur
from light.
(355) (2.9.12) in 40 mL of ethanol (96 per cent) R. Sonicate
for 20 min and filter. Rinse the flask and the filter with 5 mL
of ethanol (96 per cent) R3 combine the filtrate and washings
Peppermint Leaf
and dilute to 50.0 mL with the same solvent. Filter through (Ph. Eur. monograph 0406)
a membrane filter (nominal pore size 0.45 pm). Preparation
Reference solution (a) Dissolve 15.0 mg of piperine CRS in Peppermint Leaf Dry Extract
ethanol (96 per cent) R and dilute to 100.0 mL with the same PhEur__________________________
solvent.
DEFINITION
Reference solution (b) Disperse 0.250 g of long pepper for system
suitability HRS (355) (2.9.12) in 40 mL of ethanol Whole or cut dried leaves of Mentha X piperita L.
(96 per cent) R. Sonicate for 20 min and filter. Rinse the flask Content
and the filter with 5 mL of ethanol (96 per cent) R3 combine Minimum 12 mL/kg of essential oil for the whole drug and
the filtrate and washings and dilute to 50.0 mL with the minimum 9 mL/kg of essential oil for the cut drug.
same solvent. Filter through a membrane filter (nominal pore CHARACTERS
size 0.45 pm). Characteristic and penetrating odour.
Column'.
Characteristic aromatic taste.
— size', z = 0.15 m, 0 = 4.6 mm;
Peppermint leaf is green or brownish-green, with brownish-
violet veins in some varieties. The petioles are green or
brownish-violet.
Mobile phase:
— mobile phase A: water R3 IDENTIFICATION
— mobile phase B: acetonitrile R; A. The leaf is entire, broken or cut, thin, fragile and often
crumpled; the entire leaf is 3-9 cm long and 1-3 cm wide.
The lamina is oval or lanceolate, the apex acuminate, the
margin sharply dentate and the base asymmetrical. Venation
IV-326 Peppermint Leaf 2016
is pinnate, prominent on the lower surface, with lateral veins and filter. Evaporate the filtrate to dryness at about 40 cc
leaving the midrib at about 45°. The lower surface is slightly and dissolve the residue in 0.1 mL of toluene R.
pubescent and secretory trichomes are visible under a lens Reference solution Dissolve 50 mg of menthol R, 20 pL of
(6 X ) as bright yellowish points. The petiole is grooved, cineole R} 10 mg of thymol R and 10 pL of menthyl acetate R
usually up to 1 mm in diameter and 0.5-1 cm long. in toluene R and dilute to 10 mL with the same solvent.
Mobile phase ethyl acetate R, toluene R (5:95 P7F).
Application 10 pL of the reference solution and 20 pL of the
test solution, as bands.
Drying In air until the solvent has evaporated.
test solution. Furthermore, other weak quenching zones may
solution.
Top of the plate
Thymol: a quenching zone
Quenching zones may be present

(carvone, pulegone)
Detection B Spray with anisaldehyde solution R and examine in

daylight while heating for 5-10 min at 100-105 °C.
herbal drug of peppermint leaf
Top of the plate
brownish-green. Examine under a microscope using chloral An intense violet-red zone (near
hydrate solution R. The powder shows the following diagnostic the solvent front) (hydrocarbons)
characters (Figure 0406.-1): fragments of epidermises bearing

covering and glandular trichomes; adaxial epidermis, in Menthyl acetate: a violet-blue A violet-blue zone (menthyl
surface view [B, H], having cells with sinuous-wavy walls zone acetate)
[Ha] and cuticle striated over the veins (B) associated with A greenish-blue zone (menthone)
palisade parenchyma [Hb]; abaxial epidermis [C] with Thymol: a pink zone
diacytic stomata (2.5.2) [Ca]; covering trichomes are usually Light pink or greyish-blue or
fragmented, elongated, uniseriate with 3-8 cells with striated greyish-green zones may be
cuticle [A, E]; glandular trichomes of 2 types: a) unicellular present (carvone, pulegone,
stalk with small, rounded unicellular head 15-25 pm in isomenthone)
Cineole: a violet-blue or brown A faint violet-blue or brown zone
diameter, in surface view [Ba, Cb] or in transverse section
zone (cineole)
[D], b) unicellular stalk with enlarged oval head 55-70 pm in
diameter composed of 8 radiating cells, in surface view [Bb]
or in transverse section [Ga]; fragments from near the leaf Menthol: an intense blue or violet An intense blue or violet zone
zone (menthol)
margin [F] with isodiametric cells whose anticlinal walls are
more-or-less straight and beaded [Fa] and short, conical,
unicellular or bicellular covering trichomes [Fb]; dorsiventral
mesophyll fragments, in transverse section [G], with a single
palisade layer [Gc] and 4-6 layers of spongy parenchyma TESTS
[Gb]. Yellowish crystals of menthol under the cuticle of Foreign matter (2.5.2)
secretory cells may be present. Maximum 5 per cent stems, whose diameter is not greater
than 1.5 mm; maximum 2 per cent foreign elements;
not more than 8 per cent of the leaves show brown stains
Test solution To 0.2 g of the recently powdered herbal drug due to Puccinia menthae.
add 2 mL of methylene chloride R, shake for a few minutes
Carry out the determination using 10 g of the drug.
2016 Peppermint Oil IV-327
Water (2.2.13) Top of the plate

Maximum 110 mL/kg, determined on 20.0 g.
An intense violet-red zone (near
Total ash (2.4.16) the solvent front) (hydrocarbons)
Maximum 15.0 per cent. A brownish-yellow zone
(menthofuran)
ASSAY Menthyl acetate: a violet-blue A violet-blue zone (menthyl
zone acetate)
Essential oil (2.8.12) A greenish-blue zone (menthone)
Use 20.0 g of crushed drug, a 500 mL flask, 200 mL of Thymol: a pink zone
tcater R as the distillation liquid and 0.50 mL of xylene R in
the graduated tube. Distil at a rate of 3-4 mL/min for 2 h. Light pink or greyish-blue or
greyish-green zones may be
------------------------------------------------------------ --------------------------------------------- Ph Eur present (carvone, pulegone,
isomenthone)
1,8-Cineole: a violet-blue or A faint violet-blue or brown zone
brown zone (1,8-cineole)
Peppermint Oil ***** Menthol ะ an intense blue or violet An intense blue or violet zone
zone (menthol)
(Ph. Eur. monograph 0405) *** Reference solution Test solution
Preparations
Gastro-resistant Peppermint Oil Capsules
Peppermint Spirit B. Examine the chromatograms obtained in the test for
Ph Eur________________ _ _____________________
Results The characteristic peaks due to limonene, 1,8-cineole,
DEFINITION menthone, menthofuran, isomenthone, menthyl acetate and
Essential oil obtained by steam distillation from the fresh menthol in the chromatogram obtained with the test solution
aerial parts of the flowering plant of Mentha X piperita L. are similar in retention time to those in the chromatogram
CHARACTERS obtained with reference solution (a). Isopulegol, pulegone
and carvone may be present in the chromatogram obtained
Appearance
with the test solution.
Colourless, pale yellow or pale greenish-yellow liquid.
Characteristic odour and taste followed by a sensation of TESTS
cold. Relative density (2.2.5)
0.900 to 0.916.
Solubility
Miscible with ethanol (96 per cent) and with methylene Refractive index (2.2.6)
chloride. 1.457 to 1.467.
IDENTIFICATION Optical rotation (2.2.7)
First identification B. -30° to -10°.
Second identification A. Acid value (2.5.1)
Maximum 1.4, determined on 5.0 g diluted in 50 mL of the
A. Examine the chromatograms obtained in test A for mint
prescribed mixture of solvents.
oil.
Residts A See below the sequence of zones present in the
It complies with the test for fatty oils and resinified essential
oils.
test solution.
Mint oil
Top of the plate
Test solution Mix 0.1 g of the substance to be examined with
toluene R and dilute to 10 mL with ±e same solvent.
Thymol ะ a quenching zone Reference solution Dissolve 50 mg of menthol R, 20 pL of
cineole R, 10 mg of thymol R and 10 pL of menthyl acetate R
in toluene R and dilute to 10 mL with the same solvent.
Quenching zones may be present Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel
(carvone, pulegone)
F254 plate R (2-10 pm)].
Mobile phase ethyl acetate R) toluene R (5:95 VIV).
Reference solution Test solution Application 10 pL [or 1 pL] of the reference solution and
20 pL [or 2 pL] of the test solution, as bands of 10 mm [or
8 mm].
chromatograms obtained with the reference solution and the Development Over a path of 15 cm [or 6 cm].
test solution. Furthermore, other less intensely coloured Drying In air.
zones may be present in the chromatogram obtained with the Detection A Examine in ultraviolet light at 254 run.
test solution. Detection B Treat with anisaldehyde solution R and heat at
100-105 °C for 5-10 min; examine immediately in daylight.
IV-328 Peppermint Preparations 2016
Results B The chromatogram obtained with the test solution — menthol: 30.0 per cent to 55.0 per cent;
shows no blue zone between the zones due to 1,8-cineole — pulegone: maximum 3.0 per cent,
and menthol. — carvone: maximum 1.0 per cent;
B. Examine the chromatograms obtained in the test for — disregard limit: the area of the principal peak in the
chromatographic profile. chromatogram obtained with reference solution (b)
Results The chromatogram obtained with the test solution (0.05 per cent).
does not show a peak with the retention time of isopulegol The ratio of 1,8-cineole content to limonene content is
that has an area of more than 0.2 per cent of the total area. minimum 2.
Chromatographic profile STORAGE
Gas chromatography (2.2.28): use the normalisation At a temperature not exceeding 25 °C.
procedure. ___ ___________________________________________________________ Ph Elf
Test solution Mix 0.20 mL of the substance to be examined
with heptane R and dilute to 10.0 mL with the same solvent.
Reference solution (a) Dissolve 10 pL of limonene Ry 20 pL of
cineole Ry 40 pL of mendใone Ry 10 pL of menthofuran Ry
10 pL of isomenthone Ry 40 pL of menthyl acetate Ry 20 pL of Peppermint Spirit
isopulegol Ry 60 mg of menthol Ry 20 pL of pulegone Ry 10 pL Peppermint Essence
of piperitone R and 10 pL of carvone R in heptane R and dilute DEFINITION
to 10.0 mL with the same solvent. Peppermint Oil 100 mL
Reference solution (b) Dissolve 5 pL of isopulegol R in Ethanol (90 per cent) Sufficient to produce 1000 mL
heptane R and dilute to 10.0 mL with the same solvent.
Dilute 0.1 mL of the solution to 5.0 mL with heptane R.
Column:
— material: fused silica; Dissolve the Peppermint Oil in Ethanol (90 per cent) and
— size: I = 60 m, 0 = 0.25 mm; add sufficient Ethanol (90 per cent) to produce 1000 mL.
— stationary phase: macrogol 20 000 R (film thickness If the solution is not clear, shake with previously sterilised
0.25 pm). Purified Talc and filter.
Carrier gas helium for chromatography R. The spirit complies with the requirements stated under Spirits and
with the following requirements.
Split ratio 1:50. TESTS
Ethanol content
Temperature:
78 to 82% v/v, Appendix VIII F.
Weight per mL
Time Temperature
(°C)
Column 0 - 10 60 Content of oil
9.0 to 11.0% v/v when determined by the following method.
10 - 70 60 -> 180
Add 25 mL of the spirit and 5 mL of xylene to 90 mL of a
70 - 75 180 10% w/v solution of sodium chloride containing 1.0% v/v of
Injection port 200 hydrochloric acid in a flask of about 150 mL capacity with a
long neck graduated in 0.1 mL increments and of such a
Detector 220
diameter that a 15-cm length has a capacity of 10 mL. Shake
the mixture for about 30 minutes, allow to separate and raise
the undissolved oily layer into the graduated part of the neck
of the flask by the gradual addition of more of the acidified
Injection 1 pL. sodium chloride solution, allow to stand for 2 hours or until
Elution order Order indicated in the composition of reference there is no further change in volume of the oily layer and
solution (a); record the retention times of these substances. measure the volume of the oily layer. The volume of the oily
Identification of peaks Using the retention times determined layer, after the subtraction of 5 mL, is taken to be the
from the chromatogram obtained with reference solution (a), volume of oil.
locate the components of reference solution (a) in the
System suitability, reference solution (a):
limonene and 1,8-cineole; minimum 1.5 between the
Gastro-resistant Peppermint Oil
peaks due to piperitone and carvone. Capsules
Determine the percentage content of each of the following DEFINITION
components. The limits are within the following ranges: Gastro-resistant Peppermint Oil Capsules contain
— limonene: 1.0 per cent to 3.5 per cent; Peppermint Oil. They are enteric capsules.
— 1,8-cineole: 3.5 per cent to 8.0 per cent;
The capsules comply with the requirements stated under Capsules
— menthone: 14.0 per cent to 32.0 per cent;
------- menthofuran: 1.0 per cent to 8.0 per cent;
— isomenthone: 1.5 per cent to 10.0 per cent; IDENTIFICATION
— menthyl acetate: 2.8 per cent to 10.0 per cent; A. Carry out the method for thin-layer chromatography,
— isopulegol: maximum 0.2 per cent; Appendix in A, using the following solutions.
2016 Peppermint Oil Preparations IV-329
(1) Dissolve a quantity of the contents of the capsules menthyl acetate, 0.6 g of menthol, 0.2 g of pulegone and 0.1 g
containing 0.1 g of Peppermint Oil in sufficient toluene to of carvone in 1 mL of hexane.
produce 10 mL.
(2) Dissolve 10 mg of thymol, 10 |1L of menthyl acetate, 20 |1L (a) Use a fused-silica capillary column (60 m X 0.25 mm)
of cineole and 50 mg of menthol in toluene and dilute to
coated with macrogol 20,000 as the bonded phase.
(b) Use helium as the carrier gas at 1.5 mL per minute.
(c) Use the gradient conditions described below.
(a) Use a TLC silica gel GF254 plate.
(d) Use an inlet temperature of 220°.
(e) Use a flame ionisation detector at a temperature of 220°.
(c) Apply20 )1L of solution (1) and 10 pL of solution (2) as
bands. (f) Inject 0.2 pL of each solution.
(g) Use a split ratio of 1:100.
(d) After removal of the plate, dry in air and examine under
ultraviolet light (254 nm), spray with anisaldehyde solution and When the chromatograms are recorded in the prescribed
examine in daylight for 5 to 10 minutes while heating at 100° conditions, the components elute in the order indicated in
to 105°. the composition of solution (2). Record the retention times
of these substances.
MOBILE PHASE
5 volumes of ethyl acetate and 95 volumes of toluene.

Time Temperature Comments
CONFIRMATION
(minutes)
When examined under ultraviolet light (254 nm) the
chromatogram obtained with solution (1) may show 0-10 60° isothermal
quenching zones (carvone, pulegone) situated just below the linear gradient
10-70 60° -180°
level of the zone (thymol) in the chromatogram obtained
with solution (2). 70-75 180° isothermal
When examined in daylight the chromatogram obtained with
solution (2) shows, in order of increasing Rf value, an intense
blue to violet zone (menthol) in the lower third; a violet-blue
SYSTEM SUITABILITY
to brown zone (cineole); a pink zone (thymol); and a violet
blue zone (menthyl acetate). In the chromatogram obtained The test is not valid unless the number of theoretical plates
with solution (1) there is a zone due to menthol (the most calculated from the limonene peak at 110° is at least 30,000
intense) and a faint zone due to cineole; at Rf values between and the resolution factor between the peaks corresponding to
those of the cineole and thymol zones in the chromatogram limonene and cineole is at least 1.5 in the chromatogram
obtained with solution (2), there may be light pink or obtained with solution (2).
greyish-blue or greenish-grey zones (carvone, pulegone, DETERMINATION OF CONTENT
isomenthone); in the middle of the chromatogram, there is a Using the retention times determined from the
violet-blue zone (menthyl acetate) and just below it a chromatogram obtained with solution (2), locate the
greenish-blue zone (menthone); an intense violet-red zone components of solution (2) on the chromatogram obtained
(hydrocarbons) appears near the solvent front and below it with solution (1) (disregard the peak due to hexane).
there may be a brownish-yellow zone (menthofuran); other
Determine the percentage content of the components by the
less intensely coloured zones may also appear.
normalisation procedure.
Chromatographic profile. The retention time of the principal
Limonene 1.0 to 5.0%,
peaks in the chromatogram obtained with solution (1) is
Cineole 3.5 to 14.0%,
similar to that of the principal peaks in the chromatogram
Menthone 14.0 to 32.0%,
obtained with solution (2). Carvone and pulegone may be
Menthofuran 1.0 to 9.0%,
absent from the chromatogram obtained with solution (1).
Isomenthone 1.5 to 10.0%,
TESTS Menthyl acetate 2 8 to 10.0%,
Disintegration test Menthol 30.0 to 55.0%,
Complies with the requirements stated under Gastro-resistant Pulegon Not more than 4.0%,
Capsules. Carvone Not more than 1.0%.
Refractive index The ratio of cineole content to limonene content is greater
1.457 to 1.467, determined on the contents of the capsules, than 2.
Appendix V G.
STORAGE
Relative density Gastro-resistant Peppermint Oil Capsules should be
0.900 to 0.916, determined on the contents of the capsules, protected from light.
Appendix V E.
Composition of peppermint oil
Carry out the method for gas chromatography,
Appendix m B, .using the following freshly prepared
solutions.
(1) Use the contents of the capsules.
(2) Dissolve 0.1 g of limonene, 0.2 g of cineole, 0.4 g of
menthone, 0.1 g of menthofuran, 0.1 g of isomenthone, 0.4 g of
-330 Peppermint Leaf Dry Extract 2016
Peppermint Leaf Dry Extract ** ** Reference solution (b) Dissolve 5 mg of fendic acid R in
reference solution (a) and dilute to 50 mL with the same
(Ph. Eur. monograph 2382) *** solution.
PhEtr__________________________________________ \_ Column’.
— size’. I = 0.25 m, 0 = 4.6 mm;
DEFINITION — stationary phase’, octadecylsilyl silica gel for chromatography R
Dry’ extract produced from Peppermint leaf (0406). (5 pm).
Content Mobile phase’.
Minimum 0.5 per cent of rosmarinic add (C]SH16O8; — mobile phase A: phosphoric acid R, acetonitrile R, water R
Mr 360.33) (dried extract). (1:19:80 VIVIV);
PRODUCTION — mobile phase B: phosphoric acid R, methanol R, acetonitrile R
The extract is produced from the herbal drug by a suitable (1:40:59 VIVIV);
procedure using ethanol (30-50 per cent VIV) or water of
minimum 60 °C.
CHARACTERS 0 - 20 100 -) 55 0 -> 45
Appearance 20 - 25 55 -> 0 45 -> 100
Brown, amorphous powder.
25 - 30 0 -> 100 100 0
IDENTIFICATION
Thin-layer chromatography (2.2.27). Flow rate 1.2 mUmin.
Test solution To 0.2 g of the extract to be examined add Detection Spectrophotometer at 330 nm.
5 mL of methanol R. Sonicate for 5 min and filter.
Injection 20 pL.
Reference solution Dissolve 5 mg of rosmarinic acid R, 1 mg of
Relative retention With reference to rosmarinic acid (retention
hyperoside R and 1 mg of rutin R in 10 mL of methanol R.
time = about 11 min): ferulic acid = about 0.8.
plate R (2-10 pm)].
Mobile phase anhydrous formic acid R, water R, ethyl acetate R acid and rosmarinic acid.
(6:6:90 VIVIV).
Calculate the percentage content of rosmarinic acid using the
Application.10 pL [or 4 pL] as bands of 15 mm [or 8 mm]. following expression:
Drying In air. Al X 7ท2 X p X 0.2
Detection Heat at 100 °C for 5 min and spray the hot plate A? X 7711
with a 5 g/L solution of diphenylboric acid aminoethyl ester R in
ethyl acetate R’} examine in ultraviolet light at 365 nm. Al = area of the peak due to rosmarinic acid in the
A2 = area of the peak due to rosmarinic acid in the
in the chromatogram obtained with the test solution. ;
(a)
nil = mass of the extract to be examined used to
Top of he plate prepare the test solution, in grams;
m2 = mass of rosmarinic acid CRS used to prepare
Rosmarinic acid: a light blue A light blue fluorescent zone
fluorescent zone (rosmarinic acid)
p = percentage content of rosmarinic acid in rosmarinic
acid CRS.
_________________________________________________________ _____ PhEir
Hyperoside: an orange fluorescent

Peru Balsam ** *
Rutin: an orange fluorescent zone A yellow fluorescent zone
Ph Eur____________________________________________________________ __
Reference solation Test solation
DEFINITION
ASSAY Balsam obtained from the scorched and wounded trunk of
Liquid chromatography (2.2.29). Myroxylon balsamum (L.) Harms var. pereirae (Royle) Harms.
Test solution Use brown glass flasks. To 0.400 g of the extract Content
to be examined add 15 mL of ethanol (50 per cent V/V) R, 45.0 per cent mini to 70.0 per cent mini of esters, mainly
sonicate for 10 min and filter into a 20 mL volumetric flask. benzyl benzoate and benzyl cinnamate.
Rinse the flask and the filler with ethanol (50 per cent VIV) R CHARACTERS
and dilute to 20.0 mL with the same solvent. Appearance
Reference solution (a) Dissolve 10.0 mg of rosmarinic acid CRS Dark brown, viscous liquid which is transparent and
in ethanol (50 per cent VIV) R and dilute to 100.0 mL with yellowish-brown when viewed in a thin layer; it is not stick}’,
the same solvent. non-drying and does not form threads.
2016 Phellodendron Amurense Bark IV-331
Solubility ASSAY
Practically insoluble in water, freely soluble in anhydrous To 2.50 g in a separating funnel add 7.5 mL of dilute sodium
ethanol, not miscible with fatty oils, except for castor oil. hydroxide solution R and 40 mL of peroxide-free ether R and
IDENTIFICATION shake vigorously for 10 min. Separate the lower layer and
A. Dissolve 0.20 g in 10 mL of ethanol (96 per cent) R. shake it with 3 quantities, each of 15 mL, of peroxide-free
Add 0.2 mL of ferric chloride solution Rl. A green or ether R. Combine the ether layers, dry over 10 g of anhydrous
yellowish-green colour develops. sodium sulfate R and filter. Wash the sodium sulfate with
2 quantities, each of 10 mL, of peroxide-free ether R. Combine
the ether layers and evaporate to dryness. Dry the residue
Test solution Dissolve 0.5 g of the substance to be examined (esters) at 100-105 °C for 30 min and weigh.
in 10 mL of ethyl acetate R.
STORAGE
Reference solution Dissolve 4 mg of thymol R, 30 mg of benzyl
cinnamate R and 80 |1L of benzyl benzoate R in 5 mL of ethyl
acetate R. ____________________________________________________________ PhEur

Mobile phase glacial acetic acid R3 ethyl acetate R, hexane R
(0.5:10:90 VIVIL).
Application 10 pL, as bands of 20 mm by 3 mm. Phellodendron Amurense Bark
Development Twice over a path of 10 cm. DEFINITION
Drying In air. Phellodendron Amurense Bark is ±e dried bark of
Detection A Examine in ultraviolet light at 254 nm and mark Phellodendron amurense Ruprecht.
the quenching zones. It is collected between spring and summer and the rough
Results A The chromatogram obtained with the reference part of the bark is removed and รนทdried.
solution shows in the upper third 2 quenching zones, the It contains not less than 0.4% พ/พ of berberine
higher one due to benzyl benzoate and the lower one due to (C2oH18N04) and not less than 0.2% พ/พ palmatine
benzyl cinnamate. The chromatogram obtained with the test (C21H22NO4) calculated with reference to ±e dried material.
solution shows 2 quenching zones at the same levels and of IDENTIFICATION
approximately the same size.
A. The bark pieces are usually rectangular or shallowly
Detection B Spray with a freshly prepared 200 g/L solution of quilled, of variable length and width; between 2 and 6 mm
phosphomolybdic acid R in ethanol (96 pel' cent) Rt using thick. Prepared, cut slices are transversely sliced into
10 mL for a plate 200 mm square and examine in daylight rectangular pieces and occasionally show the remains of the
while heating at 100-105 °C for 5-10 min. corky outer bark. The outer surface is relatively smooth with
Results B The zones due to benzyl benzoate and benzyl yellow, yellowish-green or yellowish-brown inner bark
cinnamate are blue against a yellow background. (secondary phloem) interrupted by irregularly shaped remains
The chromatogram obtained with the reference solution of brown or greyish-brown outer bark (cork or rhytidoma),
shows at about the middle a violet-grey zone (thymol). In the the latter fissured or sometimes raised, angular and somewhat
chromatogram obtained with the test solution, a blue zone spongy. Lenticels are occasionally visible in the outer bark.
(nerolidol) is seen just below the level of the zone due to The inner surface is smooth and yellow or yellowish-brown
thymol in the chromatogram obtained with the reference throughout with fine longitudinal striations. The texture is
solution. Just below the zone due to nerolidol, no blue zone light and hard. The fracture is fibrous, bright yellow or
is seen corresponding to a quenching zone seen when yellowish-green.
examined in ultraviolet light at 254 nm (colophony). In the B. Reduce to a powder (355). The powder is yellow or
upper and lower part of the chromatogram obtained with the yellowish-brown. Examine under a microscope using chloral
test solution, other faint blue zones may be seen. hydrate solution. The powder shows the following diagnostic
TESTS characters: abundant fragments of fibres, yellow or pale
Relative density (2.2.5) yellow, mainly in bundles or sheets, with thickened walls and
1.14 to 1.17. a long, narrow lumen, surrounded by a crystal sheath of
parenchyma cells containing prisms of calcium oxalate.
Saponification value (2.5.6) Scattered calcium oxalate prism crystals, up to about 24 pm,
230 to 255, determined on the residue obtained in the assay. are also seen in the powder. The fibre sheets are interspersed
Artificial balsams with medullary rays composed of polygonal cells two to three
Shake 0.20 g with 6 mL of light petroleum Rl. The light cells wide. Numerous large stone cells, bright yellow, sub-
petroleum solution is clear and colourless and the whole of orbicular, fusiform or irregular in shape, some branched or
the insoluble parts of the balsam stick to the wall of the test with a pointed apex, with thickened, distinctly striated walls,
tube. are present, commonly arranged in groups. Groups of
Fatty oils tabular, colourless, cork cells in layers are present.
Shake 1 g with 3 mL of a 1000 g/L solution of chloral c. Carry out the method for thin-layer chromatography,
hydrate R. The resulting solution is as clear as the 1000 g/L Appendix in A, using the following solutions.
solution of chloral hydrate R. (1) To 0.25 g of the finely powdered drug (180) add 4 mL
Turpentine of a mixture of 20 mL of water and 80 mL of methanol.
Evaporate to dryness 4 mL of the solution obtained in the Mix with the aid of ultrasound for 10 minutes and filter.
test for artificial balsams. The residue has no odour of Extract the remaining residue in the filter with two 2-mL
turpentine. quantities of methanol. Combine the solutions and dilute to
20 mL with methanol
IV-332 Phellodendron Chinense Bark 2016
(2) 0.04% w/v each of palmatine chloride BPCRS and berberine MOBILE PHASE
chloride BPCRS in methanol. 27 volumes of acetonitrile and 73 volumes of a 1.36% w/v
CHROMATOGRAPHIC CONDITIONS solution of potassium dihydrogen orthophosphate in water.
(a) Use as the coating octadecylsilyl silica gel for HPTLC SYSTEM SUITABILITY
(Merck silica gel HPTLC plates are suitable) The test is not valid unless, in the chromatogram obtained
(b) Use the mobile phase as described below. with solution (2), the resolution between the peaks due to
(c) Apply 20 pL of each solution, as 8 mm bands. palmatine (retention time approximately 8 minutes) and
berberine (retention time approximately 9 minutes) is at least
(d) Develop the plate to 6 cm from the point of application.
2.0.
(e) After removal of the plate, dry7 in air for 5 minutes, dip
the plate in ethanolic sulfuric acid (20%) and then heat at 105° DETERMINATION OF CONTENT
until coloured bands appear. Examine under ultraviolet light Using the retention times and the peak areas from the
(366 nm). chromatograms obtained with solution (2), locate and
MOBILE PHASE
integrate the peaks due to berberine and palmatine in the
chromatogram obtained with solution (1). Calculate the
10 volumes of anhydrous formic acid, 10 volumes of water and content of berberine and of palmatine in the sample using
80 volumes of ethyl acetate. the declared content of berberine in berberine
SYSTEM SUITABILITY chloride BPCRS and palmatine in palmatine
The test is not valid unless the chromatogram obtained with chloride BPCRS and the following expression:
RESULTS A'xm=xpxl2.5
See the sequence of zones present in the chromatograms
obtained with the solution (1) and solution (2). Other faint
A2 X m,
zones may be present.
Top of the plate A] = Area of the peak due to berberine/palmatine in the
A2 = Area of the peak due to berberine/palmatine in the
ทโ1 = Weight of the drug being examined in g.
A green band (berberine) A green band (berberine) m 2 = Weight of berberine chloride!palmatine chloride in the
An orange band reference solution in g.
A green band (palmatine) A green band (palmatine)
An orange band p = Percentage content of berberine/palmatine in berberine
A blue band chloridelpalmatine chloride.
A blue band
STORAGE
p. amurense Solution (2) Phellodendron Amurense Bark should be protected from
moisture.
TESTS
Loss on drying
When dried at 100° to 105° for 2 hours, loses not more than
10.0% of its weight, Appendix DC D. Use 1 g. Phellodendron Chinense Bark
Total ash DEFINITION
Not more than 8.0%, Appendix XI J, Method II. Phellodendron Chinense Bark is the dried bark of
ASSAY Phellodendron chinense Schneid.
Carry out the method for liquid chromatography, It is collected between spring and summer and the rough
Appendix in D, using the following solutions. outer bark is removed and discarded. The remainder is
(1) To 0.1 g of powdered sample, add 80 mL of a mixture of sundried.
equal volumes of acetonitrile and 0.1% v/v solution of It contains not less than 3.0% พ/พ of berberine
orthophosphoric acid. Mix with the aid of ultrasound for (C2oH18N04), calculated with reference to the dried
40 minutes and allow to cool. Dilute to 100 mL with mobile material.
phase and filter (Whatman GF/C is suitable).
IDENTIFICATION
(2) 0.01% w/v of palmatine chloride BPCRS and 0.01% w/v A. The bark pieces are usually rectangular or shallowly
berberine chloride BPCRS in mobile phase. quilled of widely varying length and width; up to 6 mm
CHROMATOGRAPHIC CONDITIONS thick. Prepared, cut, slices are sliced transversely into
(a) Use a stainless steel column (15 cm X 4.6 mm) packed rectangular pieces. The outer surface is greyish or yellowish
with end-capped octadecylsilyl silica gel for chromatography (depending on the amount of thin outer cork bark removed),
(5 pm) (Phenomenex Luna Cl8 is suitable). with irregularly scattered raised transverse lenticels. The inner
surface is smooth and yellow to yellowish-brown throughout
(b) Use isocratic elution and the mobile phase described
with fine longitudinal striations. The texture is light and
below.
hard. The fracture is fibrous, yellow or yellowish green; older
(c) Use a flow rate of 1.2 mL per minute. barks are thicker and include a hard sponge-like zone on the
(d) Use an ambient column temperature. innermost side.
(e) Use a detection wavelength of 235 nm. B. Reduce to a powder (355). The powder is yellow or
(f) Inject 50 pl of each solution. yellowish-brown. Examine under a microscope using chloral
2016 Phellodendron Chinense Bark IV-333
hydrate solution. The powder shows the following diagnostic berberine and palmatine and no orange or blue bands below
characters: abundant fragments of short fibres, yellow or pale the band for palmatine given in the chromatogram obtained
yellow, mainly in bundles or sheets, with thickened walls and with solution (2).
a long, narrow lumen, surrounded by a crystal sheath of
Loss on drying
parenchyma cells containing prisms of calcium oxalate. When dried at 100° to 105° for 2 hours, loses not more than
Scattered calcium oxalate prism crystals, up to about 40 pm,
10.0% of its weight, Appendix IX D. Use 1 g.
are also seen in the powder. The fibre sheets are interspersed
with conspicuous medullary rays composed of polygonal cells Total ash
two to three cells wide. Numerous large stone cells, bright Not more than 8.0%, Appendix XI J, Method II.
yellow, sub-orbicular, fusiform or irregular in shape, some ASSAY
branched or with a pointed apex, with thickened, distinctly Carry out the method for liquid chromatography,
striated walls, are present, commonly arranged in groups. Appendix in D, using the following solutions.
c. Carry out the method for thin-layer chromatography, (1) To 0.1 g of powdered sample, add 80 mL of a mixture of
Appendix in A, using the following solutions. equal volumes of acetonitrile and 0.1% v/v solution of
(1) To 0.25 g of the finely powdered drug (180) add 4 mL orthophosphoric acid. Mix with the aid of ultrasound for
of a mixture of 20 mL of water and 80 mL of methanol. 40 minutes and allow to cool. Dilute to 100 mL with mobile
Mix with the aid of ultrasound for 10 minutes and filter. phase and filter (Whatman GF/C is suitable).
Extract the remaining residue in the filter with two 2-mL (2) 0.01% w/v of palmatine chloride BPCRS and 0.01% w/v
quantities of methanol. Combine the solutions and dilute to berberine chloride BPCRS in mobile phase.
20 mL with methanol.
(2) 0.04% w/v each of palmatine chloride BPCRS and berberine (a) Use a stainless steel column (15 cm X 4.6 mm) packed
chloride BPCRS in methanol.
with end-capped octadecylsilyl silica gel for chromatography
CHROMATOGRAPHIC CONDITIONS (5 pm) (Phenomenex Luna C18 is suitable).
(a) Use as the coating octadecylsilyl silica gel for HPTLC (b) Use isocratic elution and the mobile phase described
(Merck silica gel HPTLC plates are suitable). below.
(b) Use the mobile phase as described below. (c) Use a flow rate of 1.2 mL per minute.
(c) Apply 20 pL of each solution, as 8 mm bands. (d) Use an ambient column temperature.
(d) Develop the plate to 6 cm from the point of application. (e) Use a detection wavelength of 235 nm.
(e) After removal of the plate, dry in air for 5 minutes, dip (f) Inject 50 pL of each solution.
the plate in ethanolic sulfuric acid (20%) and then heat at 105° MOBILE PHASE
until coloured bands appear. Examine under ultraviolet light
(366 nm). 27 volumes of acetonitrile and 73 volumes of a 1.36% w/v
solution of potassium dihydrogen orthophosphate in water.
MOBILE PHASE
SYSTEM SUITABILITY
10 volumes of anhydrous formic acid, 10 volumes of tuater and
80 volumes of ethyl acetate.
with solution (2), the resolution between the peaks due to
SYSTEM SUITABILITY palmatine (retention time of approximately 8 minutes) and
The test is not valid unless the chromatogram obtained with berberine (retention time of approximately 9 minutes) is at
solution (2) shows two clearly separated bands. least 2.0.
RESULTS DETERMINATION OF CONTENT
See the diagram for the sequence of zones present in the Using the retention time and the peak area from the
chromatograms obtained with solution (1) and solution (2). chromatograms obtained with solution (2), locate and
Furthermore, in the chromatogram obtained with solution integrate the peak due to berberine in the chromatogram
(1) a faint or more intense green band corresponding to obtained with solution (1). Calculate the content of berberine
palmatine may be present. Other faint zones may be present. in the sample using the declared content of berberine in
berberine chloride BPCRS and the following expression:
Top of the plate
A'xm» xpxl2.5
A2 xm1
A green band (berberine) A green band (berberine)
A yellow band
A green band (palmatine) A] = Area of the peak due to berberine in the
A2 = Area of the peak due to berberine in the
Solution (2) mi = Weight of the drug being examined in g.
Solution (1)
m2 = Weight of berberine chloride in the reference solution
in g.
p = Percentage content of berberine in berberine chloride.
TESTS
Phellodendron amurense STORAGE
In identification test c, the chromatogram obtained with Phellodendron Chinense Bark should be protected from
solution (1) shows no orange band between the bands for moisture.
IV-334 Phyllanthus Emblica Pericarp 2016
Phyllanthus Emblica Pericarp Top of the plate

Dark blue band Gallic acid: a dark blue band
DEFINITION
Phyllanthus Emblica Pericarp is ±e pericarp of dried mature
fruits of Phyllanthus emblica L. (syn. Emblica officinalis
Gaertn.) Dark blue band Ellagic acid: a dark blue band
It contains not less than 6.0% tannins, expressed as
pyrogallol (C6H603. Afr 126.1), calculated with reference to
the dried drug.
IDENTIFICATION Blue/faint blue band
A. Irregular pieces of the pericarp showing a dark brownish Blue/faint blue band
to black outer surface, much wrinkled and grooved with
occasional greyish-white patches; the underlying brown Solution (1) Solution (2)
mesocarp is about 2 to 3 mm wide surrounding a thin, paler
brown endocarp. Infrequently whole seeds, or portions of the
creamish white testa may be present, attached to the TESTS
endocarp; whole seeds are subspherical, about 1 cm in Ethanol-soluble extractive
diameter and the testa is marked with 6 equidistant Not less than 15.0%, Appendix XI Bl
longitudinal ridges. Occasional whole fruits may also be Foreign matter
present; they are spherical to ovoid or irregular, about 2 cm Not more than 5% of foreign matter including seed material,
in diameter and show a depression at one end. Appendix XI D.
B. The powder is yellow-green or pale brown. It shows small Water-soluble extractive
polygonal cells from the exocarp covered with a thick cuticle, Not less than 50%, Appendix XI B2.
numerous mesocarp, some filled with amorphous grey
birefractive masses, some with mucilage, and some with Ash
calcium oxalate crystals in the form of needles, large prism or Not more than 7.0%, Appendix XI J, method II.
microsphenoids; thick-walled, pitted sclereids from the Loss on drying
mesocarp found singly and usually surrounded by Not more than 10.0%, Appendix IX D. Use 1 g.
parenchyma, and fragments of the endocarp consisting of a ASSAY
thick layer of pitted fibres and sclereids.
Carry out the determination of tannins in herbal drugs,
c. Carry out the method for thin-layer chromatography, Appendix XI M. Use 1.0 g of powdered drug.
Appendix IK A, using the following solutions.
(1) Add 25 mL of ethanol to 1.0 g of the powdered herbal
drug, sonicate for 30 minutes and filter.
(2) 0.1% w/v of ellagic acid and 0.1% w/v of gallic acid in Dwarf Pine Oil * *
methanol.
Ph Eur______________________________________________________________
(a) Use as the coating silica gel 60 F254 or high-performance
silica gel 60 F2S4 (Merck silica gel 60 F254 HPTLC plates are DEFINITION
suitable). Essential oil obtained by steam distillation of the fresh leaves
(b) Use the mobile phase as described below. and twigs of Pinus mugo Turra. A suitable antioxidant may be
added.
(c) Apply 10 pL [or 2 pL] of each solution, as bands.
(d) Develop the plate to 15 cm [or 7 cm]. CHARACTERS
(e) After removal of the plate, dry in air, spray with a 5% w/v Appearance
solution of iron(ni) chloride in ethanol and examine in daylight.
MOBILE PHASE
IDENTIFICATION
1.5 volumes of toluene, 3 volumes of acetic acid, 4 volumes of
formic acid and 30 volumes of ethyl acetate. Second identification A
B.. Thin-layer chromatography (2.2.27).
SYSTEM SUITABILITY
Test solution Dilute 1 mL of the substance to be examined to
10 mL with toluene R.
Reference solution Dissolve 10 mg of borneol R and 10 pL of
CONFIRMATION
bornyl acetate R in toluene R and dilute to 10 mL with the
The chromatogram obtained with solution (1) shows a dark same solvent.
blue band with an Rf value of 0.6 corresponding in colour
and position to the band obtained with ellagic acid in
plate J? (2-10 pm)].
solution (2) and a dark blue band with an Rf value of
approximately 0.9 corresponding in position to the dark blue Mobile phase ethyl acetate R, toluene R (5:95 VIV).
band obtained with gallic acid in solution (2). Two or three Application 10 pL [or 2 pL], as bands.
separated blue or faint blue bands with Rf values of between Development Over a path of 15 cm [or 6 cm].
0.2 and 0.3 are also present. Other bands may be present in Drying In air.
solution (1). Detection Spray with anisaldehyde solution R and heat at
2016 Pine Silvestris Oil IV-335
See below the sequence of zones present in the Temperature:

test solution. Furthermore, other zones may be present in the Time Temperature
chromatogram obtained with the test solution. (min) (°C)
Column 0 - 10 65
Top of the plate 10 - 41 65 -> 220
A pink zone 41 - 50 220
Injection port 220
Detector 250
Bornyl acetate: a brown or A brown or greyish-brown zone
greyish-brown zone (bornyl acetate)
A pink zone Detection Flame ionisation.
Injection 1 pL.
Borneol: a brown or Elution order Order indicated in the composition of reference
A cluster of violet zones
greyish-brown zone solution (a); record the retention times of these substances.
— resolution: minimum 1.5 between the peaks due to car-3-
ene and p-myrcene.
B. Examine the chromatograms obtained in the test for determined from the chromatogram obtained with reference
chromatographic profile. solution (a), locate the components of reference solution (a)
Results The characteristic peaks in the chromatogram in the chromatogram obtained with the test solution;
obtained with the test solution are similar in retention time to the peak due to p-phellandrene is eluted after the peak due
those in the chromatogram obtained with reference to limonene with a relative retention of about 1.03 with
solution (a). reference to limonene.
TESTS
Relative density (2.2.5) — a-pinene: 10.0 per cent to 30.0 per cent;
0.857 to 0.868.
— camphene: maximum 2.0 per cent;
Refractive index (2.2.6) — p-pinene: 3.0 per cent to 14.0 per cent;
1.474 to 1.480. — car-3-ene: 10.0 per cent to 20.0 per cent;
Optical rotation (2.2.7) — p-myrcene: 3.0 per cent to 12.0 per cent;
—7° to -15°. — limonene: 8.0 per cent to 14.0 per cent;
— p-phellandrene: 10.0 per cent to 19.0 per cent;
Acid value (2.5.7)
— p-cymene: maximum 2.5 per cent;
Maximum 1.0.
— terpinolene: maximum 8.0 per cent;
Peroxide value (2.5.5) — bornyl acetate: 0.5 per cent to 5.0 per cent;
Maximum 20. — p-caryophyUene: 0.5 per cent to 5.0 per cent;
Fatty oils and resinified oils (2.5.7) — disregard limit. the area of the principal peak in the
It complies with the test. chromatogram obtained with reference solution (b)
Chromatographic profile (0.05 per cent).
Gas chromatography (2.2.25): use the normalisation STORAGE
procedure. In an inert container and at a temperature not exceeding
Test solution Dilute 200 J1L of the substance to be examined 25 °C.
to 10.0 mL with heptane R. _____________________________________________________ ■ PhEur
Reference solution (a) Dilute 30 |1L of a-pinene R, 5 mg of

camphene Ry 10 pL of p-pinene Ry 20 pL of car-3-ene Ry 5 pL
of P-myrcene Ry 10 pL of limonene Ry 5 pL of p-cymene Ry
10 pL of terpinolene Ry 5 pL of bornyl acetate R and 5 pL of
p-caryophyllene R in heptane R and dilute to 5 mL with the Pine Silvestris Oil ** **
same solvent. (Ph. Eur. monograph 1842) ***
Reference solution (b) Dissolve 5 mg of camphene R in PhEur____________________________________ _________________________
Dilute 1.0 mL of this solution to 10.0 mL with heptane R. DEFINITION
Column'. Essential oil obtained by steam distillation of the fresh leaves
— material', fused silica; and branches of Pinus sylvestris L. A suitable antioxidant may
— size: I = 60 m, 0 = 0.25 mm; be added.
— stationary phase: macrogol 20 000 R (film thickness CHARACTERS
0.25 pm). Appearance
Carrier gas helium for chromatography R. Clear, colourless or pale yellow liquid.
Flow rate 1.5 mL/min. Characteristic odour.
Split ratio 1:50. identification
IV-336 Pine Silvestris Oil 2016
Second identification A p-cymene R, 10 pL of terpinolene R, 10 pL of bornyl acetate R

A. Thin-layer chromatography (2.2.27). and 10 pL of P-caryophyllene R in 1 mL of heptane R.
Test solution Dilute 1 mL of the substance to be examined to Reference solution (b) Dissolve 10 mg of camphene R in
10 mL with toluene R. heptane R and dilute to 2 mL with the same solvent. Dilute
Reference solution Dissolve 10 mg of borneol R and 10 |1L of 0.1 mL of the solution to 1 mL with heptane R.
bornyl acetate R in toluene R and dilute to 10 mL with the Column:
same solvent. — material: fused silica,
Plate TLC silica gel plate R (5-40 |im) [or TLC silica gel — size: z = 60 m, 0 = 0.22 mm,
plate R (2-10 pm)]. — stationary phase: macrogol 20 000 R (0.2 pm).
Mobile phase ethyl acetate R, toluene R (5:95 y/l7). Carrier gas helium for chromatography R.
Application 10 pL [or 2 pL] as bands. Flow rate 1.5 mUmin.
Development Over a path of 15 cm [or 6 cm]. Split ratio 1:100.
Drying In air. Temperature:
Detection Treat with anisaldehyde solution R3 heat at
100-105 °C for 5-10 min and examine in daylight. Time Temperature
(°C)_____________
Column 0 - 10 65
test solution. Furthermore other faint zones may be present 10 - 41 65 -> 220
in the chromatogram obtained with the test solution. 41 - 50 220
Injection port 220

Top of the plate
Detector 250
A pink zone (hydrocarbons)

Bornyl acetate: a brown or A brown or grey-brown zone Injection 0.2 pL.
grey-brown zone (bornyl acetate)
Elution order Order indicated in the preparation of reference
System suitability: reference solution (a) ะ
Borneol: a brown or grey-brown A cluster of violet zones — resolution: minimum 1.5 between the peaks due to car-3-
zone ene and P-myrcene.
Reference solation Test solution determined from the chromatogram obtained with reference
solution (a), locate the components of reference solution (a)
B. Examine the chromatograms obtained in the test for The peak due to p-phellandrene is eluted after the peak due
chromatographic profile. to limonene with a relative retention of about 1.03 with
Results The characteristic peaks in the chromatogram reference to limonene.
obtained with the test solution are similar in retention time to Determine the percentage content of these components.
those in the chromatogram obtained with reference The limits are within the following ranges:
solution (a). — d-pinene: 32.0 per cent to 60.0 per cent,
— camphene: 0.5 per cent to 2.0 per cent,
TESTS
— car-3-ene: 6.0 per cent to 18.0 per cent,
0.855 to 0.875.
— p-myrcene: 1.5 per cent to 10.0 per cent,
Refractive index (2.2.6) — limonene: 7.0 per cent to 12.0 per cent,
1.465 to 1.480. — p-phellandrene: maximum 2.5 per cent,
Optical rotation (2.2.7) — p-cymene: maximum 2.0 per cent,
-9° to -30°. — terpinolene: maximum 4.0 per cent,
Acid value (2.5./) — bornyl acetate: 1.0 per cent to 4.0 per cent,
Maximum 1.0. — P-caryophyllene: 1.0 per cent to 6.0 per cent,
Peroxide value (2.5.5) chromatogram obtained with reference solution (b).
Maximum 20.
STORAGE
Fatty oils and resinified oils (2.8.7)
________PhE’j
procedure.
Test solution The substance to be examined.
Reference solution (a) Dissolve 30 pL of a-pinene R, 10 mg of
camphene R, 20 pL of p-pinene R} 10 pL of car-3-ene R,
10 pL of p-myrcene R, 20 pL of limonene R} 10 pL of
2016 Plantain IV-337
Plantain *****
(Ribwort Plantain, Ph Eur monograph 1884) ***
DEFINITION
Whole or fragmented, dried leaf and scape of Plantago
lanceolata L. ร./.
Content
Minimum 1.5 per cent of total orr/zo-dihydroxycinnamic acid
derivatives expressed as acteoside (C29H36015; Mr 624.6)
(dried drug).
IDENTIFICATION
A. The leaf is up to 30 cm long and 4 cm wide, yellowish-
green to brownish-green, with a prominent, whitish-green,
almost parallel venation on the abaxial surface. It consists of
a lanceolate lamina narrowing at the base into a channelled
petiole. The margin is indistinctly dentate and often
undulate. It has 3, 5 or 7 primary veins, nearly equal in
length and running almost parallel. Hairs may be almost
absent, sparsely scattered or sometimes abundant, especially
on the lower surface and over the veins. The scape is
brownish-green, longer than the leaves, 3-4 mm in diameter
and is deeply grooved longitudinally, with 5-7 conspicuous
ribs. The surface is usually covered with fine hairs.
B. Microscopic examination (2. <3.23). The powder is
characters (Figure 1884.-1): fragments of epidermis,
composed of cells with irregularly sinuous anticlinal walls, the
fragments of the upper epidermis of the lamina in surface
herbal drug of ribwort plantain
view [H] and in transverse section [D] are accompanied by
palisade parenchyma [Da, Ha], and those of the lower
epidermis in surface view [G] show stomata (2.8.3) mostly of Top of the plate
the diacytic type [Ga] and sometimes of the anomocytic
type [Gb]; the multicellular, uniseriate, conical covering
Acteoside: a yellow zone A yellow z วทe (acteoside)
trichomes are highly characteristic, whole [C] or mostly
fragmented [A], with a basal cell larger than the other
epidermal cells followed by a short cell supporting 2 or more
elongated cells with the lumen narrow and variable, occluded
at intervals corresponding to slight swellings in the trichome Aucubin: a blue zone A blue zon e (aucubin)
and giving a jointed appearance, the terminal cell has an Reference solution Test solution
acute apex and a filiform lumen; the glandular trichomes
have a unicellular, cylindrical stalk and a multicellular,
elongated, conical head consisting of several rows of small TESTS
cells and a single terminal cell [B, Gc]; dense groups of Digitalis lanata leaves
lignified fibro-vascular tissue with narrow, spirally and Thin-layer chromatography (2.2.27).
annularly thickened vessels and slender, moderately thickened Solvent mixture water R, methanol R (30:70 V/Vj.
fibres [F]; fragments of the scape [E] with cells with
Test solution Use a freshly prepared solution. To 1 g of the
thickened walls and a coarsely ridged cuticle, stomata [Ec],
powdered herbal drug (355) (2.9.12) in a 25 mL flask, add
multicellular, uniseriate covering trichomes [Eb] and
10 mL of the solvent mixture and shake for 30 min. Filter,
glandular trichomes [Ea] of the type previously described.
rinse the flask and the filter with 2 quantities, each of 5 mL,
c. Examine the chromatograms obtained in the test for of the solvent mixture. Dilute to 25 mL with the solvent
Digitalis lanata leaves. mixture.
Results A See below the sequence of zones present in the Reference solution Dissolve 1 mg of acteoside R and 1 mg of
chromatogram obtained with the reference solution and the aucubin R in 1 mL of the solvent mixture.
Mobile phase anhydrous formic acid R) glacial acetic acid R,
water R) ethyl acetate R (11:11:27:100 VIVIVIV).
Development Over a path of 8 cm; heat immediately after
development at about 120 °C for 5-10 min.
Detection A Examine in daylight.
IV-338 Podophyllum Resin 2016
Results B The chromatogram obtained with the test solution CHARACTERISTICS

shows no bright blue fluorescent zone just below the reddish- An amorphous powder varying in colour from light brown to
brown fluorescent zone corresponding to aucubin in the greenish yellow, or brownish grey masses; odour,
chromatogram obtained with the reference solution. characteristic; caustic.
Foreign matter (2.8.2) Partly soluble in hot water, from which it is precipitated on
Maximum 5 per cent of leaves of different colour and cooling, in chloroform, in ether and in 5m ammonia.
maximum 2 per cent of other foreign matter.
IDENTIFICATION
Loss on drying (2.2.32) Carry out the method for thin-layer chromatography,
Maximum 10.0 per cent, determined on 1.000 g of the Appendix in A, using the following solutions in methanol.
powdered herbal drug (355) (2.9.12) by drying in an oven at (1) 1% w/v of the substance being examined.
105 °C for 2 h.
(2) 0.5% w/v of podophyllotoxin.
Total ash (2.4.16)
(3) 0.1% w/v of phenazone.
ASSAY
Stock solution In a flask, place 1.000 g of the powdered herbal (a) Use as the coating silica gel GF2S4-
drug (355) (2.9.12) and add 90 mL of ethanol (b) Use the mobile phase as described below.
(50 per cent VIV) R. Boil in a water-bath under a reflux (c) Apply as bands 10 pL of each solution.
condenser for 30 min. Allow to cool and filter into a 100 mL (d) Develop the plate to 10 cm.
volumetric flask. Rinse the flask and the filter with 10 mL of (e) After removal of the plate, allow it to dry in air and
ethanol (50 per cent VIV) R. Combine the filtrate and the examine under ultraviolet light (254 nm). spray the plate with
rinsings and dilute to 100.0 mL with ethanol methanolic sulfuric acid (50%) and heat at 130° for
(50 per cent VIV) R. 10 minutes.
Test solution To a 10 mL volumetric flask add, mixing after
MOBILE PHASE
each addition, 1.0 mL of ±e stock solution, 2 mL of
0.5 M hydrochloric acid, 2 mL of a solution prepared by 1 volume of methanol and 25 volumes of chloroform.
dissolving 10 g of sodium nitrite R and 10 g of sodium CONFIRMATION
molybdate R in 100 mL of water R, and 2 mL of dilute sodium When viewed under ultraviolet light (254 nm), the
hydroxide solution R. Dilute to 10.0 mL with water R. chromatogram obtained with solution (1) exhibits quenching
Immediately measure the absorbance (2.2.25) of the test zones corresponding in position to the principal quenching
solution at 525 nm using as compensation liquid a solution zones in the chromatograms obtained with solutions (2) and
prepared as follows: to a 10 mL volumetric flask add 1.0 mL .
(3) Other quenching zones may be present.
of the stock solution, 2 mL of 0.5 M hydrochloric acid and When viewed after spraying, the chromatogram obtained
2 mL of dilute sodium hydroxide solution R, and dilute to with solution (1) exhibits a purplish zone (podophyllotoxin)
10.0 mL with water R. corresponding in position and colour to the principal zone in
Calculate the percentage content of total ortho- solution (2) and a purplish zone
dihydroxycinnamic acid derivatives, expressed as acteoside, (4'-demethylpodophyllotoxin) corresponding in position to
using the following expression: the quenching zone found in solution (3). Other coloured
zones may be present.
A X 1000 TESTS
185 X m Matter insoluble in ethanol (96%)
Shake 1 g, finely powdered, with 20 mL of ethanol (96%) for
i.e. taking the specific absorbance to be 185 for acteoside at 5 minutes, filter through a sintered-glass crucible (ISO 4793,
525 nm. porosity grade 2, is suitable), wash the filter with ethanol
A = absorbance of the test solution at 525 nm; (96%)) and dry at 105°. The residue weighs not more than
m = mass of the substance to be examined, in grams. 25 mg.
________________________________________________________________ Ph Eur
Matter insoluble in 5m ammonia
Shake 0.5 g, finely powdered, with 30 mL of 5m ammonia for
30 minutes at about 20°; filter through a sintered-glass
crucible (ISO 4793, porosity grade 2, is suitable) and wash
the flask and filter with 30 mL of water, the time taken for
Podophyllum Resin filtering and washing being not more than 10 minutes.
Podophyllin Dry the filter and residue to constant weight at 105°.
The residue weighs not less than 0.18 g and not more than
Action and use 0.30 g.
Used in treatment of warts. Loss on drying
Preparation When dried to constant weight at 105°, loses not more than
Compound Podophyllin Paint 5.0% of its weight. Use 1 g.
Sulfated ash
DEFINITION Not more than 1.0%, Appendix IX A.
Podophyllum Resin is the resin obtained from rhizomes and
roots of Podophyllum hexandrum Royle (P. emodi Wall.). ASSAY
It contains not less than 50.0% of total aryltetralin lignans, Dissolve 0.5 g in sufficient ethanol (96%) to produce
calculated as podophyllotoxin. 100 mL. To 10 mL of this solution in a separating funnel
add 190 mL of water and extract with six 30-mL quantities
2016 Red Poppy Petals TV-339
of dichloromethane. Combine the dichloromethane layers, TESTS

extract with 10 mL of 0.2m sodium hydroxide followed by five Weight per mL
10-mL quantities of water and wash each of the six aqueous 0.925 to 0.975 g, Appendix V G.
layers separately with the same 20-mL quantity of
dichloromethane. Combine the dichloromethane solutions, Total solids
27.0 to 33.0% w/v when determined by evaporating 1 mL to
filter through absorbent cotton and evaporate the filtrate to
dryness on a water bath and drying the residue at 105° for
dryness. Dissolve the residue in sufficient ethanol (96%)) to
4 hours.
produce 100 mL, dilute 10 mL of this solution to 50 mL
with ethanol (96%) and measure the absorbance of the 'The law and the statutory regulations governing the use of Industrial
resulting solution at the maximum at 292 nm, Methylated spirit must be observed.
Appendix n B. Calculate the content of total aryltetralin
lignans expressed as podophyllotoxin, taking 105.4 as the
value of A(l%, 1 cm) at the maximum at 292 nm.
STORAGE Red Poppy Petals * *
Podophyllum Resin should be protected from light.
On exposure to light, or to temperatures above 25°, it (Ph. Eur. monograph 1881) ***
becomes darker in colour. Ph Elf_____________________________________________________________
LABELLING DEFINITION
The label states the botanical source. Dried, whole or fragmented petals of Papaver rhoeas L.
IDENTIFICATION
A. The petal is dark red or dark violet-brown, very thin,
floppy, wrinkled, often crumpled into a ball and velvety to
Compound Podophyllin Paint the touch. It is broadly ovate with an entire margin, about
6 cm long and 4-6 cm wide, narrowing at the base where
Compound Podophyllin Cutaneous Solution
there is a black spot. The vascular bundles radiate from the
DEFINITION base and they anastomose in a continuous arc, all at the
Compound Podophyllin Paint is a cutaneous solution. same short distance from the margin.
Podophyllum Resin 150 g B. Reduce to a powder (355) (2.9.12). Examine under a
Compound Benzoin Sufficient to produce 1000 mL microscope using chloral hydrate solution R. The powder has
Tincture an intense reddish-pink colour and shows the following
In making the Compound Benzoin Tincture used to prepare diagnostic characters (Figure 1881.-1): fragments of
Compound Podophyllin Paint, the Ethanol (90 per cent) epidermis composed of elongated, sinuous-walled cells [B, D,
may be replaced by Industrial Methylated Spirit1 diluted so G] with small, rounded, anomocytic stomata (2.8.3) [Ba];
as to be of equivalent ethanolic strength. numerous vascular bundles with spiral vessels [E] embedded
The paint complies with the requirements stated under Liquids for in the parenchyma; occasional fragments of the fibrous layer
Cutaneous Application and with the following requirements. of the anthers [F]; rounded pollen grains, about 30 pm in
diameter, with 3 pores and a finely verrucose exine [A, c,
IDENTIFICATION H] .
Appendix in A, using the following solutions in methanol.
(1) 7% v/v of the paint.
(2) 0.5% w/v of podophyllotoxin.
(3) 0.1% w/v of phenazone.
(a) Use as the coating silica gel GF254-
(c) Apply 10 pL of each solution.
ultraviolet light (254 nm) to locate the quenching zone due to
phenazone in solution (3). Spray the plate with methanolic
sulfuric acid (50%) and heat at 130° for 10 minutes.
MOBILE PHASE
1 volume of methanol and 25 volumes of chloroform.

CONFIRMATION Figure 1881.-1. - Illustration for identification test B ofpowdered
The chromatogram obtained with solution (1) exhibits a herbal drug of red poppy petals
purplish zone (podophyllotoxin) corresponding in position c. Thin-layer chromatography (2.2.27).
and colour to the principal zone in solution (2) and a Test solution To 1.0 g of the powdered herbal drug (355)
purplish zone (4'-demethylpodophyllotoxin) corresponding in (2.9.12) add 10 mL of ethanol (60 per cent V/V) R- Stir for
position to the quenching zone found in solution (3). Other 15 min. Filter through a filter paper.
coloured zones are present in the chromatogram obtained Reference solution Dissolve 1 mg of quinaldme red R and 1 mg
with solution (1). of sulfan blue R in 2 mL of methanol R.
IV-340 Poria 2016
Plate TLC silica gel plate R. and smooth, square, rectangular or polyhedral pieces, with
Mobile phase anhydrous formic acid R, water Ry butanol R no brown skin, difficult to break; slices easily broken, rough
(10:12:40 VIVIV). fracture with granular or farinaceous texture.
Applicatiott 10 pL as bands. B. Microscopic examination (2.8.23). The powder is whitish
Development Over a path of 10 cm. with a pale brown hue. Examine under a microscope using
Drying In air.
diagnostic characters: irregularly shaped and occasionally
Detection Examine in daylight. branched colourless particles, which dissolve gradually in
Results See below the sequence of zones present in the chloral hydrate solution R. Examine under a microscope using
chromatograms obtained with the reference solution and the a 50 g/L solution of potassium hydroxide R. The powder
test solution. Furthermore, other zones may be present in the shows the following diagnostic characters: fragments of
chromatogram obtained with the test solution. hyphae, colourless, slender, slightly curved, sometimes with
septa, branched, 3-16 pm in diameter.
Top of the plate c. Thin-layer chromatography (2.2.27).
2 yellow zones Test solution To 1 g of the powdered herbal drug (250)
(2.9.12) add a mixture of 2 mL of ethyl acetate R and 3 mL
Quinaldine red: an orange-red of methanol R. Sonicate for 10 min, centrifuge and use the
supernatant.
Reference solution Dissolve 10 mg of 4-aminobenzoic acid Ry
A violet principal zone 10 mg of coumarin R and 10 mg of thymol R in 10 mL of
A violet zone methanol R.
A yellow zone
Sulfan blue: a blue zone
Mobile phase glacial acetic acid Ry 2-propanol Ry cyclohexane R
(10:10:80 VIVIV).
A compact group of violet zones Application 5 pL as bands of 8 mm.
Reference solution Test solution Development Over a path of 6 cm.
Drying In air.
TESTS
Foreign matter (2.8.2) Results See below the sequence of zones present in the
Maximum 2.0 per cent of capsules and maximum chromatograms obtained with the reference solution and the
1.0 per cent of other foreign matter. test solution. Furthermore, other faint quenching zones may
Loss on drying (2.2.32) solution between the zones due to 4-aminobenzoic acid and
Maximum 12.0 per cent, determined on 1.000 g of the coumarin in the chromatogram obtained with the reference
powdered herbal drug (355) (2.9.12) by drying in an oven at solution.
105 °C for 2 h.
Total ash (2.4.16)
Top of the plate
Colouring intensity
Place 1.0 g of the powdered herbal drug (355) (2.9.12) in a
250 mL flask and add 100 mL of ethanol (30 per cent VIV) R.
Thymol: a quenching zone
Allow to macerate for 4 h with frequent stirring. Filter and
discard the first 10 mL. To 10.0 mL of the filtrate add 2 mL 2 quenching zones
of hydrochloric acid R and dilute to 100.0 mL with ethanol
(30 per cent VIV) R. Allow to stand for 10 min.
The absorbance (2.2.26) measured at 523 nm using ethanol Coumarin: a quenching zone
(30 per cent VIV) R as the compensation liquid is not less 4-Aminobenzoic acid: a
than 0.6. quenching zone
________________________________________________________________ Ph Ear
Poria D. The herbal drug sticks to the pestle when moistened with
water R and pressed into a mortar.
(Ph Eur monograph 2475) E. To a small piece of the herbal drug add 1 drop of
Ph Ell_______________________ iodinated potassium iodide solution Rl. A deep red colour is
produced.
DEFINITION
Dried sclerotium without skin of Wolfiporia extensa (Peck) TESTS
Ginns (syn. Poria cocos (Schw.) Wolf; Wolfiporia cocos (F.A. Foreign matter (2.8.2)
Wolf) Ryvarden & Gilb.). Maximum 0.1 per cent of brown skins and roots of conifer
and maximum 2 per cent of other foreign matter.
IDENTIFICATION
A. Square, rectangular or polyhedral pieces, or slices, varying
in length and thickness; whitish with a pale brown hue, flat
2016 Primula Root IV-341

105 °C for 2 h.
Total ash (2.4.16)
Water-soluble extractive
100 mL of boiling water R. Allow to stand for 10 min,
shaking occasionally. Allow to cool, dilute to 100.0 mL with
water R and filter. Evaporate 25.0 mL of the filtrate to
dryness on a water-bath. Dry the residue in an oven at
100-105 °C. The residue weighs a minimum of 18.75 mg.
-------------------------------------------------- ------------------------------------------------------ Ph Eur
Primula Root *****

DEFINITION
Whole or cut, dried rhizome and root of Primula veris L.
or Primula elatior Hill.
IDENTIFICATION
A. The coarsely torose, greyish-brown rhizome is straight or
slightly curved, about 1-5 cm long and about 2-4 mm thick. Figure 1364.-1. - Illustration for identification test B of powdered
The rhizome crown often bears the remains of stems and herbal drug of primula root
leaves. Attached to the rhizome are numerous brittle roots, TESTS
about 1 mm thick and usually 6-8 cm long. The root of Vincetoxicum hirundinaria Medik. root
р. elatior is light brown or reddish-brown, that of p. veris Thin-layer chromatography (2.2.27).
light yellow or yellowish-white. The fracture is smooth. Test solution To 1.0 g of the powdered herbal drug (500)
B. Microscopic examination (2.8.23). The powder is greyish- (2.9.72) add 10 mL of ethanol (70 per cent VIV) R and heat
brown. Examine under a microscope using chloral hydrate under a reflux condenser for 15 min. Cool and filter.
solution R. The powder shows the following diagnostic Reference solution Dissolve 10 mg of aescin R in 1.0 mL of
characters (Figure 1364.-1): fragments of parenchyma from ethanol (70 per cent VIV) R.
the bark of the root or the rhizome and from the medulla of
the rhizome [G, H], consisting of rounded or ovoid cells with
irregularly thickened and pitted walls; brownish fragments Mobile phase glacial acetic acid R, water R, butanol R
from the dermal tissue of the root showing absorbent hairs (10:40:50 VIVIV)’) use the upper layer.
[C]; yellow or brownish fragments of the epidermis of the Application 20 |1L as bands.
rhizome covered by a striated cuticle, in surface view [A], or Development Over a path of 12 cm.
in transverse section [F] accompanied by parenchyma from Drying In an oven at 100-105 °C.
the bark [Fa]; reticulate vessels [B] sometimes accompanied
by spiral vessels [J]; groups of large, strongly pitted,
yellowish-green sclereids from the medullary parenchyma of Results A The chromatograms obtained with the reference
the rhizome [E], which are characteristic of p. elatior. solution and the test solution show a quenching zone (aescin)
Examine under a microscope using a 50 per cent VIV near the boundary between the lower and the middle thirds.
solution of glycerol R. The powder shows simple or Mark this zone.
compound starch granules of various shapes and sizes [D]. Detection B Examine in ultraviolet light at 365 nm.
с. Thin-layer chromatography (2.2.27) as described in the Results B In the chromatogram obtained with the test
test for Vincetoxicum hirundinaria Medik. root with the solution no zones of light-blue or greenish fluorescence occur
following modifications. below the main zone due to aescin in the chromatogram
Detection Treat with anisaldehyde solution R, heat at
100-105 °C for 5-10 min and examine in daylight. Loss on drying (2.2.32)
Results The main zone (aescin) in the chromatogram powdered herbal drug (355) (2.9.72) by drying in an oven at
obtained with the reference solution is bluish-violet and is
105 °C for 2 h.
situated near the boundary between the lower and middle
thirds. The chromatogram obtained with the test solution Total ash (2.4.16)
shows 1-2 strong dark violet zones a little below the zone due
to aescin in the chromatogram obtained with the reference Ash insoluble in hydrochloric acid (2.8.1)
solution; further pale violet, yellowish or brownish-green Maximum 3.0 per cent.
__________________ Ph Eur
zones may be visible.
IV-342 Psyllium Seed 2016
Psyllium Seed ** **
Ph Elf_______________________________________________________________
DEFINITION
Ripe, whole, dry seeds of Plantago afra L. (Plantago
psyllium L.) or Plantago indica L. {Plantago arenaria Waldstein
and Kitaibel).
CHARACTERS
Sweet taste.
IDENTIFICATION
p afra Seeds are light brown to very dark brown but never
black, smooth and shiny having an elliptical oblong shape.
They are 2-3 mm long and 0.8-1.0 mm wide, one end being
wider than the other. Towards the middle of the dorsal
surface there is a fairly marked transverse constriction of light
colour. On the ventral surface, there is a linear lighter
coloured groove in the middle of which is a clear spot
corresponding to the hilum and bounded by swollen edges.
p indica Seeds are almost identical to the seeds of p. afra, but
a little less shiny; they are 2-3 mm long and have a
maximum diameter of 1.5 mm.
TESTS
Minimum 10.
Maximum 1.0 per cent, determined on 10.0 g of the drug,
including greenish unripe seeds. Psyllium seed does not Figure 1886.-1. - Illustration for identification test B of powdered
contain seeds having a dark central spot on the groove herbal drug of pygeum africanum bark
(Plantago lanceolata L. and p. major L.) or seeds with B. Microscopic examination (2.8.23). The powder is reddish-
brownish-grey or pinkish outer coats (P. ovata Forssk. brown. Examine under a microscope using chloral hydrate
and p. sempervirens Crantz). solution R. The powder shows the following diagnostic
Loss on drying (2.2.32) characters (Figure 1886.-1): numerous sclereids, varying
Maximum 14.0 per cent, determined on 1.000 g of drug by greatly in size, up to more than 500 pm in diameter, with
drying in an oven at 105 °C for 2 h. very thick walls showing concentric striations and a reduced
Total ash (2.4.16) lumen, isolated [A] or in groups [B] sometimes accompanied
Maximum 4.0 per cent. by sclereids about 50 pm in diameter [Ba], some with
granular reddish-brown contents [Bbl; isolated cluster
STORAGE crystals of calcium oxalate of various sizes [C] and a few
Store protected from moisture. calcium oxalate prisms [F]; numerous lignified fibres, usually
------------------------------------------------------------------------------------------------------------- Ph Eur broken, thick-walled and channelled with a narrow lumen,
sometimes isolated [L], but usually in groups [G]
accompanied by rectangular cells of the medullary rays [Ga];
fragments of parenchyma with reddish-brown, polygonal or
ovoid cells [D], including some with reticulate walls [J, M];
Pygeum Bark * ** fragments of cork (surface view [HJ, side view [E]). Examine
(Pygeum Africanum Bark, Ph Eur monograph 1886) *** under a microscope using lactic reagent R. The powder shows
a few simple starch granules that stain violet-blue, rounded,
Ph Elf__________________________________________________ _____________
10-20 pm in diameter, with a punedform or Y-shaped
DEFINITION hilum [K].
Whole or fragmented, dried bark of the stems and branches c. Thin-layer chromatography (2.2.27).
of Prunus africana (Hook.f.) Kalkman (syn. Pygeum africanum Test solution Extract 15.0 g of the powdered herbal drug
Hook-f.). (250) (2.9.12) with methylene chloride R for 30 min in a
IDENTIFICATION continuous extraction apparatus (Soxhlet type). Filter.
A. The dark brown or reddish-brown bark occurs in curved, Evaporate the solvent to dryness under reduced pressure.
hard, irregular pieces. The outer surface has a wrinkled dark Dissolve the residue in 1 mL of methylene chloride R.
reddish-brown cork with areas of adhering lichen. Reference solution Dissolve 20 mg of fl-sitosterol R and 20 mg
The reddish-brown or dark brown inner surface bears of ursolic acid R in 10 mL of a mixture of equal volumes of
longitudinal striations. It may also occur in rolled fragments methanol R and methylene chloride R.
with a fibrous fracture. Plate TLC silica gel plate R.
Mobile phase methanol R, methylene chloride R (10:90 VIV).
2016 Quillaia Bark IV-343
Development Over a path of 15 cm. oxalate as glistening points. Smoothed transversely cut
Drying In air. surface appearing chequered, with delicate radial lines
Detection treat with vanillin reagent R, heat at 100-105 °C for representing medullary rays and tangential lines formed by
10 min and allow to cool; examine in daylight. alternating tangential bands of fibrous and non-fibrous
Results See below the sequence of zones present in the phloem.
chromatograms obtained with the reference solution and the B. Outer bark, when present, consisting of reddish brown
test solution. Furthermore, other zones may be present in the cork cells alternating with bands of brown parenchyma
chromatogram obtained with the test solution. containing numerous groups of phloem fibres and large
prisms of calcium oxalate. Inner bark consisting of alternating
bands of tortuous fibres, irregularly enlarged at intervals,
Top of the plate
about 500 to 1000 pm long and 20 to 50 pm wide and of
A violet zone sieve tissue mixed with parenchyma. Medullary rays mostly
Several weak violet, blue or grey three to four, but sometimes up to six cells wide, with
zones occasional pitted, subrectangular sclereids adjacent to the
bundles of phloem fibres. Starch granules 5 to 20 pm,
^'Sitosterol: a violet zone A violet zone (0-sitosterol) usually about 10 pm, in diameter, and prisms of calcium
Ursolic acid: a blue zone A blue zone (ursolic acid) oxalate usually 50 to 170 pm long and up to 30 pm wide
Several weak violet, blue or grey present in the parenchymatous cells.
zones
TESTS
Extractive soluble in ethanol (45%)
A violet zone (0-sitosterol Not less than 22.0%, Appendix XI Bl.
glucoside)
Acid-insoluble ash » ■รุ . .
Not more than 1.0%, Appendix XI K.
Foreign matter
TESTS Complies with the test for foreign matter, Appendix XI D.
Maximum 12.0 per cent, determined on 1.000 g of the Quillaia Bark *****
105 °C for 2 h. (Ph. Eur. monograph 1843) ***
Total ash {2.4.16) PhEur_____________________________________________________________

Extractable matter Whole or fragmented, dried bark, with the cork and
Minimum 0.5 per cent. underlying parenchyma removed, of Quillaja saponaria
Extract 20.0 g of the powdered herbal drug (250) {2.9.12) Molina ร.I. -..1
with methylene chloride R for 4 h in a continuous extraction Content
apparatus (Soxhlet type). Evaporate the solution to dryness Minimum 6.5 per cent of triterpene glycosides, expressed as
on a water-bath in vacuo and then dry the residue at 80 °C quillaia saponin in (0104แ168055; Afr 2298) (dried drug).
for 2 h. The residue weighs a minimum of 0.10 g. IDENTIFICATION
----------- ------------------------------------------------------ ---------------------- ะ______________________________ PhEur A. Large, flat pieces of variable length and width, 3-10 mm
thick, or smaller, splintered pieces. The outer surface is
brownish-white or pale reddish-brown, longitudinally striated
or coarsely reticulated, with occasional blackish-brown
patches of incompletely removed outer bark. The inner
Quillaia surface is yelloyvish-white and smooth. The fracture is
Quillaia Bark splintery and laminated, the surface often glistening due to
Preparation the presence of numerous’ large prisms of calcium oxalate.
Quillaia Liquid Extract B. Microscopic examination {2.8.23). The powder is pale
When Powdered Quillaia is prescribed or demanded, material pinkish-yellow. Examine under a microscope using chloral
complying with the appropriate requirements below shall be hydrate solution R. The powder shows ±e following diagnostic
dispensed or supplied. characters (Figure 1843.-1): abundant phloem fibres [E, F],
up to 1 mm long, isolated or, more usually, in groups, each
DEFINITION
fibre irregular in outline with lignified walls of varying
Quillaia is the dried inner part of the bark of Quillaja
thickness and an uneven lumen; numerous, multiseriate
saponaria Molina and of other species of Quillaja. medullary rays, spindle-shaped in tangential section [Ca, Fb],
IDENTIFICATION accompanied by either phloem fibres [Fa] or phloem
A. Pieces flat, up to about 1 metre long, 10 to 20 cm broad parenchyma [Cb]; very numerous prisms of calcium oxalate,
and 3 to 10 mm, usually 6 mm, thick. Outer surface up to 200 pm long, free, whole or, more usually, fragmented
brownish white or pale reddish brown, longitudinally striated [A] or included in phloem parenchyma cells [Cc, Cd];
or coarsely reticulated, with occasional blackish brown occasional sclereids of 2 types: the 1st type is sub-rectangular
patches of adherent outer bark; inner surface yellowish white, with pitted, slightly thickened walls, isolated [G] or included
smooth and very hard; fracture splintery and laminated, the in phloem parenchyma cells [H], while the 2nd type has an
broken surface showing numerous large prisms of calcium irregularly shaped outline and very thick walls [J], sometimes
IV-344 Quillaia Bark 2016
adjacent to the bundles of phloem fibres; occasional dark Top of the plate
brown or reddish-brown fragments of cork [D]. Examine
glycerol R. The powder show's numerous, small (5-20 pm), Quillaia saponins: 3 or more green 3 or more green or brown zones
mainly simple, spherical starch granules, either scattered or as or brown zones (quillaia saponins)
compacted masses in parenchyma cells [B]. A blue zone
Sucrose: a brown or blue zone A brown or blue zone (sucrose)
TESTS
105 °C for 2 h.
Total ash (2.4.76)
ASSAY
(355) (2.9.72) into a round-bottomed flask, add 20 mL of a
20 g/L solution of potassium hydroxide R and heat under a
reflux condenser in a water-bath for 2 h. After cooling, add
2 mL of phosphoric acid R and filter through a plug of
absorbent cotton. Add the absorbent cotton to the residue,
add 25 mL of ethanol (96 per cent) R and shake thoroughly.
Filter. Combine the filtrates and dilute to 50.0 mL with
water R. Filter through a membrane filter (nominal pore size
0.45 pm).
Reference solution (a) Dissolve 12.0 mg of quillaia saponin for
Figure 1843.-1. - Illustration for identification test B of assay CRS (containing monoammonium glycyrrhizate) in a
powdered herbal drug of quillaia bark mixture of equal volumes of ethanol (96 per cent) R and a
10 g/L solution of phosphoric acid R3 and dilute to 50.0 mL
Figure 1843.-1. - Illustration for identification test B of powdered with the same mixture of solvents.
herbal drug of quillaia bark Reference solution (b) Introduce 12 mg of purified quillaia
c. Thin-layer chromatography (2.2.27). saponins HRS into a 50 mL round-bottomed flask, add
Test solution To 1.0 g of the powdered herbal drug (355) 20 mL of a 20 g/L solution of potassium hydroxide R and heat
(2.9.72) add 5 mL of methanol R and 5 mL of water R. under a reflux condenser in a water-bath for 2 h. After
Sonicate for 10 min and filter. cooling, add 2 mL of phosphoric acid R. Add 25 mL of
ethanol (96 per cent) R and shake thoroughly. Dilute to
Reference solution Dissolve 10 mg of purified quillaia saponins R
50.0 mL with water R. Filter through a membrane filter
and 2 mg of sucrose R in 1 mL of water R and mix with 1 mL
of methanol R.
Column'.
Plate TLC silica gel plate R (2-10 pm).
— size'. I = 0.25 m, 0 = 4.6 mm;
Mobile phase anhydrous acetic acid R, ethyl acetate R, water R, — stationary phase', octadecylsilyl silica gel for chromatography R
propanol R (1.5:30:30:40 VIVIVIV). (5 pm);
Application 5 pL as bands of 6 mm. — temperature'. 30 ± 2 °C.
Development Over a path of 6 cm. Mobile phase acetonitrile R15 1 g/L solution of phosphoric acid R
Drying In hot air. (35:65 VIV).
Detection Treat with a 10 per cent VIV solution of sulfuric Flow rate 1.0 mL/min.
acid R in methanol R'i heat at 120 °C for 5 min and examine Detection Spectrophotometer at 210 nm.
in daylight. Injection 50 pL.
Results See below the sequence of zones present in the Run time 1.2 times the retention time of glycyrrhizic add.
chromatograms obtained with the reference solution and the Identification ofpeaks Use the chromatogram supplied with
test solution. Furthermore, other faint zones may be present purified quillaia saponins HRS and the chromatogram obtained
in the chromatogram obtained with the test solution. with reference solution (b) to identify the peaks due to
monodesmosidic quillaia saponins 1 and 3; a minor peak due
to monodesmosidic quillaia saponin 2 may be present
2016 Restharrow Root IV-345
between the peaks due to monodesmosidic quillaia

saponins 1 and 3. Quillaia Tincture
Retention time Monodesmosidic quillaia saponin 1 = about DEFINITION
9 nun; monodesmosidic quillaia saponin 3 = about 10 min; Quillaia Liquid Extract 50 mL
glycyrrhizic acid ะ= about 13 min. Ethanol (45 per cent) Sufficient to produce 1000 mL
Calculate the percentage content of triterpene glycosides, Extemporaneous preparation
expressed as quillaia saponin in, using the following The following directions apply.
expression: Mix, allow to stand for not less than 12 hours and filter.
Al X 7ท2 X p X 2298 X 0.6 under Extracts and with the following requirements.
A2 X 7ท1 X 957
TESTS
Ethanol content
A] = sum of the areas of the peaks due to 43 to 45% v/v, Appendix vni F, Method m.
monodesmosidic quillaia saponins (1, 2 and 3)
Dry residue
solution; 1.0 to 1.5% w/v. Use 10 mL.
A2 = area of the peak due to glycyrrhizic acid derived Relative density
from monoammonium glycyrrhizate in the 0.940 to 0.955, Appendix V G.
(a);
พ1 = mass of the herbal drug to be examined used to
n/2 ะ=ะ mass of quillaia saponin for assay CRS used to Restharrow Root * *
prepare reference solution (a), in grams; (Ph. Eur. monograph 1879) ***
p = percentage content of monoammonium
Ph Eur_____________________________________________________________
glycyrrhizate in quillaia saponin for assay CRS;
0-6 = response factor between monoammonium DEFINITION
glycyrrhizate and monodesmosidic quillaia Whole or cut, dried root of Ononis spinosa L.
saponin 3; IDENTIFICATION
2298 = molecular mass of quillaia saponin III;
A. The root is more or less flattened, twisted and branched,
957 = molecular mass of monodesmosidic quillaia
deeply wrinkled, brown and grooved longitudinally.
saponin 3.
The transversely cut surface shows a thin bark and a xylem
—------------------------------------------------------------------------------------------------------- Ph Eur cylinder with a conspicuously radiate structure. The fracture
of the root is short and fibrous.
B. Microscopic examination (2.8.23). The powder is light
brown or brown. Examine under a microscope using chloral
Quillaia Liquid Extract characters (Figure 1879.-1): brown fragments of cork
DEFINITION composed of thin-walled, polygonal cells (surface view [G]);
Quillaia Liquid Extract is prepared by extracting Quillaia groups of thick-walled, narrow fibres, often accompanied by
with Ethanol (45 per cent). a parenchymatous crystal sheath containing prisms of calcium
Extemporaneous preparation oxalate [C]; vascular fragments [D, E] consisting of vessels
The following formula and directions apply. with numerous bordered pits, often accompanied by lignified
Quillaia, in moderately fine powder 1000 g fibres with pitted walls [Ea]; thin-walled parenchymatous
Ethanol (45 per cent) A sufficient quantity cells from the bark, some containing a single prism of
calcium oxalate [H]; ligneous parenchyma cells with slightly
Exhaust the Quillaia in moderately fine powder with Ethanol thickened and pitted walls [A, B], some of which contain
(45 per cent) by percolation, Appendix XI F, and reserve the prisms of calcium oxalate [Aa]; numerous free prisms of
first 850 mL of percolate. Evaporate the subsequent calcium oxalate [F]. Examine under a microscope using a
percolate to the consistence of a soft extract, dissolve it in the 50 per cent vtv solution of glycerol R. The powder shows
reserved portion and add sufficient Ethanol (45 per cent) to very numerous, rounded starch granules, 5-10 pm in
produce 1000 mL. Allow to stand for not less than 24 hours; diameter, simple or sometimes 2-4 compound, free U] or
filter. inside parenchymatous cells [K].
The extract complies with the requirements stated under Extracts c. Thin-layer chromatography (2.2.27).
TESTS (2.9.12) add 15.0 mL of methanol R and boil under a reflux
Ethanol content condenser for 30 min. Cool and filter.
28 to 34% v/v, Appendix VIII F, Method in. Reference solution Dissolve 10 mg of resorcinol R and 50 mg of
Dry residue vanillin R in 10 mL of methanol R.
20 to 30% w/v. Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel
Relative density F254 plate R (2-10 pm)].
1.02 to 1.06, Appendix V G. Mobile phase ethanol (96 per cent) R, methylene chloride R,
toluene R (10:45:45 VIVIV).
IV-346 Rhatany Root 2016
Top of the plate
Vanillin: a greyish-violet zone
A violet zone (onocol)
Resorcinol: a red zone
TESTS
105 °C for 2 h.
Total ash (2.4.16)
Extractable matter
To 2.00 g of the powdered herbal drug (250) (2.9.12) add a
mixture of 8 g of water R and 12 g of ethanol (96 per cent) R
and allow to macerate for 2 h, shaking frequently. Filter,
evaporate 5 g of the filtrate to dryness on a water-bath and
dry in an oven at 100-105 °C for 2 h. The residue weighs a
minimum of 75 mg.
___________________________________________________________ ___ PnEtr

herbal drug of restharrow root
Development Over a path of 15 cm [or 6 cm]. Rhatany Root t *
Drying In air. Krameria ***
Detection A Examine in ultraviolet light at 254 nm and (Ph. Eur. monograph 0289)
365 nm. Preparation
Results A See below the sequence of zones present in the Rhatany Tincture
chromatograms obtained with the reference solution and the When Powdered Rhatany Root is prescribed or demanded,
test solution. Furthermore, other fluorescent zones are material complying with the requirements below with the
present in the middle third of the chromatogram obtained exception of Identification test A and the test for Foreign
with the test solution. matter shall be dispensed or supplied.
PhEur________ _______________________________________________ ______
Top of he plate
DEFINITION
Vanillin ะ a zone visible at 254 nm Dried, usually fragmented, underground organs of Krameria
triandra Ruiz et Pav., known as Peruvian rhatany.
Content
Resorcinol ะ a zone visible at An intense blue fluorescent zone
254 nm visible at 365 nm
(C6H6O3; Mt 126.1) (dried drug).
Reference solution
IDENTIFICATION
Test solution
A. The taproot is dark reddish-brown and has a thick, knotty
crown. The secondary roots are the same colour and nearly
Detection B treat with anisaldehyde solution R. Heat at straight or somewhat tortuous. The bark is rugged or scaly in
100-105 °C for 5-10 min. Examine in daylight. the older pieces and smooth with sharp, transverse fissures in
the younger pieces; it separates readily from the wood.
The fracture is fibrous in the bark and splintery in the wood.
test solution. The smooth, transversely cut surface shows a dark brownish-
red bark about one third of the radius in thickness; a dense,
pale reddish-brown and finely porous wood is present with
numerous fine meddiary rays; the central heartwood is often
darker.
B. Microscopic examination (2.8.23). The powder is reddish-
characters (Figure 0289.-1): cork cells containing dark brown
2016 Rhatany Preparations IV-347
phlobaphenes (surface view [A], side view [B]); fragments of test solution. Furthermore, other faint zones may be present
phloem [C] consisting of unlignified fibres, usually 12-30 pm in the chromatogram obtained with the test solution.
in diameter with moderately thickened walls [Ca],
parenchyma cells some containing prisms and microcrystals
of calcium oxalate [Cc], and cells of the medullary rays [Cb]; Top of the plate
fragments of vessels usually 20-60 pm in diameter with
bordered pits [E]; fragments of tracheids [D] up to 20 pm
Thymol: a brownish-yellow zone A faint violet zone
นาde with slit-shaped pits [Da] and cells of the medullary
rays [Db]; lignified parenchymatous cells with thick and
channelled walls [F]. Examine under a microscope using a
An orange zone
ว0 per cent V/V solution of glycerol R. The powder shows
rounded, simple or 2- to 4-compound starch granules, an A bluish-grey zone
individual granule measuring up to 30 pm in diameter [H]
and some granules being found in the cells of the medullary
rays and in the parenchyma [G]. Dichlorophenolindophenol: a
greyish-blue zone
An intense violet zone
Reference soludon Test solution 1
TESTS
Maximum 2 per cent of foreign matter and maximum
5 per cent of fragments of crown or root exceeding 25 mm in
diameter. Root without bark may be present in very small
quantities.
105 °C.
Total ash {2.4.16)
ASSAY
Tannins {2.8.14)
_____________________________________________________________ PhEur
Rhatany Tincture ★ *
Figure 0289.-1. - Illustration for identification test B of powdered PhEur______________________________________________________________
herbal drug of rhatany root DEFINITION
c. Thin-layer chromatography (2.2.27). Tincture produced from Rhatany root (0289).
Test solution To 1.0 g of the powdered herbal drug (355) Content
(2.9.12) add 10 mL of methanol R and sonicate for 10 min. Minimum 1.0 per cent m/m of tannins, expressed as
Centrifuge or filter. Use the supernatant or filtrate. pyrogallol (C6H6O3; 126.1).
Reference solution Dissolve 5 mg of thymol R and 20 mg of
PRODUCTION
dichlorophenolindophenol, sodium salt R in 20 mL of ethanol
The tincture is produced from 1 part of the herbal drug and
5 parts of ethanol (70 per cent V/V) by a suitable procedure.
plate R (2-10 pm)]. CHARACTERS
Appearance
Reddish-brown liquid.
IDENTIFICATION
Drying In air.
Test solution The tincture to be examined.
Detection Treat with a 5 g/L solution offast blue B salt R,
Reference solution Dissolve 5 mg of thymol R and 20 mg of
allow to dry in air and examine in daylight.
dMorophenolindophenol, sodium salt R in 20 mL of ethanol
Results See below the sequence of zones present in the (60 per cent V/V) R.
IV-348 Rhubarb 2016
Plate TLC silica gel plate R (5-40 |im) [or TLC silica gel CHARACTERS
plate R (2-10 gm)]. Characteristic, aromatic odour.
IDENTIFICATION
Application 10 pL [or 4 gL] as bands of 8 mm [or 8 mm]. A. The appearance is variable: disc-shaped pieces up to
Development Over a path of 15 cm [or 6 cm]. 10 cm in diameter and 1 cm to 5 cm in thickness; cylindrical
Drying In air. pieces; oval or planoconvex pieces. The surface has a pinkish
Detection Treat with a 5 g/L solution offast blue B salt R, tinge and is usually covered with a layer of brownish-yellow
allow to dry in air and examine in daylight. powder. It shows, especially after moistening, a reticulum of
darker lines. This structure causes the marbled appearance of
Results See below the sequence of zones present in the the drug. The fracture is granular. The transverse section of
the rhizome shows a narrow outer zone of radiating
test solution. Furthermore, other faint zones may be present brownish-red lines. These medullary rays are crossed
in the chromatogram obtained with the test solution. perpendicularly by a dark cambial ring. Inside this zone is a
ring of small star-spot formations of anomalous vascular
Top of the plate bundles. The root shows a more radiate structure.
B. Reduce to a powder (355) (2.9.12). The powder is orange
to brownish-yellow. Examine under a microscope using
Thymol: a brownish-yellow zone A violet zone
diagnostic characters: large calcium oxalate cluster crystals,
An orange zone
which may measure more than 100 gm, and their fragments;
reticulately thickened non-lignified vessels measuring up to
A bluish-grey zone 175 gm. Numerous groups of rounded or polygonal, thin
walled parenchyma cells. Sclereids and fibres are absent.
Dichlorophenolindophenol: a
greyish-blue zone solution of glycerol R. The powder shows simple, rounded or
An intense violet zone compound (2 to 4) starch granules with a star-shaped hilum,
c. Examine by thin-layer chromatography (2.2.27)5 using a
suitable silica gel as the coating substance.
Test solution Heat 50 mg of the powdered herbal drug (180)
(2.9.12) in a water-bath for 15 min with a mixture of 1 mL
of hydrochloric acid R and 30 mL of water R. Allow to cool
TESTS
and shake the liquid with 25 mL of ether R. Dry the ether
Ethanol (2.9.10) layer over anhydrous sodium sulfate R and filter. Evaporate the
63 per cent VIV to 67 per cent VIV. ether layer to dryness and dissolve the residue in 0.5 mL of
Methanol and 2-propanol (2.9.11) ether R.
Maximum 0.05 per cent VIV of methanol and maximum Reference solution Dissolve 5 mg of emodin I? in 5 mL of
0.05 per cent VIVof 2-propanol. ether R.
ASSAY Apply separately to the plate as bands 20 gL of each
Tannins (2.8.14) solution. Develop over a path of 10 cm using a mixture of
Use 2.500 g of the tincture to be examined. 1 volume of anhydrous formic acid R, 25 volumes of ethyl
________________________________________________________________ Ph Eur acetate R and. 75 volumes of light petroleum R. Allow the plate
to dry in air and examine in ultraviolet light at 365 nm.
The chromatogram obtained with the reference solution
shows in its central part a zone of orange fluorescence
(emodin). The chromatogram obtained with the test solution
Rhubarb * * shows: a zone due to emodin; above the emodin zone, two
zones of similar fluorescence (physcione and chrysophanol, in
order of increasing Rp value); below the emodin zone, also
Preparation two zones of similar fluorescence (rhein and aloe-emodin, in
Compound Rhubarb Tincture order of decreasing Rp value), spray with a 100 g/L solution
When Powdered Rhubarb is prescribed or demanded, of potassium hydroxide R in methanol R. All the zones become
material complying with the requirements below with the red to violet.
exception of Identification test A and the test for Foreign D. To about 50 mg of the powdered herbal drug (180)
matter shall be dispensed or supplied. (2.9.12) add 25 mL of dilute hydrochloric acid R and heat the
Ph Eur____________________________________________ __ ________________ mixture on a water-bath for 15 min. Allow to cool, shake
with 20 mL of ether R and discard the aqueous layer. Shake
DEFINITION the ether layer with 10 mL of dilute ammonia Rl.
Rhubarb consists of the whole or cut, dried underground The aqueous layer becomes red to violet.
parts of Rheum palmatum L. or of Rheum officinale Bailion or
of hybrids of these two species or of a mixture. TESTS
The underground parts are often divided; the stem and most Rheum rhaponticum
of the bark with the roodets are removed. It contains not less Examine by thin-layer chromatography (2.2.27)i using silica
than 2.2 per cent of hydroxyanthracene derivatives, expressed gel G R as the coating substance.
as rhein (CisHgOe, Mr 284.2), calculated with reference to Test solution To 0.2 g of the powdered herbal drug (180)
the dried drug. (2.9.12) add 2 mL of methanol R and boil for 5 min under a
2016 Roselle IV-349
reflux condenser. Allow to cool and filter. Use the filtrate as

the test solution. Compound Rhubarb Tincture
Reference solution Dissolve 10 mg of rhaponticin J? in 10 mL of DEFINITION
mahanol R. Rhubarb, in moderately 100 g
Apply separately to the plate, as bands not more than 20 mm coarse powder
by 3 mm, 20 pL of each solution. Develop over a path of Cardamom Oil 0.40mL
12 cm using a mixture of 20 volumes of methanol R and Coriander Oil 0.03mL
80 volumes of methylene chloride R. Allow the plate to dry in Glycerol 100 mL
air and spray with phosphomolybdic acid solution R. Ethanol (60 per cent) Sufficient to produce 1000 mL
The chromatogram obtained with the test solution does not Extemporaneous preparation
show a blue zone near the line of application (rhaponticin) The following directions apply.
corresponding to the zone in the chromatogram obtained Moisten the Rhubarb with a sufficient quantity of Ethanol
with the reference solution. (60 per cent) and prepare 850 mL of tincture by percolation,
Loss on drying (2.2.22) Appendix XI F. Add the Cardamom Oil, the Coriander Oil
Not more than 12.0 per cent, determined on 1.000 g of the and the Glycerol and sufficient Ethanol (60 per cent) to
powdered herbal drug (180) (2.9.72) by drying in an oven at produce 1000 mL. Mix and filter, if necessary.
105 °C. The tincture complies with the requirements for Tinctures stated
Total ash (2.4.76) under Extracts and with the following requirements.
Not more than 12.0 per cent. TESTS
Ash insoluble in hydrochloric acid (2. ร. 7) Ethanol content
Not more than 2.0 per cent. 48 to 53% v/v, Appendix VUI F, Method in.
ASSAY Glycerol
Carry out the assay protected from blight light. 9.0 to 11.0% v/v when determined by the following method.
Dilute 20 mL to 100 mL with water, to 10 mL of this
Introduce 0.100 g of the powdered herbal drug (180)
solution add 100 mL of water and 1 g of activated charcoal
(2.9.72) into a 100 mL flask. Add 30.0 mL of water R, mix
and boil under a reflux condenser for 15 minutes. Filter,
and weigh. Heat in a water-bath under a reflux condenser for
wash the filter and charcoal with sufficient water to produce
15 min. Allow to cool, add 50 mg of sodium hydrogen
150 mL, add 0.25 mL of bromocresol purple solution and
carbonate R, weigh and adjust to the original mass with
neutralise with 0.1m sodium hydroxide or 0.05m sulfuric acid
water R. Centrifuge and transfer 10.0 mL of the liquid to a
to the blue colour of the indicator. Add 1.4 g of sodium
100 mL round-bottomed flask with a ground-glass neck.
periodate, allow to stand for 15 minutes, add 3 mL of
Add 20 mL of ferric chloride solution R1 and mix. Heat under
propane-1,2-diol, shake and allow to stand for 5 minutes.
a reflux condenser on a water-bath for 20 min, add 1 mL of
Add 0.25 mL of bromocresol purple solution and titrate with
hydrochloric acid R and heat for a further 20 min, shaking
0.1m sodium hydroxide KS to the same blue colour. Each mL
frequently. Cool, transfer to a separating funnel and shake
of 0.1m sodium hydroxide vs is equivalent to 9.210 mg of
with three quantities, each of 25 mL, of ether R previously
glycerol. Calculate the percentage v/v of glycerol, taking
used to rinse the flask. Combine the ether extracts and wash
1.260 g as its weight per mL.
with two quantities, each of 15 mL, of water R. Filter the
ether extracts through a plug of absorbent cotton into a Relative density
volumetric flask and dilute to 100.0 mL with ether R. 0.958 to 0.977, Appendix V G.
Evaporate 10.0 mL carefully to dryness on a water-bath and
dissolve the residue in 10.0 mL of a 5 g/L solution of
magnesium acetate R in methanol R. Measure the absorbance
(2.2.25) at 515 nm, using methanol R as the compensation
liquid. Roselle
Calculate the percentage content of rhein from the (Ph. Eur. monograph 1623)
expression: Ph Eur_______________________
DEFINITION
A X 0.64
Whole or cut dried calyces and epicalyces of Hibiscus
m
sabdariffa L. collected during fruiting.
Content
i.e. taking the specific absorbance of rhein to be 468, Minimum 13.5 per cent of acids, expressed as citric acid
calculated on the basis of the specific absorbance of
(C6H8O7;Afr 192.1) (dried drug).
barbaloin.
A = absorbance at 515 nm, CHARACTERS
m = mass of the herbal drug used, in grams. Acidic taste.
______________________________________________________________ _ Ph Eur IDENTIFICATION
A. The calyx is joined in the lower half to form an urceolate
structure, the upper half dividing to form 5 long acuminate
recurved tips. The tips have a prominent, slightly protruding
midrib and a large, thick nectary gland about 1 mm in
diameter. The epicalyx consists of 8-12 small, obovate
leaflets, which are adnate to the base of the calyx. The calyx
and epicalyx are fleshy, dry, easily fragmented and bright red
IV-350 Roselle 2016
or deep purple, somewhat lighter at the base of the inner Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
side. plate R (2-10 pm)].
B. Microscopic examination (2.8.23). The powder is red or Mobile phase anhydrous formic acid R, water R, butanol R
violet-red. Examine under a microscope using chloral hydrate (10:12:40 VIVIV).
solution R. The powder shows the following diagnostic Application 5 pL [or 2 pL] as bands of 10 mm [or 8 mm].
characters (Figure 1623.-1): predominantly red
fragments [A, F] consisting of polygonal epidermal cells with
very’ irregularly thickened walls, in surface view [Ac, Fa], Drying In air.
some containing cluster crystals of calcium oxalate [Fb], with Detection Examine immediately in daylight.
underlying parenchyma consisting of ovoid cells with slightly Results See below the sequence of zones present in the
thickened walls [Aa], some containing cluster crystals of chromatograms obtained with the reference solution and the
calcium oxalate [Ab] whilst others are filled with mucilage, test solution. Furthermore, other faint zones may be present
unicellular, long, flexuous, twisted covering trichomes [Ad], in the chromatogram obtained with the test solution.
rigid, straight, unicellular covering trichomes, simple or in
groups of 2-4 [Fd], glandular trichomes with a unicellular
Top of the plate
stalk and a globular or oval, multicellular and biseriate
head [Fe] and stomata usually of the anisocytic type
(2.8.3) [Fc]; numerous fragments of vascular bundles [D]
Quinaldine red: an orange-red
with spiral or reticulate vessels [Da], sometimes accompanied
by sclerenchymatous fibres with a wide lumen [Db], and An intense violet zone
parenchyma [De], of which some cells contain cluster crystals
Sulfan blue: a blue zone
of calcium oxalate [Dd], whilst others are mucilage-
filled [De]; rare, rectangular, parenchymatous sclereids [H]; An intense violet-blue zone
numerous fragments of rigid [C, G] or flexuous [J] covering
trichomes; free cluster crystals of calcium oxalate [B] and
glandular trichomes [E]; exceptionally, spherical pollen Reference solution Test solution
grains, about 200 pm in diameter, with a spiny exine.
TESTS
Maximum 2 per cent of fragments of fruits (red funicles and
parts of the 5-cavemed capsule with yellowish-grey pericarp,
whose thin walls consist of several layers of differently
directed fibres; flattened, reniform seeds with a dotted
surface) and maximum 2 per cent of other foreign matter.
105 °C for 2 h.
Total ash (2.4.16)
Colouring intensity
Reduce 100 g to a coarse powder (1400) (2.9.12) and
homogenise. Reduce about 10 g of this mixture to a very fine
powder (355) (2.9.12). To 1.0 g of this powder in a 100 mL
flask add 25 mL of boiling water R and heat for 15 min on a
water-bath with frequent shaking. Filter the hot mixture into
a 50 mL graduated flask; rinse successively the 100 mL flask
and the filter with 3 quantities, each of 5 mL, of warm
water R. After cooling, dilute to 50 mL with water R. Dilute
5 mL of this solution to 50 mL with water R. Measure the
absorbance (2.2.25) at 520 nm using water R as the
compensation liquid. The absorbance is not less than 0.350
for the whole drug and not less than 0.250 for the cut drug.
ASSAY
Shake 1.00 g of the powdered herbal drug (355) (2.9.12)
with 100.0 mL of carbon dioxide-free water R for 15 min.
Figure 1623.-1. - Illustration for identification test B of powdered Filter. To 50.0 mL of the filtrate add 100 mL of carbon
herbal drug of roselie dioxide-free water R. Titrate with 0.1 M sodium hydroxide to
c. Thin-layer chromatography (2.2.27). pH 7.0, determining the end-point potentiometrically
Test solution To 1 g of the powdered herbal drug (355) (2.2.20).
(2.9.12) add 10 mL of ethanol (60 per cent V/V) R. Shake for 1 mL of 0.1 M sodium hydroxide is equivalent to 6.4 mg
15 min and filter. of citric acid.
Reference solution Dissolve 2.5 mg of quinaldine red R and ____ _________________________________________ PhEj
2.5 mg of sulf071 blue R in 10 mL of tnethanol R.
2016 Rosemary Leaf IV-351
Rosemary Leaf ** **
PtiEir_________________
DEFINITION
Whole, dried leaf of Rosmarinus officinalis L.
Content
— minimum 12 mI7kg of essential oil (anhydrous drug);
minimum 3 per cent of total hydroxycinnamic derivatives,
expressed as rosmarinic acid (C18H16o8; Afr 360.3)
(anhydrous drug).
CHARACTERS
Strongly aromatic odour.
IDENTIFICATION
A. The leaves are sessile, tough, linear or linear-lanceolate,
1-4 cm long and 2-4 mm wide, with recurved edges.
The upper surface is dark green, glabrous and grainy, the
lower surface is greyish-green and densely tomentose with a
prominent midrib.
green or yellowish-green. Examine under a microscope using
diagnostic characters (Figure 1560.-1): fragments of the
lower epidermis in surface view [B, J] with straight or
sinuous-walled cells [Ba] and numerous diacytic stomata
(2.8.3) [Bb] and glandular trichomes [Ja] or covering
trichomes or their scars [Be, Bd]; numerous multicellular,
mostly branched, covering trichomes of the lower epidermis, Figure 1560.-1. - Illustration for identification test B of powdered
usually fragmented [A, c, D]; fragments of the upper herbal drug of rosemary leaf
epidermis in surface view [F] with cells with straight,
thickened and pitted walls [Fa], and an underlying
Top of the plate
hypodermis composed of large, irregular cells with thickened
and beaded anticlinal walls [Fb]; fragments of the lamina in
transverse section [G], showing the epidermis covered by a Bornyl acetate: a yellowish-brown A yellowish-brown zone of low
very thick cuticle [Ga], hypodcrmal cells extending across the zone intensity
mesophyll [Gb] at intervals, separating 1 or 2 layers of A coloured zone of low intensity
palisade parenchyma into large, crescent-shaped areas [Gc];
Cineole: a violet zone A violet zone
glandular trichomes of 2 types, the majority with a short,
unicellular stalk and a radiate head composed of 8 cells, in Coloured zones of low intensity
surface view [E] and in side view [H], others, less abundant, A violet-brown zone
Borneol: a violet-brown zone
with a uni- or bicellular stalk and a spherical, unicellular
head [Ja, K]. A coloured zone of low intensity
c. Thin-layer chromatography (2.2.27). Reference solution Test solution

Test solution Dissolve 20 |1L of the oil obtained in the assay in
1 mL of hexane R.
D. Thin-layer chromatography (2.2.27).
Reference solution Dissolve 5 mg of borneol R, 5 mg of bornyl
acetate R and 10 pL of cineole R in 1 mL of hexane R. Test solution Grind 1.0 g of the herbal drug in 10 mL of
methanol R and filter.
Reference solution Dissolve 1.0 mg of caffeic acid R and 5.0 mg
Mobile phase ethyl acetate R3 toluene R (5:95 VIV).
of rosmarinic acid R in 10 mL of methanol R.
Application 10 |1L as bands. Plate TLC silica gel plate R.
Development Over a path of 15 cm. Mobile phase anhydrous formic acid R, acetone R> methylene
Drying In air. chloride R (8.5:25:85 VIV/V).
Detection Treat with anisaldehyde solution R, heat at Application 10 |1L of the test solution and 20 pL of the
100-105 °C for 10 min and examine in daylight. reference solution, as bands.
Results See below the sequence of zones present in the Development Over a path of 8 cm.
Drying In air.
test solution.
test solution.
IV-352 Rosemary Oil 2016
Top of the plate

Rosemary Oil * *
A pink fluorescent zone
Caffeic acid: a light blue A blue fluorescent zone of low
fluorescent zone intensity PhEur_____________________________________________ _ _______________
Rosmarinic acid: a light blue An intense light blue fluorescent DEFINITION

fluorescent zone
Essential oil obtained by steam distillation from the flowering
Reference solution Test solution aerial parts of Rosmarinus officinalis L.
CHARACTERS
TESTS Appearance
Foreign matter (2.8.2) Clear, mobile, colourless or pale yellow liquid.
Maximum 5 per cent of stems and maximum 2 per cent of Characteristic odour.
other foreign matter. IDENTIFICATION
Water (2.2.13) First identification: B.
Maximum 100 mL/kg, determined on 20.0 g of the Second identification A
Total ash (2.4.16) Test solution Dissolve 0.5 mL of the substance to be
Maximum 9.0 per cent. examined in toluene R and dilute to 10 mL with the same
ASSAY solvent.
Total hydroxycinnamic derivatives Reference solution Dissolve 50 mg of borneol R, 50 mg of bornyl
Stock solution To 0.200 g of the powdered herbal drug (355) acetate R and 100 pL of cineole R in toluene R and dilute to
(2.9.12) add 80 mL of ethanol (50 per cent VIV) R. Boil in a 10 mL with the same solvent.
water-bath under a reflux condenser for 30 min. Allow to Plate TLC silica gel plate R.
cool and filter. Rinse the filter with 10 mL of ethanol
Mobile phase ethyl acetate R3 toluene R (5:95 VII7).
(50 per cent VIV) R. Combine the filtrate and the rinsings in
a volumetric flask and dilute to 100.0 mL with ethanol Application 10 pL, as bands.
(50 per cent VIV) R. Development Over a path of 15 cm.
Test solution To 1.0 mL of the stock solution add 2 mL of Drying In air.
0.5 M hydrochloric acid, 2 mL of a solution prepared by Detection Spray the plate with vanillin reagent R and heat the
dissolving 10 g of sodium nitrite R and 10 g of sodium plate at 100-105 °C for 10 min. Examine immediately in
molybdate R in 100 mL of water R, and ±en add 2 mL of daylight.
dilute sodium hydroxide solution R and dilute to 10.0 mL with Results See below the sequence of the zones present in the
water R-, mix. chromatograms obtained with the reference solution and the
Compensation solution Dilute 1.0 mL of the stock solution to test solution. Furthermore, several violet-blue to violet-grey
10.0 mL with water R. zones of medium intensity (terpene alcohols) are present in
Measure immediately the absorbance (2.2.25) of the test the lower third of the chromatogram obtained with the test
solution at 505 nm. solution.
Calculate the percentage content of total hydroxycinnamic
derivatives, expressed as rosmarinic acid, using the following Top of the plate
expression:
An intense violet zone
A X 2.5 A violet-grey zone
m
Bornyl acetate: a bluish-grey zone A bluish-grey zone of low
i.e. taking the specific absorbance of rosmarinic acid to be of low intensity intensity (bornyl acetate)
400. A violet-pink zone
A = absorbance of the test solution at 505 nm;
Cineole: an intense blue zone An intense blue zone (cineole)
Use 25.0 g of the crushed herbal drug, a 1000 mL flask and Borneol: a violet-blue zone of A violet -blue zone of medium
medium intensity intensity (borneol)
300 mL of water R as the distillation liquid. Distil at a rate of
2-3 mUmin for 3 h.
________________________________________________________________ PhEur

solution.
TESTS
0.895 to 0.920.
2016 Round Amomum Fruit IV-353
Refractive index (2.2.6) — bornyl acetate: 0.5 per cent to 2.5 per cent,
1.464 to 1.473. — a-terpineol: 1.0 per cent to 3.5 per cent,
Optical rotation (2.2.7) — borneol: 2.0 per cent to 4.5 per cent,
-5° to + 8°. — verbenone: 0.1 per cent to 2.5 per cent.
Acid value (2.5.1) For rosemary oil, Moroccan and Tunisian type, the
Maximum 1.0. percentages are within the following ranges:
— ซ-pinene: 9.0 per cent to 14.0 per cent,
— camphene: 2.5 per cent to 6.0 per cent,
— p-pinene: 4.0 per cent to 9.0 per cent,
procedure.
Test solution Dissolve 0.20 mL of the substance to be — limonene: 1.5 per cent to 4.0 per cent,
examined in hexane R and dilute to 10.0 mL with the same — cineole: 38.0 per cent to 55.0 per cent,
solvent. — p-cymene: 0.8 per cent to 2.5 per cent,
Reference solution Dissolve 20 j.iL of ซ-pinene R3 10 mg of — camphor. 5.0 per cent to 15.0 per cent,
camphene R) 20 pL of p-pinene R3 10 pL of p-myrcene R, — bornyl acetate: 0.1 per cent to 1.5 per cent,
20 pL of limonene R3 50 pL of cineole R, 10 pL of p-cymene R3 — a-terpineol: 1.0 per cent to 2.6 per cent,
50 mg of camphor R3 30 mg of bornyl acetate R, 10 mg of a- — borneol: 1.5 per cent to 5.0 per cent,
terpineol R3 10 mg of borneol R and 10 pL of verbenone R in — verbenone: maximum 0.4 per cent.
hexane R and dilute to 10.0 mL with the same solvent.
STORAGE
Column:
size: l ะ= 30 m (a film thickness of 1 pm may be used) to LABELLING
60 m (a film thickness of 0.2 pm may be used), The label states that the content is Spanish type or
0 = 0.25-0.53 mm, Moroccan and Tunisian type.
— stationary phase: macrogol 20 000 R. _____________________________________________________________Ph Eur

Flow rate 1 mL/min.
Split ratio 1:50.
Temperature: Round Amomum Fruit * \
(Ph. Eur. monograph 2555) *★*
Time Temperature
Ph Eur_____________________________________________________________
(min) co
Column 0 - 10 50 DEFINITION
10 - 85 50 -> 200 Dried, whole, peeled or unpeeled ripe fruit of Amomum
krervanh Pierre ex Gagnep. or Amomum compactum Sol.
85-110 200
ex Maton.
Injection port 200
Content
Detector 250 — essential oil: minimum 50 mUkg for A. krervanh
(anhydrous drug) and minimum 40 mUkg for
A. compactum (anhydrous drug);
Detection Flame ionisation. — 1,8-cineole (CioH180; Mr 154.3): minimum 65.0 per cent
Injection 1 pL. of the essential oil.
Elution order Order indicated in the composition of the IDENTIFICATION
reference solution. Record the retention times of these A. A. krervanh. The fruit is a trilocular, indehiscent capsule,
substances. subspherical, about 1.5-2 cm in diameter. The outer surface
System suitability: reference solution: is whitish-yellow or pale brownish-yellow, smooth, and shows
— resolution: minimum 1.5 between the peaks due to 3 deep longitudinal furrows. The apex bears a prominent
limonene and cineole and minimum 1.5 between the stylopodium; the base shows the dentate scar of the stalk.
peaks due to a-terpineol and borneol. Both ends and the hollows of the furrows are covered by a
Using the retention times determined from the pale brown pubescence. The thin, brittle pericarp is easily
chromatogram obtained with the reference solution, locate broken, showing 3 locules each containing 4-10 seeds.
the components of the reference solution in the The seeds are hard, irregularly polyhedral, with a slightly
chromatogram obtained with the test solution. raised dorsal surface, 3-4 mm in diameter, brown on the
Determine the percentage content of these components. surface, finely wrinkled, bearing the remains of the fine,
membranous aril.
For rosemary oil, Spanish type, the percentages are within
the following ranges: A compactum. The fruit is a trilocular, indehiscent capsule,
— ซ.-pinene: 18 per cent to 26 per cent, subspherical, about 1-2 cm in diameter. The outer surface is
— camphene: 8.0 per cent to 12.0 per cent, whitish-yellow or pale brownish-yellow, sometimes slightly
reddish, and shows about 15 longitudinal furrows, 3 of which
are deep. The apex bears a prominent stylopodium; the stalk
is usually fragmented. Both ends and the hollows of the
furrows are covered by a brownish-yellow pubescence.
— cineole: 16.0 per cent to 25.0 per cent,
The thin, brittle pericarp is easily broken, showing 3 locules
— p-cymene: 1.0 per cent to 2.2 per cent,
each containing 6-12 seeds. The seeds are hard, irregularly
— camphor. 13.0 per cent to 21.0 per cent,
IV-354 Round Amomum Fruit 2016
polyhedral, 2-3 mm in diameter, blackish-brown on the Reference solution Dissolve 10 pL of bornyl acetate R3 10 pL of
surface, wrinkled, bearing the remains of the transparent, cineole R and 10 mg of borneol R in 1 mL of toluene R.
membranous aril. Plate TLC silica gel plate R (2-10 pm).
B. A. krervanh. Microscopic examination (2.8.23). Mobile phase ethyl acetate R} toluene R (7:93 VIV).
The powder is greyish-brown. Examine under a microscope
following diagnostic characters: fragments of epicarp Development Over a path of 6 cm.
consisting of polyhedral cells, numerous scars of covering Drying In air.
trichomes with thick, channelled walls and rare stomata, Detection A Examine in ultraviolet light at 366 nm.
paracytic or anomocytic (2.8.3)'3 covering trichomes, mostly Results A See below the sequence of zones present in the
unicellular and usually fragmented, with regularly thickened chromatogram obtained with the test solution. The reference
walls, up to 800 pm in length; fragments of mesocarp solution shows no spots at 366 nm. Furthermore, other faint
composed of round cells with spaces between them, fluorescent zones may be present in the chromatogram
containing fine acicular crystals or prisms of calcium oxalate; obtained with the test solution.
groups of ovoid sclereids with thick, channelled walls, about
50 pm in diameter, from the inner layers of the mesocarp;
vascular bundles composed of spiral or reticulate vessels Top of the plate
accompanied by fibres with thick, channelled walls and
sclereids; fragments of the aril consisting of very fine cells,
some of which contain small crystals; fragments of the outer —
testa consisting of elongated cells with distinct, yellow, finely A blue fluorescent zone
and regularly thickened walls and rounded ends,
accompanied by an underlying layer consisting of rectangular
or polyhedral cells with orange-yellow contents,
perpendicular to the previous layer; fragments of the reddish- A blue fluorescent zone
brown inner testa composed of very thick-walled cells,
regularly polyhedral in surface view' and U-shaped in side Reference solution Test solution
view; fragments of the endosperm with round cells. Examine
Detection B Treat with anisaldehyde solution R3 heat at
glycerol R. The powder shows numerous round cells of the
endosperm, filled with small starch granules aggregated into 100-105 °C for 3 min and examine in daylight.
masses and free aggregates of starch granules. Results B See below the sequence of zones present in the
A coinpactum. Microscopic examination (2.8.23). The powder
is yellowish-brown. Examine under a microscope using chloral
characters: fragments of epicarp consisting of polyhedral cells,
numerous scars of covering trichomes with thick, channelled Top of the plate
walls and rare stomata, usually paracytic (2.8.3); unicellular
A reddish-brown zone
covering trichomes, usually fragmented, with regularly
thickened walls, up to 800 pm in length; fragments of
mesocarp composed of round cells with spaces between A bluish-violet zone
them, containing fine acicular crystals or prisms of calcium
oxalate, and oil cells; groups of ovoid sclereids with thick, Bornyl acetate: a greyish-brown
channelled walls, about 50 pm in diameter, from the inner
layers of the mesocarp; vascular bundles composed of spiral
or reticulate vessels accompanied by fibres with thick,
channelled walls and sclereids; fragments of the aril
consisting of very fine cells, some of which contain small
crystals; fragments of the pale yellow outer testa consisting of 1,8-Cineole: a bluish-violet zone A bluish-violet zone (1,8-cineole)
elongated cells with indistinct walls and rounded ends, A greyish-brown zone
accompanied by an underlying layer consisting of rectangular
or polyhedral cells with dark orange-red contents,
perpendicular to the previous layer; fragments of the reddish-
brown inner testa composed of thick-walled cells, regularly
Borneol: a greyish-brown zone at
polyhedral in surface view and U-shaped in side view; the border between the middle
fragments of the endosperm, with polyhedral cells. Examine and lower thirds
glycerol R. The powder shows numerous fragments of the A bluish-violet zone
endosperm with polyhedral cells, filled with small starch
granules aggregated into masses and free aggregates of starch
granules.
c. Thin-layer chromatography (2.2.27). 1 or 2 bluish-violet zones
(2.9.12) add 5 mL of methylene chloride R. Sonicate for
10 min. Centrifuge and use the supernatant.
2016 Safflower Flower IV-355
TESTS
Water (2.2.13) Safflower Flower ;* **
Maximum 120 mL/kg, determined by distillation on 20.0 g (Ph. Eur. monograph 2386) * **
of the powdered herbal drug (355) (2.9.12).
Ph Eu_________________________ ____ _____________________________
Total ash (2.4.16)
Dried flower of Carthamus tinctorius L.
ASSAY
Essential oil (2.8.12) Content
Minimum 1.0 per cent of total flavonoids, expressed as
Use 10.0 g of the herbal drug reduced to a coarse powder
hyperoside (C21H20012; Mr 464.4) (dried drug).
(1400) (2.9.12) immediately before the assay, a 500 mL
round-bottomed flask, 200 mL of water R as the distillation IDENTIFICATION
liquid and 0.5 mL of trintethylpentane R in the graduated A. The orange-yellow or reddish-orange, tubular,
tube. Distil at a rate of 3-3.5 mL/min for 5 h. gamopetalous, actinomorphic florets are separate from the
1,8-Cineole capitulum. Each floret consists of a long, filiform tube, about
Gas chromatography (2.2.2S): use the normalisation 1 cm long divided into 5 equal, narrow, lanceolate lobes,
procedure. about 0.5 cm long. From the opening of the tube emerges
the hollow cylinder formed by the fused yellow anthers, in
Test solution Dilute a volume of the essential oil-
which the filiform style persists, thickened near ±e apex.
trimethylpentane mixture obtained in the assay of essential oil
corresponding to 150 pL of the essential oil in heptane R and B. Microscopic examination (2.8.23). The powder is orange
dilute to 10.0 mL with the same solvent. yellow. Examine under a microscope using chloral hydrate
Reference solution (a) Dilute 10 pL of p-pinene R and 15 pL of characters (Figure 2386.-1): fragments of the corolla tube [E]
cineole R in heptane R and dilute to 5.0 mL with the same with epidermis consisting of elongated, thin-walled, finely
solvent.
striated cells whose margins are lobed [B, Ea, J], and with
Reference solution (b) Dilute 5 pL of cineole R to 100.0 mL parenchyma consisting of small polygonal cells containing
with heptane R. Dilute 1.5 mL of the solution to 10.0 mL prisms of calcium oxalate [Eb]; outer epidermis bearing
with heptane R. glandular trichomes, usually sheared off, with only the
Column: base [Ja] persisting on the epidermis; these trichomes are
— material: fused silica; isolated, biseriate with a multicellular stalk and a bicellular
— size: z = 60 m, 0 = 0.25 mm; head [C]; fragments of the lobes of the corolla showing at
— stationary phase: macrogol 20 000 R (film thickness their apices a large number of small, rounded, very
0.25 pm). prominent papillae [G]; fragments of parenchyma containing
Carrier gas helium for chromatography R. vascular bundles [Ed] surrounded by secretory canals with
Flow rate 0.9 mL/min. reddish-brown contents [Ec]; fragments of the filaments of
the anthers consisting of elongated, thick-walled, pitted
Split ratio 1:50.
cells [K] and fragments of the characteristic layer of the
Temperature: anther whose walls show thickenings in bands [H]; fragments
of the stigma, covered with rather long, conical papillae [D],
usually accompanied by pollen grains; rounded or elliptical
Time Temperature
pollen grams up to 60 pm in diameter with 3 pores and an
(°C)
echinulate exine [A]; isolated prisms of calcium oxalate [F].
Column 0 - 60 60 -> 210
Injection port 230 Test solution To 1.0 g of the powdered herbal drug (355)
Detector 250 (2.9.12) add 10 mL of methanol R. Sonicate for 10 min and
centrifuge.
Reference solution Dissolve 1 mg of rutin R and 5 mg of
quercetin dihydrate R in 50 mL of methanol R.
Injection 1 pL.
Identification of peaks Use the chromatogram obtained with plate R (2-10 Jim)].
reference solution (a) to identify the peaks due to P-pinene
Mobile phase acetic acid R, anhydrous formic acid R} water R,
and 1,8-cineole.
ethyl acetate R (11:11:27:100 VIVIVIV).
Relative retention With reference to p-pinene (retention
time ะะะ about 11 min): 1,8-cineole ะ= about 1.3.
— resolution: minimum 5 between the peaks due to p-pinene Drying In air.
and 1,8-cineole. Detection A Examine in daylight.
Calculate the percentage content of 1,8-cineole. Disregard Results A See below the sequence of zones present in the
any peak due to the solvent or with an area less than the area chromatograms obtained with the reference solution and the
of the principal peak in the chromatogram obtained with test solution. Furthermore, other faint zones may be present
reference solution (b) (0.05 per cent). in the chromatogram obtained with the test solution.
LABELLING
The label states the species present.
___________________________________________________________ ___ PhEur
IV-356 Safflower Flower 2016
Top of the plate

Quercetin: an orange fluorescent
zone
Rutin: a yellow fluorescent zone
TESTS
Absorbance (2.2.25)
A. Yellow pigment’. macerate 0.1 g of the powdered herbal
drug (355) (2.9.72) in 150 mL of water R) stir for 1 h, filter
through a sintered-glass filter (40) (2.1.2) and dilute to
500.0 mL, washing the residue, with water R.
The absorbance is not less than 0.40 at 401 nm.
B. Red pigment’, to 0.25 g of the powdered herbal drug (355)
Figure 2386.-1. - Illustration for identificatioti lest B of powdered (2.9.72) add 50 mL of a mixture of 20 volumes of water R
herbal drug of safflower flower and 80 volumes of acetone R. Heat on a water-bath at 50 °C
for 90 min. Allow to cool, filter through a sintered-glass filter
(40) (2.7.2) and dilute to 100.0 mL, washing the residue
Top of he plate
with a mixture of 20 volumes of water R and 80 volumes of
Quercetin: a light yellow zone acetone R. The absorbance is not less than 0.40 at 518 nm.
Rutin ะ a light yellow zone 105 °C for 2 h.
Total ash (2.4.16)
A yellow zone
ASSAY
Solution A Place 0.250 g of the powdered herbal drug (180)
(2.9.72) in a 250 mL flask and add 95 mL of methanol R.
Detection B Heat at 100 °C for 3 min; treat the plate whilst Heat under a reflux condenser on a water-bath for 30 min.
still hot with a 10 g/L solution of diphenylboric acid aminoethyl Allow to cool and filter. Rinse the filter with 5 mL of
ester R in methanol R and then with a 50 g/L solution of methanol R. Combine the filtrate and the rinsing solution in a
macrogol 400 R in methanol R'} allow to dry in air for about volumetric flask and dilute to 100.0 mL with methanol R.
30 min; examine in ultraviolet light at 365 nm. Test solution Place 5.0 mL of solution A in a volumetric flask
Results B See below the sequence of zones present in the and dilute to 20.0 mL with a 20 g/L solution of aluminium
chromatograms obtained with the reference solution and the chloride R in methanol R.
test solution. Furthermore, other faint zones may be present Compensation solution Place 5.0 mL of solution A in a
in the chromatogram obtained with the test solution. volumetric flask and dilute to 20.0 mL with methanol R.
After exactly 15 min, measure the absorbance (2.2.25) of the
test solution at 420 nm by comparison with the
compensation solution. Calculate the percentage content of
total flavonoids, expressed as hyperoside, using the following
expression:
A
m
2016 Sage Leaf IV-357
taking the specific absorbance of hyperoside at 420 nm to be

400.
= absorbance of the test solution at 420 nm;
พ = mass of the herbal drug to be examined, in grams.
------------------------------------------------------- -------------------------------------------------Ph Eur
Sage Leaf
(Sage Leaf (Salvia officinalis),
Preparation
Sage Tincture
DEFINITION
Whole or cut, dried leaves of Salvia officinalis L.
Essential oil content-.
— for the whole drug, minimum 12 mL/kg (anhydrous
drug);
— for the cut drug, minimum 10 mUkg (anhydrous drug).
IDENTIFICATION
A. The lamina of whole sage leaf (Salvia officinalis) is about
2-10 cm long and 1-2 cm wide, oblong-ovate, elliptical.
The margin is finely crenate to smooth. The apex is rounded
or subacute and the base is shrunken at the petiole and
rounded or cordate. The upper surface is greenish-grey and
finely granular; the lower surface is white and pubescent and
shows a dense network of raised veinlets. Figure 1370.-1. - Illustration for identification test B of powdered
B. Microscopic examination (2.8.23). The powder is light herbal drug of sage leaf
grey or brownish-green. Examine under a microscope using Detection treat with a 200 g/L solution of phosphomolybdic
chloral hydrate solution R. The powder shows the following acid R in ethanol R and heat at 100-105 °C for 10 min;
diagnostic characters (Figure 1370.-1): very numerous examine in daylight.
articulated and bent covering trichomes with narrow Results See below the sequence of zones present in the
elongated cells and a base cell with very thick walls, whole chromatograms obtained with the reference solution and the
[Be] or fragmented, either isolated [C, G, H] or on an test solution. Furthermore, other zones are present in the
epidermis (surface view [Be], transverse section [Ab]); chromatogram obtained with the test solution.
glandular trichomes of lamiaceous type, with a unicellular
stalk and an 8- to 12-celled head covered by a common
Top of the plate
cuticle, isolated (side view [D]) or on an epidermis (surface
view [Fa]); small glandular trichomes with a unicellular [Aa, A blue zone (near the solvent
Bd] or multicellular [Fb] stalk and a unicellular head, usually front)
on an epidermis; more rarely, glandular trichomes (surface
view [Eb, Ec], side view [Ed]) with a unicellular stalk [Ec] a-Thujone and P-thujone: 2 pinkish-violet zones (d-thujone
and a bicellular head [Eb, Ed]; fragments of the upper 2 pinkish-violet zones and P-thujone)
epidermis (surface view [E], transverse section [A]) with Cineole: a blue zone A blue zone (cineole)
pitted, somewhat polygonal cells [Ea], covering trichomes
and glandular trichomes, sometimes accompanied by 1 or 2
layers of palisade parenchyma [Ac, Ee]; some diacytic Blue zones
stomata (2.8.3) may be present; fragments of the lower Reference solution Test solution
epidermis [B, F] with sinuous cells [Ba] and numerous
diacytic stomata (2.8.3) [Bb].
Test solution Shake 0.5 g of the freshly powdered herbal drug Foreign matter (2.8.2)
(355) (2.9.12) with 5 mL of ethanol R for 5 min. Maximum 3 per cent of stems and maximum 2 per cent of
Reference solution Dissolve 20 pL of thujone R and 25 pL of other foreign matter.
cineole R in 20 mL of ethanol R. Water (2.2.13)
Plate TLC silica gel plate R. Maximum 100 mL/kg, determined on 20.0 g.
Mobile phase ethyl acetate R, toluene R (5:95 VIV). Total ash (2.4.16)
Application 20 pl, as bands. Maximum 10.0 per cent.
Drying In air.
IV-358 Sage Tincture 2016
ASSAY ASSAY
Essential oil (2.8.12) In a 500 mL round-bottomed flask, place 30.0 g of the
Use 20.0 g of the herbal drug, cut, if necessary, immediately tincture and add 100 mL of water R. Distil, using a
before the assay, a 500 mL flask and 250 mL of water R as descending condenser, into a separating funnel which has
the distillation liquid. Add 0.50 mL of xylene R in the been marked beforehand at 50 mL. Stop the distillation
graduated tube. Distil at a rate of 2-3 mL/min for 2 h. process as soon as the distillate reaches the 50 mL mark.
------------------------------------------------------------------------ ---------------------------- ------ Ph Eur Rinse the condenser with 10 mL of pentane R. Dissolve in
the distillate sufficient sodium chloride R to produce a
saturated solution. Shake with 3 quantities, each of 20 mL,
of pentane R. Dry the combined pentane layers, including the
Sage Tincture * ** pentane from rinsing the condenser, over anhydrous sodium
sulfate R and filter through a plug of absorbent cotton into a
(Ph. Eur. monograph 1889) * ** weighed 100 mL round-bottomed flask. Wash the sodium
Ph Eur______________________________________________ ___ _____________ sulfate several times with small quantities of pentane R.
Remove the pentane carefully at a temperature not exceeding
DEFINITION
40 °C. Dry the residue in a desiccator over diphosphorus
Tincture produced from Sage leaf (Salvia officinalis) (1370).
pentoxide R and hard paraffin at atmospheric pressure and at
Content room temperature for 2 h. Weigh the residue (essential oil).
Minimum 0.1 per cent m/m of essential oil. ______________________________________________________________ Ph Eu
PRODUCTION
The tincture is produced from 1 part of comminuted drug
and 10 parts of ethanol (70 per cent V/V) by a suitable
procedure.
CHARACTERS
Three-lobed Sage Leaf * ‘
Appearance (Ph. Eur. monograph 1561) ***
Brownish liquid with a characteristic odour. Ph Eur______________________________________________________________
IDENTIFICATION
DEFINITION
Whole or cut, dried leaves of Salvia fructicosa Mill. (syn.
Test solution The tincture to be examined. Salvia triloba L. fil).
Reference solution Dissolve 20 J1L of thujone R and 25 |1I. of Essential oil content".
cineole R in 20 mL of ethanol R. — for the whole drug, minimum 18 mL/kg (anhydrous
Plate TLC silica gel plate R. drug);
Mobile phase ethyl acetate R} toluene R (5:95 V/V). — for the cut drug, minimum 12 mL/kg (anhydrous drug).
Application 20 pL, as bands. CHARACTERS
Development Over a path of 15 cm. Spicy odour when ground, similar to eucalyptus oil.
Drying In air. IDENTIFICATION
Detection Spray with a 200 g/L solution of phosphomolybdic A. The lamina of whole three-lobed sage leaf is about
acid R in ethanol R and heat at 100-105 °C for 10 min. 8-50 mm long and about 4-20 mm wide, and oblong-ovate
Examine in daylight. or lanceolate. The margin is finely crenate and undulate but
Results See below the sequence of the zones present in the indistinct owing to the dense, hairy covering on both
chromatograms obtained with the reference solution and the surfaces. The base is obtuse and sometimes bears 1 or 2
test solution. Furthermore, other zones are present in the more or less developed lobes. The upper surface is grey-
chromatogram obtained with the test solution. tomentose pubescent, the lower surface is densely white-
tomentose pubescent; the venation is indistinct. The densely
Top of he plate white-tomentose pubescent petiole is about 1 mm in
diameter.
A blue zone (near the solvent front)
green and tomentose. Examine under a microscope using
a-Thujone and P-thujone: 2 2 pinkish-violet zones (a-thujone chloral hydrate solution R. The powder shows the following
pinkish-violet zones and P'thujone) diagnostic characters (Figure 1561.-1): very numerous
Cineole ะ a blue zone A blue zone (cineole) covering and glandular trichomes, whole and attached to
fragments of the epidermises [A, D, G, H] or fragmented
Blue zones and free [B, c, E, F]; uniseriate covering trichomes, either
Reference solution
unicellular [Ab] or multicellular articulated and thick-walled
Test solution
[Ad]; those on the upper epidermis are straight [Ga], those
on the lower epidermis are tortuous [Da]; glandular
TESTS trichomes of 2 types: some with a unicellular [Hd] or
Ethanol content (2.9.10) multicellular [Ca, Gb, He] stalk and a unicellular [Cb, Hd]
64 per cent V/V to 69 per cent V/V. or bicellular [Cc] head; others of lamiaceous type, with a
Methanol and 2-propanol (2.9.11) unicellular stalk and a head composed of 8-12 radiating cells
Maximum 0.05 per cent V/V of methanol and maximum with a raised common cuticle [Ae, B]; the upper epidermis
0.05 per cent of 2-propanol. (surface view [A], transverse section [G]) with pitted and
Dry residue (2.8.16) beaded cells [Aa], somewhat polygonal, with a few diacync
Minimum 2.0 per cent m/m, determined on 3.00 g. stomata (2.8.3)3 covering trichomes [Ab, Ad, Ga] or their
2016 Sage Oil IV-359
scars [Ac] and glandฟar trichomes [Ae, Af, Gb]; the lower Foreign matter (2.8.2)
epidermis (surface view [H], transverse section [D]) with Maximum 8 per cent of stems and maximum 2 per cent of
sinuous or wavy-walled cells [Ha] and numerous diacytic other foreign matter.
stomata (2.8.3) [Hb], glandular trichomes [Db, Hd, He] and
Water (2.2.13)
covering trichomes [Da, He], some of which are unicellular
Maximum 100 mUkg, determined on 20.0 g.
and short, with finely pitted walls [De].
Total ash (2.4.16)
ASSAY
Use 20.0 g of the herbal drug, cut, if necessary, immediately
before the assay, a 500 mL flask and 250 mL of water R as
the distillation liquid. Add 0.50 mL of xylene R in the
graduated tube. Distil at a rate of 2-3 mL/min for 2 h.
____________________________________________________________ PhEur
Sage Oil ★ *
(Clary Sage OilJ Ph Eur monograph 1850) *★*
Ph Elf_____________________________________________________________
DEFINITION
Essential oil obtained by steam distillation from the fresh or
dried flowering stems of Salvia sclarea L.
CHARACTERS
Appearance
Colourless or brownish-yellow liquid, usually pale yellow.
Characteristic odour.
IDENTIFICATION
Figure 1561.-1. —Illustration for identification test B of Test solution Dissolve 1 mL of the substance to be examined
powdered herbal drug of three-lobed sage leaf in toluene R and dilute to 10 mL with the same solvent.
c. Examine the chromatograms obtained in the test for Reference solution Dissolve 60 pL of linalol R, 200 pL of linalyl
thujone. acetate R and 60 pL of x-terpineol R in toluene R and dilute to
shows a blue zone due to cineole, equal or greater in size and Plate TLC silica gel plate R.
intensity to the zone in the chromatogram obtained with the Mobile phase ethyl acetate R3 toluene R (5:95 VIV).
reference solution. Further zones are present. Application 5 pL of the test solution and 10 pL of the
TESTS reference solution, as bands.
Thujone Development Over a path of 15 cm.
Thin-layer chromatography (2.2.27). Drying In air.
Test solution Shake 0.3 g of the freshly powdered herbal drug Detection Spray with vanillin reagent R and heat at
(355) (2.9.12) with 5.0 mL of anhydrous ethanol R for 5 min. 100-105 °C for 5-10 min; examine in daylight within 5 min.
Reference solution Dilute 20 pL of thujone R and 25 pL of Results See below the sequence of the zones present in the
cineole R in 20 mL of anhydrous ethanol R. chromatograms obtained with the reference solution and the
Plate TLC silica gel plate R. test solution. Furthermore, other faint zones are present in
Top of the plate
a-Terpineol: a dark violet zone A dark violet zone
Drying In air.
Detection treat with a 200 g/L solution of phosphomolybdic
acid R in anhydrous ethanol R and heat at 100-105 °C for Linalyl acetate: a dark violet zone A dark violet zone
10 min. Examine in daylight.
Linalol: a dark violet zone A dark violet zone
solution shows in the middle part a blue zone (cineole) and
in the upper part a pink-blue zone (thujone). Reference solution Test solution
The chromatogram obtained with the test solution shows no
zone or a very faint pink-blue zone due to thujone.
IV-360 Sage Oil 2016
B. Examine the chromatograms obtained in the test for chromatogram obtained with the test solution by its relative
chromatographic profile. retention of 1.23 with reference to linalol under the described
Results The chromatogram obtained with the test solution operating conditions.
shows 5 peaks similar in position to ±e 5 peaks in the The percentages are within the following ranges:
chromatogram obtained with the reference solution. The 2 — a- and P-thujone: maximum 0.2 per cent,
peaks corresponding to a- and P-thujone may be absent. — linalol: 6.5 per cent to 24 per cent,
TESTS — linalyl acetate: 56 per cent to 78 per cent,
Relative density (2.2.5) — CL-terpineol: maximum 5.0 per cent,
0.890 to 0.908. — germacrene-D: 1.0 per cent to 12 per cent,
— sclareol: 0.4 per cent to 2.6 per cent.
1.456 to 1.466. STORAGE
—26° to -10° _______________________________________________________________ PhEtr
Acid value (2.5.7)

Maximum 1.0.
Gas chromatography (2.2.25): use the normalisation Spanish Sage Oil
procedure. (Ph. Eur. monograph 1849) ***
Test solution The substance to be examined. Ph Eur_______________________________________________ _ ____________
Reference solution To 1 g of hexane R) add 5 pL of thujone R,
DEFINITION
5 pL of linalol R, 100 pL of linalyl acetate R3 10 pL of โt-
Essential oil obtained by steam distillation from the aerial
terpineol R and 25 mg (± 20 per cent) of sclareol R.
parts of Salvia lavandulifolia Vahl, collected at the flowering
Mix thoroughly by stirring.
stage.
Column'.
— material', fused silica, CHARACTERS
— size', z = 30 m (a film thickness of 1 pm may be used) to Appearance
60 m (a film thickness of 0.2 pm may be used), Clear, colourless or pale yellow, mobile liquid.
0 = 0.25-0.53 mm, Camphor-like odour.
IDENTIFICATION
Carrier gas helium for chromatography R. First identification: B.
Split ratio 1:100. Second identification A
Temperature:
Test solution Dissolve 0.1 mL of the essential oil to be
Time Temperature examined in 10 mL of toluene R.
(min) (°C)
Reference solution Dissolve 20 pL of thujone R and 30 pL of
Column 0-10 60
cineole R in 10 mL of toluene R.
10-75 60 -> 190 Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
75 - 120 190 plate J? (2-10 pm)].
Injection port 220 Mobile phase ethyl acetate R} toluene R (5:95 VIV).
Detector 240
Drying In air.
Detection: spray with a freshly prepared 200 g/L solution of
Injection 0.2 pL. phosphomolybdic acid R in ethanol (96 per cent) R and heat at
Elution order Order indicated in the composition of the 105 °C for 10 min; examine in daylight.
reference solution. Record the retention times of these Results See below the sequence of zones present in the
substances. chromatograms obtained with the reference solution and the
System suitability: reference solution: test solution. Furthermore, other faint zones may be present
— resolution: minimum 1.5 between the peaks due to linalol in the chromatogram obtained with the test solution.
and linalyl acetate,
Using the retention times determined from the Top of the plate
A blue zone
chromatogram obtained with the test solution (disregard any
peak due to hexane). Thujone R is a mixture of a- and Thujone ะ 2 pinkish-violet zones
P-thujone. a-Thujone elutes before P-thujone under the
described conditions. Cineole: a blue zone A blue zone (cineole)

components.
3 blue zones
Also determine the percentage content of germacrene-D.
The germacrene-D peak can be identified m ±e
2016 Salvia Miltiorrhiza Root and Rhizome IV-361
B, Examine the chromatograms obtained in the test for Determine the percentage content of each of these
chromatographic profile. components. The percentages are within the following
Results The characteristic peaks in the chromatogram ranges:
obtained with the test solution are similar in retention time to — a-pinene: 4.0 per cent to 11.0 per cent;
those in the chromatogram obtained with reference — sabinene: 0.1 per cent to 3.5 per cent;
solution (a). — limonene: 2.0 per cent to 6.5 per cent;
TESTS — 1,8-cineole: 10.0 per cent to 30.5 per cent;
— thujone: maximum 0.5 per cent;
— camphor. 11.0 per cent to 36.0 per cent;
0.907 to 0.932.
— linalol: 0.3 per cent to 4.0 per cent;
Refractive index (2.2.6) — linalyl acetate: maximum 5.0 per cent;
1.465 to 1.473. — terpinen-4-ol: maximum 2.0 per cent;
Optical rotation (2.2.7) — sabinyl acetate: 0.5 per cent to 9.0 per cent;
+ 7° to + 17°. — a-terpinyl acetate: 0.5 per cent to 9.0 per cent;
Acid value (2.5.1) — borneol: 1.0 per cent to 7.0 per cent;
Maximum 2.0, determined on 5.00 g.
Solubility in alcohol (2.8.10) (0.05 per cent).
1 volume is soluble in 2 volumes and more of ethanol
(80 per cent V/V) R. STORAGE
_____________________________________________________________ Ph Eur
procedure.
Test solution Dissolve 0.200 g of the essential oil to be
solvent. Salvia Miltiorrhiza Root and * *
Reference solution (a) Dissolve 0.200 g of Spanish sage oil for
peak identification CRS in heptane R and dilute to 10.0 mL
Rhizome *****
Ph Eur_____________________________________________________________
Reference solution (b) Dissolve 5 pL of limonene R in heptane R
and dilute to 50.0 mL with the same solvent. Dilute 0.5 mL DEFINITION
of this solution to 5.0 mL with heptane R. Dried, whole or fragmented rhizome and root of Salvia
Column: miltiorrhiza Bunge, collected in spring or autumn.
— material: fused silica; Content
— size: I = 60 m, 0 = 0.25 mm; — salvianolic acid B (C36H30O16; Mr 719): minimum
— stationary phase: macrogol 20 000 R (film thickness 3.0 per cent (dried drug);
0.25 pm). — tanshinone IIA (บ!9ผ11803; Mr 294.3): minimum
Carrier gas helium for chromatography R. 0.12 per cent (dried drug).
Flow rate 1.5 mUmin. IDENTIFICATION
Split ratio 1:50. A. The rhizome is short and thick, sometimes with stem
Temperature: remnants at the apex. The roots are numerous, about
10-20 cm long and 0.3-1 cm in diameter, cylindrical and
slightly curved; some are branched, with secondary roots and
Time Temperature
rootlets. The outer surface is reddish-brown or dark reddish-
(°C)
0 - 43 60 -> 232
brown, marked with longitudinal striations. The bark of old
Column
roots comes off usually as purplish-brown scales. The texture
Injection port 250 is hard and fragile. The fracture is soft, fissured or slightly
Detector 250 even and dense, with a reddish-brown outer part and a
greyish-yellow or purplish-brown wood, showing bundles of
yellowish-white vessels, arranged radially.
Detection Flame ionisation. Cultivars are relatively stout, about 0.5-1.5 cm in diameter.
Injection 1 pL. The outer surface is brownish-red, longitudinally wrinkled.
System suitability: reference solution (a): The bark adheres closely to the wood and is difficult to
— the chromatogram obtained is similar to the remove. The texture is compact; the fracture is relatively
chromatogram supplied with Spanish sage oil for peak even.
identification CRS; B. Microscopic examination (2.8.23). The powder is
— resolution: minimum 1.5 between the peaks due to brownish-red. Examine under a microscope using chloral
limonene and 1,8-cineole and minimum 1.5 between the hydrate solution R. The powder shows the following diagnostic
peaks due to a-terpinyl acetate and borneol. characters: fragments of cork in surface view, consisting of
Use the chromatogram supplied with Spanish sage oil for peak subrectangular or polygonal cells, up to 150 pm in diameter,
identification CRS and the chromatogram obtained with containing yellowish-brown pigment; fragments of
reference solution (a) to locate the peaks due to a-pinene, parenchyma consisting of polygonal or elongated, thin-walled
sabinene, limonene, 1,8-cineole, thujone, camphor, linalol, cells that may contain yellowish-brown pigment; xylem fibres
linalyl acetate, terpinen-4-ol, sabinyl acetate, a-terpinyl usually in bundles, long and fusiform, with pitted walls
acetate and borneol. showing oblique or criss-cross striations; very numerous
IV-362 Salvia Miltiorrhiza Root and Rhizome 2016
reticulate or pitted vessels, 3-120 pm in diameter, free, in TESTS

bundles or sometimes accompanying the fibres. Loss on drying (2.2.22)
c. Thin-layer chromatography (2.2.27). Maximum 10.0 per cent, determined on 1.000 g of the
Test solution To 1 g of the powdered herbal drug (355) powdered herbal drug (355) (2.9.72) by drying in an oven at
(2.9.72) add 40 mL of methanol R. Sonicate for 15 min. 105 °C for 2 h.
Filter. Evaporate the filtrate to 1 mL. Total ash {2.4.16)
Reference solution Dissolve 2 mg of salvianolic acid B R and Maximum 10.0 per cent.
2 mg of tanshinone Z7A R in 1 mL of methanol R. Ash insoluble in hydrochloric acid (2.5.7)
Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel Maximum 3.0 per cent.
F254 plate R (2-10 pm)]. ASSAY
Mobile phase methanol R, anhydrous formic acid R, toluene R3 Liquid chromatography (2.2.29). Protect the solutions from
methylene chloride R, ethyl acetate R light.
(5:20:20:30:40 F/F7F/F/F). Test solution Disperse 0.30 g of the powdered herbal drug
Application 5 pL [or 5 pL] as bands of 8 mm [or 8 mm]. (355) {2.9.12) in 50.0 mL of a 70 per cent F/F solution of
Development Over a path of 8 cm [or 6 cm]. methanol R. Sonicate for 1 h. Filter through a membrane
Drying In air. filter (nominal pore size 0.45 pm).
Detection A Examine in daylight. Reference solution (a) Dissolve 5.0 mg of tanshinone ZZA CRS
Results A See below the sequence of zones present in the in methanol R and dilute to 50.0 mL with the same solvent.
chromatograms obtained with the reference solution and the Dilute 2.0 mL of the solution to 10.0 mL with methanol R.
test solution. Furthermore, other faint zones may be present Reference solution (b) Dissolve 5.0 mg of salvianolic
in the upper third and middle part of the chromatogram acid B CRS in methanol R and dilute to 25.0 mL with the
obtained with the test solution. same solvent.
Reference solution (c) Dissolve 1 mg of rosmarinic acid R in
methanol R, add 5 mL of reference solution (b) and dilute to
Top of the plate
Tanshinone IIA: a prominent red A prominent red zone Column-.
(tanshinone IIA)
— size-. I — 0.25 m, 0 = 4.6 mm;
An orange zone
— stationary phase-, end-capped octadecylsilyl silica gel for
A faint brownish-green zone
Mobile phase-.
— mobile phase A: 0.1 per cent F/F solution of anhydrous
Salvianolic acid B: a faint grey A faint grey zone (salvianolic formic acid R-,
acid B)
— mobile phase B: acetonitrile for chromatography R-,

Reference solution Test solution (min) (per cent V/V) (per cent V/V)
0 - 10 79 -> 71 21 -> 29
10 - 15 71 -> 65 29 -> 35
Results See below the sequence of zones present in the 15-25 65 -> 28 35 -> 72
chromatograms obtained with the reference solution and the 25 - 37 28 -> 0 72 -> 100
in the upper third and middle part of the chromatogram
obtained with the test solution. Flow rate 1.0 mL/min.
Top of the plate Injection 10 pL.
Relative retention With reference to tanshinone IIA (retention
Tanshinone IIA: a prominent A prominent quenching zone
quenching zone (tanshinone IIA) time = about 33 min): rosmarinic acid = about 0.3;
A quenching zone salvianolic acid B = about 0.4.
System suitability-, reference solution (c):
— resolution’, minimum 5.0 between the peaks due to
A quenching zone rosmarinic acid and salvianolic acid B.
Salvianolic acid B ะ a prominent A prominent quenching zone
Calculate the percentage content of tanshinone nA using the
quenching zone (salvianolic and B) following expression:
Al X 7712 X Pl
A2 X 7ท1 X 5
A1 = area of the peak due to tanshinone nA in the

A2 = area of the peak due to tanshinone IIA in the
;
(a)
2016 Salvia Miltiorrhiza rhizome and Root IV-363
W1 = mass of the herbal drug to be examined used to microscope. Numerous mainly bordered or reticulated
prepare the test solution, in grams; vessels, 3 to 120 pm in diameter.
= mass of tanshinone IIA CRS used to prepare c. Carry out the method for thin-layer chromatography,
reference solution (a), in grams; Appendix III A, using the following solutions.
Pl = percentage content of tanshinone nA in tanshinone
(1) Place 2 g of the powdered drug (355) in a cellulose
IIA CRS.
fingerstall in a continuous extraction apparatus (Soxhlet
Calculate the percentage content of salvianolic acid B using type). Add 75 mL of methanol and heat for 1 hour.
the following expression: Evaporate the extract to 20 mL, cool and filter if necessary.
(2) 0.1% w/v each of tanshinone IIA CRS, rosmarinic acid CRS
A3 X 7713 X P2 X 2 and salvianolic acid B CRS in methanol.
A4 X mi CHROMATOGRAPHIC CONDITIONS
■^3 = area of the peak due to salvianolic acid B in the (a) Use as the coating silica gel F254.
chromatogram obtained with the test solution; (b) Use the mobile phase described below.
A.I = area of the peak due to salvianolic acid B in the (c) Apply as bands 8 pL of solution (1) and 5 pL of
chromatogram obtained with reference solution solution (2).
;
(b) (d) Develop the plate to 15 cm.
= mass of the herbal drug to be examined used to (e) After removal of the plate, dry in air and examine under
ultraviolet light (366 nm).
พ3 = mass of salvianolic acid B CRS used to prepare
reference solution (b), in grams; MOBILE PHASE
Pl = percentage content of salvianolic acid B in 10 volumes of water, 13.5 volumes of methanol and
salvianolic acid B CRS. 100 volumes of ethyl acetate.
————————------------------------------------------------------------------ Ph Eur SYSTEM SUITABILITY
The chromatogram obtained with solution (2) shows three
clearly separated bands.
CONFIRMATION
Processed Salvia Miltiorrhiza Rhizome The blue fluorescent bands with Rf values of approximately
0.7 (tanshinone 11^, 0.2 (rosmarinic acid) and 0.06
and Root (salvianolic acid B) in the chromatogram obtained with
DEFINITION solution (1) correspond in colour and position to those in the
Processed Salvia Miltiorrhiza Rhizome and Root is Salvia chromatogram obtained with solution (2). Other bands may
Miltiorrhiza Rhizome and Root that has been processed. be present in the chromatogram obtained with solution (1) as
It contains not less than 0.04% of tanshinone IIA shown below.
(C19H18o3), not less than 0.17% of rosmarinic acid
(C18H16O8) and not less than 3.0% of salvianolic acid B Top of the plate
(C36H36016), calculated with reference to the dried material.
PRODUCTION A fluorescent band Tanshinone llA: a fluorescent
It is collected in spring or autumn, separated from soil, band
washed clean, softened thoroughly, sliced longitudinally or
transversely and dried. It may be stir baked with wine. Several fluorescent bands
IDENTIFICATION
A. The longitudinally-sliced pieces are up to about 5 cm
A fluorescent band Rosmarinic acid: a
long, 1.5 cm wide and 1 to 2 mm thick; those cut from the fluorescent band
thinner roots show the dark brown striated cork covering one
longitudinal surface and the yellowish to cream inner tissues A fluorescent band Salvianolic acid B: a
on ±e other; slices cut from the thicker rhizomes are usually fluorescent band
cut obliquely so that parts of the outer and inner tissues are Solution (1) Solution (2)
included on both longitudinal surfaces. The transversely-cut
slices are irregularly elliptical to nearly circular, 4 to 12 mm
wide and 2 to 3 mm thick; the outer surface is dark brown
and uneven; the smoothed transverse surface shows the outer TESTS
layers about 1 to 2 mm wide separated by a darker line from Total ash
the yellowish white, radiate vascular tissue; some pieces show Not more than 10%, Appendix XI J.
a small, light brown central pith. Acid-insoluble ash
B. Reduce to a powder (355). The powder is reddish-brown. Not more than 2.0%, Appendix XI K.
Loss on drying
The powder shows a surface view of cork cells almost When dried for 2 hours at 105°, loses not more than 12.0%
rectangular or polygonal, containing yellowish-brown of its weight. Use 1 g.
pigment, 12 to 151 pm in diameter. Parenchymatous cells in
cortex squarish or polygonal, containing reddish-brown ASSAY
pigmental sediments. Xylem fibres usually in bundles, long For tanshinone IIA
fusiform, with oblique or criss-cross striations, 11 to 60 pm Carry out the method for liquid chromatography,
in diameter, vivid yellow when examined under a polarizing Appendix III D, using the following solutions.
IV-364 Salvia Miltiorrhiza rhizome and Root 2016
(1) Finely powder about 5.0 g of the herbal drug being (2) 0.003% w/v each of rosmarinic acid CRS and ferulic acid in
examined. Transfer 0.5 g of the powder into a 25 mL water.
volumetric flask and add 20 mL of the mobile phase given (3) 0.06% w/v of salvianolic acid B CRS in the mobile phase.
below. Shake and mix with the aid of ultrasound for
30 minutes, shaking intermittently. Add the mobile phase to CHROMATOGRAPHIC CONDITIONS
give a total volume of 25 mL. Filter through a 0.45-pm filter. (a) Use a stainless steel column (25 cm X 4.6 mm) packed
(2) 0.002% w/v of tanshinone IIA CRS in the mobile phase. with octadecylsilyl silica gel for chromatography (5 pm)
(Nucleosil ODS is suitable).
(a) Use a stainless steel column (25 cm X 4.6 mm) packed below.
with octadecylsilyl silica gel for chromatography (5 Jim)
(c) Use a flow rate of 1 mL per minute.
(Nucleosil ODS is suitable).
below. (e) Use a detection wavelength of 330 nm.
(c) Use a flow rate of 1 mL per minute. (f) Inject 20 pL of each solution.
(d) Use an ambient column temperature. MOBILE PHASE
(e) Use a detection wavelength of 270 nm. 22 volumes of acetonitrile and 78 volumes of 0.4% v/v of
(f) Inject 20 pL of each solution. formic acid.
SYSTEM SUITABILITY
MOBILE PHASE
0.01m sodium octyl sulfonate in a mixture of 25 volumes of The test is not valid unless, in the chromatogram obtained
water and 75 volumes of methanol adjusted to pH 5.0 with with solution (2), the resolution factor between the two main
peaks, rosmarinic acid and ferulic acid, is at least 5.0.
acetic acid.
SYSTEM SUITABILITY
The test is not valid, unless in the chromatogram obtained
Rosmarinic acid Using the retention time and peak area
from the chromatogram obtained with solution (2), locate
with solution (2):
and integrate the peak due to rosmarinic acid in the
— the symmetry factor of the peak due to tanshinone nA is
less than 1.2; chromatogram obtained with solution (1).
— the number of theoretical plates is not less than 4500. Calculate the content of rosmarinic acid in the sample using
the declared content of rosmarinic acid (C]8H16O8) in
rosmarinic acid CRS and the following expression:
Using the retention time and peak area from the
chromatograms obtained น'ith solution (2), locate and Al ทบ V! 100
integrate the peak due to tanshinone IIA in the A: x Vโxทิบx
ิ px 100-d
Calculate the content of tanshinone IIA in the sample using Al = Area of the peak due to rosmarinic acid in the
the declared content of tanshinone IIA (C19H18O3) in chromatogram obtained with solution (1).
tanshinone 11a CRS and the following expression: A2 ะ= Area of the peak due to rosmarinic acid in the
ฅ1 = Weight of the drug in mg.
Al ทบ Vi 100 m2 = Weight of rosmarinic acid CRS in mg.
A?x v?x ทบิxpx 100-d V1 = Dilution volume of solution (1) in mL.
V2 = Dilution volume of solution (2) in mL.
p = Percentage content of rosmarinic acid in rosmarinic
Aj = Area of the peak due to tanshinone nA in the
acid CRS.
d = Percentage loss on drying of the herbal drug being
A2 = Area of the peak due to tanshinone IIA in the
examined.
m 1 = Weight of the drug in mg. Salvianolic acid B Using the retention time and peak area
m2 = Weight of tanshinone IIA CRS in mg. from the chromatogram obtained with solution (3), locate
V1 = Dilution volume of solution (1) in mL. and integrate the peak due to salvianolic acid B in the
V2 = Dilution volume of solution (2) in mL. chromatogram obtained with solution (1).
p = Percentage content of tanshinone IIA in tanshinone Calculate the content of salvianolic acid B in the sample
IIA CRS. using the declared content of salvianolic acid B (C36H36O]6)
d = Percentage loss on drying of the herbal drug being in salvianolic add B CRS and the following expression:
examined.
For rosmarinic acid and salvianolic acid B Al ทบ Vi 100
A?x Vโx ทบิxpx 100-d
Carry out the method for liquid chromatography.
Appendix in D, using the following solutions prepared
Al = Area of the peak due to salvianolic acid in the
immediately before use.
(1) Finely powder about 5.0 g of the herbal drug being A2 = Area of the peak due to salvianolic acid in the
examined. Transfer 0.5 g of the powder into a 25-mL chromatogram obtained with solution (3).
volumetric flask and add 20 mL of the mobile phase given m 1 = Weight of the drug in mg.
below. Shake and mix with ultrasound for 30 minutes, m2 = Weight of salvianolic add B CRS in mg.
shaking intermittently. Add the mobile phase to give a total V] = Dilution volume of solution (1) in mL.
volume of 25 mL. Filter through a 0.45-pm filter. v2 = Dilution volume of solution (2) in mL.
2016 Saw Palmetto Extract IV-365
p = Percentage content of salvianolic acid B in salvianolic Top of the plate 1

acid B CRS.
A blue zone
examined. A blue zone
STORAGE
Processed Salvia Miltiorrhiza Rhizome and Root should be P-Amyrin: a blue zone A strong bluish-violet zone
protected from moisture.
0-SitosteroI ะ a blue zone
Saw Palmetto Extract ******

(Ph. Eur. monograph 2579) * ** Reference solution Test solution
Ph Elf_________ ___________________________________
DEFINITION B. Examine the chromatograms obtained in the assay of total

fatty acids.
Extract produced from Saw palmetto fruit (1848).
Results The peaks due to methyl caproate, methyl caprylate,
Content
methyl caprate, methyl laurate, methyl myristate, methyl
total fatty acids', minimum 80.0 per cent (anhydrous palmitoleate, methyl palmitate, methyl linoleate, methyl
extract);
linolenate, methyl oleate and methyl stearate in the
lauric acid (C12H24O2; Afr 200.3): minimum chromatogram obtained with the test solution are similar in
23.0 per cent (anhydrous extract); retention time to the corresponding peaks in the
— total sterols, expressed as p-sitosterol (C29H50O; MT 414.7): chromatogram obtained with reference solution (b);
minimum 0.20 per cent (anhydrous extract); the principal peaks are due to methyl laurate and methyl
P-sitosterol (C29H50O; Mr 414.7): minimum 0.10 per cent oleate.
(anhydrous extract).
TESTS
PRODUCTION
Water (2.5J 2, Method A)
The extract is produced from the herbal drug by a suitable Maximum 3.0 per cent, determined on 0.5 g.
procedure using ethanol (minimum 90 per cent VIV), or
supercritical carbon dioxide or a mixture of mainly n-hexane Relative density (2.2.5)
and methylpentanes (bp: 65-70 °C). 0.850 to 0.950.
CHARACTERS
1.40 to 1.50.
Appearance
The ethanol extract is a dark greenish-brown, oily liquid; Acid value (2.5J)
the supercritical carbon dioxide extract is a yellowish-brown 150.0 to 220.0.
or orange-brown, oily liquid; the hexane extract is a Iodine value (2.5.4, Method A)
yellowish-green to orange-yellow, oily liquid. 30.0 to 60.0.
Odour Peroxide value (2.5.5)
Strong but not rancid. Maximum 5.0.
IDENTIFICATION Saponification value (2.5.6)
First identification B. 220.0 to 250.0.
Second identification A. Solvents
A. Thin-layer chromatography (2.2.27). Residual solvents are controlled as described in chapter 5.4,
unless otherwise justified and authorised.
20 mL of ethanol (96 per cent) R. ASSAY
Reference solution Dissolve 4 mg of p-amyrin R and 10 mg of Total fatty acids
p-sitosterol R in 10 mL of ethanol (96 per cent) R. Gas chromatography (2.2.28).
Plate TLC silica gel plate R (2-10 pm). Internal standard solution Dissolve 0.47 g of methyl
margarate R in 20 mL of dimethylformamide R and dilute to
Mobile phase anhydrous acetic acid R, ethyl acetate R, toluene R
(1:30:70 VIVIV).
Test solution Disperse 0.25 g of the extract to be examined in
10 mL of dimethylformamide R. Add 4.0 mL of the internal
Development Over a path of 6 cm. standard solution and dilute to 25.0 mL with
Drying In air. dimethylformamide R. Mix 0.4 mL of this solution and
Detection Treat with anisaldehyde solution R, heat at 0.6 mL of a 18.84 g/L solution of trimethylsulfonium
100-105 °C for 5-10 min and examine in daylight. hydroxide R in methanol R.
Results See below the sequence of zones present in the Reference solution (a) Dissolve 0.699 g of lauric acid CRS and
chromatograms obtained with the reference solution and the 0.870 g of oleic acid CRS in dimethylformamide R and dilute to
test solution. Furthermore, other faint zones may be present, 10.0 mL with the same solvent. To 1.0 mL of the solution
especially in the lower third, in the chromatogram obtained add 4.0 mL of the internal standard solution and dilute to
with the test solution. 25.0 mL with dimethylformamide R. Mix 0.4 mL of this
solution and 0.6 mL of a 18.84 g/L solution of
trimethylsulfonium hydroxide R in methanol R.
IV-366 Saw Palmetto Extract 2016
Reference solution (b) Disperse 0.25 g of saw palmetto A5 = sum of the areas of the peaks due to methyl
extract HRS in 10 mL of dimethylformamide R. Add 4.0 mL linoleate, methyl linolenate and methyl oleate in
of the internal standard solution and dilute to 25.0 mL with the chromatogram obtained with the test solution;
dimethylformamide R. Mix 0.4 mL of this solution and Ab = area of the peak due to methyl oleate in the
0.6 mL of a 18.84 g/L solution of trimethylsulfonium chromatogram obtained with reference solution
hydroxide R in methanol R. (a);
Column’. m1 = mass of the extract to be examined used to
— material: fused silica; prepare the test solution, in grams;
พ2 = mass of lauric acid CRS used to prepare reference
— stationary phase: poly (dimethyl) siloxane R (film thickness solution (a), in grams;
0.33 pm). nij = mass of oleic acid CRS used to prepare reference
Carrier gas helium for chromatography R. solution (a), in grams;
Flow rate 0.5 mL/min. Pl = percentage content of lauric acid in lauric acid
CRS’,
Split ratio 1:40. p2 = percentage content of oleic acid in oleic acid CRS.
Temperature:
Calculate the percentage content of lauric acid using the
Time Temperature following expression:
(min) (°C)
Column 0-2 150
Al X A4 X 7ท2 X p X 0.1
2-7 150 -* 190 A2 X A3 X 7711
7 - 12 190
A1 = area of the peak due to methyl laurate in the
12 - 22 190 -> 220
22 - 32 220 A2 = area of the peak due to methyl laurate in the
Injection port 300
(a);
Detector 300 A3 = area of the peak due to methyl margarate in the
Detection Flame ionisation. A4 = area of the peak due to methyl margarate in the
Injection 1 pL. chromatogram obtained with reference solution
Identification of peaks Use the chromatogram supplied with (a);
saw palmetto extract HRS and the chromatogram obtained m1 = mass of the extract to be examined used to
with reference solution (b) to identify the peaks due to prepare the test solution, in grams;
methyl caproate, methyl caprylate, methyl caprate, methyl พ2 = mass of lauric acid CRS used to prepare reference
laurate, methyl myristate, methyl palmitoleate, methyl solution (a), in grams;
palmitate, methyl linoleate, methyl linolenate, methyl oleate, p = percentage content of lauric acid in lauric acid
methyl stearate and methyl margarate. CRS.
System suitability: reference solution (b) ะ Total sterols
— peak-to-valley ratio: minimum 1.2, where Hp = height Gas chromatography (2.2.28).
above the baseline of the peak due to methyl linolenate Derivatisation solution (a) chlorotrimethylsilane R, N,O-
and Hv = height above the baseline of the lowest point of
bis (trimethylsilyl) acetamide R, N-trimethylsilylimidazole R
the curve separating this peak from the peak due to
(2:3:3 VIVIV):
me±yl linoleate.
Derivatisation solution (b) Derivatisation solution (a), Ar,O-
Calculate the percentage content of total fatty acids, where bis (trimethylsilyl) trifluoroacetamide R, pyridine R (1:1:1 VIVIV).
caproic, caprylic, capric, lauric, myristic, palmitoleic, palmitic
Internal standard solution Dissolve 0.25 g of cholesterol R in
and stearic acids are expressed as lauric acid (C12H24O2; Mr
200.3) and linoleic, linolenic and oleic acids are expressed as 25.0 mL of methylene chloride R.
oleic acid (C]8H34O2; Mr 282.5), using the following Test solution Introduce 1.0 mL of the internal standard
expression: solution into a 50 mL round-bottomed flask and evaporate to
dryness. Place 3.35 g of the extract to be examined,
Al X A4 X 7712 X Pl X 0.1 As X A4 X 7713 X p2 X 0.1 accurately weighed, into the round-bottomed flask and add
A2 X A3 X 7711 Ae X A3 X 7711 20 mL of a solution prepared as follows: dissolve 130 g of
potassium hydroxide R in 200 mL of water R and dilute to
Al = sum of the areas of the peaks due to methyl 1000 mL with methanol R. Heat under reflux for 2 h, transfer
caproate, methyl caprylate, methyl caprate, methyl quantitatively to a flask and dilute to 25.0 mL with water R.
laurate, methyl myristate, methyl palmitoleate, Apply 3.0 mL of this solution to a cartridge containing
methyl palmitate and methyl stearate in the diatomaceous earth R capable of holding 3 mL of aqueous
chromatogram obtained with the test solution; phase. Absorb the solution into the column by applying
A2 = area of the peak due to methyl laurate in the vacuum. Maintain the vacuum for at least 20 min, until the
chromatogram obtained with reference solution column returns to room temperature, indicating that the
(a); methanol is completely evaporated. Rinse the column with
Aj = area of the peak due to methyl margarate in the 90 mL of methylene chloride R and evaporate the eluate to
chromatogram obtained with the test solution; dryness. Dissolve the residue in 1.0 mL of derivatisation
A4 = area of the peak due to methyl margarate in the solution (b).
chromatogram obtained with reference solution Reference solution (a) To 9.0 mg of p-sitosterol CRS add
(a); 1.0 mL of the internal standard solution and dilute to
2016 Saw Palmetto Fruit IV-367
ว.0 mL with methylene chloride R. Evaporate 0.6 mL of this A3 ะะะarea of the peak due to the trimethylsilyl
solution to dryness under a stream of nitrogen R. Dissolve the derivative of cholesterol in the chromatogram
residue in 1.0 mL of derivatisation solution (b). obtained with the test solution;
Reference solution (b) Introduce 1.0 mL of the internal A4 ะ= area of the peak due to the trimethylsilyl
standard solution into a 50 mL round-bottomed flask and derivative of cholesterol in the chromatogram
evaporate to dryness. Place 3.35 g of saw palmetto obtained with reference solution (a);
extract HRS) accurately weighed, into the round-bottomed mi = mass of the extract to be examined used to
flask and add 20 mL of a solution prepared as follows: prepare the test solution, in grams;
dissolve 130 g of potassium hydroxide R in 200 mL of water R m2 = mass of P-sitosterol CRS used to prepare reference
and dilute to 1000 mL with methanol R. Heat under reflux solution (a), in grams;
for 2 h, transfer quantitatively to a flask and dilute to p = percentage content of p-sitosterol in P-sitosterol
25.0 mL with water R. Apply 3.0 mL of this solution to a CRS.
cartndge containing diatomaceous earth R capable of holding Calculate the percentage content of P-sitosterol using the
3 mL of aqueous phase. Absorb the solution into the column following expression:
by applying vacuum. Maintain the vacuum for at least
20 min, until the column returns to room temperature, Al X A4 X 7712 X p
indicating that the methanol is completely evaporated. Rinse A2 X A3 X 7711
the column with 90 mL of methylene chloride R and evaporate
the eluate to dryness. Dissolve the residue in 1.0 mL of A1 = area of the peak due to the trimethylsilyl
derivatisation solution (b). derivative of p-sitosterol in the chromatogram
Column: obtained with the test solution;
— material: fused silica; A2 = area of the peak due to the trimethylsilyl
size: l = 25 m, 0 ะ= 0.20 mm; derivative of P-sitosterol in the chromatogram
— stationary phase: poly (dimethyl) siloxane R (film thickness obtained with reference solution (a);
0.33 pm). A3 = area of the peak due to the trimethylsilyl
Carrier gas helium for chromatography R. derivative of cholesterol in the chromatogram
A4 = area of the peak due to the trimethylsilyl
Split ratio 1:40. derivative of cholesterol in the chromatogram
Temperature: obtained with reference solution (a);
Time Temperature prepare the test solution, in grams;
(°C) m2 = mass of P-sitosterol CRS used to prepare reference
Column 0- 3 200 solution (a), in grams;
p = percentage content of P-sitosterol in P-sitosterol
3 - 13 200 -» 300
CRS.
13 - 35 300
_____________________________________________________________ Ph Eur
Injection port 325
Detector 325

Saw Palmetto Fruit * *
Injection 1 |1L.
Identification of peaks Use the chromatogram supplied with Ph Eur______________________________________________ __ —-—— ------
saw palmetto extract HRS and the chromatogram obtained DEFINITION

with reference solution (b) to identify the peaks due to the Dried ripe fruit of Serenoa repens (พ. Bartram) Small (syn.
trimethylsilyl derivatives of cholesterol, campesterol, Sabal serrulata (Michaux) T. Nuttal ex Schultes & Schultes).
stigmasterol, p-sitosterol and stigmastanol.
Content
System suitability: reference solution (b): Minimum 11.0 per cent of total fatty acids (dried drug).
— resolution: minimum 1.6 between the peaks due to the
trimethylsilyl derivatives of P-sitosterol and stigmastanol. CHARACTERS
Calculate the percentage content of total sterols (campesterol, Odour, strong but not rancid.
stigmasterol, p-sitosterol and stigmastanol), expressed as IDENTIFICATION
P-sitosterol, using the following expression: First identification A, B) D
Al X A4 X 7ท2 X p A. The fruit is an ovoid or subspherical drupe, with a dark
A2 X A3 X 7711 brown or blackish, roughly wrinkled surface and more or less
coppery sheen, up to 2.5 cm long and 1.5 cm in diameter.
A1 = sum of the areas of the peaks due to the The apex sometimes bears the remains of the style and
trimethylsilyl derivatives of campesterol, tubular calyx, with 3 teeth, and the base bears a small
stigmasterol, p-sitosterol and stigmastanol in the depression with the scar of the stalk. The epicarp and
chromatogram obtained with the test solution; underlying mesocarp form a thin fragile layer, which partially
A2 = area of the peak due to the trimethylsilyl peels off, revealing the thin, hard, pale brown endocarp,
derivative of P-sitosterol in the chromatogram which is fibrous and easily separable. The seed is irregularly
obtained with reference solution (a); spherical or ovoid, up to 12 mm long and 8 mm in diameter,
IV-368 Saw Palmetto Fruit 2016
with a hard, smooth or finely pitted surface which is reddish- D. Examine the chromatograms obtained in the assay of total
brown with a paler, raised and membranous area over the fatty acids.
raphe and micropyle; cut transversely, the seed has a thin Results The peaks due to caproic, caprylic, capric, lauric,
testa, narrow perisperm and a large area of dense, homy, myristic, palmitoleic, palmitic, linoleic, linolenic, oleic and
greyish-white endosperm, with the embryo positioned to one stearic acids in the chromatogram obtained with the test
side. solution are similar in retention time to the corresponding
B. Microscopic examination (2.8.23). Reduce to a powder peaks in the chromatogram obtained with reference
(710) (2.9.12). The powder is reddish or blackish-brown and solution (b); the principal peaks are due to lauric acid and
oily. Examine under a microscope using chloral hydrate oleic acid.
TESTS
characters: fragments of epicarp composed of several layers of
thin-walled, reddish-brown, pigmented, polyhedral cells
(10-40 pm) which are strongly cuticularised; those of ±e
outer layers are much smaller than those of the inner layers.
105 °C for 2 h.
Parenchyma cells of the mesocarp may be large and filled
with oil droplets, or smaller and containing nodules of silica. Total ash (2.4.16)
Groups of xylem tissue of the mesocarp show small lignified, Maximum 5.0 per cent.
annular or spirally thickened vessels. Stone cells of the ASSAY
mesocarp (20-200 pm) may be found scattered, usually Total fatty acids
singly but sometimes in small groups, the walls are Gas chromatography (2.2.28).
moderately thickened, distinctly striated and finely pitted.
Internal standard solution Dissolve 0.47 g of methyl
Fragments of endocarp contain groups of elongated sclereids
margarate R in 20.0 mL of dimethylformamide R and dilute to
about 300 pm long, with strongly thickened walls and
numerous pits. The seed testa consists of small, thin-walled
cells with brownish contents and underlying sclereids; Test solution Reduce 50 g of the herbal drug to a powder
albumen cells are thick-walled with large conspicuous pits (200) (2.9.12). Disperse 4.00 g of the powdered herbal drug
and contain aleurone grains and fixed oil. in 60 mL of dimethylformamide R. Sonicate for 15 min and
then shake for 30 min. Dilute to 100.0 mL with
dimethylformamide R. Allow to stand for a few minutes and
Test solution To 1.5 g of the powdered herbal drug (710) filter. To 20.0 mL of this solution add 4.0 mL of the internal
(2.9.12), add 20 mL of ethanol (96 per cent) R and stir for standard solution and dilute to 25.0 mL with
15 min. Filter. dimethylformamide R. Mix 0.4 mL of this solution and
Reference solution Dissolve 4 mg of fl-amyrin R and 10 mg of 0.6 mL of an 18.84 g/L solution of trimethylsulfonium
fl-sitosterol R in 10 mL of ethanol (96 per cent) R. hydroxide R in methanol R.
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel Reference solution (a) Dissolve 0.699 g of lauric acid CRS and
plate R (2-10 pm)]. 0.870 g of oleic acid CRS in dimethylformamide R and dilute to
Mobile phase acetic acid R3 ethyl acetate R, toluene R 10.0 mL with the same solvent. To 1.0 mL of the solution
(1:30:70 VIVIV). add 4.0 mL of the internal standard solution and dilute to
Applicatioj-I 10 pL [or 2 pL] as bands of 10 mm [or 8 mm]. 25.0 mL with dimethylformamide R. Mix 0.4 mL of this
solution and 0.6 mL of an 18.84 g/L solution of
trimethylsulfonium hydroxide R in methanol R.
Drying In air.
Reference solution (b) Disperse 0.25 g of saw palmetto
Detection Treat with anisaldehyde solution R', heat at extract HRS in 10 mL of dimethylformamide R. Add 4.0 mL
100-105 °C for 5-10 min; examine in daylight. of the internal standard solution and dilute to 25.0 mL with
Results See below the sequence of zones present in the dimethylformamide R. Mix 0.4 mL of this solution and
chromatograms obtained with the reference solution and the 0.6 mL of an 18.84 g/L solution of trimethylsulfonium
test solution. Furthermore, other faint zones may be present, hydroxide R in methanol R.
especially in the lower third, in the chromatogram obtained Column'.
with the test solution. — material', fused silica;
— size', z = 25 m, 0 = 0.20 mm;
Top of he plate
— stationary phase: poly (dimethyl) siloxane R (film thickness
0.33 pm).
A strong blue zone
2 faint blue zones Split ratio 1:40.
0-Amyrin ะ a blue zone
A strong bluish-violet zone
p-Sitosterol: a blue zone A faint blue zone
A faint blue zone

2016 Schisandra Fruit IV-369
Temperature:
Schisandra Fruit
Time Temperature
(min) (°C)
Column 0 -2 Ph Elf _ _____________________
150
2-7 150 -» 190 DEFINITION
7 - 12
Whole, dried or steamed and dried, ripe fruit of Schisandra
190
chinensis (Turcz.) Bail!.
12 - 22 190 -> 220
Content
22 - 32 220 Minimum 0.40 per cent of schisandrin (C24H32O7;
Injection port 300 Mr 432.5) (dried drug).
Detector 300 IDENTIFICATION
A. The berry is more or less spherical, up to 8 mm in
diameter; red, reddish-brown or blackish outer surface,
Detection Flame ionisation. sometimes covered in a whitish frost; strongly shrivelled
Injection 1 pL. pericarp; presence of 1-2 reniform, yellowish-brown, lustrous
Identification of peaks Use the chromatogram supplied with seeds, wi± thin seed-coat.
saw palmetto extract HRS and the chromatogram obtained B. Reduce to a powder (355) (2.9.72). The powder is
with reference solution (b) to identify the peaks due to reddish-brown. Examine under a microscope using chloral
caproic, caprylic, capric, lauric, myristic, palmitoleic, hydrate solution R. The powder shows the following diagnostic
palmitic, linoleic, linolenic, oleic and stearic acids and methyl characters: reddish-brown fragments of pericarp, consisting of
margarate. 1 layer of thin-walled epicarp cells, accompanied by sparse oil
System suitability: reference solution (b): cells and several layers of ovoid, more-or-less flattened
— peak-to-valley ratio: minimum 1.2, where Hp — height mesocarp cells; fragments of the outer testa of the seed
above the baseline of the peak due to linolenic acid and consisting of thick-walled, finely channelled sclereids,
Hv = height above the baseline of the lowest point of the polygonal in surface view (15-50 pm in diameter) and in
curve separating this peak from the peak due to linoleic palisade arrangement in side view; fragments of the inner
acid. testa with sclereids, isolated or in small groups, about 80 pm
Calculate the percentage content of total fatty acids, where in diameter, with slightly thickened and markedly channelled
caproic, caprylic, capric, lauric, myristic, palmitoleic, palmitic walls; fragments of endosperm consisting of polyhedral cells
and stearic acids are expressed as lauric acid (C12H24O2; containing oil droplets and aleurone grains. Examine under a
200.3) and linoleic, linolenic and oleic acids are expressed as microscope using a 50 per cent v/v solution of glycerol R: the
oleic acid (C18H34O2; 282.5), using the following expression: powder shows parenchymatous cells of the mesocarp
containing numerous small, round starch granules.
41 X /14 X 777.2 X Pl X 0.5 A5 X A4 X m3 X P2 X 0.5 c. Examine the chromatograms obtained in the test for
An X A3 X 7ท1 Aq X A3 X 7ท1
Schisandra sphenanthera.
Results A See below the sequence of quenching zones present
Al ะ= sum of the areas of the peaks due to caproic, in the chromatograms obtained with the reference solution
caprylic, capric, lauric, myristic, palmitoleic, and the test solution. Furthermore, other weak quenching
palmitic and stearic acids in the chromatogram zones may be present in the chromatogram obtained with the
obtained with the test solution; test solution.
A2 = area of ±c peak due to lauric acid in the
chromatogram obtained with reference solution Top of the plate
(a);
A} = area of the peak due to methyl margarate in the y-Schisandrin: a quenching zone A quenching zone (y-schisandrin)
A4 = area of the peak due to methyl margarate in the
(a);
>15 = sum of the areas of the peaks due to linoleic, Schisandrin ะ a quenching zone A quenching zone (schisandrin)
linolenic and oleic acids in the chromatogram
obtained with the test solution; Reference solution Test solution
Ab — area of the peak due to oleic acid in the
(a);
TH\ = mass of the herbal drug to be examined used to
test solution. Furthermore, o±er faint zones may be present
พ2 = mass of lauric acid CRS used to prepare reference
= mass of oleic acid CRS used to prepare reference
solution- (a), in grams;
Pl = percentage content of lauric acid in lauric acid
CRS;
P2 = percentage content of oleic acid in oleic acid CRS.
___ __ _______________________________________________ PhEur
IV-370 Scutellariae Baicalensis Root 2016

y-Schisandrin: a brown zone A brown zone (y-schisandrin)
0 - 10 100 0
10 - 16 100 -> 58 0 -> 42
16 - 26 58 42
Schisandrin: an intense, An intense, brownish-green zone Flow rate 1 โทบทโนใ.

brownish-green zone (schisandrin)
Reference solution
Test solution
Injection 10 pL.
Retention time Schisandrin = about 8 min.
TESTS System suitability:
Schisandra sphenanthera — number of theoretical plates: minimum 5000, calculated for
Thin-layer chromatography (2.2.27). the peak due to schisandrin in the chromatogram
Test solution To 2.5 g of the powdered herbal drug (355) obtained with the reference solution.
(2.9.72) add 10 mL of methanol R. Extract at 25 °C in an Calculate the percentage content of schisandrin using the
ultrasonic bath for 5 min and centrifuge. following expression:
Reference solution Dissolve 5 mg of schisandrin R and 5 mg of
y-schisandrin R in 5 mL of methanol R. Al X 7712 X p
A2 X 7711
Plate TLC silica gel F254 plate X (5-40 pm) [or TLC silica gel
F254 plate R (2-10 pm)].
Mobile phase acetic acid R} ethyl acetate R) toluene R A1 = area of the peak due to schisandrin in the
(2:22:46 FZJZ/F). chromatogram obtained with the test solution;
A2 = area of the peak due to schisandrin in the
Development Over a path of 10 cm [or 7 cm]. solution;
Drying In air. nil = mass of the herbal drug to be examined used to
Detection B Spray with a 100 g/L solution of sulfuric acid R in m2 = mass of schisandrin R used to prepare the
methanol R and heat in an oven at 120 °C for 7 min; examine reference solution, in grams;
in daylight p = percentage content of schisandrin in schisandrin R.
Results B The chromatogram obtained with the test solution _______________________________________________________________ Ph Elf
shows a zone due to schisandrin and a zone due to

Y-schisandrin; the chromatogram shows no intense violet
pink zone in the middle third.
Scutellariae Baicalensis Root ★ **
powdered herbal drug (355) (2.9.72) by drying in an oven at (Baical Skullcap Root) Ph Eur monograph 2438)
105 °C for 2 h. PhEur_________________________________________________________ -____
Total ash (2.4.16) DEFINITION
Maximum 6.0 per cent. Dried, peeled, usually fragmented root of Scuteliana
ASSAY baicalensis Georgi without rootlets. It is collected in spring or
Liquid chromatography (2.2.29). autumn.
Test solution Weigh 1.250 g of the powdered herbal Content
drug (355) (2.9.72) into a 250 mL conical flask, add 90 mL Not less than 9.0 per cent of baicalin (C2]Hi80n; Mr 446.4)
of methanol R and sonicate for 30 min. Filter the solution (dried drug).
into a volumetric flask, add 10 mL of methanol R whilst IDENTIFICATION
rinsing the filter and dilute to 100.0 mL with the same
A. The root is conical, twisted and, if not reduced in size,
solvent.
8-25 cm long and 1-3 cm in diameter. The outer surface is
Reference solution Dissolve 5.0 mg of schisandrin R in brownish-yellow or dark yellow, bearing sparse, warty traces
methanol R and dilute to 100.0 mL with the same solvent. of rootlets, the upper part rough, with twisted longitudinal
Column'. wrinkles or irregular reticula, the lower part with longitudinal
— size: I = 0.25 m, 0 = 4.6 mm; striations and fine wrinkles. Texture hard and fragile, easily
— stationary phase: end-capped octadecylsilyl silica gel for broken, fracture yellow, reddish-brown in the centre;
chromatography R; the central part of an old root dark brown or brownish-black,
— temperature: 25 °C. withered or hollowed.
Mobile phase: B. Microscopic examination (2.8.23). The powder is yellow
— mobile phase A: water R, methanol R (35:65 VIV)', or light brown. Examine under a microscope using chloral
— mobile phase B: methanol R; hydrate solution R. The powder shows the following diagnostic
characters: phloem fibres, single or in bundles, fusiform,
60-250 pm long, 9-33 pm in diameter, with thick, channelled
walls; stone cells sub-spherical, square or rectangular with
rounded edges, with thickened walls, sometimes heavily; cork
2016 Selfheal Fruit-Spike IV-371
cells polygonal and brownish-yellow; numerous reticulated small volume of ethanol (70 per cent VIV) R and filter the
vessels, 24-72 pm in diameter; lignified fibres frequently washings into the same flask. Dilute to 100.0 mL with
broken, about 12 pm in diameter, with sparse, oblique pits. ethanol (70 per cent VIV) R. Mix well. Dilute 1.0 mL of the
Examine under a microscope using a 50 per cent VIV solution to 10.0 mL with methanol R. Mix well.
solution of glycerol R. The powder shows abundant starch Reference solution (a) Dissolve 5.0 mg of baicalin CRS in
granules, simple, spheroidal, 2-10 pm in diameter, with a methanol R and dilute to 100.0 mL with the same solvent.
distinct hilum, or compound with 2-3 components.
Reference solution (b) Dissolve 2 mg of methyl
c. Thin-layer chromatography (2.2.27). parahydroxybenzoate R in methanol Ry add 20 mL of reference
Test solution To 1 g of the powdered herbal drug (355) solution (a) and dilute to 100 mL with methanol R.
(2.9.72) add 10 mL of methanol R and sonicate for 10 min. Column:
Centrifuge and use the supernatant. — size: I = 0.125 m, 0 = 4 mm;
Reference solution Dissolve 1 mg of baicalin R and 1 mg of — stationary phase: octadecylsilyl silica gel for chromatography R
acteoside R in 10 mL of methanol R. (5 pm).
Plate TLC silica gel F254 plate R (2-10 pm). Mobile phase:
Mobile phase acetic acid Ry formic acid Ry water Ry ethyl — mobile phase A: 0.1 per cent V/V solution of phosphoric
acetate R (1:1:2:15 VIVIVIV). acid Ry
— mobile phase B: acetonitrile R'y
Drying In air.
Detection Heat at 100-105 °C for 3 min, treat with a 10 g/L 0 - 30 90-» 60 10 -» 40
solution of diphenylboric acid aminoethyl ester R in methanol Ry
then treat with a 50 g/L solution of macrogol 400 R in
methanol Ry allow to dry in air for 30 min and examine in Flow rate 1.0 mL/min.
ultraviolet light at 365 nm. Detection spectrophotometer at 280 nm.
Results See below the sequence of zones present- in the Injection 10 pL.
chromatograms obtained with the reference solution and the Retention time Methyl parahydroxybenzoate = about 15 min; ■
test solution. Furthermore, other faint blue fluorescent zones baicalin = about 16 min.
solution.
— resolution: minimum 3 between the peaks due to methyl
parahydroxybenzoate and baicalin.
Top of the plate
Calculate the percentage content of baicalin using the
3-4 fluorescent zones following expression:
7ท2 X ร1 X 10 X p
2 fluorescent zones
ร2 X 7711
Verbascoside: a blue fluorescent A strong blue fluorescent zone m1 = mass of the herbal drug, in grams;
zone m2 = mass of baicalin used to prepare reference solution
A blue fluorescent zone (a), in grams;
Baicalin: a black zone A black zone ร1 = area of the peak due to baicalin in the
ร2 = area of the peak due to baicalin in the
A weak yellow fluorescent zone chromatogram obtained with reference solution
(a); ...............
p = percentage content of baicalin in baicalin CRS.
STORAGE
TESTS Protected from moisture.
Loss on drying (2.2.22) _ ______________________________________ ______________________ PhEur
105 °C for 2 h
Total ash (2.4.76)
Maximum 6.0 per cent. Selfheal Fruit-Spike
Ash insoluble in hydrochloric acid (2.8.7) (Common Selfheal Fruit-Spike,
Maximum 2.0 per cent. Ph Eur monograph 2439)
ASSAY PhEur
DEFINITION
Test solution To 0.300 g of the powdered herbal drug (355) Dried fruit-spike of Prunella vulgaris L.
(2.9.72) add 40 mL of ethanol (70 per cent VIV) Ry heat
under a reflux condenser on a water bath for 3 h, cool and Content
Minimum 0.12 per cent of the sum of oleanolic acid
filter. Transfer the filtrate to a 100 mL volumetric flask.
(C3oH4803; Mr 456.7) and ursolic add (CsoHigCh;
Wash both the container and the residue several times with a
-372 Selfheal Fruit-Spike 2016
Mr 456.7), expressed as ursolic acid, of which not less than Top of the plate
70.0 per cent consists of ursolic acid (dried drug).
A pale violet fluorescent zone
IDENTIFICATION
A. Cylindrical, somewhat flattened, 1.5-8 cm long,
0.8-1.5 cm in diameter, accompanied by remains of the stem 2 faint yellow fluorescent zones
up to 15 cm long, pale brown or brownish-red. The whole 0-sitosterol: a violet fluorescent
spike is composed of up to 10 or more whorls of persistent
calyx and bracts, each whorl with 2 opposite bracts, fan Ursolic acid: a yellowish-orange A yellowish-orange fluorescent
shaped, apex acuminate, striations of vein distinct, the outer fluorescent zone zone (ursolic acid)
surface with white hairs. Each bract is accompanied by
3 flowers, with a persistent bilabiate calyx, and whose corolla
is often missing, and by 4 small brown ovoid nutlets, white
2 faint green fluorescent zones
and convex at the acute end. Calyx closed in the fruit stage.
B. Microscopic examination (2.8.23). The powder is reddish- Reference solution Test solution
brown or brown. Examine under a microscope using chloral
hydrate solution R. The powder show's the following diagnostic TESTS
characters: very numerous covering trichomes, multicellular, Foreign matter (2.8.2)
scattered, usually broken, sometimes exceeding 1 mm long Maximum 5 per cent of stems longer than 15 cm and
and 125 pm wide at the base, with spiny walls, upper cell maximum 2 per cent of other foreign matter.
usually short and acuminate, fine needle-shaped crystals may Loss on drying (2.2.32)
be visible in the cells; fragments of the bracts, in surface Maximum 12.0 per cent, determined on 1.000 g of the
view, with lobed epidermal cells, trichomes mostly unicellular powdered herbal drug (355) (2.9.12) by drying in an oven at
and occasionally bi- or tricellular, conical, acute, short, 105 °C.
serrate; diacytic stomata (2.8.3) usually accompanied by
Total ash (2.4.16)
2 subsidiary cells very' unequal in size and rare glandular
trichomes with a unicellular stalk and a bicellular head;
fragments of the bracts and/or calyx margins with numerous Ash insoluble in hydrochloric acid (2.8.1)
serrate trichomes pointing towards the same direction; Maximum 4.0 per cent.
fragments of the calyx, in surface view, composed of lobed ASSAY
cells strongly thickened and deeply grooved; fragments of Liquid chromatography (2.2.29).
reticulate or bordered pitted vessels from the stems; rare Solvent mixture methanol R, 1,1-dimethylethyl methyl ether R
fragments of the nucules having a pericarp composed of (20:80 VIV).
palisade-like mucilaginous cells accompanied by polygonal
cells with thickened walls and granular coloured contents;
(355) (2.9.12) in 20 mL of the solvent mixture, heat under
fragments of endosperm with oily contents; very numerous
reflux at 80 °C for 30 min and filter. Repeat the extraction
oil droplets; glandular trichomes of laminaceous type with
twice. Combine the filtrates and dilute to 100.0 mL with the
4 secretory cells may be present.
solvent mixture. Evaporate 50.0 mL of this solution to
c. Thin-layer chromatography (2.2.27). dryness at 40 °C. Dissolve the residue in 1.0 mL of
Test solution To 0.5 g of the powdered herbal drug (355) 1,1-dimethylethyl methyl ether R. Rinse the flask 4 times with
(2.9.12) add 5 mL of methanol R, sonicate for 10 min and 1.0 mL of 1,1-dimethylethyl methyl ether R. Pre-condition a
centrifuge; use the supernatant. 3 mL solid phase extraction column, containing 500 mg of
Reference solution Dissolve 1 mg of P-sitosterol R and 1 mg of aminopropylsilyl silica gel for chromatography R1, using 2 mL of
ursolic acid R in 2 mL of methanol R. methanol R followed by 2 mL of ใ, 1-dimethylethyl methyl
Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel ether R. Subsequently apply the solution and the washings to
7*254 plate R (2-10 pm)]. the pre-conditioned column. Wash the column with 1.0 mL
of 1,1-dimethylethyl methyl ether R followed by 5 quantities,
Mobile phase glacial acetic acid R, ethyl acetate R, cyclohexane R
each of 1.0 mL, of methanol R. Apply 1.0 mL of a
(0.5:8:20 VIVIV). 2 per cent VIV solution of anhydrous formic acid R in
Application 10 pL [or 4 pL] as bands of 10 mm [or 8 mm]. methanol R and elute after 5 min. Repeat the elution 3 times
Development Over a path of 12 cm [or 6 cm]. and dilute the eluates to 5.0 mL with a 2 per cent VIV
Drying In air. solution of anhydrous formic acid R in methanol R.
Detection Treat with a 10 per cent VIV solution of sulfuric Solution A Dissolve 10.0 mg of ursolic acid CRS in methanol R
acid R in anhydrous ethanol R and heat at 100 °C for 3 min; and dilute to 10.0 mL with the same solvent.
examine in ultraviolet light at 365 nm. Reference solution (a) Dilute 1.0 mL of solution A to 10.0 mL
Results See below the sequence of zones present in the with a 2 per cent VIV solution of anhydrous formic acid R in
chromatograms obtained with the reference solution and the methanol R.
test solution. Furthermore, other faint zones may be present Reference solution (b) Dissolve 10.0 mg of oleanolic acid R in
in the chromatogram obtained with the test solution. methanol R and dilute to 10.0 mL with the same solvent.
Mix 1.0 mL of the solution and 1.0 mL of solution A and
dilute to 10.0 mL with a 2 per cent VIV solution of
anhydrous formic acid R in methanol R.
Column'.
— size-, z = 0.15 m, 0 = 4.6 mm;
— stationary phase', octadecylsilyl silica gel for chromatograph}' R
(5 pm).
2016 Senega Root IV-373
.Mobile phase Mix 25 volumes of a 4.6 g/L solution of CHARACTERS

ammonium dihydrogen phosphate R adjusted to pH 6.0 with Faint, sweet odour, slightly rancid or reminiscent of methyl
strong sodium hydroxide solution R, 35 volumes of methanol R1 salicylate.
and 40 volumes of acetonitrile Rl.
Reduced to a powder, it is irritant and sternutatory. Shaken
Flow rate 1.0 mUmin. with water, the powder produces a copious froth.
IDENTIFICATION
Injection 20 |1T. A. The root crown is greyish-brown and wider than the root;
Run time 1.1 times the retention time of ursolic acid. it forms an irregular head consisting of numerous remains of
Elution order Oleanolic acid, ursolic acid. stems and tightly packed purplish-brown buds. The taproot
Relative retention With reference to ursolic acid (retention is brown or yellow, occasionally branched, sometimes
time = about 28 min): oleanolic acid = about 0.9. flexuous, usually tortuous and without secondary roots,
except in the Japanese varieties and species, which contain
รุ)1stem suitability: reference solution (b):
numerous fibrous rootlets. The diameter is usually 1-8 mm
resolution: minimum 1.5 between the peaks due to
at the crown, gradually tapering to the tip; the surface is
oleanolic acid and ursolic acid.
transversely and longitudinally striated and often shows a
Calculate the percentage contents of ursolic acid and more or less distinct decurrent, elongated spiral keel.
oleanolic acid, expressed as ursolic acid, using the following The fracture is short and shows a yellowish cortex of varying
equations: thickness surrounding a paler central woody area somewhat
circular or irregular in shape depending on the species.
41 X m2 X p X 0.1
ท1 = ——
B. Examine under a microscope using chloral hydrate
42 X 7721 solution R. The transverse section of the root shows the
4ร X 7ท2 X p X 0.1 following diagnostic characters: cork formed from several
ท2 —42-----------
X 7721
7- - .. ------------
layers of thin-walled cells, phelloderm of slightly
collenchymatous cells containing droplets of oil; the phloem
H| = percentage content of ursolic acid; and xylem arrangement is usually normal, especially near the
ท2 = percentage content of oleanolic acid; crown but where a keel is present this is formed by increased
41 = area of the peak due to ursolic acid in the development of phloem; other anomalous secondary
chromatogram obtained with the test solution; development sometimes occurs, resulting in the formation of
A2 = area of the peak due to ursolic acid in the 1 or 2 large wedge-shaped rays in the phloem and xylem, the
chromatogram obtained with reference solution parenchymatous cells of which contain droplets of oil.
The xylem is usually central and consists of vessels up to
;
(a)
43 = area of the peak due to oleanolic acid in the 60 pm in diameter associated with numerous thin-walled
chromatogram obtained with the test solution; tracheids and a few small lignified parenchymatous cells.
ni] = mass of the herbal drug to be examined used to c. Reduce to a powder (355) (2.9.12). The powder is light
prepare the test solution, in grams; brown. Examine under a microscope using chloral hydrate
tn2 = mass of ursolic acid CRS used to prepare solution solution R. The powder shows the following diagnostic
A, in grams; characters: longitudinal fragments of lignified tissue made up
p = assigned percentage content of ursolic acid in of pitted tracheids and somewhat larger vessels with
ursolic acid CRS. numerous bordered pits or with reticulate thickening;
yellowish parenchyma and collenchymatous cells containing
Calculate the sum of the percentage contents of ursolic acid
droplets of oil; occasional fragments of cork, and of
and oleanolic acid (ท 1 + ท2) and the relative content of
epidermal tissue with stomata and unicellular trichomes from
ursolic acid using the following expression:
the bud scales. Crystals and stone cells are absent.
D. Thin-layer chromatography (2.2.27).
ท1 X 100
(ท1 4- ท2)
(2.9.12) add 10 mL of ethanol (70 per cent VIV) R, boil under
a reflux condenser for 15 min, filter and allow to cool.
_______________________________________________________________ Ph Eur
Reference solution Dissolve 10 mg of aescin R in ethanol
(70 per cent VIV) R and dilute to 10 mL with the same
solvent.
Senega Root ** * Mobile phase The upper layer of a mixture of 10 volumes of
glacial acetic acid R, 40 volumes of water R and 50 volumes of
Senega ***
butanol R.
(Ph. Eur. monograph 0202) Application 10 pL of the test solution and 10 pL and 40 pL
When Powdered Senega Root is prescribed or demanded, of the reference solution, as bands of 20 mm by 3 mm.
material complying with the requirements below with the Development Over a path of 12 cm.
Detection A spray with about 10 mL of anisaldehyde solution R
PhEo______________ - _____________________
for a plate 200 mm square and heat again at 100-105 °C
DEFINITION until red zones due to saponosides appear in the
Dried and usually fragmented root and root crown of chromatogram obtained with the test solution.
Polygala senega L. or of certain other closely related species or
of a mixture of these Polygala species.
IVO74 Senna Fruit 2016
Results A In the chromatogram obtained with the test view [N]) and fragments of the hypodermis of the seed
solution, 3-5 red zones appear in the lower and middle parts, forming rings (surface view [A]); fragments of cotyledons
similar in position to the grey-violet zones due to aescin in (surface view [F], transverse section [E]) consisting of small
the chromatogram obtained with the reference solution. cells of the epidermis [Ea, Fa] and of the palisade
Detection B Spray with about 10 mL of a 200 g/L solution of tissue [Eb, Fb]; prisms and cluster crystals of calcium
phosphomolybdic acid R in anhydrous ethanol R and heat at oxalate, free [De, Ga] or included in parenchyma [G];
100-105 °C until the zones due to saponosides become blue. fragments of vascular bundles [L] with spiral vessels [La] and
Results B The intensity and size of ±e zones in the fibres with moderately thickened and pitted walls [Lb];
chromatogram obtained with the test solution are between sclereids [M, O] and fibres [H] accompanied by crystal
those of the 2 bands due to aescin in the chromatograms shea±s of calcium oxalate [Ha] from fruit stalks.
obtained with 10 pL and 40 pL of the reference solution.
TESTS
Total ash (2.4.16)
STORAGE
Store protected from humidity.
-------------------------------------------------------------------------------------------------------- -- Ph Eur
Alexandrian Senna Fruit *

(Alexandrian Senna Pods, Ph Eur monograph 0207) ***
Preparations
Senna Liquid Extract
Standardised Senna Granules
Senna Tablets
When Powdered Alexandrian Senna Fruit is prescribed or
demanded, material complying with the requirements below,
wnth the exception of Identification test A and the test for
Foreign matter, shall be dispensed or supplied.
Ph Eur_______________________________________________________________
DEFINITION
Dried fruit of Cassia senna L. (syn. Cassia acutifolia Delile).
Content
Minimum 3.4 per cent of hydroxyanthracene glycosides, Figure 0207.-1. - Illustration for identification test B of powdered
expressed as sennoside B (042แ38020; MT 863) (dried drug). herbal drug of Alexandrian senna pods
IDENTIFICATION
A. Flattened reniform pods, green or greenish-brown with Test solution To 0.5 g of the powdered herbal drug (180)
brown patches at the positions corresponding to the seeds, (2.9.12) add 5 mL of a mixture of equal volumes of ethanol
usually 40-50 mm long and at least 20 mm wide. At one end (96 per cent) R and water R and heat to boiling. Centrifuge
is a stylar point and at the other a short sulk. The pods and use the supernatant liquid.
conuin 6-7 flattened and obovate seeds, green or pale Reference solution Dissolve 10 mg of senna extract CRS in
brown, with a continuous network of prominent ridges on 1 mL of a mixture of equal volumes of ethanol (96 per cent) R
the tesu. and water R (a slight residue remains).
B. Microscopic examination (2.8.23). The powder is brown. Plate TLC silica gel plate R.
Examine under a microscope using chloral hydrate solution R. Mobile phase glacial acetic acid Ry water Ry ethyl acetate Ry
The powder shows the following diagnostic characters propanol R (1:30:40:40 VIVIVIV).
(Figure 0207.-1): fragments of the epicarp (surface Application 10 |1L as bands of 20 mm.
view [B, K]) with polygonal cells, anomocytic [Ba] or
paracytic [Bb, Ka] stomau (2.8.3) and covering
trichomes [Kb] or their scars [Be]; isolated conical warty Drying In air.
covering trichomes, usually bent [C]; fragments of the Detection'. Treat with a 20 per cent VIV solution of nitric
mesocarp (surface view [D]) with fibres [Da] in 2 crossed acid R and heat at 120 °C for 10 min. Allow to cool and
layers accompanied by a layer of cells conuining calcium treat with a 50 g/L solution of potassium hydroxide R in
oxalate prisms [Db] and sometimes cells of the underlying ethanol (50 per cent VIV) R until the zones appear.
endocarp [De]; fragments of the outer layers of the testa Results The principal zones in the chromatogram obtained
(transverse section [J]) covered by a thick cuticle Qa] with with the test solution are similar in position (sennosides B, A,
palisade cells about 50 Jim long, with thick walls and a D and c in order of increasing Rp value), colour and size to
reduced lumen [Jb], accompanied by the hypodermis with the principal zones in the chromatogram obtained with the
pillar-like cells Qc]; palisade cells of the testa (surface reference solution; between the zones due to sennosides D
2016 Senna Fruit FV-375
and c a red zone due to rhein-8-glucoside may be visible;

the zones due to sennosides D and c are faint in the Tinnevelly Senna Fruit * *
chromatogram obtained with the test solution. (Tinnevelly Senna Pods, Ph Eur monograph 0208) ***
D. Place about 25 mg of the powdered herbal drug (180) Preparations
(2.9.12) in a conical flask and add 50 mL of water R and Senna Liquid Extract
2 mL of hydrochloric acid R. Heat in a water-bath for 15 min,
Senna Tablets
cool and shake with 40 mL of ether R. Separate the ether
layer, dry over anhydrous sodium sulfate R, evaporate 5 mL to When Powdered Tinnevelly Senna Fruit is prescribed or
dryness and to the cooled residue add 5 mL of dilute demanded, material complying with the requirements below
ammonia Rl. A yellow or orange colour develops. Heat on a with the exception of Identification test A and the test for
water-bath for 2 min. A reddish-violet colour develops. Foreign matter shall be dispensed or supplied.
Ph Eur __________________________ :_________________________ 1____________________
TESTS
Foreign matter (2.8.2) DEFINITION
Maximum 1 per cent. Dried fruit of- Cassia angustifolia Vahl.
Loss on drying (2.2.32) Content
Maximum 12.0 per cent, determined on 1.000 g of the Minimum12.2 per cent of hydroxyanthracene glycosides,
powdered herbal drug (355) (2.9.12) by drying in an oven at expressed!as sennoside B (C42H38O2(b Mr 863) (dried drug).
105 °C for 2 h.
IDENTIFICATION
Total ash (2.4.16) A. Flattened, slightly reniform pods, yellowish-brown or
Maximum 9.0 per cent. brown with dark brown patches at the positions
Ash insoluble in hydrochloric acid (2.8.1) corresponding to the seeds, usually 35-60 mm long and
Maximum 2.0 per cent. 14-18 mm wide. At one end.is a stylar point and at the other
a short stalk. The pods contain 5-8 flattened and obovate
ASSAY
seeds, green or pale brown, with incomplete, wavy, transverse
Carry out the assay protected from bright light. ridges on the testa.
Place 0.150 g of the powdered herbal drug (180) (2.9.12) in B. Microscopic examination, (2.8.23). The powder is brown.
a 100 mL flask. Add 30.0 mL of water R, mix, weigh and Examine under a microscope using chloral hydrate solution R.
place in a water-bath. Heat under a reflux condenser for The powder shows the following diagnostic characters
15 min. Allow to cool, weigh and adjust to the original mass (Figure 0208.-1): fragments of the epicarp (surface
with water R. Centrifuge and transfer 20.0 mL of the view [B, K]) with polygonal cells, anomocytic [Ba] or
supernatant liquid to a 150 mL separating funnel. paracytic [Bb, Ka] stomata (2.8.3) and covering
Add 0.1 mL of dilute hydrochloric acid R and shake with trichomes [Kb] or their scars [Be]; isolated conical warty
3 quantities, each of 15 mL, of chloroform R. Allow to covering trichomes, usually bent [C]; fragments of the
separate and discard the chloroform layer. Add 0.10 g of mesocarp (surface view [D]) with fibres [Da] in 2 crossed
sodium hydrogen carbonate R and shake for 3 min. Centrifuge layers accompanied by a layer of cells containing calcium
and transfer 10.0 mL of the supernatant liquid to a 100 mL oxalate prisms [Db] and sometimes cells of the underlying
round-bottomed flask with a ground-glass neck. Add 20 mL endocarp [De]; fragments of the outer layers of the testa
of ferric chloride solution Rl and mix. Place the flask in a (transverse section [J]) covered by a thick cuticle [Ja] with
water-bath so that the water level is above that of the liquid palisade cells about 50 |im long, with thick walls and a
in the flask, and heat under a reflux condenser for 20 min. reduced lumen [Jb], accompanied by the hypodermis with
Add 3 mL of hydrochloric acid R and heat for a further pillar-like cells Qc]; palisade cells of the testa (surface
20 min, with frequent shaking, to dissolve the precipitate. view [N]) and fragments of the hypodermis of the seed
Cool, transfer the mixture to a separating funnel and shake forming rings (surface view [A]); fragments of cotyledons
with 3 quantities, each of 25 mL, of ether R previously used (surface view [F], transverse section [E]) consisting of small
to rinse the flask. Combine the 3 ether layers and wash with cells of the epidermis [Ea, Fa] and of the palisade
2 quantities, each of 15 mL, of water R. Transfer the tissue [Eb, Fb]; prisms and cluster crystals of calcium
combined ether layers to a volumetric flask and dilute to oxalate, free [De, Ga] or included in parenchyma [G];
100.0 mL with ether R. Evaporate 10.0 mL carefully to fragments of vascular bundles [L] with spiral vessels [La]
dryness and dissolve the residue in 10.0 mL of a 5 g/L and fibres with'moderately thickened and pitted walls [Lb];
solution of magnesium acetate R in methanol R. Measure the sclereids [M, O] and fibres [H] accompanied by crystal
absorbance (2.2.25) at 515 nm using methanol R as the sheaths of calcium oxalate [Ha] from fruit stalks.
compensation liquid. c. Thin-layer chromatography (2.2.27).
Calculate the percentage content of hydroxyanthracene Test solution To 0.5 g of the powdered herbal drug (180)
glycosides, expressed as sennoside B, using the following (2.9.12) add 5 mL of a mixture of equal volumes of ethanol
expression: (96 per cent) R and water R and heat to boiling. Centrifuge
A X 1.25 and use the supernatant liquid.
m Reference solutionSy\^Q\NQ 10 mg of senna extract CRS in
1 mL of a mixture of equal volumes of ethanol (96 per cent) R
i.e. taking the specific absorbance of sennoside B to be 240. and water R (a slight residue remains). 1 .1
A ะะะ absorbance at 515 nm; Plate TLC silica gel plate R.
m = mass of the substance to be examined, in grams. Mobile phase glacial acetic acid R> water R, ethyl acetate R,
STORAGE propanol fl (1:30:40:40 VIVIVIV).
Protected from moisture. Application 10 |1L as bands of 20 mm.
Ph Eur
IV-376 Senna Preparations 2016
ASSAY
Cany out the assay protected from bright light.
Place 0.150 g of the powdered herbal drug (180) (2.9.72) in
a 100 mL flask. Add 30.0 mL of water R, mix, weigh and
place in a water-bath. Heat under a reflux condenser for
15 min. Allow to cool, weigh and adjust to the original mass
with water R. Centrifuge and transfer 20.0 mL of the
supernatant liquid to a 150 mL separating funnel.
Add 0.1 mL of dilute hydrochloric acid R and shake with
3 quantities, each of 15 mL, of chloroform R. Allow to
separate and discard the chloroform layer. Add 0.10 g of
sodium hydrogen carbonate R and shake for 3 min. Centrifuge
and transfer 10.0 mL of the supernatant liquid to a 100 mL
round-bottomed flask with a ground-glass neck. Add 20 mL
of ferric chloride solution Rl and mix. Place the flask in a
water-bath so that the water level is above that of the liquid
in the flask, and heat under a reflux condenser for 20 min.
Add 3 mL of hydrochloric acid R and heat for a further
20 min, with frequent shaking, to dissolve the precipitate.
Cool, transfer the mixture to a separating funnel and shake
with 3 quantities, each of 25 mL, of ether R previously used
to rinse the flask. Combine the 3 ether layers and wash with
2 quantities, each of 15 mL, of water R. Transfer the
combined ether layers to a volumetric flask and dilute to
100.0 mL with ether R. Evaporate 10.0 mL carefully to
dryness and dissolve the residue in 10.0 mL of a 5 g/L
solution of magnesium acetate R in methanol R. Measure the
absorbance (2.2.25) at 515 nm using methanol R as the
compensation liquid.
Figure 0208.-1. - Illustration for identification test B of powdered Calculate the percentage content of hydroxyanthracene
herbal drug of Tinnevelly senna pods glycosides, expressed as sennoside B, using the following
Development Over a path of 10 cm. expression:
Drying In air.
Detection’. Treat with a 20 per cent v/v solution of nitric A X 1.25
acid R and heat at 120 °C for 10 min. Allow to cool and 771
treat with a 50 g/L solution of potassium hydroxide R in

ethanol (50 per cent VIV) R until the zones appear. i.e. taking the specific absorbance of sennoside B to be 240.
Results The principal zones in the chromatogram obtained A = absorbance at 515 nm;
with the test solution are similar in position (sennosides B, A, m = mass of the substance to be examined, in grams.
D and c in order of increasing Rp value), colour and size to
STORAGE
the principal zones in the chromatogram obtained with the
Protected from moisture.
reference solution; between the zones due to sennosides D
and c a red zone due to rhein-8-glucoside may be visible; ___________________________________________ ____ PnEtr
the zones due to sennosides D and c are faint in the
D. Place about 25 mg of the powdered herbal drug (180)
(2.9.12) in a conical flask and add 50 mL of water R and Senna Liquid Extract
2 mL of hydrochloric acid R. Heat in a water-bath for 15 min,
cool and shake with 40 mL of ether R. Separate the ether DEFINITION
layer, dry over anhydrous sodium sulfate R, evaporate 5 mL to Senna Fruit, Alexandrian or 1000 g
dryness and to the cooled residue add 5 mL of dilute Tinnevelly, crushed
ammonia Rl. A yellow or orange colour develops. Heat on a Coriander Oil
Ethanol (90 per cent) 250 mL
water-bath for 2 min. A reddish-violet colour develops.
Purified Water, freshly boiled and A sufficient quantity
TESTS cooled
Maximum 1 per cent. Extemporaneous preparation
Macerate the crushed Senna Fruit in 5 litres of Purified
Water for 8 hours, decant the clear liquid and strain; repeat
the process twice using 2 litres of Purified Water for each
105 °C for 2 h.
maceration. Lightly press the marc, strain the expressed
Total ash (2.4.16) liquid, mix the strained liquid with the previously decanted
Maximum 9.0 per cent. liquid and heat the combined liquids at 80° for 3 minutes in
Ash insoluble in hydrochloric acid (2.8.1) a covered vessel. Allow to stand for not less than 24 hours;
Maximum 2.0 per cent. filter.
2016 Senna Preparations IV-377
Evaporate the filtrate to 750 mL under reduced pressure at a (e) Use a detection wavelength of 350 nm.
temperature not exceeding 60°. Separately, dissolve the (f) Inject 20 gL of each solution.
Conander Oil in the Ethanol (90 per cent), add the solution
to the evaporated filtrate and add sufficient Purified Water to MOBILE PHASE
produce 1000 mL. Allow to stand for not less than 24 hours; 17 volumes of acetonitrile and 83 volumes of a 1% v/v
filter. solution of glacial acetic acid.
The extract complies with the requirements stated under Extracts DETERMINATION OF CONTENT
and with the following requirements. Calculate the total content of sennosides A and B as
TESTS sennoside B in the granules using the declared content of
Ethanol content sennosides in Alexandrian senna fruit powder BPCRS.
21 to 24% v/v, Appendix VIII F, Method m. STORAGE
Dry residue Standardised Senna Granules should be kept in an airtight
17 to 25% w/v. container.
Relative density LABELLING
1.02 to 1.09, Appendix V G. The quantity of active ingredient is stated in terms of the
equivalent amount of sennoside B.
Standardised Senna Granules Senna Tablets

DEFINITION
Standardised Senna Granules contain Alexandrian Senna DEFINITION
Fruit in powder form with suitable excipients. The granules Senna Tablets contain the powdered pericarp of Senna Fruit,
contain 0.55% พ/พ of sennosides, calculated as sennoside B. Alexandrian or Tinnevelly.
The granules comply with the requirements stated under Granules The tablets comply with the requirements stated under Tablets and
and with the following requirements. with the following requirements.
Content of sennosides calculated as sennoside B Content of total sennosides, calculated as sennoside B
0.467 to 0.633% พ/พ. 85.0 to 115.0% of the stated amount.
IDENTIFICATION IDENTIFICATION
A. To 25 mg, in No. 180 powder, add 50 mL of water and The powdered tablets exhibit diagnostic structures of senna
2 mL of hydrochloric acid, heat in a water bath for pericarp. External epidermis of isodiametric cells with very
15 minutes, allow to cool and shake with 40 mL of ether. thick outer walls. Occasional stomata. Trichomes few,
Dry the ether layer over anhydrous sodium sulfate, filter, unicellular, conical and warty. Parenchymatous cells from
evaporate 5 mL of the filtrate to dryness, cool and add 5 mL inner part of a two-to five-layered zone subjacent to the
of 6m ammonia to the residue; a yellow or orange colour is epidermis, each containing a single prism of calcium oxalate.
produced. Heat on a water bath for 2 minutes; a reddish Thick-walled fibres in two to four layers, the fibres of the
violet colour is produced. outer and inner zones respectively with their long axes at
right angles to each other. Sutural vascular strands sheathed
B. In the Assay, the chromatogram obtained with solution by cells containing prisms of calcium oxalate; elements of
(1) exhibits two peaks corresponding to the peaks due to seed tissue may also be present.
sennoside A and sennoside B in the chromatogram obtained
with solution (2). TESTS
Loss on drying Disintegration
Maximum time, 60 minutes, Appendix XU A.
When dried at 105° for 5 hours, lose not more than 2.0% of
their weight. Use 5 g. ASSAY
ASSAY Weigh and powder 20 tablets. Carry out the following
procedure protected from light. To a quantity of the powder
containing the equivalent of 7.5 mg of total sennosides add
Appendix m D, using the following solutions.
30 mL of water, weigh, heat under a reflux condenser on a
(1) Shake 2 g of the granules with 50 mL of a 0.3% v/v water bath for 15 minutes, allow to cool, weigh and restore
solution of acetic acid adjusted to pH 5.9 with Im sodium the original weight with water. Centrifuge, transfer 20 mL of
hydroxide for 30 minutes, centrifuge, filter through a glass the supernatant liquid to a separating funnel, add 0.1 mL of
fibre paper (Whatman GF/C is suitable) and use the filtrate. 2m hydrochloric acid, shake with two 15 mL quantities of
(2) Prepare solution (2) in the same manner as solution (1) chloroform, allow to separate and discard the chloroform
but using a quantity of Alexandrian senna fruit powder BPCRS layers. Add 0.10 g of sodium hydrogen carbonate and shake for
containing the equivalent of 11 mg of sennoside B. 3 minutes; centrifuge and transfer 10 mL of the liquid to a
CHROMATOGRAPHIC CONDITIONS round-bottomed flask fined with a ground-glass neck. Add a
mixture of 8 mL of iron(jti) chloride solution and 12 mL of
water and mix. Heat under a reflux condenser on a water
with end-capped octadecylsilyl silica gel for chromatography
bath for 20 minutes, add 1 mL of hydrochloric acid and
(5 pm) (Spherisorb ODS 2 is suitable).
continue heating for a further 20 minutes, shaking frequently,
(b) Use isocratic elution and the mobile phase described until the precipitate is dissolved. Allow to cool, transfer the
below. mixture to a separating funnel and extract with three 25-mL
(c) Use a flow rate of 2 mL per minute. quantities of ether previously used to rinse the flask. Wash the
(d) Use an ambient column temperature. combined ether extracts with two 15-mL quantities of water
IV-378 Senna Leaf 2016
and add sufficient ether to produce 100 mL. Evaporate sheath of prismatic crystals of calcium oxalate [Fa]; isolated
10 mL just to dryness on a water-bath and dissolve the prisms of calcium oxalate [D]; isolated cluster crystals of
residue in 10 mL of Im potassium hydroxide, filtering if calcium oxalate [H]; fragments of median parenchyma from
necessary' through a sintered-glass filter (ISO 4793, porosity the lamina [C] with some cells containing cluster crystals of
grade 3, is suitable). Measure the absorbance of the resulting calcium oxalate [Ca], often accompanied by palisade
solution without delay at the maximum at 500 nm, parenchyma [Cb] and annular vessels [Cc].
Appendix II B. Calculate the content of total sennosides, as
sennoside B, taking 200 as the value of A(l%, 1 cm) at the
maximum at 500 nm.
LABELLING
The quantity of active ingredient is stated in terms of total
sennosides expressed as the equivalent content of
sennoside B.
Senna Leaf ★ *
Preparation
Standardised Senna Leaf Dry Extract
When Powdered Senna Leaf is prescribed or demanded,
PhEur_______________________________________________________________
DEFINITION
Dried leaflets of Cassia senna L. (syn. Cassia acutifolia Delile),
known as Alexandrian or Khartoum senna, or Cassia
angustifolia Vahl, known as Tinnevelly senna, or a mixture of
the 2 species.
Content
Minimum 2.5 per cent of hydroxyanthracene glycosides,
expressed as sennoside B (042แ38020; Afr 863) (dried drug).
IDENTIFICATION
A. c. senna occurs as greyish-green or brownish-green, thin, Figure 0206.-1. - Illustration for identification test B of powdered
fragile leaflets, lanceolate, mucronate, asymmetrical at the herbal drug of senna leaf
base, usually 15-40 mm long and 5-15 mm wide, the c. Thin-layer chromatography (2.2.27).
maximum width being at a point slightly below the centre; Test solution To 0.5 g of the powdered herbal drug (180)
the lamina is slightly undulant with both surfaces covered (2.9.12) add 5 mL of a mixture of equal volumes of ethanol
with fine, short trichomes. Pinnate venation is visible mainly (96 per cent) R and water R and heat to boiling. Centrifuge
on the lower surface, with lateral veins leaving the midrib at and use the supernatant liquid.
an angle of about 60° and anastomosing to form a ridge near
Reference solution Dissolve 10 mg of senทa extract CRS in
the margin.
1 mL of a mixture of equal volumes of ethanol (96 per cent) R
Stomatal index (2.8.3): 10-12.5-15. and water R (a slight residue remains).
c angustifolia Occurs as yellowish-green or brownish-green Plate TLC silica gel plate R.
leaflets, elongated and lanceolate, slightly asymmetrical at the
Mobile phase glacial acetic acid R, water R, ethyl acetate R,
base, usually 20-50 mm long and 7-20 mm wide at the
propanol R (1:30:40:40 VIVIVIV).
centre. Both surfaces are smooth with a very small number of
short trichomes and are frequently marked with transverse or Application 10 pL as bands of 20 mm.
oblique lines. Development Over a path of 10 cm.
Stomatal index (2.8.3): 14-17.5-20. Drying In air.
B. Microscopic examination (2.8.23). The powder is light Detection: Treat with a 20 per cent VIV solution of nitric
green or greenish-yellow. Examine under a microscope using acid R and heat at 120 °C for 10 min. Allow to cool and
chloral hydrate solution R. The powder shows the following treat with a 50 g/L solution of potassium hydroxide R in
diagnostic characters (Figure 0206.-1): fragments of ethanol (50 per cent VIV) R until the zones appear.
epidermis (C. angustifolia: [A, B], c. senna: [J, K]) with Results The principal zones in the chromatogram obtained
polygonal cells [Aa, Ka], paracytic stomata (2.8.3) [Ab, Ac, with the test solution are similar in position (sennosides B, A,
Ba, Ja, Kb] and unicellular covering trichomes, conical in D and c in the order of increasing Rf value), colour and size
shape, with warty walls (surface view [Ad], side view to the principal zones in the chromatogram obtained with the
(C. senna: [G])), or their scars [Bb, Jb], frequently reference solution; between the zones due to sennosides D
accompanied by palisade parenchyma [Ae, Jc]; isolated, and c a red zone due to rhein-8-glucoside may be visible.
fragmented covering trichomes [E]; fibres [F] with a crystal
2016 Senna Preparations IV-379
D. Place about 25 mg of the powdered herbal drug (180)

(2.9.12) in a conical flask and add 50 mL of water R and Standardised Senna Leaf Dry * *
2 mL of hydrochloric acid R. Heat in a water-bath for 15 min, Extract *****
cool and shake with 40 mL of ether R. Separate the ether (Ph. Eur. monograph 1261)
layer, dry over anhydrous sodium sulfate Ry evaporate 5 mL to
Ph Eur____________________________________________________________
dryness and to the cooled residue add 5 mL of dilute
ammonia Rl. A yellow or orange colour develops. Heat on a DEFINITION
water-bath for 2 min. A reddish-violet colour develops. Standardised dry extract produced from Senna leaf (0206).
TESTS Content
Foreign matter (2.3.2) 5.5 per cent to 8.0 per cent of hydroxyanthracene glycosides,
Maximum 3 per cent of foreign organs and maximum expressed as sennoside B (C42H38O20> Mr 863) (dried
1 per cent of foreign elements. extract). The measured content does not deviate from the
Loss on drying (2.2.32) value stated on the label by more than ± 10 per cent.
Maximum 12.0 per cent, determined on 1.000 g of the PRODUCTION
powdered herbal drug (355) (2.9.12) by drying in an oven at The extract is produced from the herbal drug by a suitable
105 °C for 2 h. procedure using e±anol (50-80 per cent V/V).
Total ash (2.4.16) CHARACTERS
Maximum 12.0 per cent. Appearance
Ash insoluble in hydrochloric acid (2.8.1) Brownish or brown powder.
IDENTIFICATION
ASSAY A. Thin-layer chromatography (2.2.27).
Carry out the assay protected from bright light. Solvent mixture ethanol (96 per cent) R, water R (50:50 V/V).
Place 0.150 g of the powdered herbal drug (180) (2.9.72) in Test solution To 0.1 g of the extract to be examined add
a 100 mL flask. Add 30.0 mL of water R, mix, weigh and 5 mL of the solvent mixture and heat to boiling. Cool and
place in a water-bath. Heat under a reflux condenser for centrifuge. Use the supernatant.
15 min. Allow to cool, weigh and adjust to the original mass Reference solution Dissolve 10 mg of senna extract CRS in
with water R. Centrifuge and transfer 20.0 mL of the 1 mL of the solvent mixture (a slight residue remains).
supernatant liquid to a 150 mL separating funnel.
Add 0.1 mL of dilute hydrochloric acid R and shake with
3 quantities, each of 15 mL, of chloroform R. Allow to Mobile phase glacial acetic acid Ry water Ry ethyl acetate R,
separate and discard the chloroform layer. Add 0.10 g of propanol R (1:30:40:40 V/V/V/V).
sodium hydrogen carbonate R and shake for 3 min. Centrifuge Application 10 pL as bands.
and transfer 10.0 mL of the supernatant liquid to a 100 mL Development Over a path of 10 cm.
round-bottomed flask with a ground-glass neck. Add 20 mL Drying In' air.
of ferric chloride solution Rl and mix. Place the flask in a
Detection Treat with a 20 per cent v/v solution of nitric
water-bath so that the water level is above that of the liquid
acid R and heat at 120 °C for 10 min; allow to cool and treat
in the flask, and heat under a reflux condenser for 20 min.
with a 50 g/L solution of potassium hydroxide R in ethanol
Add 3 mL of hydrochloric acid R and heat for a further
(50 per cent V/V) R until the zones appear.
20 min, with frequent shaking, to dissolve the precipitate.
Cool, transfer the mixture to a separating funnel and shake Results The principal zones in the chromatogram obtained
with 3 quantities, each of 25 mL, of ether R previously used with the test solution are similar in position, colour and size
to rinse the flask. Combine the 3 ether layers and wash with to the principal zones in the chromatogram obtained with the
2 quantities, each of 15 mL, of water R. Transfer the reference solution. The chromatograms show in the lower
combined ether layers to a volumetric flask and dilute to third a prominent brown zone due to sennoside B and above
100.0 mL with ether R. Evaporate 10.0 mL carefully to it a yellow zone followed by another prominent brown zone
dryness and dissolve the residue in 10.0 mL of a 5 g/L due to sennoside A. In the upper half of the chromatograms
solution of magnesium acetate R in methanol R. Measure the are visible, in order of increasing RF value, a prominent
absorbance (2.2.25) at 515 nm using methanol R as the reddish-brown zone and an orange-brown zone followed by a
compensation liquid. faint pink zone and 2 yellow zones. Close to the solvent front
a dark pink zone appears, which may be followed by several
Calculate the percentage content of hydroxyanthracene
faint zones.
glycosides, expressed as sennoside B, using the following
expression: B. Place about 25 mg of the extract to be examined in a
conical flask and add 50 mL of water R and 2 mL of
hydrochloric acid R. Heat in a water-bath for 15 min, cool and
A X 1.25 shake with 40 mL of ether R. Separate the ether layer, dry
over anhydrous sodium sulfate Ry evaporate 5 mL to dryness
and to the cooled residue add 5 mL of dilute ammonia Rl.
i. e. taking the specific absorbance of sennoside B to be 240. A yellow or orange colour develops. Heat on a water-bath for
A = absorbance at 515 nm; 2 min. A reddish-violet colour develops.
m = mass of the substance to be examined, in grams. TESTS
STORAGE Loss on drying (2.8.17)
Protected from moisture. Maximum 5.0 per cent.
______________________________________________________________ _ Ph Eur ASSAY
Carry out the assay protected from bright light.
IV-380 Sesame Seed 2016
Place 0.150 g of the extract to be examined in a 100 mL which have no contents; the remainder of the testa is
flask, disperse in water R and dilute to 100.0 mL with the composed of collapsed cells. Endosperm 3 layered, rarely 2
same solvent. Filter the solution, discarding the first 10 mL layered, consisting of polygonal cells containing fixed oils and
of the filtrate. Transfer 20.0 mL of the filtrate to a 150 mL small aleurone grains. Epidermis of cotyledons, a single layer
separating funnel. Add 0.1 mL of dilute hydrochloric acid R covered with a thin cuticle, with an underlying single layer of
and shake with 3 quantities, each of 15 mL, of ether R. Allow palisade- like cells; endosperm tissue composed of polygonal,
the layers to separate and discard the ether layer. Add 0.10 g parenchyma cells containing fixed oil and aleurone grains.
of sodium hydrogen carbonate R to the aqueous layer and shake Cluster crystals of calcium oxalate and fragments may be
for 3 min. Centrifuge and transfer 10.0 mL of the found scattered in the powder.
supernatant to a 100 mL round-bottomed flask with a c. Carry out the test for the identification of fatty oils by
ground-glass neck. Add 20 mL offerric chloride solution Rl thin-layer chromatography, Appendix X N, using the
and mix. Heat for 20 min under a reflux condenser in a following solutions.
water-bath with the water level above that of the liquid in the
(1) Add 100 mL of dichloromethane to 10.0 g of the
flask; add 3 mL of hydrochloric acid R and heat for a further
powdered herbal drug. Shake for 24 hours on a mechanical
30 min with frequent shaking to dissolve the precipitate.
shaker, filter and evaporate to dryness at 40°. Dissolve 60 mg
Cool, transfer the mixture to a separating funnel and shake
of the oily residue in 9 mL of dichloromethane.
with 3 quantities, each of 25 mL, of ether R previously used
to rinse the flask. Combine the ether layers and wash with (2) 0.67% w/v of sesame oil in dichloromethane.
2 quantities, each of 15 mL, of water R. Transfer the ether CHROMATOGRAPHIC CONDITIONS
layers to a volumetric flask and dilute to 100.0 mL with (a) Use as the coating octadecylsilyl silica gel for HPTLC
ether R. Evaporate 10.0 mL carefully to dryness and dissolve (Merck silica gel 60 RP-18 HPTLC plates are suitable).
the residue in 10.0 mL of a 5.0 g/L solution of magnesium
(b) Use mobile phase A and mobile B described below.
acetate R in methanol R. Measure the absorbance (2.2.25) at
515 nm using methanol R as the compensation liquid. (c) Apply 1 |1L of each solution as 6 mm bands.
Calculate the percentage content of hydroxyanthracene (d) Develop the plate twice to 0.5 cm in mobile phase A,
glycosides expressed as sennoside B using the following drying the plate after each application. Develop the plate a
expression: further two times to 8 cm in mobile phase B, drying the plate
after each application.
i.e. taking the specific absorbance of sennoside B to be 240.
(e) After removal of the plate, dry in air and spray with a
A X 4.167 10% w/v solution of
ethanolic phosphomolybdic acid solution, heat at 120° for
3 minutes and examine in daylight.
A = absorbance at 515 nm; MOBILE PHASE
m = mass of the extract to be examined, in grams. Mobile phase A ether
LABELLING Mobile phase B 20 volumes of dichloromethane, 40 volumes
The label states the content of hydroxyanthracene glycosides. of glacial acetic acid and 50 volumes of acetone.
_______________________________________________________________ Ph Eur SYSTEM SUITABILITY
solution (2) shows 4 clearly separated bands.
Sesame Seed CONFIRMATION
DEFINITION The chromatogram obtained with solution (1) shows 4 grey

bands with varying intensities. The 4 principal bands are
Sesame seeds are the dried seeds of Sesamum indicum L.
similar in position, colour and size to the bands obtained
IDENTIFICATION with solution (2). Other bands may be present.
A. The seed is a flattened pyriform shape. Size, testa colour
TESTS
and surface texture vary according to cultivar: sizes range
from 1.5 to 4 mm long, 1 to 2 mm wide and 0.5 to 1 mm
Loss on drying
When dried for 2 hours at 100° to 105°, loses not more than
thick; the dull testa may be white, cream, yellow, grey,
5.0% of its weight, Appendix IX D. Use 1 g.
brown, red or black; medicinal cultivars from China and
India are predominantly yellow, dark brown or black; Total Ash
and surface texture ranges from smooth to finely reticulate or Not more than 9%, Appendix XI J, Method II.
rugose. The wider end is rounded and the narrow end tapers
to a short point with a punctiform white or brown hilum.
When viewed with a hand lens the seed margin is smooth or
narrowly 1- or 2-ridged throughout all or part of its length,
and a longitudinal line may be visible on one of the flat
Sophora Flower ★ :
surfaces, notably in those cultivars with a pale coloured testa. (Ph. Eur. monograph 2639) *
Inside the thin testa, a narrow whitish endosperm and an
Ph Eur------------------------------------------------------------------------ --------------------------------
embryo comprised of a two large, yellowish and oily
cotyledons are present. DEFINITION
B. Reduce to a powder. The powder varies in colour from Dried, opened flower of Styphnolobium japonicum (L.) Schott
yellow to brown or blackish. Epidermis of the testa, a single (syn. Sophora japonica L.).
layer of palisade-like cells thin-walled, with yellowish Content
coloured contents and an embedded cluster crystal of — minimum 8.0 per cent of total flavonoids, expressed as
calcium oxalate in each cell, except in those of the ridges rutin (บ27พ30016; Mt 611) (dried drug);
2016 Sophora Flower IV-381
— minimum 6.0 per cent of rutin (C27H3o016; M, 611)

(dried drug).
IDENTIFICATION
A. The opened flower is crumpled, rolled, and has a very
thin and short pedicel. The dark green or brown,
campanulate calyx is about 3-4 mm long and consists of
วิ fused sepals with longitudinal striations at the base, divided
31 the apex into 5 slightly bilabiate lobes. The pale yellow or
light yellowish-brown, papilionaceous type corolla is often
broken and measures about 10-15 mm; the upper petal is the
largest, subrounded, with a reflexed apex and a bright yellow
unguis at its internal base. The other 4 petals are oblong.
There are 10 free stamens surrounding a cylindrical and
curved central style.
B. Microscopic examination (2.&2S). The powder is
characters (Figure 2639.-1): roundish [La] or triangular [Lb]
pollen grains [L] with 3 pores and a smooth exine, about
18 pm in diameter; isolated covering trichomes [A, B, F, O]
of varying lengths (60-660 pm), slightly flexed, usually
consisting of 1 or 2 basal cells and a long pointed distal cell,
with smooth or slightly warty walls; fragments of sepals [C]
composed of anomocytic stomata (2.8.3) with 4-8 subsidiary
cells [Ca], covering trichomes [Cb] or their scars [Cc];
fragments of petals [G, H] with cells covered by a finely
striated cuticle [Ga, Ha], sometimes accompanied by fine
annular or spiral vessels [Hb] and parenchyma with some
cells containing crystalline masses of rutin [He]; fragments of
parenchyma [E] from the sepals containing prisms of calcium Figure 2639.-1. - Illustration for identification test B of powdered
oxalate [Ea] and crystalline masses of rutin [Eb]; fragments herbal drug of sophora flower
of anthers [N] showing the characteristic fibrous layer, in
transverse section [Na] or in surface view [K], and immature Top of the plate
pollen grains [Nb]; free prisms of calcium oxalate [M].
An orange-yellow zone
Examine under a microscope using chloral hydrate solution R,
without heating the preparation: brownish-yellow rutin
crystals are visible, free or included in cells, as crystalline
A brown zone
masses [Eb, He, J] or in fan-shaped aggregates of very fine
needles [D, Ec, Gb]. Hyperoside: a yellowish-orange
zone
2 green zones
(2.9.72) add 5.0 mL of methanol R, sonicate for 10 min and
filter. Rutin: an orange-yellow zone A very intense orange-yellow
zone (rutin)
Reference solution Dissolve 10 mg of hyperoside R and 10 mg
of ทItin R in 10 mL of methanol R.
plate R (2-10 pm)]. TESTS
Mobile phase anhydrous formic acid R> water R, ethyl acetate R Foreign matter (2.8.2)
(10:10:80 VIVIV). Maximum 5 per cent of flower buds and maximum
Application 10 pL [or 5 pL] as bands of 10 mm [or 8 mm]. 2 per cent of o±er foreign matter.
Development Over a path of 10 cm [or 6 cm]. Loss on drying (2.2.32)
Drying In air. Maximum 11.0 per cent, determined on 1.000 g of the
105 °C for 2 h.
solution of macrogol 400 R in methanol R) allow to dry in air Total ash (2.4.16)
for about 30 min, and examine in ultraviolet light at 365 nm. Maximum 9.0 per cent.
Results See below the sequence of fluorescent zones present ASSAY
in the chromatograms obtained with the reference solution Total flavonoids
and the test solution. Furthermore, other faint fluorescent Stock solution Place 2.000 g of the powdered herbal
zones may be present in the chromatogram obtained with the drug (355) (2.9.12) in the cartridge of a continuous-
test solution. extraction apparatus (Soxhlet type). Add 100 mL of
heptane R and heat under a reflux condenser until the
extraction liquid is colourless. Allow to cool and discard the
heptane. Add 90 mL of methanol R and continue the
IV-382 Sophora Flower-Bud 2016
extraction with heating under a reflux condenser until the System suitability: reference solution (b):
extraction liquid is colourless. Allow to cool. Transfer the — resolution: minimum 1.5 between the peaks due to rutin
methanolic solution to a 100 mL volumetric flask. Rinse the and apigenin 7-glucoside.
extraction flask with a few millilitres of methanol R. Combine Calculate the percentage content of rutin using the following
the methanolic solutions and dilute to 100.0 mL with expression:
methanol R. Dilute 10.0 mL of this solution to 100.0 mL
with water R and shake vigorously. Al X m2 X p X 5
Test solution Dilute 10.0 mL of the stock solution to A2 X 7711
100.0 mL with a 20 g/L solution of aluminium chloride R in
methanol R. A1 = area of the peak due to rutin in the chromatogram
Compensation solution Dilute 10.0 mL of the stock solution to obtained with the test solution;
100.0 mL with methanol R. 142 = area of the peak due to rutin in the chromatogram
Measure the absorbance (2.2.25) of the test solution after obtained with reference solution (a);
15 min by comparison with the compensation solution at nil — mass of the herbal drug to be examined used to
425 nm. prepare the test solution, in grams;
Calculate the percentage content of total flavonoids, m2 = mass of naoside trihydrate CRS used to prepare
expressed as rutin, using the following expression: reference solution (a), in grams;
p = assigned percentage content of rutin in naoside
i.e. taking the specific absorbance of rutin to be 370.
trihydrate CRS.
A X 1000 ________ Ph Elf
7ท X 37
A = absorbance of the test solution at 425 nm;

Sophora Flower-Bud * *,
Rutin
Liquid chromatography (2.2.29). (Ph Eur monograph 2427) *
Test solution Place 0.500 g of the powdered herbal drug (355) Ph Eur______________________________________________ ________________
(2.9.72) in a conical flask and add 50.0 mL of methanol R. DEFINITION

Weigh, sonicate for 30 min and allow to cool. Weigh and Whole, dried flower bud of Styphnolobium japonicuni (L.)
compensate for the loss of solvent with methanol R. Shake Schott (syn. Sophora japonica L.).
vigorously, filter, and dilute 2.0 mL of the filtrate to 10.0 mL
Content
with methanol R.
— minimum 20.0 per cent of total flavonoids, expressed as
Reference solution (a) Dissolve 10.0 mg of naoside rutin (C27H30Oi 6; Mr 611) (dried drug);
trihydrate CRS in 2 mL of methanol R and dilute to 10.0 mL — minimum 15.0 per cent of rutin (C27H3oOi6; Mr 611)
with a 50 per cent VIV solution of methanol R. Dilute 2.0 mL (dried drug).
of this solution to 10.0 mL with a 50 per cent VIV solution
of methanol R. IDENTIFICATION
A. The flat flower bud, ovoid or ellipsoid, has a very thin and
Reference solution (b) Dissolve 10.0 mg of apigenin
short pedicel and is about 7-10 mm long and 3-4 mm thick.
7-glucoside R and 10.0 mg of rutin R in 30 mL of methanol R
The dark green or brown calyx, forming the lower part of the
and dilute to 50.0 mL with a 50 per cent VIV solution of
bud, is about 3-4 mm long and consists of 5 fused sepals
methanol R.
with longitudinal striations at the base. The pale yellow or
Column’. brownish-yellow corolla, unopened, delicate, extends beyond
— size’. I = 0.25 m, 0 = 4.6 mm;
the calyx and contains 10 free stamens surrounding a central
— stationary phase’, octadecylsilyl silica gel for chromatography R
style.
(5 gm).
B. Microscopic examination (2.5.25). The powder is pale
Mobile phase’.
yellow. Examine under a microscope using chloral hydrate
— mobile phase A’. 1 per cent VIV solution of glacial acetic
acid Rj
characters (Figure 2427.-1): roundish [La] or triangular [Lb]
— mobile phase B: methanol R’y
pollen grains [L] with 3 pores and a smooth exine, about
18 pm in diameter; isolated covering trichomes [A, B, F, O)
Time Mobile phase A Mobile phase B of varying lengths (60-660 pm), slightly flexed, usually
(min) (per cent V/V) (per cent V/V) consisting of 1 or 2 basal cells and a long pointed distal cell,
0-5 68 32 with smooth or slightly warty walls; fragments of sepals [C]
5 - 20 68 50 32 -> 50 composed of anomocytic stomata (2.5.3) with 4-8 subsidiary
cells [Ca], covering trichomes [Cb] or their scars [Cc];
20 - 30 50 ■> 0 50 -> 100
fragments of petals [G, H] with cells covered by a finely
30 - 35 0 100 striated cuticle [Ga, Ha], sometimes accompanied by fine
annular or spiral vessels [Hb] and parenchyma with some
cells containing crystalline masses of rutin [He]; fragments of
parenchyma [E] from the sepals containing prisms of calcium
Detection Spectrophotometer at 350 nm. oxalate [Ea] and crystalline masses of rutin [Eb]; fragments
Injection 20 pL. of anthers [N] showing the characteristic fibrous layer, in
Relative retention With reference to rutin (retention transverse section [Na] or in surface view [K], and immature
time = about 17 min): apigenin 7-glucoside = about 1.1. pollen grains [Nb]; free prisms of calcium oxalate [M].
2016 Sophora Flower-Bud IV-383
Examine under a microscope using chloral hydrate solution Ry

Top of the plate
Without heating the preparation: brownish-yellow rutin
crystals are visible, free or included in cells, as crystalline An orange-yellow zone
masses [Eb, He, J] or in fan-shaped aggregates of very fine

needles [D, Ec, Gb].
A brown zone
Hyperoside: a yellowish-orange
zone
2 green zones
Rutin: an orange-yellow zone A very intense orange-yellow

zone (rutin)
TESTS
Maximum 5 per cent of opened flowers and maximum
2 per cent of other foreign matter.
105 °C for 2 h
Total ash (2.4.16)
ASSAY
Total flavonoids
Stock solution Place 1.00 g of the powdered herbal drug (355)
(2.9.12) in the cartridge of a continuous-extraction apparatus
(Soxhlet type). Add 100 mL of heptane R and heat under a
reflux condenser until the extraction liquid is colourless.
Allow to cool and discard the heptane. Add 90 mL of
methanol R and continue the extraction with heating under a
reflux condenser until the extraction liquid is colourless.
Allow to cool. Transfer the methanolic solution to a 100 mL
Figure 2427.-1. - Illustration for identification test B of powdered volumetric flask. Rinse the extraction flask with a few
herbal drug of sophora flower-bud millilitres of methanol R. Combine ±e methanolic solutions
and dilute to 100.0 mL with methanol R. Dilute 10.0 mL of
this solution to 100.0 mL with water R and shake vigorously.
Test solution Dilute 10.0 mL of the stock solution to
(2.9.72) add 5.0 mL of methanol Ry sonicate for 10 min and
100.0 mL with a 20 g/L solution of aluminium chloride R in
filter.
methanol R.
Reference solution Dissolve 10 mg of hyperoside R and 10 mg Compensation solution Dilute 10.0 mL of the stock solution to
of rutin R in 10 mL of methanol R.
Plate TLC silica gel plate R (5-40 Jim) [or TLC silica gel Measure the absorbance (2.2.25) of the test solution after
plate R (2-10 Jim)].
15 min by comparison with the compensation solution at
Mobile phase anhydrous formic acid R, water Ry ethyl acetate R 425 nm.
(10:10:80 VIVIV). Calculate the percentage content of total flavonoids,
Application 10 pL [or 5 |1L] as bands of 10 mm [or 8 mm]. expressed as rutin, using the following expression:
Drying In air. A X 1000
Detection Treat with a 10 g/L solution of diphenylboric acid mx37
solution of macrogol 400 R in methanol Ry allow to dry in air i.e. taking the specific absorbance of rutin to be 370.
for about 30 min, and examine in ultraviolet light at 365 nm. A = absorbance of the test solution at 425 nm;
Results See below the sequence of fluorescent zones present m = mass of the herbal drug to be examined, in grams.
in the chromatograms obtained with the reference solution Rutin
and the test solution. Furthermore, other faint fluorescent Liquid chromatography (2.2.29).
zones may be present in the chromatogram obtained with the Test solution Place 0.200 g of the powdered herbal drug (355)
test solution. (2.9.12) in a conical flask and add 50.0 mL of methanol R.
Weigh, sonicate for 30 min and allow to cool. Weigh and
compensate for the loss of solvent with methanol R. Shake
vigorously, filter, and dilute 2.0 mL of the filtrate to 10.0 mL
with methanol R.
IV-384 Spearmint Oil 2016
Reference solution (a) Dissolve 10.0 mg of rutoside IDENTIFICATION

trihydrate CRS in 2 mL of methanol R and dilute to 10.0 mL Examine the chromatograms obtained in the test for
with a 50 per cent V/V solution of methanol R. Dilute 2.0 mL Chromatographic profile. The retention times of the principal
of this solution to 10.0 mL with a 50 per cent V/V solution peaks in the chromatogram obtained with solution (1) are
of methanol R. similar to those of the principal peaks in the chromatogram
Reference solution (b) Dissolve 10.0 mg of apigenin obtained with solution (2).
7-glucoside R and 10.0 mg of rutin R in 30 mL of methanol R TESTS
and dilute to 50.0 mL with a 50 per cent V/V solution of
Optical rotation
methanol R. American-type oil, -45° to -60°; Chinese-type oil, -50°
Column'. to -62°; Appendix V F.
— size. I = 0.25 m, 0 = 4.6 mm;
Refractive index
— stationary phase, octadecylsilyl silica gel for chromatography R
(5 ^m)-
Mobile phase. Solubility in ethanol
— mobile phase A’. 1 percent V/V solution of glacial acetic Soluble, at 20°, in 1 part of ethanol (80%), Appendix X M.
acid R; The solution may become cloudy when diluted.
— mobile phase B: methanol R; Weight per mL
American-type oil, 0.917 to 0.934 g; Chinese-type oil,
Mobile phase A Mobile phase B 0.935 to 0.952 g, Appendix V G.
0-5 68 32
Carry’ out the method for gas chromatography,
5 - 20 68 -» 50 32 -> 50 Appendix III B, using the following solutions. Solution (1) is
20 - 30 50 -> 0 50 -> 100 the substance being examined. For solution (2) mix carefully
0.1 g of limonene, 0.2 g of cineole, 0.4 g of menthone, 0.1 g of
30 - 35 0 100
(+)-isomenthone, 0.4 g of menthyl acetate, 0.2 g of pulegone,
0.6 g of menthol and 0.1 g of carvone with 1 g of hexane.
Flow rate 1.3 mL/min. The chromatographic procedure may be carried out using
Detection Spectrophotometer at 350 nm. (a) a glass capillary column (25 m to 60 m X about
Injection 20 pL. 0.25 mm) coated with polyethylene glycol 20,000 as bonded
phase (Carbowax 20M is suitable) and (b) helium as the
Relative retention With reference to rutin (retention
carrier gas at a flow rate of 1.5 mL per minute. Maintain the
time = about 17 min): apigenin 7-glucoside = about 1.1.
temperature of the column at 55° for 6 minutes then increase
System suitability, reference solution (b): it at the rate of 4° per minute to 180c; keep the injection port
— resolution', minimum 1.5 between the peaks due to rutin temperature at 220° and the detector at 230°.
and apigenin 7-glucoside.
Inject 0.1 pL of solution (2). When the chromatograms are
Calculate the percentage content of rutin using the following recorded in the prescribed conditions, the components elute
expression: in the order indicated in the composition of the reference
solution. Record the retention times of these substances.
Al X 7ท2 X p X 5 The test is not valid unless the number of theoretical plates
A2 X mi calculated from the limonene peak is at least 30,000 and the
resolution factor between the peaks corresponding to limonene
/4] = area of the peak due to rutin in the chromatogram and cineole is at least 1.5.
obtained with the test solution; Inject 0.1 pL of solution (1). Using the retention times
A2 = area of the peak due to rutin in the chromatogram determined from ±e chromatogram obtained with solution
obtained with reference solution (a); (2) locate the components of the reference solution on the
พ1 = mass of the herbal drug to be examined used to chromatogram obtained with solution (1) (disregard the peak
prepare the test solution, in grams; due to hexane). Determine the percentage content of the
m2 = mass of rutoside trihydrate CRS used to prepare components by normalisation. The percentages are within the
reference solution (a), in grams; following ranges:
p = assigned percentage content of rutin in rutoside
Limonene 2.0 to 25.0%.
trihydrate CRS.
Cineole less than 2.5%.
________________________________________________________________ Ph Eur
Menthone less than 2.5%.
Isomenthone less than 1.0%.
Menthyl acetate less than 1.0%.
Pulegone less than 0.5%.
Spearmint Oil
Menthol less than 2.0%.
DEFINITION
Carvone Not less than 55.0%.
Spearmint Oil is obtained by distillation from fresh flowering
plants of Mentha spicata L. and Mentha X cardiaca (Gray) STORAGE
Bak. Spearmint Oil should be kept in a well-filled container and
protected from light.
CHARACTERISTICS
A clear, colourless, pale yellow or greenish yellow liquid LABELLING
when recently distilled, but becoming darker and viscous on The label states whether the oil is American-type oil or
keeping; visibly free from water; odour, that of spearmint. Chinese-type oil.
2016 Squill Preparations IV-385
Squill CHARACTERISTICS
Odourless or almost odourless.
Preparations
Squill Liquid Extract Macroscopical
Curved or irregularly shaped strips, about 10 to 50 mm long,
Squill Oxymel
3 to 10 mm wide and 1 to 3 mm thick, frequently tapering
When Powdered Squill is prescribed or demanded, material towards the ends, occasionally grouped three or four together
complying with the appropriate requirements below shall be and attached to a portion of the axis; ridged in the direction
dispensed or supplied. of their length and varying in colour from pale yellowish
DEFINITION brown to buff; brittle when dry, but tough and flexible when
Squill consists of the bulb of Drimia maritima (L.) Steam, exposed to air.
collected soon after the plant has flowered, divested of its Microscopical
dry, outer, membranous coats, cut into transverse slices and Epidermis: cells tetrahedral to hexahedral, thin-walled, three
dried. It is known in commerce as white squill. to five times longer than wide, having a thick, striated cuticle;
IDENTIFICATION stomata rare, anomocytic, Appendix XI H, circular in outline,
40 to 42 pm in diameter; mesophyll of thin-walled polygonal
A. Transverse slices, about 5 to 8 mm thick, occurring as
cells containing mucilage, some cells also containing bundles
straight or curved triangular pieces about 5 to 50 mm long
of acicular crystals of calcium oxalate, 20 to 900 pm in
and 3 to 8 mm wide at mid-point, tapering towards each
length; vascular bundles collateral, scattered throughout the
end, yellowish white, texture homy, somewhat translucent,
mesophyll; xylem vessels with spiral and annular wall
breaking with an almost glassy fracture when quite dry, but
thickening; trichomes and starch absent.
readily absorbing moisture when exposed to the air and
becoming tough and flexible; transversely cut surface showing IDENTIFICATION
a single row of prominent, vascular bundles near the concave The mucilage contained in the cells of the mesophyll is
edge and numerous smaller bundles scattered throughout the stained red with alkaline corallin solution and reddish purple
mesophyll. with 0.01m iodine.
B. Epidermis: cells polygonal and axially elongated, 1 to TESTS
2 times longer than wide, cuticle thick, stratified; stomata Ash
very' rare, anomocytic, Appendix XI H, and nearly circular in Not more than 6.0%, Appendix XI J.
outline, about 50 to 60 pm in diameter; mesophyll of
colourless, thin-walled parenchyma containing very STORAGE
occasional starch granules, many cells containing bundles of Indian Squill should be stored in a dry7 place.
acicular crystals of calcium oxalate embedded in mucilage,
crystals up to about 1 mm long and about 1 to 15 pm wide;
other cells containing sinistrin; vascular bundles collateral,
scattered throughout the mesophyll; xylem vessels with spiral
and annular wall thickening; trichomes absent.
Squill Liquid Extract
c. The mucilage contained in the cells of the mesophyll is DEFINITION
stained red with alkaline corallin solution but produces no red Squill Liquid Extract is prepared by extracting Squill with
colour with ruthenium red solution and no purple colour with Ethanol (70 per cent).
0.01m iodine. Extemporaneous preparation
TESTS The following formula and directions apply.
Squill, in coarse powder 1000 g
Acid-insoluble ash
Ethanol (70 per cent) A sufficient quantity
Not more than 1.5%, Appendix XI K, Method I.
Exhaust the Squill, in coarse powder, with Ethanol
Extractive soluble in ethanol (60%)
(70 per cent) by percolation, Appendix XI F. Reserve the first
Not less than 68.0%, Appendix XI Bl. Use material that has
850 mL of the percolate; evaporate the subsequent percolate
been dried for 1 hour at 105° and powdered.
to the consistence of a soft extract and dissolve it in the
STORAGE reserved portion. Add sufficient Ethanol (70 per cent) to
Squill should be stored in a dry place. produce 1000 mL and filter.
The extract complies with the requirements stated under Extracts
TESTS
Indian Squill Ethanol content
Preparation 34 to 50% v/v, Appendix vni F, Method m.
Squill Oxymel Dry residue
When Powdered Indian Squill is prescribed or demanded, 40 to 55% w/v.
material complying with the appropriate requirements below Relative density
shall be dispensed or supplied. 1.00 to 1.14, Appendix V G.
DEFINITION
Indian Squill consists of the bulb of Drimia indica (Roxb.) J
p Jessop, collected soon after the plant has flowered, divested
of dry, outer membranous coats and usually cut
longitudinally into slices and dried.
FV-386 Squill Preparations 2016
Squill Oxymel a mixture of equal volumes of ethanol (96%) and water.

Evaporate the combined extracts, extract the residue with
DEFINITION 10 mL of calcium hydroxide solution, filter and wash the filter
Squill, bruised or Indian 50 g with 10 mL of calcium hydroxide solution. To the combined
Squill, bruised filtrate and washings add 0.1 g of ammonium sulfate, extract
Acetic Acid (33 per cent) 90 mL or a sufficient quantity with two 10 mL quantities of ethanol-free chloroform, wash the
Purified Water, freshly 250 mL combined extracts with 10 mL of water and discard the
boiled and cooled chloroform solution. To the combined alkaline liquid and
Purified Honey A sufficient quantity aqueous washings add 10 mL of 1M hydrochloric acid, heat on
Extemporaneous preparation a water bath to remove any chloroform, cool and dilute to
The following directions apply. 100 mL with water. To 20 mL of this solution add 8 mL of a
freshly prepared 1.0% w/v solution of sodium nitrite, allow to
Macerate the Squill or the Indian Squill with the Acetic Acid
stand for 15 minutes, add 12 mL of 5m ammonia, dilute to
(33 per cent) and the Purified Water for 7 days with
50 mL with water and measure the absorbance of a 4-cm layer
occasional agitation, strain, press out the liquid, heat the
of the resulting solution at the maximum at 442 nm,
mixed liquids to boiling, filter whilst hot, cool, determine the
Appendix n B, using in the reference cell a solution prepared
content of acetic acid, add sufficient Acetic Acid
in the same manner and at the same time but using 8 mL of
(33 per cent) to the remainder of the filtrate to produce a
water in place of the solution of sodium nitrite. Calculate the
solution containing about 8.5% w/v of acetic acid and mix.
content of C17H19NO3 from a calibration curve prepared
To every three volumes of the resulting solution add seven
using quantities of 2, 4, 6 and 8 mL of a 0.008% w/v
volumes of Purified Honey and mix thoroughly.
solution of anhydrous morphine in 0.1m hydrochloric acid, each
Content of acetic acid, C2H4O2 diluted to 20 mL with 0.1m hydrochloric acid and using the
2.2 to 2.7% w/v. method described above beginning at the words ‘add 8 mL
TESTS ...’. Determine the weight per mL of the linctus,
Optical rotation Appendix V G, and calculate ±c content of C17H19NO3,
4-0.6° to -3.0°, Appendix V F, when measured in a 25% w/v weight in volume.
solution in water decolourised, if necessary, with activated
charcoal.
Weight per mL
1.260 to 1.270 g, Appendix V G. Paediatric Opiate Squill Linctus
ASSAY Opiate Linctus for Infants; Paediatric Opiate Squill Oral
Dilute 20 mL with 20 mL of carbon dioxide-free water and Solution
titrate wi± Im sodium hydroxide ES using phenolphthalein DEFINITION
solution Rl as indicator. Each mL of Im sodium hydroxide KS
Paediatric opiate Squill Linctus is an oral solution containing
is equivalent to 60.05 mg of C2H4O2. 6% v/v each of Squill Oxymel and Camphorated opium
Tincture in a suitable vehicle with a tolu flavour.
The following formula applies.
Opiate Squill Linctus Squill Oxymel 60 mL
Compound Squill Linctus; Gee’s Linctus; Opiate Squill Oral Camphorated Opium Tincture 60 mL
Solution Tolu Syrup 60 mL
DEFINITION Glycerol 200 mL
Opiate Squill Linctus is an opalescent oral solution containing Syrup Sufficient to produce 1000
33% v/v each of Squill Oxymel and Camphorated Opium mL
Tincture in a suitable vehicle with a tolu flavour. The linctus complies with the requirements stated under Oral
Extemporaneous preparation Liquids and with the following requirements.
The following formula applies. Content of anhydrous morphine, C17H19NO3
Squill Oxymel 300mL 0.0024 to 0.0036% w/v.
Camphorated Opium Tincture 300mL
ASSAY
Tolu Syrup 300mL
To 32 g add 5 mL of water and 1 mL of 5m ammonia and
The linctus complies with the requirements stated under Oral extract with 30 mL of a mixture of equal volumes of ethanol
Liquids and with the following requirements. (96%) and chloroform and then with two 22.5-mL quantities
Content of anhydrous morphine, C17H19NO3 of a mixture of 2 volumes of chloroform and 1 volume of
0.013 to 0.020% w/v. ethanol (96%), washing each extract with the same 20 mL of
a mixture of equal volumes of ethanol (96%) and water.
TESTS
Evaporate the combined extracts, extract the residue with
Ethanol content 10 mL of calcium hydroxide solution, filter and wash the filter
18.0 to 22.0% v/v, Appendix vm F. with 10 mL of calcium hydroxide solution. To the combined
ASSAY filtrate and washings add 0.1 g of ammonium sulfate, extract
To 12 g add 5 mL of water and 1 mL of 5m ammonia and with two 10 mL quantities of ethanol-free chloroform, wash the
extract with 30 mL of a mixture of equal volumes of ethanol combined extracts with 10 mL of water and discard the
(96%) and chloroform and then with two 22.5-mL quantities chloroform solution. To the combined alkaline liquid and
of a mixture of 2 volumes of chloroform and 1 volume of aqueous washings add 5 mL of Im hydrochloric acid, heat on
ethanol (96%), washing each extract with the same 20 mL of a water bath to remove any chloroform, cool and dilute to
2016 St. John’s Wort IV-387
50 mL with water. To 20 mL of this solution add 8 mL of a [Ea] associated with palisade parenchyma [Eb] and small
freshly prepared 1.0% w/v solution of sodium nitrite, allow to vessels [Ec]; elongated cells of fragments of the petal
stand for 15 minutes, add 12 mL of 5m ammonia, dilute to epidermis with straight or wavy anticlinal walls [J];
50 mL with water and measure the absorbance of a 4-cm layer vessels [D] with reticulate or pitted walls [Da] and groups of
of the resulting solution at the maximum at 442 nm, thick-walled fibres [Db]; fragments of the central parenchyma
Appendix II B, using in the reference cell a solution prepared of the stems [K] with lignified and pitted rectangular
in the same manner and at the same time but using 8 mL of cells [Ka] sometimes associated with vessels [Kb]; fragments
water in place of the solution of sodium nitrite. Calculate the of the anthers [F] showing the central part consisting of small
content of c 17H19NO3 from a calibration curve prepared cells containing cluster crystals of calcium oxalate [Fb] and
using quantities of 2, 4, 6 and 8 mL of a 0.008% w/v cells from the fibrous layer [Fa]; fragments of the staminal
solution of anhydrous morphine in 0.1m hydrochloric acid, each filament with elongated, thin-walled cells with a striated
diluted to 20 mL with 0.1m hydrochloric acid and using the cuticle [C]; numerous pollen grains with 3 germinal pores
method described above beginning at the words ‘add 8 mL and a smooth exine, occurring singly [G] or in dense groups.
Determine the weight per mL of the linctus,
Appendix V G, and calculate the content of C17H19NO3,
weight in volume.
St. John’s Wort ** **

Hypericum ***
Preparation
St. John’s Wort Dry Extract, Quantified
Ph Elf___________
DEFINITION
Whole or fragmented, dried flowering tops of Hypericum
perforatum L., harvested during flowering time.
Content
Minimum 0.08 per cent of total hypericins, expressed as
hypericin (C30H,608; Mr 504.4) (dried drug).
IDENTIFICATION
A. The branched and bare stem shows 2 more or less
prominent longitudinal ridges. The leaves are opposite,
sessile, exstipulate, oblong-oval and 15-30 mm long; present
on the leaf margins are glands which appear as black dots
and over all the surface of the leaves many small, strongly
translucent excretory glands which are visible in transmitted
light. The flowers are regular and form corymbose clusters at
the apex of the stem. They have 5 green, acute sepals, with
black secretory glands on the margins; 5 orange-yellow
petals, also with black secretory glands on the margins;
herbal drug of St. John’s wort
3 staminal blades, each divided into many orange-yellow
stamens and 3 carpels surmounted by red styles. c. Thin-layer chromatography (2.2.27).
The drug may also show the following: immature and ripe Test solution Stir 0.5 g of the powdered herbal drug (500)
fruits and seeds. Immature fruits are green or yellowish, seeds (2.9.12) in 10 mL of methanol R in a water-bath at 60 °C for
are whitish. Occasional ripe fruits may be present; these are 10 min and filter.
dry trilocular capsules containing numerous seeds, brown, Reference solution Dissolve 5 mg of hyperoside R and 5 mg of
broad or small-ovate, 5-10 mm long, with broad linear or rutin R in methanol R, then dilute to 5 mL with the same
punctiform glands, irregularly striated ducts, conducting solvent.
secretions. Ripe seeds are 1-1.3 mm long, cylindrical or Plate TLC silica gel plate R.
trigonous, shortly pointed at both ends, brown or almost Mobile phase anhydrous formic acid R, water R, ethyl acetate R
black, minutely pitted longitudinally. (6:9:90 VIVIV).
B. Microscopic examination (2.8.23). The powder is Application 10 pL of the test solution and 5 pL of the
greenish-yellow. Examine under a microscope using chloral reference solution, as bands of 10 mm.
characters (Figure 1438.-1): fragments of the leaf epidermis
[A, B] or stems [H] with paracytic [Ab, Ha], anisocytic [Ac, Drying At 100-105 °C for 10 min.
Bb, Hb] or anomocytic [Ae] stomata (2.8.3)’, fragments of Detection Treat with a 10 g/L solution of diphenylboric acid
the leaf epidermis often accompanied by palisade aminoethyl ester R in methanol R and then with a 50 g/L
parenchyma [Ad, Be]; polygonal cells of the upper epidermis solution of macrogol 400 R in methanol R. After about 30 min,
with thickened and beaded walls [Ba]; more or less sinuous, examine in ultraviolet light at 365 nm.
thin-walled cells of the lower epidermis [Aa]; fragments of Results The chromatogram obtained with the reference
the leaf and sepal [E] with large, red-pigmented oil glands solution shows in the lower third a zone due to rutin and
IV-388 St. John’s Wort Preparations 2016
above it a zone due to hyperoside, both with yellow-orange — flavonoids, expressed as rutin (บ27บ30016; A4r 610.5):
fluorescence. The chromatogram obtained with the test minimum 6.0 per cent (anhydrous extract);
solution shows in the lower third 2 reddish-orange — hyperforin (C35H52O4; MT 536.8): maximum 6.0 per cent
fluorescent zones due to rutin and hyperoside, and in the (anhydrous extract) and not more than the content stated
lower part of the upper third a zone due to pseudohypericin on the label.
and above it a zone due to hypericin, both with red
PRODUCTION
fluorescence. o±er yellow or blue fluorescent zones are
The extract is produced from the herbal drug by a suitable
visible.
procedure using ethanol (50-80 per cent VIV) or methanol
TESTS (50-80 per cent VIV).
CHARACTERS
Appearance
Brownish-grey powder.
Maximum 10.0 per cent, determined on 1.000 g of the IDENTIFICATION
powdered herbal drug (500) (2.9.12) by drying in an oven at Thin-layer chromatography (2.2.27).
105 °C for 2 h. Test solution Disperse 0.25 g of the extract to be examined in
Total ash (2.4.16) 5 mL of methanol R.
Maximum 7.0 per cent. Reference solution Dissolve 5 mg of rutin R and 5 mg of
hyperoside R in methanol R and dilute to 10 mL with the same
ASSAY
solvent.
Test solution In a 100 mL round-bottomed flask, introduce
0.800 g of the powdered herbal drug (500) (2.9.12) 3 60 mL
plate R (2-10 pm)].
of a mixture of 20 volumes of water R and 80 volumes of
tetrahydrofuran R and a magnetic stirrer. Boil the mixture in a Mobile phase anhydrous formic acid R, water R, ethyl acetate R
water-bath at 70 °C under a reflux condenser for 30 min. (6:9:90 VIV/V).
Centrifuge (2 min at 700 g) and decant the supernatant into Application 10 pL [or 5 pL] as bands of 10 mm [or 8 mm].
a 250 mL flask. Take up the residue with 60 mL of a Development Over a path of 10 cm [or 7.5 cm].
mixture of 20 volumes of water R and 80 volumes of Drying At 100-105 °C for 10 min.
tetrahydrofuran R. Heat again under a reflux condenser for
30 min. Centrifuge (2 min at 700 g) and decant ±e
supernatant. Combine the extracts and evaporate to dryness.
solution of macrogol 400 R in methanol R. Examine after
Take up the residue with 15 mL of methanol R with the help
about 30 min in ultraviolet light at 365 nm.
of ultrasound and transfer to a 25 mL measuring flask. Rinse
the 250 mL flask with methanol R and dilute to 25.0 mL with Results Sec below the sequence of zones present in the
the same solvent. Centrifuge again, filter 10 mL through a chromatograms obtained with the reference solution and the
syringe filter (0.2 pm). Discard the first 2 millilitres of the test solution. Furthermore, other fluorescent zones may be
filtrate. Introduce 5.0 mL of the filtrate into a measuring present in the chromatogram obtained with the test solution.
flask and dilute to 25.0 mL with methanol R.
Compensation liquid methanol R. Top of the plate
Measure the absorbance (2.2.25) at 590 nm of the test A yellowish-orange fluorescent

solution, by comparison with the compensation liquid.
2 red fluorescent zones (hypericin
Calculate the percentage content of total hypericins, and pseudohypericin)
expressed as hypericin, using the following expression:
A X 125
m X 870 3 yellowish-orange fluorescent
i.e. taking the specific absorbance of hypericin to be 870.

Hyperoside: a yellowish-orange A yellowish-orange fluorescent
_______________________________________________________________ Ph Eur fluorescent zone zone (hyperoside)
Yellow and blue possibly
superimposed fluorescent zones
Rutin: a yellowish-orange A yellowish-orange fluorescent
fluorescent zone zone (rutin)
Quantified St. John’s Wort Dry * * Reference solution Test solation
Extract *****
(Ph. Eur. monograph 1874) TESTS
Ph Eur_______________________________________________________________ Water (2.5.12)
DEFINITION Maximum 4.0 per cent, determined on 0.5 g.
Quantified dry extract obtained from St. John's wort (1438). ASSAY
Content Total hypericins
— total hypericinsJ expressed as hypericin (C30H16O8; Afr Liquid chromatography (2.2.29).
504.5): 0.10 per cent to 0.30 per cent (anhydrous Test solution Dissolve 70.0 mg of the extract to be examined
extract); in 25.0 mL of methanol R. Sonicate and centrifuge the
2016 St. John’s Wort Preparations IV-389
solution. Expose the solution to a xenon lamp at about Mobile phase:

765 w/m2 for 8 min. — mobile phase A: phosphoric acid Ry water R (3:1000 VIV);
Reference solution Dissolve a quantity of St. John's wort dry — mobile phase B: phosphoric acid Ry acetonitrile R
extract HRS corresponding to 0.15 mg of hypericin in (3:1000 V/V);
25.0 mL of methanol R. Sonicate and centrifuge. Expose the
solution to a xenon lamp at about 765 w/m2 for 8 mm. Time Mobile phase A Mobile phase B Flow rate
Column: (per cent V/V) (per cent V/V) (mL/min)
size: l ะะะ 0.15 m, 0 = 4.6 mm; 0-8 82 18 0.8
stationary phase: octadecylsilyl silica gel for chromatography R
8 - 18 82-» 47 18-» 53 0.8
(5 urn);
— temperature: 40 °C. 18 - 18.1 47-» 3 53 -» 97 0.8
Mobile phase Mix 39 volumes of ethyl acetate Ry 41 volumes 18.1 - 19 3 97 0.8 -> 1.2
of a 15.6 g/L solution of sodium dihydrogen phosphate R 19-31 3 97 1.2
adjusted to pH 2 with phosphoric acid R and 160 volumes of
methanol R.
Flow rate 1.0 mL/min. Detection Spectrophotometer at 360 nm, then at 275 nm after
Detection Spectrophotometer at 590 nm. the elution of biapigenin (about 22 min).
Injection 20 pL. Injection 10 pL.
Run time 15 min. Identification of peaks Use the chromatogram supplied with
Sr. John's wort dry extract HRS and the chromatogram
Identification of peaks Use the chromatogram supplied with obtained with reference solution (b) to identify the peaks due
•S7. John's wort dry extract HRS and the chromatogram to rutin, hyperoside, isoquercitroside, quercitroside,
obtained with the reference solution to identify the peaks quercetin, biapigenin, hyperforin and adhyperforin.
due to pseudohypericin and hypericin.
System suitability: reference solution (๖):
รุ)’stem suitability: reference solution:
— the chromatogram obtained is similar to the chromatogram supplied with St. John's wort dry
chromatogram supplied with St. John's wort dry extract HRS;
extract HRS;
— resolution: minimum 2.0 between the peaks due to rutin
— resolution: minimum 2 between the peaks due to and hyperoside, and minimum 2.0 between the peaks due
pseudohypericin and hypericin. to hyperforin and adhyperforin.
Calculate the percentage content of total hypericins, Calculate the percentage content of hyperforin using the
expressed as hypericin, using the following expression: following expression:
(Al 4- A2) X m2 X p A4 X 7724 X p X 2.3

A3 X 7721 A5 X m3 X 10
Al = area of the peak due to pseudohypericin in the A4 = area of ±e peak due to hyperforin in the
chromatogram obtained with the test solution; chromatogram obtained with the test solution;
A2 = area of the peak due to hypericin in the A5 = area of the peak due to rutin in the chromatogram
chromatogram obtained with the test solution; obtained with reference solution (a);
A3 = area of the peak due to hypericin in the m3 = mass of the extract to be examined used to
chromatogram obtained with the reference prepare the test solution, in grams;
solution; rn4 = mass of rutoside trihydrate CRS used to prepare
m1 = mass of the extract to be examined used to reference solution (a), in grams;
prepare the test solution, in grams; 2.3 = correction factor for hyperforin with respect to
m2 ะะะ mass of Sr. John's wort dry extract HRS used to rutin;
prepare the reference solution, in grams; p = percentage content of rutin in rutoside trihydrate
p = percentage content of hypericin in St. John's wort CRS
dry extract HRS.
Hyperforin and flavonoids rutin, using the following expression:
Liquid chromatography (2.2.29). Carry out the assay protected
from light.
7724 X p X (As 4- A7 4- As 4- A9 4- A10 4- All)
Solvent mixture water R, methanol R (20:80 V/V).
7713 X A5 X 10
Test solution Dissolve 75.0 mg of the extract to be examined
in 20.0 mL of the solvent mixture. Sonicate and centrifuge.
A5 = area of the peak due to rutin in the chromatogram
Reference solution (a) Dissolve 20.0 mg of rutoside obtained with reference solution (a);
trihydrate CRS in 200.0 mL of the solvent mixture. Afy = area of the peak due to rutin in the chromatogram
Reference solution (b) Dissolve 75.0 mg of St. John's wort dry obtained with the test solution;
extract HRS in 20.0 mL of the solvent mixture. Sonicate and Aq = area of the peak due to hyperoside in the
centrifuge. chromatogram obtained with the test solution;
Column: A3 = area of the peak due to isoquercitroside in the
— size: z = 0.15 m, 0 = 4.6 mm; chromatogram obtained with the test solution;
— stationary phase: octadecylsilyl silica gel for chromatography R A9 = area of the peak due to quercitroside in the
(3 pm). chromatogram obtained with the test solution;
IV-390 Stephania Tetrandra Root 2016
A10 = area of the peak due to quercetin in the Detection Treat with a 5 g/L solution of iodine R in ethanol
chromatogram obtained with the test solution; (96 per cent) R until the background becomes yellow;
An = area of the peak due to biapigenin in the examine in daylight after the yellow colour has disappeared.
chromatogram obtained with the test solution; Residts See below the sequence of zones present in the
พร = mass of the extract to be examined used to chromatograms obtained with the reference solution and the
prepare the test solution, in grams; test solution. Furthermore, other faint zones may be present
m4 ะ= mass of rutoside trihydrate CRS used to prepare in the chromatogram obtained with the test solution.
p = percentage content of rutin in rutoside trihydrate
CRS. Top of the plate
LABELLING Protopine: an orange zone
The label states the content of hyperforin.

------------------------------------------------------------------------------------- - . ■ _____Ph Eur Tetrandrine: an orange zone An orange zone (tetrandrine)
An orange zone
Stephania Tetrandra Root *

(Fourstamen Stephania Root, ***
Ph Eur________________________________________________________ ______ TESTS
DEFINITION Aristolochia fangchi
Scraped, cut and dried root of Stephania tetrandra s.Moore. Test for aristolochic acids in herbal drugs (2.5.27). The drug
to be examined complies with method A.
Content
Minimum 1.6 per cent of the sum of tetrandrine and Loss on drying (2.2.52)
fangchinoline, expressed as tetrandrine (C38H42N2O6; Maximum 10.0 per cent, determined on 1.000 g of the
Mr 623) (dried drug). powdered herbal drug (355) (2.9.72) by drying in an oven at
105 °C for 2 h.
IDENTIFICATION
A. The root is found as slices or irregularly cylindrical or
Total ash {2.4.16)
semi-cylindrical pieces, mosdy tortuous, about 0.5-1 cm thick Maximum 4.0 per cent.
and 1-5 cm in diameter. The greyish-yellow outer surface Ash insoluble in hydrochloric acid {2.8.1)
usually shows deep and sinuous transversal striations; Maximum 1.0 per cent.
the curved parts are knotty and bumpy. The texture is dense ASSAY
and compact. The cut surface is greyish-white and shows Tetrandrine and fangchinoline
radial striations. Liquid chromatography (2.2.29).
B. Reduce to a powder (355) (2.9.72). The powder is Test solution In a 50 mL round-bottomed flask, weigh 0.500 g
whitish-grey. Examine under a microscope using chloral of the powdered herbal drug (355) (2.9.72). Add 25 mL of a
hydrate solution R. The powder shows the following diagnostic 2 per cent VIV solution of hydrochloric acid R in methanol R.
characters: numerous fragments of parenchyma with cells Weigh. Heat under a reflux condenser on a water-bath at
having slightly thickened and moniliform walls; reticulate or 60 °C for 30 min. Cool and weigh. Adjust to the initial
pined xylem vessels accompanied by fibres; fragments of weight using a 2 per cent VIV solution of hydrochloric acid R
phelloderm containing sclereids; rare cork fragments; rare, in methanol R. Filter. Dilute 5.0 mL of the filtrate to
fine, rod-shaped calcium oxalate crystals. Examine under a 10.0 mL with the mobile phase.
microscope using a 50 per cent VIV solution of glycerol R.
Reference solution Dissolve 10.0 mg of tetrandrine CRS in
The powder shows very many round or truncated, simple or
5 mL of methanol R and dilute to 10.0 mL with the mobile
2- or 3-compound starch granules, 10-20 pm in diameter,
phase.
with a punctiform hilum.
Column’.
— size’. I = 0.25 m, 0 = 4.6 mm;
Test solution To 0.4 g of the powdered herbal drug (355) — stationary phase', octadecylsilyl silica gel for chromatography R
(2.9.72) add 10 mL of a mixture of 1 volume of anhydrous (5 pm).
formic acid R, 9 volumes of water R and 40 volumes of
Mobile phase 4.1 g/L solution of sodium laurylsulfonate for
methanol R. Sonicate at 25 °C for 10 min and filter.
chromatography R in a mixture of 1 volume of glacial acetic
Reference solution Dissolve 10 mg of protopine hydrochloride R acid R, 30 volumes of methanol R, 30 volumes of พater R and
and 10 mg of tetrandrine R in methanol R and dilute to 40 volumes of acetonitrile R.
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gd
plate R (2-10 pm)].
Injection 20 pL.
Mobile phase concentrated ammonia R, methanol R, ethyl
acetate Ry toluene R (0.3:5:10:10 VIVIVIV). Run time 30 min.
Application 10 pL [or 5 pL] as bands of 10 mm [or 8 mm]. Relative retention With reference to tetrandrine (retention
time = about 18 min): fangchinoline = about 0.7.
Devdopment Over a path of 10 cm [or 6 cm].
Drying In a current of warm air for 5 min.
2016 Sterculia Preparations IV-391
resolution', minimum 3.0 between the peaks due to TESTS

fangchinoline and tetrandrine. Acid-insoluble ash
Calculate the percentage content of tetrandrine and Not more than 1.0%, Appendix XI K.
fangchinoline, expressed as tetrandrine, using the following Foreign matter
expression: 1 Complies with the test for foreign matter, Appendix XI D.
(Al + As) X 7722 X p X 5 Ash
A2 X 7721 Not more than 7.0%, Appendix XI J.
Volatile acid
Al = area of the peak due to tetrandrine in the
Not less than 14.0%, calculated as acetic acid, C2H4O2,
chromatogram obtained with the test solution; when determined by the following method. To 1 g contained
A2 = area of the peak due to tetrandrine in the
in a 700 mL Kjeldahl flask add 100 mL of water and 5 mL
chromatogram obtained with the reference of orthophosphoric acid, allow to stand for several hours, or
solution;
until the gum is completely swollen, and boil gently under a
A3 ะ= area of the peak due to fangchinoline in the reflux condenser for 2 hours. Steam distil until 800 mL of
chromatogram obtained with the test solution; distillate is obtained and ±e acid residue measures about
mi = mass of the herbal drug to be examined used to 20 mL and titrate the distillate with 0.1m sodium hydroxide
prepare the test solution, in grams; ES using phenolphthalein solution Rl as indicator. Repeat the
m2 = mass of tetrandrine CRS used to prepare the operation without the substance being examined.
reference solution, in grams; The difference between the titrations represents the amount
p = assigned percentage content of tetrandrine in of alkali required to neutralise the volatile acid. Each mL of
tetrandrine CRS. 0.1m sodium hydroxide ES is equivalent to 6.005 mg of
---------------------------------------------------------------------------------------------------------- Ph Eur volatile acid, calculated as C2H4O2.
Microbial contamination
1.0 g is free from Escherichia coli, Appendix XVI Bl.
STORAGE
Sterculia Sterculia should be stored in a dry place.
Sterculia Gum; Karaya Gum
Preparation
Sterculia Granules
When Powdered Sterculia is prescribed or demanded, Sterculia Granules
material complying with the appropriate requirements below DEFINITION
and containing not less than 10.0% of volatile acid shall be
Sterculia Granules are Sterculia in granule form.
The granules comply with the requirements stated under Granules
DEFINITION and with the following requirements.
Sterculia is the gum obtained from Sterculia urens Roxb.
an4 other species of Sterculia. CHARACTERISTICS
White or buff with a distinct odour of acetic acid;
CHARACTERISTICS transparent, irregular shaped granules of about 1 to 4 mm
It has the macroscopical and microscopical characters which swell when treated with water.
described under Identification tests A and B.
IDENTIFICATION
Sparingly soluble in water, but swells into a homogeneous, A. Irregular or vermiform pieces, about 5 to 20 mm thick;
adhesive, gelatinous mass. Practically insoluble in ethanol greyish white with a brown or pink tinge; surface striated.
(96%).
B. When powdered and mounted in ethanol (96%) it appears
IDENTIFICATION as small, transparent, angular particles of various sizes and
A Irregular or vermiform pieces, about 5 to 20 mm thick; shapes; the particles lose their sharp edges when water is
greyish white with a brown or pink tinge; surface striated. added and each gradually swells until a large, indefinite,
B. When powdered and mounted in ethanol (96%) it appears almost structureless mass results; when mounted in ruthenium
as small, transparent, angular particles of various sizes and red solution the particles are stained red; no blue coloured
shapes; the'particles lose their sharp edges when water is particles (starch) are visible when mounted in iodine
added and each gradually swells until a large, indefinite, solution Rl.
almost structureless mass results; when mounted in ruthenium c. Add 1 g to 80 mL of water and allow to stand for
red solution the particles are stained red; no blue coloured 24 hours, shaking occasionally. A tacky and viscous granular
particles (starch) are visible when mounted in iodine mucilage is produced. Retain the mucilage for use in test D.
solution Rl. D. Boil 4 mL of the mucilage obtained in test c with
c. Add 1 g to 80 mL of water and allow to stand for 0.5 mL of hydrochloric acid, add 1 mL of 5m sodium hydroxide,
24 hours, shaking occasionally. A tacky and viscous granular filter, add 3 mL of cupri-tartaric solution Rl to the filtrate and
mucilage is produced. Retain the mucilage for use in test D. heat. A red precipitate is produced.
D. Boil 4 mL of the mucilage obtained in test c with TESTS
0.5 mL of hydrochloric acid, add 1 mL of 5m sodium hydroxide, Acid-insoluble ash
filter, add 3 mL of cupri-tartaric solution Rl to the filtrate and Not more than 1.0%, Appendix XI K.
heat. A red precipitate is produced.
Ash
E. Warm 0.5 g with 2 mL of 5m sodium hydroxide. A brown Not more than 7.0%, Appendix XI J.
colour is produced.
IV-392 Stramonium Leaf 2016
Volatile acid accompanied by palisade [Aa] and spongy [Ca] parenchyma;

Not less than 13.0%, calculated as acetic acid, C2H4O2, anisocytic [Ac, Cb] and anomocytic [Ab] stomata (2.8.3),
when determined by the following method. To 1 g contained more frequent on the lower epidermis; fragments of covering
in a 700 mL Kjeldahl flask add 100 mL of water and 5 mL trichomes, conical [E], uniseriate with 3-5 cells with warty
of orthophosphoric acid, allow to stand for several hours, or walls, some of them collapsed [Ea]; glandular trichomes,
until the granules are completely swollen, and boil gently short and clavate, in side view [B] with heads formed by 2-7
under a reflux condenser for 2 hours. Steam distil until cells; dorsiventral mesophyll in transverse section [F], with a
800 mL of the distillate is obtained and the acid residue single layer of palisade cells [Fa] and a spongy
measures about 20 mL and titrate the distillate with parenchyma [Fb] containing cluster crystals of calcium
0.1m sodium hydroxide KS1 using phenolphthalein solution Rl as oxalate [Fc]; fragments of spongy parenchyma [D] with some
indicator. Repeat the operation without the substance being cells containing small cluster crystals of calcium oxalate [Db],
examined. The difference between the titrations represents associated with annularly and spirally thickened vessels [Da],
the amount of alkali required to neutralise the volatile acid. in surface view. The powdered herbal drug may also show:
Each mL of 0.1m sodium hydroxide FS is equivalent to fibres and reticulately thickened vessels from the stems;
6.005 mg of volatile acid, calculated as subspherical pollen grains about 60-80 pm in diameter With
Loss on drying 3 germinal pores and a nearly smooth exine [G]; fragments
The powdered granules, when dried to constant weight at of the corolla [H] with wavy-walled cells [Ha] and underlying
105°, lose not more than 20.0% of their weight. Use 1 g. mesophyll [Hb] with some cells containing prisms [He] or
cluster crystals [Hd] of calcium oxalate; seed fragments
Microbial contamination containing yellowish-brown, sinuous, thick-walled sclereids of
1.0 g is free from Escherichia coli, Appendix XVI Bl. the testa [J], and occasional prisms and microsphenoidal
STORAGE crystals of calcium oxalate.
Sterculia Granules should be stored in a dry place.
Stramonium Leaf * *
Preparation
Prepared Stramonium
When Stramonium Leaf or Powdered Stramonium Leaf is
prescribed, Prepared Stramonium shall be dispensed.
Ph Eur_______________________________________________________________
DEFINITION
Dried leaf or dried leaf and flowering, and occasionally fruit
bearing, tops of Datura stramonium L. and its varieties.
Content
hyoscyamine (017บ23พ03; Mr 289.4) (dried drug).
The alkaloids consist mainly of hyoscyamine with varying
proportions of hyoscine (scopolamine).
CHARACTERS
Unpleasant odour.
IDENTIFICATION
A. The leaves are dark brownish-green or dark greyish-green
with a short petiole, often much twisted and shrunken during
drying, thin and brittle, ovate or triangular-ovate, dentately
lobed with an acuminate apex and often unequal at the base.
Young leaves are pubescent on the veins, older leaves are
nearly glabrous. Stems are green or purplish-green, slender,
curved and twisted, wrinkled longitudinally and sometimes Figure 0246.-1. - Illustration for identification test B of powdered
wrinkled transversely, branched dichasially, with a single herbal drug of stramonium leaf
flower or an immature fruit in the fork. Flowers, on short c. Examine the chromatograms obtained in the
pedicels, have a gamosepalous calyx with 5 lobes and chromatography test.
trumpet-shaped brownish-white or purplish corolla. The fruit Results The principal zones in the chromatograms obtained
is a capsule, usually covered with numerous short, stiff with the test solution are similar in position, colour and size
emergences; seeds are brown or black with a minutely pitted to the principal zones in the chromatogram obtained with the
testa. same volume of the reference solution.
B. Microscopic examination (2.8.23). The powder is greyish- D. Shake 1 g of the powdered herbal drug (180) (2.9.12)
green. Examine under a microscope using chloral hydrate with 10 mL of 0.05 M sulfuric acid for 2 min. Filter and add
solution R. The powder shows the following diagnostic to the filtrate 1 mL of concentrated ammonia R and 5 mL of
characters (Figure 0246.-1): fragments of upper [A] and water R. Shake cautiously with 15 mL of peroxide-free ether R,
lower [C] epidermises of the lamina, in surface view, showing avoiding the formation of an emulsion. Separate the ether
cells with slightly wavy anticlinal walls and a smooth cuticle layer and dry over anhydrous sodium sulfate R. Filter and
2016 Stramonium IV-393
evaporate the ether in a porcelain dish. Add 0.5 mL of nitric b) Moisten 10.0 g of the powdered herbal drug (180)
dcid R and evaporate to dryness on a water-bath. Add 10 mL (2.9.12) with a mixture of 5 mL of ammonia R, 10 mL of
of acetone R and, dropwise, a 30 g/L solution of potassium ethanol (96 per cent) R and 30 mL of peroxide-free ether R and
hydroxide R in ethanol (96 per cent) R. A deep violet colour mix thoroughly. Transfer the mixture to a suitable percolator,
develops. if necessary with the aid of the extracting mixture. Allow to
TESTS macerate for 4 h and percolate with a mixture of 1 volume of
chloroform R and 3 volumes of peroxide-free ether R until the
Chromatography
alkaloids are completely extracted. Evaporate to dryness a
few millilitres of the liquid flowing from the percolator,
Test solution To 1.0 g of the powdered herbal drug (180) dissolve the residue in 0.25 M sulfuric acid and verify the
(2.9.72) add 10 mL of 0.05 M sulfuric acid, shake for 15 min absence of alkaloids using potassium tetraiodomercurate
and filter. Wash the filter with 0.05 M sulfuric acid until solution R. Concentrate the percolate to about 50 mL by
25 mL of filtrate is obtained. To the filtrate add 1 mL of distilling on a water-bath and transfer it to a separating
concentrated ammonia R and shake with 2 quantities, each of funnel, rinsing with peroxide-free ether R. Add a quantity of
10 mL, of peroxide-free ether R. If necessary, separate by peroxide-free ether R equal to at least 2.1 times the volume of
centrifugation. Dry the combined ether layers over anhydrous the percolate to produce a liquid of a density well below that
sodium sulfate R, filter and evaporate to dryness on a water of water. Shake the solution with no fewer than 3 quantities,
bath. Dissolve the residue in 0.5 mL of methanol R. each of 20 mL, of 0.25 M sulfuric acid, separate the 2 layers
Reference solution Dissolve 50 mg of hyoscyamine sulfate R in by centrifugation if necessary and transfer the acid layers to a
9 mL of methanol R. Dissolve 15 mg of hyoscine 2"d separating funnel. Make the acid layer alkaline with
hydrobromide R in 10 mL of methanol R. Mix 3.8 mL of the ammonia R and shake with 3 quantities, each of 30 mL, of
hyoscyamine sulfate solution and 4.2 mL of the hyoscine chloroform R. Combine the chloroform layers, add 4 g of
hydrobromide solution and dilute to 10 mL with methanol R. anhydrous sodium sulfate R and allow to stand for 30 min with
Plate TLC silica gel G plate R. occasional shaking. Decant the chloroform and wash the
Mobile phase concentrated ammonia R, water R, acetone R anhydrous sodium sulfate with 3 quantities, each of 10 mL,
(3:7:90 VIVIV). of chloroform R. Add the washings to the chloroform extract,
evaporate to dryness on a water-bath and heat in an oven at
Application 10 pL and 20 pL of each solution, as bands of
20 mm by 3 mm, leaving 1 cm between the bands.
millilitres of chloroform R, add 20.0 mL of 0.01 M sulfuric acid
Development Over a path of 10 cm. and remove the chloroform by evaporation on a water-bath.
Drying At 100-105 °C for 15 min; allow to cool. Titrate the excess of acid with 0.02 M sodium hydroxide using
Detection A Spray with potassium iodobismuthate solution R2, methyl red mixed solution R as indicator.
using about 10 mL for a plate 200 mm square, until the Calculate the percentage content of total alkaloids, expressed
orange or brown zones become visible against a yellow as hyoscyamine, using the following expression:
background.
Results A The zones in the chromatograms obtained with the 57.88 X (20 - ท)
test solution are similar in position (hyoscyamine in the lower (100 - d)xm~
third, hyoscine in the upper third of the chromatograms) and
colour to those in the chromatograms obtained with the d = loss on drying, as a percentage;
reference solution. The zones in the chromatograms obtained ท = volume of 0.02 M sodium hydroxide, in millilitres;
with the test solution are at least equal in size to the m = mass of the powdered herbal drug, in grams.
corresponding zones in the chromatogram obtained with the
same volume of the reference solution. Faint secondary zones STORAGE
may appear, particularly in the middle of the chromatogram Protected from moisture.
obtained with 20 pL of the test solution or near the point of _________ ____________ ____________________Ph Eur
application in the chromatogram obtained with 10 pL of the
test solution.
coating is transparent; examine after 15 min.
Results B The zones due to hyoscyamine in the Prepared stramonium * **
chromatograms obtained with the reference solution and the (Ph. Eur. monograph 0247) **
test solution change from brown to reddish-brown but not to
Ph Eur_____ _ __________________________ ____________________________
greyish-blue (atropine) and any secondary zones disappear.
Foreign matter (2.5.2) DEFINITION
Maximum 3 per cent of stems with a diameter greater than Stramonium leaf powder (180) (2.9.12) adjusted, if
5 mm. necessary, by the addition of powdered lactose or
stramonium leaf of lower content of total alkaloids.
Total ash (2.4.16)
Maximum 20.0 per cent. Content
0.23 per cent to 0.27 per cent of total alkaloids, expressed as
Ash insoluble in hydrochloric acid (2.8.1) hyoscyamine (C17H23NO3; Mr 289.4) (dried drug).
CHARACTERS
ASSAY Appearance
a) Determine the loss on drying (2.2.52) on 2.000 g of the Greyish-green powder.
Unpleasant odour.
105 °C.
IV-394 Stramonium 2016
IDENTIFICATION orange or brown zones become visible against a yellow

A. Examine under a microscope using chloral hydrate background.
solution R. The powder shows the following diagnostic Results A The zones in the chromatograms obtained with the
characters: fragments of leaf lamina showing epidermal cells test solution are similar in position (hyoscyamine in the lower
with slightly wavy anticlinal walls and smooth cuticle; third, hyoscine in the upper third of the chromatogram) and
stomata are more frequent on the lower epidermis (anisocytic colour to those in the chromatograms obtained with the
and anomocytic) (2.8.3)-, covering trichomes are conical, reference solution. The zones in the chromatograms obtained
uniseriate with 3-5 cells and warty walls; glandular trichomes with the test solution are at least equal in size to the
are short and clavate with heads formed by 2-7 cells; corresponding zones in the chromatogram obtained with the
dorsiventral mesophyll, with a single layer of palisade cells same volume of ±e reference solution. Faint secondary zones
and a spongy parenchyma containing cluster crystals of may appear, particularly in the middle of the chromatogram
calcium oxalate; annularly and spirally thickened vessels. obtained with 20 pL of the test solution or near the point of
The powdered herbal drug may also show the following application in the chromatogram obtained with 10 pL of the
diagnostic characters: fibres and reticulately thickened vessels test solution.
from the stems; subspherical pollen grains usually about
60-80 pm in diameter with 3 germinal pores and nearly
coating is transparent; examine after 15 min.
smooth exine; fragments of the corolla with papillose
epidermis; seed fragments containing yellowish-brown, Results B The zones due to hyoscyamine in the
sinuous, thick-walled sclereids of testa; occasional prisms and chromatograms obtained with the test solution and the
microsphenoidal crystals of calcium oxalate. Examined in reference solution change from brown to reddish-brown but
glycerol (85 per 0๓ท) Ry it may be seen to contain lactose not to greyish-blue (atropine) and any secondary zones
crystals. disappear.
B. Examine the chromatograms obtained in the Loss on drying (2.2.22)
Chromatography test. Maximum 5.0 per cent, determined on 1.000 g by drying in
an oven at 105 °C.
Results The principal zones in the chromatogram obtained
with the test solution are similar in position, colour and size Total ash (2.4.16)
to the principal zones in the chromatogram obtained with the Maximum 20.0 per cent.
same volume of the reference solution. Ash insoluble in hydrochloric acid (2.8.1)
c. Shake 1 g with 10 mL of 0.05 M sulfuric acid for 2 min. Maximum 4.0 per cent.
Filter and add to the filtrate 1 mL of concentrated ammonia R ASSAY
and 5 mL of water R. Shake cautiously with 15 mL of a) Determine the loss on drying (2.2.22) on 2.000 g by
peroxide-free ether Ry avoiding the formation of an emulsion. drying in an oven at 105 °C.
Separate the ether layer and dry over anhydrous sodium
b) Moisten 10.0 g with a mixture of 5 mL of ammonia Ry
sulfate R. Filter and evaporate the ether in a porcelain dish.
10 mL of ethanol (96 per cent) R and 30 mL of peroxide-free
Add 0.5 mL of nitric acid R and evaporate to dryness on a
ether R and mix thoroughly. Transfer the mixture to a
water-bath. Add 10 mL of acetone R and, dropwise, a 30 g/L
suitable percolator, if necessary with the aid of the extracting
solution of potassium hydroxide R in ethanol (96 per cent) R.
mixture. Allow to macerate for 4 h and percolate with a
A deep violet colour develops.
mixture of 1 volume of chloroform I? and 3 volumes of
TESTS peroxide-free ether R until the alkaloids are completely
Chromatography extracted. Evaporate to dryness a few millilitres of the liquid
Thin-layer chromatography (2.2.27). flowing from the percolator, dissolve the residue in 0.25 M
Test solution To 1.0 g of the drug to be examined add 10 mL sulfuric acid and verify the absence of alkaloids using
of 0.05 M sulfuric addy shake for 15 min and filter. Wash the potassium tetraiodomercurate solution R. Concentrate the
filter with 0.05 M sulfuric acid until 25 mL of filtrate is percolate to about 50 mL by distilling on a water-bath and
obtained. To the filtrate add 1 mL of concentrated ammonia R transfer it to a separating funnel, rinsing with peroxide-free
and shake with 2 quantities, each of 10 mL, of peroxide-free ether R. Add a quantity of peroxide-free ether R equal to at
ether R. If necessary, separate by centrifugation. Dry the least 2.1 times the volume of the percolate to produce a
combined ether layers over anhydrous sodium sulfate Ry filter liquid of a density well below that of water. Shake the
and evaporate to dryness on a water-bath. Dissolve the solution with no fewer than 3 quantities, each of 20 mL, of
residue in 0.5 mL of methanol R. 0.25 M sulfuric addy separate the 2 layers by centrifugation if
necessary and transfer the acid layers to a 2nd separating
9 mL of methanol R. Dissolve 15 mg of hyoscine funnel. Make the acid layer alkaline with ammonia R and
hydrobromide R in 10 mL of methanol R. Mix 3.8 mL of the shake with 3 quantities, each of 30 mL, of chloroform R.
hyoscyamine sulfate solution and 4.2 mL of the hyoscine Combine the chloroform layers, add 4 g of anhydrous sodium
sulfate R and allow to stand for 30 min with occasional
hydrobromide solution and dilute to 10 mL with methanol R.
shaking. Decant the chloroform and wash the sodium sulfate
with 3 quantities, each of 10 mL, of chloroform R. Add the
Mobile phase concentrated ammonia Ry water Ry acetone R washings to the chloroform extract, evaporate to dryness on a
(3:7:90 VIV/V). water-bath and heat in an oven at 100-105 °C for 15 min.
Application 10 pL and 20 pL of each solution as bands of Dissolve the residue in a few millilitres of chloroform Ry add
20 mm by 3 mm, leaving 1 cm between the bands. 20.0 mL of 0.01 M sulfuric add and remove the chloroform
Development Over a path of 10 cm. by evaporation on a water-bath. Titrate the excess of acid
with 0.02 M sodium hydroxide using methyl red mixed
Drying At 100-105 °C for 15 min and allow to cool.
solution R as indicator.
Detection A spray with potassium iodobismuthate solution R2y
using about 10 mL for a plate 200 mm square, until the
2016 Tea-tree Oil IV-395
Calculate the percentage content of total alkaloids, expressed B. Examine the chromatograms obtained in the test for
as hyoscyamine, using the following expression: chromatographic profile.
57.88 (20 - ท) Results The characteristic peaks in the chromatogram
(100 — d) m
d ะะะ loss on drying, as a percentage; solution.
71 = voltime of 0.02 M sodium hydroxide, in millilitres; TESTS
771 = mass of the herbal drug, in grams. Relative density (2.2.5)
STORAGE 0.885 to 0.906.
In an airtight container. Refractive index (2.2.6)
------------------------ -- ------------------------ ------------------------------------------------------Ph Eur
1.475 to 1.482.
+ 5° to + 15°.
Tea Tree Oil ** ** Gas chromatography (2.2.28): use the normalisation
procedure.
Melaleuca Oil ***
Test solution Dissolve 0.15 mL of the substance to be
(Ph, Eur. monograph 1837) examined in 10 mL of hexane R.
PnEir_________________
Reference solution Dissolve 5 pL of a-pinene R, 5 pL of
DEFINITION sabinene R> 15 pL of a-terpinene R, 5 pL of limonene R, 5 pL
Essential oil obtained by steam distillation from the foliage of cineole R, 30 pL of y-terpinene R, 5 pL of p-cymene R, 5 pL
and terminal branchlets of Melaleuca altemifolia (Maiden and of terpinolene R, 60 pL of terpinen-4-ol R, 5 pL of
Betch) Cheel, M. linariifolia Smith, M. dissitiflora F. Mueller aromadendrene R and 5 mg of a-terpineol R in 10 mL of
and/or other species of Melaleuca. hexane R.
Column:
CHARACTERS
Appearance — size: I = 30 m (a film thickness of 1 pm may be used) to
Clear, mobile, colourless or pale yellow liquid. 60 m (a film thickness of 0.2 pm may be used),
Characteristic odour. 0 = 0.25-0.53 mm,
IDENTIFICATION — stationary phase: macrogol 20 000 R.
First identification: B. Carrier gas helium for chromatography R.
Second identification A Flow rate 1.3 mL/min.
A. Thin-layer chromatography (2.2.27). Split ratio 1:50.
Test solution Dissolve 0.1 mL of the substance to be Temperature:
examined in 5 mL of heptane R.
Reference solution Dissolve 30 pL of cineole R, 60 pL of Time Temperature
terpinen-4-ol R and 10 mg of (/.-terpineol R in 10 mL of (min) (°C)
Column 0- 1 50
heptane R.
Plate TLC silica gel plate R. 1 - 37 50 -> 230
Mobile phase ethyl acetate R, heptane R (20:80 VIV). 37 - 45 230
Application 10 pL, as bands. Injection port 240

Development Over a path of 10 cm. Detector 240
Drying In air.
Detection Spray with anisaldehyde solution R. Heat at
100-105 °C for 5-10 min while observing. Examine in
daylight. Injection 1 pL.
Residts See below the sequence of the zones present in the Elution order Order indicated in the composition of the
chromatograms obtained with the reference solution and the reference solution. Record the retention times of these
test solution. Furthermore, other zones are present in the substances.
chromatogram obtained with the test solution. System suitability: reference solution:
Top of the plate terpinen-4-ol and aromadendrene.
Cineole: a violet-brown zone A violet-brown zone, less intense
(cineole) chromatogram obtained with the reference solution, locate
Terpinen-4-ol: a brownish-violet A brownish-violet zone the components of the reference solution in the
terpinen-4-ol) chromatogram obtained with the test solution. Disregard the
a-terpineol: a violet or A violet or brownish-violet zone peak due to hexane.
brownish-violet zone (a-terpineol)
Determine the percentage content of these components.
— sabinene: maximum 3.5 per cent,
— a-terpinene: 5.0 per cent to 13.0 per cent,
IV-396 Terminalia Arjuna Stem Bark 2016
— limonene-. 0.5 per cent to 4.0 per cent, (2) 0.01% w/v each of arjunolic acid and gallic acid in absolute
— cineole', maximum 15.0 per cent, ethanol.
— y-terpinene-. 10.0 per cent to 28.0 per cent,
— p-cymene'. 0.5 per cent to 12.0 per cent,
— terpinolene: 1.5 per cent to 5.0 per cent, (a) Use as the coating high performance silica gel (Merck
— terpinen-4-ol: minimum 30.0 per cent, silica gel 60 HPTLC plates are suitable).
— aromadendrene'. maximum 7.0 per cent, (b) Use the mobile phase described below.
— a-terpineok 1.5 per cent to 8.0 per cent. (c) Apply as bands 8 |1L of each solution.
STORAGE (d) Develop the plate to 8 cm.
At a temperature not exceeding 25 cc. (e) Remove the plate and allow it to dry in air for 5 minutes.
------------------------------------------------------------------------------------------ --------------- Ph Eur
Spray the plate with anisaldehyde solution, heat at 100° to
105° for 5 minutes and examine in daylight.
MOBILE PHASE
15 volumes of ethyl acetate, 15 volumes of formic acid and

70 volumes of toluene.
Terminalia Arjuna stem Bark
SYSTEM SUITABILITY
DEFINITION
Terminalia Arjuna Stem Bark consists of cut dried bark of
the stems of Terminalia arjuna พ. and A. It contains not less
than 6% of tannins, expressed as pyrogallol, calculated with CONFIRMATION
reference to the dried drug. The chromatogram obtained with solution (1) shows a band
IDENTIFICATION with an Rf value of approximately 0.30 corresponding in
colour and position to the band obtained for arjunolic acid in
A. Irregularly flattened or slightly curved or recurved pieces,
solution (2); two clearly separated dark bands with
up to about 8 cm long, 4 cm wide and 1 cm thick; outer
an Rf value of approximately 0.2; a band with an Rf value of
surface uneven, dark brown or sometimes mottled greyish-
0.63 is present. Other bands may be present.
brown, smooth or, more frequently, irregularly striated
longitudinally with occasional transverse ridges; inner surface
pink to reddish brown with longitudinal striations and Top of the plate
occasional paler brown patches. Fracture short and starchy in
the inner part, the outer part frequendy laminated.
B. Reduce to a powder (355). The powder is reddish-brown.
The powder shows a variety of parenchymatous cells, some
thin-walled, square or round and others yellowish, polygonal
and thick-walled. Rectangular or polygonal, pitted, thin
walled, light brown, lighdy lignified cells from the cork layer Dark band
or outer areas of the cortex are present.
Fibres occur singly and in small or large groups, individual
cells narrowing to highly pointed ends, and possibly showing
wavy invaginations of the walls where surrounding cells have
become detached; degree of lignification varies; walls are Dark band Dark band: arjunolic
acid
yellowish brown, some pitted, others not. Single fibres may
be complete, but those in groups are usually fragmented, Two separated dark Yellow band: gallic
bands acid
individual cells being straight or noticeably curved in places.
Rounded cells of the medullary rays, which are one cell wide,
intersperse the fibres. Solution (1) Solution (2)
Small, calcium oxalate cluster crystals occur scattered
throughout as well as being found within parenchymatous
cells, some forming a crystal sheath alongside the fibres.
Other crystals are very large and less well defined, and
usually free. TESTS
Examine under a microscope using 50% v/v of glycerol. Ash
Starch granules are frequent, but not abundant, mainly free, Not more than 25%, Appendix XI J.
but some in parenchymatous cells. They are small, simple, Loss on drying
round, oval or irregular in shape, and occasionally in 2 to 3 When dried for 2 hours at 100° to 105°, loses not more than
compound granules, without visible hila. More or less 10% of its weight. Use 1 g.
frequent scattered lumps of brown pigment may be found
Water soluble extractive
sometimes in parenchymatous cells.
Not less than 20%, Appendix XI B2.
Appendix in A, using the following solutions. ASSAY
Carry out the determination of tannins in herbal drugs,
(1) Shake 1.0 g of the powdered drug with 10 mL of absolute
Appendix XI M. Use 1.0 g of powdered drug.
ethanol, centrifuge at 3000 rpm for 5 minutes and filter
(Whatman GF/C is suitable).
2016 Terminalia Belerica Fruit IV-397
Terminalia Belerica Fruit CONFIRMATION

Under ultraviolet light (254 nm) the chromatogram obtained
DEFINITION
with solution (1) shows bands with Rf values of
Terminalia Belerica Fruit consists of pericarp of dried ripe approximately 0.11 and 0.15 corresponding in colour and
fruits of Terminalia belerica Roxb. It contains not less than position to the bands obtained with ellagic acid and gallic
10/O of tannins, expressed as pyrogallol, calculated with acid in solution (2) and light blue bands with Rf values of
reference to the dried drug. approximately 0.04 and 0.36. Other bands may be present.
IDENTIFICATION
A* The dried fruits are spherical to subspherical, about 3 to
ว cm in diameter, slightly depressed at the upper end and
more or less tapering to the scar of the pedicel at the lower
end. The surface is brown to yellowish brown with a grey
velvety sheen, irregularly wrinkled and sometimes with faint,
incomplete, longitudinal ridges. Cut transversely, the fruit
shows the pericarp about 4 to 5 mm thick enclosing a very
hard, yellowish-white seed.
B. Reduce to a powder (355). The powder is light-brown.
rhe powder shows many free, unicellular, straight or slightly
bent trichomes from the epicarp. A variety of thick-walled,
heavily pitted, lignified fibro-sclereids of elongated and
spherical shapes occur in large and small groups; occasional
medium sized reticulate vessels; heavily pitted, lignified
parenchymatous cells and others with reticulate thickenings;
many lignified, pitted, thin walled sclereids and groups of
fragmented, thick-walled, pitted, fibres are present. Rarely oil
globules, starch granules, and calcium oxalate crystals may be
found in the parenchymatous cells from the embryo.
Examine under a microscope using 50% v/v of glycerol.
The powder shows minute, either single or 2 to 4 compound
starch granules, some scattered, but mainly filling
parenchymatous cells. Some have slit or stellate hila; simple
granules are often not perfectly spherical. Larger calcium
oxalate crystals, with fewer small ones, are scattered or in Under daylight after spraying with anisaldehyde solution the
parenchymatous cells. chromatogram obtained with solution (1) shows a band with
an Rf value of approximately 0.15 corresponding in colour
and position to the band obtained gallic acid in solution (2);
Appendix LU A, using the following solutions.
a dark band with an Rf value of 0.36; several dark bands in
(1) Add 10 mL of absolute ethanol to 1.0 g of the powdered the upper part of the plate.
drug, centrifuge at 3000 rpm for 5 minutes and filter
(2) 0.01% w/v each of arjunolic acid, gallic acid and ellagic acid Top of the plate
in absolute ethanol.
(a) Use as the coating high performance silica gel F254 Several dark bands
(Merck silica gel 60 F254 HPTLC plates are suitable).
(b) Use the mobile phase described below.
(c) Apply as bands 8 J1L of each solution.
(e) Remove the plate and allow it to dry in air for 5 minutes.
Examine under ultraviolet light (254 nm). spray the plate with
anisaldehyde solution, heat at 100° to 105° for 5 minutes and
Dark band
MOBILE PHASE Dark band: arjunolic
15 volumes of ethyl acetate, 15 volumes offormic acid and acid
70 volumes of toluene. Light brown band Light brown band:
SYSTEM SUITABILITY gallic add

solution (2) shows two clearly separated bands under both
ultraviolet light (254 nm) and daylight. Solution (1) Solution (2)
IV-398 Terminalia Chebula Fruit 2016
TESTS CHROMATOGRAPHIC CONDITIONS

Ash (a) Use as the coating high performance silica gel F254
Not more than 7%3 Appendix XI J. (Merck silica gel 60 F254 HPTLC plates are suitable).
Loss on drying (b) Use the mobile phase described below.
When dried for 2 hours at 100° to 105°, loses not more than (c) Apply as bands 8 |1L of each solution.
Water soluble extractive (e) Remove the plate and allow it to dry in air for 5 minutes.
Not less than 45%, Appendix XI B2.
Examine under ultraviolet light (254 nm). spray the plate with
ASSAY anisaldehyde solution, heat at 100° to 105° for 5 minutes and
Carry out the determination of tannins in herbal drugs3 examine in daylight.
Appendix XI M. Use 1.0 g of powdered drug. MOBILE PHASE
A mixture of 15 volumes of ethyl acetate, 15 volumes oiformic

acid and 70 volumes of toluene.
SYSTEM SUITABILITY
Terminalia Chebula Fruit The test is not valid unless the chromatogram obtained with
DEFINITION solution (2) shows two clearly separated bands under both
Terminalia Chebula Fruit consists of pericarp of mature ultraviolet light (254 nm) and daylight.
fruits of Terminalia chebula Retz. It contains not less than VALIDITY
20% of tannins, expressed as pyrogallol, calculated with When examined under ultraviolet light (254 nm) the
reference to the dried drug. chromatogram obtained with solution (1) shows bands
IDENTIFICATION with Rf values of approximately 0.11 and 0.15 corresponding
A. The dried fruits are sub-globular to ovoid, 3 to 4 cm long in colour and position to the bands obtained with gallic acid
and 1.5 to 2 cm wide, bluntly pointed at the tip and tapering and ellagic acid in solution (2).
towards the base. The surface is yellowish to greenish,
sometimes brown, shiny and more or less wrinkled and has
Top of the plate
distinct longitudinal ridges. Cut transversely, the fruit shows
the pericarp about 3 to 4 mm thick, non-adherent to the very
hard, creamy-white seed.
B. Reduce to a powder (355). The powder is yellowish
solution. The powder shows round, oval or elongated, thin
walled parenchymatous cells in groups. Occasional narrow
walled, unpitted, lightly lignified fibres occur in small or
larger groups, some forming wave-like arrangements. Large
groups of fragmented, heavily lignified and pitted fibres also
occur. A variety of thick-walled, heavily pitted, lignified
fibro-sclereids of elongated, rectangular, and irregular shapes
occur in large and small groups. Fewer pitted, lignified
parenchyma, or thinner-walled, less lignified and pitted
sclereids, with broad lumens, are also found. There are very Blue band Blue band: gallic acid
occasional small, spiral, lignified vessel fragments. Small, Dark band Dark band: ellagic acid
greenish, thick-walled polygonal epicarp cells are seen in
surface view. Parenchymatous cells with reticulate thickenings
across the surface are rare, as are others without such
thickenings, but containing oil globules. Examine under a
microscope using 50% v/v of glycerol. The powder shows
minute, either single or 2 to 4 compound granules, some
scattered, but mainly filling parenchymatous cells. Some have When examined under daylight after spraying with
slit or stellate hila; simple granules are often not perfectly anisaldehyde solution the chromatogram obtained with solution
spherical. Larger calcium oxalate crystals, with fewer small (1) shows a band with an Rf value of approximately 0.15
ones, are scattered or in parenchymatous cells. corresponding in colour and position to the band obtained
c. Carry out the method for thin-layer chromatography3 for gallic acid in solution (2) and a dark band with
Appendix in A, using the following solutions. an Rf value of approximately 0.30. There may be some faint
(1) To 1.0 g of the powdered drug, add 10 mL of absolute brown bands with Rf values of approximately 0.40 and 0.70.
ethanol, centrifuge at 3000 rpm for 5 minutes and filter
(2) 0.01% w/v each of arjunolic acid, gallic acid and ellagic acid
in absolute ethanol.
2016 Thyme IV-399
_______ Top of the plate lower is longer and has 2 hairy teeth. After flowering, the
calyx tube is closed by a crown of long, stiff hairs.
The corolla, about twice as long as the calyx, is usually
brownish in the dry state and is slightly bilabiate.
The leaf of Thymus zygis is usually 1.7-6.5 mm long and
0.4-1.2 mm wide; it is acicular or linear-lanceolate and the
edges are markedly rolled towards the abaxial surface. Both
surfaces of the lamina are green or greenish-grey and the
midrib is sometimes violet; the edges, in particular at the
base, have long, white hairs. The dried flowers are very
similar to those of T. vulgaris.
B. Microscopic examination (2.8.23). The powder of both
species is greyish-green or greenish-brown. Examine under a
Dark band Dark band: arjunolic shows the following diagnostic characters (Figure 0865.-1
acid
and Figure 0865.-2): fragments of the outer epidermis of the
Light brown band Light brown band: corolla (surface view [A, c, F]), consisting of cells with wavy
gallic acid and slightly thickened [Fc] or unthickened [Ac] walls,
numerous uniseriate, multicellular, covering trichomes, often
with 1 cell collapsed [Aa], glandular trichomes with a
Solution (1) Solution (2) unicellular head and a unicellular [Ca, Fb] or
multicellular [Ab] stalk, diacytic stomata (2.8.3) [Fa] and
glandular trichomes generally with 12 cells [D]; cells of the
epidermis from the base of the corolla, isodiametric with
slightly thickened walls [C]; pollen grains, relatively rare,
TESTS spherical and smooth, with 6 germinal slit-like pores,
Ash measuring about 35 pm in diameter [B]; the powder of T.
Not more than 5%, Appendix XI J. zygis also contains numerous thick bundles of fibres from the
Loss on drying main veins and from fragments of stems; the epidermises of
When dried for 2 hours at 100° to 105°, loses not more than the leaves (surface view [G, K]) have cells with anticlinal
10% of its weight. Use 1 g. walls that are sinuous and beaded [Ga, Ka], and diacytic
stomata (2.8.3) [Gb]; numerous glandular trichomes made
Water soluble extractive up of 12 secretory cells, the cuticle of which is generally
Not less than 50%, Appendix XI B2. raised by the secretion to form a globular or ovoid, bladder
ASSAY like covering [Kb]; glandular trichomes with a unicellular
Cany out the determination of tannins in herbal drugs, stalk and a globular or ovoid head [Kc]; in both species, the
Appendix XI M. Use 1.0 g of powdered drug. adaxial epidermis bears covering trichomes with warty walls
that are shaped as pointed teeth [Gc], and is usually
associated wi± underlying palisade parenchyma [Gd, Kd];
the abaxial epidermis (transverse section [H, L]) bears
covering trichomes of different types: unicellular, straight or
Thyme slightly curved [Ha, La]; bicellular or tricellular, articulated
and most often elbow-shaped [Hb, J] (T. vulgaris)’, bicellular
or tricellular, more or less straight [N], or very large,
multicellular [M], at the base of the lamina (T. zygis)’,
DEFINITION fragments of calyx covered by numerous, uniseriate trichomes
Whole leaves and flowers separated from the previously dried with 5-6 cells and a weakly striated cuticle (surface view [E]).
stems of Thymus vulgaris L. or Thymus zygis L. or a mixture c. Thin-layer chromatography (2.2.27).
of both species. Test solution To 0.5 g of the powdered herbal drug (355)
Content (2.9.12) add 5 mL of methanol R. Sonicate for 10 min.
— essential oil: minimum 12 mL/kg (anhydrous drug); Centrifuge or filter; use the supernatant or the filtrate.
— sum of the contents of thymol and carvacrol (both c 10H14O; Reference solution Dissolve 1 mg of rutin R and 1 mg of
Afr 150.2): minimum 40 per cent in the essential oil. rosmarinic acid R in 5 mL of methanol R.
CHARACTERS Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica gel
Strong odour reminiscent of thymol. F254 plate R (2-10 pm)].
Mobile phase anhydrous formic acid R, water R, ethyl acetate R
IDENTIFICATION
(1:1:15 VIVIV).
A. The leaf of Thymus vulgaris is usually 4-12 mm long and
up to 3 mm wide, sessile or with a very short petiole.
The lamina is tough, entire, lanceolate or ovate, covered on Development Over a path of 15 cm [or 6 cm].
both surfaces by a -grey or greenish-grey indumentum; Drying In air.
the edges are markedly rolled up towards the abaxial surface.
The midrib is depressed on the adaxial surface and is very
prominent on the abaxial surface. The calyx is green, often
with violet spots and is tubular; at the end are 2 lips of which
the upper one is bent back and at the end has 3 lobes, the
IV-400 Thyme 2016
Detection Heat at 100 °C for 3 min, treat the still-hot plate

with a 5 g/L solution of diphenylboric acid aminoethyl ester R in
ethyl acetate R, then treat with a 50 g/L solution of macrogol
400 R in methylene chloride R; examine in ultraviolet light at
365 nm.
solution.
Top of he plate
2 red fluorescent zones
Rosmarinic acid: a blue A blue fluorescent zone

fluorescent zone (rosmarinic acid)
1 or 2 blue fluorescent zones
2 yellow or orange fluorescent

zones
A green fluorescent zone may be
present
Rutin: an orange-yellow
fluorescent zone
Figure 0865.-1. - Illustration for identification test B of powdered D. Examine the chromatograms obtained in the assay for
herbal drug of thyme thymol and carvacrol.
obtained with the test solution arc similar in retention time to
those in the chromatogram obtained with reference
solution (a).
TESTS
Foreign matter (2. ร. 2)
Maximum 10 per cent of stems and maximum 2 per cent of
other foreign matter. Stems must not be more than 1 mm in
diameter and 15 mm in length.
Thymus serpyllum L.
Adulteration with T. serpyllum L. is indicated by the
presence of leaves with long trichomes at their base and with
weakly pubescent other parts.
Water (2.2.13)
Maximum 100 mUkg, determined on 20.0 g of the
Total ash (2.4.16)
ASSAY
Use 30.0 g of the herbal drug, a 1000 mL round-bottomed
flask and 400 mL of water R as the distillation liquid. Distil
at a rate of 2-3 mUmin for 2 h without xylene R in the
graduated tube.
Thymol and carvacrol
procedure.
Figure 0865.-2. - Illustration for identification test B of powdered Test solution Filter the essential oil obtained in the
herbal drug of thyme determination of essential oil over a small amount of
2016 Thyme Oil, Thymol Type IV-401
anhydrous sodium sulfate R and dilute to 5.0 mL with IDENTIFICATION

heptane R by rinsing the apparatus and the anhydrous sodium First identification B.
sulfate. Dilute a volume of the filtered solution corresponding
to 100 pL of the essential oil to 5.0 mL with heptane R.
Reference solution (a) Dissolve 0.20 g of thymol R and 50 mg
of carvacrol R in heptane R and dilute to 5.0 mL with the Test solution Dissolve 0.2 mL of the substance to be
same solvent. examined in methylene chloride R and dilute to 10 mL with
the same solvent.
Reference solution (b) Dilute 10 pL of carvacrol R to 10.0 mL
with heptane R. Dilute 100 pL of the solution to 10.0 mL Reference solution Dissolve 5 mg of thymol R and 10 pL of
with heptane R. carvacrol R in methylene chloride R and dilute to 10 mL with
the same solvent.
Column:
— material: fused silica; Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
size: I = 30-60 m, 0 ะ= 0.25 mm; plate R (2-10 pm)].
stationary phase: macrogol 20 000 R (film thickness Mobile phase methylene chloride R.
0.25 pm). Application 10 pL [or 4 pL] as bands of 10 mm [or 8 mm].
Carrier gas nitrogen for chromatography R or helium for Development Over a path of 12 cm [or 6 cm].
chromatography R. Drying In air.
Flow rate 1-2 mL/min. Detection Treat with anisaldehyde solution R and heat at
split ratio 1:100. 100-105 °C for 5-10 min; examine in daylight.
Temperature: Results See below the sequence of zones present in the
Time Temperature
(°C)
Column 0 - 45 40 -> 220
Top of the plate
Injection port 190
Detector 210
Thymol: an orange-brown zone An intense orange-brown zone

Detection Flame ionisation. (thymol)
Injection 0.2 pL. Carvacrol: an orange-grey zone A faint orange-grey zone
(carvacrol) may be present
solution (a); record the retention times of these substances.
— resolution: minimum 1.5 between the peaks due to thymol
and carvacrol.
A brownish-grey zone
chromatogram obtained with reference solution (a), locate Reference solution Test solution
chromatogram obtained with the test solution. B. Examine the chromatograms obtained in the test for
Determine the percentage content of thymol and carvacrol. chromatographic profile.
Disregard any peak due to the solvent or with an area less Results The characteristic peaks in the chromatogram
than the area of the principal peak in the chromatogram obtained with the test solution are similar in retention time to
obtained with reference solution (b) (0.05 per cent). those in the chromatogram obtained with reference
------- ---------------------------------------------------------------------------------------------------Ph Eur solution (a).
TESTS
0.915 to 0.935.
Thyme Oil, Thymol Type * * Refiractive index (2.2.6)
1.490 to 1.505.
(Ph Eur monograph 1374) ***
PhEtr_______________________________________________________________ Gas chromatography (2.2.28): use the normalisation
DEFINITION procedure.
Essential oil obtained by steam distillation from the fresh Test solution Dissolve 200 pL of the substance to be examined
flowering aerial parts of Thymus vulgaris L.J T. zygis L. or a in heptane R and dilute to 10.0 mL with the same solvent.
mixture of both species. Reference solution (a) Dissolve 5 pL of P-myrcene R} 5 pL of
CHARACTERS a-terpinene R, 20 pL of p-cymene R3 10 pL of y-terpinene Rj
Appearance 5 pL of linalol R> 5 pL of terpinen-4-ol R3 40 mg of thymol R
Clear, yellow or very dark reddish-brown, mobile liquid. and 5 pL of carvacrol R in 5 mL of heptane R.
Odour reminiscent of thymol. Reference solution (b) Dissolve 10 pL of carvacrol R in
Solubility Dilute 100 pL of the solution to 10.0 mL with heptane R.
Miscible with anhydrous ethanol and with light petroleum.
IV-402 Thyme 2016
Column'. reddish or purplish, the older stems brown and woody, the
— material', fused silica; younger stems pubescent. The leaves are opposite, 3-12 mm
long and up to 4 mm wide, elliptical to ovate-lanceolate with
— stationary phase: poly (dimethyl) (diphenyl) siloxane R (film an obtuse apex, cuneate and shortly petiolate at the base;
thickness 0.25 pm). the margin is entire and markedly ciliate, especially near the
Carrier gas helium for chromatography R. base; both surfaces are more or less glabrous but distinctly
Flow rate 1.5 mL/min. punctate. The inflorescence is composed of about 6-12
flowers in rounded to ovoid, terminal heads. The calyx is
Split ratio 1:50.
tubular, 2-lipped with the upper lip dividing to form 3 teeth,
Temperature: the lower lip with 2 teeth, edged with long hairs; the inner
Time Temperature surfaces are strongly pubescent, the hairs forming a closed
(min) ___________ (°C) tube after flowering. The corolla is purplish-violet or red,
Column 0 - 75 65 -> 215 2-lipped, the lower lip with 3 lobes and the upper lip
Injection port 230 notched, the inner surface is strongly pubescent; 4
epipetalous stamens project from the corolla tube.
Detector 250
Detection Flame ionisation. green or greenish-brown. Examine under a microscope using
Injection 1 pL. chloral hydrate solution R. The powder shows the following
diagnostic characters (Figure 1891.-1): fragments of the leaf
Elution order Order indicated in the composition of reference epidermises [A, B, F] covered by a finely striated cuticle and
solution (a); record the retention times of these substances. consisting of cells with sinuous anticlinal walls [Aa, Ba, Fa]
รุ)’stem suitability: reference solution (a): and diacytic stomata (2.8.3) [Ab, Bb, Fb]; cells of the adaxial
— resolution: minimum 1.5 between the peaks due to thymol leaf epidermis [B] with wavy, irregularly thickened anticlinal
and carvacrol. walls [Ba]; numerous covering trichomes on both epidermises
Identification of peaks Using the retention times determined and the leaf margins, with some of the cells containing very
from the chromatogram obtained with reference solution (a)j small crystals of calcium oxalate [Af, Ca, Fd], the majority
locate the components of reference solution (a) in the are short, conical, unicellular, with thickened and warty walls
chromatogram obtained with the test solution. The peak due (surface view [Be], side view [Fc]); fewer multicellular
to a-thujene elutes with a relative retention of about 0.8 with covering trichomes, long, tapering to a point, composed of
reference to p-myrcene. The peak due to carvacrol methyl up to 8 cells, slightly swollen at the joints, with finely pitted
ether elutes with a relative retention of about 0.9 with walls, on an epidermis [Ae] or fragmented [C]; abundant
reference to thymol. glandular trichomes, mostly multicellular of the lamiaceous
Determine the percentage content of these components. type [Ac] with a unicellular stalk and a glandular head
The limits are within the following ranges: consisting of 12 inconspicuous cells, others with a unicellular
— a-thujene: 0.2 per cent to 1.5 per cent; stalk and a unicellular globular or ovoid head [Ad]; purplish-
— P-myrcene: 1.0 per cent to 3.0 per cent; violet fragments of the corolla whose inner epidermis consists
— a-terpinene: 0.9 per cent to 2.6 per cent; of cells with rounded papillae [D] and whose outer epidermis
— p-cymene: 14.0 per cent to 28.0 per cent; [E], with a striated cuticle, consists of cells with lobed walls
— y-terpinene: 4.0 per cent to 12.0 per cent; [Ea], unicellular [Eb] or multicellular [Ec] uniseriate covering
— linalol: 1.5 per cent to 6.5 per cent; trichomes, glandular trichomes with a unicellular head and a
— terpinen-4-ol: 0.1 per cent to 2.5 per cent; unicellular stalk [Ed] and glandular trichomes of the
— carvacrol methyl ether. 0.05 per cent to 1.5 per cent; lamiaceous type; relatively rare pollen grains, spherical, about
— thymol: 31.0 per cent to 55.0 per cent; 30 pm in diameter, with a finely pitted exine and 6 germinal
— carvacrol: 0.5 per cent to 5.5 per cent; pores [G].
— disregard limit:, the area of the principal peak in the c. Thin-layer chromatography (2.2.27).
chromatogram obtained with reference solution (b) Test solution To 0.5 g of the powdered herbal drug (355)
(0.05 per cent). (2.9.12) add 5 mL of methanol R. Sonicate for 10 min.
STORAGE Centrifuge or filter; use the supernatant or the filtrate.
At a temperature not exceeding 25 °C. Reference solution Dissolve 1 mg of rutin R and 1 mg of
_______________________________________________________________ Ph Eur rosmarinic acid R in 5 mL of methanol R.
F254 plate R (2-10 pm)].
Mobile phase anhydrous formic acid R} water R} ethyl acetate R
Wild Thyme * (1:1:15 VIVIV).
(Ph. Eur. monograph 1891) ’ Application 20 pL [or 5 pL] as bands of 20 mm [or 8 mm].
Ph Eur__________________________________________________ _______ Development Over a path of 15 cm [or 6 cm].
DEFINITION Drying In air.
Whole or cut, dried, flowering aerial parts of Thymus Detection Heat at 100 °C for 3 min; treat the still-hot plate
serpyUum L. with a 5 g/L solution of diphenylboric acid aminoethyl ester R in
ethyl acetate R, then treat with a 50 g/L solution of macrogol
Content 400 R in methylene chloride R'} examine in ultraviolet light at
Minimum 3.0 mITkg of essential oil (dried drug).
365 nm.
IDENTIFICATION Results See below the sequence of zones present in the
A. The stem is much branched, up to about 1.5 mm in chromatograms obtained with the reference solution and the
diameter, cylindrical or indistinctly quadrangular, green,
2016 Tolu IV-403
test solution. Furthermore, other faint fluorescent zones may abaxial surface showing many types of warty covering
be present in the chromatogram obtained with the test trichomes: unicellular, straight or slightly curved, bicellular or
solution. tricellular, often elbow-shaped, and bicellular or tricellular,
more or less straight.
105 °C for 2 h.
Rosmarinic acid: a blue A blue fluorescent zone Total ash (2.4.16)
fluorescent zone (rosmarinic acid) Maximum 10.0 per cent. '
1 or 2 blue fluorescent zones Maximum 3.0 per cent.
ASSAY
Use 50.0 g of the cut herbal drug, a 1000 mL round-
A yellow or orange fluorescent bottomed flask and 500 mL of water R as the distillation
liquid. Distil at a rate of 2-3 mL/min for 2 h without xylene R
A green or blue fluorescent zone
may be present in the graduated tube.
Rutin: an orange-yellow _____________________________________________________________ Ph Eur
fluorescent zone
Tolu Balsam * *
Ph Eur_____________________________________________________________ _
DEFINITION
Oleo-resin obtained from the trunk of Myroxylon
balsamum (L.) Harms var. balsamum.
Content
25.0 per cent to 50.0 per cent of free or combined acids,
expressed as cinnamic acid (CgHsO^ Mf 148.2) (dried
drug).
CHARACTERS
Appearance
Hard, friable, brownish to reddish-brown mass; thin
fragments are brownish-yellow when examined against the
light.
Reminiscent odour of vanillin.
Solubility
Practically insoluble in water, very soluble to freely soluble in
alcohol, practically insoluble in light petroleum.
IDENTIFICATION
Test solution Stir 0.40 g of the fragmented drug with 10 mL
of methylene chloride R for 5 min and filter.
Reference solution Dissolve 50 mg of benzyl cinnamate R in
methylene chloride R3 add 50 pL of benzyl benzoate R and
dilute to 10 mL with methylene chloride R.
Mobile phase light petroleum Rj toluene R (5:95 VIV).
Figure 1891.-1.- Illustration for identification testB of powdered Application 20 pL, as bands.
herbal drug of wild thyme
Foreign matter (2.8.2) Detection Spray with vanillin reagent R and heat at
iMaximum 3 per cent, determined on 30 g. 100-105 °C for 5 min. Examine in daylight.
Thymus vulgaris L. or Thymus zygis L Results See below the sequence of the zones present in the
Adulteration with T. vulgaris L. or T. zygis L. is indicated by chromatograms obtained with the test and reference
the presence of acicular to linear-lanceolate leaves with a solutions. Furthermore, other coloured zones are present in
strongly bent margin, the adaxial surface showing covering the chromatogram obtained with the test solution.
trichomes shaped as pointed teeth with warty walls, the
IV-404 Tolu Preparations 2016
Top of the plate
Benzyl benzoate: a greyish-blue a greyish-blue zone

Tolu-flavour Solution
DEFINITION
Benzyl cinnamate: a greyish-green a greyish-green zone Cinnamic Acid 5.0 g
Benzoic Acid 2.5 g
Reference solution Test solution Ethyl Cinnamate 0-3 g
Vanillin 0-1 g
Cinnamon Oil 0-02 mL
TESTS Sucrose 500 g
Acid value Ethanol (96 per cent) 350 mL
100 to 160. Water Sufficient to produce 1000 mL
Dissolve 0.5 g of the fragmented drug in 50 mL of alcohol R.
Add 0.5 mL of acid blue 93 solution R and 5.0 mL of 0.5 M
alcoholic potassium hydroxide. Stir vigorously and titrate with
0.5 M hydrochloric acid until the colour changes from Dissolve the Sucrose in 320 mL of Water. Add 250 mL of
brownish-red to blackish-green («1 mL of 0.5 M hydrochloric the Ethanol (96 per cent), with mixing. Dissolve the
acid). Carry out a blank test in the same manner (ท2 mL of Cinnamic Acid, Benzoic Acid, Ethyl Cinnamate, Vanillin and
0.5 M hydrochloric acid). Calculate the acid value in the same Cinnamon Oil in the remaining 100 mL of Ethanol
manner as the saponification value (2.5.6). (96 per cent), add this solution to the sucrose solution with
mixing, dilute to 1000 mL with Water and mix. Allow to
Matter insoluble in alcohol stand for a few hours before use.
Maximum 5 per cent.
Boil 2.0 g of the fragmented drug with 25 mL of alcohol IDENTIFICATION
(90 per cent V/V) R and filter. Wash the residue with alcohol Carry out the method for thin-layer chromatography,
(90 per cent V/V) R, boiling until completely extracted, then Appendix m A, using ±e following solutions.
dry the residue at 100-105 °C. Weigh the residue. (1) The solution being examined.
Loss on drying (2.2.32) (2) 0.5% w/v of cinnamic acid, 0.25% w/v of benzoic acid and
Maximum 5.0 per cent, determined on 2.000 g of the 0.03% v/v of ethyl cinnamate in ethanol (90%).
fragmented drug by spreading on a flat evaporating dish CHROMATOGRAPHIC CONDITIONS
9 cm in diameter and allowing to dry in vacuo for 4 h. (a) Use as the coating silica gel GF254.
Total ash (2.4.16) (b) Use the mobile phase as described below.
(c) Apply 5 |1L of each solution.
ASSAY (d) Develop the plate to 15 cm.
Boil 1.500 g under a reflux condenser with 25 mL of 0.5 M (e) After removal of the plate, dry in air for 15 minutes and
alcoholic potassium hydroxide for 1 h. Evaporate the ethanol repeat the development using the same mobile phase.
and heat the residue with 50 mL of water R until the Remove the plate, allow the solvent to evaporate and
substance is homogeneously distributed. After cooling, add examine under ultraviolet light (254 nm).
80 mL of water R and a solution of 1.5 g of magnesium
MOBILE PHASE
sulfate R'\ท!50 mL of water R. Mix, and allow to stand for
10 min. Filter through a pleated filter paper and wash the 15 volumes of glacial acetic acid, 25 volumes of hexane and
residue with 20 mL of water R. Combine the filtrate and the 75 volumes oin-pentane.
washings, acidify with hydrochloric acid R and extract with CONFIRMATION
4 quantities, each of 40 mL, of ether R. Discard the aqueous The spots in the chromatogram obtained with solution (1)
layer. Combine the organic extracts and wash with are similar in size and correspond in position to those in the
2 quantities, each of 20 mL, and with 3 quantities, each of chromatogram obtained with solution (2).
10 mL, of a 50 g/L solution of sodium hydrogen carbonate R.
Discard the ether layer. Combine the aqueous extracts, TESTS
acidify with hydrochloric acid R and stir once with 30 mL, Ethanol content
twice with 20 mL and once with 10 mL of methylene 31 to 36% v/v, Appendix VUI F.
chloride R. Dry the combined methylene chloride extracts Weight per mL
over anhydrous sodium sulfate R. Filter through a pleated filter 1.125 to 1.155 g, Appendix V G.
and wash the residue with 10 mL of methylene chloride R.
Reduce the combined methylene chloride extracts to 10 mL
by distillation and eliminate the remaining methylene
chloride in a current of air. Dissolve the residue with heating
in 10 mL of alcohol R previously neutralised to phenol red Tolu Syrup
solution R. After cooling, titrate with 0.1 M sodium hydroxide, DEFINITION
using the same indicator. Tolu-flavour Solution 100 mL
1 mL of 0.1 M sodium hydroxide is equivalent to 14.82 mg of Syrup Sufficient to produce 1000 mL
total acids, expressed as cinnamic acid.
STORAGE Liquids and with the following requirement.
Do not store in powdered form. Weight per mL
_____ _ _____________________________________ _ __________________ PhEur 1.29 to 1.32 g, Appendix V G.
2016 Tormentil Preparations IV-405
Paediatric Compound Tolu Linctus Reference solution Dissolve 1.0 mg of catechin R in 1.0 mL of
methanol R.
Paediatric Compound Tolu Oral Solution
DEFINITION
Mobile phase glacial acetic acid R, ether R, hexane R, ethyl
Paediatric Compound Tolu Linctus is an oral solution
acetate R (20:20:20:40 VIVIVIV).
containing 0.6% w/v of Citric Acid Monohydrate in a
suitable vehicle with a tolu flavour. Application 10 pL as bands.
The linctus complies with the requirements stated under Oral Development Over a path of 10 cm.
Liquids and with the following requirements. Drying In air for 10-15 min.
Content of total acid, calculated as citric acid Detection Spray with a freshly prepared 5 g/L solution of fast
monohydrate, C6H8O7,H2O blue B salt R. Reddish zones appear. Expose the plate to
0.60 to 0.66% w/v. ammonia vapour, the zones become more intense turning
reddish-brown. Examine in daylight.
ASSAY
To 15 g add 100 mL of water and titrate with 0.1m sodium
hydroxide ES using phenolphthalein solution Rl as indicator.
test solution. Furthermore, other fainter zones are present in
Each mL of 0.1m sodium hydroxide vs is equivalent to
7.005 mg of C6H8O7,H2O. Determine the weight per mL of
the linctus, Appendix V G, and calculate the content of Top of the plate
C6H8O7,H2O, weight in volume.
Tormentil ***** Catechin: an intense

reddish-brown zone
A more intense reddish-brown
zone (catechin)
Preparation A fainter zone
Tormentil Tincture
An intense zone
Ph Eir______________________________________________________________
Fainter zones
DEFINITION
Whole or cut, dried rhizome, freed from the roots, of
Potentilla erecta (L.) Raeusch. (P. tormentilia Stokes).
Content TESTS
Minimum 7 per cent of tannins, expressed as pyrogallol Foreign matter (2.5.2)
Maximum 3 per cent of root and stems as well as rhizomes
(C6 H6O3; Mr 126.1) (dried drug).
with black fracture and maximum 2 per cent of other foreign
IDENTIFICATION matter.
A. The rhizome is cylindrically spindle-shaped, with a very
Cadmium (2.4.27)
irregular appearance, often forming, twisted, knotty tubers,
Maximum 2.0 ppm.
up to 10 cm long and 1-2 cm thick, very hard and scarcely
branched. The surface is brown to reddish-brown, rugose Loss on drying (2.2.52)
and has remains of roots and transversely elongated Maximum 12.0 per cent, determined on 1.000 g of the
depressed whitish scars from the stems. At the top of the
rhizome the remains of numerous aerial stems may be 105 °C for 2 h.
present. The fracture is short and granular, dark red to Total ash {2.4.16)
brownish-yellow. Maximum 5.0 per cent.
B. Reduce to a powder (355) (2.9.72). The powder is ASSAY
reddish-brown. Examine under a microscope using chloral Tannins {2.8.14)
hydrate solution R. The powder shows the following diagnostic Use 0.500 g of the powdered herbal drug (180) (2.9.72).
characters: coarsely serrate cluster crystals of calcium oxalate, ________________________________________ _____________________ PhEur
up to 60 pm in diameter; fragments of thin-walled
parenchyma containing reddish-brown tannin; groups of
narrow, bordered-pitted vessels with lateral pores; thick
walled and pitted, polygonal parenchyma; groups and Tormentil Tincture * *
fragments of sclerenchymatous thick-walled fibres; occasional
fragments of cork with thin-walled, brown, tabular cells.
Examine under a microscope using a 50 per cent VIV PhEur______________________ __ ____________ ———————
solution of glycerol R. The powder shows spherical or
DEFINITION
elliptical starch granules, up to about 20 pm in length.
Tincture produced from Tormentil (1478).
Content
Minimum 1.5 per cent mlm of tanninSj expressed as
(2.9.12) add 10 mL of water R, shake for 10 min and filter.
pyrogallol (C$H6O3; Afr 126.1).
Shake the filtrate with 2 quantities, each of 10 mL, of ethyl
acetate R and filter the combined upper phases over 6 g of PRODUCTION
anhydrous sodium sulfate R. Evaporate the filtrate to dryness The tincture is produced from 1 part of comminuted drug
under reduced pressure and dissolve the residue in 1.0 mL of and 5 parts of ethanol (70 per cent VIV) by a suitable
ethyl acetate R. procedure.
IV-406 Trachyspermum ammi 2016
CHARACTERS surface warty; commissural surface flat; vittae visible as two

Red or reddish-brown liquid. darker longitudinal bands.
IDENTIFICATION B. Reduce to a powder (355). The powder is greenish
Thin-layer chromatography (2.2.27). brown. Examine under a microscope using chloral hydrate
solution. The powder contains numerous fragments of the
Test solution Mix 1.0 mL of the tincture to be examined with
papillose epicarp, also showing cuticular striations, with
1.0 mL of alcohol (70 per cent VIV) R.
attached or detached whole or fragmented unicellular, warty-
Reference solution Dissolve 1.0 mg of catechin R in 1.0 mL of walled trichomes; parquetry layer of endocarp in surface
methanol R. view; endosperm of thick-walled cells containing oil globules
Plate TLC silica gel plate R. and aleurone grains with embedded microrosette crystals of
Mobile phase ether R, glacial acetic acid R, hexane R, ethyl calcium oxalate; fragments of yellowish-brown septate vittae;
acetate R (20:20:20:40 VIVIVIV). bicollateral vascular bundles with associated lignified,
Application 10 pL as bands. reticulate or pitted parenchyma.
Development Over a path of 10 cm. c. Examine the chromatograms obtained in the test for
Chromatographic profile. The retention times of the principal
Drying In air for 10-15 min.
peaks in the chromatogram obtained with solution (1) are
Detection Spray with a freshly prepared 5 g/L solution of fast similar to those in the chromatogram obtained with
blue B salt R. Reddish zones appear. Expose the plate to solution (3).
ammonia vapour, the zones become more intense, turning
reddish-brown. Examine in daylight. TESTS
Water
Not more than 10% พ/พ, Appendix IX c. Use 10.0 g.
test solution. Total Ash
Not more than 10%, Appendix XI J, Method II.
Top of the plate Chromatographic profile
Carr}' out the method for gas chromatography,
Appendix III B, using the following solutions.
Catechin: an intense zone An intense zone (catechin)
(1) Use the essential oil-toluenc mixture obtained in the
determination of essential oil.
A fainter zone (2) 0.4% v/v each of ๅ-terpinene and p-cymene in toluene.
An intense zone (3) 0.1% v/v each of p-cymene and Y-terpinene and 0.1% w/v
Fainter zones of thymol in toluene.
(4) 0.01% v/v of Y-terpinene in toluene.
Reference solution Test solation
TESTS (a) Use a fused silica column (30 m X 0.53 mm) bonded
Ethanol content (2.9.10) with a 1 pm film thickness and coated with polyethylene glycol
64 per cent VIV to 69 per cent VIV. 20,000 as the bonded phase (DB-Wax is suitable).
Methanol and 2-propanol (2.9.11) (b) Use helium as the carrier gas at 1.5 mL per minute.
Maximum 0.05 per cent VIV of methanol and maximum (c) Use the temperature gradient described below.
0.05 per cent VIV of 2-propanol. (d) Inject 1.0 J1L of each solution.
ASSAY (e) Use a split ratio of 1:50.
Tannins (2.8.14) (f) Record the chromatogram for a sufficient length of time
Use 2.50 g of the tincture to be examined. to elute all the peaks in the
________________________________________________________________ Ph Eur
Time Temperature (°)

(Minutes)
Trachyspermum Ammi
DEFINITION column 0->5 60
Trachyspermum Ammi is the dried ripe fruit of 5->68 60-»250
Trachyspermum ammi (L.) Sprague (syn. c. [Carum] copticum
(L) Benth. & Hook.f. ex C.B. Clarke). 68—>75 250
Content Inject port 250
It contains not less than 2.5% v/w of essential oil calculated
with reference to the anhydrous drug. Detector 260
IDENTIFICATION
A. The dried fruits occur mainly as entire cremocarps with
carpophore present, yellowish green, ovoid, laterally
SYSTEM SUITABILITY
compressed, 1 to 3 nun in length and 1 to 2.8 mm in
diameter, usually with pedicel attached; styles remaining as a The test is not valid unless in the chromatogram obtained
curved, bifid stylopod at the apex. Each fruit composed of with solution (2), the resolution factor between the peaks due
two mericarps, dorsal surface convex with five distinct ridges, to y-terpinene and p-cymene is at least 2.5.
2016 Turmeric IV-407
In the chromatogram obtained with solution (3) 3 the peaks

elute in the following order:
/•-terpinene, p-cymene and thymol.
chromatogram obtained with solution (3), locate the
components of solution (3) in the chromatogram obtained
with solution (1) and calculate the content ofp-cymene,
y-terpinene and thymol by normalisation.
Limits:
— p-cymene 10 to 25%,
— Y-terpinene 10 to 30%,
— thymol 45 to 70%.
Disregard any peak with an area less than the peak in the
ASSAY
Essential oil
Carry out the method for Essential Oils in Herbal Drugs,
Appendix XI E, using 15 g of the powdered drug with
1000 mL of water as distillation liquid. Distil at a rate of 2 to
3 mL per minute for 2 hours using 0.5 mL of toluene in the
graduated tube. Measure the quantity of essential oil distilled
and use for the test for Chromatographic profile.
Javanese Turmeric ** **
(Ph. Eur. monograph 1441) * ** Figure 1441.-1. - Illustration for identification test B of powdered
PnEu_____________ _________________________________________________
herbal drug ofJavanese turmeric
c. Thin-layer chromatography (2.2.27). Examine the
DEFINITION chromatograms obtained in the test for Curcuma longa L.
Dried rhizome, cut in slices, of Curcuma zanthorrhiza Roxb.
(syn. c. zanthorrhiza D. Dietrich).
Content test solution. Furthermore, other faint zones may be present
— essential oik minimum 50 mL/kg (anhydrous drug); in the chromatogram obtained with the test solution.
— dicinnamoyl methane derivatives, expressed as curcumin
(C21H20o6; Mr 368.4): minimum 1.0 per cent
(anhydrous drug). Top of he plate
CHARACTERS
Aromatic odour. Curcuminoids: a greenish A greenish fluorescent zone
fluorescent zone (curcuminoids)
IDENTIFICATION
A. Orange-yellow or yellowish-brown or greyish-brown slices,
mostly peeled 1.5-6 mm thick and 15-50 mm, more rarely Curcuminoids: 2 greenish A greenish fluorescent zone
fluorescent zones (curcuminoids)
up to 70 mm, in diameter. Fragments of the brownish-grey
cork are sporadically present. The transverse surface is yellow
with dark spots in the paler centre. The fracture is short and Reference solution Test solution
finely grained.
B. Microscopic examination (2.8.23). The powder is orange
yellow or yellowish-brown. Examine under a microscope Results B See below the sequence of zones present in the
using chloral hydrate solution R. The powder shows the chromatograms obtained with the reference solution and the
following diagnostic characters (Figure 1441.-1): fragments of test solution. Furthermore, other faint zones may be present
parenchyma [C, E] with large, rounded or ovoid cells
containing orange-yellow or yellowish-brown secretory cells
[Ea]; fragments of spiral [Fa, G] or reticulate [F] vessels; rare
fragments of cork (surface view [B], side view [D]);
fragments of epidermis [H] with fragments of thick-walled
unicellular covering trichomes [Ha] with a pointed tip [Hb].
solution of glycerol R. The powder shows numerous ovoid or
irregular starch granules [A], about 30-50 pm long and about
10-30 pm wide, with a more or less visible eccentric hilum.
IV-408 Turmeric Rhizome 2016
Top of the plate Calculate the percentage content of dicinnamoyl methane

faint pink
derivatives, expressed as curcumin, using the following
expression:
An intense reddish
dark zone A X 5000

1607 X m
Thymol: a dark
i.e. taking the specific absorbance of curcumin to be 1607.
pinkish-red zone A = absorbance at 425 nm,
Curcuminoids: a brown brown zone (curcuminoids) m = mass of the herbal drug to be examined, in grams.
_____________________________________________ _ _______________ Ph Eis
Curcuminoids: 2 yellow yellow zone (curcuminoids)

Turmeric Rhizome ** \
TESTS Ph Eur____________________________________________ _ __________ ______
Curcuma longa L
DEFINITION
Whole, cured (by boiling or steaming), dried rhizome of
Test solution To 1 g of the freshly powdered herbal drug Curcuma longa L. (syn. Curcuma domestica Valeton) with
(355) (2.9.12) add 10 mL of ethanol (96 per cent) R, shake, roots and outer surface removed.
allow to stand for 30 min with occasional shaking and filter;
use the filtrate. Content
— essential oil: minimum 25 mL/kg (anhydrous drug);
Reference solution Dissolve 20 mg of curcuminoids R and — dicinnamoyl methane derivatives, expressed as curcumin
10 mg of thymol R in 10 mL of ethanol (96 per cent) R.
(€2iH20O6; Mr 368.4): minimum 2.0 per cent
Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica (anhydrous drug).
CHARACTERS
Mobile phase glacial acetic acid R, toluene R (20:80 VIV).
Spicy odour.
IDENTIFICATION
A. The rhizome is ovate, oblong-ovoid, pyriform or
Drying In air. cylindrical, often shortly branched, up to 6 cm long and
Detection A Examine in ultraviolet light at 365 nm. 15 mm thick. The primary rhizome shows scars from the
Results A The chromatogram obtained with the test solution lateral branches. The surface is slightly dusty, spotted and
does not show a very intense greenish fluorescent zone in the brownish-yellow, yellow or brownish-grey, finely striated.
lower third. The fracture is granular, smooth, non-fibrous, slightly glossy,
Detection B Treat with anisaldehyde solution R and heat at uniformly orange-yellow; it shows a narrow cortex that is
100-105 °C for 10 min; examine in ultraviolet light at darker on the outside.
365 nm. B. Microscopic examination (2.8.23). The powder is orange
yellow. Examine under a microscope using chloral hydrate
Water (2.2.13)
solution R. The powder shows die following diagnostic
Maximum 120 mLTkg, determined on 20.0 g of the
powdered herbal drug (500) (2.9.12). characters (Figure 2543.-1): fragments of parenchyma
including some secretory cells that contain masses of
Total ash (2.4.16) brownish-yellow oil [G]; reticulate or pitted vessels [B, D];
Maximum 8.0 per cent. rare fragments of epidermis (surface view [F]), with cells
ASSAY whose walls are slightly and irregularly thickened [Fa] and
Essential oil (2.8.12) scars of covering trichomes [Fb]; occasional long and
Use 2.5 g of the freshly powdered herbal drug (500) (2.9.12) 3 flexuous, thick-walled, unicellular covering trichomes,
a 2 L round-bottomed flask, 400 mL of water R as the fragmented and free [E] or attached to fragments of
distillation liquid and 0.5 mL of xylene R in the graduated epidermis U]; rare fragments of cork (surface view [A], side
tube. Distil at a rate of 2 mL/min for 3 h. view [H]), sometimes covered by epidermis [Ha]. Examine
Dicinnamoyl methane derivatives
glycerol R. The powder shows starch granules, free or
Disperse 0.500 g of the powdered herbal drug (500) (2.9.12)
included in parenchymatous cells, usually gelatinised and
in 30 mL of ethanol (96 per cent) R in a 100 mL round-
agglomerated in a starchy paste; occasional ovoid starch
bottomed flask. Heat under a reflux condenser for 2.5 h.
granules, often deformed by curing [C], are also present.
Cool and filter into a volumetric flask, rinse the round-
bottomed flask and the filter with ethanol (96 per cent) R and c. Thin-layer chromatography (2.2.27). Examine the
dilute to 100.0 mL with the same solvent. Dilute 1.0 mL of chromatograms obtained in the test for Curcuma zanthorrhiza
the solution to 50.0 mL with ethanol (96 per cent) R. Measure Roxb.
the absorbance (2.2.25) at 425 nm using ethanol
(96 per cent) R as the compensation liquid.
2016 Turmeric Rhizome IV-409
Top of the plate
A faint pink zone
An intense reddish zone
Thymol ะ a dark zone
Curcuminoids: a brown zone A brown zone (curcuminoids)
Curcuminoids: 2 yellow zones 2 yellow zones (curcuminoids)
TESTS
Curcuma zanthorrhiza Roxb. (syn. Curcuma zanthorrhiza
D. Dietr.) Thin-layer chromatography (2.2.29).
Test solution To 1 g of the freshly powdered herbal drug
(355) (2.9.72) add 10 mL of ethanol (96 per cent) R, shake,
allow to stand for 30 min with occasional shaking and filter;
use the filtrate.
Reference solution Dissolve 20 mg of curcuminoids R and
10 mg of thymol R in 10 mL of ethanol (96 per cent) R.
Plate TLC silica gel F254 plate R (5-40 pm) [or TLC silica
Mobile phase glacial acetic acid R, toluene R (20:80 VIV).
Figure 2543.-1. - Illustration for identification test B of powdered Application 10 pL [or 3 pL] as bands of 10 mm [or 8 mm].
herbal drug of turmeric rhizome
Results A See below the sequence of zones present in the Drying In air.
chromatograms obtained with the reference solution and the Detection A Examine in ultraviolet light at 365 run.
test solution. Furthermore, other faint zones may be present Detection B Treat with anisaldehyde solution R and heat at
in the chromatogram obtained with the test solution. 100-105 °C for 10 min; examine in ultraviolet light at
365 nm.
Top of the plate
shows no dark zone just above the zone due to thymol in the
Water (2.2.75)
Curcuminoids: a greenish A greenish fluorescent zone
fluorescent zone (curcuminoids) powdered herbal drug (500) (2.9.72).
Total ash (2.4.16)
Curcuminoids: 2 greenish 2 greenish fluorescent zones Maximum 7.0 per cent.
fluorescent zones (curcuminoids)
ASSAY
Reference solution Test solution Use 2.5 g of the freshly powdered herbal drug (500) (2.9J2),
a 2 L round-bottomed flask, 400 mL of water R as the
distillation liquid and 0.5 mL of xylene R in the graduated
Results B See below the sequence of zones present in the tube. Distil at a rate of 2 mL/min for 3 h.
Dicinnamoyl methane derivatives
Disperse 0.500 g of the powdered herbal drug (500) (2.9.72)
in 30 mL of ethanol (96 per cent) R in a 100 mL round-
bottomed flask. Heat under a reflux condenser for 2.5 h.
Cool and filter into a volumetric flask, rinse the round-
bottomed flask and the filter with ethanol (96 per cent) R and
dilute to 100.0 mL with the same solvent. Dilute 1.0 mL of
the solution to 50.0 mL with ethanol (96 per cent) R. Measure
the absorbance (2.2.25) at 425 nm using ethanol
(96 per cent) R as the compensation liquid.
Calculate the percentage content of dicinnamoyl methane
derivatives, expressed as curcumin, using the following
expression:
IV-410 Turpentine Oil 2016
A X 5000
1607 X m chromatographic profile.
i.e. taking the specific absorbance of curcumin to be 1607. Results The peaks in the chromatogram obtained with the test
A = absorbance at 425 nm; solution are similar in retention time to those in the
JU ะ= mass of the herbal drug to be examined, in grams. chromatogram obtained with reference solution (a).
--------------------------------------------------------------------------------------------- ------------- Ph Eur TESTS
0.856 to 0.872.
Turpentine Oil 1.465 to 1.475.
Turpentine Oil, Pinus Pinaster Type Optical rotation (2.2.7)
(Ph Eur monograph 1627) -40° to -28°.
Preparations Acid value (2.5.1)
White Liniment Maximum 1.0.
PhEir____________________________________ Peroxide value (2.5.5, Method B)
DEFINITION Maximum 20.
Essential oil obtained by steam distillation, followed by Fatty oils and resinified essential oils (2.5.7)
rectification at a temperature below 180 °C, from the It complies with the test.
oleoresin obtained by tapping Pinus pinaster Aiton and/or Chromatographic profile
Pinus massoniana D.Don. A suitable antioxidant may be Gas chromatography (2.2.25): use the normalisation
added. procedure.
CHARACTERS Test solution. Dilute 1.0 mL of the oil to be examined in
Appearance heptane R and dilute to 10.0 mL with the same solvent.
Clear, colourless or pale yellow liquid. Reference solution (a) Dissolve 30 pL of ซ.-pinene R, 10 mg of
Odour reminiscent of a-pinene and P-pinene. camphene R, 20 pL of P-pinene R, 10 pL of car-3-ene R)
IDENTIFICATION 10 pL of P-myrcene R, 20 pL of limonene R, 10 pL of
First identification B longifolene R, 10 pL of p-caryophyllene R and 10 mg of
caryophyllene oxide 7? in 1.0 mL of heptane R.
A. Thin-layer chromatography (2.2.27). Reference solution (ใ)) Dissolve 5 pL of p-caryophyllene R in
Test solution Dilute 1 mL of the oil to be examined in 10 ml
Dilute 0.1 mL of the solution to 1.0 mL with heptane R.
of toluene R.
Column'.
Reference solution Dissolve 10 pL of p-caryophyllene R and
— material', fused silica;
10 pL of caryophyllene oxide R in 10 mL of toluene R.
— size'. I = 60 m, 0 = 0.25 mm;
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel — stationary phase', macrogol 20 000 R (film thickness
plate R (2-10 pm)]. 0.25 pm).
Mobile phase ethyl acetate R, toluene R (5:95 V/V). Carrier gas helium for chrotnatography R.
Application 10 pL [or 2 pL] as bands of 10 mm [or 8 mm]. Flow rate 1.0 mL/min.
Development Over a path of 15 cm [or 6 cm]. Split ratio'. 1:200.
Drying In air. Temperature'.
Time Temperature
Results See below the sequence of zones present in the (min) (°C)
chromatograms obtained with the reference solution and the Column 0 - 10 60
test solution. Furthermore, other faint zones may be present 10 - 80 60-> 200
below the zone due to caryophyllene oxide in the 80 - 120 200
chromatogram obtained with the test solution. Injection port 200
Detector 250
Top of the plate
P-Caryophyllene: a pink zone A pink zone

Injection 1.0 pL.
Caryophyllene oxide: a pink zone A pink zone (caryophyllene solution (a); record the retention times of these substances.
oxide)
— resolution: minimum 1.5 between the peaks due to car-3-
ene and P-myrcene.
A brownish-violet zone
the components of reference solution (a) in the
Reference solution Test solution chromatogram obtained with the test solution.
2016 Valerian IV-411
Determine the percentage content of these components. cells (longitudinal section [K], transverse section [J]); spiral,
The limits are within the following ranges: reticulate or pitted lignified vessels, isolated or in small
a-pinene: 70.0 per cent to 85.0 per cent; groups [D, G]; thin-walled, elongated cells of the piliferous
camphene: 0.5 per cent to 2.0 per cent; layer (surface view [A], transverse section [B]), some with
— p-pinene: 5.0 per cent to 20.0 per cent; root hairs [Aa, Ba] or their scars [Ab]; the piliferous layer is
— car-3-ene: maximum 1.0 per cent; usually accompanied by an underlying layer of cells with
P-myrcene: 0.4 per cent to 1.5 per cent; slightly thickened and elongated walls [Ac, Bb]; fragments of
— limonene'. 1.0 per cent to 7.0 per cent; dermal tissue from the rhizome composed of 1 or 2 layers of
— longifolene: 0.2 per cent to 4.0 per cent; polygonal cells with irregularly thickened walls [F]; a few
p-caryophyllene: 0.1 per cent to 3.0 per cent; groups of sclereids with thick walls and a narrow lumen [E]
caryophyllene oxide', maximum 1.0 per cent; from the pith of the rhizome. Examine under a microscope
disregard limit. the area of the peak in the chromatogram using a 50 per cent VIV solution of glycerol R. The powder
obtained with reference solution (ไว) (0.05 per cent). shows numerous starch granules, simple or 2- to
Residue on evaporation (2.8.9) 6-compound, but frequently separated, rounded or irregular
Maximum 2.5 per cent, determined after heating on a water and up to about 15 pm in diameter, most of the granules
bath for 3 h. show a rather indistinct cleft or radiate hilum [C].
STORAGE
----------- - ---------------------------- - -------------------------------------------------------------- PhEur
Valerian
(Valerian Root, Ph. Eur. monograph 0453) ***
Preparations
Valerian Dry Extract
Valerian Dry Hydroalcoholic Extract
Valerian Tincture
When Powdered Valerian is prescribed or demanded,
material complying with the appropriate requirements below
shall be dispensed or supplied.
Pn Elf________ ______________________________________________________
DEFINITION
Dried, whole or fragmented underground parts of Valeriana
officinalis L. ร./., including the rhizome surrounded by the
roots and stolons.
Content
— essential oil', minimum 4 mUkg (dried drug);
— sesquiterpenic acids', minimum 0.17 per cent พ/พ,
expressed as valerenic acid (C15H22O2; Mr 234.3) (dried
drug);
IDENTIFICATION
A. The rhizome is yellowish-grey or pale brownish-grey, Figure 0453.-1. - Illustration for identification test B of powdered
obconical or cylindrical, up to about 50 mm long and 30 mm herbal drug of valerian root
in diameter; the base is elongated or compressed, usually
entirely covered by numerous roots. The apex usually
exhibits a cup-shaped scar from the aerial parts; stem bases Test solution Suspend 1 g of the powdered herbal drug (355)
are rarely present. When cut longitudinally, the pith exhibits (2.9.12) in 10 mL of methanol R and sonicate for 10 min.
a central cavity transversed by septa. The roots are Filter the supernatant through a membrane filter (nominal
numerous, almost cylindrical, of the same colour as the pore size 0.45 pm). Use the filtrate.
rhizome, 1-3 mm in diameter and sometimes more than Reference solution Dissolve 5 mg of acetoxyvalerenic acid R and
100 mm long. A few filiform fragile secondary roots are 5 mg of valerenic acid R in 20 mL of methanol R.
present. The fracture is short. The stolons show prominent Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
nodes separated by longitudinally striated internodes, each plate R (2-10 pm)].
20-50 mm long, with a fibrous fracture. Mobile phase glacial acetic acid R> ethyl acetate R, cyclohexane R
B. Microscopic examination (2.8.23). The powder is pale (2:38:60 VIVIV).
yellowish-grey or pale greyish-brown. Examine under a Application 20 pL [or 5 pL] as bands of 10 mm [or 8 mm].
occasional groups of rectangular sclereids with moderately Drying In air.
thickened walls and a large lumen, from the stem base [H]; Detection: Treat with anisaldehyde solution R and heat at
very numerous fragments of parenchyma with large ovoid 100-105 °C for 5-10 min; examine in daylight.
IV-412 Valerian 2016
Resides See below the sequence of zones present in the Time Mobile phase A Mobile phase B
chromatograms obtained with the reference solution and the (min) (per cent V/V) (per cent V/V)
test solution. Furthermore, other violet zones may be present 0- 5 55 45
in the chromatogram obtained with the test solution. 5 - 18 55 -» 20 45 -> 80
18 - 22 20 80
Top of the plate

Valerenic acid: a violet zone A violet zone (valerenic acid) Detection spectrophotometer at 220 nm.
Acetoxyvalerenic acid: a violet A violet zone (acetoxyvalerenic Injection 20 pL.
acid) Peak identification Use the chromatogram supplied with
valerian dry extract HRS and the chromatogram obtained with
2 faint or very faint violet zones the reference solution to identify the peaks due to
acetoxyvalerenic acid and valerenic acid.
— relative retention with reference to valerenic acid (retention
time = about 19 min): acetoxyvalerenic acid = about 0.5.
TESTS
Calculate the percentage content of sesquiterpenic acids,
expressed as valerenic acid, using the following expression:
Maximum 5 per cent of stem bases and maximum 2 per cent
of other foreign matter.
(Al + A2) X 7712 X p X 5
A3 X 7711
Maximum 12.0 per cent, determined on 1.000 g of well-
homogenised powdered herbal drug (355) (2.9.12) by drying
A\ = area of the peak due to acetoxyvalerenic acid in the
in an oven at 105 °C for 2 h.
Total ash (2.4.16) A2 = area of the peak due to valerenic acid in the
Maximum 12.0 per cent. chromatogram obtained with the test solution;
Ash insoluble in hydrochloric acid (2.8.1) A3 — area of the peak due to valerenic acid in the
Maximum 5.0 per cent. chromatogram obtained with the reference solution;
ASSAY ni\ = mass of the herbal drug to be examined used to
ไท2 = mass of valerian dry extract HRS used to prepare the
Use 40.0 g of freshly powdered herbal drug (500) (2.9.12), a
2000 mL flask, 500 mL of water R as the distillation liquid
p = percentage content of valerenic acid in valerian dry
extract HRS.
rate of 3-4 mIJmin for 4 h.
______________ — Pn Eir
Sesquiterpenic acids
(2.9.72) in a 100 mL round-bottomed flask with a ground
glass neck. Add 20 mL of methanol Rl. Mix and heat on a Cut Valerian * *
cool and filter. Place the filter with the residue in the 100 mL (Valerian Root, Cut, Ph. Eur. monograph 2526)
round-bottomed flask. Add 20 mL of methanol Rl and heat Ph Eur___________________________________ ________________ ____________
on a water-bath under the reflux condenser for 15 min.
DEFINITION
Allow to cool and filter. Combine the filtrates and dilute to
Dried, cut underground parts of Valeriana officinalis L. S.L,
50.0 mL with methanol Rl, rinsing the round-bottomed flask
including the rhizome, roots and stolons.
and the filter.
It is produced from Valerian root (0453) for the purpose of
Reference solution Dissolve an amount of valerian dry
being used in herbal teas.
extract HRS corresponding to 1.0 mg of valerenic acid in
methanol Rl and dilute to 10.0 mL with the same solvent. Content
Sonicate for 10 min and filter through a membrane filter — essential oil: minimum 3 mL/kg (dried drug);
(nominal pore size 0.45 pm). — sesquiterpenic acids: minimum 0.10 per cent m/m expressed
Column: as valerenic acid (C15H22O25 M.1. 234.3) (dried drug).
— size: I -= 0.25 m, 0 = 4.6 mm; IDENTIFICATION
— stationary phase: octadecylsilyl silica gel for chromatography R A. Microscopic examination (2.8.23). The powder is pale
(5 pm). yellowish-grey or pale greyish-brown. Examine under a
Mobile phase: microscope using chloral hydrate solution R. The powder
— mobile phase A: acetonitrile Rl, 5 g/L solution of phosphoric shows the following diagnostic characters (Figure 2526.-1):
acid R (20:80 V!V)-, occasional groups of rectangular sclereids with moderately
— mobile phase B: 5 g/L solution of phosphoric acid R, thickened walls and a large lumen, from the stem base [HJ;
acetonitrile Rl (20:80 FZP); very numerous fragments of parenchyma with large ovoid
cells (longitudinal section [K], transverse section [J]); spiral,
reticulate or pitted lignified vessels, isolated or in small
groups [D, G]; thin-walled, elongated cells of the piliferous
2016 Valerian IV-413
layer (surface view [A], transverse section [B]), some with test solution. Furthermore, other violet zones may be present
root hairs [Aa, Ba] or their scars [Ab]; the piliferous layer is in the chromatogram obtained with the test solution.
usually accompanied by an underlying layer of cells with
slightly thickened and elongated walls [Ac, Bb]; fragments of
dermal tissue from the rhizome composed of 1 or 2 layers of Top of the plate
polygonal cells with irregularly thickened walls [F]; a few
groups of sclereids with thick walls and a narrow lumen [E]
Valerenic acid: a violet zone A violet zone (valerenic acid)
from the pith of the rhizome. Examine under a microscope
using a 50 per cent VIV solution of glycerol R. The powder Acetoxyvalerenic acid: a violet A violet zone (acetoxyvalerenic
shows numerous starch granules, simple or 2- to zone acid)
6-compound, but frequently separated, rounded or irregular

and up to about 15 pm in diameter; most of the granules 2 faint or very faint violet zones
show a rather indistinct cleft or radiate hilum [C].
TESTS
Foreign matter {2.8.2}
Maximum 5 per cent of stem bases and maximum 2 per cent
of other foreign matter, determined on the herbal drug prior
to cutting.
Loss on drying {2.2.32}
Maximum 12.0 per cent, determined on 1.000 g of well-
homogenised powdered herbal drug (355) {2.9.12} by drying
in an oven at 105 °C for 2 h.
Total ash {2.4.16}
Ash insoluble in hydrochloric acid {2.8.1}
ASSAY
Essential oil {2.8.12}
Use 40.0 g of freshly powdered herbal drug (500) (2.9. /2), a
2000 mL flask, 500 mL of water R as the distillation liquid
rate of 3-4 mUmin for 4 h.
Sesquiterpenic acids
{2.9.12} in a 100 mL round-bottomed flask with a ground
glass neck. Add 20 mL of methanol Rl. Mix and heat on a
cool and filter. Place the filter with the residue in the 100 mL
round-bottomed flask. Add 20 mL of methanol Rl and heat
Figure 2526.-1. - Illustration for identification test A of powdered on a water-bath under the reflux condenser for 15 min.
herbal drug of cut valerian root Allow to cool and filter. Combine the filtrates and dilute to
50.0 mL with methanol Rl 3 rinsing the round-bottomed flask
and the filter.
Test solution Suspend 1 g of the powdered herbal drug (355)
Reference solution Dissolve an amount of valerian dry
{2.9.12} in 10 mL of methanol R and sonicate for 10 min.
Filter the supernatant through a membrane filter (nominal
methanol Rl and dilute to 10.0 mL with the same solvent.
pore size 0.45 pm). Use the filtrate.
Sonicate for 10 min and filter through a membrane filter
Reference solution Dissolve 5 mg of acetoxyvalerenic acid R and (nominal pore size 0.45 pm).
5 mg of valerenic acid J? in 20 mL of methanol R. Column:
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel — size: I = 0.25 m, 0 = 4.6 mm;
plate R (2-10 pm)]. — stationary phase: octadecylsilyl silica gel for chromatography R
Mobile phase glacial acetic acid R3 ethyl acetate R, cyclohexane R (5 pm).
(2:38:60 VIVIV). Mobile phase:
Application 20 pL [or 5 pL] as bands of 10 mm [or 8 mm]. — mobile phase A: acetonitrile Rl3 5 g/L solution oiphosphoric
Development Over a path of 10 cm [or 6 cm]. acid R {2^ VtV}3
— mobile phase B: 5 g/L solution of phosphoric acid R3
Drying In air.
acetonitrile Rl (20:80 V/V);
Detection: Treat with anisaldehyde solution R and heat at
IV-414 Valerian Preparations 2016
Time Mobile phase A Mobile phase B Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
(per cent V/V) (per cent V/V) plate R (2-10 pm)].
0-5 55 45
5 - 18 55 -> 20 45 -> 80 (2:38:60 VIVIV).
18 - 22 20 80 Application 20 pL [or 5 pL] as bands of 10 mm [or 8 mm].
Flow rate 1.5 ทาบmin. Drying In air.
Detection Spectrophotometer at 220 nm. Detection Spray with anisaldehyde solution R and heat at
Injection 20 pL.
Peak identification Use the chromatogram supplied with
valerian dry extract HRS and the chromatogram obtained with
test solution. A faint violet zone due to valerenic acid may be
the reference solution to identify the peaks due to
acetoxyvalerenic acid and valerenic acid.
Furthermore, other zones may be present in the
System suitability. reference solution: chromatogram obtained with the test solution.
— relative retention with reference to valerenic acid (retention
time = about 19 min): acetoxyvalerenic acid = about 0.5. Top of the plate
expressed as valerenic acid, using the folloxring expression:
Valerenic acid: a violet zone
(41 -r A2) X 7ท2 XpX5 Acetoxyvalerenic acid: a violet zone A violet zone (acetoxyvalerenic
A3 X mi acid)
/4] = area of the peak due to acetoxyvalerenic acid in the A violet zone (hydroxyvalerenic
chromatogram obtained with the test solution; acid)
A2 = area of the peak due to valerenic acid in the
chromatogram obtained with the test solution; Reference solution Test solution
Aj = area of the peak due to valerenic acid in the
พ] = mass of the herbal drug to be examined used to TESTS
prepare the test solution, in grams; Loss on drying (2.8.17)
m2 = mass of valerian dry extract HRS used to prepare the Maximum 6.0 per cent.
reference solution, in grams; ASSAY
p = percentage content of valerenic acid in valerian dry Liquid chromatography (2.2.29).
extract HRS.
Solvent mixture methanol R, water R (50:50 P7F).
______________________________________________________________ _ Ph Eur Test solution In a 300 mL conical flask suspend 1.00 g of the
extract to be examined in 40 mL of water R whilst swirling.
Add 40 mL of methanol R and swirl for 1 h at 200 r/min.
Filter the suspension into a volumetric flask and rinse the
conical flask with 3 quantities, each of 5 mL, of the solvent
Valerian Dry Aqueous Extract * * mixture. Dilute to 100.0 mL with the solvent mixture.
(Ph. Eur. monograph 2400) *** Reference solution (a) Dissolve a quantity of valerian dry extract
Ph Ew_______________________________________________________________ HRS corresponding to 1.0 mg of valerenic acid in methanol R
and dilute to 10.0 mL with the same solvent. Sonicate for
DEFINITION
10 min and filter through a membrane filter (nominal pore
Extract produced from Valerian root (0453). size 0.45 pm).
Content Reference solution (b) Dilute 1.0 mL of reference solution (a)
Minimum 0.02 per cent of sesquiterpenic acids, expressed as to 50.0 mL with methanol R.
valerenic acid (0]5บ2202; Mr 234.3) (dried extract). Column-.
PRODUCTION — size’. I = 0.25 m, 0 = 4 mm;
The extract is produced from the herbal drug by a suitable — stationary phase’, octadecylsilyl silica gel for chromatography R
procedure using water at not less than 60 °C. (5 pm).
CHARACTERS Mobile phase’.
— mobile phase A: acetonitrile R13 5 g/L solution of phosphoric
Appearance
Brown or brownish, hygroscopic powder. acid R (20:80 V/Vfi
— mobile phase B: 5 g/L solution of phosphoric acid R,
IDENTIFICATION acetonitrile R1 (20:80 V!V}'3
Test solution Suspend 1.0 g of the extract to be examined in Time Mobile phase A Mobile phase B
10 mL of methanol R and sonicate for 10 min. Filter ±e (min) (per cent V/V) (per cent V/V)
supernatant through a membrane filter (nominal pore size 0-5 55 45
0.45 pm). Use the filtrate as the test solution. 5- 18 55 -> 20 45->80
Reference solution Dissolve 5 mg of acetoxyvalerenic acid R and 18 - 22 20 80
5 mg of valerenic acid R in 20 mL of methanol R.
2016 Valerian Preparations IV-415
Flow rale 1.5 mlVmin. Mobile phase glacial acetic acid R) ethyl acetate R, cyclohexane R
Detection Spectrophotometer at 220 nm. (2:38:60 VIVIV).
Injection 20 pL. Application 20 pL [or 5 pL] as bands of 10 mm [or 8 mm].
Identification of peaks Use the chromatogram supplied with Development Over a path of 10 cm [or 6 cm].
valerian dry extract HRS and the chromatogram obtained with Drying In air.
reference solution (a) to identify the peaks due to Detection treat with anisaldehyde solution R and heat at
acetoxyvalerenic acid and hydroxyvalerenic acid. 100-105 °C for 5-10 min; examine in daylight
Relative retention With reference to valerenic acid (retention Results See below the sequence of zones present in the
time = about 19 min): hydroxyvalerenic acid = about 0.2; chromatograms obtained with the reference solution and the
acetoxyvalerenic acid = about 0.5. test solution. Furthermore, other violet zones may be present
Calculate the percentage content of sesquiterpenic acids, in the chromatogram obtained with the test solution.
Top of the plate
(-41 4- A2) X 7ท2 X p x0.2
A3 X 7711
A\ =ะ area of the peak due to hydroxyvalerenic acid in Acetoxyvalerenic acid: a violet zone A violet zone (acetoxyvalerenic
acid)
the chromatogram obtained with the test solution;
A2 = area of the peak due to acetoxyvalerenic acid in
the chromatogram obtained with the test solution; 2 faint or very faint violet zones
A3 ะ= area of the peak due to valerenic acid in the Reference solution Test solution
(b);
rni = mass of the extract to be examined used to TESTS
prepare the test solution, in grams; Water (2.5.12)
tn2 — mass of valerian dry extract HRS used to prepare Maximum 5.0 per cent, determined on 0.5 g.
ASSAY
extract HRS.
Test solution Suspend 1.00 g of the extract to be examined in
------------------------------ ---------------------------------------------------------------------------- PhEur 50.0 mL of methanol R13 sonicate for 10 min and filter
through a membrane filter (nominal pore size 0.45 pm).
Reference solution Dissolve a quantity of valerian dry
Valerian Dry Hydroalcoholic / ** methanol R1 and dilute to 10.0 mL with the same solvent.
Extract ***** (nominal pore size 0.45 pm).
(Ph Eur monograph 1898) Column'.
Ph Elf._______________________________________________________ — size-. I = 0.25 m, 0 = 4.6 mm;
DEFINITION (5 pm).
Extract produced from Valerian root (0453).
Mobile phase:
Content — mobile phase A: acetonitrile Rl, 5 g/L solution of phosphoric
Minimum 0.25 per cent mini of sesquiterpenic acids, acid R (20:80 VIV)',
expressed as valerenic acid (C15H22O2; Mr 234.3) — mobile phase B: 5 g/L solution of phosphoric acid R,
(anhydrous extract). acetonitrile R1 (20:80 VIV);
PRODUCTION
The extract is produced from the herbal drug by a suitable Top of the plate
procedure using ethanol (30-90 per cent VIV) or methanol
(40-55 per cent VIV).
Valerenic acid ะ a violet zone A violet zone (valerenic acid)
CHARACTERS
Acetoxyvalerenic acid: a violet zone A violet zone (acetoxyvalerenic
Appearance acid)
Brown, hygroscopic powder.
IDENTIFICATION 2 faint or very faint violet zones
Thin-layer chromatography (2.2.27). Test solution
Reference solution
Test solution Suspend 1 g of the extract to be examined in
10 mL of methanol R and sonicate for 10 min. Filter the
supernatant through a membrane filter (nominal pore size Flow rate 1.5 mL/min.
0.45 pm). Detection Spectrophotometer at 220 nm.
Reference solution Dissolve 5 mg of acetoxyvalerenic acid R and Injection 20 pL.
5 mg of valerenic acid R in 20 mL of methanol R. Identification of peaks Use the chromatogram supplied with
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel valerian dry extract HRS and the chromatogram obtained with
plate 7? (2-10 pm)]. the reference solution to identify the peaks due to
IV-416 Valerian Preparations 2016
hydroxyvalerenic acid, acetoxyvalerenic acid and valerenic Results See below the sequence of zones present in the
acid. chromatograms obtained with the reference solution and the
System suitability: reference solution: test solution. Furthermore, other violet zones may be present
— relative retention with reference to valerenic acid (retention in the chromatogram obtained with the test solution.
time = about 19 min): hydroxyvalerenic acid = about
0.2; acetoxyvalerenic acid = about 0.5.
Top of the plate
(Al + A2 + A3) X 7ท2 X p X 5 Acetoxyvalerenic acid: a violet zone A violet zone (acetoxyvalerenic
A4 X mi acid)
Al = area of the peak due to hydroxyvalerenic acid in 2 faint or very faint violet zones
A2 = area of the peak due to acetoxyvalerenic acid in
A3 = area of the peak due to valerenic acid in the
TESTS
Ethanol (2.9.10)
A.I = area of the peak due to valerenic acid in the
95 per cent to 105 per cent of the quantity stated on the
label.
solution;
nil = mass of the extract to be examined used to ASSAY
prepare the test solution, in grams; Liquid chromatography (2.2.29).
m2 = mass of valerian dry extract HRS used to prepare Test solution Dilute 10.0 g of the tincture to be examined to
the reference solution, in grams; 50.0 mL with methanol Rl.
p = percentage content of valerenic acid in valerian dry Reference solution Dissolve an amount of valerian diy extract
extract HRS. HRS corresponding to 1.0 mg of valerenic acid in
____________________________________________________ __ ________ PhEur methanol Rl and dilute to 10.0 mL with the same solvent.
Column:
Valerian Tincture * * — size: I = 0.25 m, 0 ะะะ 4.6 mm;
(Ph. Eur. monograph 1899) *** (5 pm).
PhEur______________________________________________________ _________ Mobile phase:
DEFINITION — mobile phase A: acetonitrile Rl 3 5 g/L solution of phosphoric
acid R (20:80 V/V),
Tincture produced from Valerian root (0453).
— mobile phase B: 5 g/L solution of phosphoric acid R3
Content acetonitrile Rl (20:80 V/V)'3
Minimum 0.015 per cent m/m of sesquiterpenic acids,
expressed as valerenic acid (C15H22O2; Mr 234.3).
PRODUCTION (min) (per cent V/V) (per cent V/V)
The tincture is produced from 1 part of the drug and 5 parts 0- 5 55 45
of ethanol (60 to 80 per cent V/V) by an appropriate
5 - 18 55 -> 20 45 -> 80
procedure.
18 - 22 20 80
CHARACTERS
Appearance
Brown liquid. Flow rate 1.5 mUmin.
IDENTIFICATION Detection Spectrophotometer at 220 nm.
Thin-layer chromatography (2.2.27). Injection 20 pL.
Test solution Dilute 5 mL of the tincture to be examined with Peak identification Use the chromatogram supplied with
5 mL of ethanol (70 per cent V/V) R. valerian dry extract HRS and the chromatogram obtained with
Reference solution Dissolve 5 mg of acetoxyvalerenic acid R and the reference solution to identify the peaks due to
5 mg of valerenic acid R in 20 mL of methanol R. acetoxyvalerenic acid and valerenic acid.
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel System suitability: reference solution:
plate R (2-10 pm)]. — relative retention with reference to valerenic acid (retention
time = about 19 min): acetoxyvalerenic acid = about 0.5.
(2:38:60 V/V/V). Calculate the percentage content of sesquiterpenic adds,
Drying In air.
(Al 4- A2) X 7ท2 X p X 5
A3 X 7711
2016 Verbena Herb IV-417
/I I = area of the peak due to acetoxyvalerenic acid in

^2 = area of the peak due to valerenic acid in the
■^3 = area of the peak due to valerenic acid in the
solution;
พ! = mass of the tincture to be examined used to
พ2 = mass of valerian dry extract HRS used to prepare
the reference solution, in grams;
extract HRS.
---- ------------------------------------------------------- Ph Eur
Verbena Herb **
Ph Eir______________________________________________________________
DEFINITION
Whole or fragmented, dried aerial parts of Verbena
officinalis L. collected during flowering.
Content
Minimum 1.5 per cent of verbcnalin (C17H24O10; A4r 388.4)
(dried drug).
IDENTIFICATION
A. The stem is greenish-brown, quadrangular, longitudinally Figure 1854.-1. - Illustration for identification test B of powdered
grooved and roughly hairy, especially on the angles. herbal drug of verbena herb
The larger leaves are petiolate and deeply pinnately lobed, Results See below the sequence of zones present in the
with bluntly dentate margins, the smaller leaves are sessile, chromatograms obtained with the reference solution and the
not lobed, with crenate or dentate margins; the surfaces are test solution. Furthermore, other zones may be present in the
rough and covered with bristly hairs, particularly over the chromatogram obtained with the test solution.
veins, which are prominent on the lower surface. The flowers
are numerous, arranged in a slender spike in the axils of leaf Top of the plate
like bracts; the tubular calyx has 5 acutely pointed lobes with
the pale pink or lilac corolla forming a tube about twice as
long as the calyx. A brown or green zone
Arbutin: a blue or brown zone
greenish-brown. Examine under a microscope using chloral
hydrate solution R. The powder shows the following diagnostic An intense brownish-grey zone
characters (Figure 1854.-1): fragments of the leaves, which in Rutin ะ a dark brownish-yellow
surface view [C] show sinuous-walled epidermal cells [Ca]
with anisocytic [Cb] or anomocytic [Cc] stomata (2.8.3) 3
more numerous on the lower epidermis; fragments of stem
epidermis [A] consisting of long, polygonal or rectangular
epidermal cells [Aa] with thickened walls and stomata [Ab];
covering trichomes, unicellular, thick-walled, up to 500 pm TESTS
long, wide at the base and arising from the centre of a single Aloysia citrodora
ring of domed, spherical epidermal cells, in surface view [B] A. A lemon-like odour indicates the presence of Aloysia
or in side view [D]; occasional glandular trichomes of 2 citrodora.
types: (a) long stalk with a flattened head about 35 pm in
diameter and consisting of 4-8 radiating cells in side view [E]
or in surface view of the head [G], and (b) short unicellular Test solution To 0.5 g of the powdered herbal drug (710)
stalk and an enlarged ovate head composed of 4 radiating (2.9.12) add 5 mL of methanol R. Heat in a water-bath at
cells in surface view [Cd] or in transverse section [K]; 60 °C for 10 min. Cool and filter.
triangular-ovoid or rounded pollen grains about 30 pm in Reference solution Dissolve 10 mg of arbutin R and 10 mg of
diameter, with 3 pores and a smooth exine [J]; many rutin R in methanol R and dilute to 10 mL with the same
fragments of stems [F] consisting of groups of fibres [Fb], solvent.
vessels [Fa] and fragments of parenchyma [Fc]; isolated Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
fragments of fibres [H]. plate R (2-10 pm)].
c. Examine the chromatograms obtained in test B for Aloysia Mobile phase anhydrous formic acid R} glacial acetic acid R>
citrodora. water R, ethyl acetate R (11:11:27:100 VIVIVIV).
IV-418 Verbena Herb 2016
1. verbenalin 2. ferulic acid 3. unknown substance (may be absent) 4. acteoside
Figure 1854.-2. - Chromatogram for the assay of verbena herb: test solution
Application 20 pL [or 5 pL] as bands of 10 mm [or 8 mm]. Mobile phase:

Development Over a path of 12 cm [or 6 cm]. — mobile phase A: 0.3 per cent VIV solution Qi phosphoric
Drying In air. acid R'i
100-105 °C for about 10 min; examine in daylight. Time Mobile phase A Mobile phase B
Results The chromatogram obtained with the test solution (min) (per cent V/V) (per cent V/V)
shows no intense blue or violet zone approximately at the 0-20 93 -> 83 7 —> 17
position of rutin in the chromatogram obtained with the 17
20-30 83
reference solution.
30-35 83 -> 75 17 -> 25
powdered herbal drug (710) (2.9.12) by drying in an oven at Flow rate 1.0 mUmin.
105 °C for 2 h. Detection Spectrophotometer at 240 nm.
Total ash (2.4.16) Injection 20 pL.
Maximum 10.0 per cent. System suitability Test solution:
Ash insoluble in hydrochloric acid (2.8.1) — the chromatogram obtained is similar to the
Maximum 2.0 per cent. chromatogram shown in Figure 1854.-2;
ASSAY acid and acteoside.
Calculate the percentage content of verbenalin using the
Internal standard solution Dissolve 10.0 mg of ferulic acid R in following expression:
ethanol (60 per cent V/V) R and dilute to 100.0 mL with the
same solvent. Al X A4 X 7ท2 X 1000
Test solution To 1.00 g of the powdered herbal (hug (710) A2 X A3 X mi
(2.9.12) add 50.0 mL of the internal standard solution and
stir with a magnetic stirrer for 2 h. Centrifuge for 15 min and A1 = area of the peak due to verbenalin in the
filter the supernatant using a membrane filter (nominal pore chromatogram obtained with the test solution;
size 0.45 pm). A2 = area of the peak due to verbenalin in the
Reference solution Dissolve the contents of a vial of chromatogram obtained with the reference
verbenalin CRS in the internal standard solution and dilute to solution;
5.0 mL with the same solution. A3 = area of the peak due to ferulic acid in the
Precolumn’. chromatogram obtained with the test solution;
— size: I = 0.01 m, 0 = 4.0 mm; A4 = area of the peak due to ferulic acid in the
— stationary phase: octadecylsilyl silica gel for chromatography R chromatogram obtained with the reference
(5 pm). solution;
Column: m1 = mass of the herbal drug used to prepare the test
— size: I = 0.25 m, 0 = 4.0 mm; solution, in grams;
— stationary phase: octadecylsilyl silica gel for chromatography R m2 = mass of verbenalin in the reference solution, in
(5 pm); grams.
— temperature: 20 °C. ______
2016 Willow Bark IV-419
Willow Bark *****

Preparation
Willow Bark Dry Extract
_
definition
Whole or fragmented dried bark of young branches or whole
dried pieces of current-year twigs of various species of genus
•SaJix including ร. purpurea L., ร. daphnoides Vill.
and ร. fragilis L.
Content
Minimum 1.5 per cent of total salicylic derivatives, expressed
as salicin (C13H18O7; Mr 286.3) (dned drug).
IDENTIFICATION
A. The bark is 1-2 mm thick and occurs in flexible,
elongated, quilled or curved pieces. The outer surface is
smooth or slightly wrinkled longitudinally and greenish-
yellow or brownish-grey. The inner surface is smooth or
finely striated longitudinally and white, pale yellow or
reddish-brown, depending on the species. The fracture is
short in the outer part and coarsely fibrous in the inner
region. The diameter of current-year twigs is not greater than
10 mm. The wood is white or pale yellow.
B. iMicroscopic examination (2.8.23}. The powder is pale
yellow, greenish-yellow or light brown. Examine under a
bundles [B, C] of narrow fibres [Ba, Ca], up to about
Figure 1583.-1. - Illustration for identification test B of
600 Jim long, with very thick walls and surrounded by a powdered herbal drug of willow bark
crystal sheath containing prisms of calcium oxalate [Bb, Cb];
parenchymatous cells of the cortex [D, J], with thick, pitted
and deeply beaded walls [Da], and containing large cluster Results See below the sequence of zones present in the
crystals of calcium oxalate [Ga, Ja]; some parenchyma cells chromatograms obtained with the reference solution and test
are collenchymatous [G]; uniseriate medullary rays, in solutions (a) and (b). Furthermore, other zones may be
tangential section [Db]; thickened cork cells, in surface present in the chromatograms obtained with test solutions (a)
view [F]; numerous scattered prism crystals [E] and cluster and (b).
crystals [A] of calcium oxalate; fragments of brownish
collenchyma from the buds may also be present. Twigs show,
Top of the plate
additionally, wood fragments [H] composed of lignified
fibres [Ha] and vessels [Hb], sometimes accompanied by
medullary rays [He].
Several reddish-violet
c. Thin-layer chromatography (2.2.27). zones may be present
Test solution (a) To 1.0 g of the powdered herbal drug (355)
(2.9.12} add 10 mL of methanol R. Heat on a water-bath at Salidn: a reddish- A weak reddish-violet A reddish-violet zone
violet zone zone (salicin) (salicin)
about 50 °C, with frequent shaking, for 10 min. Cool and
filter.
Test solution (b) To 5.0 mL of test solution (a) add 1.0 mL of Chlorogenic add: a
a 50 g/L solution of anhydrous sodium carbonate R and heat in brown zone
a water-bath at about 60 °C for 10 min. Cool and filter if
necessary. Reference solution Test solution (a) Test solution (b)
Reference solution Dissolve 2 mg of salicin R and 2 mg of

chlorogenic acid R in 1.0 mL of methanol R.
TESTS
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel Foreign matter (2.8.2}
plate 7? (2-10 Jim)]. Maximum 3 per cent of twigs with a diameter greater than
Mobile phase water R, methanol R, ethyl acetate R 10 mm and maximum 2 per cent of other foreign matter.
(8:15:77 VIVIV}.
Cadmium (2.4.27}
Application 10 jiL [or 2 J1L] as bands. Maximum 2.0 ppm.
Development Over a path of 15 cm [or 6 cm]. Loss on drying (2.2.32}
Drying In a current of warm air. Maximum 11 per cent, determined on 1.000 g of the
Detection Treat with a mixture of 5 volumes of sulfuric acid R powdered herbal drug (355) (2.9.12} by drying in an oven at
and 95 volumes of methanol R. Heat at 100-105 °C for 5 min 105 °C for 2 h.
IV-420 Willow Preparations 2016
Total ash (2.4.16)

Maximum 10 per cent. Willow Bark Dry Extract
ASSAY (Ph. Eur. monograph 2312)
Liquid chromatography (2.2.29). Ph Eur___ ______________________________
Test solution To 1.000 g of the powdered herbal drug (355) DEFINITION

(2.9.12) add 40 mL of methanol R and 40.0 mL of a 4.2 g/L Dry extract produced from Willow bark (1583).
solution of sodium hydroxide R. Heat in a water-bath at about
60 °C under a reflux condenser, with frequent shaking, for Content
about 1 h. After cooling, add 4.0 mL of a 103.0 g/L solution Minimum 5.0 per cent of total salicylic derivatives, expressed
of hydrochloric acid R. Filter the suspension into a 100 mL as salicin (C13H18O7; Mr 286.3) (dried extract).
volumetric flask, wash and dilute to 100.0 mL with a mixture PRODUCTION
of equal volumes of methanol R and water R. Filter through a The extract is produced from the herbal drug by a suitable
membrane filter (nominal pore size 0.45 pm). procedure using either water or a hydroalcoholic solvent
Reference solution Dissolve 5.0 mg of picein R in 25.0 mL of a equivalent in strength to a maximum of 80 per cent VIV
mixture of 20 volumes of water R and 80 volumes of ethanol.
methanol R (solution A). Dissolve 15.0 mg of salicin CRS in CHARACTERS
25 mL of a mixture of 20 volumes of water R and Appearance
80 volumes of methanol R‘3 add 5.0 mL of solution A and Yellowish-brown amorphous powder.
dilute to 50.0 mL with water R.
Column’. IDENTIFICATION
— size: 7 = 0.10 m, 0 = 4.6 mm; Thin-layer chromatography (2.2.27).
— stationary phase: octadecylsilyl silica gel for chromatography R Test solution (a) To 0.200 g of the extract to be examined
(3 pm). add 5 mL of methanol R. Sonicate for 5 min, filter and dilute
Mobile phase: to 10 mL with methanol R.
— mobile phase A: tetrahydrofuran R3 0.5 per cent VIV Test solution (b) To 5.0 mL of test solution (a) add 1.0 mL of
solution of phosphoric acid R (1.8:98.2 VIV); a 50 g/L solution of anhydrous sodium carbonate R and heat in
— mobile phase B: tetrahydrofuran R'3 a water-bath at about 60 °C for 10 min. Cool and filter if
necessary.
Reference solution Dissolve 2.0 mg of salicin R and 2.0 mg of
chlorogenic acid R in 1.0 mL of methanol R.
0- 15 100 0 plate R (2-10 pm)].
15-17 100 -> 90
Mobile phase water R3 methanol R, ethyl acetate R
0 -> 10
(8:15:77 VIVIV).
17-23 90 10 Application 10 pL [or 2 pL] as bands.
Flow rate 1.0 mL/min. Detection Spray with a mixture of 5 volumes of sulfuric acid R
and 95 volumes of methanol R. Heat at 100-105 °C for 5 min
Injection 10 pL.
Retention lime Salicin = about 6.4 min; picein = about chromatograms obtained with the reference solution and test
7.7 min. solutions (a) and (b). Furthermore, other zones may be
System suitability: reference solution: present in the chromatogram obtained with test solutions (a)
— resolution: minimum 1.5 between the peaks due to salicin and (b).
and picein.
Top of the plate
Calculate the percentage content of total salicylic derivatives,
expressed as salicin, using the following expression:
Several reddish-violet
Al X m2 X p X 2 zones may be present
Salicin: a reddish-violet A weak reddish-violet A reddish-violet zone
A2 X mi
zone (salicin) (salicin)
Al = area of the peak due to salicin in the

Chlorogenic acid: a
chromatogram obtained with the test solution; brown zone
A2 = area of the peak due to salicin in the Reference solution Test solution (a) Test solution (b)
solution; ASSAY
m1 = mass of the herbal drug to be examined used to Liquid chromatography (2.2.29).
m2 = mass of salicin CRS used to prepare the reference
40 mL of methanol R and 40.0 mL of 0.1 M sodium
solution, in grams;
hydroxide. Heat in a water-bath at about 60 °C under a reflux
p = percentage content of salicin in salicin CRS.
condenser, with frequent shaking, for about 1 h. After
---------------------------- ------------------------------------ ---------------------------_____ Ph Eur cooling, add 4.0 mL of 1 M hydrochloric acid. Filter the
2016 Withania Somnifera Root IV-421
suspension into a 100 mL volumetric flask, then wash and IDENTIFICATION

dilute to 100.0 mL with a mixture of equal volumes of A. Pieces of root, cut into lengths of up to 8 cm, varying in
water R and methanol R. Filter through a membrane filter diameter from 2 mm to 1 cm, with some narrower pieces of
(nominal pore size 0.45 pm). rhizome, often cut at the transition zone. Outer surface pale
Reference solution Dissolve 5.0 mg of picein R in 25.0 mL of a greyish-brown, somewhat darker brown in larger specimens.
mixture of 20 volumes of water R and 80 volumes of Fracture short, showing a whitish interior. The cut surface of
methanol R (solution A). Dissolve 15.0 mg of salicin CRS in the root may show a distinction between the xylem and other
25 mL of a mixture of 20 volumes of water R and tissues marked by a faint yellow-green cambial ring.
80 volumes of methanol R. Add 5.0 mL of solution A and B. Reduce to a powder (355). Examine under a microscope
dilute to 50.0 mL with water R. using chloral hydrate solution. Cork cells in surface view
Column: polygonal, in sectional view rectangular, thin-walled,
size: I = 0.10 m, 0 = 4.6 mm; yellowish brown, often broken. Parenchymatous cells in
stationary phase: octadecylsilyl silica gel for chromatography R groups, elongated, rectangular, or oval to round, filled with
(3 pm). starch; some pitted, lightly lignified, found alongside vascular
Mobile phase: fragments; parenchyma of the medullary rays, one or two
mobile phase A: tetrahydrofuran R, 0.5 per cent v/v cells wide shown crossing xylem elements at right angles;
solution of phosphoric acid R (1.8:98.2 V/Vy, Occasional fragments of spiral, scalariform or pitted vessels
mobile phase B: tetrahydrofuran R', with broad lumen; tracheids and vessels usually heavily
lignified, reticulate or bordered pitted, single or in small
Time Mobile phase A Mobile phase B groups. Fibres often accompanying vessels, thick walled,
(min) (per cent V/V) (per cent V/V) heavily pitted, and lignified; others less pitted and lignified,
0 - 15 100 0 thin walled, either found singly or in groups of two or three.
15 - 17 100 -» 90 0 -> 10 Microcrystals of calcium oxalate scattered or occasionally in
17 - 23 90
idioblasts. Examine under a microscope using a 50% v/v
10
solution of glycerol. Starch granules abundant, simple or 2 to
23 - 25 90 ■» 100 10-» 0 4 compound, round to oval, with a point, stellate or cleft
25 - 40 100 0 hilum.
c. Carry out the method for thin-layer chromatography.
Flow rate 1.0 mL/min. Appendix in A, using the following solutions.
Detection Spectrophotometer at 270 nm. (1) Shake 0.5 g of freshly powdered (355) drug with 1 mL of
Injection 10 pL. dilute ammonia R4, add 10 mL of methanol, sonicate for
Retention time Salicin = about 6.4 min; 10 seconds, heat on a water-bath for 3 minutes, cool, filter,
picein = about 7.7 min. evaporate the filtrate to dryness at 60° and dissolve the dried
System suitability: reference solution: residue in 1 mL of methanol. Filter through a 0.45 pm filter.
— resolution: minimum 1.5 between the peaks due to salicin (2) 0.1% w/v each of withaferin A CRS, withanolide B CRS
and picein. and p-sitosterol in methanol.
Calculate the percentage content of total salicylic derivatives, CHROMATOGRAPHIC CONDITIONS
expressed as salicin, from the following expression: (a) Use a silica gel F254 precoated high performance plate
(Merck silica gel F254 HPTLC plates are suitable).
Al X 7712 X pX 2
A2 X 7721
(c) Apply 2 pL of each of solution, as 6 mm bands.
Ax ะ= area of the peak due to salicin in the (d) Develop the plate to 8 cm.
A2 = area of the peak due to salicin in the
ultraviolet light (254 nm). Immerse the plate in 5% v/v of
methanolic sulfuric acid for 1 second, allow to dry in air, heat
solution;
at 110° for 2 minutes and examine immediately in daylight.
พ1 = mass of the extract to be examined used to
Examine the derivatised plate under ultraviolet light (366 nm).
m2 = mass of salicin CRS used to prepare the reference MOBILE PHASE
solution, in grams; 5 volumes of anhydrous formic acid, 15 volumes of ethyl acetate

p = percentage content of salicin in salicin CRS. and 50 volumes of toluene.
__ ____________________________________________________________ Ph Eur SYSTEM SUITABILITY
solution (2) shows three clearly separated bands.
CONFIRMATION
Withania Somnifera Root The bands with Rf values of approximately 0.1 (withaferin
definition A), 0.26 (withanolide B) and 0.57 (P-sitosterol) in the
Withania Somnifera Root consists of the dried mature roots chromatogram obtained with solution (1) correspond in
of Withania somnifera (L.) Dunal. colour and position to those in the chromatogram obtained
It contains not less than 0.01% withaferin A (บ28พ3806) and with solution (2).
not less than 0.01% withanolide A (C29 H42O7). TESTS
PRODUCTION Absence of Withania coagulans
It is collected in winter, washed, dried and cut into short In Identification test c, the derivatised plate under ultraviolet
pieces. light (366 nm) shows no orange band with an Rf of
IV-422 Wormwood 2016
approximately 0.2 in the chromatogram obtained with Al ทา2 Vi 100

solution (1).
A:X V: x ทไ1 XPX 100-d
Ash
A1 = Area of the peak due to withaferin A in the
Acid-insoluble ash
Not more than 1.0%, Appendix XI K.
A2 = Area of the peak due to withaferin A in the
Loss on drying chromatogram obtained with solution (2).
When dried for 2 hours at 105°, loses not more than 12.0% m 1 = Weight of the drug being examined in mg.
of its weight. Use 1 g. 1ท2 = Weight of withaferin A CRS in mg.
ASSAY V1 = Dilution volume of solution (1) in mL.
Carry out the method for liquid chromatography, V2 = Dilution volume of solution (2) in mL.
Appendix in D, using the following solutions. p = Percentage content of withaferin A in withaferin A
CRS.
(1) Extract 1 g of the powdered drug with 3.0 mL of
methanol with the aid of น]trasound for 10 minutes, centrifuge
examined.
at 3000 rpm for 5 minutes and retain the supernatant
extract. The extraction is repeated twice as described. Withanolide A Using the retention time and peak area of
Combine the three supernatant extracts, adjust the total the peak due to withanolide A in the chromatogram obtained
volume of ±e combined extracts to 20.0 ml with methanol with solution (2), locate and integrate the peak due to
and filter through a 0.45-pm filter. withanolide A in the chromatogram obtained with
solution (1).
(2) 0.02% w/v each of withaferin A CRS and withanolide A
CRS and 0.01% w/v of withanolide B CRS in methanol. Calculate the content of withanolide A in the sample using
the declared content of withanolide A (C29H42O7) in
withanolide A CRS and the following expression:
with dodecylsilyl silica gel for chromatography (4 pm)
(Phenomenex Synergi Max-RP 80A is suitable). Al ๓2 V, 100
(b) Use gradient elution and the mobile phases described A:X V2X ๓1 xpxioo-d
below. Equilibrate the column with a mixture of 65% mobile
phase A and 35% mobile phase B for at least 15 minutes. Aj = Area of the peak due to withanolide A in the
(c) Use a flow rate of 1 mL per minute. chromatogram obtained with solution (1).
(d) Use a column temperature of 50°. A2 ะะะ Area of the peak due to withanolide A in the
(e) Use a detection wavelength of 230 nm. chromatogram obtained with solution (2).
mi = Weight of the drug in mg.
(f) Inject 20 |1L of each solution.
m2 = Weight of withanolide A CRS in mg.
MOBILE PHASE V1 = Dilution volume of solution (1) in mL.
Mobile phase A water. v2 = Dilution volume of solution (2) in mL.
Mobile phase B Equal volumes of ethanol (96%)) and p = Percentage content withanolide A in withanolide A
methanol. CRS.
examined.
Time Mobile phase A Mobile phase B Comments
(Minutes) % v/v % v/v STORAGE
0-5 65 35
Withania Somnifera Root should be protected from moisture.
isocratic
5-30 65->55 35—>45 linear gradient
30-31 55->0 45->100 linear gradient
31-36 0 100 isocratic
Wormwood * *
Ph Eur________________________________________________ __ ____________
DEFINITION
SYSTEM SUITABILITY Basal leaves or slightly leafy, flowering tops, or mixture of
The test is not valid unless, in the chromatogram obtained these dried, whole or cut organs of Artemisia absinthium L.
with solution (2), the resolution factor between the first two
Content
main peaks, of withaferin A and withanolide A is at least 5.0 Minimum 2 mL/kg of essential oil (dried drug).
and the symmetry factor for both peaks is less than 1.3.
IDENTIFICATION
A. The leaves are greyish or greenish, densely tomentose on
Withaferin A Using the retention time and peak area of the both surfaces. The basal leaves, with long petioles, have
peak due to withaferin A in the chromatogram obtained with triangular or oval bipinnatisect or tripinnatisect lamina, with
solution (2), locate and integrate the peak due to withaferin rounded or lanceolate segments. The cauline leaves are less
A in the chromatogram obtained with solution (1). segmented and the apical leaves are lanceolate. The stem of
Calculate the content of withaferin A in the sample using the the flower-bearing region is greenish-grey, tomentose, up to
declared content withaferin A (C2sH38O6) in withaferin 2.5 mm in diameter and usually with 5 flattened longitudinal
A CRS and the following expression: grooves. The capitula are arranged as loose, axillary panicles,
2016 Wormwood IV-423
inserted at the level of the lanceolate or slightly pinnatisect c. Thin-layer chromatography (2.2.27).
leaves; they are spherical or flattened hemispherical, 2-4 mm
Test solution Place 2 g of the powdered herbal drug (355)
in diameter and consist of a grey, tomentose involucre, the (2.9.12) in 50 mL of boiling water R and allow to stand for
outer bracts linear, inner layer ovate, blunt at the apices with 5 min, shaking the flask several times. After cooling, add
scarious margins, a receptacle with very long paleae up to 5 mL of a 100 g/L solution of lead acetate R. Mix and filter.
1 mm or more long, numerous yellow, tubular, Rinse the flask and the residue on the filter with 20 mL of
hermaphroditic florets about 2 mm long and few yellow, ray water R. Shake the filter with 50 mL of methylene chloride R.
florets.
Separate the organic layer, dry over anhydrous sodium
B. Microscopic examination (2.8.23). The powder is sulfate R, filter and evaporate the filtrate to dryness on a
greenish-grey. Examine under a microscope using chloral water-bath. Dissolve the residue in 0.5 mL of ethanol
hydrate solution R. The powder shows the following diagnostic (96 per cent) R.
characters (Figure 1380.-1.): many T-shaped trichomes [A] Reference solution Dissolve 2 mg of methyl red R and 2 mg of
with a short uniseriate stalk consisting of 1-5 small cells, resorcinol R in 10.0 mL of methanol R.
perpendicularly capped by a very long, undulating terminal
ceน tapering at the ends; fragments of epidermises in surface
view [D] with sinuous or wavy walls, anomocytic stomata Mobile phase acetone R3 glacial acetic acid R> toluene R,
(2.8.3) [Da], covering trichomes [Db] and glandular methylene chloride R (10:10:30:50 VIVIVIV).
trichomes containing oil [De] or not containing oil [Dd], Application 10 pL as bands.
each with a short, biseriatc, 2-celled stalk and a biseriate Development Over a path of 15 cm.
head with 2-4 cells; free glandular trichomes in side view [C]; Drying In air.
fragments of the corollas of the tubular and ray florets, some
Detection A Spray with acetic anhydride - sulfuric acid solution R
containing small cluster crystals of calcium oxalate [H];
numerous paleae each composed of a small cell forming a
stalk and a very long, cylindrical and thin-walled terminal cell Results A The chromatogram obtained with the test solution
about 1-1.5 mm long, either whole [E] or limited to the shows a blue zone due to artabsin shortly above a red zone
distal part [B]; spheroidal pollen grains, about 30 pm in due to methyl red in the chromatogram obtained with the
diameter, with 3 pores and a finely warty exine [G]; reference solution.
fragments of vascular tissue from the leaves [F] or the Detection B Examine in daylight while heating at 100-105 °C
stems [J] consisting of vessels with spiral or annular for 5 min.
thickenings [Fa], or with bordered pits [Ja], fibres [Fb, Jb] Results B The chromatogram obtained with the reference
and parenchymatous cells with pitted, moderately thickened solution shows in the middle third a red zone due to methyl
walls [Fc, Jc]. red and below it a light pink zone due to resorcinol.
The chromatogram obtained with the test solution shows an
intense red or brownish-red zone due to absinthin with a
similar Rp value to that of the zone due to resorcinol in the
chromatogram obtained with the reference solution. Other
zones are visible, but less intense than that due to absinthin.
TESTS
Minimum 10 000.
105 °C for 2 h
Total ash (2.4.16)
ASSAY
Use 50.0 g of the cut drug, a 1000 mL round-bottomed flask
and 500 mL of water R as the distillation liquid. Add 0.5 mL
of xylene R in the graduated tube. Distil at a rate of
2-3 mL/min for not less than 3 h.
_____________________________________ ________________________ PhEur

herbal drug of wormwood
IV-424 Yarrow 2016
Yarrow
Ph Ear______________________________________________________________
DEFINITION
Whole or cut, dried flowering tops of Achillea millefolium L.
Content
— essential oil', minimum 2 mL/kg (dried drug);
— proazulenes, expressed as chamazulene (C14H 16; Mr 184.3):
minimum 0.02 per cent (dried drug).
IDENTIFICATION
A. The leaves are green or greyish-green, faintly pubescent
on the upper surface and more pubescent on the lower
surface, 2-3 pinnately divided with linear lobes and a finely
pointed whitish tip. The capitula are arranged in a corymb at
the end of the stem. Each capitulum, 3-5 mm in diameter,
consists of the receptacle, usually 4-5 ligulate ray-florets and
3-20 tubular disk-florets. The involucre consists of 3 rows of
imbricate lanceolate, pubescent green bracts arranged with a
brownish or whitish, membranous margin. The receptacle is
slightly convex and, in the axillae of paleae, bears ligulate
ray-florets with a three-lobed, whitish or reddish ligule and
tubular disk-florets with a radial, five-lobed, yellowish or light
brownish corolla. The pubescent green, partly brown or
violet stems are longitudinally furrowed, up to 3 mm 1hick
with a light-coloured medulla.
B. Microscopic examination (2.8.23). The powder is green or
greyish-green. Examine under a microscope using chloral
characters (Figure 1382.-1): fragments of the stem epidermis
herbal drug ofyarrow
(surface view [K]), with cells having a smooth cuticle and
anomocytic stomata (2.8.3)', fragments of leaf and bract Mobile phase ethyl acetate R, toluene R (5:95 V/V).
epidermises (surface view [B]), with cells having wavy and Application 20 pL as bands.
irregularly thickened walls, a finely striated cuticle and Development Over a path of 10 cm.
anomocytic stomata (2.8.3)', very rare glandular trichomes Dying In air.
with a short stalk and a head formed of 2 rows of 3-5 cells
Detection treat with anisaldehyde solution R, heat at
enclosed in a bladder-like membrane [H]; uniseriate, whole
100-105 °C for 5-10 min and examine in daylight.
or fragmented covering trichomes [A] consisting of 4-6 small,
more or less isodiametric cells at the base and a thick-walled, Results The chromatogram obtained with the reference
often somewhat tortuous terminal cell, about 400 pm to solution shows in the upper part a red zone (guaiazulene)
greater than 1000 pm long; fragments of the ligulate corolla and in the middle part a blue or greyish-blue zone (cineole).
with papillary epidermal cells [D]; fragments of the corolla The chromatogram obtained with the test solution shows a
tubes, with sinuous epidermal cells, covered by a thin striated violet zone a little above the zone due to guaiazulene in the
cuticle (surface view [F]); small-celled parenchyma from the chromatogram obtained with the reference solution; below
corolla tubes containing cluster crystals of calcium this zone a reddish-violet zone; below which, 1-2 not clearly
oxalate [E]; groups of lignified and pitted cells from the separated greyish-violet or greyish zones (which changes to
bracts [G]; spherical pollen grains, about 30 pm in diameter, greenish-grey after a few hours) and a reddish-violet zone a
with 3 germinal pores and a spiny exine [C]; groups of little above the zone due to cineole in the chromatogram
sclerenchymatous fibres and small vessels with spiral or obtained with the reference solution. Furthermore, other faint
annular thickening, from the stem [J]. zones may be present.
c. To 2.0 g of the powdered herbal drug (710) (2.9.12) add TESTS
25 mL of ethyl acetate R, shake for 5 min and filter. Foreign matter (2.8.2)
Evaporate to dryness on a water-bath and dissolve the Maximum 5 per cent of stems with a diameter greater than
residue in 0.5 mL of toluene R (solution A). To 0.1 mL of 3 mm and maximum 2 per cent of other foreign matter.
this solution add 2.5 mL of dimethylaminobenzaldehyde Loss on drying (2.2.32)
solution R8 and heat on a water-bath for 2 min. Allow to Maximum 12.0 per cent, determined on 0.500 g of the
cool. Add 5 mL of light petroleum R and shake the mixture powdered herbal drug (355) (2.9.12) by drying in an oven at
vigorously. The aqueous layer shows a blue or greenish-blue 105 °C for 2 h.
colour.
Total ash (2.4.16)
D. Thin-layer chromatography (2.2.27). Maximum 10.0 per cent.
Test solution Use solution A prepared in identification test c. Ash insoluble in hydrochloric acid (2.8.1)
Reference solution Dissolve 10 mg of cineole R and 10 mg of Maximum 2.5 per cent.
guaiazulene R in 20 mL of toluene R.
2016 Yarrow IV-425
ASSAY
Essential oil {2.8.12}
Use 20.0 g of cut herbal drug, a 1000 mL round-bottomed
flask and 500 mL of a mixture of 1 volume of water R and
9 volumes of ethylene glycol R as the distillation liquid.
Add 0.50 mL of 13234-trimethylbenzene R in the graduated
tube. Distil at a rate of 3-4 mL/min for 4 h.
Stop cooling at the end of distillation and continue distilling
until the blue, steam-volatile components have reached the
lower end of the cooler. Immediately start cooling again, to
avoid warming the separation space. Stop the distillation after
10 min.
Proazulenes
To ensure that as little water as possible is transferred,
transfer the blue mixture of essential oil and
1 ^^-trimethylbenzene obtained in the assay of essential oil
into a 50 mL volumetric flask with the aid of small portions
of xylene R, rinsing the graduated tube of the apparatus with
xylene R, and dilute to 50.0 mL with the same solvent.
Measure the absorbance (2.2.25) at 608 nm using xylene R as
the compensation liquid.
Calculate the percentage content of proazulenes, expressed as
chamazulene, using the following expression:
A X 2.1
m
i.e. taking the specific absorbance of chamazulene to be 23.8.

_______________________________ ______________________________ _ Ph Eur
Monographs
Materials for use in the

Manufacture of Homoeopathic
Preparations
2016 Homoeopathic Preparations FV-429
Homoeopathic Preparations ***** Glycerol macerates are liquid preparations obtained from raw
materials of botanical, zoological or human origin by using
(Ph. Eur. monograph 1038) *** glycerol or a mixture of glycerol and either ethanol of a
PtiEir________ _____________________________________________________ suitable concentration or a solution of sodium chloride of a
suitable concentration.
DEFINITION
Homoeopathic preparations are prepared from substances, Potentisation
Dilutions and triturations are obtained from stocks by a
products or preparations called stocks, in accordance with a
process of potentisation in accordance with a homoeopathic
homoeopathic manufacturing procedure. A homoeopathic
manufacturing procedure: this means successive dilutions and
preparation is usually designated by the Latin name of the
succussions, or successive appropriate triturations, or a
stock, followed by an indication of the degree of dilution
combination of the 2 processes.
and/or potentisation, if applicable.
The potentisation steps are usually one of the following:
Raw materials
— 1 part of the stock plus 9 parts of the vehicle; they may
Raw materials for the production of homoeopathic
be designated as ‘D', ‘DH' or ‘X' (decimal);
preparations may be of natural or synthetic origin.
— 1 part of the stock plus 99 parts of the vehicle; they may
For raw materials of zoological or human origin, adequate be designated as ‘C' or ‘CH' (centesimal).
measures are taken to minimise the risk of agents of The number of potentisation steps defines the degree of
infection, including viruses (5./. 7), in the homoeopathic
dilution; for example, ‘D3', ‘3 DH' or ‘3X' means 3 decimal
preparations. For this purpose, it is demonstrated that:
potentisation steps, and ‘C3', ‘3 CH' or ‘3C' means
the method of production includes a step or steps that 3 centesimal potentisation steps.
have been shown to remove or inactivate agents of
infection; ‘LM' potencies are manufactured according to a specific
procedure with a 50 000 dilution factor by alternate steps of
— where applicable, raw materials of zoological origin
liquid dilution and impregnation of pillules. The number of
comply with the monograph Products with risk of
potentisation steps defines the degree of dilution, for
transmitting agetits of animal spongiform
example, 3rd LM means 3 successive LM dilutions.
encephalopathies (1483);
— where applicable, the animals and the tissues used to Dosage forms
obtain the raw materials comply with the health A dosage form of a homoeopathic preparation complies with
requirements of the competent authorities for animals for any relevant dosage form monograph in ±e European
human consumption; Pharmacopoeia, and with the following:
— for materials of human origin, the donor follows the — for the purpose of dosage forms for homoeopathic use,
recommendations applicable to human blood donors and ‘active substances' are considered to be ‘dilutions or
to donated blood (see Human plasma for triturations of homoeopathic stocks’ or ‘homoeopathic
fractionation (0853)) > unless otherwise justified and stocks’ (in case of a mother tincture or a glycerol
authorised. macerate);
A raw material of botanical, zoological or human origin may — these dosage forms can contain one or more ‘active
be used either in the fresh state or in the dried state. Where substances’;
appropriate, fresh material may be kept deep-frozen. — they are prepared using appropriate excipients.
Raw materials of botanical origin comply with the Homoeopathic dosage form 'pillule'
requirements of the monograph Herbal drugs for homoeopathic Pillules for homoeopathic use are solid preparations obtained
preparations (2045). from sucrose, lactose or other suitable excipients. Pillules for
Where justified and authorised for transportation or storage homoeopathic preparations (2153) are intended for
purposes, fresh plant material may be kept in ethanol impregnation or coating with one or more homoeopathic
(96 per cent) or in ethanol of a suitable concentration, preparations. The impregnated pillules comply with the
provided the whole material including the storage medium is requirements of the monograph Impregnated homoeopathic
used for processing. pillules (2079). They are intended for sublingual or oral use.
Raw materials comply with any requirements of the relevant Homoeopathic dosage form ‘tablet'
monographs of the European Pharmacopoeia. Tablets for homoeopathic use are solid preparations obtained
Vehicles from sucrose, lactose or other suitable excipients according to
Vehicles are excipients used for the preparation of certain the monograph Tablets (0478). They may be prepared either
stocks or for the potentisation process. They may include, for by compressing one or more ‘active substances’ with the
example: purified water, ethanol of a suitable concentration, excipients or by impregnating preformed tablets with one or
glycerol and lactose. more liquid ‘active substances’. The preformed tablets for
impregnation are obtained from sucrose, lactose or other
Vehicles comply with any requirements of the relevant
suitable excipients according to the monograph
monographs of the European Pharmacopoeia.
Tablets (0478). Tablets for homoeopathic use are intended
Stocks for sublingual or oral use.
Stocks are substances, products or preparations used as Homoeopathic dosage forms 'parenteral preparation', 'eye
starting materials for the production of homoeopathic preparation', 'nasal preparation'
preparations. A stock is usually one of the following: a
For the last potentisation step(s), an ethanol-free vehicle is
mother tincture or a glycerol macerate, for raw materials of
used to minimise the content of ethanol in the final
botanical, zoological or human origin, or the substance itself,
preparation.
for raw materials of chemical or mineral origin.
The residual ethanol content (2.9.10) is not greater than
Mother tinctures comply with the requirements of the
1 per cent v/v unless otherwise justified and authorised.
monograph Mother tinctures for homoeopathic
preparations (2029). Manufacturing methods
IV-430 Homoeopathic Preparations 2016
Homoeopathic preparations are manufactured using a range Broken Describes a herbal drug for homoeopathic
of methods of preparation and are presented in various preparations in which the more fragile parts of the plant have
dosage forms (covered by general dosage form monographs). broken during drying, packaging or transportation.
The methods of preparation are described in the monograph For dried herbal drugs for homoeopathic preparations, cut
Methods of preparation of homoeopathic stocks and potentisation describes size reduction, other than powdering, that reduces
(2371). The use of certain preparations obtained using the the particle size below that which is described in the
methods listed below is restricted to certain dosage forms as macroscopic identity of the herbal drug for homoeopathic
indicated in Table 1038.-1. preparations.
PRODUCTION
Table 1038.-1.
Herbal drugs for homoeopathic preparations are obtained
Manufacturing methods Dosage forms from cultivated or wild plants. Suitable collection, cultivation,
2.1.2 Eye drops harvesting, sorting, drying, fragmentation and storage
Solutions for injection conditions are essential to guarantee the quality of herbal
Nasal preparations drugs for homoeopathic preparations.
2.2.1, 2.2.2, 2.2.3 Eye drops Herbal drugs for homoeopathic preparations are, as far as
Coated homoeopathic pillules
possible, free from impurities such as soil, dust, dirt and
Solutions for injection
other contaminants such as fungal, insect and other animal
Nasal preparations
contaminants. They do not present signs of decay.
Ointments, creams and gels
Oral powders (triturations) If a decontaminating treatment has been used, it is necessary
Suppositories to demonstrate that the constituents of the plant are not
2.2.4 Solutions for injection affected and that no harmful residues remain. The use of
ethylene oxide is prohibited for the decontamination of
Eye drops
herbal drugs for homoeopathic preparations.
Coated homoeopathic pillules
Solutions for injection Fresh herbal drugs are processed as rapidly as possible after
Nasal preparations harvesting. Where justified and authorised for transportation
Ointments, creams and gels or storage purposes, fresh plant material may be deep-frozen;
Suppositories it may also be kept in ethanol (96 per cent) or in ethanol of a
suitable concentration, provided the whole material including
The competent authority has the right to accept or reject the storage medium is used for processing.
particular combinations of manufacturing method and Adequate measures have to be taken in order to ensure that
substance. the microbiological quality of homoeopathic preparations
_______________________________________________________________ Ph Eur
containing 1 or more herbal drugs comply with the
recommendations given in general chapter 5.1.4.
Microbiological quality of non-sterile pharmaceutical preparations
and substances for pharmaceutical use.
IDENTIFICATION
Herbal Drugs for Homoeopathic ** \ Herbal drugs for homoeopathic preparations are identified
using their macroscopic and, where necessary, microscopic
Preparations ***** descriptions and any further tests that may be required (for
Herbal Drugs for Homoeopathic Use example, thin-layer chromatography).
TESTS
Ph Elf_______________________________________________________________
The tests for foreign matter and loss on drying should be
DEFINITION performed before any further processing of the fresh plant.
Herbal drugs for homoeopathic preparations are mainly Foreign matter (2.8.2)
whole plants or parts of plants, fragmented or broken, and Where a fresh plant is used as a starting material for the
include algae, fungi or lichens, in an unprocessed state, manufacture of homoeopathic preparations, the content of
usually in fresh form. The state, fresh or dried, in which the foreign matter is as low as possible; if necessary, the
drug is used, is defined in the individual monograph of the maximum content of foreign matter is indicated in the
European Pharmacopoeia or, in its absence, in the individual individual monograph.
monograph of an official national pharmacopoeia of a Where a dried plant is used as a starting material for the
member state. In the absence of such a monograph, the state manufacture of homoeopathic preparations, carry out a test
in which the herbal drug is used has to be defined. Certain for foreign matter, unless otherwise prescribed in the
exudates that have not been subjected to a specific treatment individual monograph. The content of foreign matter is not
are also considered to be herbal drugs for homoeopathic more than 2 per cent พ/พ, unless otherwise prescribed or
preparations. Herbal drugs for homoeopathic preparations justified and authorised.
are precisely defined by the botanical scientific name of the
source species according to the binomial system (genus, Adulteration
species, variety and author). A specific appropriate test may apply to herbal drugs for
homoeopathic preparations liable to be falsified.
พhole Describes a herbal drug for homoeopathic preparations
that has not been reduced in size and is presented, dried or Loss on drying (2.2.32)
undried, as harvested. Carry out a test for loss on drying on dried herbal drugs for
homoeopathic preparations.
Fragmented Describes a herbal drug for homoeopathic
preparations that has been reduced in size after harvesting to
permit ease of handling, drying and/or packaging.
2016 Homoeopathic Preparations IV-431
If a fresh plant is processed more than 24 h after harvesting, described in an official national pharmacopoeia of a Member
a test for loss on drying should be carried out. The minimum State may equally be used.
limit is indicated in the individual monograph. Where material of animal origin is to be used, particular
Water (2.2.13) reference is made to the requirements concerning the use of
A determination of water is carried out on herbal drugs for raw material of zoological or human origin in the monograph
homoeopathic preparations with a high essential oil content. Homoeopathic preparations (1038).
Pesticides {2.8.13) In the preparation of liquid dilutions, the ethanol of the
Herbal drugs for homoeopathic preparations comply with the concentration prescribed in the method may, if necessary, be
requirements for pesticide residues. The requirements take replaced by ethanol (36 per cent V/V) [ethanol
into account the origin and the nature of the plant, where (30 per cent m/m)] or ethanol (18 per cent V/V) [ethanol
necessary the preparation in which the plant might be used (15 per cent m/m)].
and, where available, knowledge of the complete record of When the individual monograph allows that the mother
treatment of the batch of the plant. Where justified, the test tincture be prepared from more than one plant species, the
for pesticides may be performed on the mother tincture mother tincture can be prepared from the specified parts of
according to the requirements of the general monograph an individual plant species or from any mixture thereof.
Mother tinctures for homoeopathic preparations (2029). Unless otherwise stated, mother tinctures are prepared by
If appropriate, herbal drugs for homoeopathic preparations comply maceration. Maceration lasts not less than 10 days and not
with other tests, such as the following, for example. more than 30 days.
Total ash {2.4.16) Maceration may be replaced by long maceration (maximum
Bitterness value {2.8.15) 60 days) or very long maceration (maximum 180 days),
provided it is demonstrated that the quality of the resulting
Heavy metals {2.4.27)
mother tincture is the same as that of the mother tincture
Unless otherwise stated in an individual monograph or unless
prepared by maceration.
otherwise justified and authorised:
— cadmium', maximum 1.0 ppm; Unless otherwise stated in the individual monograph, the
— leadะ maximum 5.0 ppm; term ‘part(s)’ denotes ‘mass part(s)’. Unless otherwise stated
— mercury-, maximum 0.1 ppm. in the method, the maximum temperature for the preparation
is 25 °C.
If justified by the nature or origin of the herbal drug or if
required by the competent authority, suitable limits for the 1. MOTHER TINCTURES
content of other heavy metals such as arsenic or nickel are METHOD 1.1
defined. METHOD 1.1.1 (EQUIVALENT TO HOMOOPATHISCHES ARZNEIBUCH (HAB)
1A: MOTHER TINCTURES AND LIQUID DILUTIONS)
Where justified, the test for heavy metals may be performed
on the mother tincture according to the requirements of the Method 1.1.1 is used for fresh herbal drugs containing
general monograph Mother tinctures for homoeopathic generally more than 70 per cent of expressed juice and no
preparations (2029). essential oil or resin or mucilage. Mother tinctures prepared
Aflatoxin B1 {2.8.18) according to Method 1.1.1 are mixtures of equal parts of
Where appropriate, limits for aflatoxins may be required. expressed juices and ethanol (90 per cent V/V) [ethanol
(86 per cent m/m)].
Ochratoxin A {2.8.22)
Express the comminuted herbal drug. Immediately mix the
Where appropriate, a limit for ochratoxin A may be required.
expressed juice with an equal mass of ethanol
Radioactive contamination (90 per cent V/V) [ethanol (86 per cent m/m)]. Allow to
In some specific circumstances, the risk of radioactive stand in a closed container at a temperature not exceeding
contamination is to be considered. 20 °C for not less than 5 days, then filter.
ASSAY Adjustment to any value specified in the individual
Where applicable, herbal drugs for homoeopathic monograph
preparations are assayed by an appropriate method. Determine the percentage dry residue {2.8.16) or, where
STORAGE prescribed, the percentage assay content of the above-
mentioned filtrate. Calculate the amount (/41), in kilograms,
Store dried herbal drugs protected from light.
of ethanol (50 per cent V/V) [ethanol (43 per cent m/m)]
_______________________________________________________________ Ph Eur required, using the following expression:
m X (Nx - No)
No
Methods of Preparation of ★ *J
m = mass of filtrate, in kilograms;
Homoeopathic Stocks and ***** No = percentage dry residue or percentage assay content
Potentisation as required in the individual monograph;
Nx = percentage dry residue or percentage assay content
of the filtrate.
Ph Eir_______________________________________________________________
Mix the filtrate with the calculated amount of ethanol
Homoeopathic stocks are prepared, using suitable methods,
(50 per cent V/V) [ethanol (43 per cent m/m)]. Allow to
from raw materials that comply with the requirements of the
stand at a temperature not exceeding 20 °C for not less than
monograph Homoeopathic preparations (1038). The methods
5 days, then filter if necessary.
described below, combined with established methods for
potentisation, are examples of methods, but other methods
Potentisation than 60 per cent moisture (loss on drying) and no essential

The 1st ‘decimal’ dilution (Dl) is made from: oil or resin.
— 2 parts of the mother tincture; Mother tinctures prepared according to Method 1.1.3
— 8 parts of ethanol (50 per cent VIV) [ethanol (ethanol content approximately 50 per cent VIV or
(43 per cent mhn)]. 43 per cent mini) are prepared by maceration as described
The 2nd decimal dilution (D2) is made from: below.
— 1 part of the 1st ‘decimal’ dilution; Comminute the herbal drug. Take a sample and determine
— 9 parts of ethanol (50 per cent VIV) [ethanol the loss on drying (2.2.22). Unless otherwise prescribed,
(43 per cent พ/พ)]. determine the loss on drying on 2.00-5.00 g of comminuted
Subsequent decimal dilutions are produced as stated for D2. raw material in a flat-bottomed tared vessel, 45-55 mm in
The lsl ‘centesimal’ dilution (Cl) is made from: diameter, that has been previously dried as indicated for the
— 2 parts of the mother tincture; raw material. Dry the raw material at 105 °C for 2 h then
— 98 parts of ethanol (50 per cent VIV) [ethanol allow to cool in a desiccator.
(43 per cent พ/พ)]. To the comminuted herbal drug immediately add not less
The 2nd centesimal dilution (C2) is made from: than half the mass of ethanol (90 per cent VIV) [ethanol
— 1 part of the 1st ‘centesimal’ dilution; (86 per cent พ/พ)] and store in well-closed containers at a
— 99 parts of ethanol (50 per cent VIV) [ethanol temperature not exceeding 20 °C.
43 per cent พ/พ)]. Use the following expression to calculate the amount (z42), in
Subsequent centesimal dilutions are produced as stated for kilograms, of ethanol (90 per cent U/I7) [ethanol
C2. (86 per cent พ/พ)] required for the mass (พ) of raw material,
METHOD 1.1.2 (EQUIVALENT TO HAB IB: MOTHER TINCTURES AND LIQUID
then subtract the amount of ethanol (90 per cent VIV)
DILUTIONS)
[ethanol (86 per cent พ/พ)] already added and add the
difference to the mixture.
Method 1.1.2 is used where the latex of a herbal drug is to
be processed.
Mo±er tinctures prepared according to Method 1.1.2 are TSF
mixtures of fresh plant latex with ethanol (36 per cent VIV)
[ethanol (30 per cent พ/พ)]. Mix the fresh latex with 2 parts พ = mass of raw material, in kilograms;
by mass of ethanol (36 per cent VIV) [ethanol T = percentage loss on drying of the sample.
(30 per cent พ/พ)] and filter.
Allow to stand at a temperature not exceeding 20 cc for not
Adjustment to any value specified in the individual
less than 10 days, swirling from time to time, then express
monograph
the mixture and filter the resulting liquid.
Determine the percentage dry residue (2.&Z6) or, where
prescribed, the percentage assay content of the above- Adjustment to any value specified in the individual
mentioned filtrate. Calculate the amount (A 1), in kilograms, monograph
of ethanol (36 per cent VIV) [ethanol (30 per cent พ/พ)] Determine the percentage dry residue (2.8.16) or, where
required, using the following expression: prescribed, the percentage assay content of the above-
mentioned filtrate. Calculate the amount (Al) 3 in kilograms,
of ethanol (50 per cent V/V) [ethanol (43 per cent milพ)]
mx (Al- No) required, using the following expression:
No
m X (Nx - No)
No
No = percentage dry residue or percentage assay content
as required in the individual monograph; ni = mass of filtrate, in kilograms;
Nx = percentage dry residue or percentage assay content No = percentage dry residue or percentage assay content
of the filtrate. as required in the individual monograph;
Mix the filtrate with the calculated amount of ethanol Nx = percentage dry residue or percentage assay content
(36 per cent VIV) [ethanol (30 per cent พ/พ)]. Allow to of the filtrate.
stand at a temperature not exceeding 20 °C for not less than Mix the filtrate with the calculated amount of ethanol
5 days, then filter if necessary. (50 per cent VIV) [ethanol (43 per cent พ/พ)]. Allow to
Potentisation stand at a temperature not exceeding 20 °C for not less than
The 1st ‘decimal’ dilution (Dl) is made from: 5 days, then filter if necessary.
— 3 parts of the mother tincture; Potentisation
— 7 parts of ethanol (36 per cent VIV) [ethanol The 1st ‘decimal’ dilution (Dl) is made from:
(30 per cent พ/พ)]. — 2 parts of the mother tincture;
The 2nd decimal dilution (D2) is made from: — 8 parts of ethanol (50 per cent VIV) [ethanol
— 1 part of the 1st ‘decimal’ dilution; (43 per cent พ/พ)].
— 9 parts of ethanol (18 per cent VIV) [ethanol The 2nd decimal dilution (D2) is made from:
(15 per cent พ/พ)]. — 1 part of the 1 ‘decimal’ dilution;
Subsequent decimal dilutions are produced as stated for D2. — 9 parts of ethanol (50 per cent VIV) [ethanol
METHOD 1.1.3 (EQUIVALENT TO HAB 2A: MOTHER TINCTURES AND LIQUID (43 per cent พ/พ)].
DILUTIONS) Subsequent decimal dilutions are produced as stated for D2.
Method 1.1.3 is used for fresh herbal drugs containing The 1st ‘centesimal’ dilution (Cl) is made from:
generally less than 70 per cent of expressed juice and more — 2 parts of the mother tincture;
98 parts of ethanol (50 per cent VIV) [ethanol Potendsadon

(43 per cent พ/พ)]. The 1st ‘decimal’ dilution (DI) is made from:
The 2nd centesimal dilution (C2) is made from: — 2 parts of the mother tincture;
1 part of the 1st ‘centesimal’ dilution; — 8 parts of ethanol (36 per cent VIV} [ethanol
99 parts of ethanol (50 per cent VIV) [ethanol (30 per cent พ/พ)].
(43 per cent พ/พ)]. The 2nd decimal dilution (D2) is made from:
Subsequent centesimal dilutions are produced as stated for — 1 part of the 1st ‘decimal’ dilution;
C2. — 9 parts of ethanol (18 per cent VIV) [ethanol
METHOD 1 1.4 (EQUIVALENT TO HAB 2B. MOTHER TINCTURES AND LIQUID (15 per cent พ/พ)].
DILUTIONS) Subsequent decimal dilutions are produced as stated for D2.
Method 1.1.4 is used for fresh herbal drugs containing METHOD 1.1.5 (EQUIVALENT TO HAB 3A: MOTHER TINCTURES AND LIQUID
generally less than 70 per cent of expressed juice and more DILUTIONS)
than 60 per cent moisture (loss on drying) and no essential Method 1.1.5 is used for fresh herbal drugs containing
oil or resin. essential oil or resin, or generally less than 60 per cent
Mother tinctures prepared according to Method 1.1.4 moisture (loss on drying).
(ethanol content approximately 36 per cent VIV or Mother tinctures prepared according to Method 1.1.5
30 per cent ไทเท!} are prepared by maceration as described (ethanol content approximately 68 per cent VIV or
below. 60 per cent พ/พ) are prepared by maceration as described
Comminute the herbal drug. Take a sample and determine below.
the loss on drying (2.2.52). Unless otherwise prescribed, Comminute the herbal drug. Take a sample and determine
determine the loss on drying on 2.00-5.00 g of comminuted the loss on drying (2.2.32}. Unless otherwise prescribed,
raw material in a flat-bottomed tared vessel, 45-55 mm in determine the loss on drying on 2.00-5.00 g of comminuted
diameter, that has been previously dried as indicated for the raw material in a flat-bottomed tared vessel, 45-55 mm in
raw material. Dry' the raw material at 105 °C for 2 h then diameter, that has been previously dried as indicated for the
allow to cool in a desiccator. raw material. Dry the raw material at 105 °C for 2 h then
To the comminuted herbal drug immediately add not less allow to cool in a desiccator.
than half the mass of ethanol (70 per cent P7P) [ethanol To the comminuted herbal drug immediately add not less
(62 per cent พ/พ)] and store in well-closed containers at a than half the mass of ethanol (90 per cent VIV) [ethanol
temperature not exceeding 20 °C. (86 per cent พ/พ)] and store in well-closed containers at a
Use the following expression to calculate the amount (/12), in temperature not exceeding 20 °C.
kilograms, of ethanol (70 per cent VIV) [ethanol Use the following expression to calculate the amount (ฬ3), in
(62 per cent พ/พ)] required for the mass (ไท} of raw material, kilograms, of ethanol (90 per cent V/V) [ethanol
then subtract the amount of ethanol (70 per cent VIV) (86 per cent พ/พ)] required for the mass (พ) of raw material,
[ethanol (62 per cent ท!/ท!}] already added and add the then subtract the amount of ethanol (90 per cent VIV)
difference to the mixture. [ethanol (86 per cent ไท/ไท}] already added and add the
100
ท! = mass of raw material, in kilograms;
T = percentage loss on drying of the sample. พ = mass of raw material, in kilograms;
Allow to stand at a temperature not exceeding 20 °C for not T = percentage loss on drying of the sample.
less than 10 days, swirling from time to time, then express Allow to stand at a temperature not exceeding 20 °C for not
the mixture and filter the resulting liquid. less than 10 days, swirling from time to time, then express
Adjustment to any value specified in the individual the mixture and filter the resulting liquid.
monograph Adjustment to any value specified in the individual
Determine the percentage dry residue (2.8.16} or, where monograph
prescribed, the percentage assay content of the above- Determine ±e percentage dry residue (2.8.16) or, where
mentioned filtrate. Calculate the amount (A 1), in kilograms, prescribed, the percentage assay content of the above-
of ethanol (36 per cent V/V) [ethanol (30 per cent พ/พ)] mentioned filtrate. Calculate the amount (A 1), in kilograms,
required, using the following expression: of ethanol (70 per cent VIV) [ethanol (62 per cent ท!/ท!}]
required, using the following expression:
mx(Nx- No)
No mx(Nx — No)
No
ไท = mass of filtrate, in kilograms;
Nq = percentage dry residue or percentage assay content
ท! = mass of filtrate, in kilograms;
as required in the individual monograph;
Nx = percentage dry residue or percentage assay content Nq = percentage dry residue or percentage assay content
as required in the individual monograph;
of the filtrate.
Nx = percentage dry residue or percentage assay content
Mix the filtrate with the calculated amount of ethanol of the filtrate.
(36 per cent V/V) [ethanol (30 per cent พ/พ)]. Allow to
(70 per cent VIV) [ethanol (62 per cent พ/พ)]. Allow to
5 days, then filter if necessary.
stand at a temperature not exceeding 20 °C for not less than mentioned filtrate. Calculate the amount (Al), in kilograms,
5 days, then filter if necessary. of ethanol (50 per cent P7P) [ethanol (43 per cent พ/พ)]
Potentisation required, using the following expression:
The 1st ‘decimal’ dilution (DI) is made from:
— 3 parts of the mother tincture; m X (Nx - No)
— 7 parts of ethanol (70 per cent VIV) [ethanol No
(62 per cent พ/พ)].
The 2nd decimal dilution (D2) is made from: พ = mass of filtrate, in kilograms;
— 1 part of the 1st ‘decimal’ dilution; No = percentage dry residue or percentage assay content
— 9 parts of ethanol (70 per cent VIV) [ethanol as required in the individual monograph;
(62 per cent พ/พ)]. Nx = percentage dry residue or percentage assay content
of the filtrate.
Subsequent decimal dilutions are produced as stated for D2.
Use ethanol (50 per cent VIV) [ethanol (43 per cent พ/พ)] Mix the filtrate with the calculated amount of ethanol
for dilutions from D4 onwards. (50 per cent VIV) [ethanol (43 per cent พ/พ)]. Allow to
The 1st ‘centesimal’ dilution (Cl) is made from:
— 3 parts of the mother tincture; 5 days, then filter if necessary.
— 97 parts of ethanol (70 per cent VIV) [ethanol Potentisation
(62 per cent พ/พ)]. The 1st ‘decimal’ dilution (DI) is made from:
The 2nd centesimal dilution (C2) is made from: — 3 parts of the mother tincture;
— 1 part of the 1st ‘centesimal’ dilution; — 7 parts of ethanol (50 per cent V/V) [ethanol
— 99 parts of ethanol (50 per cent VIV) [ethanol (43 per cent ทใ/1พ)].
(43 per cent พ/พ)]. The 2nd decimal dilution (D2) is made from:
Subsequent centesimal dilutions are produced as stated for — 1 part of the 1st ‘decimal’ dilution;
C2. — 9 parts of ethanol (36 per cent V/V) [ethanol
METHOD 1.1.6 (EQUIVALENT TO HAB 3B: MOTHER TINCTURES AND LIQUID
DILUTIONS)
The 3rd decimal dilution (D3) is made from:
— 1 part of the 2nd decimal dilution;
Method 1.1.6 is used for fresh herbal drugs containing
— 9 parts of ethanol (18 per cent V/V) [ethanol
essential oils or resins or generally less than 60 per cent
moisture (loss on drying).
Mother tinctures prepared according to Method 1.1.6
METHOD 1.1.7 (EQUIVALENT TO HAB 3C: MOTHER TINCTURES AND
(ethanol content approximately 50 per cent VIV or
LIQUID DILUTIONS)
43 per cent ทใ/ทใ) are prepared by maceration as described
below. Method 1.1.7 is used for fresh herbal drugs containing
Comminute the herbal drug. Take a sample and determine generally less than 60 per cent moisture (loss on drying).
the loss on drying (2.2.32). Unless otherwise prescribed, Mother tinctures prepared according to Method 1.1.7
determine the loss on drying on 2.00-5.00 g of comminuted (ethanol content approximately 36 per cent v/v or
raw material in a flat-bottomed tared vessel, 45-55 mm in 30 per cent ทใ/ทใ) are prepared by maceration as described
diameter, that has been previously dried as indicated for the below.
raw material. Dry the raw material at 105 °C for 2 h then Comminute the herbal drug. Take a sample and determine
allow to cool in a desiccator. the loss on drying (2.2.32). Unless otherwise prescribed,
To the comminuted herbal drug immediately add not less determine the loss on drying on 2.00-5.00 g of comminuted
than half the mass of ethanol (80 per cent VIV) [ethanol raw material in a flat-bottomed tared vessel, 45-55 mm in
(73 per cent พ/พ)] and store in well-closed containers at a diameter, that has been previously dried as indicated for the
temperature not exceeding 20 °C. raw material. Dry the raw material at 105 °C for 2 h then
Use the following expression to calculate the amount (z43), in allow to cool in a desiccator.
kilograms, of ethanol (80 per cent VIV) [ethanol To the comminuted herbal drug immediately add not less
(73 per cent พ/พ)] required for the mass (พ) of raw material, than half the mass of ethanol (50 per cent P7P) [ethanol
then subtract the amount of ethanol (80 per cent VIV) (43 per cent ?พ/?พ)] and store in well-closed containers at a
[ethanol (73 per cent พ/พ)] already added and add the temperature not exceeding 20 °C.
difference to the mixture. Use the following expression to calculate the amount (Al), in
kilograms, of ethanol (50 per cent V/V) [ethanol
(43 per cent ?พ/?พ)] required for the mass (?พ) of raw material,
100 then subtract the amount of ethanol (50 per cent V/V)
[ethanol (43 per cent ?พ/?พ)] already added and add the
พ = mass of raw material, in kilograms;
T = percentage loss on drying of the sample. 2 X m XT
Allow to stand at a temperature not exceeding 20 °C for not 100
less than 10 days, swirling from time to time, then express
the mixture and filter the resulting liquid. พ = mass of raw material, in kilograms;
Adjustment to any value specified in the individual T = percentage loss on drying of the sample.
monograph Allow to stand at a temperature not exceeding 20 °C for not
Determine the percentage dry residue (2.8.16) or, where less than 10 days, swirling from time to time, then express
prescribed, the percentage assay content of the above the mixture and filter the resulting liquid.
Adjustment to any value specified in the individual m = mass of percolate or macerate, in kilograms;
monograph No = percentage dry residue or percentage assay content
Determine the percentage dry residue (2.8.16) or, where as required in the individual monograph;
prescribed, the percentage assay content of the above- Nx ะ= percentage dry residue or percentage assay content
mentioned filtrate. Calculate the amount (/11), in kilograms, of the percolate or macerate.
of ethanol (36 per cent VIV) [ethanol (30 per cent mhพ)]
Mix the macerate or percolate with the calculated amount of
required, using the following expression:
ethanol of the appropriate concentration. Allow to stand at a
temperature not exceeding 20 °C for not less than 5 days,
m X (Nx - Nq) then filter if necessary.
No Potentisation
The mother tincture corresponds to the 1st decimal dilution
(0 = DI).
No = percentage dry residue or percentage assay content
as required in the individual monograph; The 2nd decimal dilution (D2) is made from:
Nx = percentage dry residue or percentage assay content — 1 part of the mother tincture (DI);
of the filtrate. — 9 parts of ethanol of the same concentration.
The 3rd decimal dilution (D3) is made from:
— 1 part of the 2nd decimal dilution;
(36 per cent U/I-O [ethanol (30 per cent )ทเพ/)]. Allow to
— 9 parts of ethanol of the same concentration.
5 days, then filter if necessary'. Unless a different ethanol concentration is specified, use
ethanol (50 per cent V/V) [ethanol (43 per cent พ//พ/)] for
Potentisation
subsequent decimal dilutions from D4 onwards and proceed
The 1st ‘decimal’ dilution (DI) is made from:
as stated for D3.
— 3 parts of the mother tincture;
— 7 parts of ethanol (36 per cent VIV) [ethanol The 1st ‘centesimal’ dilution (Cl) is made from:
(30 per cent nilพ/)]. — 10 parts of the mother tincture (DI);
The 2nd decimal dilution (D2) is made from:
— 1 part of the 1st ‘decimal’ dilution; The 2nd centesimal dilution (C2) is made from:
— 1 part of the 1st ‘centesimal’ dilution;
— 9 parts of ethanol (18 per cent VIV) [ethanol
(15 per cent nil'พ/)]. — 99 parts of ethanol (50 per cent VIV) [ethanol
(43 per cent พ//พ/)], unless a different ethanol
Subsequent decimal dilutions are produced as stated for D2. concentration is specified.
METHOD 1.1.8 (EQUIVALENT TO HAB 1A. MOTHER TINCTURES AND LIQUID
Subsequent centesimal dilutions are produced as stated for
DILUTIONS)
C2
Method 1.1.8 is generally used for dried herbal drugs. METHOD 1.1.9 (EQUIVALENT TO HAB 4B: MOTHER TINCTURES AND LIQUID
Mother tinctures prepared according to Method 1.1.8 are DILUTIONS)
prepared by maceration or percolation as described below, Method 1.1.9 is generally used for animal matter.
using 1 part of dried herbal drug and 10 parts of ethanol of
Mother tinctures prepared according to Method 1.1.9 are
the appropriate concentration (anhydrous, 96 per cent VIV -
prepared by maceration or percolation as described below,
94 per cent พ//พ/, 90 per cent VIV - 86 per cent พ//พ/,
using 1 part of animal matter and 10 parts of ethanol of the
80 per cent VIV - 73 per cent mlm, 70 per cent VIV -
appropriate concentration (anhydrous, 96 per cent VIV -
62 per cent พ//พ/, 50 per cent VIV - 43 per cent พ//พ/,
94 per cent พ//พ/, 90 per cent V/V - 86 per cent mlm,
36 per cent VIV - 30 per cent mlm, 18 per cent VIV -
80 per cent V/V - 73 per cent mlm, 70 per cent VIV -
15 per cent mlm), unless otherwise prescribed in the
62 per cent mlm, 50 per cent VIV - 43 per cent mlm,
individual monograph.
36 per cent V/V - 30 per cent mini, 18 per cent VIV -
Production by maceration Unless otherwise prescribed, 15 per cent m/m), unless otherwise prescribed in ±e
comminute the herbal drug, mix thoroughly with ethanol of individual monograph.
the appropriate concentration and allow to stand in a closed
Production by maceration Unless otherwise prescribed,
container for an appropriate time. Separate the residue from
comminute the animal matter, mix thoroughly with ethanol
the ethanol and, if necessary, press out. In the latter case,
of the appropriate concentration and allow to stand in a
combine the 2 liquids obtained.
closed container for an appropriate time. Separate the residue
Production by percolation If necessary, comminute the herbal from the ethanol and, if necessary, press out. In the latter
drug. Mix thoroughly with a portion of ethanol of the case, combine the 2 liquids obtained.
appropriate concentration and allow to stand for an
Production by percolation If necessary, comminute the animal
appropriate time. Transfer to a percolator and allow the
matter. Mix thoroughly with a portion of ethanol of the
percolate to flow slowly, at room temperature, making sure
appropriate concentration and allow to stand for an
that the herbal drug to be extracted is always covered with
appropriate time. Transfer to a percolator and allow the
the remaining ethanol. The residue may be pressed out and
percolate to flow slowly at room temperature, making sure
the expressed liquid combined with the percolate.
that the animal matter to be extracted is always covered with
If adjustment to a given concentration is necessary, calculate the remaining ethanol. The residue may be pressed out and
the amount (ฬ 1), in kilograms, of ethanol of the appropriate the expressed liquid combined with the percolate.
concentration required to obtain the concentration specified If adjustment to a given concentration is necessary, calculate
or used for production, using the following expression: the amount (z41), in kilograms, of ethanol of the appropriate
concentration required to obtain the concentration specified
m X (Nx — No) or used for production, using the following expression:
No
TV-436 Homoeopathic Preparations 2016
m X (7Vg No)
Subsequent decimal dilutions are produced as stated for D2,
No using ethanol of the appropriate concentration.
The 1st centesimal dilution (Cl) is made from:
ไท = mass of percolate or macerate, in kilograms; — 1 part of the mother tincture;
No = percentage dry residue or percentage assay content — 99 parts of ethanol of the appropriate concentration.
as required in the individual monograph; The 2nd centesimal dilution (C2) is made from:
Nx ะ= percentage dry residue or percentage assay content — 1 part of the 1st centesimal dilution;
of the percolate or macerate. — 99 parts of ethanol of the appropriate concentration.
Mix the macerate or percolate with the calculated amount of Subsequent centesimal dilutions are produced as stated
ethanol of the appropriate concentration. Allow to stand at a for C2, using ethanol of the appropriate concentration.
temperature not exceeding 20 cc for not less than 5 days, METHOD 1.1.11 (FRENCH PHARMACOPOEIA)
then filter if necessary.
Method 1.1.11 is generally used for animal matter.
Potentisation
Mother tinctures prepared according to Method 1.1.11 are
The mother tincture corresponds to the 1st decimal dilution
prepared by maceration.
(0 = DI).
The mass ratio of raw material to mother tincture is usually
1 to 20. To the raw material, appropriately comminuted, add
— 1 part of the mother tincture GDI);
the quantity of ethanol of the appropriate concentration
required to produce a 1 in 20 mother tincture. Allow to
The 3rd decimal dilution (D3) is made from: macerate for at least 10 days, with sufficient shaking. Decant
— 1 part of the 2nd decimal dilution; and filter. Allow to stand for 48 h and filter again.
— 9 parts of ethanol of the same concentration. For mother tinctures with a required assay content,
Unless a different ethanol concentration is specified, use adjustment may be carried out, if necessary, by adding
ethanol (50 per cent P7F) [ethanol (43 per cent พ/พ)] for ethanol of the same concentration as used for the preparation
subsequent decimal dilutions from D4 onwards and proceed of the tincture.
as stated for D3. Potentisation
The 1st ‘centesimal’ dilution (Cl) is made from: The 1st decimal dilution (DI) is made from:
— 10 parts of the mother tincture (DI); — 1 part of tile mother tincture;
— 90 parts of ethanol of the same concentration. — 9 parts of ethanol of the appropriate concentration.
The 2nd centesimal dilution (C2) is made from: The 2nd decimal dilution (D2) is made from:
— 1 part of the 1st ‘centesimal’ dilution; — 1 part of the 1st decimal dilution;
— 99 parts of ethanol (50 per cent VIV) [ethanol — 9 parts of ethanol of the appropriate concentration.
(43 per cent ทไเไท)]3 unless a different ethanol Subsequent decimal dilutions are produced as stated for D2,
concentration is specified. using ethanol of the appropriate concentration.
Subsequent centesimal dilutions are produced as stated for The 1st centesimal dilution (Cl) is made from:
C2. — 1 part of the mother tincture;
METHOD 1.1.10 (FRENCH PHARMACOPOEIA) — 99 parts of ethanol of the appropriate concentration.
Method 1.1.10 is generally used for herbal drugs. The state The 2nd centesimal dilution (C2) is made from:
of the herbal drug, fresh or dried, is specified in the — 1 part of the 1st centesimal dilution;
individual monograph. — 99 parts of ethanol of the appropriate concentration.
Mother tinctures prepared according to Method 1.1.10 are Subsequent centesimal dilutions are produced as stated
prepared by maceration. for C2, using ethanol of the appropriate concentration.
Comminute appropriately the herbal drug. Take a sample 2. GLYCEROL MACERATES
and determine the loss on drying at 105 °C for 2 h (2.2.32) METHOD 2.1
or the water content (2.2.13). Taking this value into account, Method 2.1 is used for maceration of raw materials of animal
calcdate and add to the herbal drug the quantities of ethanol or herbal origin in glycerol (85 per cent) or glycerol/ethanol
of the appropriate concentration required to produce, unless mixtures of appropriate concentration. Pathological material
otherwise prescribed, a 1 in 10 mother tincture (1:10 mother is excluded.
tincture) with a suitable ethanol content. Allow to macerate
The raw materials are finely minced before use, where
for at least 10 days, with sufficient shaking.
appropriate.
Separate the residue from the ethanol and strain under
METHODS 2.1.1, 2.1.2 (EQUIVALENT TO HAB 42A AND 42B: MOTHER
pressure if necessary. Allow the combined liquids to stand for
TINCTURES AND LIQUID DILUTIONS THEREOF)
48 h and filter. For mother tinctures with a required assay
content, adjustment may be carried out, if necessary, by Raw materials of animal origin - freshly killed animals or
adding ethanol of the same concentration as used for the parts thereof - are used. Animals are processed immediately
preparation of the tincture. after being killed.
Potentisation Maceration
The 1st decimal dilution (DI) is made from: Disperse 1 part of finely minced animal material in:
— 1 part of the mother tincture; — 9 parts (decimal dilutions) or 99 parts (centesimal
— 9 parts of ethanol of the appropriate concentration. dilutions) of glycerol (85 per cent) for Method 2.1.1, or
— 2.1 parts of glycerol (85 per cent) for Method 2.1.2.
— 1 part of the 1“ decimal dilution; Allow to macerate for at least 2 h, then succuss. Filter when
— 9 parts of ethanol of the appropriate concentration. necessary.
Where justified, 1 part of glycerol (85 per cent) may be METHOD 2.2
added to 1 part of animal material before mincing. Where METHODS 2.2.1, 2.2.2, 2.2.3, 2.2.4 (EQUIVALENT TO HAB 4!A, 41B, 41C AND
very small amounts of animal material are used, the dilution 41D: GL MOTHER TINCTURES AND LIQUID DILUTIONS THEREOF)
may be prepared by dispersing 1 part of finely minced animal Method 2.2 is used for maceration of raw materials of animal
material in 99 parts of glycerol (85 per cent) (Cl or ‘D2’ if origin in a glycerol solution containing sodium chloride.
to be used for further decimal dilutions). Pathological material is excluded.
Potentisation Raw materials from freshly killed animals, parts or secretions
Method 2.1.1 thereof are used in Methods 2.2.1, 2.2.2 and 2.2.3. Lower
The 2nd decimal dilution (D2) is made from: animals are killed with carbon dioxide in a covered vessel.
1 part of the glycerol macerate DI; All animals are processed immediately after being killed.
9 parts of glycerol (85 per cent) or ethanol Blood components from live horses are used in
(18 per cent V/V) [ethanol (15 per cent mini)]. method 2.2.4.
Subsequent decimal dilutions are produced as stated for D2 Sample collection and/or pre-treatment
but with ethanol (18 per cent V/V) [ethanol The raw materials used in Methods 2.2.1, 2.2.2 and 2.2.3
(15 per cent พ/พ)] as the vehicle. are finely minced before use, where appropriate.
The 2nd centesimal dilution (C2) is made from:
The blood used in Method 2.2.4 is collected by a
— 1 part of the glycerol macerate Cl; veterinarian. Blood obtained from animals killed by bleeding
99 parts of ethanol (18 per cent VIV) [ethanol must not be used. Take 200 mL of this blood and add 15 IU
15 per cent พ/พ)]. of heparin sodium and 0.625 mL of a 9 g/kg solution of
Subsequent centesimal dilutions are produced as stated for sodium chloride per millilitre. Separate the blood
C2. components by fractional centrifugation and resuspend each
Method 2.1.2 individual cell sediment in 1.1 mL of a 9 g/kg solution of
The 1st ‘decimal’ dilution (DI) is made from: sodium chloride. These cell suspensions are processed into
— 3 parts of the glycerol macerate; the glycerol macerate.
— 7 parts of water for injections. Maceration
The 2nd decimal dilution (D2) is made from: Mix 1 part of finely minced animal material, secretions or
— 1 part of DI; blood cell suspensions, according to the method used, with 5
— 9 parts of water for injections. parts of a sodium chloride solution of the appropriate
concentration (see Table 2371.-1) and 95 parts of glycerol.
Allow to stand protected from light for at least 7 days, then
METHOD 2.1.3 (FRENCH PHARMACOPOEIA)
decant. If necessary for Methods 2.2.1, 2.2.2 and 2.2.3,
Raw materials of herbal or animal origin are used. centrifuge before decanting, then filter the supernatant if
Maceration necessary. The decanted liquid or the filtrate respectively is
Comminute the raw material appropriately. Take a sample the glycerol macerate.
and determine the loss on drying at 105 °C for 2 h (2.2.32) Any sediment present must be resuspended before processing
or the water content (2.2.13). Taking this value into account, the glycerol macerate.
calculate and add to the raw material the quantity of the
ethanol/glycerol mixture of the appropriate concentration to Table 2371.-1
produce, unless otherwise prescribed, a 1 in 20 glycerol
Methods 2.2.1 and Method 2.2.3
macerate. Allow to macerate for at least 3 weeks, with Method 2.2.2
2.2.4
sufficient shaking. Decant and strain under pressure if 15 g/kg solution of 40 g/kg solution of 80 g/kg solution of
necessary. Allow the combined liquids to stand for 48 h and sodium chloride in sodium chloride in sodium chloride เท
filter. purified water purified water purified water
Potentisation
The 1st decimal dilution (DI) is made from: Vehicle
— 1 part of the glycerol macerate; 0.2 parts of sodium hydrogen carbonate and 8.8 parts of
— 9 parts of a water/ethanol/glycerol mixture of appropriate sodium chloride in 991 parts of water for injections or
concentration. purified water as appropriate.
The 2nd decimal dilution (D2) is made from: Potentisation
— 1 part of the 1st decimal dilution; The glycerol macerate corresponds to the 2nd decimal
— 9 parts of a water/ethanol/glycerol mixture of appropriate dilution (‘D2’) or the 1st centesimal dilution (Cl).
concentration. The 3rd decimal dilution (D3) is made from:
Subsequent decimal dilutions are produced as stated for D2 — 1 part of the 2nd decimal dilution;
or using another appropriate vehicle. — 9 parts of the appropriate vehicle.
The 1st centesimal dilution (Cl) is made from: Subsequent decimal dilutions are produced as stated for D3.
— 1 part of the glycerol macerate; Where appropriate, the 4th decimal dilution (D4) is made
— 99 parts of a water/ethanol/glycerol mixture of appropriate from 1 part of the 3rd decimal dilution, 5.6 parts of the
concentration. vehicle and 3.4 parts of water for injections.
The 2nd centesimal dilution (C2) is made from: The 2nd centesimal dilution (C2) is made from:
— 1 part of the 1st centesimal dilution; — 1 part of the 1st centesimal dilution;
— 99 parts of a water/ethanol/glycerol mixture of appropriate — 99 parts of the appropriate vehicle.
concentration. Subsequent centesimal dilutions are produced as stated
Subsequent centesimal dilutions are produced as stated for C2.
for C2 or using another appropriate vehicle.
3. LIQUID DILUTIONS for injections (or purified water, as appropriate) for

METHOD 3.1 Method 3.1.2.
Methods 3.1.1, 3.1.2 and 3.1.3 are used for dissolution of Subsequent centesimal dilutions are produced as stated
any suitable inorganic or organic starting material, for for C2.
example minerals or venoms. Additives
Unless otherwise specified, dissolve 1 part of ±e starting For Method 3.1.1, if a reaction such as precipitation is
material in 9 parts (Dl) or 99 parts (Cl) of the liquid vehicle observed in the final dilution, the following additives may be
and succuss. used to enhance stability and/or solubility, unless otherwise
Where justified and authorised, in case of insufficient specified:
solubility of the starting material in the specified vehicle, — glacial acetic acid;
directly produce the first possible dilution. For example, if — concentrated hydrochloric acid;
the starting material is slightly soluble, dissolve 1 part of the — lactic acid;
starting material in 99 parts of the vehicle (Cl or ‘D2’ if to — sodium hydroxide.
be used for further decimal dilutions). Where solutions or dilutions have been pH-adjusted, they
METHODS 3.1.1, 3.1.2 (EQUIVALENT TO HAB 5A, 5B: SOLUTIONS, AQUEOUS must not be potentised further.
SOLUTIONS) METHOD 3.1 3
Vehicles Vehicles
The vehicles in Table 2371.-2 may be used. Suitable vehicles, for example, ethanol of an appropriate
concentration, glycerol or purified water may be used alone
Table 2371.-2 or combined.
Method 3.1.1 Method 3.1.2 Potentisation
Anhydrous ethanol Water for injections Unless otherwise specified, the 2nd decimal dilution (D2) is
Ethanol (96 per cent V/V) [ethanol (94 per Purified water made from:
cent m/m)] — 1 part of the 1st decimal dilution (Dl);
Ethanol (90 per cent V/V) [ethanol (86 per — 9 parts of the appropriate vehicle.
cent m/m)]
Ethanol (80 per cent V/V) [ethanol (73 per
cent m/m)] Unless otherwise specified, the 2nd centesimal dilution (C2)
Ethanol (70 per cent V/V) [ethanol (62 per is made from:
cent m/m)] — 1 part of the lsl centesimal dilution (Cl);
Ethanol (50 per cent V/V) [ethanol (43 per — 99 parts of the appropriate vehicle.
cent m/m)]
Subsequent centesimal dilutions are produced as stated
Ethanol (36 per cent V/V) [ethanol (30 per
cent m/m)] for C2.
Ethanol (18 per cent V/V) [ethanol (15 per METHOD 3.2
cent m/m)]
Method 3.2 is generally used to produce liquid dilutions of
Purified water
triturations of substances that for the most part are sparingly
Glycerol (85 per cent) soluble to practically insoluble.
METHODS 3.2.1, 3.2.2 (EQUIVALENT TO HAB 8A, 8B: LIQUID PREPARATION’S
For Method 3.1.1, if ethanol (18 per cent VIV) [ethanol MADE FROM TRITURATIONS, AQUEOUS PREPARATIONS MADE FROM
(15 per cent mini)] is used, the starting material may be TRITURATIONS)
dissolved in 7.58 parts of purified water and the ethanol Preparations made according to Method 3.2.1 and
concentration adjusted by adding 1.42 parts of ethanol Method 3.2.2 are produced from triturations D4, D5 and D6
(96 per cent VIV) [ethanol (94 per cent m/m)] to the or from triturations C4, C5 and C6, prepared according to
solution, for decimal dilutions. For centesimal dilutions, use method 4.1.1 by at least 2 potentisation steps.
83.4 parts of purified water for 15.6 parts of ethanol Vehicles
(96 per cent VIV) [ethanol (94 per cent mini)]. The vehicles in Table 2371.-3 may be used.
For Method 3.1.2, if the starting material is not stable and/or
soluble in water, glycerol (85 per cent) may be added at a Table 2371.-3
concentration of not more than 35 per cent of the vehicle, for Method 3.2.1 Method 3.2.2
potentisation up to D4. 1** potentisation: All potentisations:
Potentisation Purified water Water for injections
Purified water
Unless otherwise specified, the 2nd decimal dilution (D2) is
made from:
— 1 part of the 1st decimal dilution (Dl); 2nd potentisation:
Ethanol (36 per cent V/V) [ethanol
— 9 parts of ethanol (50 per cent VIV) [ethanol (30 per cent m/m)]
(43 per cent mini)] for Method 3.1.1 or 9 parts of water
for injections (or purified water, as appropriate) for
Method 3.1.2. Further potentisations:
Subsequent decimal dilutions are produced as stated for D2. (43 per cent m/m)]
Unless otherwise specified, the 2nd centesimal dilution (C2)
is made from:
— 1 part of the 1st centesimal dilution (Cl); Potentisation
— 99 parts of ethanol (50 per cent VIV) [ethanol For the first liquid potentisation, dissolve 1 part of the
(43 per cent m/m)] for Method 3.1.1 or 99 parts of water trituration in 9 parts (decimal dilutions) or 99 parts
(centesimal dilutions) of the specified vehicle (see (decimal trituration) or 100 parts of the trituration
Table 2371.-3) and succuss. For further potentisations, (centesimal trituration) from 1 part of the raw material
proceed in the same manner with 1 part of the previous (replace the mass of water lost from the fresh plant by an
dilution. equivalent amount of the vehicle). A suitable gentle drying
The D6, D7, C6 and C7 dilutions produced by the above process may need to be applied to the solid dilution.
method are not to be used for the preparation of further Where justified and authorised, it may be necessary to
dilutions. directly produce a Cl or ‘D2’ if to be used for further
METHOD 3.2.3 decimal triturations as the first solid trituration, made from
Preparations made according to Method 3.2.3 are produced 1 part of raw material and 99 parts of vehicle.
from triturations D2 onwards and from triturations Cl, C2, Trituration
C3 and C4, prepared according to method 4.1.2. บnless otherwise justified and authorised, the method
Vehicles consists of dividing the vehicle into 3 equal parts and adding
Suitable vehicles such as ethanol of an appropriate the raw material to the first part, then adding the second and
concentration or purified water may be used. third part of the vehicle, thoroughly triturating after each
addition.
Potentisation
Unless otherwise specified, the first liquid decimal For mechanical trituration, use a machine allowing the
dilution (Dn-1) is made from: requirements for particle size of the first decimal or
— 1 part of the decimal trituration Dn-2; centesimal solid trituration to be met. A machine fitted with
a scraping device may be used to ensure even trituration.
9 parts of purified water or of another suitable vehicle in
The time required to prepare one trituration is at least 1 h,
appropriate proportions.
uni ess otherwise justified and authorised.
The following decimal dilution (Dn) is made from:
For manual trituration, divide the vehicle into 3 equal parts
1 part of the first liquid decimal dilution Dn-1;
— 9 parts of a suitable vehicle. and briefly triturate the first part in a porcelain mortar.
Add the raw material, triturate the mixture for 6 min, scrape
Subsequent decimal dilutions are produced as stated for Dn. down for 4 min with an appropriate non-metallic device (for
Unless otherwise specified, the first liquid centesimal example, a porcelain spatula). Triturate for a further 6 min,
dilution (Cn-1) is made from: scrape down again for a further 4 min, then add the second
— 1 part of the centesimal trituration Cn-2; part of the vehicle and continue as above. Proceed in the
— 99 parts of purified water or of another suitable vehicle in same manner with the rest of the vehicle. The minimum time
appropriate proportions. required for the whole process is thus 1 h. Carry out the
The following centesimal dilution (Cn) is made from: whole process again for each subsequent solid dilution.
— 1 part of the first liquid centesimal dilution Cn-1; Triturations from D5 or C5 onwards may also be prepared
— 99 parts of a suitable vehicle. by intense mechanical treatment by a suitable mixing
Subsequent centesimal dilutions are produced as stated machine as follows: add the solid trituration to one thfrd of
for Cn. the vehicle and mix. Add the second third of the vehicle, mix
and proceed in the same manner with the last third of the
4. TRITURATIONS
vehicle. The whole process lasts minimum 1 hour, unless
METHOD 4.1
otherwise justified and authorised.
Method 4.1 is used for triturations, that is solid dilutions, of
In all cases, it is possible to change to a liquid medium from
raw materials or of triturations prepared according to
the 4th, 5th and 6th decimal or centesimal triturations, as
Methods 4.2.1 or 4.2.2. The duration and intensity of the
described in Methods 3.2.1 and 3.2.2.
trituration are such that homogeneity and potentisation are
achieved. METHOD 4.1.2 (FRENCH PHARMACOPOEIA)
Vehicle Trituration
Unless otherwise specified, lactose monohydrate is used. Triturations are prepared as follows:
METHOD 4 1.1 (EQUIVALENT TO HAB 6: TRITURATIONS) Decimal triturations
Triturations are prepared manually or mechanically. Reduce 1 part of the homoeopathic stock to a powder.
Mechanical trituration must be used for quantities exceeding Triturate carefully with a small quantity of the vehicle.
1 kg. The resulting particle size of the raw material in the Add the vehicle in small quantities until 9 parts of this
first decimal or centesimal dilution does not exceed 100 |im, vehicle have been used. The resulting trituration is the 1st
unless otherwise prescribed in the individual monograph. decimal trituration (Dl).
Ratios of raw material to vehicle Triturate as described above 1 part of this trituration with 9
parts of the vehicle. The resulting trituration is the 2nd
Centesimal triturations
decimal trituration (D2).
Decimal triturations
In all cases, it is possible to change to a liquid medium after
The 1” decimal trituration (Dl) is The 1" centesimal trituration (Cl)
made from: is made from:
the 7th decimal trituration (D7) as described in
1 part of the raw material 1 part of the raw material
Method 3.2.3.
Centesimal triturations
9 parts of the vehicle 99 parts of the vehicle
Proceed in the same manner but following a centesimal
Subsequent decimal Subsequent centesimal
triturations (Cn) are produced
series.
triturations (Dn) are produced
as stated for Dl, using 1 part of the as stated for Cl, using 1 part of the In all cases, it is possible to change to a liquid medium after
previous trituration (Dn-1). previous trituration (Cn-1). the 3rd centesimal trituration (C3) as described in
Method 3.2.3.
Where fresh plant material is used, the quantity of vehicle
added is such so as to obtain 10 parts of the trituration
METHOD 4.2 5. OTHER PREPARATIONS

Method 4.2 is used for triturations, that is solid dilutions, of METHOD 5.1
liquid preparations such as mother tinctures and solutions, Method 5.1 is used for preparing homoeopathic preparations
their dilutions, mixtures and co-potentised mixtures. by co-potcntising 2 or more stocks and/or dilutions thereof,
Gradually impregnate the total amount of vehicle, gently dry where co-potentisation consists of mixing several stocks or
the moist mixture, mill and sieve if necessary, then mix and dilutions of stocks then potentising them together in one or
triturate until homogeneity and potentisation are achieved. more potentisation steps.
Trituration is further carried out as described for METHODS 5.1 1, 5.1.2, 5.1.3 (EQUIVALENT TO HAB 40A, 40B, 40C:
Method 4.1.1 or Method 4.1.2. CO-POTENTISED MIXTURES)
Vehicle The stocks and/or dilutions in Table 2371.-5 may be used.

Unless otherwise specified, lactose monohydrate is used. Table 2371.-5
METHOD 4.2.1 (EQUIVALENT TO HAB 7: TRITURATIONS) Method 5.1.2 Method 5.1.3
Method 5.1.1
Ratios of starting material to vehicle Stocks Aqueous preparations Triturations
The quantity of vehicle added must always be such so as to
obtain 10 parts of the trituration (decimal trituration) or Solutions Glycerol macerates
and aqueous dilutions
100 parts of the trituration (centesimal trituration) from the thereof
required number of parts of the liquid preparation (see Triturations Triturations
Table 2371.-4), taking the mass of the dry residue into Liquid dilutions
consideration. Where the dry residue is considered negligible,
the quantity of vehicle added is 10 parts (decimal trituration) Mother tinctures
whose method of
or 100 parts (centesimal trituration), for 1 part of the liquid production specifies
preparation. a 1/10 (or 1/100)
dilution
Table 2371.-4
Vehicles
Decimal triturations Centesimal triturations The choice of the vehicle is determined by and must comply
Mother tinctures prepared according to Methods 1.1.1, 1.1.3 and 1.1.4 with any special requirement for the particular stock as well
as the dosage form (sec table Table 2371.-6).
The 1“ 'decimal' trituration (DI) is The I” ‘centesimal’ trituration (Cl)
made from: is made from ะ Table 2371.-6
2 parts of the mother tincture 2 parts of the mother tincture Method 5.1.1 Method 5.1.2 Method 5.1.3
maximum 100 parts of the vehicle, Ethanol (96 per cent V/V) [ethanol Water for Lactose
taking the mass of the dry residue taking the mass of the dry residue (94 per cent m/m)l injections monohydrate
into consideration into consideration
Ethanol (90 per cent V/V) [ethanol Purified water
Mother tinctures prepared according to Methods 1.1.2, 1.1.5, 1.1.6 (86 per cent m/m)]
and 1.1.7
Ethanol (80 per cent V/V) [ethanol Sugar syrup
The 1* ‘decimal’ trituration (DI) is The 1“ ‘centesimal’ trituration (Cl) (73 per cent m/m)] (sucrose,
made from ะ is made from ะ purified water
3 parts of the mother tincture 3 parts of the mother tincture (64:36))
maximum 100 parts of the vehicle,
(62 per cent m/m)]
taking the mass of the dry residue taking the mass of the dry residue
into consideration into consideration Ethanol (50 per cent V/V) [ethanol
(43 per cent m/m)]
Mother tinctures prepared according to Methods 1.1.8 and 1.1.9
The mother tincture corresponds to the lu decimal dilution (DI) (30 per cent m/m)]
The 2nd decimal trituration (D2) is The 1” ‘centesimal’ trituration (Cl)
(15 per cent m/m)]
made from: is made from:
1 part of the mother tincture 10 parts of the mother tincture For Method 5.1.1, when starting from a trituration and
maximum 10 parts of the vehicle, maximum 100 parts of the vehicle, where justified, purified water is used for the 1st potentisation
taking the mass of the dry residue taking the mass of the dry residue step.
into consideration into consideration
For Method 5.1.2, when starting from a glycerol macerate
Solutions prepared according to Method 3.1.1 or liquid dilutions,
mixtures and co-potentised mixtures containing sodium chloride, unless otherwise justified and
Decimal trituration n+1 (Dn+1) is Centesimal trituration n+1 (Cn+1)
authorised, the following vehicle is used: 0.2 parts of sodium
made from ะ is made from: hydrogen carbonate and 8.8 parts of sodium chloride in
1 part of the dilution (Dn) 1 part of the dilution (Cn) 991 parts of water for injections.
maximum 10 parts of the vehicle, maximum 100 parts of the vehicle, Potentisation
taking the mass of the dry residue taking the mass of the dry residue For each potentisation step, combine and succuss or triturate
into consideration into consideration 1 part of the given mixture with 9 parts (decimal dilutions)
or 99 parts (centesimal dilutions) of the appropriate vehicle.
METHOD 4.2.2
METHOD 5.1.4
Ratios of starting material to vehicle
Vehicles
Mother tinctures prepared according to Methods 1.1.10 and 1.1.11
Ethanol of an appropriate concentration, purified water or
lactose monohydrate may, for example, be used.
The 1“ decimal trituration (DI) is The 1* centesimal trituration (Cl)
made from: is made from: Potentisation
1 part of the mother tincture 1 part of the mother tincture
Potentisation may be performed as prescribed for
Methods 5.1.1, 5.1.2 and 5.1.3, either on the last step or on
10 parts of the vehicle 100 parts of the vehicle
several successive steps.
METHOD 5.1.5 CHARACTERS

Vehicle Where appropriate, the appearance and odour of the mother
Ethanol of an appropriate concentration, purified water or tincture are determined.
lactose monohydrate may, for example, be used. IDENTIFICATION
Potentisation Where applicable, at least 1 chromatographic identification
For a co-potentisation of centesimal dilutions, each test is carried out.
dilution (Cn-1) represents 1 per cent of the final product and
TESTS
the proportion of vehicle to be added is reduced by the
The limits in an individual monograph are set to include
proportion of the active substances
official methods of production. Specific limits will apply to
[i.e. 100 per cent -(1 per cent X the number of active
each defined method of production.
substances)]. The same procedure applies, in the appropriate
proportions, when co-potentising decimal dilutions. If the test for relative density is carried out, the test for ethanol
need not be carried out, and vice versa.
-------------------------------------- - ---------------------------------------------------------------- Ph Eur
The mother tincture for homoeopathic preparations complies
with the limits prescribed in the monograph.
Ethanol (2.9.10)
Mother Tinctures for * ** The ethanol content complies with that prescribed in the
Homoeopathic Preparations ***** monograph.
(Ph. Eur. monograph 2029) Methanol and 2-propanol (2.9.11)
Maximum 0.05 per cent VIV of methanol and maximum
PhEir____________
0.05 per cent VIV of 2-propanol, unless otherwise prescribed.
DEFINITION Dry residue (2.8.16)
Mother tinctures for homoeopathic preparations are liquid Where applicable, the mother tincture for homoeopathic
preparations obtained by the solvent action of a suitable preparations complies with the limits prescribed in the
vehicle upon raw materials. The raw materials are usually in monograph.
the fresh form but may be dried. Mother tinctures for
homoeopathic preparations may also be obtained from plant
Pesticides (2.8.13)
Where applicable, the mother tincture for homoeopathic
juices, with or without the addition of a vehicle. For some
preparations complies with the test. This requirement is met
preparations, the matter to be extracted may undergo a
if the herbal drug has been shown to comply with the test.
preliminary treatment.
Justification is provided in cases where the test for pesticides
PRODUCTION is performed on the mother tincture, instead of on the herbal
Mother tinctures for homoeopathic preparations are prepared drug according to the requirements of the general monograph
by maceration, percolation, digestion, infusion, decoction, Herbal drugs for homoeopathic preparations (2045). Limits will
fermentation or as described in the individual monographs, be set, taking into consideration the nature and the origin of
usually using ethanol of suitable concentration. the herbal drug. The dilution factor of the mother tincture
Mother tinctures for homoeopathic preparations are obtained and the limit of detection of the method are also taken into
using a fixed proportion of raw material to solvent, taking the account when fixing these limits.
moisture content of the raw material into account, unless Heavy metals (2.4.27)
otherwise justified and authorised. Justification is provided in cases where the test for heavy
If fresh plants are used, suitable procedures are used to metals is performed on the mother tincture, instead of on the
ensure freshness. The competent authorities may require that herbal drug according to the requirements of the general
the freshness is demonstrated by means of a suitable test. monograph Herbal drugs for homoeopathic preparations (2045).
Mother tinctures for homoeopathic preparations are usually Limits will be set, taking into consideration the nature and
clear. A slight sediment may form on standing and that is the origin of the herbal drug. The dilution factor of the
acceptable as long as the composition of the tincture is not mother tincture and the limit of detection of the method are
changed significantly. also taken into account when fixing these limits.
The manufacturing process is defined so that it is If required by the competent authority, suitable limits for the
reproducible. content of other heavy metals such as arsenic or nickel may
be defined.
Production by maceration
Unless otherwise prescribed, reduce the matter to be ASSAY
extracted to pieces of suitable size, mix thoroughly and Where applicable, an assay with quantitative limits is
extract according to the prescribed extraction method with performed.
the prescribed extraction solvent. Allow to stand in a closed
STORAGE
vessel for the prescribed time. The residue is separated from
Protected from light. A maximum storage temperature may
the extraction solvent and, if necessary, pressed out. In the
be specified.
latter case, the 2 liquids obtained are combined.
LABELLING
Adjustment of the contents
Adjustment of the content of constituents may be carried out The label states:
— that the product is a mother tincture for homoeopathic
if necessary, either by adding the extraction solvent of
suitable concentration, or by adding another mother tincture preparations (designated as ‘TM' or ‘0');
— the name of the raw material using the Latin title of the
for homoeopathic preparations of the vegetable or animal
European Pharmacopoeia monograph where one exists;
matter used for the preparation.
— the method of preparation;
— the ethanol content or other solvent content, correspond to the potency/potencies of the individual
in per cent V/V, in the mother tincture; preparations used in the mixture.
— the ratio of raw material to mother tincture;
TESTS
— where applicable, the storage conditions.
Uniformity of mass.
Ph Eur Carn7 out the test using 20 coated homoeopathic pillules to
constitute 1 unit. Weigh individually 20 units taken at
random and determine the individual and average masses.
Not more than 2 of the individual masses deviate from the
Coated Homoeopathic Pillules * * average mass by more than 10 per cent and none deviate by
more than 20 per cent.
Microbial contamination.
PhEtr_______________________________________________________________ Unless otherwise justified, authorised and labelled, coated
DEFINITION homoeopathic pillules are intended for sublingual
administration and the following acceptance criteria apply.
Solid preparations prepared from sucrose Pillules for
homoeopathic preparations (2153) (category 5), by coating with TAMC: acceptance criterion 102 CFU/g (2.6.12).
a syrup made from homoeopathic preparations either TYMC: acceptance criterion 10* CFU/g (2.6.12).
potentised or mixed with sucrose syrup. Triturations can be Absence of Staphylococcus aureus (2.6.13).
incorporated separately. Coated homoeopathic pillules
Absence of Pseudomonas aeruginosa (2.6.13).
possess a suitable mechanical strength to resist handling
PhEu
without crumbling or breaking. They are intended for
sublingual or oral use. Coated homoeopathic pillules may
also be called ‘globuli velati’.
PRODUCTION
In the manufacture, packaging, storage and distribution of Impregnated Homoeopathic
coated homoeopathic pillules, suitable measures are taken to
ensure their microbiological quality; recommendations on this
Pillules
aspect are provided in general chapter 5.1.4. Microbiological
quality of non-sterile pharmaceutical preparations and substances Ph Eur________________________________________ _
for pharmaceutical use. DEFINITION

For coating, use homoeopathic stocks (in case of a mother Preparations of solid consistency obtained from sucrose,
tincture or a glycerol macerate) and/or decimal dilutions lactose or other suitable excipients. They possess a suitable
thereof or decimal triturations and a syrup made from 64 mechanical strength to resist handling without crumbling or
parts of sucrose and 36 parts of purified water. breaking. Impregnated homoeopathic pillules are prepared by
The homoeopathic preparations used are prepared according impregnation of Pillules for homoeopathic preparation (2153)
to the general monograph Methods of preparation of with one or more liquid homoeopathic preparations. They
homoeopathic stocks and potentisation (2371). are intended for sublingual or oral use.
To prepare 100 parts of coated homoeopathic pillules, the PRODUCTION
pillules are coated with the syrup using one of the following Impregnation takes place using liquid preparations containing
procedures, depending upon the type of homoeopathic ethanol usually at a concentration of at least 68 per cent VIV
preparation used. (60 per cent mini) in proportions of 1 mass part of liquid to
Aqueous dilutions 100 mass parts of pillules.
Mix 1 part of an aqueous preparation with 9 parts of the In the manufacture, packaging, storage and distribution of
syrup and potentise by succussion; evenly apply the mixture homoeopathic pillules, suitable measures are taken to ensure
to (100 — x) parts of pillules (where X is the mass of sucrose their microbiological quality; recommendations on this aspect
in the syrup). The potency of the coated homoeopathic are provided in chapter 5.1.4. Microbiological quality of non-
pillules corresponds to the potency of the liquid preparation sterile pharmaceutical preparations and substances for
used for coating. pharmaceutical use.
Triturations CHARACTERS
Mix 10 parts of a trituration with 20 parts of the syrup;
Appearance
evenly apply the mixture to (100 - X- y) parts of pillules White, almost white or slightly coloured spheroids.
(where X is the mass of sucrose in the syrup and y is the
mass of lactose monohydrate in the incorporated trituration). Solubility
The potency of the coated homoeopathic pillules corresponds Usually freely soluble in water.
to the potency of the trituration used. TESTS
Mixtures Microbial contamination
Unless otherwise justified and authorised, prepare a mixture Unless otherwise justified, authorised and labelled, the
obtained from 10 parts of a liquid preparation (obtained pillules are intended for sublingual administration and the
from aqueous preparations, triturations or aqueous dilutions following acceptance criteria apply.
of glycerol macerates) and 90 parts of the syrup, adding TAMC: acceptance criterion 102 CFU/g (2.6.12).
sufficient purified water to evenly apply the mixture to TYMC: acceptance criterion 101 CFU/g (2.6.12).
(100 - X - y) parts of pillules (where X is the mass of
Absence of Staphylococcus aureus (2.6.13).
sucrose in the syrup and y is the mass of lactose
monohydrate in the incorporated triturations). Absence of Pseudomonas aeruginosa (2.6.13).
The potency/potencies of the coated homoeopathic pillules
Pillules for Homoeopathic ***** Fineness (2.9.55)

Not less than 90 per cent mint of the pillules are between the
Preparations ***** lower and upper limits of the corresponding category as
(Ph. Eur. monograph 2153) indicated in Table 2153.-1.
PnEir______________________________________________________________ Impregnation
Use an approved method. The average for the results is
DEFINITION
within a validated range.
Preparations of solid consistency obtained from sucrose,
lactose or other suitable excipients. They possess a suitable Microbial contamination
mechanical strength to resist handling without crumbling or TAMC: acceptance criterion 102 CFU/g (2.6.12).
breaking. They are intended for impregnation or coating with TYMC: acceptance criterion 101 CFU/g (2.6.12).
one or more homoeopathic preparations. The impregnated Absence of Staphylococcus aureus (2.6.13).
pillules comply with the requirements of the monograph Absence of Pseudomonas aeruginosa (2.6.13).
Homoeopathic pillules, impregnated (2079).
LABELLING
PRODUCTION The label states:
In the manufacture, packaging, storage and distribution of — the composition of the pillules;
pillules for homoeopathic preparations, suitable measures are — where applicable, the size of the pillules.
taken to ensure their microbiological quality;
PhEur
recommendations on this aspect are provided in chapter
5.1.4. Microbiological quality of non-sterile pharmaceutical
preparations and substances for pharmaceutical use.
If a system of sizing is used, the indications in Table 2153.-1
are used. Agaricus Phalloides for * *
Table 2153.-1. - Classification of pillules according to their
Homoeopathic Preparations *****
mass and size (Ph. Eur. monograph 2290)
Category Number of pillules Mass Fineness Ph Elf______________________________________________________________
for homoeopathic (g) (pm)
preparations DEFINITION
1 470 - 530 1.0 1000 - 1600 Whole, fresh mushroom (fruiting body) Amanita phalloides
(Vaill. ex Fr.) Link.
2 160 - 333 1.0 1400 - 2000
IDENTIFICATION
3 no - 130 1.0 1800 - 2500
A. In young specimens, the cap, 50-150 mm in diameter, is
4 70 - 90 1.0 2000 - 2800 hemispherical to campanulate with margins rolled inwards
5 40 - 50 1.0 2500 - 3350 and is still covered by the white universal veil; in mature
specimens, the cap is expanded, umbrella-like, convex to
6 16 - 30 1.0 3150 - 4500
nearly plane and occasionally depressed; its colour is pale
7 10 0.9 - 1.1 4000 - 5600 green along the margin and elsewhere greyish-green to
8 5 0.9 - 1.1 5600 - 6700 yellowish-green, typically with fine grey streaks growing
thicker towards the centre; the cuticle is peelable up to the
9 3 0.9 - 1.1 7100 - 8000
middle of the cap as a fine membrane; the underlying flesh
10 2 0.9 - 1.1 8000 - 9500 appears greenish-yellow, becoming more intense towards the
NOTE: for categories 7-10, the mass is obtained by weighing the
centre of the cap, while in the other parts of the mushroom
specified number of pillules. the flesh is uniformly whitish; lamellae are free, closely
packed, white with a slight yellowish tinge; lamellulae are
truncate; the stipe, about 10 cm high and 2 cm in diameter,
CHARACTERS is thin and solid, with a whitish, membranous, striate annulus
Appearance and an enlarged and bulbous base that almost always shows
White or almost white spheroids. a tough, white, membranous sac-like volva tom into irregular
Solubility lobes at the top. In old specimens, the stipe is hollow,
Usually freely soluble in water. whitish and often covered with small greenish scales around
the annulus. The spores are white.
IDENTIFICATION
The excipients used for the manufacture of pillules for B. Examined under a lens (xlO), the upper surface is shiny,
homoeopathic preparations are identified by one or more dry, appearing somewhat uneven, with no remains of the veil,
suitable test(s). c. Examine under a microscope using a solution containing
1.5 g of iodine p, 5 g of potassium iodide R and 100 g of
TESTS
chloral hydrate R in 100 mL of water R. The spores are
If the test for fineness is carried out, the test for uniformity of mass blackish-blue (starch reaction), short elliptical to
need not be carried out, and vice versa. subspherical, 8-11 pm long and 7-9 pm in diameter.
Uniformity of mass
TESTS
Carry out the test using 20 pillules to constitute 1 unit.
Weigh individually 20 units taken at random and determine
Maximum 5 per cent.
the individual and average masses. Not more than 2 of the
individual masses deviate from the average mass by more Loss on drying (2.2.32)
than 10 per cent and none deviate by more than 20 per cent. Minimum 85.0 per cent, determined on 5.0 g of the finely
cut drug by drying in an oven at 105 °C for 2 h.
Other Amanita species Top of the plate

Veil remnants on caps are typical for most Amanita species,
but not for A. phaUoides. Therefore all mushrooms with veil
remnants on the cap have to be discarded. The presence of Rutin: a quenching Rutin: a yellow zone
veil remnants (patches) on the cap indicates adulteration with A violet zone
A. citrina (Schaeff.) Pers, (whitish-yellow cap with whitish to
brownish patches) or with A. ท111รcaria (L.: Fr.) Lamarck, A.
Caesarea (Scop.: Fr.) Pers., or A. rubescens Pers, (orange to
bright red cap with white patches); a brownish cap indicates
Sennoside B: a A violet zone
adulteration with A. pantherina (DC.) Krombh.; a greenish-
quenching zone
white cap and a white stipe with a labile annulus indicates
A violet zone
adulteration with A. verna (Bull.: Fr.) Lamarck.
2 faint greyish-violet
zones may be present
MOTHER TINCTURE
The mother tincture complies vrith ±e requirements of the Reference solution Reference solution Test solution
general monograph Mother tinctures for homoeopathic (detection A) (detection B) (detection B)
preparations (2029).
DEFINITION B. Thin-layer chromatography (2.2.27).
Content Test solution The mother tincture to be examined.
0.001 per cent mhn to 0.010 per cent mini of ct-amanitine Reference solution Dissolve 2 mg of gramine R and 2 mg of
(C39H54N1o014S; Mt 919). rutin R in methanol R and dilute to 10 mL with the same
PRODUCTION solvent.
The mother tincture is prepared according to the following Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
methods as prescribed in the monograph Methods of plate R (2-10 pm)].
preparation of homoeopathic stocks and potentisation (237ไ)-. Mobile phase glacial acetic acid Ry water Ry methanol Ry
— method 1.1.5; methylene chloride 7? (4:6:30:60 VIVIVIV).
— method 1.1.10, using 5 parts of the cut drug for 100
parts of ethanol (45 per cent V/V) and maceration for
3 weeks. Development Over a path of 10 cm [or 6 cm].
CHARACTERS
Detection Treat with a 5 per cent VIV solution of cinnamic
Appearance
aldehyde R in methanol R and expose to hydrochloric acid R
Brownish-yellow, yellowish or green liquid.
vapour for 30 min; examine in daylight.
IDENTIFICATION Results See below the sequence of zones present in the
Carry out test A when method 1.1.5 is used and carry out chromatograms obtained with the reference solution and the
test B when method 1.1.10 is used. test solution. Furthermore, other faint zones may be present
A. Thin-layer chromatography (2.2.27). in the chromatogram obtained with the test solution.
Test solution Evaporate to dryness 20 mL of the mother
tincture under vacuum at about 40 °C. Dissolve the residue Top of the plate
in 1 mL of water R and add 1 mL of methanol R. Filter
immediately.
Reference solution Dissolve 10 mg of rutin R and 10 mg of Gramine: a yellow zone
sennoside B R in methanol R and dilute to 40 mL with the Rutin: a yellow zone
same solvent.
Mobile phase glacial acetic acid Ry water R, butanol R A pink zone
(17:17:66 VIVIV).
Application 40 pL as bands. An orange-yellow zone
Results A Locate a quenching zone (sennoside B) in the lower TESTS
third and a quenching zone (rutin) in the middle third of the Relative density (2.2.5)
chromatogram obtained with the reference solution. 0.895 to 0.915, where method 1.1.5 is used.
Detection B Treat immediately with a 1 per cent v/v solution Ethanol {2.9.10)
of cinnamic aldehyde R in methanol R and allow to dry. Treat 40 per cent v/v to 50 per cent V/Vy where method 1.1.10 is
with hydrochloric acid R-y examine in daylight. used.
Results B See below the sequence of zones present in the Dry residue {2.8. ไ 6)
chromatograms obtained with the reference solution and ±e Minimum 0.8 per cent.
test solution. Furthermore, other faint zones may be present Mother tincture of Amanita musearia
Test solution The mother tincture to be examined.
Reference solution Dissolve 10 mg of leucine R and 10 mg of

threonine 1? in 5 mL of water R and dilute to 20 mL with
Allium Sativum for Homoeopathic *****
ethanol (96 per cent) R. Preparations *****
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel Garlic for Homoeopathic Preparations
plate R (2-10 gm)].
Mobile phase glacial acetic acid R, water Ry acetone R, butanol R
PhEur_____________________________________________________________
(10:20:35:35 VIVIVIV).
Application 20 pL [or 10 pL] as bands. DEFINITION
Fresh bulb of Allium sativum L.
Drying In a current of warm air. CHARACTERS
It has a characteristic odour after cutting.
Detection Treat with a 1 g/L solution of ninhydrin R in
butanol R and heat at 105 °C for 5-10 min; examine in IDENTIFICATION
daylight. The bulb is generally 3 cm to 5 cm broad and almost
Results The presence of noticeable zones in the spherical; the flat base bears the remnants of numerous short
chromatogram obtained with the test solution, in the same greyish-brown adventitious roots. The bulb consists of about
position as the zones due to leucine and threonine in the 10 daughter bulbs (cloves) arranged roughly in a circle
chromatogram obtained with the reference solution, indicates around a central axis. Individual daughter bulbs are 1 cm to
adulteration with mother tincture of Amanita muscaria (L. 3 cm long, laterally compressed and convex on the dorsal
ex Fries) Hooker. side. Each daughter bulb has a tough, white or reddish skin
around a fleshy tubular leaf, investing a more or less rounded
ASSAY elongated cone of leaf primordia and vegetative apex.
TESTS
Test solution Dissolve 8.000 g of the mother tincture to be
examined in the mobile phase and dilute to 10.0 mL with
Water {2.2.13)
Minimum 55.0 per cent, determined on 10.0 g of the finely
the mobile phase.
cut drug, if performed to demonstrate the freshness of the
Reference solution (a) Dissolve 10.0 mg of tryptophan CRS in drug.
the mobile phase and dilute to 20.0 mL with the mobile
phase.
Reference solution (b) Mix 5.0 mL of reference solution (a)
MOTHER TINCTURE
with 5.0 mL of the test solution. The mother tincture complies with the requirements of the
Column: general monograph Mother tinctures for homoeopathic
— size: I = 0.25 m, 0 = 4.6 mm; preparations (2029).
— stationary phase: octadecylsilyl silica gel for chromatography R PRODUCTION
(5 gm); The mother tincture of Allium sativum L. is prepared by
— temperature: 30 °C. maceration of the cut drug using alcohol of a suitable
Mobile phase methanol R, water R (25:75 VIV). concentration.
Flow rate 1.0 mUmin. CHARACTERS
Detection Spectrophotometer at 303 nm. Appearance
Injection 10 gL. Brownish-yellow liquid.
Run time 40 min. It has a peculiar and unpleasant aromatic odour.
Relative retention With reference to a-amanitine (retention IDENTIFICATION
time = about 11 min): tryptophan = about 0.6. A. To 2 mL of the mother tincture to be examined, add
Systent suitability: reference solution (b): 0.2 mL of dilute sodium hydroxide solution R. A yellowish-
— resolution: minimum 4.0 between the peaks due to white precipitate develops.
tryptophan and a-amanitine. B. Thin-layer chromatography (2.2.27).
Calculate the percentage content m/m of a-amanitine using Test solution Extract 5 mL of the mother tincture to be
the following expression: examined with 2 quantities, each of 10 mL, of ether R.
Combine the ether layers and dry over anhydrous sodium
sulfate R. Filter and evaporate the filtrate in a water-bath at
low temperature. Dissolve the residue in 0.4 mL of
methanol R.
A1 = area of the peak due to tryptophan in the Reference solution Dissolve 10 mg of resorcinol Ry 10 mg of
chromatogram obtained with reference solution (a); thymol R and 30 mg of gallic acid R in 10 mL of methanol R.
A2 = area of the peak due to a-amanitine in the Plate TLC silica gel F254 plate R.
Mobile phase anhydrous formic acid Ry toluene Ry di-isopropyl
m1 = mass of tryptophan CRS used to prepare reference
ether R (10:40:50 VIVIV).
m2 = mass of the mother tincture to be examined used to Application 40 pL of the test solution and 10 pL of the
prepare the test solution, in grams; reference solution.
p = percentage content of tryptophan in tryptophan CRS; Development Over a path of 10 cm.
0.1 = correction factor between a-amanitine and Drying In air.
tryptophan. Detection Examine in ultraviolet light at 254 nm and identify
_____________________________ _________________________________ PhEur gallic acid; spray with anisaldehyde solution R) heat to
FV-446 Homoeopathic Preparations 2016
105-110 °C for 5-10 min. Examine in daylight within Test solution To 1.0 g of suitably cut herbal drug, add 10 mL
10 min. of ethanol (90 per cent V/V) R. Heat under reflux on a water
Results See below the sequence of the zones present in the bath at 60 °C for 15 min. Allow to cool and filter.
chromatograms obtained with the reference solution and the Reference solution Dissolve 5 mg of gallic acid R and 5 mg of
test solution. Other zones may also be visible in the caffeic acid R in methanol R and dilute to 10 mL with the
chromatogram obtained with the test solution. same solvent.
Top of the plate Mobile phase methanol R, toluene R (15:85 V/V).
An intense reddish-violet zone Application 20 pL of the test solution and 10 pL of the
Thymol: an orange-red zone
An intense reddish-violet zone
Drying In air.
A violet zone Detection Spray with a solution containing 10 g/L of
A yellowish or greenish zone diphenylboric acid aminoethyl ester R and 50 g/L of macrogol
400 R in methanol R. Examine in ultraviolet light at 365 run.
Resorcinol: an intense orange-red chromatograms obtained with the reference solution and the
zone
test solution. Furthermore, other fainter zones may be
Gallic acid: a yellow zone A violet zone
(UV at 254 nm: a fluorescent A greenish-yellow zone Top of the plate

quenching zone)
A violet zone may be present A greenish-blue fluorescent zone

Several violet-blue fluorescent
zones
TESTS A yellow fluorescent zone
Caffeic acid: a violet-blue
0.885 to 0.960. fluorescent zone
Ethanol (2.9.10)
50 per cent v/v to 70 per cent v/v.
Gallic acid: a violet-blue A violet-blue fluorescent zone
Dry residue (2.8.16) fluorescent zone (gallic acid)
STORAGE Reference solution Test solution
---------------------------------------------------------------------------------- —_ _________ PhEur
TESTS
Anacardium occidentals L
Fruits of Anacardium occidental L. are not present. These are
up to 35 mm long, 30 mm large, 20 mm thick, light brown
Anacardium for Homoeopathic * \ and distinctly kidney-shaped. The pericarp is smooth or
slightly crinkled with dark marbling in places.
Preparations *****
Oriental Cashew for Homoeopathic Preparations
Maximum 12.0 per cent, determined on 1.000 g of the finely
(Ph. Eur. monograph 2094) divided herbal drug by drying in an oven at 105 °C for 2 h.
PhEur____________________________________ _________________________
Total ash (2.4.16)
DEFINITION Maximum 5.0 per cent.
Dried fruit of Semecarpus anacardium L. (Anacardium ASSAY
orientate L.). Total phenol derivatives
Content Absorption spectrophotometry (2.2.25).
Minimum 6.0 per cent m/m of total phenol derivatives Stock solution Place 4.500 g of the crushed herbal drug in a
expressed as eugenol (Ci0H12O2; M,.164.2) (dried drug). flask. Add 200 mL of ethanol (90 per cent V/V) R. Boil in a
IDENTIFICATION water-bath under reflux for 4 h. Cool the flask.
A. The dried fruit is oval and more or less heart-shaped; Quantitatively transfer into a volumetric flask. Dilute to
about 2 cm long, nearly 2 cm wide and 0.5 cm thick. 250.0 mL with ethanol (90 per cent V/V) R. Filter the liquid
Its surface is smooth, shiny and blackish. A transverse section through a paper filter 125 mm in diameter. Discard the first
shows a rather well developed, tough pericarp riddled with 50 mL of the filtrate. Dilute 5.0 mL of filtrate to 50.0 mL
rather wide lacunae containing an abundant thick reddish- with ethanol (90 per cent V/V) R and shake. Dilute 5.0 mL of
brown juice. The pericarp covers a white kernel under a this solution to 10.0 mL with ethanol (90 per cent V/V) R and
reddish skin. The fruit may include the blackish, fleshy, shake.
wrinkled, cupuliferous receptacle. Test solution To 2.0 mL of stock solution add 1.0 mL of
B. Thin-layer chromatography (2.2.27). phosphomolybdotungstic reagent R and 10 mL of water R) mix
and dilute to 25.0 mL with a 290 g/L solution of sodium Stock solution Place 8.000 g of the mother tincture to be
carbonate R. Wait exactly 3 min then filter the solution examined in a volumetric flask and dilute to 250.0 mL with
through a fibre-glass filter with a 1 pm mesh aperture, ethanol (90 per cent VIV) R. Dilute 5.0 mL of this solution to
discarding the first 5 mL. 20.0 mL with ethanol (90 per cent VIV) R.
Reference solution Dissolve 80.0 mg of eugenol R in ethanol Test solution To 2.0 mL of stock solution add 1.0 mL of
(90 per cent VIV) R and dilute to 250.0 mL with the same phosphomolybdotungstic reagent R and 10 mL of water R, mix
solvent. Dilute 5.0 mL of the solution to 25.0 mL with and dilute to 25.0 mL with a 290 g/L solution of sodium
ethanol (90 per cent VIV) R. To 2.0 mL of this solution add carbonate R. Wait exactly 3 min then filter the solution
1.0 mL of phosphomolybdotungstic reagent R and 10 mL of through a fibre-glass filter with a 1 pm mesh aperture,
water R} mix and dilute to 25.0 mL with a 290 g/L solution discarding the first 5 mL.
of sodium carbonate R. Wait exactly 3 min then filter the Reference solution Dissolve 80.0 mg of eugenol R in ethanol
solution through a fibre-glass filter with a 1 pm mesh (90 per cent VIV) R and dilute to 250.0 mL with the same
aperture, discarding the first 5 mL. solvent. Dilute 5.0 mL of the solution to 25.0 mL with
Measure the absorbance (2.2.25) of the test solution and the ethanol (90 per cent VIV) R. To 2.0 mL of this solution add
reference solution at 755 nm after 30 min using water R as 1.0 mL of phosphomolybdotungstic reagent R and 10 mL of
compensation liquid. water R) mix and dilute to 25.0 mL with a 290 g/L solution
Calculate the percentage content ทใเทใ of total phenol of sodium carbonate R. Wait exactly 3 min then filter the
derivatives, expressed as eugenol, from the following solution through a fibre-glass filter with a 1 pm mesh
expression: aperture, discarding the first 5 mL.
Measure the absorbance (2.2.25) of the test solution and the
Al X rrt2 X 400 reference solution at 755 nm after 30 min, using water R as
A2 X mi compensation liquid.
A] = absorbance of the test solution; Calculate the percentage content mini of total phenol
A2 = absorbance of the reference solution; derivatives expressed as eugenol, using the following
m1 = mass of the drug to be examined, in milligrams; expression:
f,t2 = mass of eugenol in the reference solution, in
milligrams. Al X m2 X 80
A2 X mi
MOTHER TINCTURE
A1 = absorbance of the test solution;
The mother tincture complies with the requirements of the A2 = absorbance of the reference solution;
general monograph Mother tinctures for homoeopathic nii = mass of the mother tincture to be examined, in
preparations (2029). milligrams;
Ph Eur______________________________________________________________ ท12 = mass of eugenol in the reference solution, in
DEFINITION milligrams.
The mother tincture of Anacardium is prepared by ______________________________________________________________ Ph Eur
maceration using ethanol of a suitable concentration from the
dried fruit of Semecarpus anacardium L. (Anacardium
orientate L.).
Content
0.5 per cent mini to 1.0 per cent mini of total phenol
Apis for Homoeopathic * *
derivatives expressed as eugenol. Preparations *****
CHARACTERS Honey Bee for Homoeopathic Preparations
Appearance (Ph. Eur. monograph 2024)
Yellowish-brown or reddish-brown liquid. Ph Eur-------------------------------------------------------------------------- --------------------------------
IDENTIFICATION DEFINITION
Thin-layer chromatography (2.2.27) as described under Live worker honey bee (Apis mellifera L.).
Identification B of the drug with the following modification.
CHARACTERS
Test solution The tincture to be examined. Characters described under Identification.
Results See identification B for the drug.
PRODUCTION
TESTS If the bee has been exposed to treatment to prevent or cure
Relative density (2.2.5) diseases, appropriate measures are taken to ensure that the
0.815 to 0.845. levels of residues are as low as possible.
Ethanol (2.9.10) IDENTIFICATION
85 per cent VIV to 95 per cent VIV. The body is about 15 mm long, black, with a silky sheen,
Dry residue (2.8.16) and covered with red hairs with a touch of grey. The broad
Minimum 1.50 per cent mini. tibiae are without spines. The posterior margins of the
segments and legs are brown, with gradual transition to
ASSAY
orange-red. The claws are two-membered, the maxillary
Total phenol derivatives
palps single-membered. On the hind legs are baskets or
Absorption spectrophotometry (2.2.25) as described in the
scoops invested with bristles. The wings have 3 complete
assay of the drug to be examined with the following
cubital cells, with the radial cell twice as long as it is wide;
modifications.
the 3 cells on the lower margin and the 3 middle cells are
closed. A duct connects the barbed sting with the poison sac. Apomorphine Hydrochloride for
Homoeopathic Preparations
MOTHER TINCTURE Apomorphinum Muriaticum for Homoeopathic Preparations
The mother tincture complies with the requirements of the DEFINITION
general monograph Mother tinctures for homoeopathic Apomorphinc Hydrochloride for Homoeopathic Preparations
preparations (2029). contains Apomorphine Hydrochloride Hemihydrate.
PRODUCTION PRODUCTION OF STOCK
The mother tincture of Apis mdlifera L. is prepared by The first trituration of Apomorphine Hydrochloride for
maceration using alcohol of a suitable concentration. Homoeopathic Preparations is prepared using a suitable
quantity of Lactose or Anhydrous Lactose as the vehicle and
CHARACTERS
a validated trituration method that ensures homogeneity is
Pale yellow liquid that may darken on storage.
achieved. The vehicle complies with the statement under
IDENTIFICATION Vehicles in the monograph for Homoeopathic Preparations.
Thin-layer chromatography (2.2.27). Content of apomorphine hydrochloride,
Test solution The mother tincture to be examined. C17H17NO2,HC1
Reference solution Dissolve 12 mg of 4-aminobutanoic acid. R, The first decimal trituration contains 9.5% to 10.5% of
12 mg of leucine R and 12 mg of proline R in 5 mL of water R C17H17NO2,HC1 (dried substance).
and dilute to 50 mL with alcohol R. CHARACTERISTICS
Plate TLC silica gel plate R. The first decimal trituration is a white powder.
Mobile phase water R, ethanol R (17:63 VIV). IDENTIFICATION
Application 20 pL, as bands. Dissolve 2.5 g of the substance being examined without
Development Over a path of 10 cm. heating in water and dilute to 25 mL with the same solvent
Drying In air. (solution ร).
Detection Spray with ninhydrin solution R and heat at A. To 5 mL of solution ร add a few millitres of sodium
100-105 °C for 10 min; examine in daylight. hydrogen carbonate solution until a permanent, white
precipitate is formed. The precipitate slowly becomes a
greenish colour. Add 0.25 mL of 0.05M iodine and shake.
The precipitate becomes a greyish-green colour. Collect the
solutions. Other zones may also be visible.
precipitate. The precipitate dissolves in ether giving a purple
solution, dissolves in dichloroniethane chloride giving a violet
Top of the plate blue solution and dissolves in alcohol giving a blue solution.
— B. To 2 mL of solution ร add 0.1 mL of nitric acid, mix and
filter. The filtrate yields reaction A characteristic of chlorides,
A pink zone
Appendix VI.
Leucine: a pink zone
c. Dissolve 0.25 g of the substance being examined in 5 mL
A pink zone of water. Add 5 mL of ammonia and heat in a water-bath at
A pink zone
80° for 10 minutes. A red colour develops.
ASSAY
Disperse 2.5 g of the substance being examined in a mixture
Proline: an orange-yellow zone An orange-yellow zone
of 5.0 mL of 0.0IM hydrochloric acid and 50 mL of ethanol
4-Aminobutanoic acid: a pink zone A pink zone (96%). Carry out the method for potentiometric titration,
Appendix VIII B, using 0.1M sodium hydroxide. Measure the
titrant between the first 2 points of inflexion. Each mL of
0.1M sodium hydroxide is equivalent to 30.38 mg of
Reference solution Test solution Cj7HI7NO2,HC1.
TESTS
Relative density (2.2.5) Arsenious Trioxide for
0.890 to 0.910
Ethanol (2.9.10) (Arsenicum Album for Homoeopathic Preparations,
60 per cent V/V to 70 per cent V/V.
Ph. Eur. monograph 1599)
Dry residue (2.8.16) 1327-53-3
As2O3 197.8
PhEur________________________________________________
PhEur
DEFINITION
Content
99.5 per cent to 100.5 per cent of As2O3.
CHARACTERS
Appearance
White or almost white powder.
Solubility from the central region of the bracts show polygonal,

Practically insoluble to sparingly soluble in water. It dissolves isodiametnc epidermal cells with thickened and beaded walls;
in solutions of alkali hydroxides and carbonates. fairly numerous anomocytic stomata are present on the outer
IDENTIFICATION surface only. Very small cluster crystals of calcium oxalate
A. Dissolve 20 mg in 1 mL of dilute hydrochloric acid R, add may be present in the parenchymatous cells underlying the
4 mL of water R and 0.1 mL of sodium sulfide solution R. epidermis.
The resulting yellow precipitate is soluble in dilute Groups of sclereids from the central region of the bracts
ammonia Rl. show: individual cells varying in shape but usually
B. Dissolve 20 mg in 1 mL of hydrochloric acid Rl, add 5 mL considerably elongated; the ends are square or bluntly
tapering or, occasionally, somewhat enlarged; the walls are
of hypophosphorous reagent R and heat for 15 min on a water
bath. A black precipitate develops. strongly thickened and have scattered pits. Small groups of
these sclereids are occasionally found attached to fragments
TESTS of the epidermis of the bracts.
Appearance of solution The unicellular covering trichomes are nearly always found
A 100 g/L solution in dilute ammonia Rl is clear (2.2.1) and detached; they are usually very thin-walled although slight
colourless (2.2.2, Method IP). thickening may occur in the basal region; some of these
Sulfides trichomes are very long and can be found in groups forming
Maximum 20 ppm. loosely felted, cottony masses. The typically labiate glandular
Dissolve 1.0 g in 10.0 mL of dilute sodium hydroxide trichomes are abundant on the bracts and are also found
solution R. Add 0.05 mL of lead acetate solution R. Any colour detached; each has a short, biseriate stalk, and a biseriate
in the test solution is not more intense than that in a head of two or four cells around which the cuticle is raised to
standard prepared at the same time and in the same manner form a bladder-like covering.
using a mixture of 10.0 mL of a 0.015 g/ L solution of sodium The abundant pollen grains are fairly small, spherical, with
sulfide R in dilute sodium hydroxide solution R and 0.05 mL of three pores and three furrows; the exine is finely warted.
lead acetate solution R. A large number of immature pollen grains are present,
ASSAY forming elongated, closely packed masses.
Dissolve 40.0 mg in a mixture of 10 mL of water R and c. Carry out the method for thin-layer chromatography.
10 mL of dilute sodium hydroxide solution R. Add 10 mL of Appendix in A, using the following solutions.
dilute hydrochloric acid R and 3 g of sodium hydrogen (1) Add 10 mL of ethanol (90%) to 1 g of the coarsely
carbonate R and mix. Add 1 mL of starch solution R and powdered drug, stir for one hour and filter.
titrate with 0.05 M iodine. (2) 0.1% w/v each of santonin and cineole in methanoL
1 mL of 0.05 M iodine is equivalent to 4.946 mg of As2O3. CHROMATOGRAPHIC CONDITIONS
---------------------------------------------------------------------------------------------------------- Ph Eur (a) Use as the coating silica gel H.
(c) Apply 10 pL of each solution.
Artemisia Cina for Homoeopathic (e) After removal of the plate, allow to dry in air, spray with
Preparations ethanolic phosphomolybdic acid solution, heat at 100° to 105° for
about 5 minutes and examine in daylight.
DEFINITION
Artemisia Cina for Homoeopathic Preparations is the dried, mobile phase
unexpanded flower heads of Seriphidium cinum (Berg ex 5 volumes of glacial acetic acid, 45 volumes of hexane and
Poljakov) Poljakov (Syn Artemisia cina Berg ex Poljakov) 50 volumes ethyl acetate.
Artemisia cina O.C.Berg et C.F. Schmidt. SYSTEM SUITABILITY
It contains not less than 1.0% of santonin (CI5H18O3) The test is not valid unless the chromatogram obtained with
calculated with reference to the dried material. solution (2) shows the grey-blue santonin band just between
IDENTIFICATION the lower third and middle third and the grey-blue cineole
A. The dried, slightly shiny capitula are conical to elongated- band in the upper third.
ovoid, 2 to 4 mm long and 1 to 2 mm wide, yellow-green to CONFIRMATION
brownish and composed of 3 to 6 hermaphrodite florets The chromatogram obtained with solution (1) shows a grey
enclosed in an involucre of 14 to 20 imbricated ovate to blue band below the band obtained for santonin in solution
lanceolate bracts. Each bract has a distinct keel which is most (2), a strong grey-blue band level with that of santonin in
pronounced in the ovate outer bracts near the base; the keel solution (2) and one or two grey-blue bands just above
forms the midrib and it branches freely, the veinlets santonin; one or two grey-blue bands between the bands
becoming contorted and frequently anastomising. The outer obtained for santonin and cineole in solution (2) and a
surface of the bracts is covered with glistening glandular strong grey-blue band level with the band obtained with
hairs. The florets are about 1 mm long and 0.5 mm wide; cineole.
the corolla is contracted at the base and divides at the apex
into 5 short, triangular teeth.
B. Reduce to a powder and examine under a microscope
using chloral hydrate solution. Abundant fragments of the
involucral bracts in surface view are seen. Fragments from
the margins are usually only one or two cells thick and are
composed of very thin-walled, elongated cells; fragments
Top of the plate

A-fv,* X/7X100-d
A grey-blue band Cineole: a grey-blue band
1 or 2 a grey-blue bands
Al = area of the peak due to santonin in the

1 or 2 grey-blue bands chromatogram obtained with solution (1);
a grey-blue band Santonin: a grey-blue band A2 = area of the peak due to santonin in the
a grey-blue band
chromatogram obtained with solution (2);
nil = weight of the herbal drug being examined in mg;
m2 = weight of santonin BPCRS in mg;
Solution (1) Solution (2) Vi = dilution volume of solution (1) in mL;
v2 — dilution volume of solution (2) in mL;
p = percentage content of santonin (C15H18O3) in
TESTS santonin BPCRS’,
Foreign matter d = percentage loss on drying of the herbal drug being
Not more than 5% of sections of stem and pieces of narrow- examined.
linear hairy leaves; not more than 2% of other foreign matter,
Appendix XI D.
Ash
MOTHER TINCTURE
Not more than 11.0%, Appendix XI J, Method II. The mother tincture complies with the requirements stated under
Loss on drying Mother Tinctures for Homoeopathic Preparations and with the
Not more than 10.0%, Appendix IX D. following requirements.
ASSAY DEFINITION
Carry out the method for liquid chromatography. It contains not less than 0.1% of santonin (Cj5H1803).
Appendix in D, using the following solutions. PRODUCTION
(1) To 1.0 g of the powdered herbal drug add 50 mL of The mother tincture of Artemisia cina is prepared from the
methanol and stir for 2 hours. Filter the solution using a dry powdered drug using Method 1.1.8 described in the
filter paper into a 100 mL volumetric flask, wash the filtrate monograph for Methods of Preparation of Homoeopathic
with methanol) add the washings to the filtrate and dilute to Stocks and Potentisation. Use 86% พ/พ (90% v/v) of ethanol.
100 mL with methanol and mix. Weigh approximately 5 g CHARACTERISTICS
(6.5 mL) of the solution and add 20 mL of methanol in a The mother tincture is a golden yellow to greenish liquid.
50 mL volumetric flask and dilute to volume with water.
(2) 0.005% w/v of santonin BPCRS prepared by dissolving
IDENTIFICATION
100 mg santonin BPCRS in 100 ml methanol and diluting The mother tincture complies with Identification test c
5 mL of the resulting solution to 100 mL with the mobile above using the mother tincture as solution (1).
phase. TESTS
(3) 0.005% w/v each of santonin BPCRS and methyl 4- Ethanol
hydroxybenzoate in the mobile phase. 40% to 46% พ/พ (47% to 54% v/v), Appendix vni F.
CHROMATOGRAPHIC CONDITIONS Dry residue.
(a) Use a stainless steel column (15 cm X 4.6 mm) packed Not less than 1.8% พ/พ, Appendix XI p.
with octadecylsilyl silica gel for chromatography (5 pm) Relative density
(Kromasil C18 is suitable) fitted with a stainless steel 0.835 to 0.855, Appendix V G.
guard column packed with the same material. ASSAY
(b) Use isocratic elution and the mobile phase described Carry out the method for liquid chromatography,
below. Appendix in D, as described for the herbal drug using as
(c) Use a flow rate of 1.0 mL per minute. solution (1) the mother tincture.
(d) Use a column temperature of 25c. DETERMINATION OF CONTENT
(e) Use a detection wavelength of 236 nm. Calcdate the content of c 15H1803 in the mother tincture
(f) Inject 10 pL of each solution. using the declared content of c 15H1803 in santonin BPCRS
MOBILE PHASE
Equal volumes of methanol and water.

SYSTEM SUITABILITY 4 X__พ—2 X —K“ X
X p
The test is not valid unless, in the chromatogram obtained m.
to methyl 4-hydroxybenzoate and santonin is not less than 2.0.
A1 = area of the peak due to santonin in the
chromatogram obtained with solution (1);
Calculate the content of c 15H1803 in the herbal drug using A2 = area of the peak due to santonin in the
the declared content of C15H18O3 in santonin BPCRS using chromatogram obtained with solution (2);
the following expression: m1 = weight of the herbal drug being examined in mg;
m2 = weight of santonin BPCRS in mg;
P) = dilution volume of solution (1) in mL;

IZ2 = dilution volume of solution (2) in mL; Cadmium Sulfate Hydrate for ** **
p = percentage content of santonin (C15H18O3) in Homoeopathic Preparations *****
santonin BPCRS. (Cadmium Sulfuricum for Homoeopathic preparations,
CdSO4,8/3H2O 256.5
Ph Elf______________________________________________________________
Barium Chloride Dihydrate for ** ** DEFINITION
Homoeopathic Preparations ***** Content
(Barium Chloratum for Homoeopathic Preparations, 98.0 per cent to 102.0 per cent (anhydrous substance).
Ph. Eur. monograph 2142) CHARACTERS
BaC12,2H2O 244.3 10326-27-9 Appearance
Fn Elf ______________ ____ ___ _________________
White or almost white, crystalline powder.
Solubility
DEFINITION Freely soluble in water, practically insoluble in ethanol
Content (96 per cent).
99.0 per cent to 101.0 per cent of BaCl2,2H2O.
IDENTIFICATION
CHARACTERS A. It gives reaction (a) of sulfates (2.3.1).
Appearance B. To 2 mL of solution ร (see Tests) add 2 mL of sodium
White or almost white, crystalline powder or colourless sulfide solution R. A yellow precipitate is formed.
crystals.
TESTS
Solubility Solution ร
Freely soluble in water, very slightly soluble or practically Dissolve 5.0 g in carbon dioxide-free water R and dilute to
insoluble in ethanol (96 per cent). 50 mL with the same solvent.
IDENTIFICATION Appearance of solution
A. Dissolve 0.1 g in 1 mL of water R. Add 0.3 mL of dilute Solution ร is clear (2.2.1) and colourless (2.2.2, Method II).
sulfuric acid R. A white precipitate is formed; it is insoluble in Acidity or alkalinity
dilute hydrochloric acid R and in dilute nitric acid R. To 10 mL of solution ร add 0.3 mL of methyl orange
B. It gives reaction (a) of chlorides (2.3.1). solution R. Not more than 0.5 mL of 0.01 M hydrochloric acid
TESTS or 0.01 M sodium hydroxide is required to change the colour
of the indicator.
Solution ร
Dissolve 10.0 g in water R and dilute to 100 mL with the Nitrates
same solvent. Maximum 100 ppm.
Appearance of solution Dissolve 1.0 g in water R and dilute to 20.0 mL with the
Solution ร is clear (2.2.1) and colourless (2.2.2, Method II). same solvent. To 1.0 mL of this solution add 0.2 mL of a
10 g/L solution of sulfanilic acid R in acetic acid R and 0.2 mL
Acidity or alkalinity of a recently prepared 3 g/L solution of naphthylamine R in
To 10 mL of solution ร add 0.1 mL of phenolphthalein acetic acid R. Add a turning of zinc R. A pink colour is
solution R. Not more than 0.2 mL of 0.01 M hydrochloric acid produced within 5 min. It is not more intense than that of a
or 0.01 M sodium hydroxide is required to change the colour mixture of 0.5 mL of nitrate standard solution
of the indicator. (10 ppm NOt) R and 0.5 mL of water R, prepared at the
Heavy metals (2.4.8) same time.
Maximum 10 ppm. Zinc sulfate, alkaline-earth sulfates, rare-earth sulfates
12 mL of solution ร complies with test A. Prepare the Dissolve 1.0 g in 17 mL of water R. Add 0.5 mL of
reference solution using lead standard solution (1 ppm Pb) R. hydrochloric acid R and 1 g of thioacetamide R. Heat in a
water-bath for 10 min. Dilute to 20.0 mL with water R and
ASSAY
filter. Evaporate 10.0 mL of this solution to dryness in an
Dissolve 0.200 g in 100 mL of water R. Add 100 mL of
oven. Ignite the residue at about 800 ± 50 °C to constant
methanol R, 10 mL of concentrated ammonia R and 2 mg of
mass. The residue weighs a maximum of 2 mg.
phthalein purple R. Titrate with 0.1 M sodium edetate until the
colour changes from violet to colourless. Arsenic (2.4.2, Method A)
Maximum 2 ppm, determined on 5 mL of solution ร.
1 mL of 0.1 M sodium edetate is equivalent to 24.43 mg
of BaCl2,2H2O. Water (2.5.12)
16.0 per cent to 20.0 per cent, determined on 80 mg. Shake
_______________________________________________________________ Ph Eur
for 10 min before carrying out the determination.
ASSAY
Dissolve 0.200 g in 50 mL of water R. Add 10 mL of
ammonium chloride buffer solution pH 10.0 R and 50 mg of
mordant black 11 triturate Rl. Titrate with 0.1 M sodium
edetate until the colour changes from red to green.
1 mL of 0.1 M sodium edetate is equivalent to 20.85 mg
of CdSO4.
- Ph
Calcium Iodide Tetrahydrate for ** ** Calcium Phosphate for Homoeopathic

Homoeopathic Preparations ***** Preparations
(Calcium lodatum for Homoeopathic Preparations, Calcium Phosphoricum for Homoeopathic Preparations
DEFINITION
CaI2,4H2O 366.0 13640-62-5 Calcium Phosphate for Homoeopathic Preparations contains
PhEur________________________________________________________ _____ Calcium Phosphate.
DEFINITION PRODUCTION OF STOCK
Content The first trituration of Calcium Phosphate for Homoeopathic
97.0 per cent to 102.0 per cent of Cal2 (anhydrous Preparations is prepared using a suitable quantity of a
substance). vehicle, such as Lactose, Anhydrous Lactose or Sucrose, and
a validated method for trituration that ensures homogeneity
CHARACTERS
is achieved. The vehicle complies with the statement under
Appearance Vehicles in the monograph for Homoeopathic Preparations.
White or almost white, very' hygroscopic powder.
Content of calcium Ca
Solubility The first decimal trituration contains 3.5% to 4.0% of Ca.
Very soluble or freely soluble in water and in ethanol
(96 per cent). CHARACTERISTICS
The first decimal trituration is a white powder.
IDENTIFICATION
A. Solution ร (see Tests) gives reaction (a) of calcium IDENTIFICATION
(2.3.7). Wash 5 g of the first decimal trituration of the substance
B. Solution ร (see Tests) gives reaction (b) of iodides (2.3.7). being examined with three 10-mL quantities of water. The
dried residue complies with the following tests.
TESTS
A. Dissolve 0.1 g of the dried residue in 5 mL of a 25% v/v
Solution ร solution of nitric acid. The resulting solution yields reaction B
Dissolve 10.0 g in distilled water R and dilute to 100.0 mL
of phosphates, Appendix VI.
B. The dried residue yields reaction B characteristic of
Appearance of solution calcium salts, Appendix VI. Filter before adding potassium
Solution ร is clear (2.2.7) and not more intensely coloured ferrocyanide solution.
than reference solution GY5 (2.2.2, Method II).
C. The dried residue complies with the limits of the Assay.
Free iodine, iodates D. If the preparation includes Lactose as the vehicle, it
To 5 mL of solution ร add 2 mL of methylene chloride R. complies with the following test. Dissolve 0.25 g in 5 mL of
Shake and allow to stand. The organic layer is colourless water. Add 5 mL of ammonia and heat in a water-bath at 80°
(2.2.2, Method I) (free iodine). Add 0.2 mL of dilute sulfuric
for 10 minutes. A red colour develops.
acid R. Shake and allow to stand. The organic layer remains
colourless (2.2.2, Method 7) (iodates). E. If the preparation includes Sucrose as the vehicle, it
complies with the following test. Dissolve 5.0 g in carbon
Sulfates (2.4.73) dioxide-free water and dilute to 10 mL with the same solvent.
Maximum 150 ppm. Dilute 1 mL of the solution to 100 mL with water. To 5 mL
Dilute 10 mL of solution s to 15 mL with distilled water R. of the solution add 0.15 mL of freshly prepared copper sulfate
Iron (2.4.9) solution and 2. mL of freshly prepared dilute sodium hydroxide
Maximum 10 ppm, determined on 10 mL of solution ร. solution. The solution is blue and clear and remains so after
boiling. To the hot solution add 4 mL of dilute hydrochloric
Heavy metals (2.4. ร)
acid and boil for 1 minute. Add 4 mL of dilute sodium
Maximum 10 ppm.
hydroxide solution. An orange precipitate is formed
12 mL of solution ร complies with test A. Prepare the immediately.
reference solution using lead standard solution (1 ppm Pb) R.
ASSAY
Water (2.5.72)
Dissolve 0.2 g of the residue in a mixture of 1.0 mL of
18.0 per cent to 22.0 per cent, determined on 0.100 g.
hydrochloric acid R1 and 5 mL of water. Add 25.0 mL of
ASSAY 0.1m disodium edetate and dilute to 200 mL with water.
Dissolve 0.300 g in 50 mL of water R. Add 5 mL of dilute Adjust to about pH 10 with concentrated ammonia.
nitric acid R and 25.0 mL of 0.1 M silver nitrate. Shake. Add 10 mL of ammonia buffer pH 10.0 and a few
Add 2 mL offerric ammonium sulfate solution R2 and titrate milligrams of mordant black 11 triturate. Titrate the excess
with 0.1 M ammonium thiocyanate until the colour changes to disodium edetate with 0.1m zinc sulfate until the colour
reddish-yellow. changes from blue to violet. Each mL of 0.1 M sodium
1 mL of 0.1 M silver nitrate is equivalent to 14.70 mg hydroxide is equivalent to 4.008 mg of Ca.
of Cal2.
STORAGE
_______________________________________________________________ PhEur
Cineraria Maritima for Homoeopathic SYSTEM SUITABILITY

Preparations solution (2) shows three fluorescent bands: an orange
definition fluorescent band with a low Rf value (rutin), an orange
Cineraria Maritima for Homoeopathic Preparations is the fluorescent band with an Rf value in the middle region
fresh aerial parts of Cineraria maritima L. harvested before (hyperoside) and a blue fluorescent band with an Rf value in
flowering. the upper region (scopoletin).
CONFIRMATION
IDENTIFICATION
Plant Low growing, woody-based perennial 25 to 30 cm The chromatogram obtained with solution (1) shows an
occasionally up to 100 cm high, with strong, white tomentose orange fluorescent band in a similar position to rutin, another
shoots up to 20 mm in diameter. The shoots are much fluorescent band above this orange band, another orange
branched and those bearing the flowers are elongated with fluorescent band in a similar position to hyperoside with a
some smaller leaves in the upper part; the shorter, non green fluorescent band just below, one or two green
flowering shoots remain compressed with the leaves forming fluorescent bands between the bands in similar positions to
a rosette at the top. hyperoside and scopoletin, one blue-green fluorescent band
in a similar position to scopoletin and one yellow-green to
Leaves The leaves are alternate, up to 25 cm long and
orange fluorescent band above the blue-green fluorescent
12 cm wide, ovate or oblong-ovate, the lowest coarsely
band.
toothed, the upper ones deeply pinnatified or pinnate with
4 to 6 oblong to blunt, often 3 to 5 lobed, unequal segments.
The under surface is covered with a dense white felt, the Top of the plate
upper surface is green with scancred cottony hairs. A yellow-green to
orange fluorescent band
MOTHER TINCTURE A blue-green Scopoletin: a blue
fluorescent band fluorescent band
The mother tincture complies zuith the requirements stated under
Mother Tinctures for Homoeopathic Preparations and with the
following requirements.
An orange fluorescent Hyperoside: an orange
PRODUCTION band fluorescent band
The mother tincture of Cineraria maritima L. is prepared
from the cut drug using Method 1.1.7 described in the A green fluorescent
monograph for Methods of Preparation of Homoeopathic band
Stocks and Potentisation. Use 43% พ/พ (50% v/v) of ethanol.
A fluorescent band
CHARACTERISTICS
An orange fluorescent Rutin: an orange
The mother tincture is a dark yellow, clear to slightly turbid band fluorescent band
liquid.
IDENTIFICATION
A. Carry out the method for thin-layer chromatography', Solution (1) Solution (2)
(1) Dilute 5 mL of the mother tincture with 15 mL of zvater B. Carry out the method for thin-layer chromatography.
and transfer to a cartridge containing octadecyl-bonded silica Appendix in A, using the following solutions.
sorbent (a Sep-pak C18 cartridge is suitable) previously
(1) Evaporate off the ethanol from 50 mL of the mother
washed with 10 mL of methanol followed by 10 mL of water.
tincture. Make the residue alkaline with dilute ammonia R1
Elute with 10 mL of methanol) evaporate the eluant and
and extract with three 20-mL quantities of chloroform.
dissolve the residue in 0.5 mL of methanol.
Evaporate the combined chloroform extracts to dryness and
(2) 0.05% w/v each of hyperoside and rutin and 0.01% w/v of dissolve the residue in 1 mL of ethanol (60%).
scopoletin in methanol.
(2) 0.1% w/v of reserpine in acetone.
(a) Use as the coating silica gel F254.
(a) Use as the coating silica gel F254.
(c) Apply 30 |1L of solution (1) and 10 pL of solution (2) as
(c) Apply 30 pL of solution (1) and 20 pL of solution (2) as
10 mm bands.
10 mm bands.
(e) After removal of the plate, dry in air and spray the plate (e) After removal of the plate, dry in air and spray the plate
with a 1 % w/v solution of diphenylboric acid aminoethyl ester in with a 2% w/v solution of dimethylaminobenzaldehyde in
methanol and then spray with a 5% w/v solution of ethanol and then spray with a solution of sulfuric acid. Heat at
polyethylene glycol 400 in methanol. Heat at 100° to 105° for 100° to 105° for 5 minutes and examine in daylight
5 minutes, allow to dry in air and examine immediately in
MOBILE PHASE
10 volumes of methanol and 90 volumes of chloroform.
MOBILE PHASE
10 volumes of water, 10 volumes offormic acid and SYSTEM SUITABILITY

80 volumes of ethyl acetate. The test is not valid unless the chromatogram obtained with
solution (2) shows one blue band with an Rf value of 0.80.
7-454 Homoeopathic Preparations 2016
CONFIRMATION cells with slightly collenchymatous thickening and more

The chromatogram obtained with solution (1) shows a series distinct pitted circular to oval areas, lignified, spirally or
of violet bands between the line of application and Rf value annularly thickened vessels.
0.65, one pink band at Rf value 0.75 and one red band c. Carry out the method for thin-layer chromatography,
at Rf value 0.90. Appendix in A, using the following solutions.
(1) Add 30 mL of 86% v/v ethanol to 3 g of the coarsely
powdered drug and heat under reflux for 2 hours. Allow to
Top of the plate cool and filter. Evaporate 20 mL of the filtrate to about
A red band
(2) 0.1% w/v each of caffeine, coumarin and resorcinol in
Reserpine: a blue methanol.
A pink band band CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel F254-
A series of violet (c) Apply 20 |1L of each solution.
bands between the
line of application
(e) Remove the plate, dry it in air and examine under
and Rf 0.65 ultraviolet light (254 nm).
MOBILE PHASE
1 volume of 13.5m ammonia, 9 volumes of methanol and
Solution (1) Solution (2) 90 volumes of dichloromethane.
SYSTEM SUITABILITY
TESTS The test is not valid unless the chromatogram obtained with
Ethanol solution (2) shows three clearly separated bands
25 to 35% พ/พ (31 to 42% v/v), Appendix VIII F. (approximate Rf values: resorcinol 0.31, caffeine 0.67 and
coumarin 0.87).
Dry residue
Not less than 1.0%, determined on 2 mL, Appendix XI p. CONFIRMATION
Relative density The chromatogram obtained with solution (1) shows two
dark bands at Rf values of 0.08 and 0.1 respectively between
the line of application and the band due to resorcinol, one
STORAGE dark band at an Rf value of 0.56 positioned between the
Cineraria Maritima for Homoeopathic Preparations should band due to resorcinol and that due to caffeine, and one dark
be protected from light. band at approximately Rf value of 0.78 positioned between
the band due to caffeine and that due to coumarin. Other
bands may be present.
Citrullus Colocynthis Fruit for Top of the plate

DEFINITION
Citrullus Colocynthis Fruit for Homoeopathic Preparations is Coumarin: a dark band
the dried, peeled fruits of Citrullus colocynthis (L.) Schrad.
with the seeds removed.
A dark band
IDENTIFICATION
A. The peeled fruits are spherical with a diameter of 5 to
10 cm, white to pale yellow and very light in texture, Caffeine: a dark band
consisting mainly of soft, spongy tissue from the inner cupule
and the placentae. The external surface is marked by spiral, A dark band
flattish, knife marks where the peel has been removed.
In cross section, three conspicuous fissures can be seen
radiating from the centre and dividing the fruit into three Resorcinol: a dark
parts. Each part contains two groups of seeds near the band
periphery, the remaining space being filled with pithy
parenchyma. Each fruit contains 200 to 300 seeds. A dark band
The inferior ovary is initially tripartite but as the placentae A dark band
grow out from the centre towards the circumference, each
divides into two, half curving backwards, and giving the
appearance of a hexapartite ovary.
B. Reduce to a powder. The powder is pale yellowish-buff.
The powder shows abundant, large, partly lignified, thin
walled, finely pitted, usually fragmented parenchyma; smaller
TESTS the presence of 2 narrow cavities in each of which is enclosed

Foreign matter 1 of the foliaceous cotyledons.
Not more than 2.0% of the outer part of the pericarp; B. Microscopic examination (2.8.23). Reduce to a
not more than 5.0% of seeds; not more than 2.0% of other powder (710) (2.9.12). The powder is brown. Examine
foreign matter, Appendix XI D. under a microscope using chloral hydrate solution R.
Loss on drying The powder shows the following diagnostic characters
When dried at 100° to 105° for 2 hours, loses not more than (Figure 2486.-1): fragments of the epicarp (surface view [D])
22.0% of its weight. Use 1 g. consisting of thin-walled, polygonal cells, about 30-50 pm in
Total ash diameter [Da], anomocytic stomata (2.8.3) [Db], and cells in
Not more than 13.0%, Appendix XI J, Method n. a pattern consisting of a cell with slightly thickened walls,
pitted at the centre, surrounded by 4-6 cells [De]; fragments
of the epicarp and outer layers of the mesocarp (transverse
MOTHER TINCTURE section [C]) showing the epicarp covered by a fine cuticle
The mother tincture complies with the requirements stated under [Ca] and cells of the mesocarp, ovoid or rounded, some
Mother Tinctures for Homoeopathic Preparations and with the containing prisms of calcium oxalate [Cb]; numerous
following requirements. fragments of the inner layers of the mesocarp and of the
endocarp [A] consisting of sclereids [Aa] and short fibres
PRODUCTION
with pitted walls [Ab]; fragments of the endocarp consisting
The mother tincture of Citrullus colocynthis (L.) Schrad. of layers of variously oriented fibres (surface view [E]);
is prepared from the powdered drug using Method 4a sclereids and isolated fibres [B]; vascular bundles
described in the monograph for Methods of Preparation of (longitudinal section [F]), accompanied by fibres [Fa];
Homoeopathic Stocks and Potentisation. Use 86% พ/พ fragments of the endosperm [G, H] containing very
(90% v/v) ethanol.
numerous small acicular crystals.
CHARACTERISTICS c. Thin-layer chromatography (2.2.27).
The mother tincture is a light yellow to yellow liquid. Test solution To 2.00 g of the powdered herbal drug (710)
IDENTIFICATION (2.9.12) add 20 mL of ethanol (90 per cent VIV) R, shake for
The mother tincture complies with Identification test c 2 h and then centrifuge (1000 g). Use the supernatant.
above using the mother tincture as solution (1). Reference solution Dissolve 10 mg of picrotin R and 10 mg of
TESTS picrotoxinin R in ethanol (96 per cent) R and dilute to 10 mL
Ethanol with the same solvent.
81% to 91% พ/พ (86% to 94% v/v), Appendix vni F. Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
plate R (2-10 pm)].
Dry residue
1.0% to 2.5% พ/พ, Appendix XI p. Mobile phase methanol R, ethyl acetate R) heptane R
(10:40:50 VIVIV).
Relative density
Application 40 pL [or 10 pL] as bands of 20 mm [or
10 mm].
Drying In air.
Cocculus Indicus for * * Detection spray with anisaldehyde solution R, heat at
100-105 °C for 5-10 min and examine immediately in
Homoeopathic Preparations ***** daylight.
(Cocculus for Homoeopathic Preparations, Ph. Eur. Results See below ±e sequence of zones present in the
monograph 2486) chromatograms obtained with the reference solution and the
Ph Elf______________________________________________________________ test solution. Above the zone due to picrotoxinin, several
pink or violet zones may also be visible in the chromatogram
DEFINITION
Dried, ripe fruit of Anamirta cocculus (L.) Wight & Am. (syn.
A. paniculata Colebr.).
Content Top of the plate
Minimum 0.80 per cent of picrotoxinin (C15H1606; Mr
292.3) (dried drug).
Picrotoxinin: a blue zone A blue zone (picrotoxinin)
IDENTIFICATION
First identification A, B, D
Picrotin: a blue zone A blue zone (picrotin)
A. The fruits are dark greyish-brown or black, reniform or
sub-spherical, about 6-10 mm in diameter and 9-12 mm Reference solution Test solution
long; the outer surface is irregularly wrinkled with a ridge
about 4-6 mm long running between the pale, cirailar scar
left by the stalk and the small beak of the remains of the D. Examine the chromatograms obtained in the assay.
stigma. The pericarp is hard, about 1 mm thick and the Results The peaks due to picrotoxinin and picrotin in the
inner surface is brownish-grey, hard and woody. chromatogram obtained with the test solution are similar in
Cut transversely, the fruit shows a single, cup-shaped seed retention time to the corresponding peaks in the
into the hollow of which an ingrowth of the mesocarp and chromatogram obtained with the reference solution.
endocarp projects. Cut longitudinally, the endosperm shows
Al X 7ท2 X p X 2
A2 X mi
A1 = area of the peak due to picrotoxinin in the

A2 = area of the peak due to picrotoxinin in the
nil = mass of die herbal drug to be examined used to
m2 = mass of picrotoxinin CRS used to prepare the
p = assigned percentage content of picrotoxinin in
picrotoxinin CRS.
MOTHER TINCTURE
The mother tincture complies with the requirements of the
general monograph Mother tinctures for homoeopathic
DEFINITION
Content
0.07 per cent mini to 0.15 per cent mlm of picrotoxinin
(๑15H 16๑6).
PRODUCTION
The mother tincture is prepared from the dried, ripe fruit of
A. cocculus (L.) Wight & Am. according to the following
Figure 2486.-1. - Illustration for identification test B ofpowdered methods prescribed in the monograph Methods of preparation
herbal drug of Cocculus
of homoeopathic stocks and potentisation (2371):
TESTS — method 1.1.8 using the powdered herbal drug (710)
Loss on drying (2.2.52) (2.9.72) and ethanol (90 per cent VIV), use ethanol
Maximum 10.0 per cent, determined on 1.000 g of the (70 per cent VIV) to prepare the 4th decimal dilution and
powdered herbal drug (710) (2.9.72) by drying in an oven at ethanol (50 per cent P7P) for subsequent dilutions;
105 °C for 2 h. — method 1.1.10 using the crushed drug in fragments of
Total ash (2.4.16) about 2-3 mm, ethanol (90 per cent VIV) and a
Maximum 6.0 per cent. maceration time of about 3 weeks.
ASSAY CHARACTERS
Liquid chromatography (2.2.29). Appearance
Yellow or dark yellow liquid.
(2.9.72) add 20.0 mL of ethanol (90 per cent V/V) R, shake IDENTIFICATION
for 2 h and then centrifuge at 1000 g for 5 min. Dilute A. Thin-layer chromatography (2.2.27) as described in
2.0 mL of the supernatant to 20.0 mL with ±e mobile phase identification test c for the herbal drug with the following
and filter through a membrane filter (nominal pore size modification.
0.45 pm). Test solution The mother tincture to be examined.
Reference solution Dissolve 5.0 mg of picrotin CRS and 5.0 mg B. Examine the chromatograms obtained in the assay.
of picrotoxinin CRS in 10.0 mL of acetonitrile R. Dilute Results The peaks due to picrotoxinin and picrotin in the
2.0 mL of the solution to 20.0 mL with the mobile phase. chromatogram obtained with the test solution are similar in
Column'. retention time to the corresponding peaks in the
— size: 7 = 0.125 m, 0 = 4.0 mm; chromatogram obtained with the reference solution.
TESTS
(5 pm).
Mobile phase acetonitrile Rl) water R (30:70 VIV).
0.830 to 0.845 (method 1.1.8).
Ethanol (2.9.10)
Detection Spectrophotometer at 200 nm. 85 per cent VIV to 95 per cent VIV (method 1.1.10).
Injection 10 pL. Dry residue (2.8.16)
Run time Twice the retention time of picrotoxinin CRS. Minimum 0.7 per cent.
Retention time Picrotin = about 6 min; ASSAY
picrotoxinin = about 9.5 min. Liquid chromatography (2.2.29) as described in the assay of
System suitability Reference solution: the herbal drug with the following modification.
— resolution: minimum 2.0 between the peaks due to picrotin Test solution Dilute 0.500 g of the mother tincture to be
and picrotoxinin. examined to 10.0 mL with the mobile phase and filter using
Calculate the percentage content of picrotoxinin using the a membrane filtre (nominal pore size 0.45 pm).
Calculate the percentage content of picrotoxinin using the Iron

following expression: Maximum 50 ppm.
Atomic absorption spectrometry (2.2.23) Method I).
>11 X 7712 X p Test solution Dissolve 1.00 g in 5 mL of nitric acid R and
A2 X 7ท! X 10 dilute to 50.0 mL with water R.
Reference solutions Prepare the reference solutions using iron
A] ะะะ area of the peak due to picrotoxinin in the standard solution (20 ppm Fe) R, diluted as necessary with a
chromatogram obtained with the test solution; 1 per cent VIV solution of nitric acid R.
A2 = area of the peak due to picrotoxinin in the Source Iron hollow-cathode lamp.
chromatogram obtained with the reference solution; Wavelength 248.3 nm.
พ/ = mass of the mother tincture to be examined used to
Flame Air-acetylene.
พ2 = mass of picrotoxinin CRS used to prepare the Lead
reference solution, in grams; Maximum 100 ppm.
p = assigned percentage content of picrotoxinin in Atomic absorption spectrometry (2.2.23, Method I).
picrotoxinin CRS. Test solution Use the test solution prepared for the test for
—————_PhEur
iron.
Reference solutions Prepare the reference solutions using lead
standard solution (0.1 per cent Pb) R, diluted as necessary with
a 1 per cent VIV solution of nitric acid R.
Source Lead hollow-cathode lamp.
Copper for Homoeopathic ** ** Wavelength 283.3 nm.
Preparations ***** Flame Air-acetylene.
Copper for Homoeopathic Use Zinc
(Cuprum Metallicum for Homoeopathic Preparations, Ph. Eur. Maximum 50 ppm.
monograph ไ 610) Atomic absorption spectrometry (2.2.23, Method I).
Cu 63.5 7440-50-8 Test solution Use the test solution prepared for the test for
Ph Elf_______ _______________________________________________________ iron.
Reference solutions Prepare the reference solutions using zinc
DEFINITION
standard solution (100 ppm Zn) R, diluted as necessary with a
Content
1 per cent VIV solution of nitrie acid R.
99.0 per cent to 101.0 per cent of Cu.
Source Zinc hollow-cathode lamp.
CHARACTERS
Wavelength 213.9 nm.
Appearance
Reddish-brown powder. Flame Air-acetylene.
Solubility ASSAY
Practically insoluble in water, soluble in hydrochloric acid Dissolve 0.100 g in 5 mL of nitric acid R. Heat to expel the
and in nitric acid, practically insoluble in ethanol nitrous fumes. Add 200 mL of water R and neutralise (2.2.3)
(96 per cent). with dilute ammonia Rl. Add 1 g of ammonium chloride R and
3 mg of murexide R. Titrate with 0.1 M sodium edetate until
IDENTIFICATION the colour changes from green to violet.
A. To 2 mL of solution ร (see Tests) add 0.5 mL of 1 mL of 0.1 M sodium edetate is equivalent to 6.354 mg of
potassium ferrocyanide solution R. A reddish-brown precipitate
Cu.
is formed.
____________________________________________________ — PhEur
B. To 5 mL of solution ร add 0.6 mL of ammonia R. A blue
precipitate is formed. Add 2 mL of ammonia R.
The precipitate disappears; the solution has an intense blue
colour. Copper Acetate Monohydrate for *****
TESTS
Solution ร
Homoeopathic Preparations *****
(Cuprum Acedcum for Homoeopathic Preparations,
Dissolve 2.0 g in 10 mL of nitric acid R. After nitrous fumes
are no longer evolved, dilute to 60 mL with distilled water R. Ph. Eur. monograph 2146)
Acidity or alkalinity Cu(C2H3O2)2î2O 199.7 6046-93-1

To 5.0 g add 20 mL of carbon dioxide-free water R. Boil for Ph Eur__ ______________________________ -_____________________________
1 min. Cool. Filter and dilute to 25.0 mL with carbon DEFINITION
dioxide-free water R. To 10 mL of the solution add 0.1 mL of Content
bromothymol blue solution Rl. Not more than 0.5 mL of 99.0 per cent to 101.0 per cent of Cu(C2H3O2)2jH2O.
0.01 M hydrochloric acid or 0.01 M sodium hydroxide is
required to change the colour of the indicator. CHARACTERS
Appearance
Chlorides (2.4.4) Greenish-blue crystals or green powder.
Maximum 100 ppm, determined on solution ร.
Solubility
Sulfates (2.4.13) Soluble in water, slightly soluble or very slightly soluble in
Maximum 300 ppm, determined on solution ร. ethanol (96 per cent).
IDENTIFICATION
A. It gives reaction (a) of acetates (2.3.1). Crocus for Homoeopathic ** X
B. Dissolve 0.1 g in 10 mL of water R and add dilute Preparations *****
ammonia R1 dropwise. A dark blue colour is produced. Saffron for Homoeopathic Use; Saffron for
TESTS Homoeopathic Preparations
Solution ร (Ph. Eur. monograph 1624)
Dissolve 3.0 g in a mixture of 40 mL of distilled water R and Ph Eur_____________________________________ __ ______________________
0.6 mL of glacial acetic acid R, Hath heating at 70 °C. Cool
DEFINITION
and dilute to 45 mL with distilled water R.
Dried stigmas of Crocus sativus L. usually joined by the base
Appearance of solution to a short style.
Solution ร is clear (2.2./).
CHARACTERS
Impurities not precipitating with hydrogen sulfide
Maximum 0.1 per cent, calculated as sulfates.
To 2.000 g add 92 mL of water R and 8.0 mL of dilute IDENTIFICATION
sulfuric acid R. Heat to 70 °C. Pass a current of hydrogen A. The dark brick-red stigmas, when dry, are 20 mm to
sulfide R until there is no longer precipitation of copper 40 mm long and after soaking with water, about 35 mm to
sulfide. Allow to cool and stand, then filter. Evaporate to 50 mm long. The tubes, gradually widening at the top, are
dryness 50.0 mL of the filtrate in a crucible. Ignite the incised on one side, the upper margin is open and finely
residue at about 600 ± 50 °C to constant mass. crenated. The style connecting the 3 stigmas is pale yellow
and not more than 5 mm long.
Chlorides (2.4.4)
Maximum 50 ppm, determined on solution ร. B. Examine under a microscope using chloral hydrate
solution R. It shows the following diagnostic characters:
Sulfates (2.4.13) elongated epidermal cells, frequently with a short, central
Maximum 150 ppm, determined on solution ร. papilla; in water they release a yellow colouring matter;
Iron (2.4.9) the upper border of the stigma has finger-shaped papillae, up
Maximum 20 ppm. to 150 pm long; between them are single, globular pollen
Dissolve 0.500 g in 10 mL of water R. Transfer to a grains, about 100 pm wide, with a finely pitted exine,
separating funnel. Add 20 mL of hydrochloric acid R1 and vascular bundles with small spirally thickened vessels and no
10 mL of methyl isobutyl ketone R. Shake vigorously for fibres.
3 min. Allow to stand. Transfer the organic layer to a second c. Carefully crush pieces of the herbal drug to coarse
separating funnel and add 10 mL of water R. Shake particles and moisten with 0.2 mL of phosphomolybdic acid
vigorously for 3 min. Allow to stand. The aqueous layer solution R. The particles turn blue within 1-2 min or they
complies with the limit test for iron. have a blue areole around them.
Nickel D. Thin-layer chromatography (2.2.27).
Maximum 10 ppm. Test solution Carefully crush 0.1 g of the herbal drug with a
To the residue obtained in the test for impurities not glass rod and moisten with 0.2 mL of water R. After 3 min
precipitating with hydrogen sulfide, add 2.0 mL of add 5 mL of methanol R, allow to stand for 20 min,
hydrochloric acid R and 1.0 mL of sulfuric acid R. Evaporate to protected from light, and filter through a plug of glass wool.
dryness. Dissolve the residue in a mixture of 3.0 mL of dilute Reference solution Dissolve 5 mg of naphthol yellow R in 5 mL
sulfuric acid R and 17.0 mL of water R. To 4.0 mL of this of methanol R and add a solution of 5 mg of Sudan red G R
solution add 4.0 mL of พater Ry 5.0 mL of bromine water R, in 5 mL of methylene chloride R.
7.0 mL of dilute ammonia R1 and 3.0 mL of a 10 g/L Plate TLC silica gel F25.1 plate R.
solution of dimethylglyoxime R in ethanol (90 per cent VIV) R.
Mobile phase water Ry 2-propanol Ry ethyl acetate R
This solution is not more intensely coloured within 1 min
(10:25:65 VIVIV).
than a solution prepared as follows: mix 4.0 mL of a 1 ppm
solution of nickel (Ni) prepared from nickel standard solution Application 10 pL of the test solution and 5 pL of the
(10 ppm Ni) R, 4.0 mL of water R and 5.0 mL of bromine reference solution as bands.
water R-y carefully add 7.0 mL of dilute ammonia R1 and Development Over a path of 10 cm.
3.0 mL of a 10 g/L solution of dimethylglyoxime R in ethanol Drying In air.
(90 per cent VIV) R. Detection A’, examine in daylight.
ASSAY Results A: see below the sequence of zones present in the
Dissolve 0.400 g in water R and dilute to 50 mL with the chromatograms obtained with the reference solution and the
same solvent. Add 6.0 mL of glacial acetic acid Ry 10.0 g of test solution.
potassium iodide R and 1 mL of starch solution R. Titrate with
0.1 M sodium thiosulfate. Top of the plate
1 mL of 0.1 M sodium thiosulfate is equivalent to 19.97 mg of A red zone

Cu(C2H3O2)2,H2O.
A yellow zone
------------------------------------------------------------------------------------ ------------------------ Ph Eur
2 yellow zones
An intense yellow zone (crodne)
Detection B examine in ultraviolet light at 254 nm.

Results B See below the sequence of zones present in the flakes on the surface and in the spaces between the seeds;
chromatograms obtained with the reference solution and the four-sided, one arched, one often distinctly ridged and two
test solution. larger and flattened; pointed at one end, where the hilum
occurs as a paler spot, obtuse at the other extremity, where
Top of the plate the chalaza is situated. Cut transversely, the seed shows a
very narrow endosperm surrounding two yellowish-white
A red zone 1 or 2 quenching zones
cotyledons.
A yellow zone A quenching zone
TESTS
Reference solution Test solution Total ash
Not more than 5%, Appendix XI J, Method II.
Detection c Treat with anisaldehyde solution R and examine in
daylight while heating at 100-105 °C for 5-10 min. MOTHER TINCTURE
Results c See below the sequence of zones present in the The mother tincture complies with the requirements stated under
chromatograms obtained with the reference solution and the Mother Tinctures for Homoeopathic Preparations and with the
test solution. following requirements.
PRODUCTION
Top of the plate The mother tincture of Cydonia oblonga Mill, is prepared
A red zone 1 or 2 red to reddish-violet zones from the powdered drug using Method 1.1.8 described in the
monograph for Methods of Preparation of Homoeopathic
A blue to bluish-green zone A red to reddish-violet zone
Stocks and Potentisation. Use glycerol.
2 blue to bluish-green zones
CHARACTERISTICS
An intense blue to bluish-green The mother tincture is a pale yellow, clear or slightly turbid
zone (crocine) viscous liquid.
IDENTIFICATION
E. Dilute 0.1 mL of the test solution (see Identification D) Appendix in A, using the following solutions.
with 1 mL of methanol R. Deposit 0.1 mL of this solution on (1) Dilute 5 mL of the mother tincture with 5 mL of water,
a filter paper, allow to dry and spray with a 10 g/L solution mix thoroughly and transfer the diluted tincture to a
of diphenylboric acid aminoethyl ester R in methanol R. Examine cartridge containing octadecyl-bonded silica sorbent (a Sep-pak
in ultraviolet light at 365 nm. The spot shows an intense C18 cartridge is suitable) previously washed with 10 mL of
orange-yellow fluorescence. methanol followed by 10 mL of water. Wash the cartridge
TESTS with 15 mL of water and elute with 10 mL of methanol.
Colouring intensity Evaporate the eluant to dryness using a rotary evaporator.
Introduce 0.10 g into a 5 mL volumetric flask and dilute to Dissolve the residue in 0.5 mL of methanol.
5.0 mL with distilled water R. Close the flask and shake every (2) 0.1% w/v of hyperoside, 0.1% w/v of rutin and 0.01% w/v
30 min for 8 h. Then allow to stand for 16 h. Dilute 1.0 mL of scopoletin in methanol.
to 500.0 mL with distilled water R. The absorbance (2.2.25)
measured at 440 nm using distilled water R as the
compensation liquid, is not less than 0.44. (a) Use as the coating silica gel 60 F2S4-
Foreign matter
Examine the herbal drug microscopically. No parts with (c) Apply 40 pL of solution (1) and 10 pL of solution (2), as
rough walls, no crystals and no pollen grains containing 12 mm bands.
3 germinal pores are present. (d) Develop the plate to 15 cm.
Loss on drying (2.2.22) (e) After removal of the plate, dry in air and spray the plate
Maximum 10.0 per cent, determined on 0.200 g by drying in with a 1 % w/v solution of diphenylboric acid aminoethyl ester in
an oven at 105 °C. methanol, and then with a 5% w/v solution of polyethylene
glycol 400 in methanol and examine under ultraviolet light
Total ash (2.4.16)
(365 nm).
Maximum 7.0 per cent, determined on the residue obtained
in the test for loss on drying. MOBILE PHASE
______________________________________________________________ PhEur 15 volumes of anhydrous formic acid, 15 volumes of water and

70 volumes of ethyl acetate.
SYSTEM SUITABILITY
Cydonia Oblonga for Homoeopathic solution (2) shows two clearly separated orange fluorescent
bands and one blue fluorescent band at a higher Rf value.
Preparations In order of increasing Rf value the bands are: rutin,
DEFINITION hyperoside and scopoletin.
Cydonia Oblonga for Homoeopathic Preparations is the CONFIRMATION
seeds of Cydonia oblonga Mill. The chromatogram obtained with solution (1) shows three
IDENTIFICATION yellow fluorescent bands in the lower third, a blue fluorescent
The seeds are 6 to 7 mm long, reddish-brown to dark band just below the rutin standard, a blue fluorescent band
brown, frequently cohering by a white mucilage appearing in just below the hyperoside standard and a blue fluorescent
band with the same Rf value of the scopoletin standard. PRODUCTION

Other bands may be present. The mother tincture of Hedera helix L. is prepared by
maceration using ethanol of a suitable concentration.
Top of the plate Content
Minimum 0.15 per cent mint of hederacoside c (C59H96O26;
A blue fluorescent band Scopoletin- a blue
fluorescent band
Mr 1221).
CHARACTERS
Appearance
Hyperoside: an orange Dark greenish-brown liquid.
fluorescent band
A blue fluorescent band IDENTIFICATION
Rutin: an orange Reference solution Dissolve 1 mg of a-hederin R and 1 mg of
A blue fluorescent band fluorescent band
A yellow fluorescent band hederacoside c R in methanol R and dilute to 2 mL with the
A yellow fluorescent band same solvent.
A yellow fluorescent band
Mobile phase glacial acetic acid R, water R, butanol R
Solution (1) Solution (2) (1:1:4 VIVIV).
Application 20 |1L as bands.
TEST Development Over half of the plate.
Refractive index Drying In air.
1.468 to 1.475, Appendix V E. Detection Spray with a 10 per cent VIV solution of sulfuric
acid R in methanol R and heat at 100-105 °C for 10 min.
Examine in daylight.
Hedera Helix for Homoeopathic ♦ * test solution. Other faint zones may also be present in the
Preparations ***** chromatogram obtained with the test solution.
Ph Eur______________________________________________________________ _ Top of the plate
DEFINITION
Fresh, young, fully developed but not yet lignified branch of a-Hederin: a violet zone A violet zone
Hedera helix L., harvested immediately before or at the (a-hederin)
beginning of flowering. Hederacoside C: a brown zone A brown zone
(hederacoside C)
IDENTIFICATION A greyish-brown ----------
The fresh, young branches of Hedera helix L. are thin and zone
A yellow zone
flexible, climbing; they cling to their support by stem-roots.
The leaves are alternate, simple and petiolate. The petiole
shows a cylindrical section. The upper surface of the leaves is Reference solution Test solution
glabrous and shiny, darker than the lower surface.
The lamina is usually divided into 3-5 more or less deeply
cut lobes on sterile branches; it is oval, with a pointed apex TESTS
on fertile branches. The inflorescences are arranged in a
simple semi-globular corymb and grouped in terminal
0.890 to 0.925.
clusters. The pedicels of the umbel are covered in whitish
hairs. Each flower shows 5 small teeth formed by the upper Ethanol (2.9.10)
part of the sepals and 5 petals covered in very small inverted 60 per cent VI1Z to 70 per cent P7K
hairs. Dry residue (2.8.16)
TESTS
Foreign matter (2.8.2) ASSAY
If required by the competent authority, maximum 5 per cent. Liquid chromatography (2.2.29).
Loss on drying (2.2.32) Test solution In a 20.0 mL volumetric flask, dilute 3.000 g of
If required by the competent authority, minimum the mother tincture to be examined to 20.0 mL with the
50 per cent, determined on 5.0 g of the finely cut drug by mobile phase.
drying in an oven at 105 °C for 2 h. Reference solution In a 50.0 mL volumetric flask, dissolve
MOTHER TINCTURE 20.0 mg of hederacoside c R in the mobile phase and dilute to
general monograph Mother tinctures for homoeopathic Column'.
preparations (2029). — size'. I = 0.25 m, 0 = 4 mm;
(5 gm).
Mobile phase Mix 35 volumes of water Ry adjusted to pH 3 Reference solution Immediately before use, dissolve 5 mg of
with phosphoric acid Ry and 65 volumes of methanol R. hydrastine hydrochloride R and 5 mg of berberine chloride R in
Flow rate 1 mUmin. 10 mL of methanol R.
Detection Spectrophotometer at 205 nm. Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
Injection 20 pL. plate R (2-10 pm)].
Retention time Hederacoside c = about 8 min. Mobile phase anhydrous formic acid R, water Ry ethyl acetate R
(10:10:80 VIVIV).
Calculate the percentage content mini of hederacoside c
using the following expression: Application 20 pL [or 5 pL] as bands.
Al X 7772 X c X 0-4 Drying In air.
A2 X 7771 Detection Examine in ultraviolet light at 365 nm.
A1 ะ= area of the peak due to hederacoside c in the in the chromatograms obtained with the reference solution
chromatogram obtained with the test solution; and the test solution. Furthermore, other faint fluorescent
A2 = area of the peak due to hederacoside c in the zones may be present in the chromatogram obtained with the
chromatogram obtained with the reference test solution.
solution;
ni\ = mass of the mother tincture in the test solution, in Top of the plate
grams;
JtI2 = mass of hederacosideCRin the reference solution,
in grams; Berberine: a bright yellow A bright yellow fluorescent zone
fluorescent zone (berberine)
c = percentage content of hederacoside c R.
Hydrastine: a deep blue fluorescent A deep blue fluorescent zone
----------------------------------------------------------------------------------------------------------Ph Ear zone (hydrastine)
Hydrastis Canadensis for ★* ** TESTS

Homoeopathic Preparations ***** Relative density (2.2.5)
(Ph. Eur. monograph 2500) 0.890 to 0.905, where Method 1.1.8 is used.
Ph Elf _________________________________________________________ Ethanol (2.9.10)
The herbal drug complies with the requirements of the 60 per cent VIV to 70 per cent VIVy where Method 1.1.10 is
monograph Goldenseal rhizome (1831). used.
MOTHER TINCTURE
Minimum 1.2 per cent mini.
general monograph Mother tinctures for homoeopathic ASSAY
preparations (2029). Liquid chromatography (2.2.29).
DEFINITION Test solution Dilute about 1.000 g, accurately weighed, of the
The mother tincture is prepared from the whole or cut, dried mother tincture to be examined to 20.0 mL with the mobile
rhizome and roots of Hydrastis canadensis L. phase.
Reference solution Immediately before use, dissolve 10.0 mg of
Content
hydrastine hydrochloride CRS and 10.0 mg of berberine
— hydrastine (C21H21NO6; Mr 383.4): 0.10 per cent to
chloride CRS in methanol R and dilute to 100.0 mL with the
0.40 per cent;
same solvent.
— berberine (C20H18NO4; Mr 336.4): 0.20 per cent to
0.50 per cent. Column:
— size: I = 0.125 m, 0 = 4 mm;
PRODUCTION — stationary phase: end-capped octadecylsilyl silica gel for
The mother tincture is prepared by the following methods chromatography R (5 pm).
prescribed in the monograph Methods of preparation of Mobile phase Dissolve 9.93 g of potassium dihydrogen
homoeopathic stocks and potentisation (2371): phosphate 7? in 730 mL of water Ry add 270 mL of
— Method 1.1.8, using the powdered herbal drug (710) acetonitrile R and mix.
(2.9.12) and ethanol (70 per cent V/V) [or ethanol
(62 per cent mlmf\y
— Method 1.1.10, using the fragmented herbal drug (pieces Detection Spectrophotometer at 235 nm.
about 1 cm in diameter), e±anol (65 per cent V!V) and Injection 10 pL.
maceration for 3-5 weeks. Elution order Hydrastine, berberine.
CHARACTERS Identification of peaks Use the chromatogram obtained with
Appearance the reference solution to identify the peaks due to hydrastine
Yellowish-brown liquid. and berberine.
IDENTIFICATION
hydrastine and berberine.
Calculate the percentage contents mhท of hydrastine using Hyoscyamus albus L

the following expression: The presence of middle and upper leaves with a petiole and
of fruits barely swollen at the base indicates adulteration by
Al X m2 X p X 0.913 Hyoscyamus albus L.
A2 X โท1 X 5
MOTHER TINCTURE
Calculate the percentage contents mhn of berberine using the
Al X m2 X p X 0.905 PRODUCTION
A2 X 7ท1 X 5 The mother tincture of Hyoscyamus niger L. is prepared by
maceration of the drug, using ethanol of a suitable
Al = area of the peak due to hydrastine or to berberine concentration.
in the chromatogram obtained with the test Hyoscyamus niger
solution; Content
A2 = area of the peak due to hydrastine or to berberine 0.002 per cent m/m to 0.01 per cent mhn of total alkaloids,
in the chromatogram obtained with the reference expressed as hyoscyamine (C17H23NO3; Mr 289.4).
solution;
ไท 1 = mass of the mother tincture to be examined used CHARACTERS
to prepare the test solution, in grams; Appearance
m2 = mass of hydrastine hydrochloride CRS or mass of Dark greenish-brown liquid.
berberitie chloride CRS used to prepare the IDENTIFICATION
reference solution, in grams; Thin-layer chromatography (2.2.27).
p = percentage content of hydrastine hydrochloride CRS
Test solution Evaporate 10 mL of the mother tincture to be
or percentage content of berberine chloride CRS.
examined in a water-bath at 40 °C, under reduced pressure.
-----------------------------------------------------------------------------------------------------------Ph Eur Take up the residue with 1 mL of ammonia R, and shake
with 2 quantities, each of 10 mL, of ether R. Combine the
ether layers, dry over anhydrous sodium sulfate R and filter.
Evaporate on a water-bath and dissolve the residue in
0.50 mL of methanol R.
Hyoscyamus for Homoeopathic * * Reference solution (a) Dissolve 50 mg of hyoscyamine sulfate R
Preparations ***** in 10 mL of methanol R (solution A). Dissolve 15 mg of
(Ph. Eur. monograph 2091) hyoscine hydrobromide R in 10 mL of methanol R (solution B).
Ph Ell_____________________________________________________________ _
Mix 4 mL of solution A and 2 mL of solution B and dilute
to 10 mL with methanol R.
DEFINITION Reference solution (b) Dissolve 20 mg of atropine sulfate R in
Whole, fresh flowering plant of Hyoscyamus niger L. methanol R and dilute to 10 mL with the same solvent.
IDENTIFICATION Plate TLC silica gel plate R.
Hyoscyamus is an annual or biennial plant, with a well Mobile phase concentrated ammonia Ry water Ry acetone R
developed taproot. The robust, erect stem is hollow and (3:7:90 P7P7P).
subcylindrical and up to 80 cm long. The soft, viscid, dull
dark-green leaves are densely pubescent on both surfaces,
especially on the veins. The lower leaves are petiolate and are Development Over a path of 10 cm.
arranged in a rosette; the lower cauline leaves are semi- Drying At 100-105 °C for 15 min.
amplexicaul and the upper ones are completely amplexicaul. Detection A Spray with dilute potassium iodobismuthate
The lamina, up to 25 cm long, is oblong to ovate with 2 to 5 solution R until orange zones become visible. Examine in
broadly dentate lobes on each side. The midrib is well daylight.
developed. The secondary veins arise at a wide angle from Results A See below the sequence of the zones present in the
the midrib and terminate in the apices of the lobes. chromatograms obtained with the reference solutions and the
The flowering tops are densely pubescent and form a short test solution. Other faint zones may be present in the
drooping cluster. Each flower arises in the axils of a large chromatogram obtained with the test solution.
bract. The gamosepalous calyx is covered with dense cotton
like hairs and has 5 triangular-ovate lobes, each ending in a
short point ±at becomes spiny. The gamopetalous corolla, Top of the plate
with 5 nearly equal lobes, is yellowish and with a delicate, Hyoscine: an orange An orange zone
brown to blackish-violet venation. The fruit, sometimes zone (hyoscine)
present at the base of the inflorescences, is a pyxis distinctly

swollen at the base. Hyoscyamine: an Atropine ะ an orange A orange zone (hyos-
orange zone zone cyamine/atropine)
TESTS
Faint orange zones
If required by the competent authority, maximum 5 per cent. (line of application)__
Loss on drying (2.2.32) Reference Reference Test solution
If required by the competent authority, minimum solution (a) solution (b)
50 per cent, determined on 5.0 g of the finely cut drug by
drying in an oven at 105 °C for 2 h.
Detection B Subsequently spray with sodium nitrite solution R

until the yellow background disappears. Examine in daylight Hypericum for Homoeopathic * \
after 15 min. Preparations *****
Results B See test for atropine. (Ph. Eur. monograph 2028)
TESTS PhEur______________________________________________________________
DEFINITION
0.930 to 0.960.
Whole, fresh plant of Hypericum perforatum L., at the
Atropine beginning of the flowering period.
Examine the chromatograms obtained in the test for
identification.
IDENTIFICATION
The perennial plant consists of a spindle-shaped root and a
Results The zone due to hyoscyamine in the chromatogram branched rhizome, giving rise to long, decumbent runners.
obtained with the test solution changes from orange to The cylindrical, erect stem is woody at the base, 0.2 m to
reddish-brown but not to greyish-blue (atropine). 1 m long, branched in the upper part, with 2 raised
Ethanol (2.9./0) longitudinal lines.
40 per cent P7P to 50 per cent v/v. The leaves are opposite, sessile, exstipulate, oblong-oval and
Dry residue (2.8.16) 15 mm to 30 mm long. The leaf margins show black
Minimum 1.2 per cent. glandular dots, and many small translucent oil glands are
ASSAY present on the entire surface and are visible by transmitted
light.
Evaporate 100.0 g of the mother tincture to be examined, at
a low temperature under reduced pressure, until a residue of The flowers are regular and form corymbose clusters at the
about 10 g is obtained. Quantitatively transfer the residue to apex of the stem. They have 5 green, lanceolate sepals with
a separating funnel using a few millilitres of ethanol acuminate apices, and black oil glands near the entire
(70 per cent V/V) R. Add 5 mL of concentrated ammonia R margins; 5 orange-yellow petals, much longer than the sepals,
and 25 mL of water R. Extract with successive fractions of a with black oil glands near the terminal margins only;
mixture of 1 volume of chloroform R and 3 volumes of 3 staminal blades, each divided into many orange-yellow
peroxide-free ether R until the alkaloids are completely stamens and 3 carpels surmounted by red styles. Each petal
extracted. Evaporate to dryness a few millilitres of the last is asymmetrically linear-ovate in shape, with one of the
organic fraction. Take up the residue in 0.25 M sulfuric acid margin entire and the other dentate.
and verify the absence of alkaloids using potassium TESTS
tetraiodomercurate solution R. Combine the organic layers and Foreign matter (2.8.2)
extract several times with 0.25 M sulfuric acid. Separate the Maximum 4 per cent of fruits and maximum 1 per cent of
layers by centrifugation if necessary and transfer the acid other foreign matter.
layers to a second separating funnel. Make the acid layer Loss on drying (2.2.82)
alkaline with ammonia R and shake with at least 3 quantities, If performed to demonstrate the freshness of the drug,
each of 30 mL, of chloroform R. Combine the chloroform minimum 55 per cent, determined on 5.0 g of finely cut drug
layers, add 4 g of anhydrous sodium sulfate R and allow to
by drying in an oven at 105 °C.
stand for 30 min with occasional shaking. Decant the
chloroform and wash the anhydrous sodium sulfate with MOTHER TINCTURE
3 quantities, each of 10 mL, of chloroform R. Combine the The mother tincture complies with the requirements of the
chloroform fractions, evaporate to dryness on a water-bath general monograph Mother tinctures for homoeopathic
and dry in an oven at 100-105 °C for 15 min. Dissolve ±e preparations (2029).
residue in a few millilitres of chloroform R, add 10.0 mL of PRODUCTION
0.005 M sulfuric acid and remove the chloroform by The mother tincture of Hypericum perforatum L. is prepared
evaporation on a water-bath. Titrate the excess of acid with by maceration using alcohol of a suitable concentration.
0.01 M sodium hydroxide using methyl red mixed solution R as
indicator. CHARACTERS
Dark red to brownish red liquid.
Calculate the percentage content mini of total alkaloids,
expressed as hyoscyamine, from the expression: IDENTIFICATION
0.2894(10 —ท) Test solution The mother tincture to be examined.
m Reference solution Dissolve 5 mg of rutin R, 1 mg of
hypericin R and 5 mg of hyperoside R in methanol R and dilute
ท = volume of 0.01 M sodium hydroxide used, in to 5 mL with the same solvent.
millilitres; Plate TLC silica gel plate R.
m = mass of the mother tincture used, in grams. Mobile phase anhydrous formic acid R) water R) ethyl acetate R
_______________________ _______________________________________PhEur (6:9:90 VIVIV).
Application 10 pL of the test solution and 5 |1L of the
reference solution, as 10 mm bands.
Detection spray with a 10 g/L solution of diphenylboric acid
aminoethyl ester R in methanol R and then a 50 g/L solution of
macrogol 400 R in methanol R. Examine the plates after B. CAUTION: take all necessary handling precautions when
30 min in ultraviolet light at 365 nm. reducing this toxic herbal drug to a powder.
Results See below the sequence of the zones present in the Wash the herbal drug rapidly in cold water, then expose to
chromatograms obtained with the reference solution and the steam; once sufficiently softened, cut into thin slices and
test solution. In the chromatogram obtained with the test crush in a suitable apparatus. Allow to dry, finish reducing to
solution, the zone due to rutin may be weak or even absent. a powder (710) (2.9.12) and pass through a covered sieve.
The chromatogram obtained with the test solution shows a Microscopic examination (2.8.23). The powder is light
group of zones that may be blue or yellow, with a RF similar brown. Examine under a microscope using chloral hydrate
to that of the zone due to hyperoside in the chromatogram solution R. The powder shows the following diagnostic
obtained with the reference solution. Other weak zones may characters (Figure 2513.-1): oil droplets [D]; fragments of
also be visible. endosperm [B, c, F] consisting of thick-walled cells of
various sizes, the smallest located at the periphery of the
Top of lie plate endosperm [Cb] and the largest towards the centre of the
seed [F]; a few fragments of the outer layer of the endosperm
A yellow to blue zone
(surface view [J], transverse section [Ca]), with polygonal
Hypericin: a red zone 2 red zones cells sometimes associated with the inner layer of the testa,
composed of cells with indistinct walls (surface view [E],
transverse section [Cd]); sclerified covering trichomes [A, K],
Several zones sheared off, not enlarged at the base [Aa] and with walls
composed of small, oblique, sclerified strips, tightly fused
longitudinally [Ab, Ka]; numerous fragments of strips [G, H]
Hyperoside: a yellow to orange Blue or yellow zones
and rare rounded tips of covering trichomes [I<].
Rutin: a yellow to orange zone A yellow to orange zone
TESTS
0.900 to 0.920.
Ethanol (2.9.10)
60 per cent v/v to 75 per cent v/v.
_______________________________________________________________ PhEur
Ignatia for Homoeopathic * *

Preparations *****
PhEur______________________________________________________________
DEFINITION
Dried, ripe seed of Strychnos ignatii P.J. Bergius.
Content
Minimum 1.80 per cent for the sum of the contents of
brucine (บ23บ26พ204; Mr 394.5) and strychnine
(C21H22N2O2; Mr 334.4), of which minimum 65 per cent
consists of strychnine (dried drug).
IDENTIFICATION Figure 2513.-1. - Illustration for identification test B ofpowdered
A. The seed is grey, brown and dull, up to 3 cm long and herbal drug of Ignatia
10-25 mm thick. It is irregular, with 3-5 distinct sides: one of c. Thin-layer chromatography (2.2.27).
these is usually wider, convex and glabrous; the others are
angular and flattened and show the remains of testa hairs
(2.9.12) add 20 mL of ethanol (70 per cent V/V) R3 allow to
forming lighter zones in the depressions. The stony granular macerate for 15 min at room temperature, with stirring, and
texture resembles that of pebbles from a river bed; the hilum
centrifuge. Use the supernatant.
is found on the most rounded end and forms a small, light
Reference solution Dissolve 10 mg of brucine R and 10 mg of
brown depression. The fracture shows a compact, semi-
translucent, homy endosperm; the embryo is located in the strychnine R in 10 mL of ethanol (96 per cent) R.
centre and is about 10-15 mm long, with a foliaceous Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
cotyledon. plate J? (2-10 pm)].
Mobile phase concentrated ammonia R, methanol R, methylene — stationary phase', ethylene-bridged octadecylsilyl silica gel for
chloride R (1:5:95 V/V/V)', use the lower layer. chromatography (hybrid material) R (3.5 pm);
Application 10 pL [or 5 |1L] as bands. — temperature'. 35 °C.
Development Over a path of 15 cm [or 6 cm]. Mobile phase:
— mobile phase A: triethylamine R, acetonitrile for
Drying In air, then in an oven at 105-110 °C for 15 min;
allow to cool. chromatography R, methanol R2,
tris (hydroxymethyl) aminomethane buffer solution pH 9.0 R
Detection Spray with iodoplatinate reagent R and examine (0.1:7.5:7.5:85 V/V/V/V)’,
immediately in daylight. — mobile phase B: triethylamine R,
Results See below the sequence of zones present in the tris (hydroxymethyl) aminomethane buffer solution pH 9.0 R,
chromatograms obtained with the reference solution and the acetonitrile for chromatography R, methanol R2
test solution. Furthermore, other faint zones may be present (0 1:15:42.5:42.5 V/V/V/V)’,
Top of the plate (min) (per cent V/V) (percent V/V)
0-5 100 0
5 - 25 100 -> 70 0 -> 30
25 - 30 70 -> 65 30 -> 35
Strychnine: a violet zone A violet zone (strychnine)
30 - 31 65 -> 0 35 -> 100
Brucine: a blue zone A blue zone (brucine)
31 - 32 0 100

TESTS Injection 10 pL.
CAUTION: when the powdered herbal drug (710) (2.9.12) is Elution order Brucine, strychnine.
used, take the necessary precautions as indicated under System suitability, reference solution:
Identification B. — resolution: minimum 3.0 between the peaks due to brucine
Foreign matter (2.8.2) and strychnine.
Maximum 1.0 per cent. Calculate the percentage contents of brucine and strychnine
Strychnos nux-vomica L using the following expression:
The presence of flattened discoid seeds and the presence in
the powdered herbal drug, examined under a microscope, of Al X 7712 X p X 2
testa cells transformed into hairs, with a sclerified base and a A2 X 7711
lignified tip, bent at a right angle and with 7-10 lignified
ridges, and of numerous sclerified rods, indicate adulteration
A1 = area of the peak due to brucine or strychnine in the
with Strychnos nux-vomica L.
Loss on drying (2.2.32) A2 = area of the peak due to brucine or strychnine in the
Maximum 12.0 per cent, determined on 1.00 g of the chromatogram obtained with the reference solution;
powdered herbal drug (710) (2.9.12) by drying in an oven at m 1 = mass of the herbal drug to be examined used to
105 °C for 2 h. prepare the test solution, in grams;
Total ash (2.4.16) m2 = mass of brucine CRS or strychnine CRS used to
Maximum 3.5 per cent. prepare the reference solution, in grams;
Aflatoxins (2.8.18) p = assigned percentage content of brucine in brucine
Maximum 2 pg/kg (aflatoxin B 1) and maximum 4 pg/kg CRS or strychnine in strychnine CRS.
(sum of aflatoxins Bj, B2, G! and G2).
ASSAY MOTHER TINCTURE
Liquid chromatography (2.2.29). The mother tincture complies with the requirements of the
Test solution To 1.000 g of the powdered herbal drug (710) general monograph Mother tinctures for homoeopathic
(2.9.12) add 10.0 mL of ethanol (60 per cent V/V) R. Boil preparations (2029).
gently, with stirring, under a reflux condenser. After 30 min,
DEFINITION
cool and filter into a 20.0 mL volumetric flask. Wash the
The mother tincture is prepared from the dried, ripe seed of
filter with ethanol (60 per cent V/V) R and dilute to 20.0 mL
Strychnos ignatii P.J.Bergius.
with the same solvent. Dilute 1.0 mL of the solution to
20.0 mL with mobile phase A. Content
0.18 per cent m/m to 0.36 per cent mhn for the sum of the
Reference solution Dissolve 10.0 mg of brucine CRS and
contents of brucine (C23H26N2O4; Mr 394.5) and strychnine
10.0 mg of strychnine CRS in acetonitrile R and dilute to
(C21H22N2O2; Mr 334.4), of which minimum 65 per cent is
10.0 mL with the same solvent. Dilute 1.0 mL of the
strychnine.
solution to 20.0 mL with mobile phase A.
Column’.
— size'. I ะ= 0.15 m, 0 = 4.6 mm;
PRODUCTION CHARACTERS
The mother tincture is prepared from the powdered herbal Appearance
drug (710) (2.9.12) by the following methods prescribed in Fine, blackish-grey powder, without metallic lustre.
the general monograph Methods of preparation of homoeopathic Solubility
stocks and potentisation (2371)'.
Practically insoluble in water and in ethanol (96 per cent).
— method 1.1.8, using ethanol (70 per cent F/P); It dissolves with heating in dilute mineral acids.
— method 1.1.10, using ethanol (65 per cent VIV) and a
maceration time of 3-5 weeks. IDENTIFICATION
Dissolve 50 mg in 2 mL of dilute sulfuric acid R and dilute to
CHARACTERS
10 mL with water R. The solution gives reaction (a) of iron
Appearance
(2.3.1).
Brownish-yellow liquid.
TESTS
IDENTIFICATION
Solution ร
Thin-layer chromatography (2.2.27) as described in To 10.0 g add 40 mL of water R. Boil for 1 min. Cool, filter
identification test C for ±e herbal drug with the following and dilute to 50.0 mL with water R.
modification.
Alkalinity
To 10 mL of solution ร add 0.1 mL of bromothymol blue
TESTS solution Rl. Not more than 0.1 mL of 0.01 M hydrochloric
Relative density (2.2. ร) acid is required to change the colour of the indicator to
0.890 to 0.904 (method 1.1.8). yellow.
Ethanol (2.9.10) Substances insoluble in hydrochloric acid
60 per cent VIV to 70 per cent VIV (method 1.1.10). Dissolve 2.00 g in 40 mL of hydrochloric acid R. Heat on a
Dry residue (2.8.16) water-bath. As soon as fumes arc no longer evolved, filter
Minimum 1.2 per cent. through a sintered-glass filter (16) (2.1.2). Rinse with
water R. Dry the residue in an oven at 100-105 °C for 1 h.
ASSAY The residue weighs a maximum of 20 mg (1.0 per cent).
Liquid chromatography (2.2.29) as described in the assay of
Substances soluble in water
the herbal drug with the following modification.
Evaporate 10.0 mL of solution s on a water-bath and dry at
Test solution Dilute 2.000 g of the mother tincture to be 100-105 °C for 1 h. The residue weighs a maximum of 2 mg
examined to 20.0 mL with ethanol (60 per cent VIV) R. (0.1 per cent).
Calculate the percentage contents of brucine and strychnine Chlorides (2.4.4)
using the following expression: Maximum 50 ppm.
Dilute 5 mL of solution s to 15 mL with water R.
Sulfides and phosphides
In a 100 mL conical flask carefully mix 1.0 g with 10 mL of
dilute hydrochloric acid R. Within 30 ร lead acetate paper R
moistened with water R and placed over the mouth of the
flask is not coloured more intensely than light brown by the
resulting fumes.
ทใ\ = mass of the mother tincture to be examined used to Arsenic (2.4.2)
prepare the test solution, in grams; Maximum 5 ppm.
m2 = mass of brucine CRS or strychnine CRS used to Boil 0.2 g in 25 mL of dilute hydrochloric acid R until
prepare the reference solution, in grams; completely dissolved. The solution complies with limit test A.
p = assigned percentage content of brucine in bruciทe Copper
CRS or strychnine in strychnine CRS. Maximum 50 ppm.
_______________________________________________________________ Ph Eur Atomic absorption spectrometry (2.2.23, Method โ).
Test solution Dissolve 1.00 g in a mixture of 60 mL of dilute
hydrochloric acid R and 10 mL of dilute hydrogen peroxide
solution R. Reduce to a volume of 5 mL and dilute to
Iron for Homoeopathic ****** 50.0 mL with water R.
Reference solutions Prepare the reference solutions using copper
Preparations ***** standard solution (0.1 per cent Cu) R, diluting with a
Iron for Homoeopathic Use 1 per cent VIV solution of hydrochloric acid R.
(Ferrum Metallicum for Homoeopathic Preparations, Ph. Eur. Source Copper hollow-cathode lamp.
monograph 2026) Wavelength 324.8 nm.
Fe 55.85 7439-89-6 Flame Air-acetylene.
Ph Eu_______________________________________________________________ Lead
DEFINITION Maximum 50 ppm.
Iron obtained by reduction or sublimation as a fine blackish- Atomic absorption spectrometry (2.2.23, Method โ).
grey powder. Test solution In a separating funnel, place 20 mL of the test
Content solution prepared for the test for copper. Add 25 mL of lead-
97.5 per cent to 101.0 per cent. free hydrochloric acid R. Stir with 3 quantities, each of 25 mL,
of di-isopropyl ether R. Collect the aqueous layer. Add 0.10 g acid Rl. Allow to stand, separate the aqueous layer and
of sodium sulfate decahydrate R. Evaporate to dryness. Take evaporate to half its volume, allow to cool and dilute to
up the residue with 1 mL of lead-free nitric acid R and dilute 35 mL with water. Neutralise 7.5 mL of this solution to
to 20 mL with water R. litmus paper using dilute ammonia Rl and dilute to 15 mL
Reference solutions Prepare the reference solutions using lead with water. 12 mL of the resulting solution complies with
standard solution (0.1 per cent Pb) R, diluting with a limit test heavy metals, Appendix vn, Method A (70 ppm).
10 per cent v/v solution of nitric acid R containing 5 g/L of Use lead standard solution (1 ppm Pb) to prepare the standard.
sodium sulfate decahydrate R. Loss on drying
Source Lead hollow-cathode lamp. When dried to constant weight at 200°, loses not less ±an
Wavelength 217 nm. 28% and not more than 33% of its weight, Appendix IX D.
Flame Air-acetylene. Use 1 g.
ASSAY ASSAY
Dissolve 0.45 g in 3 mL of hydrochloric acid Rl in an iodine
Stir for 10 min 0.100 g in a hot solution of 1.25 g of copper
flask, add 10 mL of water and 6.0 g of potassium iodide, close
sulfate R in 20 mL of water R in a 100 mL conical flask with
the flask and allow to stand protected from light for
a ground-glass stopper. Filter rapidly and wash the filter.
30 minutes. Add 100 mL of water and 1 mL of starch solution
Combine the filtrate and the washings, acidify with dilute
and titrate with 0.1m sodium thiosulfate vs. Each mL of 0.1m
sulfuric acid R and titrate with 0.02 M potassium permanganate
until a pink colour is obtained. sodium thiosulfate US is equivalent to 22.29 mg of
FePO4,4H2O.
1 mL of 0.02 M potassium permanganate is equivalent to
5.585 mg of Fe. PRODUCTION OF STOCK
The first decimal trituration of Hydrated Iron(m) Phosphate
LABELLING
for Homoeopathic Preparations is prepared using a suitable
The label indicates whether the substance is obtained by quantity of Lactose or Anhydrous Lactose as the vehicle and
reduction or sublimation. a validated trituration method that ensures homogeneity is
————_____ PhEur achieved. The vehicle complies with the statement under
Vehicles in ±e monograph for Homoeopathic Preparations.
Content of hydrated iron(iii) phosphate FePO4, 4H2O
The first decimal trituration contains 9.0% to 11.0% of
Hydrated Iron(m) Phosphate for FePO4, 4H2O.
Homoeopathic Preparations CHARACTERISTICS
The first decimal trituration is a yellowish powder.
FePO4,4H2O 222.8 10045-86-0
(anhydrous) IDENTIFICATION
Dissolve, with wanning, 1.5 g of the first decimal trituration
DEFINITION in a mixture of 1.5 mL of dilute hydrochloric acid and 9 mL of
Hydrated Iron(m) Phosphate for Homoeopathic Preparations water (solution Si).
contains hydrated iron(m) phosphate. It contains not less A. Solution ร 1 yields reactions B and c characteristic of iron
than 96.0% and not more than 106.5% of FePO4,4H2O. and iron salts, Appendix VI.
CHARACTERISTICS B. Solution SI yields reaction B characteristic of phosphates,
A yellow to pale ochre powder. Appendix VI.
Insoluble in water; soluble in dilute mineral acids. C. Dissolve 0.25 g of the substance being examined in 5 mL
IDENTIFICATION of water. Add 5 mL of ammonia and heat in a water-bath at
80° for 10 minutes. A red colour develops.
Dissolve 0.5 g of the substance being examined in 5 mL of
dilute hydrochloric acid, with warming. Dilute the resulting ASSAY
solution to 35 mL with water and filter if necessary (solution Dissolve 4.0 g of the first decimal trituration in 3 mL of
ร). hydrochloric acid Rl in an iodine flask, add 10 mL of water
A. Solution ร yields reactions B and c characteristic of iron and 8.0 g of potassium iodide, close the flask and allow to
and iron salts, Appendix VI. stand protected from light for 30 minutes. Add 100 mL of
B. Solution ร yields reaction B characteristic of phosphates, water and 1 mL of starch solution and titrate with 0.1m sodium
Appendix VI. thiosulfate US. Each mL of 0.1m sodium thiosulfate PS is
equivalent to 22.29 mg of FePO4,4H2O.
TESTS
STORAGE
Clarity of solution
Hydrated Iron(m) Phosphate for Homoeopathic Preparations
Solution ร is clear, Appendix rv A, Method II.
should be protected from light.
Chloride
To 0.05 g of the substance being examined add 1 mL of
dilute nitric acid. Heat, dilute with 14 mL of water and filter.
The filtrate complies with the limit test for chlorides,
Appendix vn (0.1%).
Heavy metals
hydrochloric acid if necessary with heating. Extract the solution
using five 20-mL quantities of a mixture of 100 mL of
freshly distilled methyl isobutyl ketone and 1 mL of hydrochloric
B. Yields the reactions characteristic of phosphates,

Hydrated Iron(n) and Iron(m) Phosphate Appendix VI.
for Homoeopathic Preparations c. Dissolve 0.25 g in 5 mL of water. Add 5 mL of ammonia
DEFINITION and heat in a water-bath at 80° for 10 minutes. A red colour
Hydrated Iron(n) and Iron(m) Phosphate for Homoeopathic develops.
Preparations contains a mixture of hydrated iron(n) ASSAY
phosphate and iron (in) phosphate and some hydrated oxides Dissolve 3.0 g of the first decimal trituration in 3 mL of
of iron. It contains not less than 16.0% of Fe2+, equivalent to orthophosphoric acid and 10 mL of a 14% v/v solution of
not less than 47.9% of Fe3(PO4)2,8H2O. sulfuric acid in water. Add 100 mL of water and titrate with
CHARACTERISTICS 0.1m potassium permanganate. Each mL of 0.1m potassium
A slate blue amorphous powder. permanganate PS is equivalent to 27.925 mg of Fe2+.
Insoluble in water; soluble in hydrochloric acid. STORAGE
IDENTIFICATION Hydrated Iron(n) and Iron (in) Phosphate for Homoeopathic
Dissolve 0.5 g in 5 mL of dilute hydrochloric acid with Preparations should be protected from light.
warming. Dilute the resulting solution to 35 mL with water
and filter if necessary (solution ร).
A. Solution ร yields the reactions characteristic of iron and
iron salts, Appendix VI. Magnesium Phosphate for ** ไ
B. Solution ร yields the reactions characteristic of phosphates, Homoeopathic Preparations *****
Appendix VI.
(Magnesium Phosphoricum for Homoeopathic
TESTS Preparations, Ph. Eur. monograph 2505)
Heavy metals MgHPO4,3H2O 174.3 7782-75-4
PhEur--------------------------------------------------------------------------- --------------------------------
hydrochloric acid. Extract the solution using five 20-mL
quantities of a mixture of 100 mL of freshly distilled methyl DEFINITION
isobutyl ketone with 1 mL of hydrochloric acid Rl. Allow to Content
stand, separate the aqueous layer and evaporate to half its 98.0 per cent to 102.0 per cent.
volume, allow to cool and dilute to 35 mL with water.
CHARACTERS
Neutralise 7.5 mL of this solution to litmus paper using dilute
Appearance
ammonia Rl and dilute to 15 mL with water. 12 mL of the
White powder.
resulting solution complies with limit test A for heavy metals,
Appendix vn (70 ppm). Use lead standard solution Solubility
(1 ppm Pb) to prepare the standard. Very slightly soluble in water, practically insoluble in ethanol
(96 per cent). It dissolves in dilute acids.
Sulfates
Dissolve 0.25 g of the substance being examined in water. IDENTIFICATION
Add 3 mL of dilute hydrochloric acid and dilute to 15 mL with A. Dissolve 0.1 g in a mixture of 2 mL of dilute nitric acid R
water. The resulting solution complies with the Unlit test for and 8 mL of water R. The solution gives reaction (b) of
sulfates, Appendix vn. phosphates {2.3.1).
ASSAY B. Dissolve 0.1 g in a mixture of 2 mL of dilute nitric acid R
Dissolve 0.3 g of the substance being examined in 3 mL of and 8 mL of-water R. Add 10 mL of ammonium molybdate
orthophosphoric acid and 10 mL of a 14% v/v solution of solution R and filter. The filtrate gives the reaction of
sulfuric acid in water. Add 100 mL of water and titrate with magnesium {2.3.1).
0.1m potassium permanganate. Each mL of potassium TESTS
permanganate vs is equivalent to 27.925 mg of Fe2+. Solution ร
PRODUCTION OF STOCK Dissolve 5.0 g in 30 mL of dilute hydrochloric acid R and
The first decimal trituration of Hydrated Iron(n) and Iron(in) dilute to 50.0 mL with the same acid.
Phosphate for Homoeopathic Preparations is prepared using Arsenic {2.4.2, Method B)
a suitable quantity of Lactose or Anhydrous Lactose as the Maximum 5 ppm, determined on 1.0 g.
vehicle and a validated trituration method that ensures Chlorides {2.4.4)
homogeneity is achieved. The vehicle complies with the Maximum 200 ppm.
statement under Vehicles in the monograph for
Dissolve 0.25 g in 5 mL of dilute nitric acid R and dilute to
Homoeopathic Preparations.
15 mL with water R.
Content of hydrated iron(ii) and iron(iii) phosphate Magnesium dihydrogen phosphate and magnesium
The first decimal trituration contains 4.5% to 5.0% of
phosphate
Fe3(PO4)2, 8H2O. Dissolve 2.00 g in 30.0 mL of 1 M hydrochloric acid.
CHARACTERISTICS Add 20 mL of water R and 0.05 mL of methyl orange
The first decimal trituration is a light grey powder. solution R. Titrate the excess of hydrochloric acid with 1 Af
sodium hydroxide. The volume of 1 M hydrochloric acid used is
IDENTIFICATION
between 11.0 mL and 12.5 mL.
A. Yields the reactions characteristic of iron and iron salts,
Appendix VI. Sulfates {2.4.13)
Maximum 300 ppm.
Dilute 5 mL of solution s to 15 mL with distilled water R-
Iron (2.4.9) CHROMATOGRAPHIC CONDITIONS

Maximum 50 ppm. (a) Use as the coating silica gel 60 F254 (Merck silica gel 60
Dilute 2 mL of solution s to 10 mL with water R. precoated plates are suitable).
Heavy metals (2.4.5) (b) Use the mobile phase described below.
Maximum 40 ppm. (c) Apply 10 pL of each solution as 3 mm bands.
Dilute 10 mL of solution ร to 20 mL with dilute hydrochloric (d) Develop the plate to 15 cm.
acid R. 12 mL of the solution complies with test A. Prepare (e) After removal of the plate dry in air and examine under
the reference solution using lead standard solution ultra-violet light (365 nm).
(2 ppm Pb) R.
(f) Spray the plate with anisaldehyde solution and heat at 105°
ASSAY for 5 minutes and examine under ultra-violet light (365 nm).
Dissolve 0.280 g in a mixture of 1 mL of hydrochloric acid Rl MOBILE PHASE
and 5 mL of water R. Add 25.0 mL of 0.1 M sodium edetate
10 volumes of methanol and 90 volumes of toluene.
and dilute to 200 mL with water R. Neutralise with
concentrated ammonia Rj add 10 mL of ammonium chloride SYSTEM SUITABILITY
buffer solution pH 10.0 R and about 50 mg of mordant black The test is not valid unless, in the chromatogram obtained
11 trimrate R. Titrate the excess of 0.1 M sodium edetate with with solution (2), two clearly separated bands are observed
0.1 M zinc sulfate until the colour changes from pure blue to under ultra-violet light (365 nm) before and after spraying
violet. with anisaldehyde solution.
1 mL of 0.1 M sodium edetate is equivalent to 17.43 mg of CONFIRMATION
MgHPO4,3H2O. Under ultra-violet light, the chromatogram obtained with
-- ------------------------------------------------------------------------------------------------------- Ph Eur solution (1) shows the following fluorescent bands: one blue
or pink fluorescent band close to the origin, followed by a
blue or pink fluorescent band and then a blue band below
the band obtained for formononetin in solution (2), followed
by one or two blue bands approximately level with
Medicago Sativa for Homoeopathic formononetin and a red band between the bands obtained
for formononetin and coumarin in solution (2). Other bands
Preparations may be present in the chromatogram obtained with
DEFINITION solution (1).
Medicago Sativa for Homoeopathic Preparations is the fresh
whole flowering plant of Medicago sativa L. Ultraviolet light (365 กทา)
Top of the plate
IDENTIFICATION
Plant A herbaceous perennial, reaching up to 100 cm.
Leaves Alternate, petiolate, trifoliolate, leaflets mucronate,
approximately 2 cm long, 1 cm broad, typically oblanceolate
or oblong, with a toothed or entire margin, glabrous on the
upper surface and sparsely pubescent on the lower surface;
stipules lanceolate, up to 1 cm long, toothed to entire.
Stems 4-angled, branching, glabrous to pubescent.
Flowers Compact, axillary, racemes of up to 40 flowers, Coumarin: a turquoise blue
peduncle up to 3 cm long, typically pubescent; corolla fluorescent band
papilionaceous, up to 1 cm long, 5 mm broad, purple to Red fluorescent band
whitish; calyx tube approximately 5 mm in length, 2 mm in
diameter, 5-lobed, typically glabrous, lobes equal or One or two blue
subequal, up to 4 mm long. fluorescent bands Formononetin: a turquoise
blue fluorescent band with
MOTHER TINCTURE
slight tailing
The mother tincture complies with the requirements stated under Blue fluorescent band
Mother Tinctures for Homoeopathic Preparations and with the Blue or pink fluorescent
following requirements. band
PRODUCTION Blue or pink fluorescent
band
The mother tincture of Medicago sativa L. is prepared from
the herbal drug using method 1.1.5 described in the
Stocks and Potentisation. Use 86% พ/พ (90% v/v) of ethanoL The chromatogram obtained with solution (1) sprayed with
CHARACTERISTICS the anisaldehyde solution shows the following fluorescent
The mother tincture is a greenish-brown liquid. bands: a faint blue band close to the origin, followed by a
faint purple or blue fluorescent band and a blue band below
IDENTIFICATION the band obtained for formononetin in solution (2), followed
Carry out the method for thin-layer chromatographyJ by three yellow to orange bands between the bands obtained
Appendix in A, using the following solutions. for formononetin and coumarin in solution (2) and an orange
(1) The mother tincture. band between the band obtained for coumarin and the
(2) 0.1% w/v coumarin BPCRS and 0.05% w/v of solvent front Other bands may be present in the
formononetin BPCRS in methanol. chromatogram obtained with solution (1).
Anisaldehyde solution spray and ultraviolet light (365 nm) on the edge of the seed (the micropyle). A grey homy
Top of the plate endosperm makes up most of the seed. The embryo, which is
located in the central cavity, is small (about 6 mm long),
with 2 cotyledons; the radicle is turned towards the
Orange fluorescent
micropyle.
band B. CAUTION: lake all necessary handling precautions when
reducing this toxic herbal drug to a powder.
Wash the herbal drug rapidly in cold water, then expose to
vapour from boiling water; once sufficiently softened, cut into
thin slices and crush in a suitable apparatus. Allow to dry',
finish reducing to a powder (710) (2.9.72) and pass through
Coumarin: a faint indigo a covered sieve.
blue fluorescent band
Microscopic examination (2.8.23). The powder is grey.
Yellow orange
fluorescent band Examine under a microscope using chloral hydrate solution R.
Faint orange
fluorescent band (Figure 2514.-1): fragments of the outer testa (surface
view [B], transverse section [C]), with cells with an enlarged,
Formononetin: a yellow sclerified, strongly thickened and channelled base [Ba, Cb],
Orange fluorescent fluorescent band
band transformed into curved or straight hairs, usually broken,
with 7-10 lignified ridges [Bb, Ca] and a rounded lignified
tip [D]; numerous rods or strips, highly variable in length
Blue fluorescent band
and 5-15 pm wide, from the lignified ridges of the hairs [A];
Faint purple or blue
numerous fragments of endosperm [F], consisting of very’
fluorescent band
thick-walled polyhedral cells, some of which contain oil and
Faint blue band
aleurone grains; a few fragments of die outer layers of the
Solution (1) Solution (2) endosperm (surface new [E], transverse section [C]);
in surface view [E] the cells are polyhedral [Ea], sometimes
associated with the brown pigmented layer of the testa
formed of cells with indistinct walls [Eb]; in transverse
CHARACTERISTICS section [C] the cells are elongated [Cc], sometimes
The mother tincture is a greenish-brown liquid. associated with the pigmented layer [Cd] and the outer testa
[Cb].
TESTS
Ethanol
55% to 65% พ/พ (63% to 72% v/v), Appendix vm F.
Dry residue
Not less than 1.0% พ/พ, Appendix XI p.
Relative density
Nux-vomica for Homoeopathic * *

Preparations *****
Ph Elf_______________________________________________________________
DEFINITION
Dried, ripe seed of Strychnos nux-vomica L.
Content
Minimum 1.50 per cent for the sum of the contents of
brucine (C23H26N2O4; A4r 394.5) and strychnine
(C21H22N2O2; Afr 334.4), of which 43 per cent to
67 per cent is strychnine (dried drug).
IDENTIFICATION
A. The seed is discoid, with a slightly raised margin,
20-25 mm in diameter and about 5 mm thick. It is not quite
flat, with one surface slightly convex and the other slightly
concave. Some seeds are irregularly curved. The colour of
the seed ranges from light grey to greenish-grey and its satiny
appearance is due to a silky down, consisting of dense hairs
radiating from a central point on each of the faces. One of Figure 2514.-1. - Illustration for identification test B ofpowdered
the surfaces has a raised point at the centre (the hilum), from herbal drug of Nux-vomica for homoeopathic preparations
which extends a radial ridge ending in a slight protuberance
c. Thin-layer chromatography (2.2.27). with the same solvent. Dilute 1.0 mL of the solution to
Test solution To 2.0 g of the powdered herbal drug (710) 20.0 mL with mobile phase A.
(2.9.72) add 20 mL of ethanol (70 per cent VIV) R, allow to Reference solution Dissolve 10.0 mg of brucine CRS and
macerate for 15 min at room temperature, with stirring, and 10.0 mg of strychnine CRS in acetonitrile R and dilute to
centrifuge. Use the supernatant. 10.0 mL with the same solvent. Dilute 1.0 mL of the
Reference solution Dissolve 10 mg of brucine R and 10 mg of solution to 20.0 mL with mobile phase A.
strychnine R in 10 mL of ethanol (96 per cent) R. Column:
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel — size: I = 0.15 m, 0 = 4.6 mm;
plate fl (2-10 pm)]. — stationary phase: ethylene-bridged octadecylsilyl silica gel for
Mobile phase concentrated ammonia R, methanol R, methylene chromatography (hybrid material) fl (3.5 pm);
chloride fl (1:5:95 VIVIV), use the lower layer. — temperature: 35 °C.
Mobile phase:
— mobile phase A: triethylamine R, acetonitrile for
Development Over a path of 15 cm [or 6 cm]. chromatography R, methanol R2)
Drying In air, then in an oven at 105-110 °C for 15 min; tris (hydroxymethyl) aminomethane buffer solution pH 9.0 fl
allow to cool. (0 1:7.5:7.5:85 VlVIVIVy,
Detection Spray with iodoplatinate reagent R and examine — mobile phase B: triethylamine R,
immediately in daylight. tris (hydroxymethyl) aminomethane buffer solution pH 9.0 R,
Results See below the sequence of zones present in the acetonitrile for chromatography R, methanol R2
chromatograms obtained with the reference solution and the (0.1:15:42.5:42.5 VIVIVIVy
in the chromatogram obtained with the test solution. Time Mobile phase A Mobile phase B
0- 5 100 0
Top of the plate
5 - 25 100 -> 70 0 -> 30
25 - 30 70 -> 65 30 -> 35
30 - 31 65 -» 0 35 -> 100
Strychnine: a violet zone A violet zone (strychnine)
31 - 32 0 100
Brucine: a blue zone A blue zone (brucine)

Reference solution Test solution Detection Spectrophotometer at 260 nm.
Injection 10 pL.
TESTS Elution order Brucine, strychnine.
When the powdered herbal drug (710) (2.9.72) is used, take System suitability: reference solution:
the necessary precautions as indicated under Identification B. — resolution: minimum 3.0 between the peaks due to brucine
and strychnine.
iMaximum 1.0 per cent. Calcdate the percentage content of brucine and strychnine
Strychnos ignatii PJ. Bergius
The presence of irregularly shaped seeds that are neither
discoid nor flattened and the presence in the powdered Al X m.2 X p X 2
herbal drug, examined under a microscope, of sclerified hairs, A2 X 7ท!
usually sheared off, not thickened at the base and with walls
composed of small, oblique, sclerified strips, tightly fused A1 = area of the peak due to brucine or to strychnine in
longitudinally, indicate adulteration with Strychnos ignatii PJ. the chromatogram obtained with the test solution;
Bergius. A2 = area of the peak due to brucine or to strychnine in
Loss on drying (2.2.52) the chromatogram obtained with the reference
Maximum 10.0 per cent, determined on 1.00 g of the solution;
powdered herbal drug (710) (2.9.72) by drying in an oven at m 1 = mass of the herbal drug to be examined used to
105 °C for 2 h. prepare the test solution, in grams;
m2 = mass of brucine CRS or of strychnine CRS used to
Total ash (2.4.16) prepare the reference solution, in grams;
Maximum 3.0 per cent. p = assigned percentage content of brucine in brucine
Aflatoxins (2.8.18) CRS, or strychnine in strychnine CRS.
Maximum 2 pg/kg (aflatoxin Bl) and maximum 4 pg/kg
(sum of aflatoxins B], B2, G1 and G2).
MOTHER TINCTURE
ASSAY
Test solution To 1.000 g of the powdered herbal drug (710) preparation (2029).
(2.9.72) add 10.0 mL of ethanol (60 per cent VIV) R. Boil
gently, under a reflux condenser, with stirring. After 30 min, DEFINITION
cool and filter into a 20.0 mL volumetric flask. Wash the The mother tincture is prepared from the dried ripe seed of
filter with ethanol (60 per cent VIV) R and dilute to 20.0 mL Strychnos nuxûomica L.
Content
0.15 per cent mini to 0.30 per cent mini for the sum of the Potassium Dichromate for * *
contents of brucine (C23H26N2O4; 394.5) and strychnine Homoeopathic Preparations *****
(C21H22N2O2; 334.4), of which 43 per cent to 67 per cent is (Kalium Bichomicum for Homoeopathic Preparations,
strychnine. Ph. Eur. monograph 2501)
PRODUCTION K2Cr2O7 294.2 7778-50-9
The mother tincture is prepared from the powdered herbal
Ph Eur 1______ _________________
drug (710) (2.9.12) by the following methods prescribed in
the monograph Methods of preparation of homoeopathic stocks DEFINITION
andpotentisadon (237ไ): Content
— method 1.1.8 using ethanol (70 per cent F7F); 99.0 per cent to 101.0 per cent of K2Cr2O7.
— method 1.1.10 using ethanol (65 per cent VIV) and a CHARACTERS
maceration time of 3-5 weeks.
Appearance
CHARACTERS Orange crystals.
Appearance Solubility
Yellow or light browท liquid. Freely soluble in water, practically insoluble in ethanol
IDENTIFICATION (96 per cent).
Thin-layer chromatography (2.2.27) as described in IDENTIFICATION
identification test c for the herbal drug with the following A. It gives reaction (b) of potassium (2.3.1).
modification.
B. Dissolve 10 mg in 5 mL of water R. Add 0.25 mL of
Test solution The mother tincture to be examined. dilute sulfuric acid R, 0.5 mL of strong hydrogen peroxide
TESTS solution R and 1 mL of ether R. Shake. The upper layer is
Relative density (2.2.5) blue.
0.888 to 0.903 (method 1.1.8). TESTS
Ethanol (2.9.10) Solution Si
60 per cent VIV to 70 per cent VIV (method 1.1.10). Dissolve 5.0 g in distilled water R and dilute to 50.0 mL with
Dry residue (2.8.16) the same solvent.
Minimum 1.0 per cent mini. Solution S2
To 20.0 mL of solution SI add 20 mL of hydrochloric acid R
ASSAY
and 50 mL of tributyl phosphate R. Stir for 2 min. Remove
Liquid chromatography (2.2.29) as described in the assay of the lower layer and shake it with 10 mL of ether R. Evaporate
the herbal drug with the following modifications.
the lower layer to dryness under reduced pressure. Dissolve
Test solution Dilute 2.000 g of the mother tincture to be the residue in 10 mL of distilled water R. Add dilute
examined to 20.0 mL with ethanol (60 per cent VIV) R. ammonia R1 until the solution is neutral to blue litmus paper R
Calculate the percentage content of brucine and strychnine and dilute to 20.0 mL with distilled water R.
using the following expression: Appearance of solution
Solution Si is clear (2.2.1).
p
Al X 7712 X
Calcium (2.4.3)
A2 X 7ท1 X 10 Maximum 500 ppm.
Dilute 2.0 niL of solution S2 to 15 mL with distilled water R.
AI = area of the peak due to brucine or to strychnine in
the chromatogram obtained with the test solution; Chlorides (2.4.4)
A2 = area of the peak due to brucine or to strychnine in Maximum 50 ppm.
the chromatogram obtained with ±e reference Dissolve 1.0 g in 15 mL of dilute nitric acid R. Use 1 mL of
solution; nitric acid R instead of the prescribed dilute nitric acid R.
nil = mass of the mother tincture to be examined used to Sulfates (2.4.13)
prepare the test solution, in grams; Maximum 150 ppm.
ทไ2 = mass of brucine CRS or of strychnine CRS used to Dilute 10 mL of solution S2 to 15 mL with distilled water R.
p = assigned percentage content of brucine in brucine ASSAY
CRS, or strychnine in strychnine CRS. Dissolve 0.100 g in 25 mL of water R. Add 2 g of potassium
iodide R and 25 mL of dilute sulfuric acid R. Allow to stand in
------------------------------------------------------------------------------------------------------------Ph Eur the dark for 10 min. Add 150 mL of water R. Titrate with
0.1 M sodium thiosulfate until the colour changes from blue to
green, adding 1 mL of starch solution R near the end of the
titration.
1 mL of 0.1 M sodium thiosulfate is equivalent to 4.903 mg of
K2Cr2O7.
2016 Homoeopathic Preparations rV-473
In addition the following bands may be present: a blue

Prunus Spinosa Fruit for Homoeopathic fluorescent band with the same Rf as scopoletin and an
Preparations orange fluorescent band with an Rf slightly higher than that
definition of scopoletin. Two or more orange fluorescent bands may be
present between the two standards.
Prunus Spinosa Fruit for Homoeopathic Preparations is the
fresh ripe fruit of Prunus spinosa L.
Top of the plate
IDENTIFICATION
The ripe fruit is nearly a globose drupe, 8 to 15 mm in
Scopoletin: a blue
diameter, bluish-black with a blue-grey bloom. The dense An orange fluorescent band
green pulp surrounds a hard, spherical to ovoid and flattened fluorescent band
stone, 6 to 9 mm long, 6 to 8 mm wide and 4 to 6 mm
thick. The stone bulges slightly and has a sharp edge. A yellow-green Chlorogenic acid: a
fluorescent band yellow-green fluorescent
band
An orange
MOTHER TINCTURE fluorescent band
The mother tincture complies with the requirements stated under
PRODUCTION Solution (1) Solution (2)
The mother tincture of Prunus spinosa L. is prepared from
the herbal drug using Method 1.1.7 described in the
monograph for Methods of Preparation of Homoeopathic TESTS
Stocks and Potentisation. Use 43% พ/พ (50% v/v) of ethanol. Ethanol
24 to 34% พ/พ (29.3 to 40.8% v/v), Appendix VUI F.
CHARACTERISTICS
The mother tincture is a purple-red to red, clear to slightly
Dry residue
turbid liquid.
Relative density
IDENTIFICATION 0.955 to 0.995, Appendix V G.
(1) Centrifuge 10 mL of ±e mother tincture at about 3000
rpm for 5 minutes. Precondition a cartridge containing
octadecyl-bonded silica sorbent (a Sep-pak C18 cartridge is
Sodium Tetrachloroaurate * *
suitable) with 10 mL of methanol followed by 10 mL of Dihydrate for Homoeopathic *****
water. Apply 4.5 mL of the clear supernatant to the column,
wash with 5 mL zoater and elute with 5 mL of methanol. Preparations
Evaporate the eluant on a rotary evaporator at 50°. Dissolve (Aurum Chloratum Natronatum for Homoeopathic
the residue in 1 mL of methanol. Preparations, Ph. Eur. monograph 2141)
(2) 0.025% w/v of chlorogenic acid and 0.005% w/v of Na[AuCl4],2H2O 397.8
scopoletin in methanol. Ph Eur________________________________________________ __ ____________
CHROMATOGRAPHIC CONDITIONS DEFINITION

(a) Use as the coating silica gel F2S4. Sodium tetrachloroaurate(l-) dihydrate.
(b) Use the mobile phase described below. Content
(c) Apply 40 |1L of each solution as 12 mm bands. 97.0 per cent to 101.0 per cent of Na[AuCU],2H2O.
(d) Develop the plate to 15 cm. CHARACTERS
(e) After removal of the plate, dry it in air and spray with a Appearance
1 % w/v solution of diphenylboric acid aminoethyl ester in Orange-yellow, hygroscopic powder or crystals.
methanol) followed by a 5% w/v solution of polyethylene glycol Solubility
400 in methanol and examine under ultraviolet light (365 nm). Very soluble or freely soluble in water and in ethanol
MOBILE PHASE (96 per cent).
15 volumes of anhydrous formic acid) 15 volumes of water and IDENTIFICATION
70 volumes of ethyl acetate. A. Dissolve 20 mg in 2.0 mL of 0.1 M nitric acid. Add 0.1 g
SYSTEM SUITABILITY of oxalic acid R and boil in a water-bath for 1 h. A deposit of
The test is not valid unless the chromatogram obtained with metallic gold is formed.
solution (2) shows a yellow-green fluorescent band B. Solution ร (see Tests) gives reaction (a) of chlorides
(chlorogenic acid) in the middle third of the plate and a blue (2.3.1).
fluorescent band (scopoletin) in the upper third of the plate. C. Solution ร gives reaction (b) of sodium (2.3.1).
CONFIRMATION - TESTS
The chromatogram obtained with solution (1) shows at least Solution ร
one orange fluorescent band below chlorogenic acid, one Ignite 0.20 g in a porcelain crucible at 600 °C ± 50 °C for
yellow-green fluorescent band with the Rf of chlorogenic acid 30 min. Allow to cool and extract with 3 mL of water R)
and one orange fluorescent band below scopoletin. heating if necessary. Use the supernatant.
Free hydrochloric acid B. Heat 0.1 g with 0.5 mL of bromine water R until
When a glass rod impregnated with concentrated ammonia R is decolourised. Add 5 mL of water R and filter. The solution
held close to the substance to be examined, no white fumes gives reaction (a) of sulfates (2.3.1).
are produced.
TESTS
Nitrates Solution ร
Maximum 200 ppm. To 5.0 g add 50 mL of carbon dioxide-free water R prepared
Dissolve 0.20 g in 10 mL of nitrate-free water R. Add 0.2 g of from distilled water R. Allow to stand for 30 min with
oxalic acid R. Heat the solution on a water-bath for 30 min, frequent shaking and filter.
allow to cool and filter. Rinse the filter with nitrate-free Appearance of solution
water R and dilute the filtrate to 20 mL with the same Solution ร is colourless (2.2.2, Method II).
solvent. To 1.0 mL of the solution obtained add 4.0 mL of
nitrate-free water R, 0.4 mL of a 100 g/L solution of potassium Odour (2.3.4)
chloride R, 0.1 mL of diphenylamine solution R and, dropwise It has no perceptible odour of hydrogen sulfide.
with shaking, วิ mL of nitrogen-free sulfuric acid R. Transfer Acidity or alkalinity
the tube to a water-bath at 50 °C. After 15 min, any blue To 5 mL of solution ร add 0.1 mL of phenolphthalein
colour in the solution is not more intense than that in a solution Rl. The solution is colourless. Add 0.2 mL of
reference solution prepared at the same time in the same 0.01 M sodium hydroxide. The solution is red. Add 0.3 mL of
manner using a mixture of 0.2 mL of nitrate standard solution 0.01 M hydrochloric acid. The solution is colourless.
(10 ppm NOจุ) R and 4.8 mL of nitrate-free water R. Add 0.15 mL of methyl red solution R. The solution is orange-
Heavy metals (2.4. ร) red.
Maximum 100 ppm. Chlorides (2.4.4)
Dissolve 0.20 g in 15 mL of water R. Add 0.25 g of hydrazine Maximum 100 ppm.
sidfate R. Heat the solution on a water-bath for 30 min, allow Dilute 5 mL of solution s to 15 mL with water R.
to cool and filter. Rinse the filter with water R and dilute the Sulfates (2.4.13)
filtrate to 20 mL with the same solvent. 12 mL of the Maximum 100 ppm, determined on solution ร.
solution complies with test A. Prepare ±e reference solution
Sulfides
using lead standard solution (1 ppm Pb) R.
To 10 mL of solution ร add 2 mL of buffer solution pH 3.5 R
ASSAY and 1 mL of a freshly prepared 1.6 g/L solution of lead
Dissolve 40.0 mg in 10 mL of potassium iodide solution R. nitrate R in carbon dioxide-free water R. Shake. After 1 min
Allow to stand for 5 min. Titrate with 0.01 M sodium any colour in the solution is not more intense than that in a
thiosulfate until decolourised. Shortly before reaching the reference solution prepared at the same time using 1 mL of
endpoint, add 0.5 mL of starch solution R. lead standard solution (10 ppm Pb) R, 9 mL of carbon dioxide
1 mL of 0.01 M sodium thiosulfate is equivalent to 1.989 mg free water R, 2 mL of buffer solution pH 3.5 R and 1.2 mL of
ofNa[AuCl4],2H2O. thioacetamide reagent R.
STORAGE Arsenic (2.4.2, Method B)
In an airtight container, protected from light. Maximum 8 ppm.
Shake 2.5 g with 50 mL of dilute ammonia Rl for 1 h and
_______________________________________________________________ P'n Eur
filter. Evaporate 25 mL of the filtrate to dryness. Add 2 mL
of water R and 3 mL of nitric acid R to the residue and
evaporate to dryness. The residue complies with the test.
Sulfur for Homoeopathic * \ Prepare the standard using 1 mL of arsenic standard solution
(10 ppm As) R.
Preparations ***** Sulfated ash (2.4.14)
(Ph. Eur. monograph 2515) Maximum 0.2 per cent, determined on 1.0 g.
ร 32.07 7704-34-9
ASSAY
Ph Eur_______________________________________________________________ Carry out the oxygen-flask method (2.5.10) 3 using 60.0 mg
DEFINITION in a 1000 mL combustion flask with a teflon joint. Absorb
Obtained by sublimation. the combustion products in a mixture of 5 mL of dilute
hydrogen peroxide solution R and 10 mL of water R. Heat to
Content
boiling, boil gently for 2 min and cool. Using 0.2 mL of
99.0 per cent to 101.0 per cent.
phenolphthalein solution R as indicator, titrate with 0.1 M
CHARACTERS sodium hydroxide until the colour changes from colourless to
Appearance red. Carry out a blank titration under the same conditions.
Yellow powder. 1 mL of 0.1 M sodium hydroxide is equivalent to 1.603 mg of
Solubility ร.
Practically insoluble in water, soluble in carbon disulfide, STORAGE
slightly soluble in vegetable oils. Protected from light.
About 120 °C.

IDENTIFICATION
A. Heated in the presence of air, it bums wi± a blue flame,
emitting sulfur dioxide, which changes the colour of
moistened blนe litmus paper R to red.
Symphytum Officinale Root for MOBILE PHASE
10 volumes of anhydrous formic acid, 10 volumes of water and

Homoeopathic Preparations 80 volumes of ethyl acetate.
DEFINITION SYSTEM SUITABILITY
Symphytum Officinale Root for Homoeopathic Preparations The test is not valid unless under ultraviolet light (365 nm)
IS the fresh root of Symphytum officinale L. the chromatogram of solution (1) shows two bluish
IDENTIFICATION fluorescent bands at Rf 0.25 and Rf 0.55 and one light-green
The strong, spirally-formed rootstock with a smooth, black fluorescent band at the solvent front.
brown cortex, subdivides at the base. It is about 5 to 8 cm in CONFIRMATION
diameter and 17 to 30 cm long, surmounted by several tops, After spraying, the chromatogram obtained with solution (2)
close together, consisting of the black residues of the previous shows the yellow allantoin band at Rf 0.35.
year ร rosettes of leaves between the current year’s new The chromatogram obtained with solution (1) shows one
growths. A ring of 10 to 15 horizontally-running, secondary, yellow band with the same Rf of 0.35 as that for allantoin.
glabrous smooth roots grows from the base of the rosettes,
the roots often reaching a length of more than 50 cm and a TESTS
diameter of approximately 1.5 cm. The middle portion of the Ethanol
rootstock, which is more or less glabrous, branches at the 35 to 45% พ/พ (42 to 53% v/v), Appendix VHI F.
lower end into several clearly separated, straight downwards Dry residue
pointing, smooth roots, each about 1.5 cm thick and bearing Not less than 1.5% พ/พ, Appendix XI P.
a few secondary rootlets 1 to 2 cm long. Relative density
The rootstock and the thick secondary roots are non 0.920 to 0.970, Appendix V G.
lignified, succulent and break off easily. In the yellowish-
white, glassy, slightly differentiated cross section, there is a
very thin, black rhizodermis, and an easily detachable layer of
cortex, 5-8 mm thick. Within this is a single dark pigmented
vascular ring. The cut surface and particularly the richly
Symphytum Officinale Root for Ethanol
exuding mucilagenous substance turn yellow to red-brown on Decoction for Homoeopathic
exposure.
Preparations
TESTS
DEFINITION
Foreign matter
Symphytum Officinale Root for Ethanol Decoction for
Not more than 2%, Appendix XI D.
Homoeopathic Preparations is the fresh root of Symphytum
officinale L.
MOTHER TINCTURE IDENTIFICATION
The mother tincture complies with the requirements stated under The strong, spirally-formed rootstock with a smooth, black
Mother Tinctures for Homoeopathic Preparations and with the brown cortex, subdivides at the base. It is about 5 to 8 cm in
following requirements. diameter and 17 to 30 cm long, surmounted by several tops,
close together, consisting of the black residues of the previous
PRODUCTION
year’s rosettes of leaves between the current year’s new
The mother tincture of Symphytum officinale L., is prepared
growths. A ring of 10 to 15 horizontally-running, secondary,
from the herbal drug using Method 1.1.3 described in the
glabrous smooth roots, grows from the base of the rosettes,
the roots often reaching a length of more than 50 cm and a
Stocks and Potentisation. Use 86% พ/พ (90% v/v) of ethanol.
diameter of approximately 1.5 cm. The middle portion of the
CHARACTERISTICS rootstock, which is more or less glabrous, branches at the
The mother tincture is a brown liquid. lower end into several clearly separated, straight downwards
pointing, smooth roots, each about 1.5 cm thick and bearing
IDENTIFICATION
a few secondary rootlets 1 to 2 cm long.
Carry out the method for thin-layer chromatography3
Appendix HI A, using the following solutions. The rootstock and the thick secondary roots are non
lignified, succulent and break off easily. In the yellowish-
(1) The mother tincture.
white, glassy, slightly differentiated cross section, there is a
(2) 0.1% w/v of allantoin in ethanol (45%). very thin, black rhizodermis, and an easily detachable layer of
CHROMATOGRAPHIC CONDITIONS cortex, 5-8 mm thick. Within this is a single dark pigmented
(a) Use as the coating silica gel. vascular ring. The cut surface and particularly the richly
exuding mucilagenous substance turn yellow to red-brown on
exposure.
(c) Apply 20 pL of solution (1) and 10 |1L of solution (2) as
10 mm bands.
MOTHER TINCTURE
(f) Spray the plate with a 5% w/v solution of
dimethylaminobenzaldehyde in hydrochloric acid and examine in PRODUCTION
daylight. The mother tincture of Symphytum officinale L. is prepared
by decoction of the herbal drug using 43% พ/พ (50% v/v) of
ethanol.
CHARACTERISTICS The leaflets are ovate to slightly rhombic and of varying size,
The mother tincture is a brown liquid. the middle leaflet being the largest, up to 20 cm long and
IDENTIFICATION 11 cm wide with long pctiolule; the two lateral leaflets are
Carry out the method for thin-layer chromatography, smaller, up to 16 cm long and 9 cm wide with short
Appendix in A, using the following solutions. petiolules. The margins of the laminae may be entire or
broadly dentate with up to 3 or more short triangular lobes
(1) The mother tincture. on each side, particularly in the apical region of the middle
(2) 0.1% w/v of allantoin in ethanol (45%). leaflets; on the lateral leaflets the margin is frequently
CHROMATOGRAPHIC CONDITIONS asymmetrical with the lobes on one side only; all the leaflets
(a) Use as the coating silica gel. are cuneate at the base and acute at the apex.
(c) Apply 20 pL of solution (1) and 10 pL of solution (2) as MOTHER TINCTURE
10 mm bands. The mother tincture complies with the requirements stated under
(d) Develop the plate to 10 cm. Mother Tinctures for Homoeopathic Preparations and with the
(e) After removal of the plate, dry it in air and examine following requirements.
under ultraviolet light (365 nm). DEFINITION
(f) Spray the plate with a 5% w/v solution of It contains not less than 0.1% พ/พ of total flavonoids
dimethylaminobenzaldehyde in hydrochloric acid and examine in expressed as quercitrin (C21H20011).
daylight.
PRODUCTION
MOBILE PHASE The mother tincture of Toxicodendron quercifolium (Michx.)
10 volumes of anhydrous formic acid, 10 volumes of water and Greene is prepared from the herbal drug using Method 1.1.3
80 volumes of ethyl acetate. described in the monograph for Methods of Preparation of
SYSTEM SUITABILITY
Homoeopathic Stocks and Potentisation. Use 86% พ/พ
(90% v/v) of ethanol.
The test is not valid unless under ultraviolet light (365 nm),
the chromatogram obtained with solution (1) shows two CHARACTERISTICS
bluish fluorescent bands at Rf value of 0.25 and Rf value of The mother tincture is a yellowish-brown to reddish-brown
0.55 and one light-green fluorescent band at the solvent liquid.
front. IDENTIFICATION
CONFIRMATION A. To 1 mL of the mother tincture add 1 granule of zinc, a
After spraying, the chromatogram obtained with solution (1) few turnings of magnesium and 1 mL of hydrochloric acid.
shows a yellow band at Rf value of 0.35 corresponding in A dark red colour is produced which can be extracted with
position and colour to that obtained with solution (2). tert-pentyl alcohol.
B. Carry out ±e method for thin-layer chromatography,
TESTS
Ethanol
25 to 35% พ/พ (31 to 42% v/v), Appendix VEH F. (1) Use the mother tincture.
Dry residue (2) Dissolve 20 mg of arbutin and 10 mg of gallic acid in
Not less than 3.0% พ/พ, Appendix XI p. 10 mL of methanol.
Relative density CHROMATOGRAPHIC CONDITIONS
0.973 to 0.982, Appendix V G. (a) Use as the coating silica gel.
(c) Apply 10 pL of each solution as bands.
(d) Develop ±e plate to 10 cm.
Toxicodendron Quercifolium for (e) Remove the plate, dry in air, spray with a 0.5% w/v
solution of fast blue B salt in water, dry briefly and spray with
Homoeopathic Preparations 0.1m alcoholic sodium hydroxide. Examine in daylight.
Rhus Toxicodendron for Homoeopathic Preparations
MOBILE PHASE
DEFINITION 10 volumes of anhydrous formic acid, 10 volumes of water and
Toxicodendron Quercifolium for Homoeopathic Preparations 80 volumes of ethyl acetate.
is the fresh, young, not yet lignified shoots, with leaves, of
SYSTEM SUITABILITY
Toxicodendron quercifolium (Michx.) Greene.
CAUTION The siIO01ร contain a yellowish-white milky sap that
is a strong cutaneous irritant and darkens the skin. Contact with solution (2) shows two well separated bands.
the skin and mucous membranes is to be avoided. CONFIRMATION
IDENTIFICATION The chromatogram obtained with solution (1) shows the

Shoots The thin shoots have a downy to cottony following reddish-brown bands: two or three bands, which lie
indumentum and may bear at their ends, and in the axils of close together at a position between those obtained for
the alternate leaves, pointed buds covered in brown, woolly arbutin and gallic acid in solution (2) and two further
pronounced bands at about the same level as that obtained
hairs.
for gallic acid in solution (2). Other bands may be present in
Leaves Dark green on the upper surface, lighter green on the the chromatogram obtained with solution (1).
lower surface and have scattered hairs, more numerous on
the veins; they are tripinnate with petioles 15 to 20 cm long.
Top of the plate Time Mobile phase Mobile phase Comment

(Minutes)
A reddish-brown band Gallic acid: a reddish-brown (% v/v) (% v/v)
A reddish-brown band band 0-2 20 80 isocratic
2 or 3 closely positioned 0->20 100->80 linear gradient
82-83
reddish-brown bands
Arbutin: a reddish-brown When the chromatograms are recorded under the prescribed
band conditions the relative retentions with reference to 4-
dodecylresorcinol (retention time about 27 minutes) are
Solution (1) Solution (2) urushiol I about 2.0 and urushiol II about 2.4. The relative
retention of urushiol I to urushiol II is about 0.8.
SYSTEM SUITABILITY
TESTS The test is not valid unless, in the chromatogram obtained
Ethanol with solution (2), the column efficiency, determined using the
36% to 46% พ/พ (43% to 54% v/v), Appendix VIII F. peak due to 4-dodecyl resorcinol, is not less than 20,000
Dry residue theoretical plates, and in the chromatogram obtained with
Not less than 3.5% พ/พ, Appendix XI p. solution (3) the signal-to-noise ratio of the peaks due to
urushiol I and urushiol II is at least 10.
Relative density
0.945 to 0.965, Appendix V G. LIMITS
Urushiols Calculate the percentage พ/พ content of urushiols in solution

Carry out the method for liquid chromatography, . (1), expressed as 4-dodecylresorcinol, using the following
Appendix in D, using the following solutions. expression:
(1) Evaporate 10.00 g of the mother tincture to dryness using
a rotary evaporator at a temperature not exceeding 40°. A1 X c2 xp X 1.20
Add 10 mL water to the residue and mix with the aid of
ultrasound for 20 minutes. Add 10 mL of heptane and shake A2 X c 1 X 100
vigorously for 15 minutes. Allow to separate, remove and
retain the heptane layer avoiding any suspended particles. A1 = sum of the peak areas due to urushiols in the
Perform the extraction twice more on the aqueous phase, chromatogram obtained with solution (1);
then discard the remaining aqueous layer and rinse the flask A2 = area of the peak due to 4-dodecylresorcinol in the
with 10 mL of heptane. Combine the extracts and washings chromatogram obtained with solution (2);
and filter through anhydrous sodium sulfate and evaporate the Cl — concentration of the mother tincture sample in
filtrate to dryness using a rotary evaporator at a temperature solution (1) in % w/v;
not exceeding 40°. Dissolve ±e residue in 2.0 mL of c2 = concentration of 4-dodecylresorcinol in solution (2)
methanol. in % w/v;
(2) To 0.5 mL of a 0.175% w/v solution of 4-dodecylresorcinol p = percentage content of 4-dodecylresorcinol in
in methanol add 0.5 mL of a solution containing 0.1% w/v 4-dodecylresorcinol',
each of urushiol I and urushiol II in methanol and dilute to 1.20 = average molar mass ratio of urushiol I and II to
5 mL with methanol. 4-dodecylresorcinol.
(3) Dilute 1 volume of solution (2) to 20 volumes with In the chromatogram obtained with solution (1):
methanol.
The total content of urushiols, expressed as
(4) 0.00025% w/v of 4-dodecylresorcinol in methanol. 4-dodecylresorcinol, is not more than 0.05%.
CHROMATOGRAPHIC CONDITIONS Disregard any peak:
(a) Use a stainless steel column (25 cm X 4.6 mm) packed with an area less than the area of the peak due to 4-dodecyl
with octadecylsilyl silica gel for chromatography (5 |im) (Waters resorcinol in solution (4) (0.00005%);
Symmetry Cl8 is suitable). with a retention time less than 0.25 times that of the peak
(b) Use gradient elution and the mobile phase described due to 4-dodecylresorcinol, or with a retention time greater
below. than the peak due to urushiol n in the chromatogram
(c) Use a flow rate of 1 mL per minute. obtained with solution (2).
(d) Use a column temperature of 30°. ASSAY
(e) Use a detection wavelength of 276 nm. Carry out the method for liquid chromatography,
(f) Inject 20 pL of each solution. Appendix in D, using the following solutions in 50 volumes
of methanol and 50 volumes of water.
MOBILE PHASE
(1) 10% w/v of the mother tincture.
Mobile phase A 0.2% v/v orthophosphoric acid.
(2) 0.018% w/v each of isoquercitrin and quercitrin BPCRS'
Mobile phase B methanol.
(3) 0.0000018% w/v of quercitrin BPCRS'
CHROMATOGRAPHIC CONDITION’S
Urtica Dioica for Homoeopathic ** *.
with octadecylsilyl silica gel for chromatography (5 pm) (Waters Preparations *****
Symmetry Cl8 is suitable). Common Stinging Nettle for Homoeopathic
(b) Use gradient elution and the mobile phase described Preparations
below. (Ph. Eur. monograph 2030)
(c) Use a flow rate of 1 mL per minute. Ph Eur---------------------------------------------------------- -------- -------------------------------------
DEFINITION
(e) Use a detection wavelength of 340 nm. Whole, fresh, flowering plant of Urtica dioica L.
CHARACTERS
MOBILE PHASE The plant causes an itching, burning sensation on the skin.
Mobile phase A water adjusted to pH to 2.3 with
IDENTIFICATION
orthophosphoric acid.
A. The perennial plant has a taproot that sends out creeping
Mobile phase B acetonitrile. subterranean rhizomes, more or less 4-angled in transverse
section, from which extend adventious secondary roots and
Time Mobile phase A Mobile phase B Comment very numerous brownish hairy rootlets. The stipes are erect,
(Minutes) (% v/v) (% v/v) generally unbranched, 3-5 mm in diameter and 0.3-1.5 m
high, rarely up to 2.5 m high, 4-angled, greyish-green and
0-2 95 5 isocratic
covered in short hairs and stinging hairs.
The decussate leaves are 30-150 mm long and 20-80 mm
18-32 87->74 13->26 linear gradient wide. The petiole is hispid and usually slightly less than one-
32-42 74 26 isocratic third the length of the lamina. The leaf blade is ovate,
acuminate, cordate or rounded at the base, and coarsely
dentate; the apical tooth is distinctly larger than the lateral
teeth. The upper side of the leaves is dark green and usually
matt, both sides bear short serried hairs intermingled with
When the chromatograms are recorded using the prescribed long stinging hairs. The 2 stipules are linear-subulate and
conditions, the retention time of isoquercitrin is about free. The inflorescences growing from the leaf axils are
31 minutes and that of quercitrin is about 34 minutes, the complex, the flowers unisexual, and, particularly in male
relative retention is about 0.9. plants, generally distinctly longer than tlie petiole. After
shedding their pollen, male inflorescences are erect at an
SYSTEM SUITABILITY oblique angle or horizontal; female inflorescences are pendent
The test is not valid unless, in the chromatogram obtained when the fruit is ripe. All flowers have long stalks.
with solution (2): The perianth of the male flowers is divided half-way down
the symmetry factor for both isoquercitrin and quercitrin is not into equal green lobes, widest at their base, with short bnstles
less than 0.8 and not more than 1.2; and stinging hairs at the margins. The stamens are equal and
the number of theoretical plates with respect to isoquercitrin opposite to the perianth segments, each with a long, whitish
is at least 200,000. filament that curves inwards before pollen is shed and
spreads out afterwards. The ovary is rudimentary, button or
cup-shaped. The perianth of the female flowers is downy or
Calculate the content of total flavonoids, expressed as bristly on the outside and consists of outer, and 2 inner
quercitrin from the chromatograms obtained and using the segments; the inner segments are about twice the length of
declared content of c21 H2oO11 in quercitrin BPCRS and the the outer ones. The hypogynous, ovate, unilocular ovary
following expression. Disregard any peak with an area less bears a large capitate stigma with a brush-like shock of hair.
than that in solution (3). As the one-seeded fruit grows ripe, the 2 inner segments of
the perianth fold around it like wings.
^1XC2X/? B. It complies with the test for Urtica urens (see Tests).
TESTS
A2 X C] Urtica urens
The margin of the lamina is not serrate with teeth twice as
A1 = Sum of the peak areas due to quercitrin and long as wide. The clusters of flowers in the axils are longer
flavonoids in the chromatogram obtained with than the petiole of the leaf. Unisexual, apetalous flowers are
solution (1); not together on the same plant and in the same cluster.
A2 = area of the peak due to quercitrin in the Foreign matter (2.5.2)
chromatogram obtained with solution (2); Maximum 5 per cent.
c1 = concentration of the mother tincture sample in
solution (1) in % w/v;
Minimum 65.0 per cent, determined on 5.0 g of finely cut
c2 = concentration of quercitrin BPCRS in solution (2) in
herbal drug by drying in an oven at 105 °C for 2 h, if
% w/v;
performed to demonstrate the freshness of the herbal drug.
p = percentage content of quercitrin in quercitrin BPCRS.
MOTHER TINCTURE
Urtica Urens Herb for Homoeopathic
general monograph Mother tinctures for homoeopathic Preparations
preparations (2029). DEFINITION
PRODUCTION Urtica Urens Herb for Homoeopathic Preparations is the
The mother tincture of Urtica dioica is prepared by fresh leaves and flowers of Urtica urens L.
maceration using ethanol of a suitable concentration. CHARACTERISTICS
CHARACTERS The plant produces an itchy, burning sensation.
Appearance IDENTIFICATION
Greenish-brown or orange-brown liquid. Plant Annual.
IDENTIFICATION Leaves Decussate with diffusely haired petiole, which in the
Thin-layer chromatography (2.2.27). lower leaves is mostly as long as the lamina; ovate to elliptic,
1 to 5 cm long and 1 to 4 cm wide lamina with incised
serrated leaf margin, blunt to cuneate at the base, acuminate
Referetice solution Dissolve 10 mg of phenylalanine R and towards the apex. The leaves are dark-green on the upper
10 mg of serine R in a mixture of equal volumes of surface, slightly shiny and paler green on the lower surface;
methanol R and water R and dilute to 10 mL with the same prominent stinging hairs occur scattered all over the upper
mixture of solvents. surface, on the lower surface they occur mostly over the
Plate TLC silica gel plate R. veins. The two stipules on each side are lanceolate and the
Mobile phase glacial acetic acid R, water R, acetone R, butanol R margins are entire.
(10:20:35:35 VIVIVIV). Flowers The complicated inflorescences consist mainly of
Application 20 |1L, as bands. female flowers and only a few male flowers; they arise from
Development Over a path of 10 cm. the leaf axils and are about 1.5 to 2 cm long and usually
Drying In air. shorter than the leaf petioles. The perigonium of the male
flowers is split into four pale green lobes of equal size; each
Detection Spray with a 1 g/L solution of ninhydrin R in ethanol one of the four stamens situated in front of one of the
(96 per cent) R, heat at 105-110 °C for 5-10 min and perigonium lobes and has a long filament which at first is
examine in daylight within 10 min. incurved and then widens out before the anther releases the
Results See below the sequence of the zones present in the pollen. The perigonium of the female flowers consists of two
chromatograms obtained with the reference solution and the outer, short, bract-like segments and two longer, inner ones,
test solution. all with ciliated margins and scattered hairs over the surfaces;
the superior ovary is ovoid with a short style and a
conspicuous, brushlike stigma. The ripe fruits are
Top of the plate
monospermic and enclosed by the two inner segments of the
perigonium.
Phenylalanine: a violet to TESTS
reddish-brown zone
Urtica dioica
4 red to violet zones
The plant is dioecious as follows:
the male and female flowers occur on separate plants;
Serine: a reddish-violet zone A violet zone the inflorescences are longer than the leaf petioles;
A violet zone the leaves, especially those on the lower part of the stem, are
Reference solution Test solution longer than their petioles.
MOTHER TINCTURE
TESTS
0.930 to 0.950.
Ethanol (2.9.70)
40 per cent VIV to 56 per cent VIV. PRODUCTION
The mother tincture of Urtica urens L.J is prepared from the
Methanol (2.9.77) herbal drug using Method 2b described in the monograph for
Maximum 0.10 per cent VIV. Methods of Preparation of Homoeopathic Stocks and
Dry residue (2.5.76) Potentisation. Use 62% พ/พ (70% v/v) of ethanol.
CHARACTERISTICS
_______________________________________________________________ Ph Eur
The mother tincture is a green to brownish-green liquid.
IDENTIFICATION
A. To 1 mL of potassium hydroxide solution add 1 mL of the
mother tincture and heat to boiling. Red litmus paper held
over the mouth of the test tube turns blue.
B. Add 1 mL of hydrochloric acid and a few crystals of
resorcinol to 1 mL of the mother tincture. Heat to boiling.
A red colour is produced.
TV-480 Homoeopathic Preparations 2016

(1) The mother tincture.
(2) Dissolve 10 mg of leucine and 10 mg of threonine in
10 mL of 50% v/v ethanol.
(a) Use as the coating silica gel G.
(c) Apply 20 pL of each solution as bands.
(e) After removal of the plate, dry in a current of warm air or
in an oven at 100° to 105°. Spray the chromatogram with a
0.5% w/v solution of ninhydrin in butan-l-ol. Heat for
10 minutes at 100° to 105° and examine in daylight.
MOBILE PHASE
10 volumes of glacial acetic acid, 20 volumes of water,

35 volumes of acetone and 35 volumes of butan-l-ol.
SYSTEM SUITABILITY
the solution (2) shows two clearly separated coloured zones
due to threonine (reddish pink) in the lower region and
leucine (pink) in the middle region.
CONFIRMATION
The chromatogram obtained with solution (1) shows a pink
spot (which may be separated into two spots) corresponding
to leucine. A reddish pink spot occurs in the lower region
corresponding to threonine. Between the spots corresponding
to threonine and leucine there is an orange pink spot and a
pink spot present, in order of increasing Rf value. Several
spots are present below the spot corresponding to threonine.
Top of the plate
A pink band (may be two Leucine a pink band

bands)
A pink band
An orange-pink band
A reddish-pink band Threonine: a reddish-pink

band
Several bands may be
TESTS
Ethanol
25% to 35% พ/พ (30% to 42% v/v), Appendix vm F.
Dry residue
Relative density
Monographs
2016 Blood-related Products FV-483
Apply separately to the plate 2 pL of each solution and

BLOOD-RELATED PRODUCTS thoroughly dry the points of application. Develop over a path
of 15 cm using a mixture of 10 volumes of water R,
15 volumes of methanol R, 25 volumes of anhydrous acetic
acid R and 50 volumes of ethylene chloride R. The volumes of
Anticoagulant and Preservative ** ** solvents have to be measured accurately since a slight excess
of water produces cloudiness. Dry the plate in a current of
Solutions for Blood ***** warm air. Repeat the development immediately, after
(Anticoagulant and Preservative Solutions for Human renewing the mobile phase. Dry the plate in a current of
Blood, Ph Eur monograph 0209) warm air and spray evenly with a solution of 0.5 g of
Pn Etf______________________ _______________________________________ thymol R in a mixture of 5 mL of sulfuric acid R and 95 mL
of alcohol R. Heat at 130 °C for 10 min. The principal spot
definition in the chromatogram obtained with the test solution is similar
Anticoagulant and preservative solutions for human blood are in position, colour and size to the principal spot in the
sterile and pyrogen-free solutions prepared with water for chromatogram obtained with reference solution (a). The test
injections, filtered, distributed in the final containers and is not valid unless the chromatogram obtained with reference
sterilised. The content of sodium citrate solution (b) shows 4 clearly separated spots.
(C6H5Na3O7,2H2O), glucose monohydrate (C6H12O6,H2O)
B. To 2 mL add 5 mL of cupri-citric solution R. Heat to
or anhydrous glucose (C6H12o6) and sodium dihydrogen
boiling. An orange precipitate is formed and the solution
phosphate dihydrate (NaH2PO4,2H2O) is not less than
becomes yellow.
95.0 per cent and not more than 105.0 per cent of that
stated in the formulae below. The content of citric acid c. To 2 mL (for formula A) add 3 mL of water R or to
monohydrate (C6H8O7,H2O) or anhydrous citric acid 4 mL (for formula B) add 1 mL of water R. The solution
(C6H807) is not less than 90.0 per cent and not more than gives the reaction of citrates (2. ร./).
110.0 per cent of that stated in the formulae below. Subject D. 0.5 mL gives reaction (b) of sodium (2.3.1).
to agreement by the competent authority, other substances, TESTS
such as red-cell preservatives, may be included in the formula pH (2.2.3)
provided that their name and concentration are stated on the The pH of the solution to be examined is 4.7 to 5.3.
label.
Hydroxymethylftirfural
Anticoagulant and preservative solutions for human blood are To 2.0 mL add 5.0 mL of a 100 g/L solution of p-toluidine R
presented in airtight, tamper-proof containers of glass (5.2./) in 2-propanol R containing 10 per cent VIV of glacial acetic
or plastic (ร. 2. ร).
acid R and 1.0 mL of a 5 g/L solution of barbituric acid R.
Anticoagulant acid-citrate-glucose solutions (ACD) The absorbance (2.2.25), determined at 550 nm after
A B allowing the mixture to stand for 2 min to 3 min, is not
Sodium citrate (0412) 22.0 g 13.2 g greater than that of a standard prepared at the same time in
Citric acid monohydrate (0456) 8.0 g 4.8 g the same manner using 2.0 mL of a solution containing
or Citric acid, anhydrous (0455) 7.3 g 4.4 g 5 ppm of hydroxymethylfurfural R for formula A or 3 ppm of
Glucose monohydrate (0178)
* 24.5 g 14.7 g hydroxymethylftirfural R for formula B.
or Glucose, anhydrous (0177)
* 22.3 g 13.4 g
Sterility (2.6. /)
Water for injections (0169) to 1000.0 mL 1000.0 mL
They comply with the test for sterility.
Volume to be used per 100 mL 15.0 mL 25.0 mL
of blood Pyrogens (2.6.8)
They comply with the test for pyrogens. Dilute with a
pyrogen-free, 9 g/L solution of sodium chloride R to obtain a
* The competent authority may require that the substances solution containing approximately 5 g/L of sodium citrate.
comply with the test for pyrogens given in the monographs Inject 10 mL of the diluted solution per kilogram of the
on Glucose Monohydrate (0178) and Glucose Anhydrous rabbit's mass.
(0177), respectively.
ASSAY
CHARACTERS
Citric acid
A colourless or faintly yellow, clear liquid, practically free To 10.0 mL (for formula A) or to 20.0 mL (for formula B)
from particles. add 0.1 mL of phenolphthalein solution Rl. Titrate with 0.2 M
IDENTIFICATION sodium hydroxide until a pink colour is obtained.
A. Examine by thin-layer chromatography (2.2.27), using 1 mL of 0.2 M sodium hydroxide is equivalent to 14.01 mg of
silica gel G R as the coating substance. C6H8O7,H2O or to 12.81 mg of C6H8O7.
Test solution Dilute 2 mL of the solution to be examined (for Sodium citrate
formula A) or 3 mL (for formula B) to 100 mL with a Prepare a chromatography column 0.10 m long and 10 mm
mixture of 2 volumes of water R and 3 volumes of in internal diameter and filled with strongly acidic ion-exchange
methanol R. resin R (300 pm to 840 pm). Maintain a l cm layer of liquid
Reference solution (a) Dissolve 10 mg oi glucose CRS in a above the resin at all times. Wash the column with 50 mL of
mixture of 2 volumes of water R and 3 volumes of methanol R de-ionised water R at a flow rate of 12-14 mL/min.
and dilute to 20 mL with the same mixture of solvents. Dilute 10.0 mL of the solution to be examined
Reference solution (b) Dissolve 10 mg each of glucose CRS, (for formula A) or 15.0 mL (for formula B) to about 40 mL
lactose CRS, fructose CRS and sucrose CRS in a mixture of with de-ionised water R in a beaker and transfer to the
2 volumes of water R and 3 volumes of methanol R and dilute column reservoir, washing the beaker 3 times with a few
to 20 mL with the same mixture of solvents. millilitres of de-ionised water R. Allow the solution to run
IV-484 Blood-related Products 2016
through the column at a flow rate of 12-14 mUmin and LABELLING

collect the eluate. Wash the column with 2 quantities, each The label states:
of 30 mL, and with one quantity of 50 mL, of de-ionised — the composition and volume of the solution,
water R. The column can be used for 3 successive — the maximum amount of blood to be collected in the
determinations before regeneration with 3 times its volume of container,
dilute hydrochloric acid R. Titrate the combined eluate and — Anticoagulant citrate phosphate-glucose solution (CPD).
washings (about 150 mL) with 0.2 M sodium hydroxide, using
0.1 mL of phenolphthalein solution Rl as indicator.
Sodium citrate (0412) 26.3 g
Calculate the content of sodium citrate in grams per litre
Citric acid monohydrate (0456) -T? g
from the following expressions:
or Citric acid, anhydrous (0455) 2.99 g
For formula A: 1.96171 - 1.40C Glucose monohydrate (0178)' 25.5 g
or 1.96171 - 1.53๙ or Glucose, anhydrous (0177)* 23.2 g
Sodium dihydrogen phosphate dihydrate (0194) 2.51 g

For formula B: 1.307n - 1.40C Water for injections (0169) to 1000.0 mL
1.30771 - 1.53๙ Volume to be used per 100 mL of blood 14.0 mL
’The competent authority may require that the substances comply

ท = number of millilitres of 0.2 M sodium hydroxide with the test for pyrogens given in the monographs on Glucose
used in the titration, monohydrate (0178) and Glucose, anhydrous (0177), respectively.
c = content of citric acid monohydrate in grams per

litre determined as prescribed above,
CHARACTERS
c = content of anhydrous citric acid in grams per litre
A colourless or faintly yellow, clear liquid, practically free
determined as prescribed above.
from particles.
Reducing sugars
IDENTIFICATION
Dilute 5.0 mL (for formula A) or 10.0 mL (for formula B) to
A. Examine by thin-layer chromatography (2.2.27), using
100.0 mL with water R. Introduce 25.0 mL of the solution
silica gel G R as the coating substance.
into a 250 mL conical flask wnth ground-glass neck and add
25.0 mL of cupri-citric solution Rl. Add a few pieces of Test solution Dilute 2 mL of the solution to be examined to
porous material, attach a reflux condenser, heat so that 100 mL with a mixture of 2 volumes of tuater R and
boiling begins U'ithin 2 min and boil for exactly 10 min. Cool 3 volumes of methanol R.
and add 3 g of potassium iodide R dissolved in 3 mL of Reference solution (a) Dissolve 10 mg of glucose CRS in a
water R. Add 25 mL of a 25 per cent mini solution of sulfuric mixture of 2 volumes of water R and 3 volumes of methanol R
acid R with caution and in small quantities. Titrate with and dilute to 20 mL with the same mixture of solvents.
0.1 M sodium thiosulfate using 0.5 mL of starch solution R, Reference solution (b) Dissolve 10 mg each of glucose CRS,
added towards the end of the titration, as indicator (ท1 mL). lactose CRS, fructose CRS and sucrose CRS in a mixture of
Cany7 out a blank titration using 25.0 mL of water R 2 volumes of water R and 3 volumes of methanol R and dilute
(ท2 mL). to 20 mL with the same mixture of solvents.
Calculate the content of reducing sugars as anhydrous Apply separately to the plate 2 pL of each solution and
glucose or as glucose monohydrate, as appropriate, from thoroughly dry the starting points. Develop over a path of
Table 0209.-1. 15 cm using a mixture of 10 volumes of water R, 15 volumes
of methanol R, 25 volumes of anhydrous acetic acid R and
Table 0209.-1 50 volumes of ethylene chloride R. The volumes of solvents
Volume of 0.1 M Anhydrous glucose
have to be measured accurately since a slight excess of water
Glucose mono
sodium thiosulfate in milligrams hydrate in
produces cloudiness. Dry the plate in a current of warm air.
(ท1- ท1 mL) milligrams Repeat the development immediately, after renewing the
8 19.8 21.6
mobile phase. Dry the plate in a current of warm air and
spray evenly with a solution of 0.5 g of thymol R in a mixture
9 22.4 24.5 of 5 mL of sulfuric acid R and 95 mL of alcohol R. Heat at
10 25.0 27.2 130 °C for 10 min. The principal spot in the chromatogram
ท 27.6
obtained with the test solution is similar in position, colour
30.2
and size to the principal spot in the chromatogram obtained
12 30.3 33.1 with reference solution (a). The test is not valid unless the
13 33.0 36.1 chromatogram obtained with reference solution (b) show's 4
14 35.7
clearly separated spots.
39.0
B. To 2 mL add 5 mL of cupri-citric solution R. Heat to
15 38.3 42.1
boiling. An orange precipitate is formed and the solution
16 41.3 45.2 becomes yellow.
c. To 2 mL add 3 mL of water R. The solution gives the
reaction of citrates (2.5.7).
STORAGE
D. 1 mL gives reaction (b) of phosphates (2.3.1).
Store in an airtight, tamper-proof container, protected from
E. 0.5 mL gives reaction (b) of sodium (2.3.1).
light.
2016 Blood-related Products IV-485
TESTS the column reservoir, washing the beaker 3 times with a few
pH {2.2.3) millilitres of de-ionised water R. Allow the solution to run
The pH of the solution is 5.3 to 5.9. through the column at a flow rate of 12-14 mL/min and
Hydroxymethylfurfural collect the eluate. Wash the column with 2 quantities, each
To 2.0 mL add 5.0 mL of a 100 g/L solution of p-toluidine R of 30 mL, and with one quantity of 50 mL, of de-ionised
m 2-pr°Pau°l K containing 10 per cent P717 of glacial acetic water R. The column can be used for 3 successive
acid R and 1.0 mL of a 5 g/L solution of barbituric acid R. determinations before regeneration with 3 times its volume of
The absorbance (2.2.25), determined at 550 nm after dilute hydrochloric acid R. Titrate the combined eluate and
allowing the mixture to stand for 2 min to 3 min, is not washings (about 150 mL) with 0.2 M sodium hydroxide, using
greater than that of a standard prepared at the same time in 0.1 mL of phenolphthalein solution Rl as indicator.
the same manner using 2.0 mL of a solution containing Calculate the content of sodium citrate in grams per litre
5 ppm of hydroxymethylfurfural R. from the following expressions:
Sterility (2.6.1)
They comply with the test for sterility. 1.961n - 1.257P - 1.40C
Pyrogens {2.6.8)
They comply with the test for pyrogens. Dilute with a 1.961n- 1.257P- 1.53๕
pyrogen-free, 9 g/L solution of sodium chloride R to obtain a
solution containing approximately 5 g/L of sodium citrate. ท = number of millilitres of 0.2 M sodium hydroxide
Inject 10 mL of the diluted solution per kilogram of the used in the titration,
rabbit's mass. p — content of sodium dihydrogen phosphate dihydrate
ASSAY in grams per litre determined as prescribed above,
c = content of citric acid monohydrate in grams per
Sodium dihydrogen phosphate
litre determined as prescribed above,
Dilute 10.0 mL to 100.0 mL with water R. To 10.0 mL of
c = content of anhydrous citric acid in grams per litre
this solution add 10.0 mL of nitro-molybdovanadic reagent R.
determined as prescribed above.
Mix and allow to stand at 20 °C to 25 °C for 30 min. At the
same time and in the same manner, prepare a reference Reducing sugars
solution using 10.0 mL of a standard solution containing Dilute 5.0 mL to 100.0 mL with water R. Introduce 25.0 mL
0.219 g of potassium dihydrogen phosphate R per litre. Measure of the solution into a 250 mL conical flask with ground-glass
the absorbance (2.2.25) of the 2 solutions at 450 nm using as neck and add 25.0 mL of cupri-citric solution Rl. Add a few
the compensation liquid a solution prepared in the same pieces of porous material, attach a reflux condenser, heat so
manner using 10 mL of zuater R. Calculate the content of that boiling begins within 2 min and bod for exactly 10 min.
sodium dihydrogen phosphate dihydrate {P) in grams per Cool and add 3 g of potassium iodide R dissolved in 3 mL of
litre from the expression: water R. Add 25 mL of a 25 per cent mini solution of sulfuric
acid R with caution and in small quantities. Titrate with
11.46 X c X Al 0.1 M sodium thiosulfate using 0.5 mL of starch solution R,
A2 added towards the end of the titration, as indicator (ท1 mL).
Carry out a blank titration using 25.0 mL of water R
c = concentration of potassium dihydrogen phosphate R {ท2 mL).
in the standard solution in grams per litre, Calculate the content of reducing sugars as anhydrous
A1 = absorbance of the test solution, glucose or as glucose monohydrate, as appropriate, from
A2 = absorbance of the reference solution. Table 0209.-1.
Citric acid STORAGE
To 20.0 mL add 0.1 mL of phenolphthalein solution Rl and Store in an airtight, tamper-proof container, protected from
titrate with 0.2 M sodium hydroxide. light.
Calculate the content of citric acid monohydrate (Q, or LABELLING
anhydrous citric acid {C ), in grams per litre from the The label states:
equations: — the composition and volume of the solution,
— the maximum amount of blood to be collected in the
c = 0.7005n - 0.4490P container.
_______________________________________________________________ Ph Eur
c = 0.6404n - 0.4105P
ท = number of millilitres of 0.2 M sodium hydroxide used

in the titration,
p ะ= content of sodium dihydrogen phosphate dihydrate
in grams per litre determined as prescribed above.
Plasma for Fractionation ★ **
(Human Plasma for Fractionation, ** *
Sodium citrate
Prepare a chromatography column 0.10 m long and 10 mm
Ph Elf________________________________________________________________
in internal diameter and filled with strongly acidic ion-exchange
resin R (300 pm to 840 |im). Maintain a 1 cm layer of liquid DEFINITION
above the resin at all times. Wash the column with 50 mL of Liquid part of human blood remaining after separation of the
de-ionised water R at a flow rate of 12-14 mL/min. cellular elements from blood collected in a receptacle
Dilute 10.0 mL of the solution to be examined to about containing an anticoagulant, or separated by continuous
40 mL with de-ionised water R in a beaker and transfer to filtration or centrifugation of anticoagulated blood in an
apheresis procedure; it is intended for the manufacture of When obtained by plasmapheresis or from whole blood (after
plasma-derived products. separation from cellular elements), plasma intended for the
PRODUCTION recovery of proteins that are labile in plasma is frozen within
DONORS 24 h of collection by cooling rapidly in conditions validated
to ensure that a temperature of -25 °C or below is attained
Only a carefully selected, healthy donor who, as far as can be
at the core of each plasma unit within 12 h of placing in the
ascertained after medical examination, laboratory blood tests
freezing apparatus.
and a study of the donor's medical history, is free from
detectable agents of infection transmissible by plasma-derived When obtained by plasmapheresis, plasma intended solely for
products may be used. Recommendations in this field are the recovery' of proteins that are not labile in plasma is frozen
made by the Council of Europe [Recommendation by cooling rapidly in a chamber at -20 °C or below within
No. R (95) 15 on the preparation, use and quality assurance of 24 h of collection.
blood components, or subsequent revision]; a directive of the When obtained from whole blood, plasma intended solely for
European Union also deals with the matter: Commission the recovery of proteins that are not labile in plasma is
Directive 2004I33IEC of 22 March 2004 implementing separated from cellular elements and frozen in a chamber at
Directive 2002I98IEC of the European Parliament and of the -20 cc or below within 72 h of collection.
Council as regards certain technical requirements for blood and It is not intended that the determination of total protein and
blood components. human coagulation factor VIII shown below be carried out on
Immunisation of donors each unit of plasma. They are rather given as guidelines for good
Immunisation of donors to obtain immunoglobulins with manufacturing practice, the test for human coagulation factor VIII
specific activities may be carried out when sufficient supplies being relevant for plasma intended for use in the preparation of
of material of suitable quality cannot be obtained from concentrates of labile proteins.
naturally immunised donors. Recommendations for such The total protein content of a unit of plasma depends on the serum
immunisations are formulated by the World Health protein content of the donor and the degree of dilution inherent in
Organization {Requirements for the collection, processing and the donation procedure. When plasma is obtained from a suitable
quality control of blood, blood components and plasma derivatives, donor and using the intended proportion of anticoagulant solution,
WHO Technical Report Series, No. 840, 1994 or subsequent a total protein content complying with the limit of 50 g/L is
revision). obtained. If a volume of blood or plasma smaller than intended IS
Records collected into the anticoagulant solution, the resulting plasma is not
Records of donors and donations made are kept in such a necessarily unsuitable for pooling for fractionation. The aim of
way that, while maintaining the required degree of good manufacturing practice must be to achieve the prescribed limit
confidentiality concerning the donor's identity, the origin of for all normal donations.
each donation in a plasma pool and the results of the Preservation of human coagulation factor VIII in the donation
corresponding acceptance procedures and laboratory tests depends on the collection procedure and the subsequent handling of
can be traced. the blood and plasma. With good practice, 0.7 HJ/mL can usually
Laboratory tests be achieved, but units of plasma with a lower activity may still be
Laboratory tests are carried out for each donation to detect suitable for use in the production of coagulation factor
the following viral markers: concentrates. The aim of all steps taken during production of
plasma is to obtain plasma of the intended quality and to conserve
1. antibodies against human immunodeficiency virus 1
labile proteins as much as possible.
(anti-HIV-1);
Total protein
2. antibodies against human immunodeficiency virus 2
Carry out the test using a pool of not fewer than 10 units.
(anti-HIV-2);
Dilute an appropriate volume of the preparation with a 9 gfL
3. hepatitis B surface antigen (HBsAg); solution of sodium chloride R to obtain a solution containing
4. antibodies against hepatitis c virus (anti-HCV). about 15 mg of protein in 2 mL. To 2.0 mL of this solution
The test methods used are of suitable sensitivity and in a round-bottomed centrifuge tube, add 2 mL of a 75 g/L
specificity and comply with the regulations in force. If a solution of sodium molybdate R and 2 mL of a mixture of
repeat-reactive result is found in any of these tests, the 1 volume of nitrogen-free sulfuric acid R and 30 volumes of
donation is not accepted. water R. Shake, centrifuge for 5 min, decant the supernatant
and allow the inverted tube to drain on filter paper.
INDIVIDUAL PLASMA UNITS
Determine the nitrogen in the residue by the method of
The plasma is prepared by a method that removes cells and sulfuric acid digestion (2.5.9) and calculate the protein
cell debris as completely as possible. Whether prepared from content by multiplying the quantity of nitrogen by 6.25.
whole blood or by plasmapheresis, the plasma is separated
The total protein content is not less than 50 g/L.
from the cells by a method designed to prevent the
introduction of micro-organisms. No antibacterial or Human coagulation factor vni (2.7.4)
antifungal agent is added to the plasma. The containers Carry out the test using a pool of not fewer than 10 units.
comply with the requirements for glass containers {3.2.1) or Thaw the samples to be examined, if necessary, at 37 °C.
for plastic containers for blood and blood components Carry out the assay using a reference plasma calibrated
{3.2.3). The containers are closed so as to prevent any against the International Standard for human coagulation
possibility of contamination. factor VIII in plasma. The activity is not less than
0.7 lU/mL.
If 2 or more units are pooled prior to freezing, the operations
are carried out using sterile connecting devices or under STORAGE AND TRANSPORT
aseptic conditions and using containers that have not Frozen plasma is stored and transported in conditions
previously been used. designed to maintain the temperature at or below -20 °C;
for accidental reasons, the storage temperature may rise
above -20 °C on one or more occasions during storage and Hepatitis A virus RNA
transport but the plasma is nevertheless considered suitable The plasma pool is tested using a validated nucleic acid
for fractionation if all the following conditions are fulfilled: amplification technique (2.6.2/). A positive control with
the total period of time during which the temperature 1.0 X 102 IU of hepatitis A virus RNA per millilitre and, to
exceeds -20 °C does not exceed 72 h; test for inhibitors, an internal control prepared by addition of
the temperature does not exceed —15 cc on more than a suitable marker to a sample of the plasma pool are included
1 occasion; in the test. The test is invalid if the positive control is non-
— the temperature at no time exceeds -5 °C. reactive or if the result obtained with the internal control
POOLED PLASMA indicates the presence of inhibitors. The pool complies with
During the manufacture of plasma products, the first the test if it is found non-reactive for hepatitis A virus RNA.
homogeneous pool of plasma (for example, after removal of Hepatitis c virus RNA
cryoprecipitate) is tested for HBsAg and for HIV antibodies The plasma pool is tested using a validated nucleic acid
using test methods of suitable sensitivity and specificity; amplification technique (2.6.21). A positive control with
the pool must give negative results in these tests. 1.0 X 102 IU of hepatitis c virus RNA per millilitre and, to
The plasma pool is also tested for hepatitis c virus RNA test for inhibitors, an internal control prepared by addition of
using a validated nucleic acid amplification technique a suitable marker to a sample of the plasma pool are included
(2.6.27). A positive control with 100 ILJ/mL of hepatitis c in the test. The test is invalid if the positive control is non-
virus RNA and, to test for inhibitors, an internal control reactive or if the result obtained with the internal control
prepared by addition of a suitable marker to a sample of the indicates the presence of inhibitors. The pool complies with
plasma pool are included in the test. The test is invalid if the the test if it is found non-reactive for hepatitis c virus RNA.
positive control is non-reactive or if the result obtained with Hepatitis c virus RNA for NAT testing BRP is suitable for use
the internal control indicates the presence of inhibitors. as a positive control.
The plasma pool complies with the test if it is found non- Hepatitis E virus RNA
reactive for hepatitis c virus RNA. The plasma pool is tested using a validated nucleic acid
Hepatitis c virus RNA for NAT testing BRP is suitable for use amplification technique (2.6.2/). A positive control with
as a positive control. 3.2 X 102 IU of hepatitis E virus RNA per millilitre and, to
test for inhibitors, an internal control prepared by addition of
CHARACTERS
a suitable marker to a sample of the plasma pool are included
Before freezing: clear or slightly turbid liquid without visible
in the test. The test is invalid if the positive control is non-
signs of haemolysis; it may vary in colour from light yellow to
reactive or if the result obtained with the internal control
green.
indicates the presence of inhibitors. The pool complies with
LABELLING the test if it is found non-reactive for hepatitis E virus RNA.
The label enables each individual unit to be traced to a B19 virus DNA
specific donor. The plasma pool contains not more than 10.0 IU/|1L.
To limit the potential burden of B19 virus in plasma pools,
the plasma pool is also tested for Bl9 virus using a validated
nucleic acid amplification technique (2.6.2/). A positive
control with 10.0 IU of B19 virus DNA per microlitre and,
to test for inhibitors, an internal control prepared by addition
Plasma (Pooled and Treated for ***** of a suitable marker to a sample of the plasma pool are
Virus Inactivation) ***** included in the test. The test is invalid if the positive control
(Human Plasma (Pooled and Treated for Virus is non-reactive or if the result obtained with the internal
Inactivation)> Ph Eur monograph 1646) control indicates the presence of inhibitors.
Ph Etf ____________________________________________________________
B19 virus DNA for NAT testing BRP is suitable for use as a
positive control.
DEFINITION
METHOD OF PREPARATION
Sterile, frozen or freeze-dried, non-pyrogenic preparation
The method of preparation is designed to minimise activation
obtained from human plasma derived from donors belonging
of any coagulation factor (to minimise potential
to the same ABO blood group. The preparation is thawed or
thrombogenicity) and includes a step or steps that have been
reconstituted before use to give a solution for infusion.
shown to inactivate known agents of infection; if substances
The human plasma used complies with the monograph are used for the inactivation of viruses during production, the
Human plasma for fractionation (0853). subsequent purification procedure must be validated to
PRODUCTION demonstrate that the concentration of these substances is
The units of plasma to be used are cooled to -30 °C or reduced to a suitable level and that any residues are such as
lower within 6 h of separation of cells and always within 24 h not to compromise the safety of the preparation for patients.
of collection. Inactivation process
The pool is prepared by mixing units of plasma belonging to The solvent-detergent process, which is one of the methods
the same ABO blood group. used to inactivate enveloped viruses, uses treatment with a
combination of tributyl phosphate and octoxinol 10; these
PLASMA POOL TESTS
reagents are subsequently removed by oil extraction or by
The pool of plasma is tested for hepatitis B surface antigen solid phase extraction so that the amount in the final product
(HBsAg) and for HIV antibodies using test methods of is less than 2 pg/mL for tributyl phosphate and less than
suitable sensitivity and specificity; the pool must give negative 5 pg/mL for octoxinol 10.
results in these tests.
No antimicrobial preservative is added.
The solution is passed through a bacteria-retentive filter, Test solution Dilute the preparation to be examined with an
distributed aseptically into the final containers and equal volume of a 9 g/L solution of sodium chloride R. Filter
immediately frozen; it may subsequently be freeze-dried. through a membrane filter (nominal pore size 0.45 pm).
Plastic containers comply with the requirements for sterile Reference solution Dissolve 0.300 g of sodium citrate R in
plastic containers for human blood and blood components water R and dilute to 100.0 mL with the same solvent.
(S.2.3). Column:
Glass containers comply with the requirements for glass — size: / = 0.3 m, 0 = 7.8 mm;
containers for pharmaceutical use {3.2.1). — stationary phase: cation-exchange resin R (9 pm).
CHARACTERS Mobile phase 0.51 g/L solution of sulfuric acid R.
Appearance Flow rate 0.5 mUmin.
— frozen preparation', clear or slightly opalescent liquid, free Detection Spectrophotometer at 215 nm.
from solid and gelatinous particles after thawing. Equilibration 15 min.
— freeze-dried preparation: almost white or slightly yellow
Injection 10 pL.
powder or friable solid mass.
Retention time Citrate = about 10 min.
Thaw or reconstitute the preparation to be examined as stated on
the label immediately before carrying out the identification, tests Limit:
and assay. — citrate: maximum 25 mmol/L.
IDENTIFICATION Calcium
Maximum 5.0 mmol/L.
A. Examine by electrophoresis {2.2.31) comparing with
normal human plasma. The electropherograms show the Atomic absorption spectrometry {2.2.23, Method I).
same bands. Source Calcium hollow-cathode lamp using a transmission
B. It complies with the test for anti-A and anti-B band preferably of 0.5 nm.
haemagglutinins (see Tests). Wavelength 622 nm.
TESTS Atomisation device Air-acetylene or acetylene-propane flame.
pH {2.2.3) Potassium
6.5 to 7.6. Maximum 5.0 mmol/L.
Osmolality {2.2.35) Atomic emission spectrometry {2.2.22, Method I).
Minimum 240 mosmol/kg. Wavelength 766.5 nm.
Total protein Sodium
Minimum 45 g/L. Maximum 200 mmol/L.
Dilute if necessary with a 9 g/L solution of sodium chloride R Atomic emission spectrometry {2.2.22, Method I).
to obtain a protein concentration of about 7.5 mg/mL. Place Wavelength 589 nm.
2.0 mL of this solution in a round-bottomed centrifuge tube
Water
and add 2 mL of a 75 g/L solution of sodium molybdate R and
Determined by a suitable method, such as the semi-micro
2 mL of a mixture of 1 volume of nitrogen-free sulfuric acid R
determination of water {2.5.12), loss on drying {2.2.32) or
and 30 volumes of water R. Shake, centrifuge for 5 min,
near-infrared spectroscopy {2.2.40), the water content is
decant the supernatant and allow the inverted tube to drain
within the limits approved by the competent authority
on filter paper. Determine the nitrogen in the residue by the
(freeze-dried product).
method of sulfuric acid digestion (2.5.9) and calculate the
quantity of protein by multiplying the result by 6.25. Sterility {2.6.1)
Activated coagulation factors (2.6.22)
It complies with the test for activated coagulation factors. Pyrogens {2.6.8) or Bacterial endotoxins {2.6.14)
Carry out the test with 0.1 mL of the preparation to be It complies with the test for pyrogens or, preferably and
examined instead of 10-fold and 100-fold dilutions. where justified and authorised, with a validated in vitro test
The coagulation time for the preparation to be examined is such as the bacterial endotoxin test.
not less than 150 ร. For the pyrogen test, inject 3 mL per kilogram of the rabbit's
Anti-A and anti-B haemagglutinins {2.6.20, Method A) mass.
The presence of haemagglutinins (anti-A or anti-B) Where the bacterial endotoxin test is used, the preparation to
corresponds to the blood group stated on the label. be examined contains less than 0.1 IU of endotoxin per
Hepatitis A virus antibodies millilitre.
Minimum 1.0 IU/mL, determined by a suitable ASSAY
immunochemical method {2.7.1). Assay of human coagulation factor VIII {2.7.4)
Human hepatitis A immunoglobulin BRP is suitable for use as a Use a reference plasma calibrated against the International
reference preparation. Standard for blood coagulation factor vni in plasma.
Irregular erythrocyte antibodies The estimated potency is not less than 0.5 IU/mL.
The preparation to be examined does not show the presence The confidence limits {P = 0.95) are not less than
of irregular erythrocyte antibodies when examined without 80 per cent and not more than 120 per cent of the estimated
dilution by an indirect antiglobulin test. potency.
Citrate Assay of human coagulation factor V
Liquid chromatography (2.2.29). Carry out the assay of human coagulation factor V described
below using a reference plasma calibrated against the
2016 Blood-related Products rV-489
International Standard for blood coagulation factor V in preparation to be examined and 0.1 mL of a suitable APTT
plasma. reagent (containing phospholipid and contact activator), both
Using imidazole buffer solution pH 7.3 R, prepare at least previously heated to 37 °C, and incubate the mixture for a
3 twofold dilutions of the preparation to be examined, recommended time at 37 °C. To each tube add 0.1 mL of a
preferably in duplicate, from 1 in 10 to 1 in 40. Test each 3.7 g/L solution of calcium chloride R previously heated to
dilution as follows: mix 1 volume of plasma substrate deficient 37 °C. Using a timer, measure the coagulation time,
III factor V R, 1 volume of the dilution to be examined, i.e. the interval between the moment of the addition of the
1 volume of thromboplastin R and 1 volume of a 3.5 g/L calcium chloride and the 1st indication of the formation of
solution of calcium chloride R-) measure the coagulation times, fibrin, which may be observed visually or by means of a
I.e. the interval between the moment at which the calcium suitable apparatus. The volumes given above may be adapted
chloride solution is added and the 1st indication of the to the APTT reagent and apparatus used. The coagulation
formation of fibrin, which may be observed visually or by time complies with the approved specification for the
means of a suitable apparatus. product.
In the same manner, determine the coagulation time of LABELLING
4 twofold dilutions (1 in 10 to 1 in 80) of human normal The label states:
plasma in imidazole buffer solution pH 7.3 R. — the ABO blood group;
Check the validity of the assay and calculate the potency of — the method used for virus inactivation.
the test preparation by the usual statistical methods ______________________________________________________________ Ph Eur
(for example, 5.3).
The estimated potency is not less than 0.5 โบ/mL.
The confidence limits (P = 0.95) are not less than
80 per cent and not more than 120 per cent of the estimated
potency. Albumin Solution * *
Assay of human coagulation factor XI (2.7.22) Albumin; Human Albumin ***
Use a reference plasma calibrated against the International
(Human Albumin Solution, Ph Eur monograph 0255)
Standard for blood coagulation factor XI in plasma.
Ph Eur______________________________________________________________
The estimated potency is not less than 0.5 lU/mL.
The confidence limits (P = 0.95) arc not less than DEFINITION
80 per cent and not more than 125 per cent of the estimated Sterile liquid preparation of a plasma protein fraction
potency. containing human albumin. It is obtained from plasma that
Coagulation factors V, VIII, XI and XIII plasma BRP is complies with the monograph Human plasma for fractionation
suitable for use as a reference preparation in the above (0853). The preparation may contain excipients such as
assays. sodium caprylate (sodium octanoate) or N-acetyltryptophan
or a combination of the two.
Assay of human protein c (2.7.30)
Use a reference plasma calibrated against the International PRODUCTION
Standard for human protein c in plasma. Separation of the albumin is carried out under controlled
The estimated potency is not less ±an 0.7 lU/mL. conditions, particularly of pH, ionic strength and temperature
The confidence limits (P = 0.95) are not less than so that in the final product not less than 95 per cent of the
80 per cent and not more than 120 per cent of the estimated total protein is albumin. Human albumin solution is prepared
potency. as a concentrated solution containing 150-250 g/L of total
protein or as an isotonic solution containing 35-50 g/L of
Assay of human protein ร (2.7.31)
total protein. No antimicrobial preservative or antibiotic is
added. The solution is passed through a bacteria-retentive
Standard for human protein ร in plasma.
filter and distributed aseptically into sterile containers which
The estimated potency is within the limits approved for the are then closed so as to prevent contamination. The solution
particular product. The confidence limits (P = 0.95) are not in its final container is heated to 60 ± 1.0 °C and
less than 80 per cent and not more than 120 per cent of the maintained at this temperature for not less than 10 h.
estimated potency. The containers are then incubated at 30-32 °C for not less
Assay of human plasmin inhibitor (2.7.25) than 14 days or at 20-25 °C for not less than 4 weeks and
(a2-antiplasmin) examined visually for evidence of microbial contamination.
Use a reference plasma calibrated against human normal CHARACTERS
plasma. Appearance
1 unit of human plasmin inhibitor is equal to the activity of Clear, slightly viscous liquid, almost colourless, yellow, amber
1 mL of human normal plasma. Human normal plasma is or green.
prepared by pooling plasma units from not fewer than IDENTIFICATION
30 donors and storing at -30 °C or lower. Examine by a suitable immunoelectrophoresis technique.
The estimated potency is not less than 0.2 units/mL. Using antiserum to normal human serum, compare normal
The confidence limits (P = 0.95) are not less than human serum and the preparation to be examined, both
80 per cent and not more than 120 per cent of the estimated diluted to contain 10 g/L of protein. The main component of
potency. the preparation to be examined corresponds to the main
Activated partial thromboplastin time (APTT) component of normal human serum. The preparation may
Use an apparatus suitable for measurement of coagulation show the presence of small quantities of other plasma
times or perform the assay with incubation tubes maintained proteins.
in a water-bath at 37 °C. Place in each tube 0.1 mL of the
TESTS range of 4-12 g/L and injection of 50-600 pg of protein are

pH (2.2.3) usually suitable.
6.7 to 7.3. Column'.
Dilute the preparation to be examined with a 9 g/L solution — size'. I = 0.6 m, 0 = 7.5 mm, or I = 0.3 m, 0 = 7.8 mm;
of sodium chloride R to obtain a solution containing 10 g/L of — stationary phase', hydrophilic silica gel for chromatography R,
protein. of a grade suitable for fractionation of globular proteins
Total protein with relative molecular masses in the range 10 000 to
If necessary, dilute an accurately measured volume of the 500 000.
preparation to be examined with a 9 g/L solution of sodium Mobile phase Dissolve 4.873 g of disodium hydrogen phosphate
chloride R to obtain a solution containing about 15 mg of dihydrate Ry 1.741 g of sodium dihydrogen phosphate
protein in 2 mL. To 2.0 mL of this solution in a round- monohydrate R, 11.688 g of sodium chloride R and 50 mg of
bottomed centrifuge tube add 2 mL of a 75 g/L solution of sodium azide R in 1 L of tuater R.
sodium molybdate R and 2 mL of a mixture of 1 volume of Flow rate 0.5 mUmin.
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake, Detection Spectrophotometer at 280 nm.
centrifuge for 5 min, decant the supernatant and allow the
The peak due to polymers and aggregates is located in the
inverted tube to drain on filter paper. Determine the nitrogen
part of the chromatogram representing the void volume.
in the residue by the method of sulfuric acid digestion (2.5.9) Disregard the peak due to the stabiliser. The area of the peak
and calculate the quantity of protein by multiplying by 6.25.
due to polymers and aggregates is not greater than
The protein content is not less than 95 per cent and not
10 per cent of the total area of the chromatogram. This
more than 105 per cent of the stated content.
represents not more than 5 per cent when expressed in
Protein composition percentage of protein considering the difference in response
Zone electrophoresis (2.2.31). factor between the albumin monomer and the polymers and
Use strips of suitable cellulose acetate gel or agarose gel as aggregates.
the supporting medium and barbital buffer solution pH 8.6 Rl Haem
as the electrolyte solution. Dilute the preparation to be examined using a 9 g/L solution
If cellulose acetate is the supporting material, the method of sodium chloride R to obtain a solution containing 10 g/L of
described below can be used. If agarose gels are used, and protein. The absorbance (2.2.25) of the solution measured at
because they are normally part of an automated system, the 403 nm using water R as the compensation liquid is not
manufacturer'ร instructions are followed instead. greater than 0.15.
Test solution Dilute the preparation to be examined with a Prekallikrein activator (2.6.15)
9 g/L solution of sodium chloride R to a protein concentration Maximum 35 lU/mL.
of 20 g/L. Aluminium
Reference solution Dilute human albumin for electrophoresis BRP Maximum 200 pg/L.
with a 9 g/L solution of sodium chloride 7? to a protein Atomic absorption spectrometry (2.2.23, Method I or II).
concentration of 20 g/L.
Use a furnace as atomic generator.
To a strip apply 2.5 pL of the test solution as a 10 mm band
Use plastic containers for preparation of the solutions and use
or apply 0.25 pL per millimetre if a narrower strip is used.
plastic equipment where possible. Wash glassware (or equipment)
To another strip, apply in the same manner the same volume
in nitric acid (200 g/L HNOtJ before use.
of the reference solution. Apply a suitable electric field such
that the most rapid band migrates at least 30 mm. Treat the Test solution Use the preparation to be examined, diluted if
strips with amido black 10B solution R for 5 min. Decolorise necessary.
wi± a mixture of 10 volumes of glacial acetic acid R and Reference solutions Prepare at least 3 reference solutions in a
90 volumes of methanol R until the background is just free of range spanning the expected aluminium concentration of the
colour. Develop the transparency of the strips with a mixture test solution, for example by diluting aluminium standard
of 19 volumes of glacial acetic acid R and 81 volumes of solution (10 ppm Al) R with a 1 g/L solution of octoxinol 10 R.
methanol R. Measure the absorbance of ±e bands at 600 nm Monitor solution Add aluminium standard solution
in an instrument having a linear response over the range of (10 ppm Al) Rota suitable certified reference material to the
measurement. Calculate the result as the mean of test solution in a sufficient amount to increase the aluminium
3 measurements of each strip. concentration by 20 pg/L.
System suitability In the electropherogram obtained with the Blank solution 1 g/L solution of octoxinol 10 R.
reference solution on cellulose acetate or on agarose gels, the Wavelength 309.3 nm or other suitable wavelength.
proportion of protein in the principal band is within the
Slit width 0.5 nm.
limits stated in the leaflet accompanying the reference
preparation. Tube Pyrolytically coated, with integrated platform.
Results In the electropherogram obtained with the test Background corrector Off.
solution on cellulose acetate or on agarose gels, not more Awmisation device Furnace; fire between readings.
than 5 per cent of the protein has a mobility different from The operating conditions in Table 0255.-1 are cited as an
that of the principal band. example of conditions found suitable for a given apparatus;
Molecular-size distribution they may be modified to obtain optimum conditions.
Size exclusion chromatography (2.2.30).
Test solution Dilute the preparation to be examined with a
9 g/L solution of sodium chloride R to a concentration suitable
for the chromatographic system used. A concentration in the
2016 Blood-related Products LV-491
Table 0255.-1. - operating conditions found suitable, cited as — that the product is not to be used if it is cloudy or if a
.________ ____________ an exampte____________________ deposit has formed;
Step Final Ramp time Hold time Gas — the name and quantity of any added substance.
temperature (ร) (ร)
- (°C) ____ PhEur
1 120 10 80 argon
2 200 5 20 argon
3 650 5 10 argon
4 1300 5 10 argon Antithrombin III Concentrate
5 1300 1 10 no gas (Human Antithrombin III Concentrate, *★*
6 2500 0.7 4 no gas Ph Eur monograph 0878)
7 2600 0.5 3 argon
Action and use
8 20 12.9 3 no gas Anticoagulant factor.
PhEur______________________________________________________________
Injection Each of the following solutions 3 times: blank DEFINITION

solution, reference solutions, test solution and monitor Sterile, freeze-dried preparation of a plasma glycoprotein
solution. fraction that inactivates thrombin in the presence of an excess
System suitability: of heparin. It is obtained from human plasma ±at complies
the recovery of aluminium added in preparation of the with ±e monograph on Human plasma for
monitor solution is within the range 80-120 per cent. fractionation (0853). The preparation may contain excipients
Prepare a cabbration curve from the mean of the readings such as stabilisers.
obtained with the reference solutions and determine the When reconstituted in the volume of solvent stated on the
aluminium content of the preparation to be examined using label, the potency is not less than 25 IU of antithrombin m
the calibration curve. per millilitre.
Potassium PRODUCTION
Maximum 0.05 mmol of K per gram of protein. The method of preparation is designed to maintain
Atomic emission spectrometry (2.2.22, Method P). functional integrity of antithrombin rn. It includes a step or
Wavelength 766.5 nm. steps that have been shown to remove or to inactivate known
Sodium agents of infection; if substances are used for inactivation of
viruses during production, the subsequent purification
Maximum 160 mmol/L and 95 per cent to 105 per cent of
procedure must be validated to demonstrate that the
the content of Na stated on the label.
concentration of these substances is reduced to a suitable
Atomic emission spectrometry' (2.2.22, Method I). level and any residues are such as not to compromise the
Wavelength 589 nm. safety of the preparation for patients.
Sterility (2.6.7) The specific activity is not less than 3 IU of antithrombin HI
It complies with the test. per milligram of total protein, excluding albumin.
Pyrogens (2.6.5) or Bacterial endotoxins (2.6.14) The antithrombin in is purified and concentrated.
It complies with the test for pyrogens or, preferably and No antimicrobial preservative or antibiotic is added.
where justified and authorised, with a validated in vitro test The antithrombin in concentrate is passed through a
such as the bacterial endotoxin test. bacteria-retentive filter, distributed aseptically into its final,
For the pyrogen test, for a solution with a protein content of sterile containers and immediately frozen. It is then freeze-
35-50 g/L, inject 10 mL per kilogram of the rabbit’s mass; dried and the containers are closed under vacuum or in an
for a solution with a protein content of 150-250 g/L, inject atmosphere of inert gas.
5 mL per kilogram of the rabbit’s mass. It shall be demonstrated that the manufacturing process
Where the bacterial endotoxin test is used, the preparation to yields a product with a consistent fraction of antithrombin in
be examined contains less than 0.5 ru of endotoxin per able to bind to heparin. It is evaluated by a suitable analytical
millilitre for solutions with a protein content not greater than procedure which is determined during process development,
50 g/L, less than 1.3 IU of endotoxin per millilitre for such as:
solutions with a protein content greater than 50 g/L but not Heparin-binding fraction Examine by agarose gel
greater than 200 g/L, and less than 1.7 IU of endotoxin per electrophoresis (2.2.31). Prepare a 10 g/L solution of agarose
millilitre for solutions with a protein content greater than for electrophoresis R containing 15 IU of heparin R per millilitre
200 g/L but not greater than 250 g/L. in barbital buffer solution pH 8.4 R. Pour 5 mL of this solution
onto a glass plate 5 cm square. Cool at 4 °C for 30 min.
STORAGE Cut 2 wells 2 mm in diameter 1 cm and 4 cm from the side
Protected from light. of the plate and 1 cm from the cathode. Introduce into one
LABELLING well 5 pL of the preparation to be examined, diluted to an
The label states: activity of about 1 IU of antithrombin in per millilitre.
— the name of the preparation; Introduce into the other well 5 pL of a solution of a marker
— the volume of the preparation; dye such as bromophenol blue R. Allow the electrophoresis to
— the content of protein expressed in grams per litre; proceed at 4 °C, using a constant electric field of 7 v/cm,
— the content of sodium expressed in millimoles per litre; until the dye reaches the anode.
Cut across the agarose gel 1.5 cm from that side of the plate Water
on which the preparation to be examined was applied and Determined by a suitable method, such as semi-micro
remove the larger portion of the gel leaving a band 1.5 cm determination of water (2.5.12), loss on drying (2.2.82) or
wide containing the material to be examined. Replace the near-infrared spectroscopy (2.2.40), the water content is
removed portion with an even layer consisting of 3.5 mL of a within the limits approved by the competent authority.
10 g/L solution of agarose for electrophoresis R in barbital buffer Sterility (2.6.1)
solution pH 8.4 R, containing a rabbit anti-human It complies with the test.
antithrombin in antiserum at a suitable concentration,
previously determined, to give adequate peak heights of at Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
least 1.5 cm. Place the plate with the original gel at the It complies with the test for pyrogens or, preferably and
cathode so that a 2nd electrophoretic migration can occur at where justified and authorised, with a validated in vitro test
right angles to the 1st. Allow this 2nd electrophoresis to such as the bacterial endotoxin test.
proceed using a constant electric field of 2 v/cm for 16 h. For the pyrogen test, inject per kilogram of the rabbit’s mass
Cover the plates with filter paper and several layers of thick a volume equivalent to 50 IU of antithrombin m.
lint soaked in a 9 g/L solution of sodium chloride R and Where the bacterial endotoxin test is used, the preparation to
compress for 2 h, renewing the saline several times. Rinse be examined contains less than 0.1 IU of endotoxin per
with water R, dry’ the plates and stain with acid blue 92 International Unit of antithrombin in.
solution R.
ASSAY
Calculate ±e fraction of antithrombin in bound to heparin, Human antithrombin III (2.7.17)
which is the peak closest to the anode, with respect to the The estimated potency is not less than 80 per cent and not
total amount of antithrombin in, by measuring the area more than 120 per cent of the stated potency.
defined by the 2 precipitation peaks. The confidence limits (P = 0.95) are not less than
The fraction of antithrombin in able to bind to heparin is 90 per cent and not more than 110 per cent of the estimated
not less than 60 per cent. potency.
CHARACTERS STORAGE
Appearance Protected from light, in an airtight container.
White or almost white, hygroscopic, friable solid or powder.
LABELLING
Recojistitute the preparation to be examined as stated on the label The label states:
immediately before carrying out the identification, tests (except — the number of International Units of antithrombin Ul in
those for solubility, total protein and water) and assay. the container;
IDENTIFICATION — the name and volume of the liquid to be used for
It complies with the limits of the assay. reconstitution;
— where applicable, the amount of albumin added as a
TESTS
stabiliser.
Solubility
_____________________________________________________ __________Ph fJ
To a container of the preparation to be examined add the
volume of liquid stated on the label at the recommended
temperature. The preparation dissolves completely under
gentle swirling within 10 min in the volume of the solvent
stated on the label, forming a clear or slightly turbid,
colourless or almost colourless solution. Dried Factor VII Fraction f **
pH (2.2.8) (Human Coagulation Factor VII,

6.0 to 7.5. Ph Eur monograph 1224)
Osmolality (2.2.85)
Action and use
Minimum 240 mosmol/kg.
Coagulation factor vn substitute.
Total protein
If necessary, dilute an accurately measured volume of the Ph Eur_________________________________________________ _ ____________
reconstituted preparation to obtain a soludon containing DEFINITION

about 15 mg of protein in 2 mL. To 2.0 mL of the solution Sterile, liquid or freeze-dried preparation of a plasma protein
in a round-bottomed centrifuge tube add 2 mL of a 75 g/L fraction containing the single-chain glycoprotein human
solution of sodium molybdate R and 2 mL of a mixture of coagulation factor vn and may also contain small amounts
1 volume of nitrogen-free sulfuric acid R and 30 volumes of of the activated form, the 2-chain derivative human
water R. Shake, centrifuge for 5 min, decant the supernatant coagulation factor VUa. It may also contain human
and allow the inverted tube to drain on filter paper. coagulation factors ท, IX and X, protein c and protein ร.
Determine the nitrogen in the residue by the method of It is obtained from human plasma that complies with the
sulfuric acid digestion (2.5.9) and calculate the amount of monograph on Human plasma for fractionation (0853).
protein by multiplying the result by 6.25. The preparation may contain excipients such as stabilisers,
Heparin (2.7.5) heparin and antithrombin.
Maximum 0.1 IU of heparin per International Unit of The potency of the preparation, reconstituted as stated on
antithrombin HI. the label, is not less than 15 IU of human coagulation
It is necessary to validate the method for assay of heparin for factor vn per millilitre.
each preparation to be examined to allow for interference by
antithrombin in.
PRODUCTION pH (2.2.2)
GENERAL PROVISIONS 6.5 to 7.5.
The method of preparation is designed to maintain Osmolality (2.2.25)
functional integrity of human coagulation factor VII and to Minimum 240 mosmol/kg.
minimise activation of any coagulation factor (to minimise
potential thrombogenicity). It includes a step or steps that Total protein
have been shown to remove or to inactivate known agents of If necessary, dilute an accurately measured volume of the
infection; if substances are used for inactivation of viruses reconstituted preparation with a 9 g/L solution of sodium
during production, the subsequent purification procedure chloride R to obtain a solution containing about 15 mg of
must be validated to demonstrate that the concentration of
protein in 2 mL. To 2.0 mL of the solution in a round-
these substances is reduced to a suitable level and that any bottomed centrifuge tube, add 2 mL of a 75 g/L solution of
residues are such as not to compromise the safety of the sodium molybdate R and 2 mL of a mixture of 1 volume of
preparation for patients. nitrogen-free sulfuric acid R and 30 volumes of water R. Shake,
The specific activity is not less than 2 IU of human inverted tube to drain on filter paper. Determine the nitrogen
coagulation factor VII per milligram of total protein, before in the residue by the method of sulfuric acid digestion (2.5.9)
the addition of any protein stabiliser. and calculate the amount of protein by multiplying the result
The human coagulation factor vn fraction is dissolved in a by 6.25.
suitable liquid. No antimicrobial preservative or antibiotic is
Activated coagulation factors (2.6.22)
For each of the dilutions, the coagulation time is not less
filter, distributed aseptically into the final containers and
than 150 ร.
immediately frozen. It is subsequently freeze-dried and the
containers are closed under vacuum or under an inert gas. Heparin (2.7.12)
If heparin has been added, the preparation to be examined
CONSISTENCY OF THE METHOD OF contains not more than the amount of heparin stated on the
PRODUCTION label and in any case not more than 0.5 IU of heparin per
It shall be demonstrated that the manufacturing process International Unit of human coagulation factor vn.
yields a product with consistent activities of human
Thrombin
coagulation factors II, IX and X, expressed in International
If the preparation to be examined contains heparin,
Units relative to the activity of human coagulation factor VII.
determine the amount present as described in the test for
This is evaluated by suitable analytical procedure(s) that is
heparin and neutralise the heparin by addition of protamine
(are) determined during process development.
sulfate R (10 pg of protamine sulfate neutralises 1 IU of
It shall be demonstrated that the manufacturing process heparin). In each of 2 test-tubes, mix equal volumes of the
yields a product with a consistent activity of human reconstituted preparation and of a 3 g/L solution of
coagulation factor Vila. This is evaluated by suitable fibrinogen R. Keep one of the tubes at 37 °C for 6 h and the
analytical procedure(s) that is (are) determined during other at room temperature for 24 h. In a 3rd tube, mix equal
process development. volumes of the fibrinogen solution and of a solution of
Activity of human coagulation factor Vila human thrombin R (1 IU/mL) and place the tube in a water
It may be determined, for example, using a recombinant bath at 37 °C. No coagulation occurs in the tubes containing
soluble tissue factor that does not activate human coagulation the preparation to be examined. Coagulation occurs within
factor vn but possesses a cofactor function specific for 30 s in the tube containing thrombin.
human coagulation factor Vila; after incubation of a mixture Human coagulation factor II (2.7.18)
of the recombinant soluble tissue factor with phospholipids The estimated content is not more than 125 per cent of the
reagent and the dilution of the test sample in human stated content. The confidence limits (P = 0.95) are not less
coagulation factor VH-deficient plasma, calcium chloride is than 90 per cent and not more than 111 per cent of the
added and the clotting time determined; the clotting time is estimated potency.
inversely related to the human coagulation factor Vila activity
of the test sample.
Human coagulation factor IX (2.7.11)
The estimated content is not more than 125 per cent of the
CHARACTERS stated content. The confidence limits (p = 0.95) are not less
Appearance ±an 80 per cent and not more than 125 per cent of the
White or almost white, pale yellow, green or blue, estimated potency.
hygroscopic powder or friable solid. Human coagulation factor X (2.7.19)
Reconstitute the preparation to be examined as stated on the label The estimated content is not more than 125 per cent of the
immediately before carrying out the identification, tests (except stated content. The confidence limits (P = 0.95) are not less
those for solubility and water) and assay. than 90 per cent and not more than 111 per cent of the
IDENTIFICATION estimated potency.
It complies with the limits of the assay. Water
TESTS
determination of water (2.5.72), loss on drying (2.2.32) or
Solubility near-infrared spectrometry (2.2.40), the water content is
To a container of the preparation to be examined add the within the limits approved by the competent authority.
volume of liquid stated on the label at the recommended
temperature. The preparation dissolves completely with Sterility (2.6.1)
gentle swirling within 10 min, giving a clear or slightly It complies with the test.
opalescent solution that may be coloured.
FV-494 Blood-related Products 2016
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)

It complies with the test for pyrogens or, preferably and Factor Vila (rDNA) Concentrated **
where justified and authorised, with a validated in vitro test Solution *****
such as the test for bacterial endotoxins. (Human Coagulation Factor Vila (rDNA)
For the pyrogen test, inject per kilogram of the rabbit’s mass Concentrated Solution, Ph. Eur. monograph 2534)
a volume equivalent to not less than 30 IU of human
coagulation factor vn. light chain
ANAFLEELRP GSLERECKEE QCSFEEAREI FKDAERTKLF 40
Where the test for bacterial endotoxins is used, the FCLPAFEGRN 80
WISYSDGDQC ASSPCQNGGS CKDQLQSYIC
preparation to be examined contains less than 0.1 IU of CETHKDDQLI CVNENGGCEQ YCSDHTGTKR SCRCHEGYSL 120
endotoxin per International Unit of human coagulation LADGVSCTPT VEYPCGKIPI LEKRNASKPQ GR 152
factor vn. heavy chain
ASSAY IVGGKVCP 160
KGECPWQVLL TLINTIWWS AAHCFDKIKH 200
Human coagulation factor vn (2.7.10) LVNGAQLCGG
WRNLIAVLGE HDLSEHDGDE QSRRVAQVII PSTYVPGTTN 240
The estimated potency is not less than 80 per cent and not
HDIALLRLHQ PVVLTDHWP LCLPERTFSE RTLAFVRFSL 280
more than 125 per cent of the stated potency.
VSGWGQLLDR GATALELMVL NVPRLMTQDC LQQSRKVGDS 320
The confidence limits (P = 0.95) are not less than HYRGTWYLTG 360
PNITEYMFCA GYSDGSKDSC KGDSGGPHAT
80 per cent and not more than 125 per cent of the estimated IVSWGQGCAT VGHFGVYTRV SQYIEWLQKL MRSEPRPGVL 400
potency. LRAPFP 406
STORAGE
disulfide bndges
In an airtight container, protected from light. 17-22. 50-61. 55-70, 72-81. 91-102, 98-112, 114-127. 135-262. 159-164,
178-194, 310-329, 340-368
LABELLING
glycosylation sites
The label states: 52. 60. 145. 322
— the number of International Units of human coagulation E (4<a?bo^GMSat position 6. 7, 14. 16, 19, 20. 25. 26. 29. 35
factor vn per container;
— the maximum content of human coagulation factor ท, potentially modified residue
D ((3R)-3-hydroxyAsp) at position 63
human coagulation factor IX and human coagulation
factor X per container, in International Units; H NH2
H NH; HO2C __
— the amount of protein per container; ๖< CO2H
— the name and quantity of any added substances, HO2C co2h HO H
including, where applicable, heparin; E = 4-carboxyGlu D = (3R}-3-hydroxyAsp
— the name and volume of the liquid to be used for
reconstitution;
^1982^3054^5600618^28 Mr approx. 50 000
— that the transmission of infectious agents cannot be totally
Ph Eur_________________________________________________ _____________
excluded when medicinal products prepared from human
blood or plasma are administered. DEFINITION
_______________________________________________________________ Ph Eur Solution containing closely related glycoproteins, which have
the same amino acid sequence (406 amino acids) and
disulfide bridges as the naturally occurring analogue (plasma-
derived activated coagulation factor vn). Human coagulation
factor VUa (rDNA) (eptacog alfa, activated) is a 2-chain
molecule, obtained by proteolytic cleavage of the peptide
bond between Arg 152 and He 153 of single-chain
coagulation factor VII, consisting of a 20 kDa light chain
(N-terminal) and a 30 kDa heavy chain (C-terminal)
connected by a disulfide bond.
Human coagulation factor Vila (rDNA) is distinguishable
from the naturally occurring analogue in terms of its post-
translational modifications, including glycosylation pattern.
Content
1.11 mg to 1.78 mg of protein per millilitre.
Potency
44 000 IU to 64 000 IU per milligram of protein.
PRODUCTION
Human coagulation factor Vila (rDNA) is produced in
mammalian cells by a method based on recombinant DNA
technology (rDNA).
Prior to release, the following tests are carried out on each batch of
the final bulk product, unless exemption has been granted by the
competent authority.
Host-cell-derived proteins
The limit is approved by the competent authority.
Host-cell- and vector-derived DNA
Peak Charged Structure Peak Charged Structure
1. No Core fucosylated biantennary - non sialylated 7. Yes Core fucosylated biantennary -

(2 N-acetylgalactosamine terminals) monosialylated (and 1 galactose
terminal)
2. No Core fucosylated biantennary - non sialylated 8. Yes Core fucosylated biantennary -
(N-acetylgalactosamineand galactose terminals) bisialylated
No Structure not determined 9. Yes High-mannose structure with
1 [Htosphate group
No Core fucosylated biantennary - non sialylated 10. Yes Core fucosylated tnantennary -
(galactose and N-acetylglucosamine terminals) trisialylated
No Core fucosylated biantennary - non sialylated 11. Yes Core fucosylated triantennary -
(2 galactose terminals) trisialylated
6. Yes Core fucosylated biantennary - monosialylated 12. Yes Structure not determined
(and 1 N-acetylgalactosamine terminal)
Figure 2534.-1. - Chromatogram for the test for glycan analysis of human coagulation factor Vila (rDNA): reference solution
Glycan analysis 37 °C. Remove the protein fraction by adding 1.5 mL of

Use a suitable method developed according to general ethanol (96 per cent) R at -20 °C to the tubes. Mix and allow
chapter 2.2.59. Glycan analysis of glycoproteins. to stand at -20 °C for 20-30 min. Centrifuge the tubes at
Glycan analysis includes the following steps: 10 000 r/min for 10 min. Collect the supernatant and
— after desalting, release of the glycans (see 2.2.59 evaporate to dryness, using for example a rotary evaporator.
section 2-3); LABELLING OF GLYCANS
— labelling of the glycans with a suitable fluorescent label Label the liberated glycans with 2-aminobenzamide using a
(Table 2.2.59.-2); suitable procedure. The procedure employs a combination of
— analysis of the labelled glycans by liquid chromatography reagents optimised and validated for the efficient labelling of
(2.2.29) with fluorometric detection. glycans, and for the subsequent extraction and recovery of
The following procedures may be used. the labelled glycans from the reaction.
Test solution Dilute the preparation to be examined in water R LIQUID CHROMATOGRAPHY (2.2.29)
to obtain a concentration of about 1.5 mg/mL. Precolumn:
Reference solution Dissolve human coagulation factor Vila — size: I = 0.05m, 0 = 4.0 mm;
(rDNA) CRS in water R to obtain a concentration of — stationary phase: strongly basic anion-exchange resin for
1.5 mg/mL. chromatography R.
DESALTING Column:
Desalt the test solution and the reference solution as — size: I = 0.25 m, 0 = 4.0 mm;
described under Identification B. The buffer used for — stationary phase: strongly basic anion-exchange resin for
desalting and elution is a 1.21 g/L solution of chromatography R-}
tris (hydroxymethyl) aminomethane R, adjusted to pH 7.5 with — temperature: 30 °C.
hydrochloric acid R. After desalting, the concentration of the Mobile phase:
solutions is about 1.0 mg/mL. — mobile phase A: 6 g/L solution of sodium hydroxide Rj
— mobile phase B: solution containing 6 g/L of sodium
SELECTIVE RELEASE OF GLYCANS hydroxide R and 40.8 g/L of sodium acetate R;
Transfer 500 pL of the desalted test solution and 500 pL of
the desalted reference solution to separate centrifuge tubes,
and add 10 pL of a 200 U/mL solution of peptide N-
glycosidase F R. Cap the tubes and incubate for 16-24 h at
Time Mobile phase A Mobile phase B Reference solution Dissolve human coagulation factor Vila
0 - 52
(rDNA) CRS in solution A to obtain a concentration of
100 -> 35 0 -> 65
1.5 mg/mL. Desalt and digest at the same time and in the
52.0 - 52.1 35 -> 0 65 -> 100 same manner as for the test solution.
52.1 - 65 0 100 CHROMATOGRAPHIC SEPARATION Liquid
65 - 65.1 0-> 100 100 -> 0
chromatography (2.2.29).
Column:
65.1 - 90 100 0
— size: I = 0.25 m, 0 = 4.0 mm;
— stationary phase: octadecylsilyl silica gel for
chromatography R (5 pm) with a pore size of 30 nm;
Flow rate 0.5 mDmin.
Detection Fluorimeter at 330 nm for excitation and 420 nm Mobile phase:
for emission.
— mobile phase A: add 0.65 mL of trifluoroacetic acid R to
Injection 100 pL, using an automatic injector maintained at 1000 mL of water R and degas;
2-8 °C. — mobile phase B: mix 0.5 mL of trifluoroacetic acid R,
System suitability: reference solution: 100 mL of water R and 900 mL of acetonitrile for
— the chromatogram obtained is similar to the chromatography R and degas;
chromatogram shown in Figure 2534.-1; peaks 1 to 12
are clearly visible;
— peak width at half-height: maximum 30 ร for peak 8.
Calculate the percentage content of charged glycans in the 0 - 100 100 -> 50 0 -> 50
reference solution using the following expression:
100 - 105 50 -> 0 50 -> 100
105 - 110 0 100
110 - 110.1 0 -> 100 100 -> 0
110.1 - 125 100 0

A = sum of the areas of the peaks due to charged glycans
(peaks 6 to 12);
B = sum of the areas of the peaks due to uncharged Flow rate 1.0 mL/min.
glycans (peaks 1 to 5).
Calculate the percentage content of charged glycans in the Injection 25 pL.
test solution accordingly.
System suitability The chromatogram obtained with the
Limit: reference solution is similar to the chromatogram supplied
— percentage of charged glycans: as authorised by the with human coagulation factor Vila (rDNA) CRS.
Results The chromatogram obtained with the test solution is
CHARACTERS similar to the chromatogram obtained with the reference
Appearance solution:
Colourless liquid. — all major peaks identified in the chromatogram
IDENTIFICATION obtained with the reference solution are present in the
A. It forms a clot when examined in the conditions described chromatogram obtained with the test solution;
under Assay (Potency). — no new major peaks are observed in the
chromatogram obtained with the test solution in
B. Peptide mapping (2.2.55). comparison with the chromatogram obtained with the
SELECTIVE CLEA VAGE OF THE PEPTIDE BONDS reference solution.
Solution A Dissolve 0.74 g of calcium chloride R and 6.06 g of c. Examine the chromatograms obtained in the test for
tris (hydroxymethyl) aminomethane R in 1000 mL of water R glycan analysis.
and adjust to pH 7.5 with hydrochloric acid R. Results The chromatogram obtained with the test solution is
Test solution Dilute the preparation to be examined with similar to the chromatogram obtained with the reference
solution A to obtain a concentration of about 1.5 mg/mL. solution.
Desalt a volume of this solution by a suitable method (for
TESTS
example using a suitable centrifugal filter unit or gel-filtration
column with solution A as elution buffer). After desalting, Degraded heavy chain and oxidised forms of human
the concentration should be about 1.0 mg/mL. Transfer the coagulation factor Vila (rDNA)
desalted solution to a polypropylene tube. Prepare a Liquid chromatography (2.2.29): use the normalisation
1 mg/mL solution of trypsin for peptide mapping R and add procedure.
10 pL to 1 mL of the desalted solution. Cap the tube and Test solution Dilute the preparation to be examined in water R
mix gently by inversion. Incubate at 37 °C for 24 h. At time to obtain a concentration of about 1.5 mg/mL.
5.5 h, add 10 pL of the trypsin solution. Remove the sample Reference solution Dissolve human coagulation factor Vila
from the incubator, place it at room temperature, add 9 pL (rDNA) CRS in water R to obtain a concentration of
of glacial acetic acid R and mix by inversion. Maintain the 1.5 mg/mL.
solution at -15 °C or below until chromatographic Column:
separation; if analysed immediately using an automatic — size: I = 0.25 m, 0 = 4.0 mm;
injector, maintain at 2-8 °C. — stationary phase: butylsilyl silica gel for chromatography R
(5 pm) with a pore size of 30 nm;
1. degraded heavy chain3. oxidised form 5. oxidised form 7. human coagulation factor Vila 9. unknown
(rDNA)
2. degraded heavy chain4. oxidised form 6. degraded heavy chains, unknown 10. unknown
Figure 2534.-2. - Chromatogram for the test for degraded heavy chain and oxidised forms of human coagulation factor Vila (rDNA):
reference solution
— temperature: 60-70 °C. Results:

Mobile phase: — the chromatogram obtained with the test solution is
— mobile phase A: mix 1 mL of trifluoroacetic acid R and similar to the chromatogram obtained with the reference
999 mL of water R and degas; solution.
— mobile phase B: mix 1 mL of trifluoroacetic acid R, 200 mL Calculate the individual percentage area (relative to the total
of water R and 800 mL of acetonitrile for chromatography R peak area) of the peaks due to the degraded heavy chain
and degas; human coagulation factor Vila (rDNA) (peaks 1, 2 and 6)
and oxidised forms of human coagulation factor Vila
Time
(rDNA) (peaks 3, 4 and 5).
Mobile phase A Mobile phase B
(per cent V/V) (per cent V/V) Limits:
0 - 30 54 -> 41 46 ■> 59 — sum of degraded heavy chain forms: maximum 11 per cent;
— sum of oxidised forms: maximum 2.2 per cent.
30 - 33 41 -» 0 59 -> 100
Gla-domainless human coagulation factor Vila (rDNA)
33 - 38 0 100
(gamma-carboxylation)
38 - 40 0 -» 54 100 -> 46 Liquid chromatography (2.2.29): use the normalisation
procedure.
Test solution Dilute the preparation to be examined in water R
to obtain a concentration of about 1.5 mg/mL.
Reference solution Dissolve human coagulation factor Vila
Injection About 20 pL, using an automatic injector (rDNA) CRS in water R to obtain a concentration of
maintained at 2-8 °C. 1.5 mg/mL.
Retention time Human coagulation factor Vila Precolumn:
(rDNA) = about 26 min. — stationary phase: styrene-divinylbenzene copolymer R with
System suitability: iminodiacetic groups, for removal of calcium.
— the chromatogram obtained with the reference solution is Column:
similar to the chromatogram shown in Figure 2534.-2; — size: I = 0.25 m, 0 = 4.0 mm;
peaks 1 to 10 are clearly visible; — stationary phase: strongly basic anion-exchange resin for
— peak-to-valley ratio: minimum 1.5, where Hp = height chromatography Rl'i
above the baseline of peak 6 and Hv = height above the — temperature: 25 °C.
baseline of the lowest point of the curve separating this
peak from peak 7.
Mobile phase: — symmetry factor, maximum 1.3 for the peak due to the
— mobile phase A: solution containing 1.2 g/L of monomer;
tris (hydroxymethyl) aminomethane R and 2.8 g/L of bis-tris — peak-to-valley ratio: minimum 1.1, where Hp — height
propane R, adjusted to pH 9.4 with glacial acetic acid R above the baseline of the peak due to dimers and
and degassed; Hv = height above the baseline of the lowest point of the
— mobile phase B: solution containing 1.2 g/L of curve separating this peak from the peak due to the
โทร(hydroxymethyl)aminomethane R, 2.8 g/L of bis-tris monomer.
propane R and 107.9 g/L of ammonium acetate R, adjusted Limit:
to pH 9.4 with concentrated ammonia R and degassed; — sum of the areas of the peaks with a retention time less than
that of the monomer, maximum 2.7 per cent.
Time Mobile phase A Mobile phase B Non-activated single-chain factor VII (rDNA)
(min) (per cent V/V) (per cent V/V) Polyacrylamide gel electrophoresis (2.2.31) Use the
0 - 2.5 100 0 normalisation procedure.
2.5 - 27.5 100 + 0 0 + 100 Gel dimensions 1 mm thick.
27.5 - 30.5 0 + 100 100 -> 0 Resolving gel 12 per cent acrylamide.
Sample buffer (reducing conditions) concentrated SDS-PAGE
sample buffer for reducing conditions R containing dithiothreitol R
Flow rate 1.0 mL/min. as the reducing agent.
Detection Spectrophotometer at 280 nm. Test solution Dilute the preparation to be examined in water R
Injection About 100 pL, using an automatic injector to obtain a concentration of about 800 pg/mL. Mix equal
maintained at 2-8 °C. volumes of this solution and the sample buffer (reducing
Relative retention With reference to human coagulation conditions).
factor Vila (rDNA) (retention time = about 14 min): Reference solution (a) Dissolve human coagulation factor Vila
Gla-domainless human coagulation factor VUa (rDNA) CRS in water R to obtain a concentration of about
(rDNA) = about 0.7. 800 pg/mL. Mix equal volumes of this solution and the
System suitability: reference solution: sample buffer (reducing conditions).
— resolution: baseline separation between the peak due to Reference solution (b) Solution of molecular mass markers
Gla-domainless human coagulation factor VUa (rDNA) suitable for calibrating SDS-polyacrylamide gels in the range
and the peak cluster due to human coagulation factor of 10-70 kDa.
vna (rDNA). Sample treatment Boil for 5 min or heat at 73 ± 3 ๐c for
Limit: 10 min.
— Gla-domainless human coagulation factor Vila (rDNA): Application 10 pL.
maximum 6.1 per cent.
Detection By Coomassie staining.
Integrate the peak cluster to baseline.
Quantification Integrating densitometer.
Dimers and related substances of higher molecular
System suitability:
mass
— the principal bands in the electropherogram obtained with
Size-exclusion chromatography (2.2.30) Use the
the test solution correspond in position to the principal
normalisation procedure.
bands in the electropherogram obtained with reference
Test solution Dilute the preparation to be examined in water R solution (a) (30 kDa, heavy chain and 20-25 kDa, light
to obtain a concentration of about 1.5 mg/mL. chain);
Reference solution Dissolve human coagulation factor Vila — reference solution (b): the validation criteria are met
(rDNA) CRS in water R to obtain a concentration of (2.2.31),
1.5 mg/mL. — a band corresponding to non-activated single-chain
Column: factor vn (rDNA) (molecular mass of 51 kDa) is visible
— size: I = 0.3 m, 0 = 7.5 mm; in the electropherogram obtained with reference
■— stationary phase: hydrophilic silica gel for chromatography R solution (a).
(10 pm) of a grade suitable for fractionation of globular Limit:
proteins in the relative molecular mass range of 10 000 to — non-activated single-chain factor VII (rDNA): maximum
500 000; 3 per cent.
— temperature: 21-25 °C. Bacterial endotoxins (2.6.14)
Mobile phase Dissolve 26.4 g of ammonium sulfate R in Less than 10 lU/mL.
approximately 900 mL of water R. Adjust first to pH 2.5 with
ASSAY
phosphoric add R and then to pH 7.0 with triethylamine R.
Protein
Add 50 mL of 2-propanol R and dilute to 1000 mL with
Size-exclusion chromatography (2.2.30) as described in the
water R.
test for dimers and related substances of higher molecular
Flow rate 0.5 mL/min. mass with the following modifications.
Detection spectrophotometer at 215 nm. Injection 10 pL, 20 pL and 30 pL of the reference solution.
Injection 20 pL, using an automatic injector maintained at Plot peak areas against injected protein content and perform
2-8 °C. a linear regression to create a standard curve.
System suitability: reference solution: Calculate the content of human coagulation factor Vila
— the chromatogram obtained is similar to the (rDNA) using the monomer peak area in the chromatogram
chromatogram supplied with human coagulation factor Vila obtained with the test solution and taking into account the
(rDNA) CRSi
assigned content of human coagulation factor Vila

(rDNA) CRS. Dried Factor VIII (rDNA) ★ **
System suitability: (Human Coagulation Factor VIII (rDNA), ***
repeatability: maximum relative standard deviation of Ph Eur monograph 1643)
2.0 per cent after 5 injections of 20 J1L of the reference
solution; Action and use
the correlation coefficient calculated for the standard Coagulation factor vm substitute.
curve (r2) is not less than 0.990.
PhEir______________________________________________________________
Potency
The principle of the assay is to measure the ability of a DEFINITION
factor Vila preparation to reduce the prolonged coagulation Human coagulation factor vni (rDNA) is a freeze-dried
time of factor Vll-deficient plasma. preparation of glycoproteins having the same activity as
coagulation factor vm in human plasma. It acts as a cofactor
The biological activity is assessed by comparing the dose
of the activation of factor X in the presence of factor IXa,
response curve of the preparation to be examined to that of a
phospholipids and calcium ions.
reference preparation calibrated in International Units.
The International Unit is the activity contained in a stated Human coagulation factor vm circulates in plasma mainly as
amount of the International Reference Preparation. a two-chain glycosylated protein with 1 heavy (relative
molecular mass of about 200 000) and 1 light (relative
The equivalence in International Units of the International
molecular mass 80 000) chain held together by divalent
Reference Preparation is stated by the World Health
metal ions. Human coagulation factor vm (rDNA) is
Organization.
prepared as full-length factor vm (octocog alfa), or as a
Method shortened two-chain structure (relative molecular mass
Use a suitable coagulation analyser or carry out the assay with 90 000 and 80 000), in which the B-domain has been
incubation tubes and reagents maintained in a water-bath at deleted from the heavy chain (moroctocog alfa).
37 °C.
Full-length human rDNA coagulation factor vm contains
Solution A Prepare a solution containing 15.12 g/L of 1,4- 25 potential N-glycosylation sites, 19 in the B domain of the
piperazinediethanesulfonic acid R, 5.73 g/L of sodium chloride R, heavy chain, 3 in the remaining part of the heavy chain
0.74 g/L of sodium edetatc R and 10 g/L of bovine albumin R-, (relative molecular mass 90 000) and 3 in the light chain
adjust to pH 7.2 with sodium hydroxide R. (relative molecular mass 80 000). The different products are
Prepare 3 different solutions of the preparation to be characterised by their molecular size and post-translational
examined and of the reference preparation, by diluting with modification and/or other modifications.
solution A, to obtain concentrations within the linearity range PRODUCTION
(0.002-0.15 lU/mL). Prepare in duplicate and use the Human coagulation factor vm (rDNA) is produced by
solutions immediately. recombinant DNA technology in mammalian cell culture.
To 40 pL of each solution, add 40 |1L of factor Vll-deficient It is produced under conditions designed to minimise
plasma R, incubate for an appropriate time at 37 °C, and add microbial contamination.
40 |1L of human tissue factor solution R. Purified bulk factor VIII (rDNA) may contain added human
Measure the coagulation time, i.e. the interval between the albumin and/or other stabilising agents, as well as other
addition of the human tissue factor solution and the first auxiliary substances to provide, for example, correct pH and
indication of the formation of fibrin. osmolality.
The volumes given above and sequence of reagents may be The specific activity is not less than 2000 ru of factor VHI:C
adapted to the human tissue factor solution and apparatus used. per milligram of total protein before the addition of any
Calculate the activity in International Units per millilitre protein stabiliser, and varies depending on purity and the
using an appropriate statistical method, for example the type of modification of molecular structure of factor vm.
parallel-line assay (5.3). The quality of the bulk preparation is controlled using one or
The confidence limits (P = 0.95) are not less than more manufacturer's reference preparations as reference.
80 per cent and not more than 125 per cent of the estimated MANUFACTURER'S REFERENCE PREPARATIONS
potency. During development, reference preparations are established
LABELLING for subsequent verification of batch consistency during
The label states: production, and for control of bulk and final preparation.
— the content of human coagulation factor Vila (rDNA), in They are derived from representative batches of purified bulk
milligrams per millilitre; factor vm (rDNA) that are extensively characterised by tests
— the specific activity, in International Units per milligram including those described below and whose procoagulant and
of protein. other relevant functional properties have been ascertained
_______________________________________________________________Ph Eur
and compared, wherever possible, with the International
Standard for factor vm concentrate. The reference
preparations are suitably characterised for their intended
purpose and are stored in suitably sized aliquots under
conditions ensuring their stability.
PURIFIED BULK FACTOR vm (RDNA)
The purified bulk complies with a suitable combination of the
following tests for characterisation of integrity of the factor VIII
(rDNA). Where any substance added during preparation of the
purified bulk interferes with a test, the test is carried out before
addition of that substance. Where applicable, the characterisation Bacterial endotoxins {2.6.14)
tests may alternatively be carried out on the finished product. Less than 3 IU in the volume that contains 100 IU of
Specific biological activity or ratio of factor VIII factor vni activity.
activity to factor vm antigen ASSAY
Cany out the assay of human coagulation factor VIII (2.7.4). Carry out the assay of human coagulation factor vni {2.7.4).
The protein content, or where a protein stabiliser is present,
the factor vไHI antigen content, is determined by a suitable
method and the specific biological activity or the ratio of
The confidence limits {P = 0.95) are not less than
factor vni activity to factor vni antigen is calculated.
Protein composition potency.
The protein composition is determined by a selection of
appropriate characterisation techniques which may include STORAGE
peptide mapping, Western blots, HPLC, gel electrophoresis, Protected from light.
capillary electrophoresis, mass spectrometry’ or other LABELLING
techniques to monitor integrity7 and purity. The protein The label states:
composition is comparable to that of the manufacturer's — the factor VIII content in International Units,
reference preparation. — the name and amount of any excipient,
Molecular size distribution — the composition and volume of the liquid to be used for
Using size-exclusion chromatography {2.2.30), the molecular reconstitution.
size distribution is comparable to that of the manufacturer's
reference preparation.
Peptide mapping (2.2.55)
There is no significant difference between the test protein
and the manufacturer's reference preparation.
Carbohydrates/sialic acid
Dried Factor VIII Fraction ; *,
To monitor batch-to-batch consistency, the monosaccharide (Human Coagulation Factor VIII, ***
content and the degree of sialylation or the oligosaccharide Ph Eur monograph 0275)
profile are monitored and correspond to those of the
manufacturer's reference preparation. Action and use
FINAL LOT Coagulation factor VIII substitute.
It complies with the requirements under Identification, Tests Ph Eur______________________________________________________ _________
and Assay.
DEFINITION
Excipients
Sterile, freeze-dried preparation of a plasma protein fraction
80 per cent to 120 per cent of the stated content, determined
containing the glycoprotein human coagulation factor VUI
by a suitable method, where applicable.
together with varying amounts of human von Willebrand
CHARACTERS factor, depending on the method of preparation. It is
Appearance prepared from human plasma that complies with the
White or slightly yellow powder or friable mass. monograph on Human plasma for fractionation (0853).
IDENTIFICATION The preparation may contain excipients such as stabilisers.
A. It complies with the limits of the assay. The potency of the preparation, reconstituted as stated on
the label, is not less than 20 IU of factor Vni:C per millilitre.
B. The distribution of characteristic peptide bands
corresponds with that of the manufacturer's reference PRODUCTION
preparation (SDS-PAGE or Western blot). GENERAL PROVISIONS
The method of preparation is designed to maintain
TESTS
functional integrity of human coagulation factor vni and to
Reconstitute the preparation as stated on the label immediately
minimise potential neoantigcnicity. It includes a step or steps
before carrying out the tests (except those for solubility and water)
that have been shown to remove or to inactivate known
and assay.
agents of infection; if substances are used for the inactivation
Solubility of viruses, the subsequent purification procedure must be
It dissolves within 5 min at 20-25 °C, giving a clear or validated to demonstrate that the concentration of these
slighdy opalescent solution. substances is reduced to a suitable level and that any residues
pH (2.2.2) are such as not to compromise the safety of the preparation
6.5 to 7.5. for patients.
Osmolality (2.2.25) The specific activity is not less than 1 IU of factor VHI:C per
Minimum 240 mosmol/kg. milligram of total protein before the addition of any protein
stabiliser.
Water
Determined by a suitable method, such as the semi-micro The human coagulation factor VUI fraction is dissolved in 3
determination of water {2.5.12), loss on drying (2.2.22) or suitable liquid. No antimicrobial preservative or antibiotic is
near-infrared spectroscopy {2.2.40), the water content is added. The solution is passed through a bacteria-retentive
within the limits approved by the competent authority. filter, distributed aseptically into the final containers and
Sterility (2.6.1) containers are closed under vacuum or under an inert gas.
It complies with the test for sterility.
CONSISTENCY OF THE METHOD OF Anti-A and anti-B haemagglutinins (2.6.20, Method A)

PRODUCTION The 1 to 64 dilution does not show agglutination. Dilute the
Products stated to have human von Willebrand factor activity reconstituted preparation with a 9 g/L solution of sodium
(products intended for treatment of von Willebrand's disease). chloride R to contain 3 IU of factor vnr.c per millilitre.
It shall be demonstrated by suitable analytical procedures Water
determined during process development that the Determined by a suitable method, such as semi-micro
manufacturing process yields a product with a consistent determination of water (2.5.12), loss on drying (2.2.32) or
composition with respect to human von Willebrand factor. near-infrared spectroscopy (2.2.4(1), the water content is
This composition may be characterised in a number of ways. within the limits approved by the competent authority.
For example, the distribution of the different human von
Willebrand factor multimers may be determined by sodium
Sterility (2.6.1)
dodecyl sulfate (SDS) agarose gel electrophoresis (about
1 per cent agarose) with or without Western blot analysis, Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
using a normal human plasma pool as reference. It complies with the test for pyrogens or, preferably and
Visualisation of the multimeric pattern may be performed where justified and authorised, with a validated in vitro test
using, for example, an immunoenzymatic technique and such as the test for bacterial endotoxins.
quantitative evaluation may be carried out by densitometric For the pyrogen test, inject per kilogram of the rabbit’s mass
analysis. a volume equivalent to not less than 50 IU of factor VHI:C.
Products that show flakes or particles after reconstitution for use If Where the test for bacterial endotoxins is used, the
a few small flakes or particles remain when the preparation is preparation to be examined contains less than 0.03 IU of
reconstituted, it shall be demonstrated during validation endotoxin per International Unit of factor VHI:C.
studies that the potency is not significantly affected after
ASSAY
passage of the preparation through the filter provided.
Human coagulation factor vni (2.7.4)
CHARACTERS The estimated potency is not less than 80 per cent and not
Appearance more than 120 per cent of the stated potency.
White or pale yellow, hygroscopic powder or friable solid. The confidence limits (P = 0.95) are not less than
Reconstitute the preparation to be examined as stated on the label 80 per cent and not more than 120 per cent of the estimated
immediately before carrying out the identification, tests (except potency.
those for solubility and water) and assay. Human von Willebrand factor (2.7.21)
IDENTIFICATION If preparations are intended for the treatment of von
Willebrand's disease, the estimated potency is not less than
It complies with the limits of the assay.
60 per cent and not more than 140 per cent of the stated
TESTS potency.
Solubility Pending the availability of an International Standard for human
To a container of the preparation to be examined, add the von Willebrand factor concentrate calibrated for use in the
volume of the liquid stated on the label at the recommended collagen-binding assay, only the ristocetin cofactor assay may be
temperature. The preparation dissolves completely with used.
gentle swirling within 10 min, giving a clear or slightly
opalescent, colourless or slightly yellow solution. STORAGE
Where the label states that the product may show a few small
flakes or particles after reconstitution, reconstitute the LABELLING
preparation as described on the label and pass it through the The label states:
filter provided: the filtered solution is clear or slightly — the number of International Units of factor VIII:C and,
opalescent. where applicable, of human von Willebrand factor in the
pH (2.2.3) container;
6.5 to 7.5. — the amount of protein in the container;
— the name and quantity of any added substance;
Osmolality (2.2.35) — the name and volume of the liquid to be used for
Minimum 240 mosmol/kg. reconstitution;
Total protein — where applicable, that the preparation may show the
If necessary, dilute an accurately measured volume of the presence of a few small flakes or particles after
reconstituted preparation with a 9 g/L solution of sodium reconstitution;
chloride R to obtain a protein concentration of about — that the transmission of infectious agents cannot be totally
7.5 mg/mL. Place 2.0 mL of this solution in a round- excluded when medicinal products prepared from human
bottomed centrifuge tube and add 2 mL of a 75 g/L solution blood or plasma are administered.
of sodium molybdate R and 2 mL of a mixture of 1 volume of _______ _________________________________________ _ _____________ Ph Eur
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake,
in the residue by the method of sulfuric acid digestion (2.5.9)
and calculate the . amount of protein by multiplying the result
by 6.25. For some products, especially those without a protein
stabiliser such as albumin, this method may not be applicable and
another validated method for protein determination must therefore
be performed.

Factor IX (rDNA) Concentrated The limit is approved by the competent authority.
Solution Glycan analysis
(Human Coagulation Factor IX (rDNA) Concentrated Use a suitable method developed according to general
Solution Ph. Eur. monograph 2522) chapter 2.2.59. Glycan analysis of glycoproteins, Section 2-3.
— Release the glycans using one of the agents described in
YNSGKLEEFV QGNLERECME EKCSFEEARE VFENTERTTE 40 Table 2.2.59.-1, for example peptide N-glycosidase F
FWKQYVDGDQ CESNPCLNGG SCKDDINSYE CWCPFGFEGK 80 (PNGase F).
NCELDVTCNI KNGRCEQFCK NSADNKWCS CTEGYRLAEN 120 — Label the released glycans with one of the fluorescent
QKSCEPAVPF PCGRVSVSQT SKLTRAEAVF PDVDYVNSTE 160 labelling agents described in Table 2.2.59.-2, for example
AETILDNITQ STQSFNDFTR WGGEDAKPG QFPWQWLNG 200 2-aminobenzamide.
KVDAFCGGSI VNEKWIVTAA HCVETGVKIT WAGEHNIEE 240 — Analyse the labelled glycans by liquid chromatography
TEHTEQKRNV IRIIPHHNYN AAINKYNHDI ALLELDEPLV 280 (2.2.29) using a high-pH-resistant column with
LNSYVTPICI ADKEYTNIFL KFGSGYVSGW GRVFHKGRSA 320 fluorescence detection.
LVLQYLRVPL VDRATCLRST KFTIYNNMFC AGFHEGGRDS 360 The following indications are given as an example.
CQGDSGGPHV TEVEGTSFLT GIISWGEECA MKGKYGIYTK 400 Test solution Dilute the preparation to be examined with the
VSRYVNWIKE KTKLT 415 formulation buffer (see Tests) to obtain a concentration of
disulfide bndges: about 2 mg/mL. Use 50 pL of this solution to proceed to
18-23, 51-62, 56-71, 73-82, 88-99, 95-109, 111-124, 132-289, glycan release and labelling. Resuspend or dilute the labelled
206-222, 336-350, 361-389
glycans in 200 pL of water R.
glycosylation sites:
Ser-53, Ser-61, Asn-157, Thr-159, Asn-167. Thr-169
Reference solution (a) Dilute human coagulation factor IX
(rDNA) CRS with the formulation buffer to obtain a
modified residues:
concentration of about 2 mg/mL. Use 50 pL of this solution
fi (4-carboxyGlu): 7, 8, 15. 17, 20. 21, 26, 27, 30, 33. 36, 40
D ((3A?}-3-hydroxyAsp): 64
to proceed to glycan release and labelling. Resuspend or
ร (๙-phosphoSer): 68, 158 dilute the labelled glycans in 200 pL of water R.
Y (๙-รนเfoTyr): 155 Reference solution (b) Use a suitable human coagulation
factor IX (rDNA) in-house reference preparation shown to
be representative of batches tested clinically and batches used
to demonstrate consistency of production. Dilute with the
£ = 4-carboxyGlu D = (3ft)-3-hydroxyAsp
formulation buffer to obtain a concentration of about
2 mg/mL. Use 50 pL of this solution to proceed to glycan
release and labelling. Resuspend or dilute the labelled glycans
in 200 pL of water R.
Blank solution Use 50 pL of the formulation buffer to proceed
ร = (๙-phosphoSer) Y = (๙-sulfoTyr)
to glycan release and labelling.
Analyse the labelled glycans by liquid chromatography
C2053B3116N558O675P2S26 approx. 55 000 (2.2.29).
PhEir______________________________________________________________ Precolumn'.
— size'. / = 0.01 m, 0 = 4.6 mm;
DEFINITION
— stationary phase', polyamine grafted poly (vinyl alcohol)
Solution containing closely related glycoproteins, which have
copolymer R.
the same amino acid sequence (415 amino acids) as the
Column'.
naturally occurring Ala 148 allelic form analogue (plasma-
derived coagulation factor IX). It is a single-chain — size". I = 0.25 m, 0 = 4.6 mm;
— stationary phase: polyamine grafted poly (vinyl alcohol)
glycoprotein with structural and functional characteristics
copolymer R (5 pm).
similar to those of the endogenous factor IX. It may contain
buffer salts and/or non-proteinaceous stabilisers. — temperature: 50 °C.
Mobile phase:
Content — mobile phase A: water R, glacial acetic acid R, acetonitrile R
Minimum 150 IU per millilitre.
(1:2:97 VIVIVy,
Potency — mobile phase B: concentrated ammonia R, glacial acetic
200 to 360 IU per milligram of protein. acid R, water R (1:3:96 VIVIVy,
PRODUCTION
Human coagulation factor IX (rDNA) is produced in Time Mobile phase A Mobile phase B
mammalian cells by a method based on recombinant DNA (min) (per cent V/V) (per cent V/V) _
technology (rDNA). The method of preparation is designed 0- 2 70 30
to maintain the functional integrity of factor IX, and to 70 -> 0 30-> 100
2 - 67
minimise the activation of human coagulation factor IX
67 - 70 0 100
(rDNA) (to minimise potential thrombogenicity).
No antibiotic or antimicrobial preservative is added. 70 - 70.1 0 -> 70 100 -> 30
Prior to release, the following tests are carried out on each batch of 70.1 - 95 70 30
the final bulk product, unless exemption has been granted by the
Host-cell-derived proteins Flow rate 0.5 mL/min.
Detection Fluorimeter at 330 nm for excitation and 420 nm — no significant peaks are observed in regions P0 to P4 in
for emission. the chromatogram obtained with the blank solution.
Injection 20 |1L, using an automatic injector maintained at Results:
2-8 °C. — the profile of the chromatogram obtained with the test
If carry-over of material is observed, running a blank gradient solution corresponds to that of the chromatogram
after each injection may be appropriate. obtained with reference solution (b);
— the relative retentions of the most prominent peaks in
Identification of peak groups Use the chromatogram in Figure
groups P0 to P3 in ±e chromatogram obtained with the
2522.-1 to identify the 5 groups of oligosaccharides
test solution correspond to those in the chromatogram
corresponding to P0 neutral, Pl mono-, P2 di-, P3 tri- and
obtained with reference solution (b);
P4 tetrasialylated oligosaccharides. Record the retention
— the tetrasialylated peak area ratio for the test solution is
times of the most prominent peaks in groups P0 to P4.
within the limits authorised by the competent authority.
Calculate the relative retentions of the most prominent peaks
in groups P0 to P3 with reference to the most prominent CHARACTERS
peak in group P4. Appearance
Calculate the tetrasialylated peak area ratio for the test Clear, colourless liquid.
solution using the following expression: IDENTIFICATION
A. It forms a clot when examined in the conditions described
Ap4 under Assay (Potency).
'รใ=0Api B. Peptide mapping (2.2.55).
SELECTIVE CLEA VAGE OF THE PEPTIDE BONDS
/Ipj = peak area of group P4;
Solution A Dissolve 143.3 g of guanidine hydrochloride R3
A Pi ะะะ peak area of groups P0 to P3.
9.086 g of tris(hydroxymethyl) aminomethane R and 0.931 g of
System suitability'. sodium edetate R in 250 mL of water R and adjust to
— the chromatogram obtained with reference solution (a) is pH 8.0 ± 0.1 with hydrochloric acid R.
qualitatively similar to the chromatogram supplied with Test solution Dilute the preparation to be examined with the
human coagulation factor IX (rDNA) CRS\ 5 groups of formulation buffer (see Tests) to obtain a concentration of
oligosaccharide peaks corresponding to P0 neutral, Pl
about 1.5 mg/mL
mono-, P2 di-, P3 tri- and P4 tetrasialylated
Reference solution Prepare at the same time and in the same
oligosaccharides are present; group P4 includes the
manner as for the test solution but using human coagulation
highest peak, and P3 the second-highest peak;
factor IX (rDNA) CRS instead of the preparation to be — all peaks identified in the chromatogram supplied with
examined. human coagulation factor IX (rDNA) CRS are visible in
Reduction and alkylation To 67 pL of the test solution add the chromatogram obtained with the reference
28 pL of water R, 100 pL of solution A, then วิ pL of a solution.
30.85 g/L solution of dithiothreitol R, mix well and centrifuge Results:
briefly. Overlay with nitrogen. Incubate in a water-bath at — the profile of the chromatogram obtained with the test
40 cc for 1 h. Add 6.6 pL of a freshly prepared 115.04 g/L solution corresponds to that of the chromatogram
solution of iodoacetic acid R, mix well and centrifuge briefly. obtained with the reference solution;
Overlay with nitrogen. Incubate at room temperature for 1 h — no new major peaks arc observed in the
protected from light. Add วิ.3 pL of a 30.85 g/L of solution chromatogram obtained with the test solution in
of dithiothreitol R and mix well. Add 188.1 pL of water R. comparison to the chromatogram obtained with the
Digestion To the reduced solution prepared previously, add reference solution.
10 pL of a freshly prepared 3.4 บ/mL solution of lysyl c. Polyacrylamide gel electrophoresis (2.2.2/).
endopeptidase R, mix well and centrifuge briefly. Overlay with Examine the electropherograms obtained in the test for
nitrogen. Incubate at 30 cc for 4 h. Mix 90 pL of the impurities with molecular masses differing from that of
digested solution and 180 pL of a 33.22 g/L solution of human coagulation factor IX (rDNA).
sodium edetate R. Calculate the relative mobility (in per cent) of the main band
Cany7 out the reduction/alkylation and digestion steps for the in the electropherogram obtained with the test solution with
reference solution in the same manner as for the test reference to the mobility of the main band in the
solution. electropherogram obtained with reference solution (a), using
CHROMATOGRAPHIC SEPARATION Liquid the following expression:
chromatography (2.2.29).
Column'.
— size: I = 0.25 m, 0 = 2.1 mm;
— stationary phase: octadecylsilyl silica gel for
chromatography R (5 pm) with a pore size of 30 nm;
M1 = molecular mass of the main band in the
electropherogram obtained with the test solution;
Mobile phase:
M2 = molecular mass of the main band in the
— mobile phase A: add 0.5 mL of trifluoroacetic acid R to electropherogram obtained with reference solution
1000 mL of water R and degas;
(a).
— mobile phase B: mix 0.5 mL of trifluoroacetic acid R,
50 mL of water R and 950 mL of acetonitrile for Results:
chromatography R and degas; — the electropherogram obtained with the test solution is
similar to the electropherogram obtained with
reference solution (a);
Time Mobile phase A Mobile phase B — the mobility of the main band in the electropherogram
(min) (per cent V/V) (per cent V/V) obtained with the test solution is within 10 per cent of
0- 5 97 3 that of the main band in the electropherogram
5 - 35 97 4 85 3 4 15
obtained with reference solution (a).
35 - 60 85 4 81 15 4 19
TESTS
Formulation buffer
60 - 81 81 4 74 19 4 26 Dissolve 19.53 g of glycine R, 1.55 g of histidine R and
81 - 101 74 4 71 26 4 29 10.00 g of sucrose R in 1000 mL of water R. Add 50 pL of
polysorbate 80 R and adjust to pH 6.8 with hydrochloric acid R.
101 - 135 71 4 60 29 4 40
Gamma-carboxyglutamic acid (Gia)
135 - 140 60 4 0 40 4 100
Liquid chromatography (2.2.29): use the normalisation
140 - 150 0 100 procedure.
150 - 150.01 0 4 97 100 4 3 Test solution Dilute the preparation to be examined with the
formulation buffer to obtain a concentration of about
150.01 - 190 97 3
1 mg/mL.
190 - 191 97 4 50 3 4 50 Reference solution Prepare in the same manner as for the test
191 - 251 50 50 solution but using human coagulation factor IX (rDNA) CRS
instead of the preparation to be examined.
Blank solution The formulation buffer.
Flow rate 0.25 mL/min. Column:
Detection spectrophotometer at 214 nm. — size: I — 0.05 m, 0 = 5 mm;
Injection 240 pL, using an automatic injector maintained at — stationary phase: strongly-basic anion exchange resin for
2-
8 °C. chromatography R (10 pm).
System suitability: Mobile phase:
— the chromatogram obtained with the reference — mobile phase A: solution containing 2.42 g/L of
solution is qualitatively similar to the chromatogram tris(hydroxymethyl)aminomethane R, adjusted to pH 9.0
supplied with human coagulation factor IX with hydrochloric acid R',
(rDNA) CRS', — mobile phase B: solution containing 2.42 g/L of
tris(hydroxymethyl)aminomethane R and 58.45 g/L of
sodium chloride R, adjusted to pH 9.0 with hydrochloric Reference solution Prepare in the same manner as for the test
acid R; solution but using human coagulation factor IX (rDNA) CRS
instead of the preparation to be examined.
Time Mobile phase A Mobile phase B Column:
(min) (per cent V/V) (per cent V/V) — size. / = 0.10 m, 0 = 4.6 mm;
0 - 40 70 -> 60 30 -> 40 — stationary phase: styrene-divinylbenzene copolymer R (10 pm)
40 - 49
with a pore size of 400 nm;
60 -» 0 40 -> 100
49 - 50 0 70 100 -> 30 Mobile phase:
50 - 71 70 30 — mobile phase A: add 1 mL of trifluoroacetic acid R to
1000 mL of water R;
— mobile phase B: mix 1 mL of trifluoroacetic acid R, 200 mL
Flow rate 0.75 mUmin. of water R and 800 mL of acetonitrile for chromatography R.
Injection 50 pL; perform at least 3 injections using an Time Mobile phase A Mobile phase B
automatic injector maintained at 2-8 °C. (per cent V/V) (per cent V/V)
Relative retention With reference to human coagulation 0 - 0.5 75 25
factor rx (rDNA) containing 12 Gia residues per molecule 0.5 - 30 75 -> 20 25 -> 80
(12 Gia, retention time = about 25 min): 9Gla = 0.60;
30 - 31 20 -> 0 80 -> 100
lOGla = 0.75; 1 IGla ะะะ 0.85. NOTE: molecular species
containing 9 or fewer Gia residues per molecule of human 31 - 33 0 100
coagulation factor IX (rDNA) may not be present in the
preparation.
— repeatability: maximum relative standard deviation of Detection Spectrophotometer at 214 nm.
3 per cent for the total area of the peak due.to human Injection 100 pL; perform at least 3 injections using an
coagulation factor IX (rDNA), determined on 3 injections automatic injector.
performed immediately before the run; Relative retention With reference to the 2nd peak of the double
— the lOGla peak is visible and is similar to the peak (retention time = about 12-14 min) due to human
corresponding peak in the chromatogram supplied with coagulation factor IX (rDNA): related protein A = about
human coagulation factor IX (rDNA) CRS; 0.75; related protein B = about 0.78; related protein
— peak-to-valley ratio: minimum 1.2, where Hp = height c = about 0.80; related protein D = about 0.85; related
above the baseline of the peak due to 1 IGla, and protein E = about 0.93.
Hv = height above the baseline of the lowest point of the System suitability: reference solution:
curve separating this peak from the peak due to 12Gla. — the chromatogram obtained is qualitatively similar to the
Results: chromatogram supplied with human coagulation factor IX
— repeatability: maximum relative standard deviation of (rDNA) CRS;
3 per cent for the total area of the peak due to human — repeatability: maximum relative standard deviation of
coagulation factor IX (rDNA), determined on 3 injections 3 per cent for the total area of the peak due to human
of the test solution; coagulation factor IX (rDNA) after 3 injections
— the profile of tire chromatogram obtained with the test performed immediately before the run;
solution corresponds to that of the chromatogram — peak-to-valley ratio: minimum 1.5, where Hp = height
obtained with the reference solution. above the baseline of the peak due to related protein E
Calculate the total Gia content using the following and Hv = height above the baseline of the lowest point of
expression: the curve separating this peak from the peak due to
human coagulation factor IX (rDNA).
Report individual relative peak areas considering the peak
2 ----------- ;----------- X i Gla.mol~ area of the entire chromatogram. Individual relative per cent
total peak area peak areas are calculated as the average of the 3 injections of
the test solution.
— A Pi’, area of the concerned peak (9Gla, lOGla, 1 Ida or Results:
12Gla); any shoulder appearing on the descending part of — the profile of the chromatogram obtained with the test
the 12Gla peak is included in the area of the 12Gla peak; solution corresponds to that of the chromatogram
— total peak area: sum of the areas of peaks 9Gla to 12Gla; obtained with the reference solution, except that minor
— i Gla.moL1: 9, 10, 11 or 12, corresponding to the peaks, which are due to impurities, may be absent in the
theoretical number of Gia residues per mole of human chromatogram obtained with the test solution.
coagulation factor IX (rDNA) for the concerned peak. Limits:
Limit: — related protein C: maximum 0.6 per cent;
— 11.0 to 12.0 moles of Gia per mole of human coagulation — total impurities (all peaks not eluted at the positions expected
factor IX (rDNA). for human coagulation factor IX (rDNA) and its related
Related proteins and impurities proteins): maximum 1.0 per cent.
Liquid chromatography (2.2.29). Impurities with molecular masses differing from that
Test solution Dilute the preparation to be examined with the of human coagulation factor IX (rDNA)
formulation buffer to obtain a concentration of about Polyacrylamide gel electrophoresis {2.2.31) using a gradient
0.5 mg/mL. gel.
Gradient gels (resolving gels) are prepared with an increasing protein bands with molecular masses of approximately
concentration of acrylamide from the top to the bottom. 54 kDa, 44 kDa, 29-32 kDa, 27 kDa and 14 kDa are
Preparation of gradient gels requires a gradient-forming present.
apparatus. System suitability.
Prepare a 3-15 per cent acrylamide gradient gel. — a clear background is obtained after destaining;
Prepare a 3 per cent acrylamide solution by mixing: — the band in the electropherogram obtained with reference
— 10 volumes of 30 per cent acrylamideIbisacrylamide (36.5: ไ) solution (b) is clearly visible;
solution R; — all expected bands in the electropherogram obtained with
— 13 volumes of 3 M tris-hydrochloride buffer solution reference solution (c) are visible;
PH8.8R-, — the bands in the electropherogram obtained with
— 72 volumes of water R; reference solution (c) are clearly separated;
— 2 volumes of a 100 g/L solution of sodium dodecyl — no band is visible in the blank lanes.
sulfate R; Results:
— 1 volume of a 0.04 mg/mL solution of putrescine R; — the electropherogram obtained with the test solution is
— 2 volumes of a 15 mg/mL solution of ammonium similar to the electropherogram obtained with reference
persulfate R. solution (a);
Prepare a 15 per cent acrylamide solution by mixing: — no new band in the electropherogram obtained with the
— 50 volumes of 30 per cent acrylamidelbisacrylamide (36.5:1) test solution has an intensity greater than that of the band
solution Ry in the electropherogram obtained with reference
— 13 volumes of 3 M tris-hydrochloride buffer solution solution (b).
pH 8.8 R; Impurities with molecular masses greater than that of
— 15 volumes of water R; human coagulation factor rx (rDNA)
— 17 volumes of an 855.8 g/L solution of sucrose R; Size-exclusion chromatography (2.2.30) Use the
— 2 volumes of a 100 g/L solution of sodium dodecyl normalisation procedure.
sulfate R; Test solution Dilute the preparation to be examined with the
— 1 volume of a 0.04 mg/mL solution of putrescine R; formulation buffer to obtain a concentration of about
— 2 volumes of a 15 mg/mL solution of ammonium 400 pg/mL.
persulfate R.
Reference solution Dilute human coagulation factor IX
Load the compartments of the gradient-forming apparatus (rDNA) CRS with the formulation buffer to obtain a
with the acrylamide solutions and proceed as per the concentration of about 400 pg/mL.
instructions of the equipment supplier to obtain the
Resolution solution Incubate a volume of the reference solution
polymerised gradient gel.
at 50 °C for 120 min in an HPLC vial. After incubation,
After polymerisation is completed, rinse the gradient gel with place the vial immediately in the autosampler. Start the
water R. Remove any excess liquid. Pour the stacking gel chromatographic run immediately afterwards.
solution into the equipment, insert a clean comb and allow
Blank solution The formulation buffer.
for polymerisation.
Precolumn:
Alternatively, commercially available gradient gels may be — size: I = 0.04 m, 0 = 6 mm;
used. — stationary phase: hydrophilic silica gel for chromatography R
Test solution Dilute the preparation to be examined with the (5 pm) of a grade suitable for the fractionation of globular
formulation buffer to obtain a concentration of about proteins in the relative molecular mass range of 10 000 to
1 mg/mL. 500 000. .
Reference solution (a) Dilute human coagulation factor IX Column:
(rDNA) CRS with the formulation buffer to obtain a — size: I = 0.30 m, 0 = 7.8 mm;
concentration of about 1 mg/mL. — stationary phase: hydrophilic silica gel for chromatography R
Reference solution (b) 0.01 mg/mL solution of bovine (5 pm) of a grade suitable for the fractionation of globular
albumin R in the formulation buffer. proteins in the relative molecular mass range of 10 000 to
Reference solution (c) A solution of molecular mass markers 500 000.
suitable for calibrating SDS-polyacrylamide gels in the range Mobile phase Dissolve 7.80 g of sodium dihydrogen phosphate R
of 5-200 kDa. and 8.77 g of sodium chloride R in 1000 mL of water R.
Sample buffer Concentrated SDS-PAGE sample buffer for Adjust to pH 7.00 ± 0.05 with phosphoric acid R.
reducing conditions R containing dithiothreitol R as reducing Flow rate 1.0 mUmin.
agent. Detection Spectrophotometer at 214 nm.
Sample treatment Incubate in a water-bath for 5 min. Injection 50 pL; perform 3 injections using an automatic
Application 35 pL. injector maintained at 2-8 °C.
Use SDS-PAGE running buffer R for running the gel. Retention time Human coagulation factor IX (rDNA) ะะะ about
For each gel, run 1 lane with reference solution (b), 2 lanes 9 min.
with reference solution (c), 2 lanes with the incubated System suitability:
reducing buffer (as blank) and at least 1 lane with reduced — the chromatogram obtained with the reference solution is
reference solution (a); use the remaining lanes for reduced qualitatively similar to the chromatogram supplied with
test solutions. human coagulation factor IX (rDNA) CRS;
— peak-to-valley ratio: minimum 1.5, where Hp = height
Detection By Coomassie staining.
above the baseline of the peak due to the high molecular
Identification of bands Human coagulation factor IX (rDNA) mass species and Hv = height above the baseline of the
has an approximate molecular mass of 55 kDa, and related lowest point of the curve separating this peak from the
peak due to human coagulation factor IX (rDNA) in the

chromatogram obtained with the resolution solution. Dried Factor IX Fraction ; **
Calculate the relative area (in per cent) of the sum of the (Human Coagulation Factor IXJ ***
peaks with retention times less than that of human Ph Eur monograph 1223)
coagulation factor IX (rDNA), with reference to the area of
the peak due to human coagulation factor IX (rDNA). Action and use
Any shoulder appearing on the descending part of the peak Coagulation factor IX substitute.
due to human coagulation factor IX (rDNA) is included in
its area. Ph Elf______________________________________________________________
Reside. DEFINITION
the profile of the chromatogram obtained with the test Sterile freeze-dried preparation of a plasma protein fraction
solution corresponds to that of the chromatogram containing coagulation factor IX. It is obtained from human
obtained with the reference solution. plasma that complies with the monograph on Human plasma
Limit: for fractionation (0853), by a method that effectively separates
sum of the peaks eluted before the principal peak: maximum human coagulation factor IX from other prothrombin
1.3 per cent. complex factors (human coagulation factors II, vn and X).
The preparation may contain excipients such stabilisers,
Microbial contamination {2.6.12) heparin and antithrombin.
Maximum 10 CFU/mL.
The potency of the preparation, reconstituted as stated on
Bacterial endotoxins {2.6.14) the label, is not less than 20 IU of human coagulation
Less than 1 IU per 100 IU of factor IX activity. factor IX per millilitre.
ASSAY
PRODUCTION
The specific biological activity of the substance is determined GENERAL PROVISIONS
before the addition of any protein stabiliser. The method of preparation is designed to maintain
Protein functional integrity of human coagulation factor IX and to
Size-exclusion chromatography {2.2.30) as described in the minimise activation of any coagulation factor (to minimise
test for impurities with molecular masses greater than that of potential thrombogenicity). It includes a step or steps that
human coagulation factor IX (rDNA) with the following have been shown to remove or to inactivate known agents of
modifications. infection; if substances are used for inactivation of viruses
Prepare triplicate dilutions of the test solution. during production, the subsequent purification procedure
Reference solutions Dilute human coagulation factor IX must be validated to demonstrate that the concentration of
these substances is reduced to a suitable level and that any
(rDNA) CRS with the formulation buffer to obtain a
concentration of 1 mg/mL. Further dilute this solution to residues are such as not to compromise the safety of the
preparation for patients.
prepare a standard curve with concentrations in the range of
100-800 pg/mL (5 concentrations, typically 100 pg/mL, The specific activity is not less than 50 IU of human
200 pg/mL, 400 pg/mL, 600 pg/mL, 800 pg/mL). coagulation factor IX per milligram of total protein, before
the addition of any protein stabiliser.
Plot peak areas versus injected protein content and perform
linear regression to create a standard curve. The human coagulation factor IX fraction is dissolved in a
suitable liquid. No antimicrobial preservative or antibiotic is
System suitability (in addition to those described in the test for
impurities with molecular masses greater than that of human
filter, distributed aseptically into the final containers and
coagulation factor IX (rDNA)):
— the correlation coefficient (r2) calculated for the standard
containers are closed under vacuum or under an inert gas.
curve is not less than 0.995.
Calculate the protein concentration of each replicate of the CONSISTENCY OF THE METHOD OF
preparation to be examined using the standard curve and the PRODUCTION
assigned content in human coagulation factor IX (rDNA) CRS. It shall be demonstrated that the manufacturing process
yields a product having a consistent composition. This is
Potency
evaluated by suitable analytical procedures that are
Assay of human coagulation factor IX {2.7.11).
determined during process development and that normally
include:
— assay of human coagulation factor IX;
The confidence limits {P = 0.95) are not less than
— determination of activated coagulation factors;
— determination of activities of human coagulation
potency.
factors n, vn and X, which shall be shown to be not
Human coagulation factor IX concentrate BRP is suitable for more than 5 per cent of the activity of human coagulation
use as a reference preparation. factor IX.
STORAGE CHARACTERS
In an airtight container, under approved conditions. Appearance
LABELLING White or pale yellow, hygroscopic powder or friable solid.
The label states the factor IX content in International Units Reconstitute the preparation to be examined as stated on the label
per millilitre and in International Units per milligram of immediately before carrying out the identification, tests (except
protein. those for solubility and water) and assay.
___________________ ___________________________________________ Ph Eur IDENTIFICATION
TESTS LABELLING
Solubility The label states:
To a container of the preparation to be examined add the — the number of International Units of human coagulation
volume of the liquid stated on the label at the recommended factor IX per container;
temperature. The preparation dissolves completely with — the amount of protein per container;
gentle swirling within 10 min, giving a clear or slightly — the name and quantity of any added substances including,
opalescent, colourless solution. where applicable, heparin;
pH (2.2.5) — the name and volume of the liquid to be used for
6.5 to 7.5. reconstitution;
— that the transmission of infectious agents cannot be totally
Osmolality’ (2.2.55)
excluded when medicinal products prepared from human
Minimum 240 mosmoFkg.
blood or plasma are administered.
Total protein _________________________________
If necessary, dilute an accurately measured volume of the
reconstituted preparation with a 9 g/L solution of sodnun
chloride R to obtain a solution containing about 15 mg of
bottomed centrifuge tube, add 2 mL of a 75 g/L solution of Dried Factor XI Fraction ** \
sodium molybdate R and 2 mL of a mixture of 1 volume of
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake, (Human Coagulation Factor XI, *
centrifuge for 5 min, decant the supernatant and allow the Ph Eur monograph 1644)
in the residue by the method of sulfuric acid digestion (2.5.9) Action and use
and calculate the amount of protein by multiplying the result Coagulation factor XI substitute.
by 6.25. For some products, especially those without a protein
Ph Eur_________________________________ ________________ -____________
stabiliser such as albumin, this method may not be applicable.
Another validated method for protein determination must therefore DEFINITION
be performed. Sterile plasma protein fraction containing coagulation factor
Activated coagulation factors (2.6.22) XI. It is prepared from Human plasma for
If necessary’, dilute the reconstituted preparation to contain fractionation (0853). The preparation may contain excipients
20 IU of human coagulation factor IX per millilitre. For each such as heparin, Cl-esterase inhibitor and antithrombin III.
of the dilutions, the coagulation time is not less than 150 ร. The potency of the preparation, reconstituted as stated on
the label, is not less than 50 units per millilitre.
Heparin (2.7.12)
If heparin has been added, the preparation to be examined PRODUCTION
contains not more than the amount of heparin stated on the The method of preparation is designed to maintain
label and in all cases not more than 0.5 IU of heparin per functional integrity of human coagulation factor XI and to
International Unit of human coagulation factor IX. minimise activation of any coagulation factor (to minimise
Water potential thrombogenicity). It includes a step or steps that
Determined by a suitable method, such as semi-micro have been shown to remove or to inactivate known agents of
determination of water (2.5.72), loss on drying (2.2.52) or infection; if substances are used for inactivation of viruses
near-infrared spectroscopy (2.2.40), the water content is during production, the subsequent purification procedure
within the limits approved by the competent authority. must be validated to demonstrate that the concentration of
these substances is reduced to a suitable level and any
Sterility (2.6.1) residues are such as not to compromise the safety of the
preparation for patients.
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14) After preparation, the factor XI fraction is dissolved in a
It complies with the test for pyrogens or, preferably and suitable liquid. No antimicrobial preservative or antibiotic is
where justified and authorised, with a validated in vitro test added. The solution is distributed into the final containers
such as the test for bacterial endotoxins. and immediately frozen. It is subsequently freeze-dried and
For the pyrogen test, inject per kilogram of the rabbit’s mass the containers are closed under vacuum or under inert gas.
a volume equivalent to not less than 50 IU of human
CHARACTERS
coagulation factor IX.
Appearance
Where the test for bacterial endotoxins is used, the
White or almost white powder or friable solid.
preparation to be examined contains less than 0.03 ru of
Reconstitute the preparation to be examined as stated on the labd
endotoxin per International Unit of human coagulation
immediately before carrying out the identification, tests (except
factor IX.
those for solubility and water) and assay.
ASSAY
IDENTIFICATION
Human coagulation factor IX (2.7.11)
more than 125 per cent of the stated potency. TESTS
The confidence limits (P = 0.95) are not less than Solubility
80 per cent and not more than 125 per cent of the estimated To a container of the preparation to be examined, add the
potency. volume of liquid stated on the label at room temperature.
The preparation dissolves completely with gentle swirling
STORAGE
In an airtight container, protected from light. within 10 min.
pH (2.2.3) Water
6.8 to 7.4. Determined by a suitable method, such as the semi-micro
Osmolality (2.2.55) determination of water (2.5.72), loss on drying (2.2.52) or
Minimum 240 mosmol/kg. near-infrared spectroscopy (2.2.40) 5 the water content is
Total protein within the limits approved by the competent authority.
If necessary, dilute an accurately measured volume of the Sterility (2.6.7)
preparation to be examined with a 9 g/L solution of sodium It complies with the test.
chloride R to obtain a protein concentration of about Pyrogens (2.6.5) or Bacterial endotoxins (2.6.74)
7.5 mg/mL. Place 2.0 mL of this solution in a round- It complies with the test for pyrogens or, preferably and
bottomed centrifuge tube and add 2 mL of a 75 g/L solution where justified and authorised, with a validated in vitro test
of sodium molybdate R and 2 mL of a mixture of 1 volume of such as the bacterial endotoxin test.
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake, For the pyrogen test, inject per kilogram of the rabbit’s mass
centrifuge for 5 min, decant the supernatant and allow the a volume equivalent to 100 ru of factor XI.
Where the bacterial endotoxin test is used, the preparation to
in the residue by the method of sulfuric acid digestion (2.5.9)
be examined contains less than 0.1 IU of endotoxin per
and calcdate the amount of protein by multiplying the result
International Unit of factor XI.
by 6.25.
Activated coagulation factors (2.6.22) ASSAY
For each of the dilutions, the coagulation time is not less Carry out the assay of human coagulation factor XI (2.7.22).
than 150 ร. The estimated potency is not less than 80 per cent and not
Heparin (2.7.72)
If heparin has been added, the preparation to be examined
contains not more than the amount of heparin stated on the
potency.
label and in all cases not more than 0.5 IU of heparin
per unit of factor XI. STORAGE
Antithrombin III (2.7.77) Protected from light, at a temperature of 2 °C to 8 °C.
If antithrombin III has been added, the preparation to be LABELLING
examined contains not more than the amount of The label states:
antithrombin in stated on the label. — the number of units per container;
Cj-esterase inhibitor — the maximum amount of protein per container;
If cresterase inhibitor has been added, the preparation to be — where applicable, the amount of heparin per container;
examined contains not more than the amount of c I-esterase — where applicable, the amount of antithrombin HI per
inhibitor stated on the label. container;
The Cl-esterase inhibitor content of the preparation to be — where applicable, the amount of c 1-esterase inhibitor per
examined is determined by comparing its ability to inhibit container;
Cl-esterase with the same ability of a reference preparation — the name and volume of the liquid to be used for
consisting of human normal plasma. 1 unit of cresterase is reconstitution.
equal to the activity of 1 mL of human normal plasma. ______ ________________________________________________________Ph Elf
Varying quantities of the preparation to be examined are

mixed with an excess of Cl-esterase and the remaining
Ci-estcrase activity is determined using a suitable
chromogenic substrate.
Method Reconstitute the preparation as stated on the label.
Dried Prothrombin Complex *****
Prepare an appropriate series of 3 or 4 independent dilutions (Human Prothrombin Complex, *
from 1 unit/mL of factor XI, for both the preparation to be Ph Eur monograph 0554)
examined and the reference preparation, using a solution
containing 9 g/L of sodium chloride R and either 10 g/L of Action and use
human albumin R or 10 g/L of bovine albumin R. Warm all Coagulation factor IX substitute. Preparations with
solutions to 37 °C in a water-bath for 1-2 min before use. appropriate activity may be used to correct deficiencies of
Place a suitable amount of Ci-esterase solution in tubes or in coagulation factors II or X.
microtitre plate wells and incubate at 37 °C. Add a suitable
amount of one of the dilutions of the reference preparation Ph Elf___-———————— -------------------------------------------
or of the preparation to be examined and incubate at 37 °C DEFINITION

for 5 min. Add a suitable amount of a suitable chromogenic Sterile plasma protein fraction containing human coagulation
substrate such as methoxycarbonyl-L-lysyl(s- factor IX together with variable amounts of human
benzyloxycarbonyl)-glycyl-L-arginine 4-nitroanilide. Read the coagulation factors n, vn and X; the presence and
rate of increase of absorbance (Ari/min) at 405 nm. Carry proportion of these additional factors depends on the method
out a blank test using tris (hydroxymethyl) aminomethane sodium of fractionation. It is obtained from human plasma that
chloride buffer solution pH 7.4 R instead of the Cl-esterase and complies with the monograph on Human plasma for
the substrate. fractionation (0853). The preparation may contain excipients
Calculate the Cj-esterase inhibitor content using the usual such as stabilisers, heparin and antithrombin.
statistical methods (for example, 5.5). The potency of the preparation, reconstituted as stated on
Anti-A and anti“B haemagglutinins (2.6.20, Method A) the label, is not less than 20 IU of human coagulation
The 1 to 64 dilution does not show agglutination. factor IX per millilitre.
If the content of any of the factors is stated as a รingle value, Activated coagulation factors (2.6.22)
the estimated potency is not less than 80 per cent and not If necessary, dilute the reconstituted preparation to contain
more than 125 per cent of the stated potency; if the content 20 IU of human coagulation factor IX per millilitre. For each
of any of the factors is stated as a range, the estimated of the dilutions, ±e coagulation time is not less than 150 ร.
potency is not less than the lower limit and not greater than Heparin (2.7.12)
the upper limit of the stated range. If heparin has been added during preparation, the
PRODUCTION preparation to be examined contains not more than the
The method of preparation is designed to maintain amount of heparin stated on the label and in all cases not
functional integrity of the relevant coagulation factors it more than 0.5 IU of heparin per International Unit of human
contains and to minimise activation of any coagulation factor coagulation factor IX.
(to minimise potential thrombogenicity). It includes a step or Thrombin
steps that have been shown to remove or to inactivate known If the preparation to be examined contains heparin,
agents of infection; if substances are used for inactivation of determine the amount present as described in the test for
viruses during production, the subsequent purification heparin and neutralise it by addition of protamine sulfate R
procedure must be validated to demonstrate that the (10 pg of protamine sulfate neutralises 1 IU of heparin).
concentration of these substances is reduced to a suitable In each of 2 test-tubes, mix equal volumes of the
level and that any residues are such as not to compromise the reconstituted preparation and of a 3 g/L solution of
safety of the preparation for patients. fibrinogen R. Keep one of the tubes at 37 °C for 6 h and the
The specific activity is not less than 0.6 IU of human other at room temperature for 24 h. In a 3rd tube, mix equal
coagulation factor IX per milligram of total protein, before volumes of the fibrinogen solution and of a solution of
the addition of any protein stabiliser. human thrombin R (1 lU/mL) and place the tube in a water
The prothrombin complex fraction is dissolved in a suitable bath at 37 °C. No coagulation occurs in the rubes containing
liquid. No antimicrobial preservative or antibiotic is added. the preparation to be examined. Coagulation occurs within
The solution is passed through a bacteria-retentive filter, 30 ร in the tube containing thrombin.
distributed aseptically into the final containers and Water
immediately frozen. It is subsequently freeze-dried and the Determined by a suitable method, such as semi-micro
containers are closed under vacuum or under an inert gas. determination of water (2.5.72), loss on drying (2.2.32) or
CHARACTERS near-infrared spectrometry (2.2.40), the water content is
within the limits approved by the competent authority.
Appearance
White or slightly coloured, very hygroscopic powder or friable Sterility (2.6.1)
solid. It complies with the test.
Reconstitute the preparation to be examined as stated on the label Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
immediately before carrying out the identification, tests (except It complies with the test for pyrogens or, preferably and
those for solubility and water) and assay. where justified and authorised, with a validated in vitro test
such as the bacterial endotoxin test.
IDENTIFICATION
It complies with the limits of the assays for human For the pyrogen test, inject per kilogram of the rabbit’s mass
coagulation factors IX and n and, where applicable, those for a volume equivalent to not less than 30 IU of human
human coagulation factors vn and X. coagulation factor IX.
Where the bacterial endotoxin test is used, the preparation to
TESTS
be examined contains less than 0.05 IU of endotoxin per
Solubility International Unit of human coagulation factor IX.
To a container of the preparation to be examined add the
volume of the liquid stated on the label at the recommended ASSAY
temperature. The preparation dissolves completely with Human coagulation factor IX (2.7.11)
gentle swirling within 10 min, giving a clear solution that The estimated potency is not less than 80 per cent and not
may be coloured. more than 125 per cent of the stated potency.
The confidence interval (P = 0.95) is not greater than
pH (2.2.3)
80 per cent to 125 per cent of the estimated potency.
6.5 to 7.5.
Human coagulation factor II (2.7.18)
Osmolality (2.2.35)
Total protein The confidence interval (P = 0.95) is not greater than
If necessary, dilute an accurately measured volume of the 90 per cent to 111 per cent of the estimated potency.
reconstituted preparation with a 9 g/L solution of sodium
The estimated human coagulation factor II potency is not
chloride R to obtain a solution containing about 15 mg of less than 70 per cent and not more than 165 per cent of the
estimated human coagulation factor IX potency.
bottomed centrifuge tube add 2 mL of a 75 g/L solution of
sodium molybdate R and 2 mL of a mixture of 1 volume of Human coagulation factor vn (2.7.10)
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake, If the label states that the preparation contains human
centrifuge for 5 min, decant the supernatant and allow the coagulation factor vn, the estimated potency is not less than
inverted tube to drain on filter paper. Determine the nitrogen 80 per cent and not more than 125 per cent of the stated
in the residue by the method of sulfuric acid digestion (2.5.9) potency. The confidence interval (P = 0.95) is not greater
and calculate the amount of protein by multiplying the result than 80 per cent to 125 per cent of the estimated potency.
by 6.25.
Human coagulation factor X (2.7.19) The protein fraction is dissolved in a suitable liquid.
If the label states that the preparation contains human No antimicrobial preservative or antibiotic is added.
coagulation factor X, the estimated potency is not less than The solution is passed through a bacteria-retentive filter,
80 per cent and not more than 125 per cent of the stated distributed aseptically into the final containers and
potency. The confidence interval (P = 0.95) is not greater immediately frozen. It is subsequently freeze-dried and the
than 90 per cent to 111 per cent of the estimated potency. containers are closed under vacuum or under an inert gas.
STORAGE CHARACTERS
In an airtight container, protected from light. Appearance
labelling White or pale yellow, hygroscopic powder or friable solid.
The label states: Reconstitute the preparation to be examined as Slated on the label
the number of International Units of human coagulation immediately before carrying out the identification, tests (except
factor IX, and the number or range of International Units those for solubility and water) and assay.
of human coagulation factor II per container; IDENTIFICATION
where applicable, the number or range of International It complies with the limits of the assay.
Units of human coagulation factor vn and human
coagulation factor X per container; TESTS
the amount of protein per container; Solubility
the name and quantity of any added substances, To a container of the preparation to be examined add the
including, where applicable, heparin and antithrombin; volume of liquid stated on the label at the recommended
the name and quantity of the liquid to be used for temperature. The preparation dissolves within 30 min at
reconstitution; 20-25 °C, forming an almost colourless, slightly opalescent
that the transmission of infectious agents cannot be totally solution.
excluded when medicinal products prepared from human pH (2.2.3)
blood or plasma are administered. 6.5 to 7.5.
------------------- -------------------------------------------------------------------------------------- Ph Eur Osmolality (2.2.35)
Stability of solution
No gel formation appears at 20-25 °C within 60 min
Dried Fibrinogen ★* ** following reconstitution.
(Human Fibrinogen, Ph Eur monograph 0024) ***

Water
Determined by a suitable method, such as semi-micro
Ph Elf______________________________________________________________ determination of water (2.5.12), loss on drying (2.2.32) or
DEFINITION near-infrared spectroscopy (2.2.40), the water content is
Sterile, freeze-dried preparation of a plasma protein fraction within the limits approved by the competent authority.
containing the soluble constituent of human plasma that is Sterility (2.6.1)
transformed to fibrin on the addition of thrombin. It is It complies with the test.
obtained from human plasma that complies with the Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
monograph on Human plasma for fractionation (0853). It complies with the test for pyrogens or, preferably and
The preparation may contain excipients such as salts, buffers where justified and authorised, with a validated in vitro test
and stabilisers. such as the test for bacterial endotoxins.
When reconstituted as stated on the label, the solution For the pyrogen test, inject per kilogram of the rabbit’s mass
contains not less than 10 g/L of fibrinogen. a volume equivalent to not less than 30 mg of fibrinogen.
PRODUCTION Where the test for bacterial endotoxins is used, the
The method of preparation is designed to maintain preparation to be examined contains less than 0.03 IU of
functional integrity of human fibrinogen. It includes a step or endotoxin per milligram of fibrinogen.
steps that have been shown to remove or to inactivate known
ASSAY
agents of infection; if substances are used for inactivation of
Mix 0.2 mL of the reconstituted preparation with 2 mL of a
suitable buffer solution (pH 6.6-6.8) containing sufficient
thrombin (approximately 3 lU/mL) and calcium
(0.05 mol/L). Maintain at 37 °C for 20 min, separate the
level and any residues are such as not to compromise the
precipitate by centrifugation (5000 g, 20 min) and wash
safety of the preparation for patients. thoroughly with a 9 g/L solution of sodium chloride R.
The specific activity (fibrinogen content with respect to total Determine the nitrogen content by sulfuric acid digestion
protein content) is not less than 80 per cent before addition (2.5.9) and calculate the fibrinogen (clonable protein)
of any protein stabiliser. The fibrinogen content is content by multiplying the result by 6.0. The content is not
determined by a suitable method such as that described less than 70 per cent and not more than 130 per cent of the
under Assay, and the total protein content is determined by a stated content of fibrinogen.
suitable method such as that described under Total protein
in Human albumin solution (0255). Albumin may also be
STORAGE
obtained with fibrinogen during fractionation, in which case a In an airtight container, protected from light.
specific determination of albumin is carried out by a suitable LABELLING
immunochemical method (2.7.1) and the quantity of albumin The label states:
determined is subtracted from the total protein content for — the content of fibrinogen in the container;
the calculation of the specific activity.
— the name and volume of the liquid to be used for B. It complies with the limits of the assay of human
reconstitution; coagulation factor XIII (where applicable).
— where applicable, the name and amount of protein
TESTS
stabiliser added in the preparation.
Solubility
------------------------------------------------------------------------------ - ------------------------ Ph Eur Freeze-dried concentrates dissolve within 20 min in the
volume of liquid and at the temperature stated on the label,
forming an almost colourless, clear or slightly turbid solution.
pH (2.2.3)
Fibrin Sealant Kit ***** 6.5 to 8.0.
(Ph. Eur. monograph 0903) *** Stability of solution
PhEir_______________________________________ ______________________
No gel formation appears at room temperature during
120 min following thawing or reconstitution.
DEFINITION
Water
Sterile, freeze-dried, frozen or liquid preparation of plasma Determined by a suitable method, such as semi-micro
protein fractions containing essentially 2 components, namely determination of water (2.5.72), loss on drying (2.2.32) or
fibrinogen concentrate (component 1), a protein fraction near-infrared spectroscopy (2.2.40), the water content is
containing human fibrinogen, and a preparation containing within the limits approved by the competent authority.
human thrombin (component 2). A fibrin clot is rapidly
formed when the 2 thawed or reconstituted components are Sterility (2.6.I)
mixed. Other ingredients (for example, human coagulation It complies with the test.
factor xni, a fibrinolysis inhibitor or calcium ions) and ASSAY
stabilisers (for example, Human albumin solution (0255)) may Fibrinogen (clottable protein)
be added. Mix 0.2 mL of the reconstituted concentrate with 2 mL of a
Human constituents are obtained from plasma that complies suitable buffer solution (pH 6.6-7.4) containing sufficient
with the monograph Human plasma for fractionation (0853). human thrombin R (approximately 3 lU/mL) and calcium
When thawed or reconstituted as stated on the label, (0.05 mol/L). Maintain at 37 °C for 20 min, separate the
component 1 contains not less than 40 g/L of clonable precipitate by centrifugation at 5000 g for 20 min, wash
protein; the thrombin activity of component 2 varies over a thoroughly with a 9 g/L- solution of sodium chloride R and
wide range (approximately 4-1000 lU/mL). determine the protein as nitrogen by รน]furic acid digestion
(2.5.9). Calculate the clottable protein content by multiplying
PRODUCTION the result by 6.0. The estimated content in milligrams of
The method of preparation is designed to maintain clottablc protein is not less than 70 per cent and not more
functional integrity of the components. It includes a step or than 130 per cent of the stated content. If for a particular
steps that have been shown to remove or to inactivate known preparation this method cannot be applied, use another
agents of infection; if substances are used for inactivation of validated method for determination of fibrinogen.
Human coagulation factor XIII
Standard for blood coagulation factor XIII in plasma. Where
level and any residues are such as not to compromise the
the label indicates that the human coagulation factor XIII
safety of the preparation for patients.
potency is greater than 10 lU/mL, the estimated potency is
The constituents or mixtures of constituents are dissolved in not less than 80 per cent and not more than 120 per cent of
a suitable liquid. No antimicrobial preservative or antibiotic is the stated potency.
added. Constituents or mixtures of constituents are passed
Make at least 3 suitable dilutions of thawed or reconstituted
through a bacteria-retentive filter and distributed aseptically
concentrate and of the reference preparation using human
into sterile containers. Containers of freeze-dried constituents
coagulation factor Xlll-deficient plasma or another suitable
are closed under vacuum or filled with a suitable inert gas,
diluent. Coagulation factors V, VIII, XI and XIII plasma BRP
such as oxygen-free nitrogen, before being closed.
is suitable for use as a reference preparation. Add to each
If the human coagulation factor XIII content in component 1 dilution suitable amounts of the following reagents:
is greater than 10 lU/mL, the assay of human coagulation — activator reagent, containing bovine or human thrombin,
factor xni is carried out. a suitable buffer, calcium chloride and a suitable inhibitor
CHARACTERS such as Gly-Pro-Arg-Pro-Ala-NH2 which inhibits clotting
Appearance of the sample but does not prevent human coagulation
— freeze-dried constituents', white or pale yellow, hygroscopic factor xni activation by thrombin;
powder or friable solid, — detection reagent, containing a suitable factor xnia-
— frozen constituents: colourless or pale yellow, opaque solid, specific peptide substrate, such as Lcu-Gly-Pro-Gly-Glu-
— liquid constituents: colourless or pale yellow liquid. Ser-Lys-Val-Ile-Gly-NH2 and glycine ethyl ester as 2nd
substrate in a suitable buffer solution;
For the freeze-dried or frozen constituents, reconstitute or thaw as
— NADH reagent, containing glutamate dehydrogenase,
stated on the label immediately before carrying out the
a-ketoglutarate and NADH in a suitable buffer solution.
identification and the tests, except those for solubility and water.
After mixing, the absorbance changes (A/4/min) are measured
at a wavelength of 340 nm, after the linear phase of the
COMPONENT• 1 (FIBRINOGEN
reaction is reached.
CONCENTRATE)
Calculate the potency of the test preparation by the usual
IDENTIFICATION statistical methods (5.3, for example). The confidence limits
A. It complies with the limits of the assay of fibrinogen.
(P = 0.95) are not less than 80 per cent and not more than
125 per cent of the estimated potency. Human Haematopoietic stem Cells ;“**
COMPONENT 2 (THROMBIN Ph Elf_____________________________________________________________
PREPARATION) This monograph provides a standard for the preparation and
IDENTIFICATION control of human haematopoietic stem cells for use in therapy.
It complies with the limits of the assay of thrombin. It does not exclude the use of alternative preparation and control
methods that are acceptable to the competent authority.
TESTS
Solubility DEFINITION
Freeze-dried preparations dissolve within 5 min in the Human haematopoietic stem cells are primitive multipotent
cells capable of self-renewal as well as differentiation and
volume of liquid stated on the label, forming a colourless,
clear or slightly turbid solution. maturation into all haematopoietic lineages. They are found
in small numbers in bone marrow, in the mononuclear cell
pH (2.2.5) fraction of circulating blood and in umbilical cord blood.
5.0 to 8.0. The preparation also contains haematopoietic progenitor
Water cells, which are capable of differentiation but not self
Determined by a suitable method, such as semi-micro renewal. The numbers of haematopoietic stem cells and
determination of water (2.5.72), loss on drying (2.2.52) or haematopoietic progenitor cells are correlated.
near-infrared spectroscopy (2.2.40), the water content is This monograph applies to haematopoietic stem cells that
within the limits approved by the competent authority. have not undergone expansion or genetic modification, and
Sterility (2.6.7) that are intended to provide a successful engraftment leading
It complies with the test. to a permanent restoration of all lineages of blood cell
production to a sufficient level and function in a recipient
ASSAY
whose haematopoiesis has been compromised by, for
Thrombin
example, disease or high doses of chemotherapy and/or
If necessary, dilute the reconstituted preparation to be radiation therapy, or has to be replaced in certain congenital
examined to approximately 2-20 IU of thrombin per millilitre diseases. The infused haematopoietic stem cells can originate
using as diluent a suitable buffer solution (pH 7.3-7.5), such from the recipient (autologous) or from another individual
as imidazole buffer solution pH 7.3 R containing 10 g/L of (allogeneic).
human albumin R or bovine albumin p. To a suitable volume
Haematopoietic stem cells are recognised by their ability to
of the dilution, add a suitable volume of fibrinogen solution
reconstitute human haematopoiesis in vivo. They also have
(1 g/L of clottable protein) warmed to 37 °C and start
the capacity to differentiate into colony-forming cells, which
measurement of the clotting time immediately. Repeat the
are able to give rise to colonies in the presence of various
procedure with each of at least 3 dilutions, in the range
growth factors. The membrane marker CD34 is commonly
stated above, of a reference preparation of thrombin,
used for the successful isolation/purification of
calibrated in International Units.
haematopoietic stem cells from crude preparations and as an
Calculate the activity of the test preparation by the usual indicator of haematopoietic stem cell content in routine
statistical methods (5.3> for example). The estimated activity is quality control.
not less than 80 per cent and not more than 125 per cent of
the stated activity. For a component with a low thrombin PRODUCTION
concentration and a nominal value of approximately DONORS
4 lU/mL, the estimated activity is not less than 50 per cent Where allogeneic cells are used, they are derived from
and not more than 150 per cent of the stated activity. carefully selected donors in accordance with donor selection
The confidence limits (P = 0.95) are not less than criteria. Directive 2004/23/EC of the European Union deals
80 per cent and not more than 125 per cent of the estimated wi± the criteria for donor selection.
activity. COLLECTION
STORAGE Peripheral blood stem cells These are collected by cytapheresis
Protected from light and, for freeze-dried components, in an after mobilisation from the bone marrow by administration of
airtight container. growth factors and/or treatment of autologous donors with
cytotoxic substances. The cells may be processed to select a
LABELLING population of interest and may be cryopreserved.
The label states:
Bone marrow Bone marrow is harvested by aspirating the cells
— the amount of fibrinogen (milligrams of clottable protein),
from the cavities of hollow bones, then removing bone
thrombin (International Units) per container, and of
fragments by filtration and, if necessary, separating the buffy
human coagulation factor xni, if the latter is greater than
coat cells after centrifugation or with commercial kits based
10 lU/mL,
on the cytapheresis principle. The cells may be processed to
— where applicable, the name and volume of liquid to be
select a population of interest and may be cryopreserved.
used to reconstitute the components.
Umbilical cord blood Placental blood haematopoietic cells are
___________________________________________________ _ Ph Eur
collected from placentae via the vein of the umbilical cord.
The cells are then cryopreserved.
CRYOPRESERVATION
Cryopreservation allows storage for long periods. The cells
are suspended in a validated medium containing a suitable
cryoprotectant (for example, dimethyl sulfoxide) and
macromolecules (for example, autologous plasma/albumin) antibodies conjugated to a fluorochrome and analysed by
and are frozen in cryobags in a manner designed to maintain flow cytometry (2.7.24).
viability of the cells by controlled cooling according to a Colony-forming cell (CFC) assay (2.7.28)
validated method. They are stored at a temperature Proliferative capacity is established by a suitable assay.
of —140 °C or lower. Where cryobags are stored under other The test is not necessarily carried out on each unit.
conditions of temperature and duration, the functionality of The correlation between the dose of CD34 and the number
the preparation must be validated. Cryobags from donors of CFCs in a given situation (pathology, packaging,
that test positive for an}’ infectious disease marker must be mobilisation) is determined. The CFC assay is carried out
stored in such a way as to avoid cross-contamination. periodically; whenever a change that could affect the quality
SUBSTANCES USED IN PRODUCTION of CD34+ cells is made to the protocol for packaging or
The quality7 of substances used in production may be critical mobilisation, it is carried out on a suitable number of units.
with respect to ±e quality7, safety and efficacy of the final Microbiological control
product, particularly for substances of biological origin. This Examine as prescribed in general method 2.6.27.
is of particular importance for: Microbiological control of cellular products. Where justified, the
— proteins, including enzymes and antibodies; product may be released before completion of the test.
— cryopreservation reagents;
— purification reagents.
Quality7 assurance
All substances must be produced within a recognised quality
management system using suitable production facilities.
Quality specifications Normal Immunoglobulin for * *
A suitable quality specification must be presented for each Intramuscular Administration *****
substance, including notably: Normal Immunoglobulin
— identity;
Normal Immunoglobulin Injection
— potency7 (where applicable);
— purity; (Human Normal Immunoglobulin for Intramuscular
— determination of bacterial endotoxins (2.6.14) (where Administration, Ph. Eur. monograph 0338)
applicable); Ph Eur_________________________________________ _________
— microbiological quality (total viable count, tests for
DEFINITION
specified micro-organisms);
Sterile liquid or freeze-dried preparation containing
— sterility (2.6.1) (where applicable).
immunoglobulins, mainly immunoglobulin G (IgG). Other
Viral safety proteins may be present. Human normal immunoglobulin for
The requirements of chapter 5.1.7 apply. intramuscular administration contains the IgG antibodies of
Transmissible spongiform encephalopathies (5.2.8) normal subjects and is intended for intramuscular
A risk assessment of the product with respect to transmissible administration. The preparation may contain excipients such
spongiform encephalopathies is carried out, and suitable as stabilisers. Multidose preparations contain an antimicrobial
measures are taken to minimise any such risk. preservative.
Water This monograph does not apply to products intentionally
Water used in the preparation of cellular products complies prepared to contain fragments of IgG or chemically modified
with the relevant monograph (Waterfor injections (0169), IgG.
Water, highly purified (1927), Purified water (0008)). Water Human normal immunoglobulin for intramuscular
incorporated into the final product complies with the section administration is obtained from plasma that complies with
on Water for injections in bulk in the monograph Water for the monograph Human plasma for fractionation (0853).
injections (0169), and in addition is sterile.
PRODUCTION
TESTS The method of preparation includes a step or steps that have
Target specifications are established for the different tests, but these been shown to remove or to inactivate known agents of
are not used as rigid acceptance criteria. infection; if substances are used for inactivation of viruses, it
Tests carried out include the following (further tests, such as shall have been shown that any residues present in the final
purging, cell depletion, allogeneic application, may be product have no adverse effects on the patients treated with
necessary depending on any treatment applied to the cells the immunoglobulin.
and on the intended recipient): The product shall have been shown, by suitable tests in
Nucleated cell count (2.7.29) animals and evaluation during clinical trials, to be well
tolerated when administered intramuscularly.
Viability (2.7.29) Any antimicrobial preservative or stabilising agent used shall
Viability is assessed for products that are not infused within have been shown to have no deleterious effect on the final
24 h of collection. product in the amount present.
CD34+ cell count Human normal immunoglobulin for intramuscular
For peripheral blood stem cells, CD34+ cell count is administration is prepared from pooled material from not
determined using a validated automated apparatus to analyse fewer than 1000 donors by a method that has been shown to
cells labelled with anti-CD34 antibodies. The apparatus and yield a product that:
method employed must be able to determine the number of — does not transmit infection;
CD34+ cells with a sensitivity, accuracy and reproducibility — at a protein concentration of 50 g/L, contains at least 2
comparable with those of immunophenotyping (2.7.23), antibodies (1 viral and 1 bacterial) for which an
where cells are labelled using anti-CD34 and anti-CD45 International Standard or reference preparation is
available, the concentration of such antibodies being at digestion (2.5.9) and calculate the content of protein by
least 3 times that in the initial pooled material; multiplying the result by 6.25.
has a defined distribution of IgG subclasses.
Protein composition
Human normal immunoglobulin for intramuscular Zone electrophoresis (2.2.31).
administration is prepared as a stabilised solution, for
Use strips of suitable cellulose acetate gel or suitable agarose
example in a 9 g/L solution of sodium chloride, a 22.5 g/L gel as the supporting medium and barbital buffer solution
solution of glycine or, if the preparation is to be freeze-dried,
pH 8.6 R1 as the electrolyte solution.
a 60 g/L solution of glycine. No antibiotic is added to the
plasma used. Single-dose preparations do not contain an If cellulose acetate is the supporting material, the method
described below can be used. If agarose gels are used, and
antimicrobial preservative. The solution is passed through a
bacteria-retentive filter. The preparation may subsequently be because they are normally part of an automated system, the
freeze-dried and the containers closed under vacuum or manufacturer's instructions are followed instead.
under an inert gas. Test solution Dilute the preparation to be examined with a
The stability of the preparation is demonstrated by suitable 9 g/L solution of sodium chloride R to obtain a protein
tests earned out during development studies. concentration of 30 g/L.
Reference solution Reconstitute human immunoglobulin for
CHARACTERS electrophoresis BRP and dilute with a 9 g/L solution of sodium
Appearance chloride R to obtain a protein concentration of 30 g/L.
liquid preparation', clear or slightly opalescent, colourless or
pale-yellow or light-brown liquid; during storage it may
or apply 0.4 pL per millimetre if a narrower strip is used.
show formation of slight turbidity or a small amount of
To another strip apply in the same manner the same volume
visible particulate matter;
of the reference solution. Apply a suitable electric field such
freeze-dried preparation', white or slightly yellow powder or
that the albumin band of normal human serum applied on a
solid friable mass, hygroscopic.
control strip migrates at least 30 mm. Stain the strip with
For the freeze-dried preparation, reconstitute as stated on the label amido black 10B solution R for 5 min. Decolourise with a
immediately before carrying out the identification and the tests, mixture of 10 volumes of glacial acetic acid R and 90 volumes
except those for solubility and water. of methanol R so that the background is just free of colour.
IDENTIFICATION Develop the transparency of the strips with a mixture of
Examine by a suitable immunoelectrophoresis technique. 19 volumes of glacial acetic acid R and 81 volumes of
Using antiserum to normal human serum, compare normal methanol R. Measure the absorbance of the bands at 600 nm
human serum and the preparation to be examined, both in an instrument having a linear response over the range of
diluted to obtain a protein concentration of 10 g/L. measurement. Calculate the result as the mean of
The main component of the preparation to be examined 3 measurements of each strip.
corresponds to the IgG component of normal human serum. System suitability In the electropherogram obtained with the
The preparation to be examined may show the presence of reference solution, the proportion of protein in the principal
small quantities of other plasma proteins; if human albumin band is within the limits stated in the leaflet accompanying
has been added as a stabiliser, it may be seen as a the reference preparation.
component. Results In the electropherogram obtained with the test
TESTS solution, not more than 10 per cent of protein has a mobility
Solubility different from that of the principal band. This limit is not
For the freeze-dried preparation, to a container of the applicable if albumin has been added to the preparation as a
preparation to be examined add the volume of the liquid stabiliser; for such preparations, a test for protein
stated on the label at the recommended temperature. composition is carried out during manufacture before
The preparation dissolves completely within 20 min at addition of the stabiliser.
20-25 °C. Molecular-size distribution
pH (2.2.3) Size-exclusion chromatography (2.2.30).
5.0 to 7.2. Test solution Dilute the preparation to be examined with a
Dilute the preparation to be examined with a 9 g/L solution 9 g/L solution of sodium chloride R to a concentration suitable
of sodium chloride R to obtain a protein concentration of for the chromatographic system used. A concentration in the
10 g/L. range of 4-12 g/L and injection of 50-600 pg of protein are
usually suitable.
Total protein
Reference solution Dilute human immunoglobulin (molecular
The preparation has a protein concentration of not less than
size) BRP with a 9 g/L solution of sodium chloride R to the
100 g/L and not more than 180 g/L and contains not less
same protein concentration as the test solution.
than 90 per cent and not more than 110 per cent of the
quantity of protein stated on the label. Column:
— size: I = 0.6 m, 0 = 7.5 mm [or I = 0.3 m,
Dilute the preparation to be examined with a 9 g/L solution
0 = 7.8 mm];
of sodium chloride R to obtain a protein concentration of — stationary phase: hydrophilic silica gel for chromatography R)
about 7.5 mg/mL. Place 2.0 mL of this solution in a round- of a grade suitable for fractionation of globular proteins
bottomed centrifuge tube and add 2 mL of a 75 g/L solution with relative molecular masses in the range 10 000 to
of sodium molybdate R and 2 mL of a mixture of 1 volume of
500 000.
Mobile phase Dissolve 4.873 g of disodium hydrogen phosphate
dihydrate R, 1.741 g of sodium dihydrogen phosphate
monohydrate R, 11.688 g of sodium chloride R and 50 mg of
in the centrifugation residue by the method of sulfuric acid
sodium azide R in 1 L of water R.
Flow rate 0.5 mL/min. Where the bacterial endotoxin test is used, the preparation to
Detection Spectrophotometer at 280 nm. be examined contains less than 5 IU of endotoxin per
Identification of peaks In the chromatogram obtained with the millilitre.
reference solution, the principal peak corresponds to the IgG STORAGE
monomer and there is a peak corresponding to the dimer In an airtight, colourless glass container, protected from light,
with a relative retention to the principal peak of about 0.85. at the temperature stated on the label.
Identify the peaks in the chromatogram obtained with the
LABELLING
test solution by comparison with the chromatogram obtained
The label states:
with the reference solution; any peak with a retention time
— for liquid preparations, the volume of the preparation in
less than that of the dimer corresponds to polymers and
the container and the protein content expressed in grams
aggregates.
per litre;
Results In the chromatogram obtained with the test solution: — for freeze-dried preparations:
— retention time: for the monomer and for the dimer, the — the quantity of protein in the container;
retention time relative to the corresponding peak in the — the name or composition and the volume of the
chromatogram obtained with the reference solution is reconstituting liquid to be added;
1 ± 0 02; — the route of administration;
— peak area: the sum of the peak areas of the monomer and — the distribution of subclasses of IgG present in the
the dimer represent not less than 85 per cent of the total preparation;
area of the chromatogram and the sum of the peak areas — where applicable, that the preparation is suitable for use
of polymers and aggregates represents not more than in the prophylaxis of hepatitis A infection;
10 per cent of the total area of the chromatogram. This — where applicable, the anti-hepatitis A virus activity in
requirement is not applicable if albumin has been added International Units per millilitre;
as a stabiliser; for such preparations, a test for molecular- — where applicable, the amount of albumin added as a
size distribution is carried out during manufacture before stabiliser;
addition of the stabiliser. — where applicable, the name and amount of antimicrobial
Antibody to hepatitis B surface antigen preservative in the preparation;
Minimum 0.5 IU per gram of immunoglobulin, determined — the maximum content of immunoglobulin A.
by a suitable immunochemical method (2.7./). ____ _______________________________ _______________ ________ PnEa
Antibody to hepatitis A virus
If intended for use in the prophylaxis of hepatitis A, it
complies with the following additional requirement.
Determine the antibody content by comparison with a
reference preparation calibrated in International Units, using
Normal Immunoglobulin for ** *1
an immunoassay of suitable sensitivity and specificity (2.7./). Intravenous Use *****
The International Unit is the activity contained in a stated (Human Normal Immunoglobulin for Intravenous
amount of the International Standard for anti-hepatitis A Administration, Ph Eur monograph 0918)
immunoglobulin. The equivalence in International Units of Ph Eur________________________________ _—————
the International Standard is stated by the World Health
Organization. DEFINITION
Human normal immunoglobulin for intravenous
Human hepatitis A immunoglobulin BRP is calibrated in
administration is a sterile liquid or freeze-dried preparation
International Units by comparison with the International
containing immunoglobulins, mainly
Standard.
immunoglobulin G (IgG). Other proteins may be present.
The stated potency is not less than 100 lU/mL. Human normal immunoglobulin for intravenous
The estimated potency is not less than the stated potency. administration contains the IgG antibodies of normal
The confidence limits (P = 0.95) are not less than subjects. This monograph does not apply to products
80 per cent and not more than 125 per cent of the estimated intentionally prepared to contain fragments or chemically
potency. modified IgG.
Immunoglobulin A Human normal immunoglobulin for intravenous
As determined by a suitable immunochemical method administration is obtained from plasma that complies with
(2.7./), the content of immunoglobulin A is not greater ±an the requirements of the monograph Human plasma for
the maximum content stated on the label. fractionation (0853). The preparation may contain excipients
Water such as stabilisers.
PRODUCTION
determination of water (2.5./2), loss on drying (2.232) or
The method of preparation includes a step or steps that have
near-infrared spectroscopy (2.2.40), the water content is
been shown to remove or to inactivate known agents of
infection; if substances are used for inactivation of viruses, it
Sterility (2.6. /) shall have been shown that any residues present in the final
It complies with the test. product have no adverse effects on the patients treated with
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14) the immunoglobulin. The method of preparation also
It complies with the test for pyrogens or, preferably and includes a step or steps that have been shown co remove
where justified and authorised, with a validated in vitro test thrombosis-generating agents. Emphasis is given to the
such as the bacterial endotoxin test. identification of activated coagulation factors and their
For the pyrogen test, inject 1 mL per kilogram of the rabbit's zymogens and process steps that may cause their activation.
mass. Consideration is also to be given to other procoagulant
agents that could be introduced by the manufacturing Total protein

process. The preparation contains not less than 30 g/L and between
The product shall have been shown, by suitable tests in 90 per cent and 110 per cent of the quantity of protein
animals and evaluation during clinical trials, to be well stated on the label.
tolerated when administered intravenously. Dilute the preparation to be examined with a 9 g/L solution
Human normal immunoglobulin for intravenous of sodium chloride R to obtain a solution containing about
administration is prepared from pooled material from not 15 mg of protein in 2 mL. To 2.0 mL of this solution in a
fewer than 1000 donors by a method that has been shown to round-bottomed centrifuge tube add 2 mL of a 75 g/L
yield a product that: solution of sodium molybdate R and 2 mL of a mixture of
— does not transmit infection; 1 volume of nitrogen-free sulfuric acid R and 30 volumes of
at an immunoglobulin concentration of 50 g/L, contains water R. Shake, centrifuge for 5 min, decant the supernatant
antibodies for at least 2 of which (1 viral and 1 bacterial) and allow the inverted tube to drain on filter paper.
an International Standard or Reference Preparation is Determine the nitrogen in the centrifugation residue by the
available, the concentration of such antibodies being at method of sulfuric acid digestion (2.5.9) and calculate the
least 3 times that in the initial pooled material; content of protein by multiplying the result by 6.25.
has a defined distribution of immunoglobulin G Protein composition
subclasses; Zone electrophoresis (2.2.5/).
complies with the test for Fc function of immunoglobulin Use strips of suitable cellulose acetate gel or suitable agarose
(2.7.9); gel as the supporting medium and barbital buffer solution
— does not exhibit thrombogenic (procoagulant) activity. pH 8.6 Rl as the electrolyte solution.
Human normal immunoglobulin for intravenous If cellulose acetate is the supporting material, the method
administration is prepared as a stabilised solution or as a described below can be used. If agarose gels are used, and
freeze-dried preparation. In both cases the preparation is because they are normally part of an automated system, the
passed through a bacteria-retentive filter. The preparation manufacturer's instructions are followed instead.
may subsequently be freeze-dried and the containers closed
under vacuum or under an inert gas. No antibiotic is added
9 g/L solution of sodium chloride R to an immunoglobulin
to the plasma used. No antimicrobial preservative is added
concentration of 30 g/L.
either during fractionation or at the stage of the final bulk
solution. Reference solution Reconstitute human immunoglobulin for
electrophoresis BRP and dilute with a 9 g/L solution of sodium
The stability of the preparation is demonstrated by suitable
chloride R to a protein concentration of 30 g/L.
tests carried out during development studies.
CHARACTERS or apply 0.4 pL per millimetre if a narrower strip is used.
Appearance To another strip apply in the same manner the same volume
— liquid preparation', clear or slightly opalescent and of the reference solution. Apply a suitable electric field such
colourless or pale yellow liquid; that the albumin band of normal human serum applied on a
— freeze-dried preparation', hygroscopic, white or slightly control strip migrates at least 30 mm. Stain the strips with
yellow powder or solid friable mass. amido black 10B solution R for 5 min. Decolourise with a
For the freeze-dried preparation, reconstitute as Slated on the label mixture of 10 volumes of glacial acetic acid R and 90 volumes
immediately before carrying out the identification and the tests, of methanol R so ±at the background is just free of colour.
except those for solubility and water. Develop the transparency of the strips with a mixture of
19 volumes of glacial acetic acid R and 81 volumes of
IDENTIFICATION
methanol R. Measure the absorbance of the bands at 600 nm
Examine by a suitable immunoelectrophoresis technique.
in an instrument having a linear response over the range of
Using antiserum to normal human serum, compare normal
measurement. Calculate the result as the mean of
human serum and the preparation to be examined, both
3 measurements of each strip.
diluted to contain 10 g/L of protein. The main component of
the preparation to be examined corresponds to the IgG System suitability In the electropherogram obtained with the
component of normal human serum. The preparation to be reference solution, the proportion of protein in the principal
examined may show the presence of small quantities of other band is within the limits stated in the leaflet accompanying
plasma proteins; if human albumin has been added as a the reference preparation.
stabiliser, it may be seen as a major component. Results In the electropherogram obtained with the test
solution, not more than 5 per cent of protein has a mobility
TESTS
different from that of the principal band. This limit is not
Solubility applicable if albumin has been added to the preparation as a
For the freeze-dried preparation, add to the container the stabiliser; for such preparations, a test for protein
volume of the liquid stated on the label at the recommended composition is carried out during manufacture before
temperature. The preparation dissolves completely within addition of the stabiliser.
30 min at 20-25 °C.
Molecular size distribution
pH (2.2.5) Size exclusion chromatography (2.2.50).
4.0 to 7.4.
Dilute the preparation to be examined with a 9 g/L solution 9 g/L solution of sodium chloride R to a concentration suitable
of sodium chloride R to obtain a solution containing 10 g/L of for the chromatographic system used. A concentration in the
protein. range of 4-12 g/L and injection of 50-600 pg of protein are
Osmolality (2.2.55) usually suitable.
Reference solution Dilute human immunoglobulin (molecular Sterility (2.6.7)

size) BRP with a 9 g/L solution of sodium chloride R to the It complies with the test.
same protein concentration as the test solution. Pyrogens (2.6.2) or Bacterial endotoxins (2.6.14)
Column: It complies with the test for pyrogens or, preferably and
— size: I — 0.6 m, 0 = 7.5 mm, or l ะะะ 0.3 m, 0 = 7.8 mm; where justified and authorised, with a validated in vitro test
— stationary phase: hydrophilic silica gel for chromatography R such as the bacterial endotoxin test.
of a grade suitable for fractionation of globular proteins For the pyrogen test, inject per kilogram of the rabbit's mass
with relative molecular masses in the range 10 000 to a volume equivalent to 0.5 g of immunoglobulin, but not
500 000. more than 10 mL per kilogram of the rabbit's mass.
Mobile phase Dissolve 4.873 g of disodium hydrogen phosphate Where the bacterial endotoxin test is used, the preparation to
dihydrate R, 1.741 g of sodium dihydrogen phosphate be examined contains less than 0.5 IU of endotoxin per
monohydrate R, 11.688 g of sodium chloride R and 50 mg of millilitre for solutions with a protein content not greater than
sodium azide R in 1 L of water R. 50 g/L, and less than 1.0 IU of endotoxin per millilitre for
Flow rate 0.5 mL'min. solutions with a protein content greater than 50 g/L but not
Detection spectrophotometer at 280 nm. greater than 100 g/L.
Identification of peaks In the chromatogram obtained with the STORAGE
reference solution, the principal peak corresponds to the IgG Liquid preparation: in a colourless glass container, protected
monomer and there is a peak corresponding to the dimer from light, at the temperature stated on the label.
with a relative retention to the principal peak of about 0.85; Freeze-dried preparation: in an airtight colourless glass
identify the peaks in the chromatogram obtained with the test container, protected from light, at a temperature not
solution by comparison with the chromatogram obtained exceeding 25 °C.
shorter than that of the dimer corresponds to polymers and
LABELLING
aggregates. The label states:
— for liquid preparations, the volume of the preparation in
Results In the chromatogram obtained with the test solution:
the container and the protein content expressed in grams
— retention time: for the monomer and for the dimer, the
per litre;
retention time relative to the corresponding peak in the
— for freeze-dried preparations, the quantity of protein in
chromatogram obtained with the reference solution is
the container;
1 ± 0.02;
— the amount of immunoglobulin in the container;
— peak area: the sum of the peak areas of the monomer and
— the route of administration;
the dimer represent not less than 90 per cent of the total — for freeze-dried preparations, the name or composition
area of the chromatogram and the sum of the peak areas
and the volume of the reconstituting liquid to be added;
of polymers and aggregates represents not more than
— the distribution of subclasses of immunoglobulin G
3 per cent of the total area of the chromatogram. This
present in tile preparation;
requirement does not apply to products where albumin
— where applicable, the amount of albumin added as a
has been added as a stabiliser; for products stabilised with
stabiliser;
albumin, a test for distribution of molecular size is carried
— the maximum content of immunoglobulin A.
out during manufacture before addition of the stabiliser.
Anticomplementary activity (2.6.17)
The consumption of complement is not greater than
50 per cent (1 CH50 per milligram of immunoglobulin).
Prekallikrein activator (2.6.75) Normal Immunoglobulin for ★ ;
Maximum 35 lU/mL, calculated with reference to a dilution
of the preparation to be examined containing 30 g/L of
Subcutaneous Administration *****
immunoglobulin. (Human Normal Immunoglobulin for Subcutaneous
Administration, Ph. Eur. monograph 2788)
Anti-A and anti-B haemagglutinins (2.6.20, Method B)
Ph Eur----------------------------------------------------- --- ---------------------------------- ----------------
It complies with the test for anti-A and anti-B
haemagglutinins (direct method). DEFINITION
Anti-D antibodies (2.6.26) Sterile liquid or freeze-dried preparation containing
It complies with the test for anti-D antibodies in human immunoglobulins, mainly immunoglobulin G (IgG). Other
immunoglobulin. proteins may be present. Human normal immunoglobulin for
subcutaneous administration contains the IgG antibodies of
Antibody to hepatitis B surface antigen
normal subjects and is intended for subcutaneous
Minimum 0.5 IU per gram of immunoglobulin, determined
administration. The preparation may contain excipients such
by a suitable immunochemical method (2.7.7).
as stabilisers.
Immunoglobulin A This monograph does not apply to products intentionally
As determined by a suitable immunochemical method prepared to contain fragments of IgG or chemically modified
(2.7.7), the content of immunoglobulin A is not greater ±an
IgG.
the maximum content stated on the label.
Human normal immunoglobulin for subcutaneous
Water administration is obtained from plasma that complies with
Determined by a suitable method, such as the semi-micro the monograph Human plasma for fractionation (0853).
determination of water (2.5.72), loss on drying (2.2.22) or
PRODUCTION
near-infrared spectroscopy (2.2.40), the water content is
The method of preparation includes a step or steps that have
been shown to remove or to inactivate known agents of
“jfecôn’ if substances are used for inactivation of viruses, it TESTS

shall have been shown that any residues present in the final Solubility
product have no adverse effects on the patients treated with For the freeze-dried preparation, to a container of the
the immunoglobulin. preparation to be examined add the volume of the liquid
The method of preparation also includes a step or steps that stated on the label at the recommended temperature.
have been shown to remove thrombosis-generating agents. The preparation dissolves completely within 20 min at
Emphasis is given to the identification of activated 20-25 °C
coagulation factors and their zymogens and process steps that pH (2.2.5)
may cause their activation. Consideration is also to be given 4.6 to 7.2.
to other procoagulant agents that could be introduced by the
manufacturing process.
of sodium chloride R to obtain a protein concentration of
The product shall have been shown, by suitable tests in 10 g/L.
animals and evaluation during clinical trials, to be well
tolerated when administered subcutaneously. Any stabilising
Total protein
The preparation has a protein concentration of not less than
agent used shall have been shown to have no deleterious
100 g/L and not more than 220 g/L and contains not less
effect on the final product in the amount present.
Human normal immunoglobulin for subcutaneous quantity of protein stated on the label.
administration is prepared from pooled material from not
fewer than 1000 donors by a method that has been shown to Dilute the preparation to be examined with a 9 g/L solution
yield a product that: of sodium chloride R to obtain a protein concentration of
about 7.5 mg/mL. Place 2.0 mL of this solution in a round-
— does not transmit infection;
bottomed centrifuge tube and add 2 mL of a 75 g/L solution
— at a protein concentration of 50 g/L, contains at least 2
of sodium molybdate R and 2 mL of a mixture of 1 volume of
antibodies (1 viral and 1 bacterial) for which an
International Standard or reference preparation is
available, the concentration of such antibodies being at
least 3 times that in the initial pooled material;
in the centrifugation residue by the method of sulfuric acid
has a defined distribution of IgG subclasses^
digestion (2.5.9) and calculate the content of protein by
complies with the test for Fc function of immunoglobulin
multiplying the result by 6.25.
(2.7.9);
does not exhibit thrombogenic (procoagulant) activity. Protein composition
Zone electrophoresis (2.2.31).
administration is prepared as a stabilised solution, for Use strips of suitable cellulose acetate gel or suitable agarose
example in a 9 g/L solution of sodium chloride, a 22.5 g/L gel as the supporting medium and barbital buffer solution
solution of glycine or, if the preparation is to be freeze-dried, pH 8.6 Rl as the electrolyte solution.
a 60 g/L solution of glycine. No antibiotic is added to the If cellulose acetate is the supporting material, the method
plasma used. Preparations do not contain an antimicrobial described below can be used. If agarose gels are used, and
preservative. The solution is passed through a bacteria- because they are normally part of an automated system, the
retentive filter. The preparation may subsequendy be freeze- manufacturer’s instructions are followed instead.
dried and the containers closed under vacuum or under an Test solution Dilute the preparation to be examined with a
inert gas. 9 g/L solution of sodium chloride R to obtain a protein
The stability of the preparation is demonstrated by suitable concentration of 30 g/L.
tests carried out during development studies. Reference solution Reconstitute human immunoglobulin for
CHARACTERS electrophoresis BRP and dilute with a 9 g/L solution of sodium
Appearance chloride R to obtain a protein concentration of 30 g/L.
— liquid preparation', clear or slightiy opalescent, colourless or To a strip apply 4.0 J1L of the test solution as a 10 mm band
pale-yellow or light-brown liquid; during storage it may or apply 0.4 |1L per millimetre if a narrower strip is used.
show formation of slight turbidity or a small amount of To another strip apply in the same manner the same volume
visible particulate matter; of the reference solution. Apply a suitable electric field such
— freeze-dried preparation', white or slighdy yellow powder or that the albumin band of normal human serum applied on a
solid friable mass, hygroscopic. control strip migrates at least 30 mm. Stain the strip with
For the freeze-dried preparation, reconstitute as stated on the label amido black 10B solution R for 5 min. Decolourise with a
immediately before carrying out the identification and the tests, mixture of 10 volumes of glacial acetic acid R and 90 volumes
except those for solubility and water. of methanol R so that the background is just free of colour.
Develop the transparency of the strips with a mixture of
IDENTIFICATION 19 volumes of glacial acetic acid R and 81 volumes of
Examine by a suitable immuno electrophoresis technique. methanol R. Measure the absorbance of the bands at 600 nm
Using antiserum to normal human serum, compare normal in an instrument having a linear response over the range of
human serum and the preparation to be examined, both measurement. Calculate the result as the mean of
diluted to obtain a protein concentration of 10 g/L. 3 measurements of each strip.
The main component of the preparation to be examined System suitability In the electropherogram obtained with the
corresponds to the IgG component of normal human serum. reference solution, the proportion of protein in the principal
The preparation to be examined may show the presence of band is within ±e limits stated in the leaflet accompanying
small quantities of other plasma proteins; if human albumin the reference preparation.
has been added as a stabiliser, it may be seen as a
Results In the electropherogram obtained with the test
component. solution, not more than 10 per cent of protein has a mobility
different from ±at of the principal band. This limit is not Water
applicable if albumin has been added to the preparation as a Determined by a suitable method, such as the semi-micro
stabiliser; for such preparations, a test for protein determination of water (2.5.12), loss on drying (2.2.32) or
composition is carried out during manufacture before near-infrared spectroscopy (2.2.40), the water content is
addition of the stabiliser. within the limits approved by the competent authority.
Molecular-size distribution Sterility (2.6.1)
Size-exclusion chromatography (2.2.30). It complies with the test.
Test solution Dilute the preparation to be examined with a Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
9 g/L solution of sodium chloride 7? to a concentration suitable It complies with the test for pyrogens or, preferably and
for the chromatographic system used. A concentration in the where justified and authorised, with a validated in vitro test
range of 4-12 g/L and injection of 50-600 pg of protein are such as the bacterial endotoxin test.
usually suitable. For the pyrogen test, inject 1 mL per kilogram of the rabbit’s
Reference solution Dilute human immunoglobulin (molecular mass.
size) BRP with a 9 g/L solution of sodium chloride R to the Where the bacterial endotoxin test is used, the preparation to
same protein concentration as the test solution. be examined contains less than 5 IU of endotoxin per
Column: millilitre.
0 = 7.8 mm]; STORAGE

— stationary phase: hydrophilic silica gel for chromatography R, In an airtight, colourless glass container, protected from light,
of a grade suitable for fractionation of globular proteins at the temperature stated on the label.
with relative molecular masses in the range 10 000 to LABELLING
500 000. The label states:
Mobile phase Dissolve 4.873 g of disodium hydrogen phosphate — for liquid preparations, the volume of the preparation in
dihydrate R, 1.741 g of sodium dihydrogen phosphate the container and the protein content expressed in grams
monohydrate R, 11.688 g of sodium chloride R and 50 mg of per litre;
sodium azide R in 1 L of water R. — for freeze-dried preparations:
Flow rate 0.5 mlVmin. — the quantity of protein in the container;
— the name or composition and die volume of the
reconstituting liquid to be added;
Identification of peaks In the chromatogram obtained with the — the route of administration;
reference solution, the principal peak corresponds to the IgG — the distribution of subclasses of IgG present in the
monomer and there is a peak corresponding to the dimer preparation;
with a relative retention to the principal peak of about 0.85. — where applicable, the amount of albumin added as a
Identify the peaks in the chromatogram obtained with the stabiliser;
test solution by comparison with the chromatogram obtained — die maximum content of immunoglobulin A.
less than that of the dimer corresponds to polymers and
aggregates.
Results In the chromatogram obtained with the test solution:
— retention time: for the monomer and for the dimer, the
retention time relative to the corresponding peak in the
chromatogram obtained with the reference solution is
Anti-D (Rho) Immunoglobulin ★ ไ
1 ± 0.02; (Human Anti-D Immunoglobulin, *
— peak area: the sum of the peak areas of the monomer and Ph Eur monograph 0557)
the dimer represent not less than 85 per cent of the total Ph Eur------------------------------------------------------------------------------------------------------- -—
area of the chromatogram and the sum of the peak areas
of polymers and aggregates represents nor more than DEFINITION
10 per cent of the total area of the chromatogram. This Sterile liquid or freeze-dried preparation containing
requirement is not applicable if albumin has been added immunoglobulins, mainly immunoglobulin G.
as a stabiliser; for such preparations, a test for molecular- The preparation is intended for intramuscular administration.
size distribution is carried out during manufacture before It contains specific antibodies against erythrocyte D-antigen
addition of the stabiliser. and may also contain small quantities of other blood-group
antibodies. Human normal immunoglobulin for intramuscular
Anti-A and anti-B haemagglutinins (2.6.20, Method B) administration (0338) and/or Human albumin solution (0255)
may be added.
Anti-D antibodies (2.6.26) It complies with the monograph Human normal
It complies with the test. immunoglobulin for intramuscular administration (0338), except
Antibody to hepatitis B surface antigen for the minimum number of donors and the minimum total
Minimum 0.5 IU per gram of immunoglobulin, determined protein content.
by a suitable immunochemical method (2.7.1). The assay of human anti-D immunoglobulin (2.7.13) is
Immunoglobulin A carried out, as prescribed below under Potency.
As determined by a suitable immunochemical method For products prepared by a method that eliminates
(2.7.1), the content of immunoglobulin A is not greater than immunoglobulins with specificities other than anti-D, where
the maximum content stated on the label. authorised, the test for antibodies to hepatitis B surface
antigen is not required.
PRODUCTION
Human anti-D immunoglobulin is preferably obtained from
Anti-D Immunoglobulin for ; *
the plasma of donors with a sufficient titre of previously Intravenous Use *****
acquired anti-D antibodies. Where necessary, in order to (Human Anti-D Immunoglobulin for Intravenous
ensure an adequate supply of human anti-D Administration, Ph Eur monograph 1527)
immunoglobulin, it is obtained from plasma derived from
Ph Eur______________________________________________________________
donors immunised with D-positivc erythrocytes that are
compatible in relevant blood group systems in order to avoid DEFINITION
formation of undesirable antibodies. Sterile liquid or freeze-dried preparation containing
immunoglobulins, mainly immunoglobulin G. It contains
ERYTHROCYTE DONORS
specific antibodies against erythrocyte D-antigen and may
Erythrocyte donors comply with the requirements for donors
also contain small quantities of other blood-group antibodies.
prescribed in the monograph Human plasma for
Human normal immunoglobulin for intravenous
fractionation (0853).
administration (0918) and/or Human albumin solution (0255)
IMMUNISATION may be added.
Immunisation of the plasma donor is carried out under It complies with the monograph Human normal
proper medical supervision. Recommendations concerning immunoglobulin for intravenous administration (0918), except
donor immunisation, including testing of erythrocyte donors, for the minimum number of donors, the minimum total
have been formulated by the World Health Organization protein content, the limit for osmolality and the limit for
{Requirements for the collection, processing and quality control of prekallikrein activator.
blood, blood components and plasma derivatives, WHO The test for anti-D antibodies (2.6.26) prescribed in the
Technical Report Series, No. 840, 1994 or subsequent monograph Human normal immunoglobulin for intravenous
revision).
administration (0918) is not carried out, since it is replaced by
POOLED PLASMA the assay of human anti-D immunoglobulin {2.7.13) as
To limit the potential B19 virus burden in plasma pools used prescribed below under Potency.
for the manufacture of anti-D immunoglobulin, the plasma For products prepared by a method that eliminates
pool is tested for B19 virus using validated nucleic acid immunoglobulins with specificities other than anti-D, where
amplification techniques {2.6.21). authorised, the test for antibodies to hepatitis B surface
B19 virus DNA antigen is not required; a suitable test for Fc function is
Maximum 10.0 lU/pL. carried out instead of that described in general chapter 2.7.9,
A positive control with 10.0 IU of B19 virus DNA per which is not applicable to such a product.
microlitre and, to test for inhibitors, an internal control PRODUCTION
prepared by addition of a suitable marker to a sample of the Human anti-D immunoglobulin is preferably obtained from
plasma pool are included in the test. The test is invalid if the the plasma of donors with a sufficient titre of previously
positive control is non-reactive or if the result obtained with acquired anti-D antibodies. Where necessary, in order to
the internal control indicates the presence of inhibitors. ensure an adequate supply of human anti-D
B19 virus DNA for NAT testing BRP is suitable for use as a immunoglobulin, it is obtained from plasma derived from
positive control. donors immunised with D-positive erythrocytes that are
If Human normal immunoglobulin for intramuscular compatible in relevant blood group systems in order to avoid
administration (0338) and/or Human albumin solution (0255) formation of undesirable antibodies.
are added to the preparation, the plasma pool or pools from ERYTHROCYTE DONORS
which they are derived comply with the above requirement Erythrocyte donors comply with the requirements for donors
for B19 virus DNA. prescribed in the monograph Human plasma for
POTENCY fractionation (0853).
Human anti-D immunoglobulin {2.7.13, Method A) IMMUNISATION
The estimated potency is not less than 90 per cent of the Immunisation of the plasma donor is carried out under
stated potency. The confidence limits {P = 0.95) are not less proper medical supervision. Recommendations concerning
than 80 per cent and not more than 120 per cent of the donor immunisation, including testing of erythrocyte donors,
estimated potency. have been formulated by the World Health Organization
Method B or c (2.7.13) may be used for potency {Requirements for the collection, processing and quality control of
determination if a satisfactory correlation with the results blood, blood components and plasma derivatives, WHO
obtained by Method A has been established for the particular Technical Report Series, No. 840, 1994 or subsequent
product. revision).
STORAGE POOLED PLASMA
See Human normal immunoglobulin for intramuscular To limit the potential B19 virus burden in plasma pools used
administration (0338). for the manufacture of anti-D immunoglobulin, the plasma
pool is tested for Bl9 virus using validated nucleic acid
LABELLING amplification techniques {2.6.21).
See Human normal immunoglobulin for intramuscular
administration (0338). B19 virus DNA
Maximum 10.0 IU/jiL.
The label states the number of International Units per
A positive control with 10.0 IU of B19 virus DNA per
container.
microlitre and, to test for inhibitors, an internal control
____________________________________________________ __________ Ph Eur
prepared by addition of a suitable marker to a sample of the
plasma pool are included in the test. The test is invalid if the The stated potency is not less than 600 lU/mL.
positive control is non-reactive or if the result obtained with The estimated potency is not less than the stated potency.
the internal control indicates the presence of inhibitors. The confidence limits (P = 0.95) are not less than
B19 virus DNA for NAT testing BRP is suitable for use as a 80 per cent and not more than 125 per cent of the estimated
positive control. potency.
If Human normal immunoglobulin for intravenous STORAGE
administration (0918) and/or Human albumin solution (0255) See Human normal immunoglobulin for intramuscular
are added to the preparation, the plasma pool or pools from administration (0338).
which they are derived comply with the above requirement
LABELLING
for B19 virus DNA.
ASSAY administration (0338).
Human anti-D immunoglobulin (2.7.13, Method A) The label states the number of International Units per
The estimated potency is not less than 90 per cent of the container.
stated potency. The confidence limits (P = 0.95) are not less _______________________Ph Elf
estimated potency.
Method B or c (2.7.13) may be used for potency
determination if a satisfactory correlation with the results
obtained by Method A has been established for the particular Hepatitis B Immunoglobulin t \
product.
(Human Hepatitis B Immunoglobulin,
STORAGE Ph Eur monograph 0722)
See Human normal immunoglobulin for intravenous
Ph Eur__ __________________ -__________
administration (0918).
DEFINITION
LABELLING
immunoglobulins, mainly immunoglobulin G.
The preparation is intended for intramuscular or
The label states the number of International Units per subcutaneous administration. It is obtained from plasma
container. from selected and/or immunised donors having antibodies
_______________________________________________________________Ph Eur against hepatitis B surface antigen. Human normal
immunoglobulin for intramuscular administration (0338) or
administration (2788) may be added.
Depending on the route of administration, it complies with
Hepatitis A Immunoglobulin * * the monograph on Human normal immunoglobulin for
(Human Hepatitis A Immunoglobulin, * ** intramuscular administration (0338) or Human normal
Ph Eur monograph 0769) immunoglobulin for subcutaneous administration (2788), except
for the minimum number of donors and the minimum total
PhEir______________________________________________________________
protein content.
DEFINITION
POTENCY
The potency is determined by comparing the antibody titre
of the immunoglobulin to be examined with that of a
The preparation is intended for intramuscular administration.
It is obtained from plasma from selected donors having
an immunoassay of suitable sensitivity and specificity (2.7./).
antibodies against hepatitis A virus. Human normal
immunoglobulin for intramuscular administration (0338) may be The International Unit is the activity contained in a stated
added. amount of the International Standard for anti-hepatitis B
immunoglobulin. The equivalence in International Units of
It complies with the monograph on Human normal
immunoglobulin for intramuscular administration (0338), except
Organization.
protein content. Human hepatitis B immunoglobulin BRP is calibrated in
POTENCY Standard.
The stated potency is not less than 100 lU/mL.
of the immunoglobulin to be examined with that of a
The estimated potency is not less than the stated potency.
an immunoassay of suitable sensitivity and specificity (2.7./). 80 per cent and not more than 125 per cent of the estimated
The International Unit is the activity contained in a stated potency.
amount of the International Standard for anti-hepatitis A
immunoglobulin. The equivalence in International Units of STORAGE
the International Standard is stated by the World Health See Human normal immunoglobulin for intramuscular
Organization. administration (0338) or Human normal immunoglobulin for
subcutaneous administration (2788).
Human hepatitis A immunoglobulin BRP is calibrated in
Standard.
LABELLING
See Human normal immunoglobulin for intramuscular Measles Immunoglobulin f **
administration (0338) QT Human normal immunoglobulin for (Human Measles Immunoglobulin, ***
subcutaneous administration (2788). Ph Eur monograph 0397)
The label states the number of International Units per Ph Eur___________________________________________________________ __
container.
DEFINITION
—------------------ --------------------- ------------------------------------------------------------ Ph Eur
immunoglobdins, maidy immunoglobdin G.
The preparation is intended for intramuscdar administration.
It is obtained from plasma containing specific antibodies
Hepatitis B Immunoglobulin for ** ** against measles virus. Human normal immunoglobulin for
intramuscular administration (0338) may be added.
Intravenous Use ***** It complies wi± the monograph on Human normal
(Human Hepatitis B Immunoglobulin for Intravenous immunoglobulin for intramuscular administration (0338), except
Administration, Ph Eur monograph 1016) for the minimum number of donors and the minimum total
Ph Eur______________ protein content.
DEFINITION POTENCY
Sterile liquid or freeze-dried preparation containing The potency of the liqdd preparation and of the freeze-dried
immunoglobulins, mainly immunoglobulin G. It is obtained preparation after reconstitution as stated on the label is not
from plasma from selected and/or immunised donors having less than 50 IU per millilitre of neutralising antibody against
antibodies against hepatitis B surface antigen. Human normal measles virus.
immunoglobulin for intravenous administration (0918) may be The potency is determined by comparing the antibody titre
added. of the immunoglobdin to be examined with that of a
It complies with the monograph Human normal reference preparation calibrated in International Units, using
immunoglobulin for intravenous administration (0918), except a challenge dose of measles virus in a sdtable cell cdture
for the minimum number of donors, the minimum total system. A method of equal sensitivity and precision may be
protein content and the limit for osmolality. used providing that the competent authority is satisfied that it
correlates with neutralising activity for the measles virus by
POTENCY comparison with the reference preparation.
The International Unit is the specific neutralising activity for
of the immunoglobdin to be examined with that of a
measles virus contained in a stated amount of the
International Standard for human anti-measles serum.
an immunoassay (2.7.1') of suitable sensitivity and specificity.
The International Unit is the activity contained in a stated Reference Preparation is stated by the World Health
amount of the International Standard for anti-hepatitis B Organization.
immunoglobulin. The equivalence in International Units of
Method
Organization. Prepare serial 2-fold dilutions of the immunoglobdin to be
examined and of the reference preparation. Mix each dilution
Human hepatitis B immunoglobulin BRP is calibrated in
with an equal volume of a suspension of measles virus
containing about 100 CCID50 in 0.1 mL and incubate
Standard.
protected from light at 37 °C for 2 h. Using not fewer than 6
The stated potency is not less than 50 IU/mL. The estimated cell cdtures per mixture, inoculate 0.2 mL of each mixture
potency is not less than the stated potency. The confidence into each of the cell cdtures allocated to that mixture and
limits (P = 0.95) are not less than 80 per cent and not more incubate for not less than 10 days. Examine the cdtures for
than 125 per cent of the estimated potency. viral activity and compare the dilution containing the smallest
STORAGE quantity of the immunoglobdin which neutralises the virus
See Human normal immunoglobulin for intravenous with that of the corresponding dilution of ±e reference
administration (0918). preparation.
Calcdate the potency of the immunoglobdin to be examined
LABELLING
in International Units per millilitre of neutralising antibody
against measles virus.
The label states the minimum number of International Units STORAGE
of hepatitis B immunoglobulin per container. See Human normal immunoglobulin for intramuscular
______________________________________________________________ Ph Eur
LABELLING
container.
Ph
Human a-1 -proteinase Inhibitor ** ** CHARACTERS

Appearance
(Ph. Eur. monograph 2387) *** — liquid preparations-, clear or slightly opalescent, colourless
PhEur_________________________________________________________ ____ or pale yellow or pale green or pale brown;
— freeze-dried preparations-, powders or solid friable masses,
DEFINITION hygroscopic, white or pale yellow or pale brown.
Sterile liquid or freeze-dried preparation of a plasma protein
If the preparation to be examined is freeze-dried, reconstitute it as
fraction containing mainly human a-1-proteinase inhibitor
stated on the label immediately before carrying out the
(also known as human a-1-antitrypsin or a-1-antiproteinase).
identification, tests (except those for solubility and water) and
Human a-1-proteinase inhibitor is a glycoprotein existing in
assay.
isoforms with different isoelectric points and is the most
abundant multifunctional serine proteinase inhibitor in IDENTIFICATION
human plasma. It complies with the limits of the assay.
It is obtained from human plasma that complies with the TESTS
monograph Human plasma for fractionation (0853)3 using a pH (2.2.2)
suitable fractionation process and further purification steps. 6.5 to 7.8.
Other plasma proteins may be present. The preparation may
Solubility'
contain excipients such as stabilisers.
For freeze-dried preparations, add to a container of the
PRODUCTION preparation to be examined the volume of the liquid stated
GENERAL PROVISIONS on the label at room temperature. The preparation dissolves
The method of preparation is designed to maintain completely, giving a clear, colourless or pale green or pale
functional integrity of a-1-proteinase inhibitor. It includes a yellow or pale brown solution.
step or steps that have been shown to remove or to inactivate Osmolality (2.2.25)
known agents of infection. The subsequent purification Minimum 210 mosmol/kg.
concentration of any substances used for inactivation of
Total protein
viruses during production is reduced to a suitable level and
of sodium chloride R to obtain a protein concentration of
that any residues are such as not to compromise the safety of
about 7.5 mg/mL. To 2.0 mL of this solution in a round-
the preparation for patients.
bottomed centrifuge tube, add 2 mL of a 75 g/L solution of
The specific activity is not less than 0.35 mg of active human sodium molybdate R and 2 mL of a mixture of 1 volume of
a-1-proteinase inhibitor per milligram of total protein. nitrogen-free sulfuric acid R and 30 volumes of water R. Shake,
The ratio of human a-1 proteinase inhibitor activity to centrifuge for 5 min, decant the supernatant and allow the
human a-1-proteinase inhibitor antigen is not less than 0.7. inverted tube to drain on filter paper. Determine the nitrogen
No antimicrobial preservative or antibiotic is added. in the residue by the method of sulfuric acid digestion (2.5.9)
The solution is passed through a bacteria-retentive filter and and calculate the protein content by multiplying by 6.25.
distributed aseptically into the final containers. It may be
Water
subsequently freeze-dried.
CONSISTENCY OF THE METHOD OF determination of water (2.5.72), loss on drying (2.2.22) or
PRODUCTION near-infrared spectroscopy (2.2.40), tile water content is
It shall be demonstrated that the manufacturing process within the limits approved by the competent authority.
yields a product with a consistent composition. It is evaluated Sterility (2.6.1)
by suitable analytical procedures that are determined during It complies with the test.
process development, and which include:
Pyrogens (2.6.8) or Bacterial endotoxins (2.6.14)
— assay of human a-1-proteinase inhibitor activity;
It complies with the test for pyrogens or, preferably and
— determination of specific human a-1-proteinase inhibitor
where justified and authorised, with a validated in vitro test
activity, expressed as the ratio of active human a-1-
such as the bacterial endotoxin test.
proteinase inhibitor to total protein;
— characterisation of isoform composition and protein For the pyrogen test, inject per kilogram of the rabbit's mass
structure by suitable methods such as isoelectric focusing a volume equivalent to not less than 60 mg of human a-1-
(2.2.54), spectrometric methods (for example, mass proteinase inhibitor. Where the bacterial endotoxin test is
spectrometry) or capillary electrophoresis (2.2.47); used, the preparation to be examined contains less than
— determination of the ratio of human a-1-proteinase 0.08 IU of endotoxin per milligram of human a-1-proteinase
inhibitor activity to human a-1-proteinase inhibitor inhibitor.
antigen; ASSAY
— characterisation of accompanying plasma proteins that Assay of human a-1-proteinase inhibitor (2.732)
might be present, by a set of suitable me±ods such as The estimated potency is not less than 80 per cent and not
SDS-PAGE, cellulose acetate electrophoresis or capillary more than 120 per cent of the stated potency.
zone electrophoresis (2.2.31) and quantitative The confidence limits (P = 0.95) are not less than
determination of relevant accompanying plasma proteins; 80 per cent and not more than 120 per cent of the estimated
— determination of molecular-size distribution, used to potency.
quantify the polymeric forms of human a-1-proteinase
STORAGE
inhibitor; consideration is given to the potential presence
of accompanying proteins that might affect the results. In an airtight and sterile container, at a temperature not
exceeding 25 °C, unless otherwise justified and authorised-
LABELLING Prepare a series of dilutions of the viral suspension. In the

The label states: chambers of cell-culture slides (8 chambers per slide), place
the potency of active (functional) human a-1-proteinase 0.1 mL of each dilution and 0.1 mL of medium and add
inhibitor per container; 0.2 mL of the cell suspension. Incubate in an atmosphere of
the name and quantity of any added substances; carbon dioxide at 37 °C for 24 h. Carry out fixation,
the quantity of protein in the container; immunofluorescence staining and evaluation as described
— the route of administration; below. Determine the end-point titre of the seed virus
where applicable, the name and volume of the liquid to suspension and prepare the working virus dilution
be used for reconstitution; corresponding to 100 CCED50 per 0.1 mL.
that the transmission of infectious agents cannot be totally For each assay, check ±e amount of virus used by
excluded when medicinal products prepared from human performing a control titration: from the dilution
blood or plasma are administered. corresponding to 100 CCID50 per 0.1 mL, make 3 tenfold
----------------- ----------------------------- --------------------------------------------------------- Ph Eur dilutions. Add 0.1 mL of each dilution to 4 chambers
containing 0.1 mL of medium and add 0.2 mL of the cell
suspension. The test is not valid unless the titre lies between
30 CCID50 and 300 CCID50.
Dilute the reference preparation to a concentration of
Rabies Immunoglobulin ** \ 2 IU/mL using non-supplemented culture medium (stock
reference dilution, stored below -80 °C). Prepare 2 suitable
(Human Rabies Immunoglobulin, ***
Ph Eur monograph 0723) predilutions (1:8 and 1:10) of the stock reference dilution so
that the dilution of the reference preparation that reduces the
Ph Etr_____________________ ________________________________________
number of fluorescent fields by 50 per cent lies within the
DEFINITION 4 dilutions of the cell-culture slide. Add 0.1 mL of the
Sterile liquid or freeze-dried preparation containing medium to each chamber, except the first in each of 2 rows,
immunoglobulins, mainly immunoglobulin G. to which add respectively 0.2 mL of the 2 predilutions of the
The preparation is intended for intramuscular administration. stock reference dilution transferring successively 0.1 mL to
It is obtained from plasma from donors immunised against the other chambers.
rabies. It contains specific antibodies neutralising the rabies Dilute the preparation to be examined 1 in 100 using non
virus. Human normal immunoglobulin for intramuscular supplemented medium (stock immunoglobulin dilution) - to
administration (033ร) may be added. reduce to a minimum errors due to viscosity of the undiluted
It complies with the monograph on Human normal preparation - and make 3 suitable predilutions so that the
immunoglobulin for intramuscular administration (0338)3 except dilution of the preparation to be examined that reduces the
for the minimum number of donors and the minimum total number of fluorescent fields by 50 per cent lies within the
protein content. 4 dilutions of ±e cell-culture slide. Add 0.1 mL of the
medium to all the chambers except the first in each of
POTENCY 3 rows, to which add respectively 0.2 mL of the 3
The potency is determined by comparing the dose of predilutions of the stock immunoglobulin dilution. Prepare a
immunoglobulin required to neutralise the infectivity of a series of 2-fold dilutions transferring successively 0.1 mL to
rabies virus suspension with the dose of a reference the other chambers.
preparation, calibrated in International Units, required to
To all the chambers containing the dilutions of the reference
produce the same degree of neutralisation (2.7.1'). The test is
preparation and the dilutions of the preparation to be
performed in sensitive cell cultures and the presence of
examined, add 0.1 mL of the virus suspension corresponding
unneutralised virus is revealed by immunofluorescence.
to 100 CCID50 per 0.1 mL (working virus dilution), shake
The International Unit is the specific neutralising activity for manually, allow to stand in an atmosphere of carbon dioxide
rabies virus in a stated amount of the International Standard at 37 °C for 90 min, add 0.2 mL of the cell suspension,
for anti-rabies immunoglobulin. The equivalence in shake manually and allow to stand in an atmosphere of
International Units of the International Standard is stated by carbon dioxide at 37 °C for 24 h.
the World Health Organization.
After 24 h, discard the medium and remove the plastic walls.
Human rabies immunoglobulin BRP is calibrated in Wash the cell monolayer with phosphate buffered saline
International Units by comparison with the International pH 7.4 R and ±en with a mixture of 20 volumes of water R
Standard. and 80 volumes of acetone R and fix in a mixture of
Method 20 volumes of water R and 80 volumes of acetone R at
Carry out the test in suitable sensitive cells. It is usual to use -20 °C for 3 min. Spread on the slides fluorescein-conjugated
the BHK.-21 cell line, grown in the medium described below, rabies antiserum R ready for use. Allow to stand in an
between the 18th and 30th passage levels counted from the atmosphere with a high level of moisture at 37 °C for
ATCC seed lot. Harvest the cells after 2 to 4 days of growth, 30 min. Wash with phosphate buffered saline pH 7.4 R and
treat with trypsin and prepare a suspension containing dry. Examine 20 fields in each chamber at a magnification of
500 000 cells per millilitre (cell suspension). 10 min before 250 X, using a microscope equipped for fluorescence
using this suspension add 10 |ig of diethylaminoethyldextran R readings. Note the number of fields with at least
per millilitre, if necessary, to increase the sensitivity of the 1 fluorescent cell. Check the test dose used in the virus
cells. titration slide and determine the dilution of the reference
preparation and the dilution of the preparation to be
Use a fixed virus strain grown in sensitive cells, such as the
examined that reduce the number of fluorescent fields by
CVS strain of rabies virus adapted to growth in the BHK.-21
50 per cent, calculating the 2 or 3 dilutions together using
cell line (seed virus suspension). Estimate the titre of the seed
probit analysis. The test is not valid unless the statistical
virus suspension as follows.
analysis shows a significant slope of the dose-response curve

and no evidence of deviation from linearity or parallelism. Rubella Immunoglobulin * \
The stated potency is not less than 150 IU/mL. (Human Rubella Immunoglobulin, *
The estimated potency is not less than the stated potency Ph Eur monograph 0617)
and is not greater than twice the stated potency. Ph Eur_____ -________________
80 per cent and not more than 125 per cent of the estimated DEFINITION
potency. Sterile liquid or freeze-dried preparation containing
CULTURE MEDIUM FOR GROWTH OF BHK-21 The preparation is intended for intramuscular administration.
(2ELLS It is obtained from plasma containing specific antibodies
Commercially available media that have a slightly different against rubella virus. Human normal immunoglobulin for
composition from that shown below may also be used. intramuscular administration (0338) may be added.
Sodium chloride 6.4 g
It complies with the monograph on Human normal
Potassium chloride 0.40 g
Calcium chloride, anhydrous 0.20 g
Magnesium sulfate, heptahydrate 0.20 g
protein content.
Sodium dihydrogen phosphate, monohydrate 0.124 g
Glucose monohydrate 4.5 g POTENCY
Ferric nitrate, nonahydrate 0.10mg The potency is determined by comparing the activity of the
L-Arginine hydrochloride 42.0mg preparation to be examined in a suitable haemagglutination
L-Cystine 24.0mg inhibition test with that of a reference preparation calibrated
L-Histidine 16.0mg in International Units.
L-Isoleucine 52.0mg The International Unit is the activity contained in a stated
L-Leucine 52.0mg amount of the International Standard for anti-rubella
L-Lysine hydrochloride 74.0mg immunoglobulin. The equivalence in International Units of
L-Phenylalanine 33.0mg the International Reference Preparation is stated by the
L-Threonine 48.0mg World Health Organization.
L-Tryptophan 8.0mg The estimated potency is not less than 4500 IU/mL.
L-Tyrosine 36.0mg The confidence limits (P = 0.95) of the estimated potency
L-Valine 47.0mg are not less than 50 per cent and not more than 200 per cent
L-Methionine 15.0 mg of the stated potency.
L-Glutamine 0.292 g
Z-Inositol 3.60mg STORAGE
Choline chloride 2.0mg See Human normal immunoglobulin for intramuscular
Folic add 2.0mg administration (0338).
Nicotinamide 2.0mg LABELLING
Calcium pantothenate 2.0mg See Human normal immunoglobulin for intramuscular
Pyridoxal hydrochloride 2.0mg administration (0338).
Thiamine hydrochloride 2.0mg
Riboflavine 0.2mg
millilitre.
Phenol red 15.0mg
_______________________________________________ ________________ P~)E-r
Sodium hydrogen carbonate 2.75 g
Water to 1000 mL
The medium is supplemented with:
Foetal calf serum (heated at 56 °C for 30 min) 10 per cent
Tryptose phosphate broth 10 per cent Tetanus Immunoglobulin * ไ
Benzylpenicillin sodium 60 mg/L (Human Tetanus Immunoglobulin, **
Streptomycin 0.1 g/L
STORAGE Ph Eur_______________________________________________ _—————
administration (0338). DEFINITION
LABELLING immunoglobulins, mainly immunoglobulin G.
See Human normal immunoglobulin for intramuscular The preparation is intended for intramuscular administration.
administration (0338). It is obtained from plasma containing specific antibodies
The label states the number of International Units per against the toxin of Clostridium tetani. Human normal
container. immunoglobulin for intramuscular administration (0338) may be
---------------------------------------------------------------------------- ------------------------------ Ph Eur
added.
It complies with the monograph Human normal
protein content.
PRODUCTION
During development, a satisfactory relationship shall be
established between the potency determined by immunoassay
as described under Potency and that determined by means of 5.0 mL. Allow the mixtures to stand, protected from light,
the following test for toxin-neutralising capacity in mice. for 60 min. Using 6 mice for each mixture, inject
Toxin-neutralising capacity in mice The potency is determined subcutaneously a dose of 0.5 mL into each mouse. Observe
by comparing the quantity necessary to protect mice against the mice for 96 h. Mice that become paralysed may be
the paralytic effects of a fixed quantity of tetanus toxin with euthanised. The mixture that contains the largest volume of
the quantity of a reference preparation of human tetanus immunoglobdin that fails to protect the mice from paralysis
immunoglobulin, calibrated in International Units, necessary contains 1 IU. This quantity is used to calcdate the potency
to give the same protection. of de immunoglobdin in International Units per millilitre.
The International Unit of antitoxin is the specific neutralising The test is not valid udess all the mice injected with
activity for tetanus toxin contained in a stated amount of the mixtures containing 2.0 mL or less of the solution of the
International Standard, which consists of freeze-dried human reference preparation show paralysis and all those injected
immunoglobulin. The equivalence in International Units of wid mixtures containing more do not.
the International Standard is stated by the World Health POTENCY
Organization. The potency is determined by comparing de antibody titre
Human tetanus immunoglobulin BRP is calibrated in of de preparation to be examined wid dat of a reference
International Units by comparison with the International preparation calibrated in International Units, using sdtable
Standard. immunochemical medods (2.7.1) such as enzyme-linked
Method immunosorbent assay (ELISA) or toxoid inhibition assay
Selection of animals Use mice weighing 16-20 g. (TIA).
Preparation of the test toxin Prepare the test toxin by a suitable The International Unit is de activity contained in a stated
method from the sterile filtrate of a culture in liquid medium amount of de International Standard for anti-tetanus
of c. tetani. The 2 methods shown below are given as immunoglobdin. The eqdvalence in International Units of
examples and any other suitable method may be used. de International Standard is stated by de World Heald
Organization.
(1) To the filtrate of an approximately 9-day culture, add
1 -2 volumes of glycerol R and store the mixture in the liquid Human tetanus immunoglobulin BRP is calibrated in
state at a temperature slightly below 0 °C. International Units and is sdtable for use as a reference
preparation.
(2) Precipitate the toxin by addition to the filtrate of
ammonium sulfate Ry dry the precipitate in vacuo over The stated potency is not less dan 100 lU/mL of tetanus
diphosphorus pentoxide Ry reduce to a powder and store dry, antitoxin. The estimated potency is not less dan de stated
either in sealed ampoules or in vacuo over diphosphorus potency. The confidence limits (P = 0.95) are not less dan
pentoxide R. 80 per cent and not more dan 125 per cent of de estimated
potency.
Determination of test dose of toxin (LpHO dose) Prepare a
The description of medods A and B below are provided as
solution of the reference preparation in a suitable liquid such
that it contains 0.5 IU of antitoxin per millilitre. If the test examples.
toxin is stored dry, reconstitute it using a suitable liquid. Method A: direct enzyme immunoassay
Prepare mixtures of the solution of the reference preparation The amount of tetanus immunoglobdin bound to tetanus
and the test toxin such that each contains 2.0 mL of the toxoid, which is coated to a microtitre plate, is determined by
solution of the reference preparation, one of a graded series means of a peroxidase-conjugated polyclonal anti-human IgG
of volumes of the test toxin and sufficient of a suitable liquid antibody.
to bring the volume to 5.0 mL. Allow the mixtures to stand, Materials
protected from light, for 60 min. Using 6 mice for each — Phosphate-buffered saline pH 7.1 (PBS). Dissolve 0.2 g of
mixture, inject a dose of 0.5 mL subcutaneously into each potassium chloride Ry 0.2 g of potassium dihydrogen
mouse. Observe the mice for 96 h. Mice that become phosphate Ry 1.15 g of anhydrous disodium hydrogen
paralysed may be euthanised. The test dose of toxin is the phosphate R and 8.0 g of sodium chloride R in water R and
quantity in 0.5 mL of the mixture made with the smallest adjust de pH (2.2.2) if necessary. Dilute to 1000 mL
amount of toxin capable of causing, despite partial wid water R.
neutralisation by the reference preparation, paralysis in all — PBS-T. PBS containing 0.05 per cent V/V of
6 mice injected with the mixture, within the observation polysorbate 20 R.
period. — Carbonate buffer pH 9.6. Dissolve 1.4 g of anhydrous
sodium carbonate R and 3.0 g of sodium hydrogen
Determination of potency of the immunoglobulin Prepare a
carbonate R in water R and adjust de pH (2.2.5) if
necessary. Dilute to 1000 mL wid water R.
that it contains 0.5 IU of antitoxin per millilitre. Prepare a
— Tetanus toxoid. Purified and chemically inactivated tetanus
solution of the test toxin in a suitable liquid such that it
contains 5 test doses per millilitre. Prepare mixtures of the toxin.
— Microtitre plate. Use a flat-bottomed microtitre plate wid
solution of the test toxin and the immunoglobdin to be
high protein-binding capacity.
examined such that each contains 2.0 mL of the solution of
the test toxin, one of a graded series of volumes of the Method
immunoglobdin to be examined and sufficient of a sdtable Distribute 100 pL of a 0.2 Lf/mL solution of tetanus toxoid
liqdd to bring the total volume to 5.0 mL. Also prepare in carbonate buffer pH 9.6 into each of de wells of de
mixtures of the solution of the test toxin and the solution of microtitre plate. Incubate at 4 °C for approximately 18 h.
the reference preparation such that each contains 2.0 mL of Wash de plate 5 times wid PBS-T. To block unbound
the solution of the test toxin, one of a graded series binding sites add 200 pL of PBS containing 5 g/L of bovine
of volumes of the solution of the reference preparation albumin R to each of de wells and incubate for 1 h at 37 °C
centred on that volume (2.0 mL) that contains 1 IU and on a plate shaker set at 120 r/min. Wash 5 times wid PBS-T.
sufficient of a sdtable liqdd to bring the total volume to
Reconstitute the reference preparation and the preparation to Prepare each dilution directly from the 0.4 IU/mL
be examined according to the instructions. For each predilution.
preparation, prepare 2 independent predilutions of Transfer 100 pL of each dilution of the dilution series to a
0.004 lU/mL in PBS by applying several dilution steps. well of the blocked plate and add 50 pL of a 0.2 Lf/mL
Using PBS, prepare from each predilution 5 serial dilutions solution of tetanus toxoid in carbonate buffer pH 9.6 into
with a dilution factor of 1.5 resulting in a dilution series of each of the wells. Incubate for approximately 18 h at 37 c
6 dilutions in the range of 0.0005-0.004 lU/mL. Depending on a plate shaker set at 120 r/min.
on the reagents used, a small modification of the dilution
To coat the ELISA plate, distribute 100 pL of a solution of a
series might be necessary to meet the conditions of the
human tetanus immunoglobulin diluted to 1 IU/mL in
statistical model used.
carbonate buffer pH 9.6 into each of the wells of the ELISA
Apply 100 pL of each of ±e samples of the dilution series to plate. Incubate for approximately 18 h at 37 °C on a plate
the plate. Incubate for 2 h at 37 °C on a plate shaker set at shaker set at 120 r/min.
120 r/min and wash the plate 5 times with PBS-T. Apply
Day 2
100 pL of a peroxidase-conjugated anti-human IgG antibody
diluted to a suitable concentration with PBS-T containing Wash the coated ELISA plate 5 times with PBS-T. To block
5 g/L of bovine albumin R to each of the wells and incubate unbound binding sites add 200 pL of PBS containing 5 gL
for 1 h at 37 °C on a plate shaker set at 120 r/min. Wash the of bovine albumin R to each of the wells and incubate for 1 h
plate 5 times with PBS-T and apply 100 pL of a suitable at 37 cc on a plate shaker set at 120 r/min. Wash the plate
3,3',5,5'-tetramethylbenzidine (TMB) substrate to each of 5 times with PBS-T. Transfer 100 pL of each mixture of
the wells and incubate at room temperature for 10 min in the toxoid and tetanus immunoglobulin from the microtitre plate
dark. To stop the reaction, add 100 pL of a 196.2 g/L to the coated ELISA plate and incubate for 2 hours at 37 c
solution of sulfuric acid R to each of the wells. Measure the on a plate shaker set at 120 r/min. Wash the plate 5 times
absorbances at 450 nm and at the reference wavelength of with PBS-T. Add 100 pL of diluted Mab to each of the
630 nm. Calculate the potencies of the preparations by the wells, incubate the plate for 1 h at 37 °C on a plate shaker
usual statistical methods (5. ร). set at 120 r/min and wash the plate 5 times with PBS-T.
Add 100 pL of the diluted peroxidase-conjugated antibody to
Method B: indirect determination by toxoid-binding each of the wells, incubate the plate for 1 h at 37 °C on a
inhibition assay plate shaker set at 120 r/min and wash the plate 5 times
The amount of unbound toxoid in a mixture of toxoid and with PBS-T. Apply 100 pL of a suitable 3,3',5,5'-
tetanus immunoglobulin is determined by an enzyme tetramethylbenzidine (TMB) substrate to each of the wells
immunoassay and is inversely proportional to the amount of and incubate at room temperature for 10 min in the dark.
tetanus immunoglobdin present. The method is performed To stop the reaction, add 100 pL of a 196.2 g/L solution of
over 2 consecutive days. sulfuric acid R to each of the wells. Measure the absorbances
Materials at 450 nm and at die reference wavelength of 630 nm.
— Phosphate-buffered saline pH 7.1 (PBS). See under Calculate the potencies of the preparations by the usual
Method A. statistical methods (5. ร).
— PBS-T. See under Method A.
— Carbonate buffer pH 9.6. See under Method A. STORAGE
— Tetanus toxoid. See under Method A. See Human normal immunoglobulin for intramuscular
— Mab. Mouse monoclonal tetanus toxoid antibody. administration (0338).
Use according to the instructions. Prepare a suitable LABELLING
dilution of Mab, e.g. 1/5000, in PBS. See Human normal immunoglobulin for intramuscular
— Peroxidase-conjugated antibody. Peroxidase-conjugated anti administration- (0338).
mouse IgG (H-rL) antibody, affinity-purified F(ab)2 The label states the number of International Units per
fragment without cross-reactivity to human serum container.
proteins. Use according to the instructions. Prepare a
suitable dilution of the peroxidase-conjugated antibody in
PBS-T containing 5 g/L of bovine albumin R.
— Microlitre plate. Use a round-bottomed microtitre plate
with medium protein-binding capacity.
— ELISA plate. Use a flat-bottomed microtitre plate with
high protein-binding capacity.
Varicella Immunoglobulin * ไ
Method (Human Varicella Immunoglobulin, *
Day 1
PhEur___________________________________________________ __ _________
To block the protein-binding sites of the microtitre plate, add
200 pL of PBS containing 5 g/L of bovine albumin R to each DEFINITION
of the wells of the microtitre plate and incubate for 1 h at Sterile liquid or freeze-dried preparation containing
37 °C on a plate shaker set at 120 r/min. Wash the plate immunoglobulins, mainly immunoglobulin G.
5 times with PBS-T. The preparation is intended for intramuscular administration.
Reconstitute the reference preparation and the preparation to It is obtained from plasma from selected donors having
be examined according to the instructions. For each antibodies against Herpesvirus varicellae. Human normal
preparation, prepare 2 independent predilutions of immunoglobulin for intramuscular administration (0338) may be
0.4 IU/mL in PBS by applying several dilution steps. Prepare added.
from each predilution a dilution series of dilutions It complies with the monograph on Human normal
containing 0.04 IU/mL, 0.10 IU/mL, 0.12 IU/mL, immunoglobulin for intramuscular administration (0338) except
0.14 IU/mL, 0.16 IU/mL, 0.18 IU/mL and 0.20 IU/mL. for the minimum number of donors, the minimum total
protein content and, where authorised, the test for antibody LABELLING
to hepatitis B surface antigen. See Human normal immunoglobulin for intravenous
POTENCY administration (0918).
The potency is determined by comparing the antibody titre The label states the number of International Units per
of the immunoglobulin to be examined with that of a container.
reference preparation calibrated in International Units, using ______________________________________________________________Ph Elf
an immunoassay of suitable sensitivity and specificity (2.7./).
The International Unit is the activity contained in a stated
amount of the International Standard for anti varicella-zoster.
Standard is stated by the World Health Organization. von Willebrand Factor * *
The stated potency is not less than 100 lU/mL. (Human von Willebrand factor, ***
The estimated potency is not less than the stated potency. Ph Eur monograph 2298)
Ph Eur______________________________________________________________
potency. DEFINITION
Sterile, freeze-dried preparation of a plasma protein fraction
STORAGE
containing the glycoprotein human von Willebrand factor
See Human normal immunoglobulin for intramuscular with varying amounts of human coagulation factor vm,
depending on the method of preparation. It is prepared from
LABELLING human plasma that complies with the monograph on Human
See Human normal immunoglobulin for intramuscular plasma for fractionation (0853). The preparation may contain
administration (0338). excipients such as stabilisers.
The label states the number of International Units per This monograph applies to preparations formulated
container. according to the human von Willebrand factor activity.
-—————— _________________ 2______ Ph Eur The potency of the preparation, reconstituted as stated on
the label, is not less than 20 ru of human von Willebrand
factor per millilitre.
PRODUCTION
Varicella Immunoglobulin for ★* ** GENERAL PROVISIONS
The method of preparation is designed to maintain
Intravenous Use ***** functional integrity of human von Willebrand factor.
(Human Varicella Immunoglobulin for Intravenous It includes steps that have been shown to remove or to
Administration> Ph Eur monograph 1528) inactivate known agents of infection; if substances are used
Ph Elf_________ for the inactivation of viruses, the subsequent purification
DEFINITION concentration of these substances is reduced to a suitable
Sterile liquid or freeze-dried preparation containing level and that any residues are such as not to compromise the
immunoglobulins, mainly immunoglobulin G. It is obtained safety of the preparation for patients.
from plasma from selected donors having antibodies against The specific activity is not less than 1 IU of human von
human herpesvirus 3 (varicella-zoster virus 1). Human normal Willebrand factor per milligram of total protein, before the
immunoglobulin for intravenous administration (0918) may be addition of any protein stabiliser.
added.
The human von Willebrand factor fraction is dissolved in a
It complies with the monograph on Human normal suitable liquid. No antimicrobial preservative or antibiotic is
immunoglobulin for intravenous administration (0918)3 except added. The solution is passed through a bacteria-retentive
for the minimum number of donors, the minimum total filter, distributed aseptically into the final containers and
protein content and the limit for osmolality. immediately frozen. It is subsequently freeze-dried and the
POTENCY containers are closed under vacuum or under an inert gas.
The potency is determined by comparing the antibody titre CONSISTENCY OF THE METHOD OF
of the immunoglobulin to be examined with that of a PRODUCTION
reference preparation calibrated in International Units, using It shall be demonstrated that the manufacturing process
an immunoassay of suitable sensitivity and specificity (2.7./). yields a product having a consistent composition with respect
The International Unit is the activity contained in a stated to human von Willebrand factor, human coagulation
amount of the International Standard for anti varicella-zoster factor vm and the proportions of human von Willebrand
immunoglobulin. The equivalence in International Units of factor and human coagulation factor vm. This is evaluated
the International Standard is stated by the World Health by suitable analytical procedures that are determined during
Organization. process development, and ±at include the following checks:
The stated potency is not less than 25 lU/mL. The estimated Human von Willebrand factor multimers
potency is not less than the stated potency. The confidence The distribution of the different human von Willebrand
limits (P = 0.95) are not less than 80 per cent and not more factor multimers is determined by a suitable method such as
than 125 per cent of the estimated potency. sodium dodecyl sulfate (SDS) agarose gel electrophoresis
STORAGE with or without Western blot analysis, using a suitable
See Human normal immunoglobulin for intravenous normal human plasma as standard. Visualisation of the
multimeric pattern may be performed using, for example, an
immunoenzymatic technique and quantitative evaluation may Water

be earned out by densitometric analysis. Determined by a suitable method, such as semi-micro
Human von Willebrand factor activity (2.7.27) determination of water (2.5.72), loss on drying (2.2.52) or
The human von Willebrand factor activity is estimated by near-infrared spectroscopy (2.2.40), the water content is
determining the ristocetin cofactor activity and by one or within the limits approved by the competent authority.
more other suitable assays such as determination of collagen- Sterility (2.6.7)
binding activity using a suitable reference preparation. It complies with the test.
Human von Willebrand factor activity/antigen ratio Pyrogens (2.6.5) or Bacterial endotoxins (2.6.14)
Consistency of the manufacturing process with respect to the It complies with the test for pyrogens or, preferably and
ratio of human von Willebrand factor activity to human von where justified and authorised, with a validated in vitro test
Willebrand factor antigen content is demonstrated. such as the test for bacterial endotoxins.
PRODUCTS THAT SHOW PARTICLES AFTER RECONSTITUTION For the pyrogen test, inject per kilogram of the rabbit’s mass
If a few particles remain when the preparation is a volume equivalent to not less than 100 IU of human von
reconstituted, it shall be demonstrated during validation Willebrand factor.
studies that the potency is not significantly affected after Where the test for bacterial endotoxins is used, the
passage of the preparation through the filter to be provided preparation to be examined contains less than 0.05 IU of
with the preparation. endotoxin per International Unit of human von Willebrand
CHARACTERS factor.
Appearance ASSAY
Hygroscopic, white or pale yellow, powder or friable solid. Human von Willebrand factor (2.7.21)
Reconstitute the preparation to be examined as stated on the label The estimated potency is not less than 80 per cent and not
immediately before carrying out the identification, tests fexcept more than 120 per cent of the stated potency.
those for solubility and water) and assay. The confidence limits (P = 0.95) are not less than
IDENTIFICATION potency.
Pending the availability of an International Standard for human
TESTS von Willebrand factor concentrate calibrated for use in the
Solubility collagen-binding assay, only the ristocetin cofactor assay may be
To a container of the preparation to be examined, add the used.
volume of the liquid stated on the label at the recommended Human coagulation factor vni (2.7.4)
temperature. The preparation dissolves completely with The assay is carried out where the human coagulation
gentle swirling within 10 min, forming a clear or slighdy factor VIII content is greater than 10 IU of human
opalescent, colourless or slighdy yellow solution. coagulation factor VIII per 100 IU of human von Willebrand
In addition, where the label states that the product may show factor activity. The estimated potency is not less than
a few particles after reconstitution, reconstitute the 60 per cent and not more than 140 per cent of the stated
preparation as described on the label and pass it through the potency. The confidence limits (P = 0.95) are not less than
filter provided: the filtered solution is clear or slighdy 80 per cent and not more than 120 per cent of the estimated
opalescent. potency.
pH (2.2.5) STORAGE
6.5 to 7.5. In an airtight container, protected from light.
Osmolality (2.2.55) LABELLING
The label states:
Total protein — the number of International Units of human von
If necessary, dilute an accurately measured volume of the Willebrand factor in the container;
reconstituted preparation with a 9 g/L solution of sodium — the number of International Units of human coagulation
chloride R to obtain a protein concentration of about factor vni in the container, or that the content of human
7.5 mg/mL. Place 2.0 mL of this solution in a round- coagulation factor VIII is less than or equal to 10 IU of
bottomed centrifuge tube and add 2 mL of a 75 g/L solution human coagulation factor VIII per 100 IU of human von
of sodium molybdate R and 2 mL of a mixture of 1 volume of Willebrand factor activity;
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake, — the amount of protein in the container;
centrifuge for 5 min, decant the supernatant and allow the — the name and quantity of any added substance;
inverted tube to drain on filter paper. Determine the nitrogen — the name and volume of the liquid to be used for
in the residue by the method of sulfuric acid digestion (2.5.9) reconstitution;
and calculate the amount of protein by multiplying the result — where applicable, that the preparation may show the
by 6.25. For some products, especially those without a protein presence of a few particles after reconstitution;
stabiliser, this method may not be applicable. Another validated — that the transmission of infectious agents cannot be totally
method for protein determination must therefore be performed excluded when medicinal products prepared from human
Anti-A and anti-B haemagglutinins blood or plasma are administered.
The 1 to 64 dilution does not show agglutination. Dilute the _____________________________________ ___________ P!>E*
reconstituted preparation with a 9 g/L solution of sodium
chloride R to contain 6 IU of human von Willebrand factor
activity per millilitre.
Monographs
2016 Immunosera IV-533
Monoclonal Antibodies for Human ***** Process validation

During development studies, the production method is
Use ***** validated for the following aspects:
(Ph. Eur. monograph 2031) — consistency of the production process including cell-
Monoclonal Antibodies for Human Use comply with the culture/fermentation, purification and, where applicable,
requirements of the European Pharmacopoeia. These requirements fragmentation method;
are reproduced below. — removal or inactivation of infectious agents;
— adequate removal of product- and process-related
The requirements of the monograph for Immunosera do not
impurities (for example, host-cell protein and DNA,
necessarily apply to the monograph for Monoclonal Antibodies for
protein A, antibiotics, cell-culture components);
Human Use.
— specificity and biological activity of the monoclonal
Ph Elf._____________________________________________________
antibody;
DEFINITION — absence of non-endotoxin pyrogens, where applicable;
Monoclonal antibodies for human use are preparations of an — reusability of purification components (for example,
immunoglobulin or a fragment of an immunoglobulin, for column material), limits or acceptance criteria being set as
example, F(ab')2, with defined specificity, produced by a a function of the validation;
single clone of cells. They may be conjugated to other — methods used for conjugation, where applicable.
substances, including for radiolabelling. Product characterisation
They can be obtained from immortalised B lymphocytes that The product is characterised to obtain adequate information
are cloned and expanded as continuous cell lines or from including: structural integrity, isotype, amino-acid sequence,
rDNA-engineered cell lines. secondary structure, carbohydrate moiety, disulfide bridges,
conformation, specificity, affinity, biological activity and
Examined under suitable conditions of visibility, they are
heterogeneity (characterisation of isoforms).
practically free from particles.
A battery of suitable analytical techniques is used including
Currently available rDNA-engineered antibodies include the
following antibodies. chemical, physical, immunochemical and biological tests (for
example, peptide mapping, N- and C-terminal amino-acid
Chimeric monoclonal antibodies The variable heavy- and light sequencing, mass spectrometry, chromatographic,
chain domains of a human antibody are replaced by those of electrophoretic and spectroscopic techniques). Additional
a non-human species that possess the desired antigen tests are performed to obtain information on cross-reactivity
specificity. with human tissues.
Humanised monoclonal antibodies The 3 short hypervariable For those products that are modified by fragmentation or
sequences (the complementarity-determining regions) of non- conjugation, the influence of the me±ods used on the
human variable domains for each chain are engineered into antibody is characterised.
the variable domain framework of a human antibody; other
sequence changes may be made to improve antigen binding.
Process intermediates
Where process intermediates are stored, an expiry date or a
Recombinant human monoclonal antibodies The variable heavy- storage period justified by stability data is established for
and light-chain domains of a human antibody are combined each.
with the constant region of a human antibody.
Biological assay
Monoclonal antibodies obtained from cell lines modified by
The biological assay is chosen in terms of its correlation with
recombinant DNA technology also comply with the
the intended mode of action of the monoclonal antibody.
requirements of the monograph Products of recombinant DNA
technology (0784). Reference preparation
A batch shown to be stable and shown to be suitable in
This monograph applies to monoclonal antibodies, including
clinical trials, or a batch representative thereof, is used as a
conjugates, for therapeutic and prophylactic use and for use
reference preparation for the identification, tests and assay.
as in vivo diagnostics. It does not apply to monoclonal
The reference preparation is appropriately characterised as
antibodies used as reagents in the manufacture of medicinal
defined under Product characterisation, except that it is not
products. Nor does it apply to monoclonal antibodies
necessary to examine cross-reactivity for each batch of
produced in ascites, for which requirements are decided by
the competent authority.
Definition of a batch
PRODUCTION Definition of a batch is required throughout the process.
GENERAL PROVISIONS
SOURCE CELLS
Production is based on a seed-lot system using a master cell
Source cells include fusion partners, lymphocytes, myeloma
bank and, if applicable, a working cell bank derived from the
cells, feeder cells and host cells for the expression of the
cloned cells. The production method is validated during
recombinant monoclonal antibody.
development studies in order to prevent transmission of
infectious agents by the final product. All biological materials The origin and characteristics of the parental cell are
and cells used in the production are characterised and are in documented, including information on the health of the
compliance with chapter 5.2.8. Minimising the risk of donors, and on the fusion partner used (for example,
transmitting animal spongiform encephalopathy agents via human myeloma cell line, human lymphoblastoid B-cell line).
and veterinary medicinal products. Where monoclonal Wherever possible, source cells undergo suitable screening for
antibodies for human use are manufactured using materials extraneous agents and endogenous agents. The choice of
of human or animal origin, the requirements of chapter 5.1.7. viruses for the tests is dependent on the species and tissue of
Viral safety also apply. Where an immunogen is used, it is origin.
characterised and the method of immunisation is
documented.
IV-534 Immunosera 2016
CELL LINE PRODUCING THE MONOCLONAL ANTIBODY Continuous-culture production (multiple harvest)
The suitability of the cell line producing ±e monoclonal Cells are continuously cultivated for a defined period (in
antibody is demonstrated by: accordance with the stability of the system and production
— documentation on ±e history of the cell line including consistency). Monitoring is necessary throughout the life of
description of the cell fusion, immortalisation or the culture; the required frequency and type of monitoring
transfection and cloning procedure; will depend on the nature of the production system.
— characterisation of the cell line (for example, phenotype, Each harvest is tested for antibody content, bioburden,
isoenzyme analysis, immunochemical markers and endotoxin and mycoplasmas. General or specific tests for
cytogenetic markers); adventitious viruses are carried out at a suitable stage
— characterisation of relevant features of the antibody; depending on the nature of the manufacturing process and
— consistency of critical quality attributes for the antibody the materials used. For processes using production at finite
up to or beyond the population doubling level or passage level (single harvest), at least 3 harvests are tested for
generation number used for routine production; adventitious viruses using a suitable range of in vitro
— for recombinant DNA products, consistency of the coding methods.
sequence of the expression construct in cells cultivated to
The acceptance criteria for harvests for further processing are
the limit of in vitro cell age for production use or beyond,
clearly defined and linked to the schedule of monitoring
by either nucleic acid testing or product analysis.
applied. If any adventitious viruses are detected, the process
CELL BANKS is carefully investigated to determine the cause of the
The master cell bank is a homogeneous suspension of the contamination and the harvest is not further processed.
cell line producing the monoclonal antibody, distributed in Harvests in which an endogenous virus has been detected are
equal volumes in a single operation into individual containers not used for purification unless an appropriate action plan
for storage. has been defined to prevent transmission of infectious agents.
A working cell bank is a homogeneous suspension of the cell PURIFICATION
material derived from the master cell bank at a finite passage Harvests or intermediate pools may be pooled before further
level, distributed in equal volumes in a single operation into processing. The purification process includes steps that
individual containers for storage. remove and/or inactivate non-enveloped and enveloped
Post-production cells are cells cultured up to or beyond the viruses. A validated purification process, for which removal
population doubling level or generation number used for and/or inactivation of infectious agents and removal of
routine production. product- and process-related impurities has been
The following tests are performed on the master cell bank: demonstrated, is used. Defined steps of the process lead to a
viability, identity, absence of bacterial, fungal and purified monoclonal antibody (active substance) of consistent
mycoplasmal contamination, characterisation of the quality and biological activity.
monoclonal antibody produced. Adventitious viral ACTIVE SUBSTANCE
contamination is tested with a suitable range of in vivo and in The test programme for the active substance depends on the
vitro tests. Retrovirus and other endogenous viral validation of the process, on demonstration of consistency
contamination is tested using a suitable range of in vitro tests. and on the expected level of product- and process-related
The following tests are performed on the working cell bank: impurities. The active substance is tested for appearance,
viability, identity, absence of bacterial, fungal and identity, bioburden and bacterial endotoxins, product-related
mycoplasmal contamination. Adventitious viral substances, product- and process-related impurities including
contamination is tested with a suitable range of in vivo and in tests for host-cell-derived proteins and host-cell- and vector-
vitro tests. For the first working cell bank, these tests are derived DNA, as well as structural integrity', protein content
performed on post-production cells, generated from that and biological activity by suitable analytical methods,
working cell bank; for working cell banks subsequent to the comparing with the reference preparation where necessary.
first working cell bank, a single in vitro and in vivo test can When the active substance is a conjugated or transformed
be done either directly on the working cell bank or on post antibody, appropriate tests must be performed before and
production cells. after the antibody conjugation/modification.
For the master cell bank and working cell bank, tests for If storage of intermediates is intended, adequate stability of
specific viruses are carried out when potentially contaminated these preparations and its impact on quality or shelf-life of
biological material has been used during preparation of the the finished product are evaluated.
cell banks, taking into account the species of origin of this FINAL BULK
material. This may not be necessary when this material is One or more batches of active substance may be combined
inactivated using validated procedures. to produce the final bulk. Suitable stabilisers and other
The following tests are performed on the post-production excipients may be added during preparation of the final bulk.
cells: absence of bacterial, fungal and mycoplasmal The final bulk must be stored under validated conditions
contamination. Adventitious viral contamination is tested with respect to bioburden and stability.
with a suitable range of in vivo and in vitro tests. Retrovirus
FINAL LOT
and other endogenous viral contamination is tested using a
The final bulk is sterile-filtered and distributed under aseptic
suitable range of in vitro tests.
conditions into sterile containers, which may subsequently be
CULTURE AND HARVEST freeze-dried.
Production at finite passage level (single harvest) As pan of the in-process control each container (vial, syringe
Cells are cultivated up to a defined maximum number of or ampoule) is inspected after filling to eliminate containers
passages or population doublings, or up to a fixed harvest that contain visible particles. During development of the
time (in accordance with the stability of the cell line). product it must be demonstrated that either the process will
Product is harvested in a single operation. not generate visible proteinaceous particles in the final lot or
2016 Immunosera FV-535
such particles are reduced to a low level as justified and Bacterial endotoxins (2.6.74)
authorised. It complies with the limits approved for the particular
CHARACTERS product.
Liqmd preparations are clear or slightly opalescent, colourless Tests applied to modified antibodies
or slightly coloured liquids. Freeze-dried products are white Suitable tests are carried out depending on the type of
or slightly coloured powders or solid friable masses. After modification.
reconstitution they show the same characteristics as liquid ASSAY
preparations.
Carry out a suitable biological assay compared to the
IDENTIFICATION reference preparation. Design of the assay and calculation of
The identity is established by suitable validated methods the results are made according to the usual principles (for
comparing the product with the reference preparation, where example, 5.5).
appropriate. The assay also contributes to identification. STORAGE
TESTS As stated on the label.
Appearance Expiry date The expiry date is calculated from the date of
Liquid or reconstituted freeze-dried preparations comply with sterile filtration, the date of filling (for liquid preparations) or
the limits approved for the particular product with regard to the date of freeze-drying (where applicable).
degree of opalescence (2.2.7) and degree of coloration
LABELLING
(2.2.2). They are without visible particles, unless otherwise
justified and authorised. The label states:
— the number of units per millilitre, where applicable;
Solubility — the quantity of protein per container;
Freeze-dried preparations dissolve completely in the — the quantity of monoclonal antibody in the container;
prescribed volume of reconstituting liquid, within a defined — for liquid preparations, the volume of the preparation in
time, as approved for the particular product. the container;
pH (2.2.5) — for freeze-dried preparations:
It complies with the limits approved for the particular — the name and ±e volume of the reconstitution liquid
product. to be added;
Osmolality (2.2.55) — the period of time within which the monoclonal
Minimum 240 mosmol/kg, unless otherwise justified and antibody is to be used after reconstitution;
authorised. — the dilution to be made before use of ±e product, where
applicable.
Extractable volume (2.9.77)
______________________________________________________________ Ph Eur
It complies with the test for extractable volume.
Total protein (2.5.55)
It complies with the limits approved for the particular
product.
Molecular-size distribution
Immunosera * *
Molecular-size distribution is determined by a suitable Antisera ***
method, for example size-exclusion chromatography (2.2.50). (Immunosera for Human Use, Animal,
It complies with the limits approved for the particular Ph Eur monograph 0084)
product.
Immunosera comply with the requirements of the European
Molecular identity and structural integrity Pharmacopoeia monograph for Immunosera for Human Use,
Depending on the nature of the monoclonal antibody, its Animal. These requirements are reproduced below.
microheterogeneity and isoforms, a number of different tests
Ph Elf--------------------------------------------------------------------- -------------------- --------- -——
can be used to demonstrate molecular identity and structural
integrity. These tests may include peptide mapping, DEFINITION
isoelectric focusing, ion-exchange chromatography, Animal immunosera for human use are liquid or freeze-dried
hydrophobic interaction chromatography, oligosaccharide preparations containing purified immunoglobulins or
mapping, monosaccharide content and mass spectrometry. immunoglobulin fragments obtained from serum or plasma
Purity of immunised animals of different species.
Tests for process- and product-related impurities are carried The immunoglobulins or immunoglobulin fragments have
out by suitable validated methods. Provided that tests for the power of specifically neutralising or binding to the
process-related impurities have been carried out on the active antigen used for immunisation. The antigens include
substance or on the final bulk with satisfactory results, they microbial or other toxins, human antigens, suspensions of
may be omitted on the final lot. bacterial and viral antigens and venoms of snakes, scorpions
and spiders. The preparation is intended for intravenous or
Stabiliser
intramuscular administration, after dilution where applicable.
Where applicable, it complies with the limits approved for
the particular product. PRODUCTION
GENERAL PROVISIONS
Water (2.5.72)
The production method shall have been shown to yield
Freeze-dried products comply with the limits approved for
consistently immunosera of acceptable safety, potency in man
the particular product.
and stability.
Sterility (2.6.7)
Any reagent of biological origin used in the production of manner as to maintain sterility' of the product. The blood cr
immunosera shall be free of contamination with bacteria, plasma collection is conducted at a site separate from the
fungi and viruses. The general requirements of chapter 5.1.7. area where the animals are kept or bred and the area where
Viral safety apply to the manufacture of animal immunosera the immunoserum is purified. If the serum or plasma is
for human use, in conjunction with the more specific stored before further processing, precautions are taken to
requirements relating to viral safety in this monograph. avoid microbial contamination.
The method of preparation includes a step or steps that have Several single plasma or serum samples may be pooled before
been shown to remove or inactivate known agents of purification. The single or pooled samples are tested before
infection. purification for the following tests.
Methods used for production are validated, effective, Tests for contaminating viruses
reproducible and do not impair the biological activity of the If an antimicrobial preservative is added, it must be
product. neutralised before carrying out the tests, or the tests are
The production method is validated to demonstrate that the carried out on a sample taken before addition of the
product, if tested, would comply with the test for abnormal antimicrobial preservative. Each pool is tested for
toxicity for immunosera and vaccines for human use (2.6.9). contaminating viruses by suitable in vitro tests.
Reference preparation A batch shown to be suitable in clinical Each pool is tested for viruses by inoculation to cell cultures
trialร, or a batch representative thereof, is used as the capable of detecting a wide range of viruses relevant for the
reference preparation for the tests for high molecular mass particular product.
proteins and purity'.
Potency
ANIMALS Carty' out a biological assay as indicated in the monograph
The animals used are of a species approved by the competent and express the result in International Units per millilitre,
authority, are healthy and are exclusively reserved for where applicable. A validated in vitro method may also be
production of immunoserum. They arc tested and shown to used.
be free from a defined list of infectious agents.
Protein content
The introduction of animals into a closed herd follows
Dilute the product to be examined with a 9 g/L solution of
specified procedures, including definition of quarantine
sodium chloride R to obtain a solution containing about 15 mg
measures. Where appropriate, additional specific agents are
of protein in 2 mL. To 2 mL of this solution in a round-
considered depending on the geographical localisation of the
bottomed centrifuge tube add 2 mL of a 75 g/L solution of
establishment used for the breeding and production of the
sodium molybdate R and 2 mL of a mixture of 1 volume of
animals. The feed originates from a controlled source and no
animal proteins are added. The suppliers of animals are
certified by the competent authority.
If the animals are treated with antibiotics, a suitable in the residue by the method of sulfuric acid digestion (2.5.9)
withdrawal period is allowed before collection of blood or and calculate the content of protein by multiplying by 6.25.
plasma. The animals are not treated with penicillin The protein content is within approved limits.
antibiotics. If a live vaccine is administered, a suitable waiting
PURIFICATION AND VIRAL INACTIVATION
period is imposed between vaccination and collection of
The immunoglobulins are concentrated and purified by
serum or plasma for immunoserum production.
fractional precipitation, chromatography, immunoadsorption
IMMUNISATION or by other chemical or physical methods. They may be
The antigens used are identified and characterised, where processed further by enzyme treatment. The methods are
appropriate; where relevant, they are shown to be free from selected and validated to avoid contamination at all steps of
extraneous infectious agents. They are identified by their processing and to avoid formation of protein aggregates that
names and a batch number; information on ±e source and affect the immunobiological characteristics of the product.
preparation are recorded. For products intended to consist of immunoglobulin
The selected animals are isolated for at least 1 week before fragments, the methods arc validated to guarantee total
being immunised according to a defined schedule, wi± fragmentation. The methods of purification used are such
booster injections at suitable intervals. Adjuvants may be that they do not generate additional components that
used. compromise the quality and the safety of the product.
Animals are kept under general health surveillance and Unless otherwise justified and authorised, validated
specific antibody production is controlled at each cycle of procedures are applied for removal and/or inactivation of
immunisation. viruses. The procedures are selected to avoid the formation
Animals are thoroughly examined before collection of blood of polymers or aggregates and, unless the product is intended
or plasma. If an animal shows any pathological lesion not to consist of Fab' fragments, to minimise the splitting of
related to the immunisation process, it is not used, nor are F(ab')2 into Fab' fragments.
any other of the animals in the group concerned, unless it is After purification and treatment for removal and/or
evident that their use will not impair the safety of the inactivation of viruses, a stabiliser may be added to the
product. intermediate product, which may be stored for a period
COLLECTION OF BLOOD OR PLASMA defined in light of stability data.
Collection of blood is made by venepuncture or Only an intermediate product that complies with the
plasmapheresis. The puncture area is shaved, cleaned and following requirements may be used in the preparation of the
disinfected. The animals may be anaesthetised under final bulk.
conditions that do not influence the quality of the product. Purity
Unless otherwise prescribed, an antimicrobial preservative Examine by non-reducing polyacrylamide gel electrophoresis
may be added. The blood or plasma is collected in such a (2.2.31), by comparison with the reference preparation.
2016 Immunosera FV-537
The bands are compared in intensity and no additional Dilute the preparation to be examined with a 9 g/L solution
bands are found. of sodium chloride R to obtain a solution containing about
FINAL BULK 15 mg of protein in 2 mL. To 2 mL of this solution in a
The final bulk is prepared from a single intermediate product round-bottomed centrifuge tube add 2 mL of a 75 g/L
or from a pool of intermediate products obtained from solution of sodium molybdate R and 2 mL of a mixture of
animals of the same species. Intermediate products with 1 volume of nitrogen-free sulfuric acid R and 30 volumes of
different specificities may be pooled. water R. Shake, centrifuge for 5 min, decant the supernatant
and allow the inverted tube to drain on filter paper.
An antimicrobial preservative and a stabiliser may be added.
Determine the nitrogen in the residue by the method of
If an antimicrobial preservative has been added to the blood
sulfuric acid digestion (2.5.9) and calculate the content of
or plasma, the same substance is used as the antimicrobial
preservative in the final bulk. protein by multiplying by 6.25.
Only a final bulk that complies with the following Molecular-size distribution
requirements may be used in the preparation of the final lot. Examine by liquid chromatography (2.2.29 or 2.2.30).
It complies with the specification approved for the particular
Antimicrobial preservative product.
Where applicable, determine the amount of antimicrobial
preservative by a suitable physico-chemical method. Antimicrobial preservative
It contains not less than 85 per cent and not more than Where applicable, determine the amount of antimicrobial
115 per cent of the amount stated on the label. preservative by a suitable physicochemical method.
The amount is not less than the minimum amount shown to
Sterility (2.6. /) be effective and is not greater than 115 per cent of that
It complies with tire test for sterility. stated on the label.
FLNAL LOT
Phenol (2.5.15)
The final bulk of immunoscrum is distributed aseptically into Maximum 2.5 g/L for preparations containing phenol.
sterile, tamper-proof containers. The containers arc closed so
as to prevent contamination. Stabiliser
Determine the amount of stabiliser by a suitable physico
Only a final lot that complies with the requirements chemical method. The preparation contains not less than
prescribed below under Identification, Tests and Assay may 80 per cent and not more than 120 per cent of the quantity
be released for use. Provided that the tests for osmolality, stated on the label.
protein content, molecular-size distribution, antimicrobial
preservative, stabiliser, purity, foreign proteins and albumin Purity
and the assay have been earned out with satisfactory results Examine by non-reducing polyacrylamide gel electrophoresis
on the final bulk, they may be omitted on the final lot. (2.2.31), by comparison with the reference preparation.
No additional bands are found for the preparation to be
Reconstitute the preparation to be examined as stated on the label
examined.
immediately before carrying out the identification, tests (except
those for solubility and water) and assay. Foreign proteins
When examined by precipitation tests with specific antisera,
IDENTIFICATION only protein from the declared animal species is shown to be
The identity is established by immunological tests and, where present, unless otherwise prescribed, for example where
necessary, by determination of biological activity. The assay material of human origin is used during production.
may also serve for identification.
Albumin
CHARACTERS Unless otherwise prescribed in the monograph, when
Immunosera are clear to opalescent and colourless to very examined electrophoretically, the content of albumin is not
faintly yellow liquids. They are free from turbidity. Freeze- greater than the limit approved for the particular product
dried products are white or slightly yellow powders or solid and, in any case, is not greater than 3 per cent.
friable masses. After reconstitution they show the same Water {2.5.12)
characteristics as liquid preparations. Maximum 3 per cent.
TESTS Sterility (2.6.1)
Solubility It complies with the test for sterility.
To a container of the preparation to be examined, add the
Pyrogens (2.6.8)
volume of the liquid for reconstitution stated on the label. Unless otherwise justified and authorised, it complies with
The preparation dissolves completely within the time stated the test for pyrogens. Unless otherwise prescribed, inject
on the label. 1 mL per kilogram of the rabbit’s body mass.
Extractable volume (2.9.17)
ASSAY
It complies with the requirement for extractable volume.
Carry out a biological assay as indicated in the monograph
pH (2.2.5) and express the result in International Units per millilitre,
The pH is within the limits approved for the particular where appropriate. A validated in vitro method may also be
product. used.
Osmolality (2.2.35) STORAGE
Minimum 240 mosmol/kg after dilution, where applicable. Protected from light, at the temperature stated on the label.
Protein content Do not allow liquid preparations to freeze.
90 per cent to 110 per cent of the amount stated on the Expiry date The expiry date is calculated from the beginning
label, and, unless otherwise justified and authorised, not of the assay.
more than 100 g/L.
LABELLING and independent cytotoxicity, cytokine release, induction

The label states: of T-cell activation, cell death,
— the number of International Units per millilitre, where — does not contain antibodies that cross-react with human
applicable; tissues to a degree that would impair clinical safety,
— the amount of protein per container; — has a defined maximum content of anti-thrombocyte
— for freeze-dried preparations: antibody activity,
— the name and volume of the reconstituting liquid to — has a defined maximum content of haemoglobin.
be added; The product has been shown, by suitable tests in animals
— that the immunoserum is to be used immediately after and evaluation during clinical trials, to be well tolerated.
reconstitution;
Reference preparation A batch shown to be suitable for
— the time required for complete dissolution;
checking the validity of the assay and whose efficacy has been
demonstrated in clinical trials, or a batch representative
— the storage conditions;
thereof.
— the expiry date, except for containers of less than 1 mL
which are individually packed; the expiry date may be ANIMALS
omitted from the label on the container, provided it is The animals used are of a species approved by the competent
shown on the package and the label on the package states authority, are healthy and exclusively reserved for production
that the container must be kept in the package until of anti-T lymphocyte immunoglobulin. They are tested and
required for use; shown to be free from a defined list of infectious agents.
— the animal species of origin; The introduction of animals into a closed herd follows
— the name and amount of any antimicrobial preservative, specified procedures, including definition of quarantine
any stabiliser and any other excipient. measures. Where appropriate, tests for additional specific
agents arc considered depending on the geographical
---------------------------------------------------------------■------ - ----------------------------------- Ph Eur
localisation of the establishment used for the breeding and
production of the animals. The feed originates from a
controlled source and no animal proteins are added.
The suppliers of animals are certified by the competent
authority.
Anti-T Lymphocyte *****
If the animals are treated with antibiotics, a suitable
Immunoglobulin for Human Use, ***** withdrawal period is allowed before collection of blood or
Animal plasma. The animals are not treated with penicillin
antibiotics. If a live vaccine is administered, a suitable waiting
period is imposed between vaccination and collection of
Ph Eur______________________________________________________________ serum or plasma for immunoglobulin production.
DEFINITION The species, origin and identification number of the animals
Sterile liquid or freeze-dried preparation containing are specified.
immunoglobulins, obtained from serum or plasma of IMMUNISATION
animals, mainly rabbits or horses, immunised with human The antigens used are identified and characterised, where
lymphocytic antigens. appropriate. They are identified by their names and a batch
The immunoglobulin has the property of diminishing the number; information on the source and preparation are
number and function of immunocompetent cells, in recorded.
particular T-lymphocytes. The preparation contains The selected animals are isolated for at least 1 week before
principally immunoglobulin G. It may contain antibodies being immunised according to a defined schedule with
against other lymphocyte subpopulations and against other booster injections at suitable intervals. Adjuvants may be
cells. The preparation is intended for intravenous used.
administration, after dilution with a suitable diluent where Animals are kept under general health surveillance and
applicable. The preparation may contain excipients such as specific antibody production is controlled at each cycle of
stabilisers. immunisation.
Applicable provisions of the monograph on Immunosera for Animals are thoroughly examined before collection of blood
human use, animal (0084) are stated below. or plasma. If an animal shows any pathological lesion not
PRODUCTION related to the immunisation process, it is not used, nor are
GENERAL PROVISIONS any other of the animals in the group concerned, unless it is
The production method has been shown to yield consistently evident that their use will not impair the safety of the
immunoglobulins of acceptable safety, potency in man and product.
stability. Human antigens such as continuously growing T-lymphocyte
Any reagent of biological origin used in production shall be cell lines or thymocytes are used to immunise the animals.
free of contamination with bacteria, fungi and viruses. Cells may be subjected to a sorting procedure.
The method of preparation includes a step or steps that have The immunising antigens are shown to be free from
been shown to remove or inactivate known agents of infectious agents by validated methods for relevant blood
infection. borne pathogens, notably hepatitis B virus (HBV),
During development studies, it shall be demonstrated that hepatitis c virus (HCV) and human immunodeficiency
virus (HIV) and other relevant adventitious agents originating
the production method yields a product that:
— does not transmit infectious agents, from the preparation of the antigen. The cells used comply
with defined requirements for purity of the cell population
— is characterised by a defined pattern of immunological
activity, notably: antigen binding, complement-dependent and freedom from adventitious agents.
2016 Inununosera IV-539
collection of blood or plasma FINAL LOT

Collection of blood is made by venepuncture or The final bulk of anti-T-lymphocyte immunoglobulin is
plasmapheresis. The puncture area is shaved, cleaned and distributed aseptically into sterile, tamper-proof containers.
disinfected. The animals may be anaesthetised under The containers are closed as to prevent contamination.
conditions that do not influence the quality of the product. Only a final lot that complies with the requirements
No antimicrobial preservative is added to the plasma and prescribed below under Identification, Tests and Assay may
serum samples. The blood or plasma is collected in such a be released for use.
manner as to maintain sterility of the product. The blood or
CHARACTERS
plasma collection is conducted at a site separate from the
Appearance
area where the animals arc kept or bred and the area where
— liquid preparation: clear or slightly opalescent, colourless or
the immunoglobulin is purified. If the serum or plasma is
pale yellow liquid;
stored before further processing, precautions arc taken to
— freeze-dried preparation: white or slightly yellow powder or
avoid microbial contamination.
solid friable mass, which after reconstitution gives a liquid
Several single plasma or serum samples may be pooled before preparation corresponding to the description above.
purification. The single or pooled samples are tested before
purification for the following tests. IDENTIFICATION
A. Using a suitable range of species-specific antisera, carry
Tests for contaminating viruses
out precipitation tests on the preparation to be examined.
Each pool is tested for contaminating viruses by suitable in
It is recommended that the test be carried out using antisera
vitro tests including inoculation to cell cultures capable of
specific to the plasma proteins of each species of domestic
detecting a wide range of viruses relevant for the particular
animal commonly used in the preparation of materials of
product. Where applicable, in vitro tests for contaminating
biological origin in the country concerned and antisera
viruses are carried out on the adsorbed pool, after the last
specific to human plasma proteins. The preparation is shown
production stage that may introduce viral contaminants.
to contain proteins originating from the animal used for the
PURIFICATION AND VIRAL INACTIVATION anti-T lymphocyte immunoglobulin production.
The immunoglobulins are concentrated and purified by B. Examine by a suitable immunoelectrophoresis technique.
fractional precipitation, chromatography, immuno-adsorption Using antiserum to normal serum of the animal used for
or by other suitable chemical or physical methods. production, compare this serum and the preparation to be
The methods are selected and validated to avoid examined, both diluted to a concentration that will allow a
contamination at all steps of processing and to avoid clear gammaglobulin precipitation arc to be obtained on the
formation of protein aggregates that effect immunobiological gel. The main component of the preparation to be examined
characteristics of the product. corresponds to the IgG component of normal serum of the
Uni ess otherwise justified and authorised, validated animal used for production.
procedures are applied for removal and/or inactivation of c. The preparation complies with the assay.
viruses.
After purification and treatment for removal and/or
TESTS
inactivation of viruses, a stabiliser may be added to the Solubility
intermediate product, which may be stored for a period For the freeze-dried preparation, to a container add the
defined in the light of stability data. volume of the liquid stated on the label. The preparation
dissolves completely within the time stated on the label.
Only an intermediate product that complies with the
following requirements may be used in the preparation of the Extractable volume (2.9. / 7)
final bulk. It complies with the requirement for extractable volume.
If the method of preparation includes a step for adsorption of pH (2.2.5)
cross-reacting anti-human antibodies using material from The pH is within the limits approved for the particular
human tissues and/or red blood cells, the human materials product.
are submitted to a validated procedure for inactivation of Osmolality (2.2.55)
infectious agents, unless otherwise justified and authorised. Minimum 240 mosmol/kg after dilution, where applicable.
If erythrocytes are used for adsorption, the donors for such Total protein (2.5.55)
materials comply with the requirements for donors of blood 90 per cent to 110 per cent of the amount stated on the
and plasma of the monograph on Human plasma for label.
fractionation (0853). If other human material is used, it is
shown by validated methods to be free from relevant blood Stabiliser
Determine the amount of stabiliser by a suitable physico
borne pathogens, notably HBV, HCV and HIV. If substances
chemical method. The preparation contains not less than
are used for inactivation or removal of viruses, it shall have
80 per cent and not more than 120 per cent of the quantity
been shown that any residues present in the final product
stated on the label.
have no adverse effects on the patients treated with the anti-
T lymphocyte immunoglobulin. Distribution of molecular size
Size-exclusion chromatography (2.2.50).
FINAL BULK
The final bulk is prepared from a single intermediate product Test solution Dilute the preparation to be examined with a
or from a pool of intermediate products obtained from 9 g/L solution of sodium chloride R to a concentration suitable
animals of the same species. No antimicrobial preservative is for the chromatographic system used. A concentration in the
added either during the manufacturing procedure or for range 2-20 g/L is usually suitable.
preparation of the final bulk solution. During manufacturing, Reference solution Dilute human immunoglobulin (molecular
the solution is passed through a bacteria-retentive filter. size) BRP with a 9 g/L solution of sodium chloride R to the
same protein concentration as the test solution.
Column: Thrombocyte antibodies

— size: I = 0.6 m, 0 = 7.5 mm, Examined by a suitable method, the level of thrombocyte
— stationary phase: silica gel for size-exclusion antibodies is shown to be below that approved for the
chromatography R3 a grade suitable for fractionation of specific product.
globular proteins in the molecular mass range of Water (2.5.12}
20 000 to 200 000. Maximum 3 per cent.
Mobile phase Dissolve 4.873 g of disodium hydrogen phosphate Sterility (2.6.1}
dihydrate R3 1.741 g of sodium dihydrogen phosphate It complies wi± the test.
monohydrate R and 11.688 g of sodium chloride R in 1 L of
Pyrogens (2.6.8)
water R.
Unless otherwise justified and authorised, it complies with
Flaw rate 0.5 mUmin. the test for pyrogens. Unless otherwise prescribed, inject
Detection Spectrophotometer at 280 nm. 1 mL per kilogram of the rabbit's body mass.
Injection 50-600 pg of protein. ASSAY
Retention time Identify the peaks in the chromatogram The biological activity is determined by measuring the
obtained with the test solution by comparison with the complement-dependent cytotoxicity on target cells. Flow
chromatogram obtained with the reference solution; any peak cytometry is performed with read-out of dead cells stained
with a retention time shorter than that of dimer corresponds using propidium iodide. The activity is expressed as the
to polymers and aggregates. concentration of anti-T lymphocyte immunoglobulin in
System suitability: milligrams per millilitre which mediates 50 per cent
— reference solution: the principal peak corresponds to IgG cytotoxicity.
monomer and there is a peak corresponding to dimer Lymphocyte separation medium Commercial separation media
with a retention time relative to monomer of with low viscosity and a density of 1.077 g/mL.
0.85 ± 0.05, Complement Commercial complement is suitable.
— test solution: the relative retentions of monomer and dimer
Buffered salt solution pH 72 Dissolve 8.0 g of sodium chloride R,
are 1 ± 0.05 with reference to the corresponding peaks
0.2 g of potassium chloride R3 3.18 g of disodium hydrogen
phosphate R and 0.2 g of potassium dihydrogen phosphate R in
Limits: water R and dilute to 1000.0 mL with the same solvent.
— total monomer and dimer, at least 95 per cent of the total
Buffer solution for flow cytometry Add 40 mL of
area of the peaks;
0.1 per cent VIV sodium azide R and 10 mL of foetal calf
— total polymers and aggregates: maximum 5 per cent of the
serum to 440 mL of buffered salt solution pH 7.2. The foetal
total area of the peaks.
calf serum is inactivated at 56 °C for 30 min prior to use.
Purity Store at 4 °C.
Polyacrylamide gel electrophoresis (2.2.31} 3 under non
Propidium iodide solution Dissolve propidium iodide R in
reducing and reducing conditions.
buffered salt solution pH 7.2, to a concentration of
Resolving gel Non-reducing conditions: 8 per cent acrylamide; 1 mg/mL. Store this stock solution at 2-8 °C and use within
reducing conditions: 12 per cent acrylamide. 1 month. For the assay, dilute this solution with buffer
Test solution Dilute the preparation to be examined to a solution for flow cytometry, to obtain a concentration of
protein concentration of 0.5-2 mg/mL. 5 pg/mL. Store at 2-8 °C and use within 3 h.
Reference solution Dilute the reference preparation to the same Microtitre plates Plates used to prepare immunoglobulin
protein concentration as the test solution. dilutions are บ- or V-bottomed polystyrene or poly(vinyl
Application 10 J1L. chloride) plates without surface treatment.
Detection Coomassie staining. Micronic tubes Suitable for flow cytometry measurement.
Results Compared with the electropherogram of the reference Cell suspension Collect blood in anticoagulant from at least
solution, no additional bands are found in the one healthy donor. Immediately isolate the peripheral blood
electropherogram of the test solution. mononuclear cells (PBMC) by gradient centrifugation in
lymphocyte separation medium so that the PBMC form a
Anti-A and anti-B haemagglutinins (2.6.203 Method A)
visible clean interface between the plasma and the separation
The 1 to 64 dilution does not show agglutination.
medium. Collect the layer containing the cells and dispense
Where applicable, dilute the preparation to be examined as into centrifuge tubes containing buffered salt solution
prescribed for use before preparing the dilutions for the test. pH 7.2. Centrifuge at 400 g at 2-8 °C for 10 min. Discard
Haemolysins the supernatant. Suspend the cell pellet in buffer solution for
Prepare a 1 to 64 dilution of the preparation to be examined, flow cytometry. Repeat the centrifugation and resuspension
diluted if necessary as stated on the label. Take 6 aliquots of procedure of the cells twice. After the third centrifugation,
the 1 to 64 dilution. To 1 volume of 3 of the aliquots, add resuspend the cell pellet in 1 mL of buffer solution for flow
1 volume of a 10 per cent VIV suspension of group Al, cytometry. Determine the number and vitality of the cells
group B and group o erythrocytes in a 9 g/L solution of using a haemocytometer. Cell viability of at least 90 per cent
sodium chloride R, respectively. To 1 volume of the remaining is required. Adjust the cell number to 7 X 106/mL by adding
3 aliquots, add 1 volume of a 10 per cent VIV suspension of buffer solution for flow cytometry. Store the cell suspension
group Al, group B and group 0 erythrocytes in a 9 g/L at 4 °C and use within 12 h.
solution of sodium chloride R> respectively, and to each aliquot If necessary, the first PBMC pellet may be resuspended in
1 volume of fresh group AB serum (as a source of buffered salt solution pH 7.2 containing 20 per cent foetal
complement). Mix and incubate at 37 °C for 1 h. Examine calf serum and stored overnight at 2 °C. Centrifuge at 400 g
the supernatant liquids for haemolysis. No signs of at 2-8 °C for 10 min. Discard the supernatant. Suspend the
haemolysis are present. cell pellet in buffer solution for flow cytometry. Determine
the number and vitality of the cells using a haemocytometer. percentage of propidium iodide-positive cells at the upper
Cell viability of at least 90 per cent is required. Adjust the asymptote of the curve is at least 80 per cent.
cell number to 7 X 106/mL by adding buffer solution for The estimated activity is 70 per cent to 130 per cent of the
flow cytometry. activity approved for the particular product.
It is also possible for cells to be immediately frozen and
stored in nitrogen using the following method. 80 per cent and not more than 125 per cent of the estimated
Buffer solution for freezing To 20 mL of cell culture medium, potency.
add 25 mL of foetal calf scrum and 5 mL of dimethyl
sulfoxide (DMSO). Store this solution at 2-8 °C and use STORAGE
within 3 h. Protected from light at the temperature stated on the label.
20 X 106 cells per ampoule are frozen. These ampoules are Expiry date The expiry date is calculated from the beginning
stored in liquid nitrogen. of the assay.
Buffer solution for thawing To 450 mL of cell culture medium, LABELLING
add 50 mL of foetal calf serum. Store this solution at 2-8 °C The label states:
and use within 3 h. — for liquid preparations, the volume of the preparation in
Each ampoule is thawed in a water-bath at 37 cc with the container and the protein content,
shaking. Cell suspension is repeated in a buffer solution for — for freeze-dried preparations:
thawing. Centrifuge at 200 g at 2-8 °C for 10 min. Discard — the name and the volume of the reconstitution liquid
the supernatant. Suspend the cell pellet in buffer solution for to be added,
flow cytometry. Repeat the procedure for centrifugation and — the quantity of protein in the container,
resuspension of cells once. After the second centrifugation, — that the immunoserum is to be used immediately after
resuspend the cells pellet in 1 mL of buffer solution for flow reconstitution,
cytometry. Determine the number and vitality of the cells — the time required for complete dissolution,
using a haemocytometer. Cell viability of at least 90 per cent — the animal species of origin,
is required. Adjust the cell number to 7 X 106/mL by adding — the name and amount of stabiliser, where applicable,
buffer solution for flow cytometry. Store the cell suspension — the dilution to be made before use of the product.
at 4 °C and use within 3 h. ______________________________________________________________ PtiEir
Test solutions For freeze-dried preparations, reconstitute as
stated on the label. Prepare 3 independent series of not fewer
than 7 dilutions using buffer solution for flow cytometry as
diluent.
Reference solutions For freeze-dried preparations, reconstitute
Botulinum Antitoxin * *
according to the instructions for use. Prepare 3 independent (Ph. Eur. monograph 0085) * **
dilution series of not fewer than 7 dilutions using buffer The label may state ‘Bot/Ser’ followed by a letter or letters
solution for flow cytometry as diluent. indicating the type or types present.
Distribute 75 |1L of each of the dilutions of the test solution When Mixed Botulinum Antitoxin or Botulinum Antitoxin is
or reference solution to each of a series of wells of a prescribed or demanded and the types to be present are not
microtitre plate. Add 25 pL of the cell suspension of PBMC stated, Botulinum Antitoxin prepared from types A, B and E
into each well. Add 25 pL of rabbit complement to each of shall be dispensed or supplied.
the wells. Incubate at 37 °C for 30 min.
Ph Elf_______________________________________________________________
Centrifuge the plates at 200 g at 4 °C for 8 min, discard the
supernatant and keep the plate on ice. Preparation for flow DEFINITION
cytometry measurement is done step-wise by using a certain Botulinum antitoxin is a preparation containing antitoxic
number of wells in order to allow labelling with propidium globulins that have the power of specifically neutralising the
iodide R solution and measurement within a defined time toxins formed by Clostridium botulinum type A, type B or
period. Resuspend carefully the cell pellet of a certain type E, or any mixture of these types.
number of wells with 200 pL of propidium iodide solution. PRODUCTION
Transfer the suspension into tubes. Incubate at 25 °C for It is obtained by fractionation from the serum of horses, or
10 min then place immediately on ice. other mammals, that have been immunised against
Proceed with fluorescence measurement in a flow cytometer. CL botulinum type A, type B and type E toxins.
Define a region including all propidium iodide-positive cells
IDENTIFICATION
on the basis of Forward-Scattered, light (FSC) and
It specifically neutralises the types of CL botulinum toxins
flourescence (FL2 or FL3 for propidium iodide). Measure
stated on the label, rendering them harmless to susceptible
the percentage of propidium iodide-positive cells, without
animals.
gating but excluding debris. Analyse at least 3000 cells for
each of the test and reference solutions. POTENCY
Use the percentages of dead cells to estimate the potency as Not less than 500 IU of antitoxin per millilitre for each of
the concentration in milligrams per millilitre of the types A and B and not less than 50 IU of antitoxin per
preparation to be examined necessary to induce 50 per cent millilitre for type E.
of cytotoxicity by fitting a sigmoidal dose response curve to The potency of botulinum antitoxin is determined by
the data obtained 'with the test and the reference preparations comparing the dose necessary to protect mice against the
and by using a 4-parameter logistic model (see, for example, lethal effects of a fixed dose of botulinum toxin with the
chapter ร. 3) and suitable software. The test is not valid quantity of the standard preparation of botulinum antitoxin
unless the percentage of propidium iodide-positive cells at the necessary to give the same protection. For this comparison a
lower asymptote of the curve is less then 15 per cent and the reference preparation of each type of botulinum antitoxin.
calibrated in International Units, and suitable preparations of mixture, inject a dose of 1.0 mL intraperitoneally into each
botulinum toxins, for use as test toxins, are required. mouse. Observe the mice for 96 h.
The potency of each test toxin is determined in relation to The mixture that contains the largest volume of antitoxin
the specific reference preparation; the potency7 of the that fails to protect the mice from death contains 0.5 IU.
botulinum antitoxin to be examined is determined in relation This quantity7 is used to calculate the potency of the antitoxin
to the potency7 of ±e test toxins by the same method. in International Units per millilitre.
International Units of the antitoxin are the specific The test is not valid unless all the mice injected with
neutralising activity for botulinum toxin type A, type B and mixtures containing 2.0 mL or less of the solution of the
type E contained in stated amounts of the International reference preparation die and all those injected with mixtures
Standards which consist of dried immune horse sera of types containing more survive.
A, B and E. The equivalence in International Units of the
International Standard is stated from time to time by the LABELLING
World Health Organization. The label states the types of Cl. botulinum toxin neutralised
Selection of animals Use mice having body masses such that by the preparation.
the difference between the lightest and the heaviest does not
exceed 5 g.
Preparation of test toxins CA UTION: Botulinum toxin is
extremely toxic: exceptional care must be taken in any procedure
in which it is employed. Prepare type A, B and E toxins from
sterile filtrates of approximately 7-day cultures in liquid Diphtheria Antitoxin ★ **
medium of Cl. botulinum types A, B and E. To the filtrates, (Ph. Eur. monograph 0086) *
add 2 volumes of glycerol, concentrate, if necessary, by The label may state ‘Dip/Ser’.
dialysis against glycerol and store at or slightly below 0 °C.
Ph Eur________________________________________ ____ -___ —
Selection of test toxins Select toxins of each type for use as test
toxins by determining for mice the L+/10 dose and the DEFINITION
LD50, the observation period being 96 h. The test toxins Diphtheria antitoxin is a preparation containing antitoxic
contain at least 1000 LD50 in an L+/10 dose. globulins that have the power of specifically neutralising the
Determination of test doses of the toxins (L-r/10 dose). Prepare toxin formed by Corynebacterium diphtheriae.
solutions of the reference preparations in a suitable liquid PRODUCTION
such that each contains 0.25 IU of antitoxin per millilitre. It is obtained by fractionation from the serum of horses, or
Using each solution in turn, determine the test dose of the other mammals, that have been immunised against diphtheria
corresponding test toxin. toxin.
Prepare mixtures of the solution of the reference preparation
IDENTIFICATION
and the test toxin such that each contains 2.0 mL of the
It specifically neutralises the toxin formed by c. diphtheriae,
solution of the reference preparation, one of a graded series
rendering it harmless to susceptible animals.
of volumes of the test toxin and sufficient of a suitable liquid
to bring the total volume to 5.0 mL. Allow the mixtures to ASSAY
stand at room temperature, protected from light, for 60 min. Not less than 1000 IU of antitoxin per millilitre for antitoxin
Using four mice for each mixture, inject a dose of 1.0 mL obtained from horse scrum. Not less than 500 IU of
intraperitoneally into each mouse. Observe the mice for 96 h. antitoxin per millilitre for antitoxin obtained from the serum
The test dose of toxin is the quantity in 1.0 mL of the of other mammals.
mixture made with the smallest amount of toxin capable of The potency of diphtheria antitoxin is determined by
causing, despite partial neutralisation by the reference comparing the dose necessary to protect guinea-pigs or
preparation, the death of all four mice injected with the rabbits against the emhrogenic effects of a fixed dose of
mixture within the observation period. diphtheria toxin with the quantity of the standard preparation
Determination of potency of the antitoxin Prepare solutions of of diphtheria antitoxin necessary to give the same protection.
each reference preparation in a suitable liquid such that each For this comparison a reference preparation of diphtheria
contains 0.25 IU of antitoxin per millilitre. antitoxin, calibrated in International Units, and a suitable
preparation of diphtheria toxin, for use as a test toxin, are
Prepare solutions of each test toxin in a suitable liquid such
required. The potency of the test toxin is determined in
that each contains 2.5 test doses per millilitre.
relation to the reference preparation; the potency of the
Using each toxin solution and the corresponding reference diphtheria antitoxin to be examined is determined in relation
preparation in turn, determine the potency of the antitoxin. to the potency of the test toxin by the same method.
Prepare mixtures of the solution of the test toxin and the
The International Unit of antitoxin is the specific neutralising
antitoxin to be examined such that each contains 2.0 mL of
activity for diphtheria toxin contained in a stated amount of
the solution of the test toxin, one of a graded series
the International Standard, which consists of a quantity of
of volumes of the antitoxin to be examined, and sufficient of
dried immune horse serum. The equivalence in International
a suitable liquid to bring the total volume to 5.0 mL. Also
Units of the International Standard is stated by the World
prepare mixtures of the solution of the test toxin and the
Health Organization.
solution of the reference preparation such that each contains
2.0 mL of the solution of the test toxin, one of a graded Preparation of test toxin Prepare diphtheria toxin from cultures
series of volumes of the solution of the reference preparation of c. diphtheriae in a liquid medium. Filter the culture to
centred on that volume (2.0 mL) that contains 0.5 IU, and obtain a sterile toxic filtrate and store at 4 °C.
sufficient of a suitable liquid to bring the total volume to Selection of test toxin Select a toxin for use as a test toxin by
5.0 mL. Allow the mixtures to stand at room temperature, determining for guinea-pigs or rabbits the lr/100 dose and the
protected from light, for 60 min. Using four mice for each minimal reacting dose, the observation period being 48 h.
The test toxin has at least 200 minimal reacting doses in the
lr/100 dose. European Viper Venom Antiserum *** **
Minimal reacting dose This is the smallest quantity of toxin (Ph. Eur. monograph 0145) ***
which, when injected intracutaneously into guinea-pigs or The only poisonous snake native to the British Isles is the
rabbits, causes a small, characteristic reaction at the site of adder or common viper, Vipera berus. In a geographical
injection within 48 h. region where other species of snake (including elapids) arc
The test toxin is allowed to stand for some months before found, antisera able to neutralise the venoms of the species of
being used for the assay of antitoxin. During this time its snake indigenous to the region should be used. When the
toxicity declines and the lr/100 dose may be increased. preparation is intended to neutralise the venom or venoms of
Determine the minimal reacting dose and the lr/100 dose at one or more snakes other than vipers, the title Snake Venom
frequent intervals. When experiment shows that the Antiserum is used.
lr/100 dose is constant, the test toxin is ready for use and Ph Eur______________________________________________________________
may be used for a long period. Store the test toxin in the
dark at 0 ๐c to 5 °C. Maintain its sterility by the addition of DEFINITION
toluene or other antimicrobial preservative that does not European viper venom antiserum is a preparation containing
cause a rapid decline in specific toxicity. antitoxic globulins that have the power of neutralising the
venom of one or more species of viper. The globulins are
Determination of test dose of toxin (lr/100 dose). Prepare a
obtained by fractionation of the serum of animals that have
been immunised against the venom or venoms.
that it contains 0.1 IU of antitoxin per millilitre.
Prepare mixtures of the solution of the reference preparation IDENTIFICATION
and of the test toxin such that each contains 1.0 mL of the It neutralises the venom of Vipera annnodytes, or Vipera aspis,
solution of the reference preparation, one of a graded series or Vipera bents, or Vipera ursinii or the mixture of these
of volumes of the test toxin and sufficient of a suitable liquid venoms stated on the label, rendering them harmless to
to bring the total volume to 2.0 mL. Allow the mixtures to susceptible animals.
stand at room temperature, protected from light, for 15 min ASSAY
to 60 min. Using two animals for each mixture^ inject a dose Each millilitre of the preparation to be examined contains
of 0.2 mL intracutaneously into the shaven or depilated sufficient antitoxic globulins to neutralise not less than
flanks of each animal. Observe the animals for 48 h. 100 mouse LD50 of Vipera amniodytes venom or Vipera aspis
The test dose of toxin is the quantity in 0.2 mL of the venom and not less than 50 mouse LD50 of the venoms of
mixture made with the smallest amount of toxin capable of other species of viper.
causing, despite partial neutralisation by the reference The potency of European viper venom antiserum is
preparation, a small but characteristic erythematous lesion at determined by estimating the dose necessary to protect mice
the site of injection. against the lethal effects of a fixed dose of venom of the
Determination of potency of the antitoxin Prepare a solution of relevant species of viper.
the reference preparation in a suitable liquid such diat it Selection of test venoms Use venoms which have the normal
contains 0.125 IU of antitoxin per millilitre. physicochemical, toxicological and immunological
Prepare a solution of the test toxin in a suitable liquid such characteristics of venoms from the particular species of
that it contains 12.5 test doses per millilitre. vipers. They are preferably freeze-dried and stored in the
Prepare mixtures of the solution of the test toxin and of the dark at 5 ± 3 °C.
antitoxin to be examined such that each contains 0.8 mL of Select a venom for use as a test venom by determining the
the solution of the test toxin, one of a graded series LD50 for mice, the observation period being 48 h.
of volumes of the antitoxin to be examined and sufficient of a Determination of the test dose of venom Prepare graded dilutions
suitable liquid to bring the total volume to 2.0 mL. Also of the reconstituted venom in a 9 g/L solution of sodium
prepare mixtures of the solution of the test toxin and the chloride R or other isotonic diluent in such a manner that the
solution of the reference preparation such that each contains middle dilution contains in 0.25 mL the dose expected to be
0.8 mL of the solution of the test toxin, one of a graded the LD50. Dilute with an equal volume of the same diluent.
series of volumes of the solution of the reference preparation Using at least four mice, each weighing 18 g to 20 g, for
centred on that volume (0.8 mL) that contains 0.1 IU and each dilution, inject 0.5 mL intravenously into each mouse.
sufficient of a suitable liquid to bring the total volume to Observe the mice for 48 h and record the number of deaths.
2.0 mL. Allow the mixtures to stand at room temperature, Calculate the LD50 using the usual statistical methods.
protected from light, for 15 min to 60 min. Using two Determination of the potency of the antiserum to be examined
animals for each mixture, inject a dose of 0.2 mL Dilute the reconstituted test venom so that 0.25 mL contains
intracutaneously into the shaven or depilated flanks of each the test dose of 5 LD50 (test venom solution).
animal. Observe the animals for 48 h.
Prepare serial dilutions of the antiserum to be examined in a
The mixture that contains the largest volume of antitoxin 9 g/L solution of sodium chloride R or other isotonic diluent,
that fails to protect the guinea-pigs from the erythematous the dilution factor being 1.5 to 2.5. Use a sufficient number
effects of the toxin contains 0.1 IU. This quantity is used to and range of dilutions to enable a mortality curve between
calculate the potency of the antitoxin in International Units 20 per cent and 80 per cent mortality to be established and
per millilitre. to permit an estimation of the statistical variation.
The test is not valid unless all the sites injected with mixtures Prepare mixtures such that 5 mL of each mixture contains
containing 0.8 mL or less of the solution of the reference 2.5 mL of one of the dilutions of the antiserum to be
preparation show erythematous lesions and at all those examined and 2.5 mL of the test venom solution. Allow the
injected with mixtures containing more there are no lesions. mixtures to stand in a water-bath at 37 °C for 30 min. Using
_______________________________________________________________ Ph Eur not fewer than six mice, each weighing 18 g to 20 g, for each
IV-544 Immuno sera 2016
mixture, inject 0.5 mL intravenously into each mouse. Units, and a suitable preparation of Cl. novyi toxin for use as
Observe the mice for 48 h and record the number of deaths. a test toxin are required. The potency of the test toxin is
Calculate the PD50, using the usual statistical methods. determined in relation to the reference preparation;
At the same time verify the number of LD50 in the test dose the potency of the gas-gangrene antitoxin (novyi) to be
of venom, using ±e method described above. Calculate the examined is determined in relation to the potency of the test
potency of the antiserum using the following expression: toxin by the same method.
The International Unit of antitoxin is the specific neutralising
(Tv - 1) activity for Cl. novyi toxin contained in a stated amount of
PD50 the International Standard, which consists of a quantity of
Tv = number of LD50 in the test dose of venom. Units of the International Standard is stated by the World
In each mouse dose of the venom-antiserum mixture at the Health Organization.
end point there is one LD50 of venom remaining Selection of animals Use mice having body masses such that
unneutralised by the antiserum and it is this unneutralised the difference between the lightest and the heaviest does not
venom that is responsible for the deaths of 50 per cent of the exceed 5 g.
mice inoculated with the mixture. The amount of venom Preparation of test toxin Prepare the test toxin from a sterile
neutralised by the antiserum is thus one LD50 less than the filtrate of an approximately 5-day culture in liquid medium of
total amount contained in each mouse dose. Therefore, as Cl. novyi. Treat the filtrate with ammonium sulfate R, collect
the potency of the antiserum is defined in terms of the the precipitate, which contains the toxin, dry in vacuo over
number of LD50 of venom that are neutralised, rather than diphosphorus pentoxide R, powder and store dry'.
the number of LD50 in each mouse dose, the expression
Selection of test toxin Select a toxin for use as a test toxin by
required in the calculation of potency is Tv - 1 rather than
determining for mice the L+ dose and the LD50, the
observation period being 72 h. The test toxin has an L-r dose
Alternatively, the quantity of test venom in milligrams that is of 0.5 mg or less and contains not less than 25 LD50 in each
neutralised by 1 mL or some other defined volume of the L+ dose.
antiserum to be examined may be calculated.
Determination of test dose of toxin (L+ dose) Prepare a solution
LABELLING of the reference preparation in a suitable liquid such that it
The label states the venom or venoms against which the contains 12.5 IU of antitoxin per millilitre.
antiserum is effective. Prepare a solution of the test toxin in a suitable liquid such
CAUTION because of the allergenic properties of viper venoms, that 1 mL contains a precisely known amount such as
inhalation of venom dust should be avoided by suitable 10 mg.
precautions. Prepare mixtures of the solution of the reference preparation
-----------------------------------------------------------------------------------------------------------Ph Eur and the solution of the test toxin such that each contains
0.8 mL of the solution of the reference preparation, one of a
graded series of volumes of the solution of the test toxin and
sufficient of a suitable liquid to bring the total volume to
Gas-gangrene Antitoxin (Novyi) * * 2.0 mL. Allow the mixtures to stand at room temperature,
protected from light, for 60 min. Using six mice for each
Gas-gangrene Antitoxin (Oedematiens) *** mixture, inject a dose of 0.2 mL intramuscularly into each
(Ph. Eur. monograph 0087) mouse. Observe the mice for 72 h.
The label may state ‘Nov/Ser’. The test dose of toxin is the quantity in 0.2 mL of the
Preparation mixture made with the smallest amount of toxin capable of
Mixed Gas-gangrene Antitoxin causing, despite partial neutralisation by the reference
preparation, the death of all six mice injected with the
Ph Elf_______________________________________________________ _
mixture within the observation period.
DEFINITION Determination of potency of the antitoxin Prepare a solution of
Gas-gangrene antitoxin (novyi) is a preparation containing the reference preparation in a suitable liquid such that it
antitoxic globulins that have the power of neutralising the contains 12.5 IU of antitoxin per millilitre.
alpha toxin formed by Clostridium novyi (Former Prepare a solution of the test toxin in a suitable liquid such
nomenclature: Clostridium oedematiens}. It is obtained by that it contains 12.5 test doses per millilitre.
fractionation from the serum of horses, or other mammals,
that have been immunised against Cl. novyi alpha toxin.
IDENTIFICATION the solution of the test toxin, one of a graded series
It specifically neutralises the alpha toxin formed by Cl. novyi, of volumes of the antitoxin to be examined and sufficient of a
rendering it harmless to susceptible animals. suitable liquid to bring the total volume to 2.0 mL. Also
ASSAY
Not less than 3750 IU of antitoxin per millilitre.
0.8 mL of the solution of the test toxin, one of a graded
The potency of gas-gangrene antitoxin (novyi) is determined series of volumes of the solution of the reference preparation
by comparing the dose necessary to protect mice or other centred on that volume (0.8 mL) that contains 10 IU and
suitable animals against the lethal effects of a fixed dose of sufficient of a suitable liquid to bring the total volume to
Cl. novyi toxin with the quantity of the standard preparation 2.0 mL. Allow the mixtures to stand at room temperature,
of gas-gangrene antitoxin (novyi) necessary to give the same protected from light, for 60 min. Using six mice for each
protection. For this comparison a reference preparation of
gas-gangrene antitoxin (novyi), calibrated in International
mixture, inject a dose of 0.2 mL intramuscularly into each observation period being 48 h. The test toxin has an L+ dose
mouse. Observe the mice for 72 h. of 4 mg or less and contains not less than 20 LD50 in each
The mixture that contains the largest volume of antitoxin L+ dose.
that fails to protect the mice from death contains 10 IU. This Determination of test dose of toxin (L+ dose) Prepare a solution
quantity is used to calculate the potency of the antitoxin in of the reference preparation in a suitable liquid such that it
International Units per millilitre. contains 5 IU of antitoxin per millilitre.
The test is not valid unless all the mice injected with Prepare a solution of the test toxin in a suitable liquid such
mixtures containing 0.8 mL or less of the solution of the that 1 mL contains a precisely known amount such as
reference preparation die and all those injected with mixtures 10 mg.
containing a larger volume survive. Prepare mixtures of the solution of the reference preparation
------- ----------------------- ------- ------------------------------------------------------------------Ph Eur and the solution of the test toxin such that each contains
2.0 mL of the solution of the reference preparation, one of a
graded series of volumes of the solution of the test toxin and
Gas-gangrene Antitoxin ** ** 5.0 mL. Allow the mixtures to stand at room temperature,
protected from light, for 60 min. Using six mice for each
(Perfringens) ***** mixture, inject a dose of 0.5 mL intravenously into each
(Ph. Eur. monograph 0088) mouse. Observe the mice for 48 h.
The label may state ‘PerffSer’. The test dose of toxin is the quantity in 0.5 mL of the
Preparation mixture made with the smallest amount of toxin capable of
Mixed Gas-gangrene Antitoxin causing, despite partial neutralisation by the reference
Ph Elt____________________________________________
mixture within the observation period.
DEFINITION Determination of potency of the antitoxin Prepare a solution of
Gas-gangrene antitoxin (perfringens) is a preparation the reference preparation in a suitable liquid such ±at it
containing antitoxic globulins that have the power of contains 5 IU of antitoxin per millilitre.
specifically neutralising the alpha toxin formed by Clostridium Prepare a solution of the test toxin in a suitable liquid such
perfringens. It is obtained by fractionation from the serum of that it contains five test doses per millilitre.
horses, or other mammals, that have been immunised against
Prepare mixtures of ±e solution of the test toxin and ±e
Cl. perfringens alpha toxin.
IDENTIFICATION the solution of the test toxin, one of a graded series of
It specifically neutralises the alpha toxin formed by volumes of the antitoxin to be examined and sufficient of a
Cl. perfringens, rendering it harmless to susceptible animals. suitable liquid to bring the total volume to 5.0 mL. Also
ASSAY
2.0 mL of the solution of the test toxin, one of a graded
The potency of gas-gangrene antitoxin (perfringens) is series of volumes of the solution of the reference preparation
determined by comparing the dose necessary to protect mice centred on that volume (2.0 mL) that contains 10 IU and
or other suitable animals against the lethal effects of a fixed sufficient of a suitable liquid to bring the total volume to
dose of Cl. perfringens toxin with the quantity of the standard 5.0 mL. Allow the mixtures to stand at room temperature,
preparation of gas-gangrene antitoxin (perfringens) necessary protected from light, for 60 min. Using six mice for each
to give the same protection. For this comparison a reference mixture, inject a dose of 0.5 mL intravenously into each
preparation of gas-gangrene antitoxin (perfringens), calibrated mouse. Observe the mice for 48 h.
in International Units, and a suitable preparation of The mixture that contains the largest volume of antitoxin
CL perfringens toxin for use as a test toxin are required. that fails to protect the mice from death contains 10 IU. This
The potency of the test toxin is determined in relation to the quantity is used to calculate the potency of the antitoxin in
reference preparation; the potency of the gas-gangrene
International Units per millilitre.
antitoxin (perfringens) to be examined is determined in
The test is not valid unless all the mice injected with
relation to the potency of the test toxin by the same method.
mixtures containing 2.0 mL or less of the solution of the
The International Unit of antitoxin is the specific neutralising reference preparation die and all those injected with mixtures
activity for CL perfringens toxin contained in a stated amount containing a larger volume survive.
of the International Standard, which consists of a quantity of
________________________________________________________________ Ph Eur
Selection of animals Use mice having body masses such that
exceed 5 g.
Preparation of test toxin Prepare the test toxin from a sterile
filtrate of an approximately 5-day culture in liquid medium of
CL perfringens. Treat the filtrate with ammonium sulfate R,
collect the precipitate, which contains the toxin, dry in vacuo
over diphosphorus pentoxide R, powder and store dry.
Selection of test toxin Select a toxin for use as a test toxin by
determining for mice the L+ dose and the LD5Q, the

Gas-gangrene Antitoxin ** ** 5.0 mL. Allow the mixtures to stand at room temperature,
(Septicum) ***** protected from light, for 60 min. Using six mice for each
(Ph. Eur. monograph 0089) mixture, inject a dose of 0.5 mL intravenously into each
The label may state ‘Sep/Ser’. mouse. Observe the mice for 72 h.
The test dose of toxin is the quantity in 0.5 mL of the
Preparation
mixture made with the smallest amount of toxin capable of
Mixed Gas-gangrene Antitoxin
causing, despite partial neutralisation by the reference
PhEur___________________________________________________
DEFINITION mixture within the observation period.
Gas-gangrene antitoxin (septicum) is a preparation Determination of potency of the antitoxin Prepare a solution of
containing antitoxic globulins that have the power of the reference preparation in a suitable liquid such that it
specifically neutralising the alpha toxin formed by Clostridium contains 5 IU of antitoxin per millilitre.
septicum. It is obtained by fractionation from the serum of Prepare a solution of the test toxin in a suitable liquid such
horses, or other mammals, that have been immunised against that it contains five test doses per millilitre.
CL septicum alpha toxin. Prepare mixtures of the solution of the test toxin and the
IDENTIFICATION antitoxin to be examined such that each contains 2.0 mL of
It specifically neutralises the alpha toxin formed by the solution of the test toxin, one of a graded series of
CL septicum, rendering it harmless to susceptible animals. volumes of the antitoxin to be examined and sufficient of a
suitable liquid to bring the total volume to 5.0 mL. Also
ASSAY
The potency of gas-gangrene antitoxin (septicum) is 2.0 mL of the solution of the test toxin, one of a graded
determined by comparing the dose necessary to protect mice series of volumes of the solution of the reference preparation
or other suitable animals against the lethal effects of a fixed centred on that volume (2.0 mL) that contains 10 IU and
dose of CL septicum toxin with the quantity of the standard sufficient of a suitable liquid to bring the total volume to
preparation of gas-gangrene antitoxin (septicum) necessary to 5.0 mL. Allow the mixtures to stand at room temperature,
give the same protection. For this comparison a reference protected from light, for 60 min. Using six mice for each
preparation of gas-gangrene antitoxin (septicum), calibrated mixture, inject a dose of 0.5 mL intravenously into each
in International Units, and a suitable preparation of mouse. Observe the mice for 72 h.
CL septicum toxin for use as a test toxin are required. The mixture that contains the largest volume of antitoxin
The potency of the test toxin is determined in relation to the that fails to protect the mice from death contains 10 IU. This
reference preparation; the potency of the gas-gangrene quantity is used to calculate the potency of the antitoxin in
antitoxin (septicum) to be examined is determined in relation
International Units per millilitre.
to the potency of the test toxin by the same method.
The International Unit of antitoxin is the specific neutralising mixtures containing 2.0 mL or less of the solution of the
activity for CL septicum toxin contained in a stated amount of reference preparation die and all those injected with mixtures
the International Standard, which consists of a quantity of
containing more survive.
Selection of animals Use mice having body masses such that
exceed 5 g. Mixed Gas-gangrene Antitoxin * *,
Preparatioit of test toxin Prepare the test toxin from a sterile
filtrate of an approximately 5-day culture in liquid medium of (Ph. Eur. monograph 0090) *
CL septicum. Treat the filtrate with ammonium sulfate R, The label may state ‘Gas/Ser’.
collect the precipitate, which contains the toxin, dry in vacuo Ph Eur______________________________________________ _________________
over diphosphorus pentoxide R, powder and store dry.
DEFINITION
Selection of test toxin Select a toxin for use as a test toxin by Mixed gas-gangrene antitoxin is prepared by mixing gas
determining for mice the Lt dose and the LD50, the gangrene antitoxin (novyi), gas-gangrene antitoxin
observation period being 72 h. The test toxin has an Lt dose (perfringens) and gas-gangrene antitoxin (septicum) in
of 0.5 mg or less and contains not less than 25 LD50 in each appropriate quantities.
L+ dose.
IDENTIFICATION
Determination of test dose of toxin (L+ dose) Prepare a solution
of the reference preparation in a suitable liquid such that it It specifically neutralises the alpha toxins formed by
contains 5 IU of antitoxin per millilitre. Clostridium novyi (former nomenclature: Clostridium
oedematiens), Clostridium perfringens and Clostridium septicum,
Prepare a solution of the test toxin in a suitable liquid such
rendering them harmless to susceptible animals.
that 1 mL contains a precisely known amount such as
20 mg. ASSAY
Gas-gangrene antitoxin (novyi), not less than 1000 IU of
antitoxin per millilitre; gas-gangrene antitoxin (perfringens),
and the solution of the test toxin such that each contains
2.0 mL of the solution of the reference preparation, one of a not less than 1000 IU of antitoxin per millilitre; gas-gangrene
graded series of volumes of the solution of the test toxin and antitoxin (septicum) not less than 500 IU of antitoxin per
millilitre.
Carry out the assay for each component, as prescribed in the and store dry, either in sealed ampoules or in vacuo over
monographs on Gas-gangrene antitoxin (novyi) (0087), Gas diphosphorus pentoxide R.
gangrene antitoxin (perfringens) (0088) and Gas-gangrene Determination of test dose of toxin (LpHO dose) Prepare a
antitoxin (septicum) (0089). solution of the reference preparation in a suitable liquid such
---------------- - ------------------------------ -------------------------------------------------------- PhEur that it contains 0.5 IU of antitoxin per millilitre.
If the test toxin is stored dry, reconstitute it using a suitable
liquid.
Tetanus Antitoxin ** ** and the test toxin such that each contains 2.0 mL of the
solution of the reference preparation, one of a graded series
(Tetanus Antitoxin for Human Use, *** of volumes of the test toxin and sufficient of a suitable liquid
Ph Eur monograph 0091) to bring the volume to 5.0 mL. Allow the mixtures to stand
The label may state ‘Tet/Ser’. at room temperature, protected from light, for 60 min. Using
Ph Eir_______ ___________
six mice for each mixture, inject a dose of 0.5 mL
subcutaneously into each mouse. Observe the mice for 96 h.
DEFINITION Mice that become paralysed may be euthanised.
Tetanus antitoxin for human use is a preparation containing The test dose of toxin is the quantity in 0.5 mL of the
antitoxic globulins that have the power of specifically mixture made with the smallest amount of toxin capable of
neutralising the toxin formed by Clostridium tetani. causing, despite partial neutralisation by the reference
PRODUCTION preparation, paralysis in all six mice injected with the mixture
It is obtained by fractionation from the serum of horses, or within the observation period.
other mammals, that have been immunised against tetanus Determination of potency of the antitoxin Prepare a solution of
toxin. the reference preparation in a suitable liquid such that it
contains 0.5 IU of antitoxin per millilitre.
IDENTIFICATION
It specifically neutralises the toxin formed by Cl. tetani, Prepare a solution of the test toxin in a suitable liquid such
rendering it harmless to susceptible animals. that it contains five test doses per millilitre.
POTENCY
Not less than 1000 IU of antitoxin per millilitre when the solution of the test toxin, one of a graded series
intended for prophylactic use. Not less than 3000 IU of of volumes of the antitoxin to be examined and sufficient of a
antitoxin per millilitre when intended for therapeutic use. suitable liquid to bring the total volume to 5.0 mL. Also
The potency of tetanus antitoxin is determined by comparing prepare mixtures of the solution of the test toxin and the
the dose necessary to protect guinea-pigs or mice against the solution of the reference preparation such that each contains
paralytic effects of a fixed dose of tetanus toxin with the 2.0 mL of the solution of the test toxin, one of a graded
quantity of the standard preparation of tetanus antitoxin series of volumes of the solution of the reference preparation
necessary to give the same protection. In countries where the centred on that volume (2.0 mL) that contains 1 IU and
paralysis method is not obligatory the lethal method may be sufficient of a suitable liquid to bring the total volume to
used. For this method the number of animals and the 5.0 mL. Allow the mixtures to stand at room temperature,
procedure are identical with those described for the paralysis protected from light, for 60 min. Using six mice for each
method but the end-point is the death of the animal rather mixture, inject into each mouse subcutaneously a dose of
than the onset of paralysis and the LU 10 dose is used 0.5 mL. Observe the mice for 96 h. Mice that become
instead of the Lp/10 dose. For this comparison a reference paralysed may be euthanised.
preparation of tetanus antitoxin, calibrated in International The mixture that contains the largest volume of antitoxin
Units, and a suitable preparation of tetanus toxin, for use as that fails to protect the mice from paralysis contains 1 IU.
a test toxin, are required. The potency of the test toxin is This quantity is used to calculate the potency of the antitoxin
determined in relation to the reference preparation; in International Units per millilitre.
the potency of the tetanus antitoxin to be examined is
determined in relation to the potency of the test toxin by the
mixtures containing 2.0 mL or less of the solution of the
same method.
reference preparation show paralysis and all those injected
The International Unit of antitoxin is the specific neutralising with mixtures containing more do not.
activity for tetanus toxin contained in a stated amount of the
_______________________________________________________ _______ Ph Eur
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
Organization.
Selection of animals If mice are used, the body masses should
be such that the difference between the lightest and the
heaviest does not exceed 5 g.
Preparation of test toxin Prepare the test toxin from a sterile
filtrate of an approximately 9-day culture in liquid medium of
CL tetani. To the filtrate add 1 to 2 volumes of glycerol and
store slightly below 0 °C. Alternatively, treat the filtrate with
ammonium sulfate R, collect the precipitate, which contains
the toxin, dry in vacuo over diphosphorus pentoxide R, powder
LV-548 Vaccines 2016
Combined vaccines Are multicomponent preparations

VACCINES ;* ** formulated so that different antigens are administered
(Vaccines for Human Use, Ph Eur monograph 0153) * ** simultaneously. The different antigenic components are
Vaccines comply with the requirements of the European intended to protect against different strains or types of the
Pharmacopoeia monograph for Vaccines for Human Use. These same organism and/or against different organisms.
requirements are reproduced below. A combined vaccine may be supplied by the manufacturer
either as a single liquid or freeze-dried preparation or as
several constituents with directions for admixture before use.
DEFINITION Where there is no monograph to cover a particular
Vaccines for human use are preparations containing antigens combination, the vaccine complies with the monograph for
capable of inducing a specific and active immunity in man each individual component, with any necessary modifications
against an infecting agent or the toxin or antigen elaborated approved by the competent authority.
by it. Immune responses include the induction of the innate Adsorbed vaccines Are suspensions and may form a sediment
and the adaptive (cellular, humoral) parts of the immune at the bottom of the container.
system. Vaccines for human use shall have been shown to
PRODUCTION
have acceptable immunogenic activity and safety in man with
the intended vaccination schedule. General provisions
The production method for a given product must have been
Vaccines for human use may contain: whole micro-organisms shown to yield consistently batches comparable with the
(bacteria, viruses or parasites), inactivated by chemical or batch of proven clinical efficacy, immunogenicity and safety’
physical means that maintain adequate immunogenic in man. Product specifications including in-process testing
properties; whole live micro-organisms that are naturally should be set. Specific requirements for production including
avirulent or that have been treated to attenuate their in-process testing arc included in individual monographs.
virulence whilst retaining adequate immunogenic properties; Where justified and authorised, certain tests may be omitted
antigens extracted from the micro-organisms or secreted by where it can be demonstrated, for example by validation
the micro-organisms or produced by genetic engineering or studies, that the production process consistently ensures
chemical synthesis. The antigens may be used in their native
compliance with the test.
state or may be detoxified or otherwise modified by chemical
Unless offerwise justified and authorised, vaccines are
or physical means and may be aggregated, polymerised or
produced using a seed-lot system. The methods of
conjugated to a carrier to increase their immunogenicity.
preparation are designed to maintain adequate immunogenic
Vaccines may contain an adjuvant. Where the antigen is
properties, to render the preparation harmless and to prevent
adsorbed on a mineral adjuvant, the vaccine is referred to as
‘adsorbed’. contamination with extraneous agents.
Where vaccines for human use are manufactured using
Terminology used in monographs on vaccines for human use
materials of human or animal origin, the general
is defined in chapter 5.2.1.
requirements of chapter 5.1.7. Viral safety apply in
Bacterial vaccines containing whole cells Are suspensions of conjunction with the more specific requirements relating to
various degrees of opacity in colourless or almost colourless viral safety in this monograph, in chapters 5.2.2. Chicken
liquids, or may be freeze-dried. They may be adsorbed. flocks free from specified pathogens for the production and quality
The concentration of living or inactivated bacteria is control of vaccines, 5.2.3. Cell substrates for the production of
expressed in terms of International Units of opacity or, where vaccines for human use and 2.6.16. Tests for extraneous agents in
appropriate, is determined by direct cell count or, for live viral vaccines for human use, and in individual monographs.
bacteria, by viable count.
Unless otherwise justified and authorised, in the production
Bacterial vaccines containing bacterial components Are of a final lot of vaccine, the number of passages of a vims, or
suspensions or freeze-dried products. They may be adsorbed. the number of subcultures of a bacterium, from the master
The antigen content is determined by a suitable validated seed lot shall not exceed that used for production of the
assay. vaccine shown to be satisfactory in clinical trials with respect
Bacterial toxoids Are prepared from toxins by diminishing to safety and efficacy or immunogenicity.
their toxicity to an acceptable level or by completely Vaccines are as far as possible free from ingredients known to
eliminating it by physical or chemical procedures whilst cause toxic, allergic or other undesirable reactions in man.
retaining adequate immunogenic properties. The toxins are Suitable additives, including stabilisers and adjuvants may be
obtained from selected strains of micro-organisms. incorporated. Penicillin and streptomycin are neither used at
The method of production is such that the toxoid does not any stage of production nor added to the final product;
revert to toxin. The toxoids are purified. Purification is however, master seed lots prepared with media containing
performed before and/or after detoxification. Toxoid vaccines penicillin or streptomycin may, where justified and
may be adsorbed. authorised, be used for production.
Viral vaccines Are prepared from viruses grown in animals, in Consistency of production is an important feature of vaccine
fertilised eggs, in suitable cell cultures or in suitable tissues, production. Monographs on vaccines for human use give
or by culture of genetically engineered cells. They are liquids limits for various tests carried out during production and on
that vary in opacity according to the type of preparation or the final lot. These limits may be in the form of maximum
may be freeze-dried. They may be adsorbed. Liquid values, minimum values, or minimum and maximum
preparations and freeze-dried preparations after reconstitution tolerances around a given value. While compliance with these
may be coloured if a pH indicator such as phenol red has limits is required, it is not necessarily sufficient to ensure
been used in the culture medium. consistency of production for a given vaccine. For relevant
Synthetic antigen vaccines Are generally clear or colourless tests, the manufacturer must therefore define for each
liquids. The concentration of the components is usually product a suitable action or release limit or limits to be
expressed in terms of specific antigen content. applied in view of the results found for batches tested
2016 Vaccines IV-549
clinically and those used to demonstrate consistency of In agreement with the competent authority, replacement of
production. These limits may subsequently be refined on a the sterility test by a bioburden test with a low bioburden
statistical basis in light of production data. limit based on batch data and process validation may be
Substrates for propagation acceptable for intermediates preceding the final bulk,
Substrates for propagation comply with the relevant provided that a sterilising filtration is performed later in the
requirements of the Pharmacopoeia (5.2.2, 5.2.5) or in the production process.
absence of such requirements with those of the competent It is a prerequisite that the intermediate is filtered through a
authority. Processing of cell banks and subsequent cell bacteria-retentive filter prior to storage, that authorised pre
cultures is done under aseptic conditions in an area where no filtration bioburden limits have been established for this
other cells are being handled. Serum and trypsin used in the filtration, and that adequate measures are in place to avoid
preparation of cell suspensions shall be shown to be free from contamination and growth of micro-organisms during storage
extraneous agents. of the intermediate.
Seed lots/cell banks Final bulk
The master seed lot or cell bank is identified by historical The final bulk is prepared by aseptically blending the
records that include information on its origin and subsequent ingredients of the vaccine. For non-liquid vaccines for
manipulation. Suitable measures are taken to ensure that no administration by a non-parenteral route, the final bulk is
extraneous agent or undesirable substance is present in a prepared by blending the ingredients of the vaccine under
master or working seed lot or a cell bank. suitable conditions.
Culture media Adjuvants One or more adjuvants may be included in the
Culture media are as far as possible free from ingredients formulation of a vaccine to potentiate and/or modulate the
known to cause toxic, allergic or other undesirable reactions immune response to the antigen(s). Adjuvants may be
in man; if inclusion of such ingredients is necessary, it shall included in the formulation of the final vaccine or presented
be demonstrated that the amount present in the final lot is separately. Suitable characterisation and quality control of the
reduced to such a level as to render the product safe. adjuvant(s), alone and in combination with the antigen(s), is
Approved animal (but not human) scrum may be used in the essential for consistent production. Quality specifications are
growth medium for cell cultures but the medium used for established for each adjuvant, alone and in combination with
maintaining cell growth during virus multiplication shall not the antigen(ร).
contain serum, unless otherwise stated. Cell culture media Adsorbents as adjuvants Vaccines may be adsorbed on
may contain a pH indicator such as phenol red and approved aluminium hydroxide, aluminium phosphate, calcium
antibiotics at the lowest effective concentration, although it is phosphate or other suitable adsorbents. The adsorbents are
preferable to have a medium free from antibiotics during prepared in special conditions that confer the appropriate
production. physical form and adsorptive properties.
Propagation and harvest Where an adsorbent is used as an adjuvant and is generated
The seed cultures are propagated and harvested under in situ during production of the vaccine, quality specifications
defined conditions. The purity of the harvest is verified by are established for each of the ingredients and for the
suitable tests as defined in the monograph. generated adsorbent in the vaccine. Quality specifications are
Control cells intended to control, in particular:
For vaccines produced in cell cultures, control cells are — qualitative and quantitative chemical composition;
maintained and tested as prescribed. In order to provide a — physical form and associated adsorptive properties, where
valid control, these cells must be maintained in conditions relevant, and particularly where the adjuvant will be
that are essentially equivalent to those used for the present as an adsorbent;
production cell cultures, including use of the same batches of — interaction between adjuvant and antigen;
media and media changes. — purity, including bacterial endotoxin content and
microbiological quality;
Control eggs — any other parameters identified as being critical for
For live vaccines produced in eggs, control eggs are functionality.
incubated and tested as prescribed in the monograph.
The stability of each adjuvant, alone and in combination with
Purification the antigen(s), particularly for critical parameters, is
Where applicable, validated purification procedures may be established during development studies.
applied. Antimicrobial preservatives Antimicrobial preservatives are used
Inactivation to prevent spoilage or adverse effects caused by microbial
Inactivated vaccines are produced using a validated contamination occurring during the use of a vaccine.
inactivation process whose effectiveness and consistency have Antimicrobial preservatives are not included in freeze-dried
been demonstrated. Where it is recognised that extraneous products. For single-dose liquid preparations, inclusion of
agents may be present in a harvest, for example in vaccines antimicrobial preservatives is not normally acceptable.
produced in eggs from healthy, non-SPF flocks, the For multidose liquid preparations, the need for effective
inactivation process is also validated with respect to a panel antimicrobial preservation is evaluated taking into account
of model extraneous agents representative of the potential likely contamination during use and the maximum
extraneous agents. A test for effectiveness of the inactivation recommended period of use after broaching of the container.
process is carried out as soon as possible after the If an antimicrobial preservative is used, it shall be shown that
inactivation process. it does not impair the safety or efficacy of the vaccine.
Test for sterility of intermediates prior to final bulk Addition of antibiotics as antimicrobial preservatives is not
Individual monographs on vaccines for human use may normally acceptable.
prescribe a test for sterility for intermediates. During development studies, the effectiveness of the
antimicrobial preservative throughout the period of validity
IV-550 Vaccines 2016
shall be demonstrated to the satisfaction of the competent yield, ratio of infectious viruses (viable bacteria) before and
authority. after freeze-drying, potency at release and real-time stability
The efficacy of the antimicrobial preservative is evaluated as under the prescribed conditions as well as thermal stability.
described in chapter 5.1.3. If neither the A criteria nor the Where there is a significant change in the manufacturing
B criteria can be met, then in justified cases the following procedure of the antigen(s) or formulation, the need for
criteria are applied to vaccines for human use: bacteria, no re-introduction of the test is considered.
increase at 24 h and 7 days, 3 log10 reduction at 14 days, no Stability During development studies, maintenance of
increase at 28 days; fungi, no increase at 14 days and potency of the final lot throughout the period of validity shall
28 days. be demonstrated; the loss of potency in the recommended
Stability of intermediates storage conditions is assessed. Excessive loss even within the
During production of vaccines, intermediates are obtained at limits of acceptable potency may indicate that the vaccine is
various stages and are stored, sometimes for long periods. unacceptable.
Such intermediates include: Expiry date Unless otherwise stated, the expiry date is
— seed lots and cell banks; calculated from the beginning of the assay or from the
— live or inactivated harvests; beginning of the first assay for a combined vaccine.
— purified harvests that may consist of toxins or toxoids, For vaccines stored at a temperature lower than that used for
polysaccharides, bacterial or viral suspensions; stability studies and intended for release without re-assay, the
— purified antigens; expiry date is calculated from the date of removal from cold
— adsorbed antigens; storage. If, for a given vaccine, an assay is not carried out,
— conjugated polysaccharides; the expiry date for the final lot is calculated from the date of
— final bulk vaccine; an approved stability-indicating test or, failing this, from the
— vaccine in the final closed container stored at a date of freeze-drying or the date of filling into the final
temperature lower than that used for final-product containers. For a combined vaccine where components are
stability studies and intended for release without re-assay. presented in separate containers, the expiry date is that of the
Except where they are used within a short period of time, component which expires first.
stability studies are carried out on the intermediates in the The expiry date applies to vaccines stored in the prescribed
intended storage conditions to establish the expected extent conditions.
of degradation. For final bulk vaccine, stability studies may Animal tests
be carried out on representative samples in conditions In accordance with the provisions of the European
equivalent to those intended to be used for storage. For each Convention for the Protection of Vertebrate Animals Used
intermediate (except for seed lots and cell banks), a period of for Experimental and Other Scientific Purposes, tests must
validity applicable for the intended storage conditions is be carried out in such a way as to use the minimum number
established, where appropriate in light of stability studies. of animals and to cause the least pain, suffering, distress or
Final lot lasting harm. The criteria for judging tests in monographs
The final lot is prepared by aseptically distributing the final must be applied in light of this. For example, if it is indicated
bulk into sterile, tamper-proof containers, which, after freeze- that an animal is considered to be positive, infected, etc.
drying where applicable, are closed so as to exclude when typical clinical signs or death occur, then as soon as
contamination. For non-liquid vaccines for administration by sufficient indication of a positive result is obtained the animal
a non-parenteral route, the final lot is prepared by in question shall be either euthanised or given suitable
distributing the final bulk under suitable conditions into treatment to prevent unnecessary suffering. In accordance
sterile, tamper-proof containers. Where justified and with the General Notices, alternative test methods may be
authorised, certain tests prescribed for the final lot may be used to demonstrate compliance with the monograph and the
carried out on the final bulk, if it has been demonstrated that use of such tests is particularly encouraged when this leads to
subsequent manufacturing operations do not affect replacement or reduction of animal use or reduction of
compliance. suffering.
Appearance TESTS
Unless otherwise justified and authorised, each container Vaccines comply with the tests prescribed in individual
(vial, syringe or ampoule) in each final lot is inspected monographs including, where applicable, the following:
visually or mechanically for acceptable appearance. pH {2.2.3}
Degree of adsorption For an adsorbed vaccine, unless otherwise Liquid vaccines, after reconstitution where applicable,
justified and authorised, a release specification for the degree comply with the limits for pH approved for the particular
of adsorption is established in light of results found for preparation.
batches used in clinical trials. From the stability data Adjuvant
generated for the vaccine it must be shown that at the end of If the vaccine contains an adjuvant, the amount is
the period of validity the degree of adsorption is not less than determined and shown to be within acceptable limits with
for batches used in clinical trials. respect to the expected amount (see also the tests for
Thermal stability When the thermal stability test is prescribed aluminium and calcium below).
in a monograph for a live attenuated vaccine, the test is Aluminium {2.5.13}
carried out on the final lot to monitor the lot-to-lot Maximum 1.25 mg of aluminium (Al) per single human dose
consistency in heat-sensitivity of viral/bacterial particles in the where an aluminium adsorbent has been used in the vaccine,
product. Suitable conditions are indicated in the individual unless otherwise stated.
monograph. The test may be omitted as a routine test for a Calcium {2.5.14}
given product once the consistency of the production process Maximum 1.3 mg of calcium (Ca) per single human dose
has been demonstrated, in agreement with the competent where a calcium adsorbent has been used in the vaccine,
authority, using relevant parameters, such as consistency in unless otherwise stated.
Free formaldehyde (2.4.18) The main virulence components of B. anthracis are the
Maximum 0.2 g/L of free formaldehyde in the final product polyglutamic aied capsule and 2 binary anthrax toxins,
where formaldehyde has been used in the preparation of the namely lethal toxin and oedema toxin, formed from the
vaccine, unless otherwise stated. respective combination of protective antigen (PA) with either
Phenol (2.5.75) lethal factor (LF) or oedema factor (EF).
Maximum 2.5 g/L in the final product where phenol has LF is a zinc-dependent endopeptidase and EF is a potent
been used in the preparation of the vaccine, unless otherwise calmodulin and calcium-dependent adenylate cyclase. Cell-
stated. free cultures of B. anthracis contain PA and because
Water (2.5.72) expression of the 3 toxin-component genes is co-ordinately
Maximum 3.0 per cent mini for freeze-dried vaccines, unless regulated, LF and EF are also present. In addition, the
otherwise stated. vaccine is likely to contain many other B. anthracis antigens,
including membrane proteins, secreted proteins, cytoplasmic
Extractable volume (2.9.77) proteins, peptidoglycans, nucleic acids and carbohydrates.
Unless otherwise justified and authorised, it complies with
the requirement for extractable volume. PRODUCTION
GENERAL PROVISIONS
Bacterial endotoxins
Unless otherwise justified and authorised, a test for bacterial Cultures are managed in a seed-lot system. The vaccine
strain is toxigenic but lacks the plasmid with the necessary
endotoxins is earned out on the final product. Where no
limit is specified in the individual monograph, the content of genes for synthesis of the capsule, an important virulence
bacterial endotoxins determined by a suitable method factor.
(2.6.14) is less than the limit approved for the particular The production method must be shown to yield a consistent
product. and active product with a safety and efficacy profile that is
adequate or equivalent to previous lots. The vaccine must
STORAGE show a level of protection against a virulent strain of B.
Store protected from light. Unless otherwise stated, the anthracis, in a suitable animal infection model, that is equal
storage temperature is 5 ± 3 °C; liquid adsorbed vaccines to or greater than that of a reference vaccine. The vaccine
must not be allowed to freeze. must not show a level of toxicity that exceeds that of a
LABELLING reference vaccine.
The label states: The production method and stability of the final lot and
— the name of the preparation; relevant intermediates are evaluated using one or more
— a reference identifying the final lot; indicator tests. Such tests include potency and specific
— the recommended human dose and route of toxicity, and may be supported by tests confirming the
administration; presence of relevant antigens and associated proteins. Release
— the storage conditions; and shelf-life specifications are established based upon the
— the expiry date; results of stability testing so as to ensure satisfactory product
— the name and amount of any antimicrobial preservative; performance during the approved period of validity.
— the name of any antibiotic, adjuvant, flavour or stabiliser SEED LOTS
present in the vaccine; The attenuated non-encapsulated strain of B. anthracis used
— where applicable, that the vaccine is adsorbed; is identified by historical records that include information on
— the name of any constituent that may cause adverse its origin and subsequent manipulation and the tests used to
reactions and any contra-indications to the use of the characterise the strain. These include morphological, cultural,
vaccine; biochemical and genetic properties of the strain. Only a
— for freeze-dried vaccines: master seed lot or, where applicable, working seed lots, that
— the name or composition and the volume of the comply with the following requirements may be used.
reconstituting liquid to be added;
Identification
— the time within which the vaccine is to be used after
Each seed lot is identified as containing B. anthracis.
reconstitution.
Phenotypic parameters
___ ___________________________________________________________ Ph Eur
Each seed lot must have a known biochemical and enzymatic
profile and have a known history of absence of antibiotic
resistance.
Anthrax Vaccine for Human Use ****** Microbial purity

Each seed lot complies with the requirements for absence of
(Adsorbed, Prepared from Culture ***** contaminating organisms. Purity of bacterial cultures is
verified by methods of suitable sensitivity.
Filtrates) Virulence test
The absence of bacterial capsule is demonstrated for each
The label may state ‘Anthrax’. seed lot by McFadyean stain and the specific toxicity
PhEis_______ __ _____________________________________________________ (oedema) test.
DEFINITION REFERENCE PREPARATION
Anthrax vaccine for human use (adsorbed, prepared from The potency and toxicity of the vaccine bulk are verified
culture filtrates) is a preparation of Bacillus anthracis antigens using reference standards derived from representative vaccine
precipitated by aluminium potassium sulfate. The antigens batches. These batches are extensively characterised for their
are prepared from a sterile culture filtrate produced by a intended purpose and are stored in suitably sized aliquots
non-encapsulated strain, either avirulent or attenuated, of B. under conditions ensuring their stability.
anthracis.
PROPAGATION AND HARVEST reaction is not greater than that observed with the reference
The attenuated strain is grown using suitable liquid media. vaccine.
At the end of cultivation, the purity of the culture is tested. Alternatively, specific in vitro assays for lethal factor and
The culture medium is separated from the bacterial mass by adenylate cyclase activity may be used, subject to validation.
filtration. The pH of the filtrate is determined after dilution
with a 0.9 g/L solution of sodium chloride R and is shown to Antimicrobial preservative
Determine the amount of antimicrobial preservative by a
be within limits suitable for stability. A suitable test for
suitable chemical method. The content is not less than the
absence of live B. anthracis, including spores, is carried out.
minimum amount shown to be effective and is not greater
Aluminium potassium sulfate or an alternative adjuvant may
than 115 per cent of the intended content.
be added at this stage. An antimicrobial preservative may be
added to the suspension to form the purified harvest. Aluminium (2.5.13)
Only a purified harvest ±at complies with the following Maximum 1.25 mg per single human dose.
requirements may be used in the preparation of the final lot. Sterility (2.6.1)
Immunological identity It complies with the test for sterility.
Confirm the presence of B. anthracis protective antigen by a ASSAY
suitable immunochemical method (2.7.1). The potency of the anthrax vaccine is determined by
Antimicrobial preservative comparing the dose required to protect guinea-pigs against
Determine the amount of antimicrobial preservative by a intradermal challenge by a virulent strain of B. anthracis with
suitable chemical method. The amount is not less than the dose of a suitable reference preparation that gives the
85 per cent and not greater than 115 per cent of the same protection. Use 9 groups of not fewer than 16 female
intended content. guinea-pigs, each weighing 250-350 g. Prepare 4 dilutions of
the vaccine and of the reference preparation containing 1.5,
FINAL BULK VACCINE
0.5, 0.17 and 0.05 human doses in 0.5 mL. Allocate each
The purified harvest is diluted aseptically with sterile saline
dilution to a separate group. The remaining group receives
solution to make the final bulk vaccine.
0.5 mL of saline and is used to verify the challenge dose.
Only a final bulk vaccine that complies with the following Inject subcutaneously into each guinea-pig 0.5 mL of the
requirement may be used in the preparation of the final lot. dilution allocated to its group on each of 2 occasions, 1 week
Sterility (2.6.1) apart. 7 days after the 2nd injection, inject intradermally into
Carry out the test for sterility, using 10 mL for each each guinea-pig 2000 spores of a virulent strain of B.
medium. anthracis (Vollum) in 0.1 mL. Observe the animals for
FINAL LOT 10 days and record the number of deaths per group. The test
The final bulk vaccine is distributed aseptically into sterile, is not valid unless all the control animals die within 5 days of
tamper-proof glass ampoules and heat-sealed to prevent challenge. Using the proportions of animals that survive in
contamination. each of the vaccinated groups, calculate the potency of the
vaccine relative to the reference preparation using the usual
Only a final lot that is satisfactory with respect to each of the
statistical methods (5.3). The vaccine complies with the test
requirements given below under Identification, Tests and
if:
Assay may be released for use. Provided the potency assay,
— the relative potency estimate exceeds 1.0, or;
the specific toxicity (oedema) test and the test for
— the 95 per cent confidence interval for the relative
antimicrobial preservative have been carried out with
potency includes 1.0, and the lower 95 per cent
satisfactory results on the purified harvest, they may be
confidence limit is not less than 50 per cent of the relative
omitted on the final lot.
potency estimate.
IDENTIFICATION
LABELLING
The presence of B. anthracis protective antigen is confirmed
The label states that the vaccine is not to be frozen.
by a suitable immunochemical method (2.7./).
TESTS
Abnormal toxicity
Inject intraperitoneally up to 4 human doses of vaccine into
each of at least 10 healthy mice, each weighing 17-22 g.
Observe the mice daily for 7 days. The vaccine complies with Bacillus Calmette-Guerin Vaccine ★* **.
the test if none of the animals shows signs of ill health. BCG Vaccine ***
Specific toxicity (oedema) test (BCG Vaccine, Freeze-dried, Ph Eur monograph 0163)
Use not fewer than 2 rabbits per test. Prepare serial two-fold The label may state ‘BCG’.
dilutions of vaccine with normal saline, corresponding to 4,
Ph Eur_______________________________________________________________
2, 1, 0.5 and 0.25 human doses. Inject intradermally 0.1 mL
of each dilution of the test and of the reference vaccine into DEFINITION
the shaved flanks of 2 rabbits. Each rabbit receives the 10 Freeze-dried BCG vaccine is a preparation of live bacteria
previously prepared injections (5 dilutions of the test vaccine derived from a culture of the bacillus of Calmette and Guerin
and 5 dilutions of the reference vaccine). In one of the (Mycobacterium bovis BCG) whose capacity to protect against
rabbits, the lower concentrations are injected at the anterior tuberculosis has been established.
end and the higher concentrations at the posterior end.
PRODUCTION
The reverse is used for the 2nd rabbit. The rabbits are
GENERAL PROVISIONS
monitored for 24 h for signs of oedema at the injection site.
BCG vaccine shall be produced by a staff consisting of
The vaccine complies with the test if the oedematous
healthy persons who do not work with other infectious
agents; in particular they shall not work with virulent strains
of Mycobacterium tuberculosis, nor shall they be exposed to a bacterial concentration in the final bulk vaccine, the
known risk of tuberculosis infection. Staff arc examined determination is carried out before addition of the stabiliser.
periodically for tuberculosis. BCG vaccine is susceptible to Only final bulk vaccine that complies with the following
sunlight: the procedures for the preparation of the vaccine requirements may be used in the preparation of the final lot.
shall be designed so that all cultures and vaccines are
protected from direct sunlight and from ultraviolet light at all Bacterial and fungal contamination
stages of manufacture, testing and storage. Carry out the test for sterility (2.6./), using 10 mL for each
medium. The final bulk vaccine complies with the test for
Production of the vaccine is based on a seed-lot system. sterility except for the presence of mycobacteria.
consistently BCG vaccines that induce adequate sensitivity to Count of viable units
tuberculin in man, that have acceptable protective potency in Determine the number of viable units per millilitre by viable
animals and are safe. The vaccine is prepared from cultures count on solid medium using a method suitable for the
which are derived from the master seed lot by as few vaccine to be examined or by a suitable biochemical method.
subcultures as possible and in any case not more than Carry out the test in parallel on a reference preparation of
8 subcultures. During the course of these subcultures the the same strain.
preparation is not freeze-dried more than once. Bacterial concentration
If a bioluminescence test or other biochemical method is Determine ±e total bacterial concentration by a suitable
used instead of viable count, the method is validated against method, either direcdy by determining the mass of the micro
the viable count for each stage of the process at which it is organisms, or indirectly by an opacity method that has been
used. calibrated in relation to the mass of the organisms; if the
bacterial concentration is determined before addition of a
bacterial seed lots
stabiliser, the concentration in the final bulk vaccine is
The strain used to establish the master seed lot is chosen for
established by calculation. The total bacterial concentration is
and maintained to preserve its characteristics, its capacity to within the limits approved for the particular product.
sensitise man to tuberculin and to protect animals against
tuberculosis, and its relative absence of pathogenicity for man The ratio of the count of viable units to the total bacterial
and laboratory animals. The strain used shall be identified by concentration is not less than that approved for the particular
historical records that include information on its origin and product.
subsequent manipulation. FINAL LOT
A suitable batch of vaccine is prepared from the first working The final bulk vaccine is distributed into sterile containers
seed lot and is reserved for use as the comparison vaccine. and freeze-dried to a moisture content favourable to the
When a new working seed lot is established, a suitable test stability of the vaccine; the containers are closed either under
for delayed hypersensitivity in guinea-pigs is carried out on a vacuum or under an inert gas.
batch of vaccine prepared from the new working seed lot; Except where the filled and closed containers are stored at a
the vaccine is shown to be not significantly different in temperature of -20 °C or lower, the expiry date is not later
activity from the comparison vaccine. Antimicrobial agent than 4 years from the date of harvest.
sensitivity testing is also carried out. Only a final lot that complies with the following requirement
Only a working seed lot that complies with the following for count of viable units and with each of the requirements
requirements may be used for propagation. given below under Identification, Tests and Assay may be
Identification released for use. Provided the test for virulent mycobacteria
has been carried out with satisfactory results on the final bulk
The bacteria in the working seed lot are identified as
vaccine, it may be omitted on the final lot. Provided the test
Mycobacterium bovis BCG using microbiological techniques,
for excessive dermal reactivity has been carried out with
which may be supplemented by molecular biology techniques
satisfactory results on the working seed lot and on
(for example, nucleic acid amplification and restriction
5 consecutive final lots produced from it, the test may be
fragment-length polymorphism).
Bacterial and fungal contamination
Count of viable units
Carry out the test for sterility (2.6./), using 10 mL for each
Determine the number of viable units per millilitre of the
medium. The working seed lot complies with the test for
reconstituted vaccine by viable count on solid medium using
sterility except for the presence of mycobacteria.
a method suitable for the vaccine to be examined or by a
Virulent mycobacteria suitable biochemical method. The ratio of the count of
Examine the working seed lot as prescribed under Tests, viable units after freeze-drying to that before is not less than
using 10 guinea-pigs. that approved for the particular product.
PROPAGATION AND HARVEST Thermal stability
The bacteria are grown in a suitable medium for not more Maintain containers of the final lot of freeze-dried vaccine in
than 21 days by surface or submerged culture. The culture the dry state at 37 ± 1 °C for 4 weeks. Determine the
medium does not contain substances known to cause toxic or number of viable units as described under Assay in parallel
allergic reactions in humans or to cause the bacteria to for the heated vaccine and for vaccine stored at the
become virulent for guinea-pigs. The culture is harvested and temperature recommended for storage. The number of
suspended in a sterile liquid medium that protects the viable units in the heated vaccine is not less than 20 per cent
viability of the vaccine as determined by a suitable method of of that in the unheated vaccine.
viable count.
IDENTIFICATION
FINAL BULK VACCINE BCG vaccine is identified by microscopic examination of the
The final bulk vaccine is prepared from a single harvest or by bacilli in stained smears demonstrating ±eir acid-fast
pooling a number of single harvests. A stabiliser may be property and by the characteristic appearance of colonies
added; if the stabiliser interferes with the determination of
grown on solid medium. Alternatively, molecular biology

techniques (for example nucleic acid amplification) may be BCG for Immunotherapy * z
used. (Ph. Eur. monograph 1929) *
TESTS Ph Eur------------------------------------------------------ ---- -----------------------------------------------
Virulent mycobacteria
DEFINITION
Inject subcutaneously or intramuscularly into each of
BCG for immunotherapy is a freeze-dried preparation of live
6 guinea-pigs, each weighing 250-400 g and having received
bacteria derived from a culture of the bacillus of Calmette
no treatment likely to interfere with the test, a quantity of
and Guerin (Mycobacterium bovis BCG) whose capacity for
vaccine equivalent to at least 50 human doses. Observe the
treatment has been established.
animals for at least 42 days. At the end of this period,
euthanise the guinea-pigs and examine by autopsy for signs It complies with the monograph Vaccines for human
of infection svith tuberculosis, ignoring any minor reactions at use (0153).
the site of injection. Animals that die during the observation PRODUCTION
period are also examined for signs of tuberculosis. GENERAL PROVISIONS
The vaccine complies with the test if none of the guinea-pigs BCG for immunotherapy shall be produced by a staff
shows signs of tuberculosis and if not more than 1 animal consisting of healthy persons who do not work with other
dies during the observation period. If 2 animals die during infectious agents; in particular they shall not work with
this period and autopsy does not reveal signs of tuberculosis virulent strains of Mycobacterium tuberculosis, nor shall they be
repeat the test on 6 other guinea-pigs. The vaccine complies exposed to a known risk of tuberculosis infection. Staff are
with the test if not more than 1 animal dies during the examined periodically for tuberculosis. BCG for
42 days following the injection and autopsy does not reveal immunotherapy is susceptible to sunlight: the procedures for
any sign of tuberculosis. production shall be so designed that all products are
Bacterial and fungal contamination protected from direct sunlight and from ultraviolet light at all
The reconstituted vaccine complies with the test for sterility stages of manufacture, testing and storage.
(2.6./) except for the presence of mycobacteria. Production is based on a seed-lot system, 'rhe production
Excessive dermal reactivity method shall have been shown to yield consistently BCG
Use 6 healthy, white or pale-coloured guinea-pigs, each products that can be used for treatment of superficial bladder
weighing not less than 250 g and having received no cancer and are safe. The product is prepared from cultures
treatment likely to interfere with the test. Inject intradermally which are separated from the master seed lot by as few
into each guinea-pig, according to a randomised plan, subcultures as possible and in any case not more than
0.1 mL of the reconstituted vaccine and of 2 tenfold serial 8 subcultures. During the course of these subcultures the
dilutions of the vaccine and identical doses of the comparison preparation is not freeze-dried more than once.
vaccine. Observe the lesions formed at the site of the If a bioluminescence test or other biochemical method is
injection for 4 weeks. The vaccine complies with the test if used instead of viable count, the method is validated against
the reaction it produces is not markedly different from that the viable count for each stage of the process at which it is
produced by the comparison vaccine. used.
Water SEED LOTS
Not more than the limit approved for the particular product, The strain used to establish the master seed lot is chosen for
determined by a suitable method. and maintained to preserve its characteristics, its capacity to
treat and prevent superficial bladder cancer, and its relative
ASSAY
absence of pathogenicity for man and laboratory animals.
Determine the number of viable units in the reconstituted
The strain used shall be identified by historical records that
vaccine by viable count on solid medium using a method
include information on its origin and subsequent
suitable for the vaccine to be examined or by a suitable
manipulation.
validated biochemical method. The number is within the
range stated on the label. Determine the number of Before establishment of a working seed lot a batch is
viable units in the comparison vaccine in parallel. prepared and reserved for use as the comparison product.
When a new working seed lot is established, a suitable test
LABELLING for delayed hypersensitivity in guinea-pigs is carried out on 3
The label states: batch of product prepared from the new working seed lot;
— the minimum and maximum number of viable units per the product is shown to be not significantly different in
millilitre in the reconstituted vaccine, activity from the comparison product. Antimicrobial agent
— that the vaccine must be protected from direct sunlight. sensitivity testing is also carried out.
— ---- ------------------------------------------------------------------- ------------------------------ PhEur Only a working seed lot that complies with the following
requirements may be used for propagation.
Identification
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 restriction
fragment-length polymorphism).
medium. The working seed lot complies with the test for
sterility, except for the presence of mycobacteria.
Virulent mycobacteria IDENTIFICATION

Examine the working seed lot as prescribed under Tests, BCG for immunotherapy is identified by microscopic
using 10 guinea-pigsT examination of the bacilli in stained smears demonstrating
PROPAGATION AND HARVEST their acid-fast property and by the characteristic appearance
The bacteria are grown in a suitable medium for not more of colonies grown on solid medium. Alternatively, molecular
than 21 days by surface or submerged culture. The culture biology techniques (for example, nucleic acid amplification)
medium does not contain substances known to cause toxic or may be used.
allergic reactions in human beings or to cause the bacteria to TESTS
become virulent for guinea-pigs. The culture is harvested and Virulent mycobacteria
suspended in a sterile liquid medium that protects the Inject subcutaneously or intramuscularly into each of
viability of the culture as determined by a suitable method of 6 guinea-pigs, each weighing 250-400 g and having received
viable count.
no treatment likely to interfere with the test, a quantity of the
FINAL BULK product to be examined equivalent to at least 1/25 of
The final bulk is prepared from a single harvest or by pooling 1 human dose. Observe the animals for at least 42 days.
a number of single harvests. A stabiliser may be added; if the At the end of this period, euthanise the guinea-pigs and
stabiliser interferes with the determination of bacterial examine by autopsy for signs of infection with tuberculosis,
concentration on the final bulk, the determination is carried ignoring any minor reactions at the site of injection. Animals
out before addition of the stabiliser. that die during the observation period are also examined for
Only final bulk that complies with the following requirements signs of tuberculosis. The product complies with the test if
may be used in the preparation of the final lot. none of the guinea-pigs shows signs of tuberculosis and if not
more than 1 animal dies during the observation period.
If 2 animals die during this period and autopsy does not
Carry out the test for sterility (2.6./), using 10 mL of final
reveal signs of tuberculosis, repeat the test on 6 other guinea-
bulk for each medium. The final bulk complies with the test
pigs. The product complies with the test if not more than
for sterility, except for the presence of mycobacteria.
1 animal dies during the 42 days following the injection and
Count of viable units autopsy does not reveal any sign of tuberculosis.
Determine the number of viable units per millilitre by viable
count on solid medium using a method suitable for the
The reconstituted product complies with the test for sterility
product to be examined or by a suitable biochemical method.
(2.6./) except for the presence of mycobacteria.
Carry out the test in parallel on a reference preparation of
the same strain. Water
Not more than the limit approved for the particular product,
Bacterial concentration
determined by a suitable method.
Determine the total bacterial concentration by a suitable
method, either directly by determining the mass of the micro ASSAY
organisms, or indirectly by an opacity method that has been Determine the number of viable units in the reconstituted
calibrated in relation to the mass of the micro-organisms; product by viable count on solid medium using a method
if the bacterial concentration is determined before addition of suitable for the product to be examined or by a suitable
a stabiliser, the concentration in the final bulk is established validated biochemical method. The number is within the
by calculation. The total bacterial concentration is within the range stated on the label. Determine the number of
limits approved for the particular product. viable units in the comparison control in parallel.
The ratio of the count of viable units to the total bacterial LABELLING
concentration is not less than that approved for the particular The label states:
product. — the minimum and the maximum number of viable units
FINAL LOT per dose in the reconstituted product;
The final bulk is distributed into sterile containers and freeze- — that the product must be protected from direct sunlight.
dried to a moisture content favourable to the stability of the _______________________________________________________________ Ph Eur
product; the containers are closed either under vacuum or
under an inert gas.
Except where the filled and closed containers are stored at a
temperature of -20 °C or lower, the expiry date is not later
than 4 years from the date of harvest.
Cholera Vaccine ** **
Only a final lot that complies with the following requirement (Ph. Eur. monograph 0154) ***
for count of viable units and with each of the requirements The label may state ‘Cholera’.
given below under Identification, Tests and Assay may be PhEir---------------------------------------------------------------------------------------- ------------------ -
released for use. Provided the test for virulent mycobacteria
has been carried out with satisfactory results on the final DEFINITION
bulk, it may be omitted on the final lot. Cholera vaccine is a homogeneous suspension of a suitable
strain or strains of Vibrio cholerae containing not less than
Count of viable units
8 X 109 bacteria in each human dose. The human dose does
Determine the number of viable units per millilitre of the
not exceed 1.0 mL.
reconstituted product by viable count on solid medium using
a method suitable for the product to be examined, or by a PRODUCTION
suitable biochemical method. The ratio of the count of The vaccine is prepared using a seed-lot system. The vaccine
viable units after freeze-drying to that before is not less than consists of a mixture of equal parts of vaccines prepared from
that approved for the particular product. smooth strains of the 2 main serological types, Inaba and
Ogawa. These may be of the classical biotype with or without
the El-Tor biotype. A single strain or several strains of each smooth strains of the 2 main serological types, Inaba and
type may be included. All strains must contain, in addition to Ogawa. These may be of the classical biotype with or without
their type o antigens, the heat-stable 0 antigen common to the El-Tor biotype. A single strain or several strains of each
Inaba and Ogawa. If more than one strain each of Inaba and type may be included. All strains must contain, in addition to
Ogawa are used, these may be selected so as to contain other their type o antigens, the heat-stable o antigen common to
o antigens in addition. The World Health Organization Inaba and Ogawa. If more than one strain each of Inaba and
recommends new strains which may be used if necessary, in Ogawa are used, these may be selected so as to contain other
accordance with the regulations in force in the signatory o antigens in addition. The World Health Organization
States of the Convention on the Elaboration of a European recommends new strains which may be used if necessary in
Pharmacopoeia. In order to comply with the requirements for accordance with the regulations in force in the signatory
vaccination certificates required for international travel, the States of the Convention on the Elaboration of a European
vaccine must contain not less than 8 X 109 organisms of the Pharmacopoeia. In order to comply with the requirements for
classical biotype. Each strain is grown separately. vaccination certificates required for international travel, the
The bacteria are inactivated either by heating the suspensions vaccine must contain not less than 8 X 109 organisms of the
(for example, at 56 °C for 1 h) or by treatment with classical biotype. Each strain is grown separately.
formaldehyde or phenol or by a combination of the physical The bacteria are inactivated either by heating the suspensions
and chemical methods. (for example, at 56 °C for 1 h) or by treatment with
The production method is validated to demonstrate that the formaldehyde or by a combination of the physical and
product, if tested, would comply with the test for abnormal chemical methods. Phenol is not used in the preparation.
toxicity for immunosera and vaccines for human use (2.6.9) The vaccine is distributed into sterile containers and freeze-
modified as follows: inject 0.5 mL of the vaccine into each dried to a moisture content favourable to the stability of the
mouse and 1.0 mL into each guinea pig. vaccine. The containers are then closed so as to exclude
contamination.
IDENTIFICATION
The production method is validated to demonstrate that the
It is identified by specific agglutination tests.
product, if tested, would comply with the test for abnormal
TESTS toxicity for immunosera and vaccines for human use (2.6.9)
Phenol (2.5.15) modified as follows: inject 0.5 mL of the vaccine into each
If phenol has been used in the preparation, the concentration mouse and 1.0 mL into each guinea pig.
is not more than 5 g/L.
IDENTIFICATION
Antibody production
The vaccine reconstituted as stated on the label is identified
Test the ability of the vaccine to induce antibodies (such as
by specific agglutination tests.
agglutinating, vibriocidal or haemagglutinating antibodies) in
the guinea-pig, the rabbit or the mouse. Administer the TESTS
vaccine to a group of at least 6 animals. At the end of the Phenol (2.5.15)
interval of time necessary for maximum antibody formation, If phenol has been used in the preparation, the concentration
determined in preliminary tests, collect sera from the animals is not more than 5 g/L.
and titrate them individually for the appropriate antibody Antibody production
using a suitable method. The vaccine to be examined passes Test the ability of the vaccine to induce antibodies (such as
the test if each serotype has elicited a significant antibody agglutinating, vibriocidal or haemagglutinating antibodies) in
response. the guinea-pig, the rabbit or the mouse. Administer the
Sterility (2.6./) reconstituted vaccine to a group of at least 6 animals. At the
It complies with the test for sterility. end of the interval of time necessary for maximum antibody
LABELLING formation, determined in preliminary tests, collect sera from
The label states: the animals and titrate them individually for the appropriate
— the method used to inactivate the bacteria, antibody using a suitable method. The vaccine to be
— the number of bacteria in each human dose. examined passes the test if each serotype has elicited a
significant antibody response.
_______________________________________________________________ Ph Eur
Sterility (2.6. /)
The reconstituted vaccine complies with the test for sterility.
LABELLING
Cholera Vaccine, Freeze-dried ★ The label states:
(Ph. Eur. monograph 0155) *** — the method used to inactivate the bacteria,
— the number of bacteria in each human dose.
The label may state ‘Dried/Cholera’. _______________________________ ____ ____________________ — FhEj
Ph Eur______________________________________________________________
DEFINITION
Freeze-dried cholera vaccine is a preparation of a suitable
strain or strains of Vibrio cholerae. The vaccine is
reconstituted as stated on the label to give a uniform
suspension containing not less than 8 X 109 bacteria in each
human dose. The human dose does not exceed 1.0 mL of
the reconstituted vaccine.
PRODUCTION
The vaccine is prepared using a seed-lot system. The vaccine
consists of a mixture of equal parts of vaccines prepared from
2016 Vaccines FV-557
Cholera Vaccine (Inactivated, Oral) ****** Identification

Master seed lots are identified by colony morphology, and by
(Ph Eur monograph 2327) *** biochemical characterisation, using suitable molecular assays
The label may state Cholera (oral) or immunoassays. Working seed lots are identified by colony
PnEur___________________________________________________________
morphology and by molecular assays or immunoassays.
Purity
DEFINITION Purity of master seed lots and working seed lots is verified by
Cholera vaccine (inactivated, oral) is a homogeneous methods of suitable sensitivity.
suspension of inactivated suitable strains of Vibrio cholerae
PROPAGATION AND HARVEST
serogroup 01, representing serotypes and biotypes of
Each strain is grown separately from the working seed lot.
epidemic strains. The vaccine may contain the B subunit of
cholera toxin (CTB). Just prior to ingestion, one dose of Cultures are checked at different stages of fermentation
vaccine suspension is mixed with a suitable buffer as stated (subcultures and main culture) for purity, identity, cell
on the label. opacity, pH and biochemical characteristics. Unsatisfactory
cultures must be discarded.
PRODUCTION
Production cultures are shown to be consistent in respect of
GENERAL PROVISIONS
growth rate, pH and yield of cells or cell products.
The production method must be validated to yield
consistently vaccines comparable with the vaccine of proven MONOVALENT CELL HARVEST
clinical efficacy and safety in man. Only a monovalent harvest that complies with established
specifications for the following tests may be used.
The production process must be validated to show that no
clinically significant quantities of active toxin are present in pH (2.2.5)
the product. Within the range approved for the particular product.
CHOICE OF VACCINE STRAIN Identification
The vaccine consists of a mixture of epidemic V. cholerae Relevant antigenic characteristics are verified by suitable
strains inactivated by a suitable method such as heat or immunological or biochemical assays.
formalin inactivation. All strains express smooth Purity
lipopolysaccharide (LPS). The CTB is produced by Samples of culture are examined by microscopy of Gram-
recombinant DNA technology in a strain that lacks the gene stained smears, by inoculation of appropriate culture media
for cholera toxin subunit A (ctx/T). Selected V. cholerae or by another suitable procedure.
strains are low cholera-toxin producers. Opacity
The World Health Organization (WHO) can recommend The absorbance at 600 nm (2.2.25) is within the range
new vaccine strains or antigens that may be used if necessary, approved for the particular product.
in accordance with the regulations in force in the signatory
INACTIVATED MONOVALENT CELL BULK
states of the Convention on the Elaboration of a European
To limit the possibility of contamination, inactivation is
Pharmacopoeia.
initiated as soon as possible after preparation. Bacteria are
SEED LOTS inactivated after washing, either by treatment with
The strains of V. cholerae used shall be identified by historical formaldehyde or by heating under conditions that ensure
records that include information on the origin of the strains inactivation.
and their subsequent manipulation. Characterisation and Only an inactivated monovalent cell bulk that complies with
maintenance of the recombinant strains and plasmids used established specifications for the following tests may be used
for production of the recombinant B subunit of cholera in the preparation of the final bulk.
toxin (rCTB) and the origin of the gene for cholera toxin
subunit B (clvB) are documented. The stability of the rCTB pH (2.2.5)
plasmid in the recombinant strain during storage and beyond Within the range approved for the particular product.
the passage level used in production is confirmed. Identification
Characterisation of the rCTB is undertaken using a variety of Verified by slide agglutination.
analytical techniques including determination of molecular Inactivation
size, charge and amino acid composition. Techniques Complete inactivation is verified by a suitable culture
suitable for such purposes include sodium dodecyl sulfate method.
polyacrylamide gel electrophoresis (SDS-PAGE) and Sterility (2.6.1)
different liquid chromatographies. The identity of the It complies with the test for sterility, carried out using 10 mL
product is confirmed by at least partial N-terminal and for each medium.
C-terminal amino acid sequencing.
Opacity
Master seed lots are grown on agar plates, which may The inactivation process may affect the accuracy of opacity
contain appropriate antibiotics. Colonies are used to produce measurements.
working seed lots in liquid media that are free from
antibiotics. Cultures derived from the working seed lot must Purity
have the same characteristics as the cultures of the strain Samples of culture are examined by microscopy of Gram-
stained smears, by inoculation of appropriate culture media
from which the master seed lot was derived.
or by another suitable procedure.
Only a seed lot that complies with the following requirements
may be used in the preparation of the monovalent cell Smooth LPS content
harvest. Verified by a suitable immunoassay (2.7.1).
Residual cholera toxin Free formaldehyde (2.4.18)

The absence of residual cholera toxin is verified by a suitable Maximum 0.2 g/L, where applicable.
immunoassay (2.7. /) or biochemical assay. Antimicrobial preservative
Free formaldehyde {2.4.18) Where applicable, determine the amount of antimicrobial
Content to be determined where formaldehyde is used for preservative by a suitable chemical or physico-chemical
inactivation. method. The amount is not less than 85 per cent and not
PURIFIED RCTB greater than 115 per cent of the intended amount.
Production of the rCTB follows ±e guidelines for assuring ASSAY
the quality of pharmaceutical and biological products Antigen content
prepared by recombinant technology and is covered by the The amount of smooth LPS, and where applicable, the
monograph Products of recombinant DPIA technology (0784). amount of rCTB, are within the limits approved for the
Prior to harvest, the cell culture is checked for purity and particular product, determined by a suitable immunoassay
opacity. rCTB is harvested by suitable filtration, concentrated (2.7.1).
by diafiltration, purified by chromatography, filter-sterilised
and stored under suitable conditions. The pH of the pooled LABELLING
eluate is adjusted prior to buffer exchange. The label states:
— the method of inactivation;
Only purified rCTB that complies with established
— the serogroup, serotypes and biotypes of vaccine strains;
specifications for the following tests may be used in the
— the number of bacteria per human dose;
preparation of the final bulk.
— the amount of rCTB.
pH (2.2.3)
Within the range approved for the particular product.
Purity
Verified by SDS-PAGE (2.2.31) and an appropriate liquid
chromatography method (2.2.29).
Sterility (2.6.1) Adsorbed Diphtheria Vaccine * *
It complies with the test for sterility, carried out using 10 mL
(Diphtheria Vaccine (Adsorbed), *
for each medium.
rCTB
Ph Eur-----------------------------------------------------------------------------------------------------------
The amount of rCTB is determined by a suitable
immunoassay (2.7.1). DEFINITION
FINAL BULK
Diphtheria vaccine (adsorbed) is a preparation of diphtheria
The final bulk vaccine is prepared by aseptically mixing a formol toxoid with a mineral adsorbent. The formol toxoid IS
suitable buffer with monovalent cell bulks. Where used, the prepared from the toxin produced by the growth of
rCTB bulk is added in appropriate amounts. Preservatives, if Corynebacterium diphtheriae.
used, may be added at this stage. PRODUCTION
Only a final bulk that complies with the following GENERAL PROVISIONS
requirements may be used in the preparation of the final lot. Specific toxicity
Sterility (2.6.1) The production method is validated to demonstrate that the
It complies with the test for sterility, carried out using 10 mL product, if tested, would comply with the following test:
for each medium. inject subcutaneously 5 times the single human dose stated
on the label into each of 5 healthy guinea-pigs, each weighing
Antimicrobial preservative
250-350 g, that have not previously been treated with any
material that will interfere with the test. If within 42 days of
preservative by a suitable chemical or physico-chemical
the injection any of the animals shows signs of or dies from
method. The amount is not less than 85 per cent and not
diphtheria toxaemia, the vaccine does not comply with the
greater than 115 per cent of the intended amount.
test. If more than 1 animal dies from non-specific causes,
FINAL LOT repeat the test once; if more than 1 animal dies in the second
The final bulk is mixed to homogeneity and filled aseptically test, the vaccine does not comply with the test.
into suitable containers.
BULK PURIFIED TOXOID
Only a final lot ±at is within the limits approved for the For the production of diphtheria toxin, from which toxoid is
particular product and is satisfactory with respect to each of prepared, seed cultures are managed in a defined seed-lot
the requirements given below under Identification, Tests and system in which toxinogenicity is conserved and, where
Assay may be released for use. necessary, restored by deliberate reselection. A highly
IDENTIFICATION toxinogenic strain of Cotynebacteriuni diphtheriae with known
Serotypes are detected by a suitable immunoassay (2.7.1) or origin and history is grown in a suitable liquid medium.
molecular assay. rCTB is detected by a suitable immunoassay At the end of cultivation, the purity of each culture is tested
(2.7.1). The antigen-content assays may also serve as an and contaminated cultures are discarded. Toxin-containing
identity test. culture medium is separated aseptically from the bacterial
mass as soon as possible. The toxin content (Lf per millilitre)
TESTS is checked (2.7.27) to monitor consistency of production.
pH (2.2.3) Single harvests may be pooled to prepare the bulk purified
Within the range approved for the particular product. toxoid. The toxin is purified to remove components likely to
Sterility (2.6.1) cause adverse reactions in humans. The purified toxin is
It complies with the test for sterility. detoxified with formaldehyde by a method that avoids
destruction of the immunogenic potency of the toxoid and Antimicrobial preservative

reversion of the toxoid to toxin, particularly on exposure to Where applicable, determine the amount of antimicrobial
heat. Alternatively, purification may be carried out after preservative by a suitable chemical method. The amount is
detoxification. not less than 85 per cent and not greater than 115 per cent
Only bulk purified toxoid that complies with the following of the intended amount.
requirements may be used in the preparation of the final bulk Sterility (2.6. /)
vaccine. Carry out the test for sterility using 10 mL for each medium.
Sterility (2.6./) FINAL LOT
Carry out the test for sterility using 10 mL for each medium. The final bulk vaccine is distributed aseptically into sterile,
Absence of toxin and irreversibility of toxoid tamper-proof containers. The containers are closed so as to
Using the same buffer solution as for the final vaccine, prevent contamination.
without adsorbent, prepare a solution of bulk purified toxoid Only a final lot that is satisfactory with respect to each of the
at 100 Lf/mL. Divide the solution into 2 equal parts. requirements given below under Identification, Tests and
Maintain 1 part at 5 ± 3 °C and the other at 37 °C for Assay may be released for use. Provided the test for
6 weeks. Carry out a test in Veto cells for active diphtheria antimicrobial preservative and the assay have been carried
toxin using 50 pUwell of both samples. The sample should out with satisfactory results on the final bulk vaccine, they
not contain antimicrobial preservatives and detoxifying agents may be omitted on the final lot.
should be determined to be below the concentration toxic to Provided the free formaldehyde content has been determined
Veto cells. Non-specific toxicity may be eliminated by on the bulk purified antigens or on the final bulk and it has
dialysis. been shown that the content in the final lot will not exceed
Use freshly trypsinised Veto cells at a suitable concentration, 0.2 g/L, the test for free formaldehyde may be omitted on the
for example 2.5 X 105 โทบ1 and a reference diphtheria final lot.
toxin diluted in 100 Lf/mL diphtheria toxoid. A suitable IDENTIFICATION
reference diphtheria toxin will contain either not less than
Diphtheria toxoid is identified by a suitable immunochemical
100 LD50/mL or 67 to 133 lr/100 in 1 Lf and 25 000 to
method (2.7./). The following method, applicable to certain
50 000 minimal reacting doses for guinea-pig skin in 1 Lf
vaccines, is given as an example. Dissolve in the vaccine to
{diphtheria toxin BRP is suitable for use as the reference
be examined sufficient sodium citrate R to give a 100 g/L
toxin). Dilute the toxin in 100 Lf/mL diphtheria toxoid to a
solution. Maintain at 37 °C for about 16 h and centrifuge
suitable concentration, for example 2 X 10"4 LftmL.
until a clear supernatant is obtained. The clear supernatant
Prepare serial twofold dilutions of the diluted diphtheria
reacts with a suitable diphtheria antitoxin, giving a
toxin and use undiluted test samples (50 pL/well). Distribute
precipitate.
them in the wells of a sterile tissue culture plate containing a
medium suitable for Vero cells. To ascertain that any TESTS
cytotoxic effect noted is specific to diphtheria toxin, prepare Aluminium (2.5. / 3)
in parallel dilutions where the toxin is neutralised by a Maximum 1.25 mg per single human dose, if aluminium
suitable concentration of diphtheria antitoxin, for example hydroxide or hydrated aluminium phosphate is used as the
100 IU/mL. Include control wells without toxoid or toxin absorbent.
and with non-toxic toxoid at 100 Lf/mL on each plate to Free formaldehyde {2.4.18)
verify normal cell growth. Add cell suspension to each well, Maximum 0.2 g/L.
seal the plates and incubate at 37 °C for 5-6 days. Cytotoxic
effect is judged to be present where there is complete
metabolic inhibition of the Vero cells, indicated by the pH
preservative by a suitable chemical method. The content is
indicator of the medium. Confirm cytopathic effect by
not less than the minimum amount shown to be effective and
microscopic examination or suitable staining such as MTT
is not greater than 115 per cent of the quantity stated on the
dye. The test is invalid if 5 X 10"5 Lf/mL of reference
label.
diphtheria toxin in 100 Lf/mL toxoid has no cytotoxic effect
on Vero cells or if the cytotoxic effect of this amount of toxin Sterility (2.6. /)
is not neutralised in the wells containing diphtheria antitoxin. The vaccine complies with the test for sterility.
The bulk purified toxoid complies with the test if no toxicity ASSAY
neutralisable by antitoxin is found in either sample. Carry out one of the prescribed methods for the assay of
Antigenic purity (2.7.27) diphtheria vaccine (adsorbed) (2.7.6).
Not less than 1500 Lf per milligram of protein nitrogen. The lower confidence limit {P = 0.95) of the estimated
FINAL BULK VACCINE potency is not less than 30 ru per single human dose.
The final bulk vaccine is prepared by adsorption of a suitable LABELLING
quantity of bulk purified toxoid onto a mineral carrier such The label states:
as hydrated aluminium phosphate or aluminium hydroxide; — the minimum number of International Units per single
the resulting mixture is approximately isotonic with blood. human dose,
Suitable antimicrobial preservatives may be added. Certain — where applicable, that the vaccine is intended for primary
antimicrobial preservatives, particularly ±ose of the phenolic vaccination of children and is not necessarily suitable for
type, adversely affect the antigenic activity and must not be reinforcing doses or for administration to adults,
used. — the name and the amount of the adsorbent,
Only a final bulk vaccine ±at complies with the following — that the vaccine must be shaken before use,
requirements may be used in the preparation of the final lot. — that the vaccine is not to be frozen.
out with satisfactory' results on the final bulk vaccine, they

Diphtheria Vaccine (Adsorbed, ** ** may be omitted on die final lot.
Reduced Antigen Content) ***** Provided the free formaldehyde content has been determined
Adsorbed Diphtheria Vaccine for Adults and on the bulk purified toxoid or on the final bulk and it has
Adolescents been shown that the content in the final lot will not exceed
(PA. Eur. monograph 0646) 0.2 g/L, the test for free formaldehyde may be omitted on the
For a vaccine for use in the United Kingdom, the amount of final lot.
toxoid used is adjusted so that the final vaccine contains not IDENTIFICATION
more than 2.0 flocculation equivalents (2.0 Lf) per dose. Diphtheria toxoid is identified by a suitable immunochemical
PhEur____________________________________________________________ _ method (2.7.7). The following method, applicable to certain
vaccines, is given as an example. Dissolve in the vaccine to
DEFINITION be examined sufficient sodium citrate R to give a 100 g/L
Diphtheria vaccine (adsorbed, reduced antigen content) is a solution. Maintain at 37 c for about 16 h and centrifuge
preparation of diphtheria formol toxoid with a mineral until a clear supernatant is obtained. The clear supernatant
adsorbent. The formol toxoid is prepared from the toxin reacts with a suitable diphtheria antitoxin, giving a
produced by the growth of Corymbacterium diphtheriae. precipitate. If a satisfactory result is not obtained with a
The amount of diphtheria toxoid per single human dose is vaccine adsorbed on aluminium hydroxide, carry out the test
reduced compared to vaccines generally used for primary' as follows. Centrifuge 15 mL of the vaccine to be examined
vaccination. and suspend the residue in 5 mL of a freshly prepared
PRODUCTION mixture of 1 volume of a 56 g/L solution of sodium edetate R
GENERAL PROVISIONS and 49 volumes of a 90 g/L solution of disodium hydrogen
phosphate R. Maintain at 37 °C for not less than 6 h and
Specific toxicity
centrifuge. The clear supernatant reacts with a suitable
diphtheria antitoxin, giving a precipitate.
product, if tested, would comply with the following test:
inject subcutaneously 5 times the single human dose stated TESTS
on the label into each of 5 healthy guinea-pigs, each weighing Aluminium (2.5.75)
250-350 g, that have not previously been treated with any Maximum 1.25 mg per single human dose, if aluminium
material that will interfere with the test. If within 42 days of hydroxide or hydrated aluminium phosphate is used as the
the injection any of the animals shows signs of or dies from adsorbent.
diphtheria toxaemia, the vaccine does not comply with the Free formaldehyde {2.4.18)
test. If more than one animal dies from non-specific causes, Maximum 0.2 g/L.
repeat the test once; if more than one animal dies in the
second test, the vaccine does not comply with the test.
BULK PURIFIED TOXOID preservative by a suitable chemical method. The content is
The bulk purified toxoid is prepared as described in the not less than the minimum amount shown to be effective and
monograph Diphtheria vaccine (adsorbed) (0443) and complies is not greater than 115 per cent of the quantity stated on the
with the requirements prescribed therein. label.
FINAL BULK VACCINE Sterility (2.6. 7)
The final bulk vaccine is prepared by adsorption of a suitable The vaccine complies with the test for sterility.
quantity of bulk purified toxoid onto a mineral carrier such
as hydrated aluminium phosphate or aluminium hydroxide; ASSAY
the resulting mixture is approximately isotonic with blood. Carry out one of the prescribed methods for the assay of
Suitable antimicrobial preservatives may be added. Certain diphtheria vaccine (adsorbed) (2.7.6).
antimicrobial preservatives, particularly those of the phenolic The lower confidence limit (P = 0.95) of the estimated
type, adversely affect the antigenic activity and must not be potency is not less than 2 IU per single human dose.
used. LABELLING
Only a final bulk vaccine that complies with the following The label states:
requirements may be used in the preparation of the final lot. — the minimum number of International Units per single
Antimicrobial preservative human dose;
Where applicable, determine the amount of antimicrobial — the name and the amount of the adsorbent;
preservative by a suitable chemical method. The amount is — that the vaccine must be shaken before use;
not less than 85 per cent and not greater than 115 per cent — that the vaccine is not to be frozen.
of the intended amount.
Sterility (2.6./)
Carry out the test for sterility using 10 mL for each medium.
FINAL LOT
The final bulk vaccine is distributed aseptically into sterile,
tamper-proof containers. The containers are closed so as to
prevent contamination.
Assay may be released for use. Provided the test for
antimicrobial preservative and the assay have been carried
Provided the free formaldehyde content has been determined

Adsorbed Diphtheria and Tetanus ** ** on the bulk purified antigens or on the final bulk and it has
Vaccine ***** been shown that the content in the find lot will not exceed
(Diphtheria and Tetanus Vaccine (Adsorbed), 0.2 g/L, the test for free formaldehyde may be omitted on the
Ph Eur monograph 0444) final lot.
The label may state ‘DT’. IDENTIFICATION
Ph El/—____________ A. Diphtheria toxoid is identified by a suitable
immunochemical method (2.7./). The following method,
DEFINITION applicable to certain vaccines, is given as an example.
Diphtheria and tetanus vaccine (adsorbed) is a preparation of Dissolve in the vaccine to be examined sufficient sodium
diphtheria formol toxoid and tetanus formol toxoid with a citrate R to give a 100 g/L solution. Maintain at 37 °C for
mineral adsorbent. The formol toxoids are prepared from the about 16 h and centrifuge until a clear supernatant is
toxins produced by the growth of Corynebacterium diphtheriae obtained. The clear supernatant reacts with a suitable
and Clostridium tetani, respectively. diphtheria antitoxin, giving a precipitate.
PRODUCTION B. Tetanus toxoid is identified by a suitable immunochemical
GENERAL PROVISIONS method (2.7./). The following me±od, applicable to certain
Specific toxicity of the diphtheria and tetanus vaccines, is given as an example. The clear supernatant
components obtained as described in identification test A reacts with a
The production method is validated to demonstrate that the suitable tetanus antitoxin, giving a precipitate.
product, if tested, would comply with the following test: TESTS
inject subcutaneously 5 times the single human dose stated Aluminium (2.5.13)
on the label into each of 5 healthy guinea-pigs, each weighing Maximum 1.25 mg per single human dose, if aluminium
250-350 g, that have not previously been treated with any hydroxide or hydrated aluminium phosphate is used as the
material that will interfere with the test. If within 42 days of adsorbent.
Free formaldehyde (2.4.18)
diphtheria toxaemia or tetanus, the vaccine does not comply
Maximum 0.2 g/L.
with the test. If more than 1 animal dies from non-specific
causes, repeat the test once; if more than 1 animal dies in the Antimicrobial preservative
second test, the vaccine does not comply with the test. Where applicable, determine the amount of antimicrobial
BULK PURIFIED DIPHTHERIA AND TETANUS TOXOIDS
The bulk purified diphtheria and tetanus toxoids are
prepared as described in the monographs on Diphtheria
label.
vaccine (adsorbed) (0443) and Tetanus vaccine
(adsorbed) (0452) and comply with the requirements Sterility (2.6.1)
prescribed therein. The vaccine complies with the test for sterility.
FINAL BULK VACCINE ASSAY
The final bulk vaccine is prepared by adsorption of suitable Diphtheria component
quantities of bulk purified diphtheria toxoid and tetanus Carry out one of the prescribed methods for the assay of
toxoid onto a mineral carrier such as hydrated aluminium diphtheria vaccine (adsorbed) (2.7.6).
phosphate or aluminium hydroxide; the resulting mixture is The lower confidence limit (P = 0.95) of the estimated
approximately isotonic with blood. Suitable antimicrobial potency is not less than 30 IU per single human dose.
preservatives may be added. Certain antimicrobial Tetanus component
preservatives, particularly those of the phenolic type, Carry out one of the prescribed methods for the assay of
adversely affect the antigenic activity and must not be used. tetanus vaccine (adsorbed) (2.7.8).
Only a final bulk vaccine that complies with the following The lower confidence limit (P = 0.95) of the estimated
requirements may be used in the preparation of the final lot. potency is not less than 40 ru per single human dose.
LABELLING
The label states:
preservative by a suitable chemical method. The amount is
— the minimum number of International Units of each
not less than 85 per cent and not greater than 115 per cent
component per single human dose,
— where applicable, that the vaccine is intended for primary
Sterility (2.6./) vaccination of children and is not necessarily suitable for
Carry out the test for sterility using 10 mL for each medium. reinforcing doses or for administration to adults,
FINAL LOT — the name and the amount of the adsorbent,
The final bulk vaccine is distributed aseptically into sterile, — that the vaccine must be shaken before use,
tamper-proof containers. The containers are closed so as to — that the vaccine is not to be frozen.
prevent contamination. _______________________________________________________________ Ph Eur

Assay may be released for use. Provided the test for
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.
Diphtheria and Tetanus Vaccine ** ** FINAL LOT

(Adsorbed, Reduced Antigen(s) ***** tamper-proof containers. The containers are closed so as to
Content) Only a final lot that is satisfactory with respect to each of the
Adsorbed Diphtheria and Tetanus Vaccine for requirements given below under Identification, Tests and
Adults and Adolescents Assay may be released for use. Provided the test for
(Ph. Eur. monograph 0647) antimicrobial preservative and the assay have been earned
The label may state ‘dT’. out with satisfactory results on the final bulk vaccine, they
For a vaccine for use in the United Kingdom, the amount of may be omitted on the final lot.
diphtheria toxoid used is adjusted so that the final vaccine Provided the free formaldehyde content has been determined
contains not more than 2.0 flocculation equivalents (2.0 Lf) on the bulk purified toxoids or on the final bulk and it has
of diphtheria toxoid per dose. been shown that the content in the final lot will not exceed
Ph Eur_____________________________________________________________
0.2 g/L, the test for free formaldehyde may be omitted on the
final lot.
DEFINITION
IDENTIFICATION
Diphtheria and tetanus vaccine (adsorbed, reduced antigen(s)
A. Diphtheria toxoid is identified by a suitable
content) is a preparation of diphtheria formol toxoid and
immunochemical method (2.7./). The following method,
tetanus formol toxoid with a mineral adsorbent. The formol
applicable to certain vaccines, is given as an example.
toxoids are prepared from the toxins produced by the growth
Dissolve in the vaccine to be examined sufficient sodium
of Coryiไebacterium diphtherias and Clostridium tetani,
citrate R to give a 100 g/L solution. Maintain at 37 cc for
respectively. The amount of diphtheria toxoid per single
about 16 h and centrifuge until a clear supernatant is
human dose is reduced compared to vaccines generally used
obtained. The clear supernatant reacts with a suitable
for primary vaccination; the amount of tetanus toxoid may
diphtheria antitoxin, giving a precipitate. If a satisfactory'
also be reduced.
result is not obtained with a vaccine adsorbed on aluminium
PRODUCTION hydroxide, carry out the test as follows. Centrifuge 15 mL of
GENERAL PROVISIONS the vaccine to be examined and suspend the residue in 5 mL
Specific toxacity of the diphtheria and tetanus of a freshly prepared mixture of 1 volume of a 56 g/L
components solution of sodium edetatc R and 49 volumes of a 90 g/L
The production method is validated to demonstrate that the solution of disodium hydrogen phosphate R. Maintain at 37 CC
product, if tested, would comply with the following test: for not less than 6 h and centrifuge. The clear supernatant
inject subcutaneously ว times the single human dose stated reacts with a suitable diphtheria antitoxin, giving a
on the label into each of 5 healthy guinea-pigs, each weighing precipitate.
250-350 g, that have not previously been treated with any B. Tetanus toxoid is identified by a suitable immunochemical
material that will interfere with the test. If within 42 days of method (2.7.1'). The following method, applicable to certain
the injection any of the animals shows signs of or dies from vaccines, is given as an example. The clear supernatant
diphtheria toxaemia or tetanus, the vaccine does not comply obtained during identification test A reacts with a suitable
with the test. If more than one animal dies from non-specific tetanus antitoxin, giving a precipitate.
causes, repeat the test once; if more than one animal dies in TESTS
the second test, the vaccine does not comply with the test. Aluminium (2.5.13)
BULK PURIFIED DIPHTHERIA TOXOID AND TETANUS Maximum 1.25 mg per single human dose, if aluminium
TOXOIDS hydroxide or hydrated aluminium phosphate is used as the
The bulk purified diphtheria and tetanus toxoids are adsorbent.
prepared as described in the monographs Diphtheria vaccine Free formaldehyde (2.4.18)
(adsorbed) (0443) and Tetanus vaccine (adsorbed) (0452) and Maximum 0.2 g/L.
comply with the requirements prescribed therein. Antimicrobial preservative
FINAL BULK VACCINE Where applicable, determine the amount of antimicrobial
The vaccine is prepared by adsorption of suitable quantities preservative by a suitable chemical method. The content is
of bulk purified diphtheria toxoid and tetanus toxoid onto a not less than the minimum amount shown to be effective and
mineral carrier such as hydrated aluminium phosphate or is not greater than 115 per cent of the quantity stated on the
aluminium hydroxide; the resulting mixture is approximately label.
isotonic with blood. Suitable antimicrobial preservatives may Sterility (2.6.1)
be added. Certain antimicrobial preservatives, particularly The vaccine complies with the test for sterility.
those of the phenolic type, adversely affect the antigenic
ASSAY
activity and must not be used.
Diphtheria component
Only a final bulk vaccine that complies with the following Carry out one of the prescribed methods for the assay of
requirements may be used in the preparation of the final lot. diphtheria vaccine (adsorbed) (2.7.6).
Antimicrobial preservative The lower confidence limit (P = 0.95) of the estimated
Where applicable, determine the amount of antimicrobial potency is not less than 2 IU per single human dose.
Tetanus component
not less than 85 per cent and not greater than 115 per cent Carry out one of the prescribed methods for the assay of
tetanus vaccine (adsorbed) (2.7.8).
Sterility (2.6.1) The lower confidence limit (P = 0.95) of the estimated
Carry out the test for sterility using 10 mL for each medium. potency is not less than 20 IU per single human dose.
LABELLING the injection any of the animals shows signs of or dies from
The label stares: diphtheria toxaemia or tetanus, the vaccine does not comply
the minimum number of International Units of each with the test. If more than 1 animal dies from non-specific
component per single human dose; causes, repeat the test once; if more than 1 animal dies in the
the name and the amount of the adsorbent; second test, the vaccine does not comply with the test.
that the vaccine must be shaken before use; PRODUCTION OF THE COMPONENTS
— that the vaccine is not to be frozen. The production of the components complies with the
-------------------------------------------------------------------------------------------------------- Ph Eur requirements of the monographs on Diphtheria vaccine
(adsorbed) (0443), Tetanus vaccine (adsorbed) (0452) and
Hepatitis B vaccine (rDNA) (1056).
FLNAL BULK VACCINE
The final bulk vaccine is prepared by adsorption, separately
Diphtheria, Tetanus and * \ or together, of suitable quantities of bulk purified diphtheria
Hepatitis B (rDNA) Vaccine ***** toxoid, tetanus toxoid and HBsAg onto a mineral carrier
such as aluminium hydroxide or hydrated aluminium
(Adsorbed) phosphate. Suitable antimicrobial preservatives may be
(Ph. Eur. monograph 2062) added.
Lable may state ‘DT/HcpB’ Only a final bulk vaccine that complies with the following
Pn Eif___ _____________ _____________________________________________ requirements may be used in the preparation of the final lot.
DEFINITION Antimicrobial preservative
Diphtheria, tetanus and hepatitis B (rDNA) vaccine Where applicable, determine the amount of antimicrobial
(adsorbed) is a combined vaccine composed of: diphtheria preservative by a suitable chemical method. The amount is
formol toxoid; tetanus formol toxoid; hepatitis B surface not less than 85 per cent and not greater than 115 per cent
antigen (HBsAg); a mineral adsorbent such as aluminium of the intended content.
hydroxide or hydrated aluminium phosphate. Sterility (2.6.1)
The formol toxoids are prepared from the toxins produced Carry out the test for sterility using 10 mL for each medium.
by the growth of Corynebacteriuni diphtheriae and Clostridium FINAL LOT
tetani, respectively. Only a final lot that is satisfactory with respect to the test for
HBsAg is a component protein of hepatitis B virus; osmolality and with respect to each of the requirements given
the antigen is obtained by recombinant DNA technology. below under Identification, Tests and Assay may be released
for use.
PRODUCTION
GENERAL PROVISIONS
Provided the test for antimicrobial preservative and the assays
The production method shall have been shown to yield for the diphtheria and tetanus components have been carried
consistently vaccines comparable with the vaccine of proven out with satisfactory results on the final bulk vaccine, they
clinical efficacy and safety in man. may be omitted on the final lot.
The content of bacterial endotoxins (2.6.14) in the bulk Provided the content of free formaldehyde has been
purified diphtheria toxoid and tetanus toxoid is determined determined on the bulk purified antigens or on the final bulk
and it has been shown that the content in the final lot will
to monitor the purification procedure and to limit the
amount in the final vaccine. For each component, the not exceed 0.2 g/L, the test for free formaldehyde may be
content of bacterial endotoxins is less than the limit approved omitted on the final lot.
for the particular vaccine and in any case the contents are If an in vivo assay is used for the hepatitis B component,
such that the final vaccine contains less than 100 IU per provided it has been carried out with satisfactory results on
single human dose. the final bulk vaccine, it may be omitted on the final lot.
Reference vaccine(ร) Provided valid assays can be performed, Osmolality (2.2.35)
monocomponent reference vaccines may be used for the The osmolality of the vaccine is within the limits approved
assays on the combined vaccine. If this is not possible for the particular preparation.
because of interaction between the components of the IDENTIFICATION
combined vaccine or because of the difference in composition A. Diphtheria toxoid is identified by a suitable
between monocomponent reference vaccine and the test immunochemical method (2.7.1). The following method,
vaccine, a batch of combined vaccine shown to be effective in applicable to certain vaccines, is given as an example.
clinical trials or a batch representative thereof is used as a Dissolve in the vaccine to be examined sufficient sodium
reference vaccine. For the preparation of a representative citrate R to give a 100 g/L solution. Maintain at 37 °C for
batch, strict adherence to the production process used for the about 16 h and centrifuge until a clear supernatant is
batch tested in clinical trials is necessary. The reference obtained. The clear supernatant reacts with a suitable
vaccine may be stabilised by a method that has been shown diphtheria antitoxin, giving a precipitate.
to have no effect on the assay procedure.
B. Tetanus toxoid is identified by a suitable immunochemical
Specific toxicity of the diphtheria and tetanus method (2.7.1). The following method, applicable to certain
components vaccines, is given as an example. The clear supernatant
The production method is validated to demonstrate that the obtained during identification test A reacts with a suitable
product, if tested, would comply with the following test: tetanus antitoxin, giving a precipitate.
inject subcutaneously 5 times the single human dose stated c. The assay or, where applicable, the electrophoretic profile,
on the label into each of 5 healthy guinea-pigs, each weighing serves also to identify the hepatitis B component of the
250-350 g, that have not previously been treated with any vaccine.
TESTS by the growth of Corynebacteriuni diphtheriae and Clostridium

Aluminium (2.5.13) tetani, respectively.
Maximum 1.25 mg per single human dose, if aluminium
PRODUCTION
hydroxide or hydrated aluminium phosphate is used as the
adsorbent. GENERAL PROVISIONS
Free formaldehyde (2.4.18) Specific toxicity of the diphtheria and tetanus

Maximum 0.2 g/L. components
Antimicrobial preservative product, if tested, would comply with the following test
Where applicable, determine the amount of antimicrobial inject subcutaneously 5 times the single human dose stated
preservative by a suitable chemical method. The content is on the label into each of 5 healthy guinea-pigs, each weighing
not less than the minimum amount shown to be effective and 250-350 g, that have not previously been treated with any
is not greater than 115 per cent of the quantity stated on the material that will interfere with the test. If within 42 days of
label. the injection any of the animals shows signs of or dies from
Sterility (2.6.1) diphtheria toxaemia or tetanus, the vaccine does not comply
It complies with the test for sterility. with the test. If more than 1 animal dies from non-specific
Pyrogens (2.6.8) causes, repeat ±e test once; if more than 1 animal dies in the
It complies with the test for pyrogens. Inject the equivalent of second test, the vaccine does not comply with the test.
1 human dose into each rabbit. BULK PURIFIED DIPHTHERIA AND TETANUS TOXOIDS,
BULK INACTIVATED B. PERTUSSIS SUSPENSION
ASSAY
The bulk purified diphtheria and tetanus toxoids and the
inactivated B. pertussis suspension are prepared as described
Carry out one of the prescribed methods for the assay of
in the monographs Diphtheria vaccine (adsorbed) (0443),
diphtheria vaccine (adsorbed) (2.7.6).
Tetanus vaccine (adsorbed) (0452) and Pertussis vaccine (whole
The lower confidence limit (P = 0.95) of the estimated cell, adsorbed) (0161)3 respectively, and comply with the
potency is not less than 30 IU per single human dose. requirements prescribed therein.
Tetanus component FINAL BULK VACCINE
Carry out one of the prescribed methods for the assay of The final bulk vaccine is prepared by adsorption of suitable
tetanus vaccine (adsorbed) (2.7.8). quantities of bulk purified diphtheria toxoid and tetanus
The lower confidence limit (P = 0.95) of the estimated toxoid onto a mineral carrier such as hydrated aluminium
potency is not less ±an 40 IU per single human dose. phosphate or aluminium hydroxide and admixture of an
Hepatitis B component appropriate quantity of a suspension of inactivated
It complies with the assay of hepatitis B vaccine (2.7.15). B. pertussis; the resulting mixture is approximately isotonic
with blood. The B. pertussis concentration of the final bulk
LABELLING vaccine does not exceed that corresponding to an opacity of
The label states: 20 IU per single human dose. If 2 or more strains of B.
— the minimum number of International Units of diphtheria pertussis are used, the composition of consecutive lots of the
and tetanus toxoid per single human dose, final bulk vaccine shall be consistent with respect to the
— the amount of HBsAg per single human dose, proportion of each strain as measured in opacity units.
— the type of cells used for production of the HBsAg Suitable antimicrobial preservatives may be added to the bulk
component, vaccine. Certain antimicrobial preservatives, particularly those
-— where applicable, that the vaccine is intended for primary of the phenolic type, adversely affect the antigenic activity
vaccination of children and is not necessarily suitable for and must not be used.
reinforcing doses or for administration to adults,
Only a final bulk vaccine that complies with the following
— the name and the amount of the adsorbent,
requirements may be used in the preparation of the final lot.
— that the vaccine must be shaken before use,
— that the vaccine is not to be frozen. Antimicrobial preservative
------------------------------------------------------------------- ------ ------------------------------- Ph Eur
Sterility (2.6.1)
Diphtheria, Tetanus and Pertussis ******* Carry out the test for sterility using 10 mL for each medium.
(Whole Cell) Vaccine (Adsorbed) ***** FINAL LOT
Diphtheria, Tetanus and Pertussis Vaccine The final bulk vaccine is distributed aseptically into sterile,
(Adsorbed) tamper-proof containers. The containers are closed so as to
The label may state ‘DTwP’.
Ph Eur__________________________________ ___________________________ Assay may be released for use. Provided the tests for specific
DEFINITION toxicity of the pertussis component, antimicrobial preservative
Diphtheria, tetanus and pertussis (whole cell) vaccine and the assay have been carried out with satisfactory results
(adsorbed) is a preparation of diphtheria formol toxoid and on the final bulk vaccine, they may be omitted on the final
tetanus formol toxoid with a mineral adsorbent to which a lot.
suspension of inactivated BordeteUa pertussis has been added. Provided the free formaldehyde content has been determined
The formol toxoids are prepared from the toxins produced on the bulk purified antigens or on the final bulk and it has
been shown that the content in the final lot will not exceed ASSAY
0.2 g/L, the test for free formaldehyde may be omitted on the Diphtheria component
final lot. Carry out one of the prescribed methods for the assay of
IDENTIFICATION diphtheria vaccine (adsorbed) (2.7.6).
A. Diphtheria toxoid is identified by a suitable The lower confidence limit (P = 0.95) of the estimated
immunochemical method (2.7./). The following method, potency is not less than 30 ru per single human dose.
applicable to certain vaccines, is given as an example. Tetanus component
Dissolve in the vaccine to be examined sufficient Carry out one of the prescribed methods for the assay of
sodium citrate R to give a 100 g/L solution. Maintain at 37 °C tetanus vaccine (adsorbed) (2.7.8).
for about 16 h and centrifuge until a clear supernatant is If the test is carried out in guinea-pigs, the lower confidence
obtained; reserve the precipitate for identification test c. limit (P = 0.95) of the estimated potency is not less than
The clear supernatant reacts with a suitable diphtheria 40 IU per single human dose; if the test is carried out in
antitoxin, giving a precipitate. mice, the lower confidence limit (P ะ= 0.95) of the estimated
B. Tetanus toxoid is identified by a suitable immunochemical potency is not less than 60 ru per single human dose.
method (2.7.1). The following method, applicable to certain Pertussis component
vaccines, is given as an example. The clear supernatant Carry out the assay of pertussis vaccine (whole cell) (2.7.7).
obtained during identification test A reacts with a suitable
The estimated potency is not less than 4.0 IU per single
tetanus antitoxin, giving a precipitate.
human dose and the lower confidence limit (P = 0.95) of the
c. Dissolve in the vaccine to be examined sufficient estimated potency is not less than 2.0 ru per single human
sodium citrate R to give a 100 g/L solution. Maintain at 37 °C dose.
for about 16 h and centrifuge to obtain a bacterial
precipitate. Other suitable methods for separating the LABELLING
bacteria from the adsorbent may also be used. Identify The label states:
pertussis vaccine by agglutination of the bacteria from the — the minimum number of International Units of each
resuspended precipitate by antisera specific to B. pertussis or component per single human dose;
by the assay. — where applicable, that the vaccine is intended for primary
vaccination of children and is not necessarily suitable for
TESTS
reinforcing doses or for administration to adults;
Specific toxicity of the pertussis component — the name and the amount of the adsorbent;
Use not fewer than 5 mice each weighing 14 - 16 g for the — that the vaccine must be shaken before use;
vaccine group and for the saline control. Use mice of the — that the vaccine is not to be frozen.
same sex or distribute males and females equally between the
__________________________________________________ Ph Eur
groups. Allow the animals access to food and water for at
least 2 h 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 Adsorbed Diphtheria, Tetanus and ******
control group with 0.5 mL of a 9 g/L sterile solution of
sodium chloride R, preferably containing the same amount of Pertussis (Acellular Component) *****
antimicrobial preservative as that injected with the vaccine.
Weigh the groups of mice immediately before the injection Vaccine
and 72 h and 7 days after the injection. The vaccine (Diphtheria, Tetanus and Pertussis (Acellular,
complies with the test if: (a) at the end of 72 h the total mass Component) Vaccine (Adsorbed),
of the group of vaccinated mice is not less than that Ph Eur monograph 1931)
preceding the injection; (b) at the end of 7 days the average The label may state ‘DTaP’.
increase in mass per vaccinated mouse is not less than Ph Eur_____________ _________________________________________________
60 per cent of that per control mouse; and (c) not more than
5 per cent of the vaccinated mice die during the test. DEFINITION
The test may be repeated and the results of the tests Diphtheria, tetanus and pertussis (acellular, component)
combined. vaccine (adsorbed) is a combined vaccine composed ofi
diphtheria formol toxoid; tetanus formol toxoid; individually
Aluminium (2.5.13) purified antigenic components of BordeteUa pertussis; a mineral
Maximum 1.25 mg per single human dose, if aluminium adsorbent such as aluminium hydroxide or hydrated
aluminium phosphate.
adsorbent.
The formol toxoids are prepared from the toxins produced
Free formaldehyde (2.4.18) by the growth of Coiynebacterium diphtheriae and Clostridium
Maximum 0.2 g/L. tetani) respectively.
Antimicrobial preservative The vaccine contains either pertussis toxoid or a pertussis-
Where applicable, determine the amount of antimicrobial toxin-like protein free from toxic properties, produced by
preservative by a suitable chemical method. The content is expression of a genetically modified form of the
not less than the minimum amount shown to be effective and corresponding gene. Pertussis toxoid is prepared from
is not greater than 115 per cent of the quantity stated on the pertussis toxin by a method that renders the latter harmless
label. while maintaining adequate immunogenic properties and
Sterility (2.6.1) avoiding reversion to toxin. The vaccine may also contain
The vaccine complies with the test for sterility. filamentous haemagglutinin, pertactin (a 69 kDa outer
membrane protein) and other defined components of
B. pertussis such as fimbrial-2 and fimbnal-3 antigens. Sterility (2.6. /)

The latter 2 antigens may be co-purified. The antigenic Carry out the test for sterility using 10 mL for each medium.
composition and characteristics are based on evidence of FINAL LOT
protection and freedom from unexpected reactions in the Only a final lot that is satisfactory with respect to the test for
target group for which ±e vaccine is intended. osmolality and with respect to each of the requirements given
PRODUCTION below' under Identification, Tests and Assay may be released
GENERAL PROVISIONS for use.
The production method shall have been shown to yield Provided the tests for residual pertussis toxin and
consistently vaccines comparable with the vaccine of proven irreversibility of pertussis toxoid, free formaldehyde and
clinical efficacy and safety in man. antimicrobial preservative and the assay have been earned
Specific toxicity of the diphtheria and tetanus out with satisfactory results on the final bulk vaccine, they
components may be omitted on the final lot.
The production method is validated to demonstrate that the Provided the free formaldehyde content has been determined
product, if tested, would comply with the following test: on the bulk purified antigens or on the final bulk and it has
inject subcutaneously 5 times the single human dose stated been shown that the content in the final lot will not exceed
on the label into each of 5 healthy guinea-pigs, each weighing 0.2 g/L, the test for free formaldehyde may be omitted on the
250-350 g, that have not previously been treated with any final lot.
material that will interfere with the test. If within 42 days of Osmolality (2.2.35)
the injection any of the animals shows signs of or dies from The osmolality of the vaccine is within the limits approved
diphtheria toxaemia or tetanus, the vaccine does not comply for the particular preparation.
causes, repeat the test once; if more than 1 animal dies in the IDENTIFICATION
second test, the vaccine does not comply with the test. A. Diphtheria toxoid is identified by a suitable
immunochemical method (2.7.1). The following method,
The content of bacterial endotoxins (2.6.14) in the bulk
purified diphtheria toxoid, tetanus toxoid and pertussis
components is determined to monitor the purification
citrate R to give a 100 g/L solution. Maintain at 37 °C for
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 and, in
any case, the contents are such that the final vaccine contains
less than 100 IU per single human dose. B. Tetanus toxoid is identified by a suitable immunochemical
method (2.7.1). The following method, applicable to certain
Reference vaccine(s) Provided valid assays can be performed,
vaccines, is given as an example. The clear supernatant
monocomponent reference vaccines may be used for the
obtained as described in identification test A reacts with a
assays on the combined vaccine. If this is not possible
suitable tetanus antitoxin, giving a precipitate.
because of interaction between the components of the
combined vaccine or because of differences in composition c. The pertussis components are identified by a suitable
between the monocomponent reference vaccine and the test immunochemical method (2.7.1). The following method,
vaccine, a batch of combined vaccine shown to be effective in applicable to certain vaccines, is given as an example.
clinical trials or a batch representative thereof is used as a The clear supernatant obtained as described in identification
reference vaccine. For the preparation of a representative test A reacts with specific antisera to the pertussis
batch, strict adherence to the production process used for the components of the vaccine.
batch tested in clinical trials is necessary. The reference TESTS
vaccine may be stabilised by a method that has been shown Residual pertussis toxin and irreversibility of pertussis
to have no effect on the assay procedure. toxoid (2.6.33)
PRODUCTION OF THE COMPONENTS The final lot complies with the test.
The production of the components complies with the Aluminium (2.5.13)
requirements of the monographs Diphtheria vaccine Maximum 1.25 mg per single human dose, if aluminium
(adsorbed) (0443), Tetanus vaccine (adsorbed) (0452) and hydroxide or hydrated aluminium phosphate is used as the
Pertussis vaccine (acellular, component, adsorbed) (1356). adsorbent.
FINAL BULK VACCINE Free formaldehyde (2.4.18)
The final bulk vaccine is prepared by adsorption of suitable Maximum 0.2 g/L.
quantities of bulk purified diphtheria toxoid, tetanus toxoid Antimicrobial preservative
and pertussis components separately or togeffier onto a Where applicable, determine the amount of antimicrobial
mineral carrier such as aluminium hydroxide or hydrated preservative by a suitable chemical method. The content is
aluminium phosphate. Suitable antimicrobial preservatives not less than the minimum amount shown to be effective and
may be added. is not greater than 115 per cent of the quantity stated on the
Only a final bulk vaccine that complies with the following label.
Sterility (2.6.1)
Antimicrobial preservative The vaccine complies with the test for sterility.
ASSAY
not less than 85 per cent and not greater than 115 per cent Diphtheria component
of the intended content.
The lower confidence limit (P = 0.95) of the estimated avoiding reversion to toxin. The vaccine may also contain
potency is not less than the minimum potency stated on the filamentous haemagglutinin, pertactin (a 69 kDa outer
label. membrane protein) and other defined components of
Unless otherwise justified and authorised, the minimum B. pertussis such as fimbrial-2 and fimbrial-3 antigens.
potency stated on the label is 30 IU per single human dose. The latter 2 antigens may be co-purified. The antigenic
Tetanus component composition and characteristics are based on evidence of
Carry out one of the prescribed methods for the assay of protection and freedom from unexpected reactions in the
tetanus vaccine (adsorbed) (2.7.5). target group for which the vaccine is intended.
The lower confidence limit (P = 0.95) of the estimated PRODUCTION
potency is not less than 40 IU per single human dose. GENERAL PROVISIONS
Pertussis component The production method shall have been shown to yield
consistently vaccines comparable with the vaccine of proven
Cany out one of the prescribed methods for the assay of
clinical efficacy and safety in man.
pertussis vaccine (acellular) (2.7.76).
Reference vaccine(ร) Provided valid assays can be performed,
The capacity of the vaccine to induce antibodies for each
included acellular pertussis antigen is not significantly
(P = 0.95) less than that of the reference vaccine.
LABELLING combined vaccine or because of differences in composition
The label states: between the monocomponent reference vaccine and the test
the minimum number of International Units of diphtheria vaccine, a batch of combined vaccine shown to be effective in
and tetanus toxoid per single human dose; clinical trials or a batch representative thereof is used as a
the names and amounts of the pertussis components per reference vaccine. For the preparation of a representative
single human dose; batch, strict adherence to the production process used for the
where applicable, that the vaccine is intended for primary batch tested in clinical trials is necessary. The reference
vaccination of children and is not necessarily suitable for vaccine may be stabilised by a method that has been shown
reinforcing doses or for administration to adults; to have no effect on the assay procedure.
— the name and the amount of the adsorbent; Specific toxicity of the diphtheria and tetanus
— that the vaccine must be shaken before use; components
— that the vaccine is not to be frozen; The production method is validated to demonstrate that the
— where applicable, that the vaccine contains a pertussis product, if tested, would comply with the following test:
toxin-like protein produced by genetic modification. inject subcutaneously 5 times the single human dose stated
---------------------------------------------------------------------------------------------------------- Ph Eur on the label into each of 5 healthy guinea-pigs, each weighing
diphtheria toxaemia or tetanus, the vaccine does not comply
Diphtheria, Tetanus and Pertussis ***** with the test. If more than 1 animal dies from non-specific
(acellular component) Vaccine ***** causes, repeat the test once; if more than 1 animal dies in the
(Adsorbed, Reduced Antigen(s) The content of bacterial endotoxins (2.6.14) in bulk purified
Content) pertussis components is determined to monitor the
(Ph. Eur. monograph 2764) purification procedure and to limit the amount in the final
vaccine. For each component, the content of bacterial
The label may state ‘dTaP’.
endotoxins is less than the limit approved for the particular
PhEir.______________________________________________________________
vaccine and, in any case, the contents are such that the final
DEFINITION vaccine contains less than 100 IU per single human dose.
Diphtheria, tetanus and pertussis (acellular, component) PRODUCTION OF THE COMPONENTS
vaccine (adsorbed, reduced antigen(s) content) is a combined The production of the components complies with the
vaccine containing: diphtheria formol toxoid; tetanus formol requirements of the monographs Diphtheria vaccine
toxoid; individually purified antigenic components of (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452) and
BordeteUa pertussis'^ a mineral adsorbent such as aluminium Pertussis vaccine (acellular, component, adsorbed) (1356).
hydroxide or hydrated aluminium phosphate. FINAL BULK VACCINE
The formol toxoids are prepared from the toxins produced The final bulk vaccine is prepared by adsorption onto a
by the growth of Corynebacterium diphtheriae and Clostridium mineral carrier such as aluminium hydroxide or hydrated
tetani respectively. aluminium phosphate, separately or together, of suitable
The amount of diphtheria toxoid per single human dose is quantities of bulk purified diphtheria toxoid, tetanus toxoid
reduced compared to vaccines generally used for primary and acellular pertussis components. Suitable antimicrobial
vaccination; the amounts of tetanus toxoid and pertussis preservatives may be added.
components may also be reduced. Only a final bulk vaccine that complies with the following
The vaccine contains either pertussis toxoid or a pertussis requirements may be used in the preparation of the final lot.
toxin-like protein free from toxic properties, produced by Antimicrobial preservative
expression of a genetically modified form of the Where applicable, determine the amount of antimicrobial
corresponding gene. Pertussis toxoid is prepared from preservative by a suitable chemical method. The amount is
pertussis toxin by a method that renders the toxin harmless
while maintaining adequate immunogenic properties and
not less than 85 per cent and not greater than 115 per cent Aluminium (2.5.73)
of the intended content. Maximum 1.25 mg per single human dose, if aluminium
Sterility (2.6. J) hydroxide or hydrated aluminium phosphate is used as the
Carry out the test using 10 mL for each medium. adsorbent.
FINAL LOT Free formaldehyde (2.4.18)
The final bulk vaccine is distributed aseptically into sterile, Maximum 0.2 g/L.
tamper-proof containers. The containers are closed so as to Antimicrobial preservative
prevent contamination. Where applicable, determine the amount of antimicrobial
Only a final lot that is satisfactory with respect to the test for preservative by a suitable chemical method. The content is
osmolality and with respect to each of the requirements given not less than the minimum amount shown to be effective and
below under Identification, Tests and Assay may be released is not greater than 115 per cent of the quantity stated on the
for use. label.
Provided the test for residual pertussis toxin and Sterility (2.6.7)
irreversibility of pertussis toxoid, the test for antimicrobial It complies with the test.
preservative and the assays for the diphtheria, tetanus and ASSAY
pertussis components have been carried out with satisfactory Diphtheria component
results on the final bulk vaccine, they may be omitted on the Carry out one of the prescribed methods for the assay of
final lot. diphtheria vaccine (adsorbed) (2.7.6).
Provided the free formaldehyde content has been determined The lower confidence limit (P = 0.95) of the estimated
on the bulk purified antigens or on the final bulk and it has potency is not less than 2 IU per single human dose.
been shown that ±e content in the final lot will not exceed
0.2 g/L, the test for free formaldehyde may be omitted on the Tetanus component
final lot.
tetanus vaccine (adsorbed) (2.7.8).
Where there is a significant change in the manufacturing
process of the antigens or their formulation, any impact on The lower confidence limit (P = 0.95) of the estimated
the in vivo and in vitro assays must be evaluated, and the potency is not less than 20 IU per single human dose.
need for revalidation considered. Pertussis component
Osmolality (2.2.35) Carr}' out one of the prescribed methods for the assay of
The osmolality of the vaccine is within the limits approved pertussis vaccine (acellular) (2.7.16).
for the particular preparation. The capacity of the vaccine to induce antibodies for each
IDENTIFICATION (P = 0.95) less than that of the reference vaccine.
immunochemical method (2.7.7). The following method, LABELLING
applicable to certain vaccines, is given as an example. The label states:
Dissolve in the vaccine to be examined sufficient sodium — the minimum number of International Units of diphtheria
citrate R to give a 100 g/L solution. Maintain at 37 °C for and tetanus toxoid per single human dose;
about 16 h and centrifuge until a clear supernatant is — the names and amounts of the pertussis components per
obtained. The clear supernatant reacts with a suitable single human dose;
diphtheria antitoxin, giving a precipitate. If a satisfactory — where applicable, that the vaccine contains a pertussis
result is not obtained with a vaccine adsorbed on aluminium toxin-like protein produced by genetic modification;
hydroxide, carry out the test as follows. Centrifuge 15 mL of — the name and the amount of the adsorbent;
the vaccine to be examined and suspend the residue in 5 mL — that the vaccine must be shaken before use;
of a freshly prepared mixture of 1 volume of a 56 g/L — that the vaccine is not to be frozen.
solution of sodium edetate R and 49 volumes of disodium
hydrogen phosphate solution R. Maintain at 37 cc for not
less than 6 h and centrifuge. The clear supernatant reacts
with a suitable diphtheria antitoxin, giving a precipitate.
method (2.7.7). The following method, applicable to certain Adsorbed Diphtheria, Tetanus, ; ไ
vaccines, is given as an example. The clear supernatant Pertussis (Acellular Component) *****
suitable tetanus antitoxin, giving a precipitate. and Haemophilus Type b
c. The pertussis components are identified by a suitable Conjugate Vaccine
immunochemical method (2.7.7). The following method, (Diphtheria, Tetanus, Pertussis (Acellular, Component)
applicable to certain vaccines, is given as an example. and Haemophilus Type b Conjugate Vaccine
The clear supernatant obtained as described in identification (Adsorbed), Ph Eur monograph 1932)
test A reacts with specific antisera to the pertussis
The label may state ‘DTaP/Hib’.
components of the vaccine.
Ph Eur______________________________________________________________
TESTS
Residual pertussis toxin and irreversibility of pertussis DEFINITION
toxoid (2.6.33) Diphtheria, tetanus, pertussis (acellular, component) and
It complies with the test. haemophilus type b conjugate vaccine (adsorbed) is a
combined vaccine composed of: diphtheria formol toxoid;
tetanus formol toxoid; individually purified antigenic
components of BordeteUa pertussis', polyribosylribitol phosphate container, the contents of the diphtheria, tetanus and
(PRP) covalently bound to a carrier protein; a mineral pertussis antigens are in any case such that the final vial for
absorbent such as aluminium hydroxide or hydrated these components contains less than 100 IU per single
aluminium phosphate. The product is presented either as a human dose.
tetravalent liquid formulation in the same container, or as a The production method is validated to demonstrate that the
trivalent liquid formulation with the haemophilus component product, if tested, would comply with the test for abnormal
in a separate container, the contents of which are mixed with toxicity for immunosera and vaccines for human use (2.6.9).
the other components immediately before use.
During development studies and wherever revalidation is
'rhe formol toxoids are prepared from the toxins produced necessary, it shall be demonstrated by tests in animals that
by the growth of Corynebacteriuni diphtheriae and Clostridium the vaccine induces a T-cell dependent B-cell immune
letani respectively. response to PRP.
The vaccine contains either pertussis toxoid or a pertussis- Where the haemophilus component is presented in a separate
toxin-like protein free from toxic properties produced by container, the production method is validated to demonstrate
expression of a genetically modified form of the that the haemophilus component, if tested, would comply
corresponding gene. Pertussis toxoid is prepared from with the test for pyrogens (2.6.ร), carried out as follows:
pertussis toxin by a method that renders the toxin harmless inject per kilogram of the rabbit’s mass a quantity of the
while maintaining adequate immunogenic properties and vaccine equivalent to: 1 pg of PRP for a vaccine with
avoiding reversion to toxin. The acellular pertussis diphtheria toxoid or CRM 197 diphtheria protein as carrier;
component may also contain filamentous haemagglutinin, 0.1 pg of PRP for a vaccine with tetanus toxoid as carrier;
pertactin (a 69 kDa outer-membrane protein) and other 0.025 pg of PRP for a vaccine with OMP (meningococcal
defined components of B. pertussis such as fimbrial-2 and group B outer membrane protein complex) as carrier.
fimbrial-3 antigens. The latter 2 antigens may be co-purified. Reference vaccine (ร) Provided valid assays can be performed,
The antigenic composition and characteristics are based on monocomponent reference vaccines may be used for the
evidence of protection and freedom from unexpected assays on the combined vaccine. If this is not possible
reactions in the target group for which the vaccine is because of interaction between the components of the
intended.
combined vaccine or because of differences in composition
PRP is a linear copolymer composed of repeated units of between the monocomponent reference vaccine and the test
P-D-ribofuranosyl-(l —> l)-ribitol-5-phosphate
3- vaccine, a batch of combined vaccine shown to be effective in
((C10HI9OI2P)„], with a defined molecular size and derived clinical trials or a batch representative thereof is used as a
from a suitable strain of Haemophilus influenzae type b. reference vaccine. For the preparation of a representative
The carrier protein, when conjugated to PRP, is capable of batch, strict adherence to the production process used for the
inducing a T-cell-dependent B-cell immune response to the batch tested in clinical trials is necessary. The reference
polysaccharide. vaccine may be stabilised by a method that has been shown
PRODUCTION to have no effect on the assay procedure.
GENERAL PROVISIONS PRODUCTION OF THE COMPONENTS
The production method shall have been shown to yield The production of the components complies with the
consistently vaccines comparable with the vaccine of proven requirements of the monographs Diphtheria vaccine
clinical efficacy and safety in man. (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), Pertussis
Where the haemophilus component is presented in a separate vaccine (acellular, component, adsorbed) (1356) and
container, as part of consistency studies the assays of the Haemophilus type b conjugate vaccine (1219).
diphtheria, tetanus and pertussis components are carried out FINAL BULK VACCINE
on a suitable number of batches of vaccine reconstituted as Different methods of preparation may be used: a final bulk
for use. For subsequent routine control, the assays of these vaccine may be prepared by adsorption, separately or
components may be carried out without mixing with the together, of suitable quantities of bulk purified diphtheria
haemophilus component. toxoid, tetanus toxoid, acellular pertussis components and
Specific toxicity of the diphtheria and tetanus PRP conjugate onto a mineral carrier such as aluminium
components hydroxide or hydrated aluminium phosphate; or 2 final bulks
The production method is validated to demonstrate that the may be prepared and filled separately, one containing the
product, if tested, would comply with the following test: diphtheria, tetanus and pertussis components, the other the
inject subcutaneously 5 times the single human dose stated haemophilus component, which may be freeze-dried. Suitable
on the label into each of 5 healthy guinea-pigs, each weighing antimicrobial preservatives may be added.
250-350 g, that have not previously been treated with any Only a final bulk vaccine that complies with the following
material that will interfere with the test. If within 42 days of requirements may be used in the preparation of the final lot.
the injection any of the animals shows signs of or dies from Antimicrobial preservative
diphtheria toxaemia or tetanus, the vaccine does not comply Where applicable, determine the amount of antimicrobial
with the test. If more than 1 animal dies from non-specific preservative by a suitable chemical method. The amount is
causes, repeat the test once; if more than 1 animal dies in the not less than 85 per cent and not greater than 115 per cent
second test, the vaccine does not comply with the test. of the intended content.
The content of bacterial endotoxins {2.6.14) in bulk purified Sterility (2.6.1)
diphtheria toxoid, tetanus toxoid, pertussis components and Carry out the test for sterility using 10 mL for each medium.
bulk PRP conjugate is determined to monitor the purification
FINAL LOT
procedure and to limit the amount in the final vaccine.
Only a final lot that is satisfactory with respect to the test for
For each component, the content of bacterial endotoxins is
osmolality shown below and with respect to each of the
less than the limit approved for the particular vaccine; where
the haemophilus component is presented in a separate
requirements given below under Identification, Tests and conjugate where the freeze-drying process may affect the component
Assay may be released for use. to be tested.
Provided the test for residual pertussis toxin and Residual pertussis toxin and irreversibility of pertussis
irreversibility of pertussis toxoid, the test for antimicrobial toxoid (2.6.33)
preservative and the assay have been carried out with The final lot complies with the test.
satisfactory’ results on the final bulk vaccine, they may be
PRP
omitted on the final lot. Minimum 80 per cent of the amount of PRP stated on the
Provided the free formaldehyde content has been determined label. PRP is determined either by assay of ribose (2.5.31) Ct
on the bulk purified antigens or the final bulk and it has been phosphorus (2.5.18), by an immunochemical me±od (2.7.1)
shown that ±e content in the final lot will not exceed or by anion-exchange liquid chromatography (2.2.29) with
0.2 g/L, the test for free formaldehyde may be omitted on the pulsed-amperometric detection.
final lot.
Aluminium (2.5.13)
Osmolality (2.2.35) Maximum 1.25 mg per single human dose, if aluminium
The osmolality’ of the vaccine, reconstituted where applicable, hydroxide or hydrated aluminium phosphate is used as the
is within the limits approved for the particular preparation. adsorbent.
pH (2.2.3) Free formaldehyde (2.4.18)
The pH of the vaccine, reconstituted if necessary, is within Maximum 0.2 g/L.
the range approved for the particular product.
Free PRP Where applicable, determine the amount of antimicrobial
Unbound PRP is determined after removal of the conjugate, preservative by a suitable chemical method. The content is
for example by anion-exchange, size-exclusion or not less than the minimum amount shown to be effective and
hydrophobic chromatography, ultrafiltration or other is not greater than 115 per cent of the quantity stated on the
validated methods. The amount of free PRP is not greater label.
than that approved for the particular product.
Water (2.5.12)
IDENTIFICATION Maximum 3.0 per cent for the freeze-dried haemophilus
Where the haemophilus component is presented in a separate component.
container: identification tests A J B and c are carried out using the Sterility’ (2.6.1)
container containing the diphtheria, tetanus and pertussis It complies with the test for sterility.
components; identification test D is carried out on the container
Bacterial endotoxins (2.6.14)
containing the haemophilus component.
The content is within the limits approved by the competent
A. Diphtheria toxoid is identified by a suitable authority’ for the hacmophilus component of the particular
immunochemical method (2.7.1). The following method, product. If any components of the vaccine prevent the
applicable to certain vaccines, is given as an example. determination of endotoxin, a test for pyrogens is carried out
Dissolve in the vaccine to be examined sufficient sodium as described under General provisions.
about 16 h and centrifuge until a clear supernatant is ASSAY
obtained. The clear supernatant reacts with a suitable Diphtheria component
diphtheria antitoxin, giving a precipitate. Carry out one of the prescribed methods for the assay of
B. Tetanus toxoid is identified by a suitable immunochemical diphtheria vaccine (adsorbed) (2.7.6).
method (2.7.1). The following method, applicable to certain The lower confidence limit (P = 0.95) of the estimated
vaccines, is given as an example. The clear supernatant potency is not less than the minimum potency stated on the
obtained as described in identification test A reacts with a label.
suitable tetanus antitoxin, giving a precipitate. Unless otherwise justified and authorised, the minimum
C. The pertussis components are identified by a suitable potency stated on the label is 30 IU per single human dose.
immunochemical method (2.7.1). The following method, Tetanus component
applicable to certain vaccines, is given as an example. Carry out one of the prescribed methods for the assay of
The clear supernatant obtained as described in identification tetanus vaccine (adsorbed) (2.7.8).
test A reacts with specific antisera to the pertussis The lower confidence limit (P = 0.95) of the estimated
components of the vaccine. potency is not less than 40 IU per single human dose.
D. The haemophilus component is identified by a suitable Pertussis component
immunochemical method (2.7.1) for PRP. Carry out one of the prescribed methods for the assay of
TESTS pertussis vaccine (acellular) (2.7.16).
Where the product is presented with the haemophilus component in The capacity of the vaccine to induce antibodies for each
a separate container the tests for residual pertussis toxin and included acellular pertussis antigen is not significantly
irreversibility of pertussis toxoid, aluminium, free formaldehyde, (P = 0.95) less than that of the reference vaccine.
antimicrobial preservative and sterility are carried out on the
LABELLING
container with the diphtheria, tetanus and pertussis components;
The label states:
the tests for PRP content, water (where applicable), sterility and — the minimum number of International Units of diphtheria
bacterial endotoxins are carried out on the container with the
and tetanus toxoid per single human dose;
haemophilus component.
— the names and amounts of the pertussis components per
If the haemophilus component is freeze-dried, some tests may be single human dose;
carried out on the freeze-dried product rather than on the bulk — the number of micrograms of PRP per single human
dose;
the type and nominal amount of earner protein per single material that will interfere with the test. If within 42 days of
human dose; the injection any of the animals shows signs of or dies from
where applicable, that the vaccine is intended for primary diphtheria toxaemia or tetanus, the vaccine does not comply
vaccination of children and is not necessarily suitable for with the test. If more than 1 animal dies from non-specific
reinforcing doses or for administration to adults; causes, repeat the test once; if more than 1 animal dies in the
the name and the amount of the adsorbent; second test, the vaccine does not comply with the test.
— that the vaccine must be shaken before use; The content of bacterial endotoxins (2.6.14) in the bulk
that the vaccine is not to be frozen; purified diphtheria toxoid, tetanus toxoid and pertussis
where applicable, that the vaccine contains a pertussis components is determined to monitor the purification
toxin-like protein produced by genetic modification. procedure and to limit the amount in the final vaccine.
----------------- ------------------------ -- ------------------------------------------------------------ Ph Eur For each component, the content of bacterial endotoxins is
less than the limit approved for the particular vaccine.
Adsorbed Diphtheria, Tetanus, ** ** because of interaction between the components of the
Pertussis (Acellular Component) ***** combined vaccine or because of differences in composition
between the monocomponent reference vaccine and the test
and Hepatitis B (rDNA) Vaccine vaccine, a batch of combined vaccine shown to be effective in
(Diphtheria, Tetanus, Pertussis (Acellular, Component) clinical trials or a batch representative thereof is used as a
and Hepatitis B (rDNA) Vaccine (Adsorbed), reference vaccine. For the preparation of a representative
Ph Eur monograph 1933) batch, strict adherence to the production process used for the
The label may state ‘DTaP/HepB’. batch tested in clinical trials is necessary. The reference
PnEis.______________________________________________________________
vaccine may be stabilised by a method that has been shown
DEFINITION PRODUCTION OF THE COMPONENTS
Diphtheria, tetanus, pertussis (acellular, component) and The production of the components complies with the
hepatitis B (rDNA) vaccine (adsorbed) is a combined vaccine requirements of the monographs Diphtheria vaccine
composed of: diphtheria formol toxoid; tetanus formol (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), Pertussis
toxoid; individually purified antigenic components of vaccine (acellular, component, adsorbed) (1356) and Hepatitis B
Bordetella pertussis, hepatitis B surface antigen; a mineral vaccine (rDNA) (1056).
adsorbent such as aluminium hydroxide or hydrated
FINAL BULK VACCINE
aluminium phosphate.
The final bulk vaccine is prepared by adsorption, separately
The formol toxoids are prepared from the toxins produced or together, of suitable quantities of bulk purified diphtheria
by the growth of Corynebacterium diphtheriae and Clostridium toxoid, tetanus toxoid, acellular pertussis components and
tetani, respectively. hepatitis B surface antigen onto a mineral carrier such as
The vaccine contains either pertussis toxoid or a pertussis aluminium hydroxide or hydrated aluminium phosphate.
toxin-like protein free from toxic properties, produced by Suit able antimicrobial preservatives may be added.
expression of a genetically modified form of the Only a final bulk vaccine that complies with the following
corresponding gene. Pertussis toxoid is prepared from requirements may be used in the preparation of the final lot.
pertussis toxin by a method that renders the latter harmless
while maintaining adequate immunogenic properties and Antimicrobial preservative
avoiding reversion to toxin. The vaccine may also contain
filamentous haemagglutinin, pertactin (a 69 kDa outer
membrane 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 Sterility (2.6.1)
composition and characteristics are based on evidence of Carry out the test for sterility using 10 mL for each medium.
protection and freedom from unexpected reactions in the FINAL LOT
target group for which the vaccine is intended. Only a final lot that is satisfactory with respect to the test for
Hepatitis B surface antigen is a component protein of osmolality and with respect to each of the requirements given
hepatitis B virus; the antigen is obtained by recombinant below under Identification, Tests and Assay may be released
DNA technology. for use.
PRODUCTION Provided the test for residual pertussis toxin and
GENERAL PROVISIONS
irreversibility of pertussis toxoid, the test for antimicrobial
The production method shall have been shown to yield preservative and the assays for the diphtheria, tetanus and
consistently vaccines comparable with the vaccine of proven pertussis components have been carried out with satisfactory
results on the final bulk vaccine, they may be omitted on the
final lot.
Specific toxicity of the diphtheria and tetanus
Provided the content of free formaldehyde has been
components
determined on the bulk purified antigens or on the final bulk
and it has been shown that the content in the final lot will
not exceed 0.2 g/L, the test for free formaldehyde may be
inject subcutaneously 5 times the single human dose stated
If an in vivo assay is used for the hepatitis B component, The lower confidence limit (P = 0.95) of the estimated
provided it has been carried out with satisfactory' results on potency is not less than 40 IU per single human dose.
the final bulk vaccine, it may be omitted on the final lot. Pertussis component
Osmolality (2.2.35) Carry out one of the prescribed methods for the assay of
The osmolality of the vaccine is within the limits approved pertussis vaccine (acellular) (2.7.16).
for the particular preparation. The capacity of the vaccine to induce antibodies for each
IDENTIFICATION included acellular pertussis antigen is not significantly
A. Diphtheria toxoid is identified by a suitable (P = 0.95) less than that of the reference vaccine.
immunochemical method (2.7.1). The following method, Hepatitis B component
applicable to certain vaccines, is given as an example. The vaccine complies with the assay of hepatitis B vaccine
Dissolve in the vaccine to be examined sufficient sodium (2.7.15).
LABELLING
The label states:
— the minimum number of International Units of diphtheria
B. Tetanus toxoid is identified by a suitable immunochemical — the names and amounts of the pertussis components per
method (2.7.1). The following method, applicable to certain single human dose;
vaccines, is given as an example. The clear supernatant — the amount of HBsAg per single human dose;
obtained as described in identification test A reacts with a — the type of cells used for production of the hepatitis B
suitable tetanus antitoxin, giving a precipitate. component;
c. The pertussis components are identified by a suitable — where applicable, that the vaccine is intended for primary
immunochemical method (2.7.1). The following method, vaccination of children and is not necessarily suitable for
applicable to certain vaccines, is given as an example. reinforcing doses or for administration to adults;
The clear supernatant obtained as described in identification — the name and the amount of the adsorbent;
test A reacts with specific antisera to the pertussis — that the vaccine must be shaken before use;
components of the vaccine. — that the vaccine is not to be frozen;
D. The assay or, where applicable, the electrophoretic profile, — where applicable, that the vaccine contains a pertussis
serves also to identify the hepatitis B component of the toxin-like protein produced by genetic modification.
vaccine.
TESTS
Residual pertussis toxin and irreversibility of pertussis
toxoid (2.6.33)
The final lot complies with the test.
Aluminium (2.5.13)
Adsorbed Diphtheria, Tetanus, * <
Maximum 1.25 mg per single human dose, if aluminium Pertussis (Acellular Component) ****’
adsorbent.
and Inactivated Poliomyelitis
Free formaldehyde (2.4.18) Vaccine
Maximum 0.2 g/L. (Diphtheria, Tetanus, Pertussis (Acellular, Component)
and Poliomyelitis (Inactivated) Vaccine (Adsorbed),
preservative by a suitable chemical method. The content is The label may state ‘DTaP/IPV’.
not less than the minimum amount shown to be effective and Ph Eur_______________________________________________________________
is not greater than 115 per cent of the quantity stated on the DEFINITION
label. Diphtheria, tetanus, pertussis (acellular, component) and
Sterility (2.6.1) poliomyelitis (inactivated) vaccine (adsorbed) is a combined
The vaccine complies with the test for sterility. vaccine containing: diphtheria formol toxoid; tetanus formol
Pyrogens (2.6.8) toxoid; individually purified antigenic components of
The vaccine complies with the test for pyrogens. Inject the BordeteUa pertussis', suitable strains of human poliovirus
equivalent of 1 human dose into each rabbit. types 1, 2 and 3 grown in suitable cell cultures and
inactivated by a validated method; a mineral adsorbent such
ASSAY as aluminium hydroxide or hydrated aluminium phosphate.
The formol toxoids are prepared from the toxins produced
by the growth of Corynebacterium diphtheriae and Clostridium
tetani respectively.
The lower confidence limit (P = 0.95) of the estimated
The vaccine contains either pertussis toxoid or a pertussis
potency is not less than the minimum potency stated on the
toxin-like protein free from toxic properties produced by
label.
expression of a genetically modified form of the
Unless otherwise justified and authorised, the minimum corresponding gene. Pertussis toxoid is prepared from
potency stated on the label is 30 IU per single human dose. pertussis toxin by a method that renders the toxin harmless
Tetanus component while maintaining adequate immunogenic properties and
Carry out one of the prescribed methods for the assay of avoiding reversion to toxin. The vaccine may also contain
tetanus vaccine (adsorbed) (2.7.8). filamentous haemagglutinin, pertactin (a 69 kDa outer
membrane protein) and other defined components of Bovine serum albumin

B. pertussis such as fimbrial-2 and fimbrial-3 antigens. Determined on the poliomyelitis components by a suitable
The latter 2 antigens may be co-purified. The antigenic immunochemical method (2.7. /) after virus harvest and
composition and characteristics are based on evidence of before addition of the adsorbent in the preparation of the
protection and freedom from unexpected reactions in the final bulk vaccine, the amount of bovine serum albumin is
target group for which the vaccine is intended. such that the content in the final vaccine will be not more
PRODUCTION than 50 ng per single human dose.
GENERAL PROVISIONS Antimicrobial preservative
The production method shall have been shown to yield Where applicable, determine the amount of antimicrobial
consistently vaccines comparable with the vaccine of proven preservative by a suitable chemical method. The amount is
clinical efficacy and safety in man. not less than 85 per cent and not greater than 115 per cent
components Sterility (2.6. /)
The production method is validated to demonstrate that the Carry out the test for sterility using 10 mL for each medium.
product, if tested, would comply with the following test: FINAL LOT
inject subcutaneously 5 times the single human dose stated Only a final lot that is satisfactory with respect to the test for
on the label into each of 5 healthy guinea-pigs, each weighing osmolality and with respect to each of the requirements given
250-350 g, that have not previously been treated with any below under Identification, Tests and Assay may be released
material that will interfere with the test. If within 42 days of for use.
the injection any of the animals shows signs of or dies from Provided the test for residual pertussis toxin and
diphtheria toxaemia or tetanus, the vaccine does not comply irreversibility of pertussis toxoid, the test for antimicrobial
with the test. If more than 1 animal dies from non-specific preservative and the assays for the diphtheria, tetanus and
causes, repeat the test once; if more than 1 animal dies in the pertussis components have been carried out with satisfactory
second test, the vaccine does not comply with the test. results on the final bulk vaccine, they may be omitted on the
The content of bacterial endotoxins (2.6.14) in bulk purified final lot.
diphtheria toxoid, tetanus toxoid, pertussis components and Provided the free formaldehyde content has been determined
purified, inactivated monovalent poliovirus harvests is on the bulk purified antigens or on the final bulk and it has
determined to monitor the purification procedure and to been shown that the content in the final lot will not exceed
limit the amount in the final vaccine. For each component, 0.2 g/L, the test for free formaldehyde may be omitted on the
the content of bacterial endotoxins is less than the limit final lot.
approved for the particular vaccine and, in any case, the
Provided that the determination of D-antigen content has
contents are such that the final vaccine contains less than
been carried out with satisfactory results during preparation
100 IU per single human dose.
of the final bulk before addition of the adsorbent, it may be
Reference vaccine (ร) Provided valid assays can be performed, omitted on the final lot.
Provided that the in vivo assay for the poliomyelitis
component has been carried out with satisfactory results on
the final bulk vaccine, it may be omitted on the final lot.
between the monocomponent reference vaccine and the test The in vivo assay for the poliomyelitis component may be
vaccine, a batch of combined vaccine shown to be effective in omitted once it has been demonstrated for a given product
clinical trials or a batch representative thereof is used as a and for each poliovirus type that the acceptance criteria for
reference vaccine. For the preparation of a representative the D-antigen determination are such that it yields the same
batch, strict adherence to the production process used for the result as the in vivo assay in terms of acceptance or rejection
batch tested in clinical trials is necessary. The reference of a batch. This demonstration must include testing of
vaccine may be stabilised by a method that has been shown subpotent batches, produced experimentally if necessary, for
to have no effect on the assay procedure. example by heat treatment or other means of diminishing the
immunogenic activity. Where there is a significant change in
PRODUCTION OF THE COMPONENTS the manufacturing process of the antigens or their
The production of the components complies with the formulation, any impact on the in vivo and in vitro assays
requirements of the monographs Diphtheria vaccine (adsorbed) must be evaluated, and the need for revalidation considered.
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine
(acellular, component, adsorbed) (1356) and Poliomyelitis Osmolality (2.2.25)
vaccine (inactivated) (0214). The osmolality of the vaccine is within the limits approved
for the particular preparation.
FINAL BULK VACCINE
The final bulk vaccine is prepared by adsorption onto a IDENTIFICATION
mineral carrier such as aluminium hydroxide or hydrated A. Diphtheria toxoid is identified by a suitable
aluminium phosphate, separately or together, of suitable immunochemical method (2.7./). The following method,
quantities of bulk purified diphtheria toxoid, tetanus toxoid, applicable to certain vaccines, is given as an example.
acellular pertussis components and admixture of suitable Dissolve in the vaccine to be examined sufficient sodium
quantities of purified monovalent harvests of human citrate R to give a 100 g/L solution. Maintain at 37 °C for
poliovirus types 1, 2 and 3 or a suitable quantity of a about 16 h and centrifuge until a clear supernatant is
trivalent pool of such purified monovalent harvests. Suitable obtained. The clear supernatant reacts with a suitable
antimicrobial preservatives may be added. diphtheria antitoxin, giving a precipitate.
Only a final bulk vaccine that complies with the following B. Tetanus toxoid is identified by a suitable immunochemical
requirements may be used in the preparation of the final lot. method (2.7.1). The following method, applicable to certain
obtained as described in identification test A reacts with a The European Pharmacopoeia Unit and the International
suitable tetanus antitoxin, giving a precipitate. Unit are equivalent.
c. The pertussis components are identified by a suitable In vivo test The vaccine complies with the in vivo assay of
immunochemical method (2.7./). The following method, poliomyelitis vaccine (inactivated) {2.7.20).
LABELLING
The clear supernatant obtained as described in identification
The label states:
test A reacts with specific antisera to the pertussis
D. The vaccine is shown to contain human poliovirus — the names and amounts of the pertussis components per
types 15 2 and 3 by a suitable immunochemical method single human dose;
(2.7.7) such as the determination of D-antigen by enzyme- — the types of poliovirus contained in the vaccine;
linked immunosorbent assay (ELISA). — the nominal amount of poliovirus of each type (1, 2
TESTS and 3), expressed in European Pharmacopoeia Units of
Residual pertussis toxin and irreversibility of pertussis D-antigen, per single human dose;
toxoid (2.6.33) — the type of cells used for production of the poliomyelitis
The final lot complies with the test. component;
Aluminium (2.5.75)
Maximum 1.25 mg per single human dose if aluminium
— the name and the amount of the adsorbent;
adsorbent.
— that the vaccine must be shaken before use;
Free formaldehyde {2.4.18) — that the vaccine is not to be frozen;
Maximum 0.2 g/L. — where applicable, that the vaccine contains a pertussis
Antimicrobial preservative toxin-like protein produced by genetic modification.
label.
Sterility (2.6.1) Diphtheria, Tetanus and ; *
It complies with the test for sterility. Poliomyelitis (Inactivated) Vaccine *****
ASSAY
(Adsorbed, Reduced Antigen(s)
Carry out one of the prescribed methods for the assay of Content)
diphtheria vaccine (adsorbed) (2.7.6). (Ph. Eur. monograph 2328)
The lower confidence limit {P ะ= 0.95) of the estimated The label may state ‘Td/IPV’.
potency is not less than the minimum potency stated on the
Ph Eur_________________________________________________ ______________
label.
Unless otherwise justified and authorised, the minimum DEFINITION
potency stated on the label is 30 ru per single human dose. Diphtheria, tetanus and poliomyelitis (inactivated) vaccine
(adsorbed, reduced antigen(s) content) is a combined vaccine
Tetanus component containing: diphtheria formol toxoid; tetanus formol toxoid;
Carry out one of the prescribed methods for the assay of suitable strains of human poliovirus types 1, 2 and 3 grown
tetanus vaccine (adsorbed) {2.7.8). in suitable cell cultures and inactivated by a validated
The lower confidence limit {P = 0.95) of the estimated method; a mineral adsorbent such as aluminium hydroxide
potency is not less than 40 IU per single human dose. or hydrated aluminium phosphate.
Pertussis component The formol toxoids arc prepared from the toxins produced
Carry out one of the prescribed methods for the assay of by the growth of Corynebacterium diphtheriae and Clostridium
pertussis vaccine (acellular) {2.7.16). tetani respectively.
The capacity of the vaccine to induce antibodies for each The amount of diphtheria toxoid per single human dose is
included acellular pertussis antigen is not significantly reduced compared to vaccines generally used for primary
{P = 0.95) less than that of the reference vaccine. vaccination; the amount of tetanus toxoid may also be
Poliomyelitis component reduced.
D-antigen content As a measure of consistency of production, PRODUCTION
determine the D-antigen content for human poliovirus GENERAL PROVISIONS
types 1, 2 and 3 by a suitable immunochemical method The production method shall have been shown to yield
(2.7.1) following desorption, using a reference preparation consistently vaccines comparable with the vaccine of proven
calibrated in European Pharmacopoeia Units of D-antigen. clinical efficacy and safety in man.
For each type, the content, expressed with reference to the Reference vaccine(s) Provided valid assays can be performed,
amount of D-antigen stated on the label, is within the limits monocomponent reference vaccines may be used for the
approved for the particular product. Poliomyelitis vaccine assays on the combined vaccine. If this is not possible
(inactivated) BRP is calibrated in European Pharmacopoeia because of interaction between the components of the
Units and intended for use in the assay of D-antigen. combined vaccine or because of the difference in composition
vaccine, a batch of combined vaccine shown to be effective in below under Identification, Tests and Assay may be released
clinical trials or a batch representative thereof is used as a for use.
reference vaccine. For the preparation of a representative Provided the test for antimicrobial preservative and the assays
batch, strict adherence to the production process used for the for the diphtheria and tetanus components have been carried
batch tested in clinical trials is necessary. The reference out with satisfactory results on the final bulk vaccine, they
vaccine may be stabilised by a method that has been shown may be omitted on the final lot.
Specific toxicity of the diphtheria and tetanus on the bulk purified antigens or on the final bulk and it has
components been shown that the content in the final lot will not exceed
The production method is validated to demonstrate that the 0.2 g/L, the test for free formaldehyde may be omitted on the
product, if tested, would comply with the following test: final lot.
inject subcutaneously 5 times the single human dose stated
Provided the determination of D-antigen content cannot be
carried out on the final lot, it is carried out during
preparation of the final bulk before addition of the adsorbent.
the injection any of the animals shows signs of or dies from Provided the in vivo assay for the poliomyelitis component
diphtheria toxaemia or tetanus, the vaccine does not comply has been carried out with satisfactory results on the final bulk
with the test. If more than one animal dies from non-specific vaccine, it may be omitted on the final lot.
causes, repeat the test once; if more than one animal dies in The in vivo assay for the poliomyelitis component may be
the second test, the vaccine does not comply with the test. omitted once it has been demonstrated for a given vaccine
The content of bacterial endotoxins (2.6.14) in bulk purified and for each poliovirus type that the acceptance criteria for
diphtheria toxoid, tetanus toxoid and inactivated monovalent the D-antigen determination are such that it yields the same
poliovirus harvests is determined to monitor the purification result as the in vivo assay in terms of acceptance or rejection
procedure and to limit the amount in the final vaccine. of a batch. This demonstration must include testing of
For each component, the content of bacterial endotoxins is subpotent batches, produced experimentally if necessary, for
less than the limit approved for the particular vaccine and, in example by heat treatment or other means of diminishing the
any case, the contents are such that the final vaccine contains immunogenic activity. Where there is a significant change in
less than 100 IU per single human dose. the manufacturing process of the antigens or their
formulation, any impact on the in vivo and in vitro assays
PRODUCTION OF THE COMPONENTS
must be evaluated, and the need for revalidation considered.
The production of the components complies with the
requirements of the monographs on Diphtheria vaccine Osmolality (2.2.35)
(adsorbed) (0443), Tetanus vaccine (adsorbed) (0452) and The osmolality of the vaccine is within the limits approved
Poliomyelitis vaccine (inactivated) (0214). for the particular preparation.
FINAL BULK VACCINE IDENTIFICATION
The final bulk vaccine is prepared by adsorption onto a A. Diphtheria toxoid is identified by a suitable
mineral carrier such as aluminium hydroxide or hydrated immunochemical method (2.7.1). The following method,
aluminium phosphate, separately or together, of suitable applicable to certain vaccines, is given as an example.
quantities of bulk purified diphtheria toxoid and tetanus Dissolve in the vaccine to be examined sufficient sodium
toxoid, and an admixture of suitable quantities of purified citrate R to give a 100 g/L solution. Maintain at 37 °C for
monovalent harvests of human poliovirus types 1, 2 and 3 or about 16 h and centrifuge until a clear supernatant is
a suitable quantity of a trivalent pool of such purified obtained. The clear supernatant reacts with a suitable
monovalent harvests. Suitable antimicrobial preservatives may diphtheria antitoxin, giving a precipitate. If a satisfactory
be added. result is not obtained with a vaccine adsorbed on aluminium
Only a final bulk vaccine that complies with the following hydroxide, carry out the test as follows. Centrifuge 15 mL of
requirements may be used in the preparation of the final lot. the vaccine to be examined and suspend the residue in 5 mL
of a freshly prepared mixture of 1 volume of a 56 g/L
Bovine serum albumin
solution of sodium edetate R and 49 volumes of a 90 g/L
Determined on the poliomyelitis components by a suitable
solution of disodium hydrogen phosphate R. Maintain at 37 °C
immunochemical method (2.7.7) after virus harvest and
for not less than 6 h and centrifuge. The clear supernatant
before addition of the adsorbent in the preparation of the
reacts with a suitable diphtheria antitoxin, giving a
final bulk vaccine, the amount of bovine serum albumin is
precipitate.
such that the content in the final vaccine will be not more
than 50 ng per single human dose. B. Tetanus toxoid is identified by a suitable immunochemical
suitable tetanus antitoxin, giving a precipitate.
of the intended content. C. The vaccine is shown to contain human poliovirus
types 1, 2 and 3 by a suitable immunochemical method
Sterility (2.6.7)
(2.7.7) such as the determination of D-antigen by enzyme-
linked immunosorbent assay (ELISA).
FINAL LOT
The final bulk vaccine is distributed aseptically into sterile, TESTS
tamper-proof containers. The containers are closed so as to Aluminium (2.5.13)
prevent contamination. Maximum 1.25 mg per single human dose, if aluminium
Only a final lot that is satisfactory with respect to the test for hydroxide or hydrated aluminium phosphate is used as the
osmolality and with respect to each of the requirements given adsorbent.
Free formaldehyde {2.4.18)

Maximum 0.2 g/L. Diphtheria, Tetanus, Pertussis * •
Antimicrobial preservative (Acellular, Component) and
Poliomyelitis (Inactivated) Vaccine
not less than the minimum amount shown to be effective and (Adsorbed, Reduced Antigen(s)
label. Content)
Sterility (2.6.1)
It complies with the test for sterility. The label may state ‘dTaP/IPV’.
Ph Eur_____________________________________ ___ ______ _______________
ASSAY
Diphtheria component DEFINITION
Carty' out one of the prescribed methods for the assay of Diphtheria, tetanus, pertussis (acellular, component) and
diphtheria vaccine (adsorbed) (2.7.6). poliomyelitis (inactivated) vaccine (adsorbed, reduced
The lower confidence limit (P = 0.95) of the estimated antigen(s) content) is a combined vaccine containing:
potency is not less than 2 IU per single human dose. diphtheria formol toxoid; tetanus formol toxoid; individually
purified antigenic components of Bordetella pertussis') suitable
Tetanus component strains of human poliovirus types 1, 2 and 3 grown in
Carry out one of the prescribed methods for the assay of suitable cell cultures and inactivated by a validated method;
tetanus vaccine (adsorbed) (2.7.8). a mineral adsorbent such as aluminium hydroxide or
The lower confidence limit (P = 0.95) of the estimated hydrated aluminium phosphate.
potency is not less ±an 20 IU per single human dose. The formol toxoids arc prepared from the toxins produced
Poliomyelitis component by the growth of Corynebacterium diphtheriae and Clostridium
D-antigen content As a measure of consistency of production, tetani respectively.
determine the D-antigen content for human poliovirus The amount of diphtheria toxoid per single human dose is
types 1, 2 and 3 by a suitable immunochemical method reduced compared to vaccines generally used for primary
(2.7.7) following desorption, using a reference preparation vaccination; the amounts of tetanus toxoid and pertussis
calibrated in European Pharmacopoeia Units of D-antigen. components may also be reduced.
For each type, the content, expressed with reference to the
The vaccine contains either pertussis toxoid or a pertussis
amount of D-antigen stated on the label, is within the limits
toxin-like protein free from toxic properties produced by
approved for the particular product. Poliomyelitis vaccine
expression of a genetically modified form of the
(inactivated) BRP is calibrated in European Pharmacopoeia
corresponding gene. Pertussis toxoid is prepared from
Units and intended for use in the assay of D-antigen.
pertussis toxin by a method that renders the toxin harmless
The European Pharmacopoeia Unit and the International
while maintaining adequate immunogenic properties and
Unit are equivalent.
avoiding reversion to toxin. The vaccine may also contain
In vivo test The vaccine complies with the in vivo assay of filamentous haemagglutinin, pertactin (a 69 kDa outer
poliomyelitis vaccine (inactivated) (2.7.20. membrane protein) and other defined components of B.
LABELLING pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter
The label states: 2 antigens may be co-purified. The antigenic composition
— the minimum number of International Units of diphtheria and characteristics are based on evidence of protection and
and tetanus toxoid per single human dose; freedom from .unexpected reactions in the target group for
— the types of poliovirus contained in the vaccine; which the vaccine is intended.
— the nominal amount of poliovirus of each type (1,2 and PRODUCTION
3), expressed in European Pharmacopoeia Units of GENERAL PROVISIONS
D-antigen, per single human dose; The production method shall have been shown to yield
— the type of cells used for production of the poliomyelitis consistently vaccines comparable with the vaccine of proven
component; clinical efficacy and safety in man.
— the name and the amount of ±e adsorbent; Reference vaccine (ร) Provided valid assays can be performed,
— that the vaccine is not to be frozen. assays on the combined vaccine. If this is not possible
——————_________ Ph Eur because of interaction between the components of the
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
components
inject subcutaneously 5 times the single human dose stated Provided the free formaldehyde content has been determined
on the label into each of 5 healthy guinea-pigs, each weighing on the bulk purified antigens or on the final bulk and it has
250-350 g, that have not previously been treated with any been shown that the content in the final lot will not exceed
material that will interfere with the test. If within 42 days of 0.2 g/L, the test for free formaldehyde may be omitted on the
the injection any of the animals shows signs of or dies from final lot.
diphtheria toxaemia or tetanus, the vaccine does not comply Provided the determination of D-antigen content cannot be
with the test. If more than 1 animal dies from non-specific carried out on the final lot, it is carried out during
causes, repeat the test once; if more than 1 animal dies in the preparation of the final bulk before addition of the adsorbent.
Provided the in vivo assay for the poliomyelitis component
The content of bacterial endotoxins (2.6.14) in bulk purified has been carried out with satisfactory results on the final bulk
diphtheria toxoid, tetanus toxoid, pertussis components and vaccine, it may be omitted on the final lot.
inactivated monovalent poliovirus harvests is determined to
The in vivo assay for the poliomyelitis component may be
monitor the purification procedure and to limit the amount omitted once it has been demonstrated for a given vaccine
in the final vaccine. For each component, the content of
and for each poliovirus type that the acceptance criteria for
bacterial endotoxins is less than the limit approved for the
the D-antigen determination are such that it yields the same
particular vaccine and, in any case, the contents are such that result as the in vivo assay in terms of acceptance or rejection
the final vaccine contains less than 100 IU per single human
of a batch. This demonstration must include testing of
dose.
subpotent batches, produced experimentally if necessary, for
PRODUCTION OF THE COMPONENTS example by heat treatment or other means of diminishing the
The production of the components complies with the immunogenic activity. Where there is a significant change in
requirements of the monographs Diphtheria vaccine (adsorbed) the manufacturing process of the antigens or their
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine formulation, any impact on the in vivo and in vitro assays
(acellular, component, adsorbed) (1356) and Poliomyelitis must be evaluated, and the need for revalidation considered.
vaccine (inactivated) (0214).
Osmolality (2.2.35)
FINAL BULK VACCINE The osmolality of the vaccine is within the limits approved
The final bulk vaccine is prepared by adsorption onto a for the particular preparation.
mineral carrier such as aluminium hydroxide or hydrated
IDENTIFICATION
aluminium phosphate, separately or together, of suitable
quantities of bulk purified diphtheria toxoid, tetanus toxoid
and acellular pertussis components, and an admixture of
suitable quantities of purified monovalent harvests of human
poliovirus types 1, 2 and 3 or a suitable quantity of a
trivalent pool of such purified monovalent harvests. Suitable
antimicrobial preservatives may be added.
Only a final bulk vaccine that complies with the following diphtheria antitoxin, giving a precipitate. If a satisfactory
requirements may be used in the preparation of the final lot. result is not obtained with a vaccine adsorbed on aluminium
Bovine serum albumin hydroxide, carry out the test as follows. Centrifuge 15 mL of
Determined on the poliomyelitis components by a suitable the vaccine to be examined and suspend the residue in 5 mL
immunochemical method (2.7.1) after virus harvest and of a freshly prepared mixture of 1 volume of a 56 g/L
before addition of the adsorbent in the preparation of the solution of sodium edetate R and 49 volumes of a 90 g/L
final bulk vaccine, the amount of bovine serum albumin is solution of disodium hydrogen phosphate R. Maintain at 37 °C
such that the content in the final vaccine will be not more for not less than 6 h and centrifuge. The clear supernatant
than 50 ng per single human dose. reacts with a suitable diphtheria antitoxin, giving a
Antimicrobial preservative precipitate.
Where applicable, determine the amount of antimicrobial B. Tetanus toxoid is identified by a suitable immunochemical
preservative by a suitable chemical method. The amount is method (2.7.1). The following method, applicable to certain
not less than 85 per cent and not greater than 115 per cent vaccines, is given as an example. The clear supernatant
of the intended content. obtained as described in identification test A reacts with a
Sterility (2.6.1) suitable tetanus antitoxin, giving a precipitate.
Carry out the test for sterility using 10 mL for each medium. c. The pertussis components are identified by a suitable
FINAL LOT
The clear supernatant obtained as described in identification
test A reacts with a specific antisera to the pertussis
D. The vaccine is shown to contain human poliovirus types
osmolality and with respect to each of the requirements given
1, 2 and 3 by a suitable immunochemical method (2.7.1)
below under Identification, Tests and Assay may be released
such as the determination of D-antigen by enzyme-linked
for use.
immunosorbent assay CELIS A).
Provided the test for residual pertussis toxin and
irreversibility of pertussis toxoid, the test for antimicrobial TESTS
preservative and the assays for the diphtheria, tetanus and Residual pertussis toxin and irreversibility of pertussis
pertussis components have been carried out with satisfactory toxoid (2.6.33)
results on the final bulk vaccine, they may be omitted on the The final lot complies with the tesL
final lot.
Aluminium (2.5.13)
Maximum 1.25 mg per single human dose, if aluminium Diphtheria, Tetanus, Pertussis ** *,
hydroxide or hydrated aluminium phosphate is used as the (Acellular, Component), *****
adsorbent.
Poliomyelitis (Inactivated) and
Maximum 0.2 g/L. Haemophilus Type b Conjugate
Antimicrobial preservative Vaccine (Adsorbed)
not less than the minimum amount shown to be effective and The label may state ‘DTaP/IPV/Hib’.
is not greater than 115 per cent of the quantity stated on the Ph Eur_———————
label. DEFINITION
Sterility (2.6.1) Diphtheria, tetanus, pertussis (acellular, component),
It complies with the test for sterility. poliomyelitis (inactivated) and haemophilus type b conjugate
ASSAY vaccine (adsorbed) is a combined vaccine composed of:
Diphtheria component diphtheria formol toxoid; tetanus formol toxoid; individually
Carry out one of the prescribed methods for the assay of purified antigenic components of BordeteUa pertussis', suitable
diphtheria vaccine (adsorbed) (2.7.6). strains of human poliovirus types 1, 2 and 3 grown in
suitable cell cultures and inactivated by a suitable method;
The lower confidence limit (P = 0.95) of the estimated
potency is not less than 2 IU per single human dose. polyribosylribitol phosphate (PRP) covalently bound to a
carrier protein; a mineral adsorbent such as aluminium
Tetanus component hydroxide or hydrated aluminium phosphate. The product is
Carry out one of the prescribed methods for the assay of presented either as a pentavalent liquid formulation in the
tetanus vaccine (adsorbed) (2.7.8). same container, or as a tctravalent liquid formulation with
The lower confidence limit (P = 0.95) of the estimated the freeze-dried haemophilus component in a separate
potency is not less than 20 IU per single human dose. container, the contents of which arc mixed with the other
Pertussis component components immediately before use.
Carty' out one of the prescribed methods for the assay of The formol toxoids are prepared from the toxins produced
pertussis vaccine (acellular) (2.7.16). by the growth of Corynebacterium diphtheriae and Clostridium
The capacity of the vaccine to induce antibodies for each letani respectively.
included acellular pertussis antigen is not significantly The vaccine contains either pertussis toxoid or a pertussis
(P = 0.95) less than that of the reference vaccine. toxin-like protein free from toxic properties produced by
Poliomyelitis component expression of a genetically modified form of the
D-antigen content As a measure of consistency of production, corresponding gene. Pertussis toxoid is prepared from
determine the D-antigen content for human poliovirus types pertussis toxin by a method that renders the toxin harmless
1, 2 and 3 by a suitable immunochemical method (2.7.1) while maintaining adequate immunogenic properties and
following desorption, using a reference preparation calibrated avoiding reversion to toxin. The acellular pertussis
in European Pharmacopoeia Units of D-antigen. For each component may also contain filamentous haemagglutinin,
type, the content, expressed with reference to the amount of pertactin (a 69 kDa outer-membrane protein) and other
D-antigen stated on the label, is within the limits approved defined components of B. pertussis such as fimbrial-2 and
for the particular product. Poliomyelitis vaccine fimbrial-3 antigens. The latter 2 antigens may be co-purified.
(inactivated) BRP is calibrated in European Pharmacopoeia The antigenic composition and characteristics are based on
Units and intended for use in the assay of D-antigen. evidence of protection and freedom from unexpected
The European Pharmacopoeia Unit and the International reactions in the target group for which the vaccine is
Unit are equivalent. intended.
In vivo test The vaccine complies with the in vivo assay of PRP is a linear copolymer composed of repeated units of
poliomyelitis vaccine (inactivated) (2.7.20). 3-P-D-ribofuranosyl-(l -> l)-ribitol-5-phosphate
LABELLING [(C10H19012P) ,1], with a defined molecular size and derived
from a suitable strain of Haemophilus influenzae type b.
The label states:
The carrier protein, when conjugated to PRP, is capable of
inducing a T-cell-dependent B-cell immune response to the
polysaccharide.
single human dose; PRODUCTION
— where applicable, that the vaccine contains a pertussis GENERAL PROVISIONS
toxin-like protein produced by genetic modification; The production method shall have been shown to yield
— the types of poliovirus contained in the vaccine; consistently vaccines comparable with the vaccine of proven
— the nominal amount of poliovirus of each type (1,2 and clinical efficacy and safety in man.
3), expressed in European Pharmacopoeia Units of Specific toxicity of the diphtheria and tetanus
D-antigen, per single human dose; components
— the type of cells used for production of the poliomyelitis The production method is validated to demonstrate that the
component; product, if tested, would comply with the following test:
— the name and the amount of the adsorbent; inject subcutaneously 5 times the single human dose stated
— that the vaccine must be shaken before use; on the label into each of 5 healthy guinea-pigs, each weighing
— that the vaccine is not to be frozen. 250-350 g, that have not previously been treated with any
______________________________ __ ______________________________ Ph Eur
material that will interfere with the test. If within 42 days of phosphate, and admixture of suitable quantities of purified,
(he injection any of the animals shows signs of or dies from monovalent harvests of human poliovirus types 1, 2 and 3 or
diphtheria toxaemia or tetanus, the vaccine does not comply a suitable quantity of a trivalent pool of such monovalent
mth the test. If more than 1 animal dies from non-specific harvests. Suitable antimicrobial preservatives may be added.
causes, repeat the test once; if more than 1 animal dies in the Where the vaccine is presented with all 5 components in the
second test, the vaccine docs not comply with the test. same container, the final bulk is prepared by addition of a
Bacterial endotoxins {2.6.14) suitable quantity of the haemophilus bulk conjugate to the
The content of bacterial endotoxins in bulk purified tetravalent bulk. Where the haemophilus component is
diphtheria toxoid, tetanus toxoid, pertussis components, presented in a separate container, the final bulk is prepared
punfied, inactivated monovalent poliovirus harvests and bulk by dilution of the bulk conjugate with suitable diluents for
PRP conjugate is determined to monitor the purification freeze-drying. A stabiliser may be added.
procedure and to limit the amount in the final vaccine. Only final bulks that comply with the following requirements
For each component, the content of bacterial endotoxins is may be used in the preparation of the final lot.
less than the limit approved by the competent authority for
the particular vaccine.
Determined on the poliomyelitis components by a suitable
Development and consistency studies immunochemical method (2.7.1) during preparation of the
During development studies and wherever revalidation is final bulk vaccine, before addition of the adsorbent, the
necessary, it shall be demonstrated by tests in animals that amount of bovine serum albumin is such that the content in
the vaccine induces a T-cell-depcndent B-cell immune the final vaccine will be not more than 50 ng per single
response to PRP. human dose.
Where the haemophilus component is presented in a separate Antimicrobial preservative
container, and as part of consistency studies, the assays of the Where applicable, determine the amount of antimicrobial
diphtheria, tetanus, pertussis and poliomyelitis components preservative by a suitable chemical method. The amount is
are earned out on a suitable number of batches of vaccine not less than 85 per cent and not greater than 115 per cent
reconstituted as for use. For subsequent routine control, the of the intended content.
assays of these components may be carried out without
Sterility (2.6.1)
mixing with the haemophilus component.
Vi'here the haemophilus component is presented in a separate
FINAL LOT
container, the production method is validated to demonstrate
that the haemophilus component, if tested, would comply Where the haemophilus component is presented in a separate
container, the final bulk of the haemophilus component is
with the test for pyrogens {2.6.8), carried out as follows:
inject per kilogram of the rabbit's mass a quantity of the freeze-dried.
vaccine equivalent to: 1 pg of PRP for a vaccine with Only a final lot that is satisfactory with respect to the test for
diphtheria toxoid or CRM 197 diphtheria protein as carrier; osmolality shown below and with respect to each of the
0.1 pg of PRP for a vaccine with tetanus toxoid as carrier; requirements given below under Identification, Tests and
0.025 pg of PRP for a vaccine with OMP (meningococcal Assay may be released for use.
group B outer membrane protein complex) as carrier. Provided that the test for residual pertussis toxin and
Reference vaccine(ร) Provided valid assays can be performed, irreversibility of pertussis toxoid, the test for antimicrobial
monocomponent reference vaccines may be used for the preservative and the assay have been carried out with
assays on the combined vaccine. If this is not possible satisfactory results on the final bulk vaccine, they may be
because of interaction between the components of the omitted on ±e final lot.
combined vaccine or because of differences in composition Provided that the free formaldehyde content has been
between the monocomponent reference vaccine and the test determined on the bulk purified antigens and the purified
vaccine, a batch of combined vaccine shown to be effective in monovalent harvests or the trivalent pool of polioviruses or
clinical trials or a batch representative thereof is used as a the final bulk and it has been shown that the content in the
reference vaccine. For the preparation of a representative final lot will not exceed 0.2 g/L, the test for free
batch, strict adherence to the production process used for the formaldehyde may be omitted on the final lot.
batch tested in clinical trials is necessary. The reference If the in vivo assay for the poliomyelitis component is used,
vaccine may be stabilised by a method that has been shown provided it has been carried out with satisfactory results on
to have no effect on the assay procedure. the final bulk vaccine, it may be omitted on the final lot.
PRODUCTION OF THE COMPONENTS The in vivo assay for the poliomyelitis component may be
The production of the components complies with the omitted once it has been demonstrated for a given product
requirements of the monographs Diphtheria vaccine (adsorbed) and for each poliovirus type that the acceptance criteria for
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine the D-antigen determination are such that it yields the same
(acellular, component, adsorbed) (1356), Poliomyelitis vaccine result as the in vivo assay in terms of acceptance or rejection
(inactivated) (0214) and Haemophilus type b conjugate vaccine of a batch. This demonstration must include testing of
(1219). subpotent batches, produced experimentally if necessary, for
example by heat treatment or other means of diminishing the
FINAL BULKS
immunogenic activity. Where there is a significant change in
The final tetravalent bulk of the diphtheria, tetanus, pertussis
the manufacturing process of the antigens or their
and poliomyelitis components is prepared by adsorption,
formulation, any impact on the in vivo and in vitro assays
separately or together, of suitable quantities of bulk purified
must be evaluated, and the need for revalidation considered.
diphtheria toxoid, bulk purified tetanus toxoid and bulk
purified acellular pertussis components onto a mineral earner Osmolality {2.2.35)
such as aluminium hydroxide or hydrated aluminium The osmolality of the vaccine, reconstituted where applicable,
is within the limits approved for the particular preparation.
Free PRP Residual pertussis toxin and irreversibility of pertussis

Where the haemophilus component is presented in liquid toxoid (2.6.33)
formulation, the presence of other components may interfere The final lot complies with the test.
in the assay and it may not be possible to separate the PRP PRP
from the adjuvant. The presence of free PRP may be Not less than 80 per cent of the amount of PRP stated on
determined on the bulk conjugate prior to the addition of the label. PRP is determined either by assay of ribose
other components or on the non-adsorbed fraction in the (2.5.31) or phosphorus (2.5.18), by an immunochemical
final combination. method (2.7.1) or by anion-exchange liquid chromatography
Where the haemophilus component is presented in a separate (2.2.29) with pulsed-amperometric detection.
container, a number of methods have been used to separate
Aluminium (2.5.13)
free PRP from the conjugate, including precipitation, gel
filtration, size-exclusion, anion-exchange and hydrophobic
chromatography, ultrafiltration and ultracentrifiigation.
adsorbent.
The free PRP can then be quantified by a range of
techniques, including high-performance anion-exchange Free formaldehyde (2.4.18)
chromatography with pulsed amperometric detection Maximum 0.2 g/L.
(HPAEC-PAD) and immunoassays with anti-PRP Antimicrobial preservative
antibodies. The amount of free PRP is not greater than that Where applicable, determine the amount of antimicrobial
approved for the particular product. preservative by a suitable chemical method. The content is
IDENTIFICATION not less than the minimum amount shown to be effective and
Identification tests A, B, c and D are earned out using the vial
label.
containing the diphtheria, tetanus, pertussis and poliomyelitis
components; identification lest E is carried out either on the vial Water (2.5.12)
containing all 5 components, or on the vial containing the Maximum 3.0 per cent for the freeze-dried haemophilus
haemophilus component alone. component.
A. Diphtheria toxoid is identified by a suitable Sterility (2.6.1)
immunochemical method (2.7.1'). The following method, It complies with the test for sterility.
applicable to certain vaccines, is given as an example. Bacterial endotoxins (2.6.14)
Dissolve in the vaccine to be examined sufficient sodium The content is within the limits approved by the competent
citrate R to give a 100 g/L solution. Maintain at 37 °C for authority for the haemophilus component of the particular
about 16 h and centrifuge until a clear supernatant is product. If any components of the vaccine prevent the
obtained. The clear supernatant reacts with a suitable determination of endotoxin, a test for pyrogens is carried out
diphtheria antitoxin, giving a precipitate. as described under General provisions.
ASSAY
obtained during identification test A reacts with a suitable
tetanus antitoxin, giving a precipitate.
Unless otherwise justified and authorised, the lower
c. The pertussis components are identified by a suitable
confidence limit (P = 0.95) of the estimated potency is not
less than 30 IU per single human dose.
The clear supernatant obtained during identification test A Tetanus component
reacts with specific antisera to the pertussis components of Carry out one of the prescribed methods for the assay of
the vaccine. tetanus vaccine (adsorbed) (2.7.8).
D. The vaccine is shown to contain human poliovirus The lower confidence limit (P = 0.95) of the estimated
types 1, 2 and 3 by a suitable immunochemical method potency is not less than 40 IU per single human dose.
(2.7. /), such as determination of D-antigen by enzyme-linked Pertussis component
immunosorbent assay (ELISA). Carry out one of the prescribed methods for the assay of
E. The haemophilus component is identified by a suitable pertussis vaccine (acellular) (2.7.16).
immunochemical method (2.7.1) for PRP. The capacity of the vaccine to induce antibodies for each
TESTS included acellular pertussis antigen is not significantly
Where the haemophilus component is presented in a separate
container, the tests for residual pertussis toxin and irreversibility of Poliomyelitis component
pertussis toxoid, aluminium, free formaldehyde, antimicrobial D-antigen content As a measure of consistency of production,
preservative and sterility are carried out on the container with the determine the D-antigen content for human poliovirus
diphtheria, tetanus, pertussis and poliomyelitis components; types 1, 2 and 3 by a suitable immunochemical method
the tests for PRP, water, sterility and bacterial entodoxins are (2.7.1) following desorption, using a reference preparation
carried out on the container with the haemophilus component calibrated in European Pharmacopoeia Units of D-antigen.
alone. For each type, the content, expressed with reference to the
Where the haemophilus component is presented in a separate
approved for the particular product. Poliomyelitis vaccine
container, some tests may be carried out on the freeze-dried
product rather than on the bulk conjugate where the freeze-drying
process may affect the component to be tested.
2016
Vaccines IV-581
The European Pharmacopoeia Unit and the International The vaccine contains either pertussis toxoid or a pertussis
Unit are equivalent. toxin-like protein free from toxic properties produced by
Zn vtvo test The vaccine complies with the in vivo assay of expression of a genetically modified form of the
poliomyelitis vaccine (inactivated) (2.7.20). corresponding gene. Pertussis toxoid is prepared from
LABELLING pertussis toxin by a method that renders the toxin harmless
The label states: while maintaining adequate immunogenic properties and
avoiding reversion to toxin. The acellular pertussis
the minimum number of International Units of diphtheria
component may also contain filamentous haemagglutinin,
pertactin (a 69 kDa outer-membrane protein) and other
the names and amounts of the pertussis components per
defined components of B. pertussis such as fimbrial-2 and
single human dose;
fimbrial-3 antigens. The latter 2 antigens may be co-purified.
the nominal amount of poliovirus of each type (1,2
The antigenic composition and characteristics are based on
and 3), expressed in European Pharmacopoeia Units of
evidence of protection and freedom from unexpected
D-antigen, per single human dose;
reactions in the target group for which the vaccine is
the type of cells used for production of the poliomyelitis
intended.
component;
the number of micrograms of PRP per single human Heparins B surface antigen is a component protein of
dose; hepatitis B virus; the antigen is obtained by recombinant
the type and nominal amount of carrier protein per single DNA technology.
human dose; PRP is a linear copolymer composed of repeated units of
where applicable, that the vaccine is intended for primary 3-p-D-ribofuranosyl-(l -> l)-ribitol-5-
vaccination of children and is not necessarily suitable for phosphate [(CioHigO^P),,], with a defined molecular size
reinforcing doses or for administration to adults; and derived from a suitable strain of Haemophilus influenzae
the name and the amount of the adsorbent; type b. The carrier protein, when conjugated to PRP, is
that the vaccine must be shaken before use; capable of inducing a T-cell-dependent B-cell immune
— that the vaccine is not to be frozen; response to the polysaccharide.
where applicable, that the vaccine contains a pertussis PRODUCTION
toxin-like protein produced by genetic modification. GENERAL PROVISIONS
------------------------- ---------------- - -------------------------------------------------------------- Ph Eur The production method shall have been shown to yield
If the vaccine is presented with the haemophilus component
in a separate container, as part of consistency studies the
Diphtheria, Tetanus, Pertussis ** \ assays of the diphtheria, tetanus, pertussis, hepatitis B and
(Acellular, Component), ***** poliomyelitis components are carried out on a suitable
number of batches of vaccine reconstituted as for use.
Hepatitis B (rDNA), Poliomyelitis For subsequent routine control, the assays of these
components may be carried out without mixing with the
(Inactivated) and Haemophilus haemophilus component.
Type b Conjugate Vaccine Specific toxicity of the diphtheria and tetanus
(Adsorbed) components
(Ph. Eur. monograph 2067) The production method is validated to demonstrate that the
The label may state ‘DTaP/HepB/IPV/Hib’. inject subcutaneously 5 times the single human dose stated
PhEur ___________________________________________________________ on the label into each of 5 healthy guinea-pigs, each weighing
DEFINITION 250-350 g, that have not previously been treated with any
Diphtheria, tetanus, pertussis (acellular, component), material that will interfere with the test. If within 42 days of
hepatitis B (rDNA), poliomyelitis (inactivated) and the injection any of the animals shows signs of or dies from
haemophilus type b conjugate vaccine (adsorbed) is a diphtheria toxaemia or tetanus, the vaccine does not comply
combined vaccine composed of: diphtheria formol toxoid;
causes, repeat the test once; if more than 1 animal dies in the
tetanus formol toxoid; individually purified antigenic
components of Bordetella pertussis, hepatitis B surface antigen
(HBsAg); human poliovirus types 1, 2 and 3 grown in The content of bacterial endotoxins (2.6.14) in bulk purified
suitable cell cultures and inactivated by a suitable method; diphtheria toxoid, tetanus toxoid and pertussis components,
polyribosylribitol phosphate (PRP) covalently bound to a hepatitis B surface antigen, purified, inactivated monovalent
carrier protein. The antigens in the vaccine may be adsorbed poliovirus harvests and bulk PRP conjugate is determined to
on a mineral carrier such as aluminium hydroxide or monitor the purification procedure and to limit the amount
hydrated aluminium phosphate. The product is presented in the final vaccine. For each component, the content of
either as a hexavalent liquid formulation in the same bacterial endotoxins is not greater than the limit approved.
container, or as a pentavalent liquid formulation with the During development studies and wherever revalidation is
haemophilus component in a separate container, the contents necessary, a test for pyrogens in rabbits (2.6.8) is carried out
of which are mixed with the other components immediately by injection of a suitable dose of the final lot. The vaccine is
before or during use. shown to be acceptable with respect to absence of pyrogenic
The formol toxoids are prepared from the toxins produced activity.
During development studies and wherever revalidation is harvests and before preparation of the final bulk vaccine,
necessary, it shall be demonstrated by tests in animals that before addition of the adsorbent, the amount of bovine
the vaccine induces a T-cell-dependent B-cell immune serum albumin is such that the content in the final vaccine
response to PRP. will be not more than 50 ng per single human dose.
The stability of the final lot and relevant intermediates is Antimicrobial preservative
evaluated using one or more indicator tests. For the Where applicable, determine the amount of antimicrobial
haemophilus component, such tests may include preservative by a suitable chemical method,. The amount is
determination of molecular size, determination of free PRP in not less than 85 per cent and not greater than 115 per cent
the conjugate and kinetics of depolymerisation. Taking of the intended content.
account of the results of the stability testing, release
Sterility (2.6. /)
requirements are set for these indicator tests to ensure that
Cany out the test for sterility using 10 mL for each medium.
the vaccine will be satisfactory’ at the end of the period of
validity. FINAL LOT
Reference vaccine (ร) Provided valid assays can be performed, Where the haemophilus component is in a separate
container, the final bulk of the haemophilus component is
freeze-dried. Only a final lot that is satisfactory with respect
to the test for osmolality shown below and with respect to
each of the requirements given below under Identification,
Tests and Assay may be released for use.
vaccine, a batch of combined vaccine shown to be effective in Provided that the test for osmolality, the test for residual
clinical trials or a batch representative thereof is used as a pertussis toxin and irreversibility' of pertussis toxoid, the test
reference vaccine. For the preparation of a representative for antimicrobial preservative and the assays for the
batch, strict adherence to the production process used for the diphtheria, tetanus and pertussis components have been
batch tested in clinical trials is necessary'. The reference carried out with satisfactory results on the final bulk vaccine,
vaccine may be stabilised by a method that has been shown they may be omitted on the final lot.
to have no effect on the assay procedure. Provided the free formaldehyde content has been determined
PRODUCTION OF THE COMPONENTS on the bulk purified antigens and the purified monovalent
The production of the components complies with the harvests or the trivalent pool of polioviruses or the final bulk
requirements of the monographs Diphtheria vaccine (adsorbed) and it has been shown that the content in the final lot will
(0443), Tetanus vaccine (adsorbed) (0452), Pertussis vaccine not exceed 0.2 g/L, the test for free formaldehyde may be
(acellular, component, adsorbed) (1356), Hepatitis B vaccine omitted on the final lot.
(rDNA) (1056), Poliomyelitis vaccine (inactivated) (0214) and Provided that the test for bovine serum albumin has been
Haemophilus type b conjugate vaccine (1219). carried out with satisfactory results on the trivalent pool of
FINAL BULKS inactivated monovalent harvests of polioviruses or on the
final bulk vaccine, it may be omitted on the final lot.
Vaccine with all components in the same container The final bulk
is prepared by adsorption, separately or together, of suitable If an in vivo assay is used for the hepatitis B component,
quantities of bulk purified diphtheria toxoid, tetanus toxoid, provided it has been carried out with satisfactory results on
acellular pertussis components and hepatitis B surface the final bulk vaccine, it may be omitted on the final lot.
antigen onto a mineral carrier such as aluminium hydroxide Provided the in vivo assay for the poliomyelitis component
or hydrated aluminium phosphate and admixture of a has been carried out with satisfactory results on the final bulk
suitable quantity of PRP conjugate and suitable quantities of vaccine, it may be omitted on the final lot.
purified and inactivated, monovalent harvests of human The in vivo assay for the poliomyelitis component may be
poliovirus types 1, 2 and 3 or a suitable quantity of a omitted once it has been demonstrated for a given product
trivalent pool of such monovalent harvests. Suitable and for each poliovirus type that the acceptance criteria for
antimicrobial preservatives may be added. the D-antigen determination are such that it yields the same
Vaccine with the haemophilus component in a separate container result as the in vivo assay in terms of acceptance or rejection
The final bulk of diphtheria, tetanus, pertussis, hepatitis B of a batch. This demonstration must include testing of
and poliovirus component is prepared by adsorption, subpotent batches, produced experimentally if necessary', for
separately or together, of suitable quantities of bulk purified example by heat treatment or other means of diminishing the
diphtheria toxoid, tetanus toxoid, acellular pertussis immunogenic activity. Where there is a significant change in
components and hepatitis B surface antigen onto a mineral the manufacturing process of the antigens or their
carrier such as aluminium hydroxide or hydrated aluminium formulation, any impact on the in vivo and in vitro assays
phosphate and admixture of suitable quantities of purified must be evaluated, and the need for revalidation considered.
and inactivated, monovalent harvests of human poliovirus Free PRP
types 1, 2 and 3 or a suitable pool of such monovalent For vaccines with all components in the same container, the
harvests. This final bulk is filled separately. Suitable free PRP content is determined on the non-absorbed
antimicrobial preservatives may be added. The final bulk of fraction. Unbound PRP is determined on the haemophilus
the haemophilus component is prepared by dilution of the component after removal of the conjugate, for example by
bulk conjugate to the final concentration with a suitable anion-exchange, size-exclusion or hydrophobic
diluent. A stabiliser may be added. chromatography, ultrafiltration or other validated methods.
Only final bulks that comply wi± the following requirements The amount of free PRP is not greater than that approved
may be used in the preparation of the final lot. for the particular product.
Bovine serum albumin Bacterial endotoxins {2.6.14)
Determined on the poliomyelitis components by a suitable Less than the limit approved for the product concerned.
immunochemical method (2.7. /) after purification of the
Osmolality (2.2.55) Aluminium (2.5.13)

The osmolality of the vaccine, reconstituted where applicable, Maximum 1.25 mg per single human dose, if aluminium
IS within the limits approved for the particular preparation. hydroxide or hydrated aluminium phosphate is used as the
identification adsorbent.
If the vaccine is presented with the haemophilus component in a Free formaldehyde (2.4.18)
separate container: identification tests A, B, c, D and E are Maximum 0.2 g/L of free formaldehyde per single human
carried out using the container with the diphtheria, tetanus, dose.
pertussis} hepatitis B and poliomyelitis components; identification Antimicrobial preservative
tor F is carried out on the container with the haemophilus Where applicable, determine the amount of antimicrobial
components. preservative by a suitable chemical method. The content is
A. Diphtheria toxoid is identified by a suitable not less than the minimum amount shown to be effective and
immunochemical method (2.7.7). The following method is is not greater than 115 per cent of the quantity stated on the
given as an example. Dissolve in the vaccine to be examined label.
sufficient sodium citrate R to give a 100 g/L solution. Water (2.5.72)
Maintain at 37 °C for about 16 h and centrifuge until a clear Maximum 3.0 per cent for the freeze-dried haemophilus
supernatant is obtained. The clear supernatant reacts with a component.
suitable diphtheria antitoxin, giving a precipitate.
Sterility (2.6.1)
B. Tetanus toxoid is identified by a suitable immunochemical It complies with the test for sterility.
method (2.7.1'). The following method is given as an
example. The clear supernatant obtained during ASSAY
identification test A reacts with a suitable tetanus antitoxin, Diphtheria component
giving a precipitate. Carry out one of the prescribed methods for the assay of
c. The clear supernatant obtained during identification
test A reacts with specific antisera to the pertussis The lower confidence limit (P = 0.95) of the estimated
components of the vaccine when examined by suitable potency is not less than the minimum potency stated on the
immunochemical methods (2.7.7). label.
D. The hepatitis B component is identified by a suitable Unless otherwise justified and authorised, the minimum
immunochemical method (2.7.1), for example the in vitro potency stated on the label is 30 IU per single human dose.
assay, or by a suitable electrophoretic method (2.2.31). Tetanus component
E. The vaccine is shown to contain human poliovirus Carry out one of the prescribed methods for the assay of
types 1, 2 and 3 by a suitable immunochemical method tetanus vaccine (adsorbed) (2.7.8).
,
(2.7.7) such as determination of D-antigen by enzyme-linked The lower confidence limit (P = 0.95) of the estimated
immunosorbent assay (ELISA). potency is not less than 40 IU per single human dose.
F. The PRP and its carrier protein are identified by a suitable Pertussis component
immunochemical method (2.7.7). Carry out one of the prescribed methods for the assay of
TESTS pertussis vaccine (acellular) (2.7.16).
If the product is presented with the haeniophilus component in a The capacity of the vaccine to induce antibodies for each
separate container, the tests for residual pertussis toxin and included acellular pertussis antigen is not significantly
irreversibility of pertussis toxoid, free formaldehyde, aluminium, (P = 0.95) less than that of the reference vaccine.
antimicrobial preservative and sterility are carried out on the Hepatitis B component
container with the diphtheria, tetanus, pertussis, poliomyelitis and The vaccine complies with the assay of hepatitis B vaccine
hepatitis B components; the tests for PRP, water, antimicrobial (2.7.15).
preservative (where applicable), aluminium (where applicable) Poliomyelitis component
and sterility are carried out on the container with the haemophilus D-antigen content As a measure of consistency of production,
component. determine the D-antigen content for human poliovirus
Some tests for the haemophilus component are carried out on the types 1, 2 and 3 by a suitable immunochemical method
freeze-dried product rather than on the bulk conjugate where the (2.7.1) following desorption, using a reference preparation
freeze-drying process may affect the component to be tested. calibrated in European Pharmacopoeia Units of D-antigen.
Residual pertussis toxin and irreversibility of pertussis For each type, the content, expressed with reference to the
toxoid (2.6.33) amount of D-antigen stated on the label, is within the limits
The final lot complies with the test. approved for the particular product. Poliomyelitis vaccine
PRP
Minimum 80 per cent of the amount of PRP stated on the
The European Pharmacopoeia Unit and the International
label, for a vaccine with the haemophilus component in a
Unit are equivalent.
separate container.
In vivo test The vaccine complies with the in vivo assay of
For a vaccine with all components in the same container: the
poliomyelitis vaccine (inactivated) (2.7.20).
PRP content determined on the non-absorbed fraction is not
less than that approved for the product. labelling
PRP is determined either by assay of ribose (2.5.31) or The label states:
phosphorus (2.5.18), by an immunochemical method (2.7.7) — the minimum number of International Units of diphtheria
or by anion-exchange liquid chromatography (2.2.29) with and tetanus toxoid per single human dose;
pulsed-amperometric detection.
single human dose;
the amount of HBsAg per single human dose; During development studies and wherever revalidation is
the nominal amount of poliovirus of each type (1,2 and necessary, it shall be demonstrated by tests in animals that
3), expressed in European Pharmacopoeia Units of the vaccine induces a T-cell-dependent B-cell immune
D-antigen, per single human dose; response to PRP.
— the types of cells used for production of the poliomyelitis As part of consistency studies the assays of the diphtheria,
and the hepatitis B components; tetanus, pertussis and poliomyelitis components are carried
— the number of micrograms of PRP per single human out on a suitable number of batches of vaccine reconstituted
dose; as for use. For subsequent routine control, the assays of these
— the type and nominal amount of carrier protein per single components may be carried out without mixing with the
human dose; haemophilus component.
For the haemophilus component, the production method is
validated to demonstrate that the haemophilus component, if
tested, would comply with the test for pyrogens (2.6.<8),
— the name and the amount of the adsorbent;
carried out as follows: inject per kilogram of the rabbit's
mass a quantity of the vaccine equivalent to: 1 pg of PRP for
— that the vaccine is not to be frozen;
a vaccine with diphtheria toxoid or CRM 197 diphtheria
— where applicable, that the vaccine contains a pertussis
protein as carrier; 0.1 pg of PRP for a vaccine with tetanus
toxin-like protein produced by genetic modification.
toxoid as carrier; 0.025 pg of PRP for a vaccine with OMP
------------------------------------------------------ —-------------------- ---------------------- Ph Eur (meningococcal group B outer membrane protein complex)
as carrier.
Diphtheria, Tetanus, Pertussis / ** assays on the combined vaccine. If this is not possible
(Whole Cell), Poliomyelitis ***** because of interaction between the components of the
combined vaccine or because of the difference in composition
(Inactivated) and Haemophilus between monocomponent reference vaccine and the test
vaccine, a batch of combined vaccine shown to be effective in
Type b Conjugate Vaccine clinical trials or a batch representative thereof is used as a
(Adsorbed) reference vaccine. For the preparation of a representative
Diphtheria, Tetanus, Pertussis, Poliomyelitis batch, strict adherence to the production process used for the
(Inactivated) and Haemophilus Type b Conjugate batch tested in clinical trials is necessary. The reference
Vaccine (Adsorbed) vaccine may be stabilised by a method that has been shown
(Ph Eur mo'ทograph 2066) to have no effect on the assay procedure.
The label may state ‘DTwP/IPV/Hib’. Specific toxicity of the diphtheria and tetanus
components
Ph Elf_______________________________________
DEFINITION product, if tested, would comply with the following test:
Diphtheria, tetanus, pertussis (whole cell), poliomyelitis inject subcutaneously 5 times the single human dose stated
(inactivated) and haemophilus type b conjugate vaccine on the label into each of 5 healthy guinea-pigs, each weighing
(adsorbed) is a combined vaccine composed of: diphtheria 250-350 g, that have not previously been treated with any
formol toxoid; tetanus formol toxoid; an inactivated material that will interfere with the test. If within 42 days of
suspension of Bordetella pertussis’, suitable strains of human the injection any of the animals shows signs of or dies from
poliovirus types 1, 2 and 3 grown in suitable cell cultures and diphtheria toxaemia or tetanus, the vaccine does not comply
inactivated by a suitable method; polyribosylribitol phosphate with the test. If more titan 1 animal dies from non-specific
(PRP) covalently bound to a carrier protein; a mineral causes, repeat the test once; if more than 1 animal dies in the
adsorbent such as aluminium hydroxide or hydrated second test, the vaccine does not comply with the test.
aluminium phosphate. The product is presented with the PRODUCTION OF THE COMPONENTS
haemophilus component in a separate container, the contents The production of the components complies with the
of which are mixed with the other components immediately requirements of the monographs Diphtheria vaccine
before use. (adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), Pertussis
The formol toxoids are prepared from the toxins produced vaccine (whole cell, adsorbed) (0161), Poliomyelitis vaccine
by the growth of Corynebacterium diphtheriae and Clostridium (inactivated) (0214) and Haemophilus type b conjugate
tetani respectively. vaccine (1219).
PRP is a linear copolymer composed of repeated units of FINAL BULKS
3-0-D-ribofuranosyl-(l -» l)-ribitol-5-phosphate The final bulk of the diphtheria, tetanus, pertussis and
[(C1 oH19012P)n], with a defined molecular size and derived poliomyelitis components is prepared by adsorption,
from a suitable strain of Haemophilus influenzae type b. separately or together, of suitable quantities of bulk purified
The carrier protein, when conjugated to PRP, is capable of diphtheria toxoid, and bulk purified tetanus toxoid onto a
inducing a T-cell-dependent B-cell immune response to the mineral carrier such as aluminium hydroxide or hydrated
polysaccharide. aluminium phosphate and admixture of suitable quantities of
PRODUCTION an inactivated suspension of B. pertussis and of purified,
monovalent harvests of human poliovirus types 1, 2 and 3 or
GENERAL PROVISIONS
a suitable quantity of a trivalent pool of such monovalent
harvests. Suitable antimicrobial preservatives may be added.
2016
Vaccines IV-585
e final bulk of the haemophilus component is prepared by IDENTIFICATION

dilution of the bulk conjugate to the final concentration with
Identification tests A, B, c and D are carried out using the vial
a suitable diluent. A stabiliser may be added. containing the diphtheria, tetanus, pertussis and poliomyelitis
Only final bulks that comply with the following requirements components; identification test E is carried out on the vial
may be used in the preparation of the final lot. containing the haemophilus component
Bovine serum albumin K. Diphtheria toxoid is identified by a suitable
Determined on the poliomyelitis components by a suitable immunochemical method (2.7.7). The following method,
immunochemical method (2.7.7) during preparation of the applicable to certain vaccines, is given as an example.
final bulk vaccine, before addition of the adsorbent, the Dissolve in the vaccine to be examined sufficient sodium
amount of bovine serum albumin is such that the content in citrate R to give a 100 g/L solution. Maintain at 37 °C for
the final vaccine will be not more than 50 ng per single about 16 h and centrifuge until a clear supernatant is
human dose. obtained. lhe clear supernatant reacts with a suitable
Antimicrobial preservative diphtheria antitoxin, giving a precipitate.
Where applicable, determine the amount of antimicrobial B. Tetanus toxoid is identified by a suitable immunochemical
preservative by a suitable chemical method. The amount is method (2.7.7). The following method, applicable to certain
not less than 85 per cent and not greater than 115 per cent vaccines, is given as an example. The clear supernatant
of the intended content. obtained during identification test A reacts with a suitable
Sterility (2.6.7) tetanus antitoxin, giving a precipitate.
Carry out the test for sterility using 10 mL for each medium. c. The centrifugation residue obtained in identification A
FINAL LOT
may be used. Other suitable methods for separating the
bacteria from the adsorbent may also be used. Identify
The final bulk of the haemophilus component is freeze-dried.
pertussis vaccine by agglutination of the bacteria from the
Only a final lot that is satisfactory with respect to the test for resuspended precipitate by antisera specific to B. pertussis or
osmolality shown below and with respect to each of the by the assay of the pertussis component prescribed under
requirements given below under Identification, Tests and Assay.
Assay may be released for use.
D. The vaccine is shown to contain human poliovirus
Provided the tests for specific toxicity of the pertussis types 1, 2 and 3 by a suitable immunochemical method
component and antimicrobial preservative, and the assays for (2.7.7)
, such as determination of D-antigen by enzyme-linked
the diphtheria, tetanus and pertussis components have been immunosorbent assay (ELISA).
earned out with satisfactory results on the final bulk vaccine,
E. The haemophilus component is identified by a suitable
they may be omitted on the final lot.
immunochemical method (2.7.7) for PRP.
on the bulk purified antigens, the inactivated B. pertussis
TESTS
suspension and the purified monovalent harvests or the The tests for specific toxicity of the pertussis component,
trivalent pool of polioviruses or on the final bulk and it has aluminium, free formaldehyde, antimicrobial preservative and
been shown that the content in the final lot will not exceed sterility are carried out on the container with diphtheria, tetanus,
0.2 g/L, the test for free formaldehyde may be omitted on the pertussis and poliomyelitis components; the tests for PRP, water,
final lot. sterility and bacterial endotoxins are carried out on the container
with the haemophilus component.
Provided the in vivo assay for the poliomyelitis component
has been carried out with satisfactory results on the final bulk Some tests for the haemophilus component may be carried out on
vaccine, it may be omitted on the final lot. the freeze-dried product rather than on the bulk conjugate where
the freeze-drying process may affect the component to be tested.
The ไท vivo assay for the poliomyelitis component may be
omitted once it has been demonstrated for a given product Specific toxicity of the pertussis component
and for each poliovirus type that the acceptance criteria for Use not fewer than 5 healthy mice each weighing 14-16 g,
the D-antigen determination are such ±at it yields the same for the vaccine group and for the saline control. Use mice of
result as the in vivo assay in terms of acceptance or rejection the same sex or distribute males and females equally between
of a batch. This demonstration must include testing of the groups. Allow the animals access to food and water for at
subpotent batches, produced experimentally if necessary, for least 2 h before injection and during the test. Inject each
example by heat treatment or other means of diminishing the mouse of the vaccine group intraperitoneally with 0.5 mL,
immunogenic activity. Where there is a significant change in containing a quantity of the vaccine equivalent to not less
the manufacturing process of the antigens or their than half the single human dose. Inject each mouse of the
formulation, any impact on the in vivo and in vitro assays control group with 0.5 mL of a 9 g/L sterile solution of
must be evaluated, and the need for revalidation considered. sodium chloride R, preferably containing the same amount of
Osmolality (2.2.55) Weigh the groups of mice immediately before the injection
The osmolality of the vaccine, reconstituted where applicable, and 72 h and 7 days after the injection. The vaccine
is within the limits approved for the particular preparation. complies with the test if: (a) at the end of 72 h the total mass
Free PRP of the group of vaccinated mice is not less than that
Unbound PRP is determined on the haemophilus component preceding the injection; (b) at the end of 7 days the average
after removal of the conjugate, for example by anion- increase in mass per vaccinated mouse is not less than
exchange, size-exclusion or hydrophobic chromatography, 60 per cent of that per control mouse; and (c) not more than
ultrafiltration or other validated methods. The amount of free 5 per cent of the vaccinated mice die during the test.
PRP is not greater than that approved for the particular The test may be repeated and the results of the tests
product. combined.
rV-586 Vaccines 2016
PRP LABELLING
Minimum 80 per cent of the amount of PRP stated on the The label states:
label. PRP is determined either by assay of ribose (2.5. ร/) or — the minimum number of International Units of diphtheria
phosphorus (2.5.18) 3 by an immunochemical method (2.7.1) and tetanus toxoid per single human dose;
or by anion-exchange liquid chromatography (2.2.29) with — the minimum number of International Units of pertussis
pulsed-amperometric detection. vaccine per single human dose;
Aluminium (2.5.18) — the nominal amount of poliovirus of each type (1,2
Maximum 1.25 mg per single human dose, if aluminium and 3), expressed in European Pharmacopoeia Units of
hydroxide or hydrated aluminium phosphate is used as the D-antigen, per single human dose;
adsorbent — the type of cells used for production of the poliomyelitis
Free formaldehyde (2.4.18) component;
— the number of micrograms of PRP per single human
Maximum 0.2 g/L.
dose;
Antimicrobial preservative — the type and nominal amount of carrier protein per single
Where applicable, determine the amount of antimicrobial human dose;
preservative by a suitable chemical method. The content is — where applicable, that the vaccine is intended for primary
not less than the minimum amount shown to be effective and vaccination of children and is not necessarily suitable for
is not greater than 115 per cent of the quantity stated on the reinforcing doses or for administration to adults;
label. — the name and the amount of the adsorbent;
Water (2.5.12) — that the vaccine must be shaken before use;
Maximum 3.0 per cent for the haemophilus component. — that the vaccine is not to be frozen.
Sterility (2.6.1) ______________________________________________ ___ _____________ PhEir

The content is within the limits approved by the competent
authority for the haemophilus component of the particular
product. If any components of the vaccine prevent the Diphtheria, Tetanus, Pertussis * *
determination of endotoxin, a test for pyrogens is carried out (Whole Cell) and Poliomyelitis *****
as described under General provisions.
(Inactivated) Vaccine (Adsorbed)
ASSAY
Diphtheria, Tetanus, Pertussis and Poliomyelitis
(Inactivated) Vaccine (Adsorbed)
diphtheria vaccine (adsorbed) (2.7.6). (Ph Eur monograph 2061)
The lower confidence limit (P = 0.95) of the estimated The label may state ‘DTwP/IPV’.
potency is not less than 30 IU per single human dose. Ph Eur__ __ _ -
Tetanus component DEFINITION

Carry out one of the prescribed methods for the assay of Diphtheria, tetanus, pertussis (whole cell) and poliomyelitis
tetanus vaccine (adsorbed) (2.7.8). (inactivated) vaccine (adsorbed) is a combined vaccine
If the test is carried out in guinea-pigs, the lower confidence containing: diphtheria formol toxoid; tetanus formol toxoid;
limit (P = 0.95) of the estimated potency is not less than an inactivated suspension of BordeteUa pertussis') suitable
40 IU per single human dose; if ±e test is carried out in strains of human poliovirus types 1, 2 and 3 grown in
mice, the lower confidence limit (P = 0.95) of the estimated suitable cell cultures and inactivated by a validated method;
potency is not less than 60 IU per single human dose. a mineral adsorbent such as aluminium hydroxide or
hydrated aluminium phosphate.
Pertussis component
Carry out the assay of pertussis vaccine (whole cell) (2.7.7). The formol toxoids are prepared from the toxins produced
The estimated potency is not less than 4.0 ru per single
human dose and the lower confidence limit (P = 0.95) of the
estimated potency is not less than 2.0 IU per single human PRODUCTION
dose. GENERAL PROVISIONS
Poliomyelitis component The production method shall have been shown to yield
D-antigen content As a measure of consistency of production, consistently vaccines comparable with the vaccine of proven
determine the D-antigen content for human poliovirus clinical efficacy and safety in man.
types 1, 2 and 3 by a suitable immunochemical method Reference vaccine(s) Provided valid assays can be performed,
(2.7. /) following desorption using a reference preparation monocomponent reference vaccines may be used for the
calibrated in European Pharmacopoeia Units of D-antigen. assays on the combined vaccine. If this is not possible
For each type, the content, expressed with reference to the because of interaction between the components of the
amount of D-antigen stated on the label, is within the limits combined vaccine or because of the difference in composition
approved for the particular product. Poliomyelitis vaccine between monocomponent reference vaccine and the test
(inactivated) BRP is calibrated in European Pharmacopoeia vaccine, a batch of combined vaccine shown to be effective in
Units and intended for use in the assay of D-antigen. clinical trials or a batch representative thereof is used as a
The European Pharmacopoeia Unit and the International reference vaccine. For the preparation of a representative
Unit are equivalent. batch, strict adherence to the production process used for the
In vivo test The vaccine complies with the in vivo assay of batch tested in clinical trialร is necessary. The reference
poliomyelitis vaccine (inactivated) (2.7.20).
2016
Vaccines IV-587
vaccine may be stabilised by a method that has been shown Provided that the in vivo assay for the poliomyelitis
to have no effect on the assay procedure. component has been carried out with satisfactory results on
Specific toxicity of the diphtheria and tetanus the final bulk vaccine, it may be omitted on the final lot.
components The in vivo assay for the poliomyelitis component may be
The production method is validated to demonstrate that the omitted once it has been demonstrated for a given product
product, if tested, would comply with the following test: and for each poliovirus type that the acceptance criteria for
inject subcutaneously 5 times the single human dose stated the D-antigen determination are such that it yields the same
on the label into each of 5 healthy guinea-pigs, each weighing result as the in vivo assay in terms of acceptance or rejection
2 ว0-350 g, that have not previously been treated with any of a batch. This demonstration must include testing of
material that will interfere with the test. If within 42 days of subpotent batches, produced experimentally if necessary, for
the injection any of the animals shows signs of or dies from example by heat treatment or other means of diminishing the
diphtheria toxaemia or tetanus, the vaccine does not comply immunogenic activity. Where there is a significant change in
With the test. If more than 1 animal dies from non-specific the manufacturing process of the antigens or their
causes, repeat the test once; if more than 1 animal dies in the formulation, any impact on the in vivo and in vitro assays
second test, the vaccine does not comply with the test. must be evaluated, and the need for revalidation considered.
PRODUCTION OF THE COMPONENTS Osmolality (2.2.35)
The production of the components complies with the The osmolality of the vaccine is within the limits approved
requirements of the monographs Diphtheria vaccine for the particular preparation.
(adsorbed) (0443), Tetanus vaccine (adsorbed) (0452), Pertussis
IDENTIFICATION
vaccine (whole cell, adsorbed) (0161) and Poliomyelitis vaccine
(inactivated) (0214).
final bulk VACCINE applicable to certain vaccines, is given as an example.
The final bulk vaccine is prepared by adsorption onto a Dissolve in the vaccine to be examined sufficient sodium
mineral earner such as aluminium hydroxide or hydrated citrate R to give a 100 g/L solution. Maintain at 37 °C for
aluminium phosphate, separately or together, of suitable about 16 h and centrifuge until a clear supernatant is
quantities of bulk purified diphtheria toxoid and bulk purified obtained. The clear supernatant reacts with a suitable
tetanus toxoid and admixture of suitable quantities of an diphtheria antitoxin, giving a precipitate.
inactivated suspension of B. pertussis and purified monovalent B. Tetanus toxoid is identified by a suitable immunochemical
harvests of human poliovirus types 1, 2 and 3 or a suitable method (2.7.1). The following method, applicable to certain
quantity of a trivalent pool of such purified monovalent vaccines, is given as an example. The clear supernatant
harvests. Suitable antimicrobial preservatives may be added. obtained during identification test A reacts with a suitable
Only a final bulk vaccine that complies with the following tetanus antitoxin, giving a precipitate.
requirements may be used in the preparation of the final lot. c. The centrifugation residue obtained in identification A
Bovdne serum albumin may be used. Other suitable methods for separating the
Determined on the poliomyelitis components by a suitable bacteria from the adsorbent may also be used. Identify
immunochemical method (2.7.1') during preparation of the pertussis vaccine by agglutination of the bacteria from the
final bulk vaccine, before addition of the adsorbent, the resuspended precipitate by antisera specific to B. pertussis or
amount of bovine serum albumin is such that the content in by the assay of the pertussis component prescribed under
the final vaccine will be not more than 50 ng per single Assay.
human dose. D. The vaccine is shown to contain human poliovirus types
Antimicrobial preservative 1, 2 and 3 by a suitable immunochemical method (2.7.1)
Where applicable, determine the amount of antimicrobial such as the determination of D-antigen by enzyme-linked
preservative by a suitable chemical method. The amount is immunosorbent assay (ELISA).
not less than 85 per cent and not greater than 115 per cent TESTS
of the intended content. Specific toxicity of the pertussis component
Sterility (2.6.1) Use not fewer than 5 healthy mice each weighing 14-16 g for
Carry out the test for sterility using 10 mL for each medium. the vaccine group and for the saline control. Use mice of the
FINAL LOT same sex or distribute males and females equally between the
Only a final lot that is satisfactory with respect to the test for groups. Allow the animals access to food and water for at
osmolality and with respect to each of the requirements given least 2 h before injection and during the test. Inject each
below under Identification, Tests and Assay may be released mouse of the vaccine group intraperitoneally with 0.5 mL,
for use. containing a quantity of the vaccine equivalent to not less
than half the single human dose. Inject each mouse of the
Provided that the tests for specific toxicity of the pertussis
control group with 0.5 mL of a 9 g/L sterile solution of
component and antimicrobial preservative, and the assays for
sodium chloride R, preferably containing the same amount of
the diphtheria, tetanus and pertussis components have been
carried out with satisfactory results on the final bulk vaccine,
Weigh the groups of mice immediately before the injection
and 72 h and 7 days after the injection. The vaccine
Provided that the free formaldehyde content has been complies with the test if: (a) at the end of 72 h the total mass
determined on the bulk purified antigens, the inactivated of the group of vaccinated mice is not less than that
B. pertussis suspension and the purified monovalent harvests preceding the injection; (b) at the end of 7 days the average
or the trivalent pool of polioviruses or on the final bulk and it increase in mass per vaccinated mouse is not less than
has been shown that the content in the final lot will not 60 per cent of that per control mouse; and (c) not more than
exceed 0.2 g/L, the test for free formaldehyde may be 5 per cent of the vaccinated mice die during the test.
The test may be repeated and the results of the tests — where applicable, that the vaccine is intended for primary
combined. vaccination of children and is not necessarily suitable for
Aluminium (2.5.75) reinforcing doses or for administration to adults;
Maximum 1.25 mg per single human dose, if aluminium — the name and the amount of the adsorbent;
hydroxide or hydrated aluminium phosphate is used as the — that the vaccine must be shaken before use;
adsorbent. — that the vaccine is not to be frozen.
Free formaldehyde (2.4.18) _______________ _ PhE'j
Maximum 0.2 g/L.

not less than ±e minimum amount shown to be effective and
Haemophilus Type b Conjugate ** *,
is not greater than 115 per cent of the quantity stated on the Vaccine *****
label. (Ph. Eur. monograph 1219)
Sterility (2.6.7) The label may state lHib’.
It complies with the test for sterility. Ph Eur________________________________________ _____________ -________
ASSAY DEFINITION
Diphtheria component Haemophilus type b conjugate vaccine is a liquid or freeze-
Carty7 out one of the prescribed methods for the assay of dried preparation of a polysaccharide, derived from a suitable
diphtheria vaccine (adsorbed) (2.7.6). strain of Haemophilus influenzae type b, covalently bound to a
The lower confidence limit (P = 0.95) of the estimated carrier protein. The polysaccharide, polyribosylribitol
potency is not less than 30 IU per single human dose. phosphate, referred to as PRP, is a linear copolymer
Tetanus component composed of repeated units of 3-P-D-ribofuranosyl-(l ->1)-
Carty' out one of the prescribed methods for the assay of ribitol-5-phosphate [(CioH]<)Oi2P)n]3 with a defined
tetanus vaccine (adsorbed) (2.7.8). molecular size. The carrier protein, when conjugated to PRP,
If the test is carried out in guinea pigs, the lower confidence is capable of inducing a T-cell-dependent B-cell immune
limit (P = 0.95) of the estimated potency is not less than response to the polysaccharide.
40 IU per single human dose; if the test is carried out in PRODUCTION
mice, the lower confidence limit (p = 0.95) of the estimated GENERAL PROVISIONS
potency is not less than 60 IU per single human dose. The production method shall have been shown to yield
Pertussis component consistently haemophilus type b conjugate vaccines of
Carry out the assay of pertussis vaccine (whole cell) (2.7.7). adequate safety and immunogenicity' in man. The production
The estimated potency is not less than 4.0 IU per single of PRP and of the carrier protein arc based on seed-lot
human dose and the lower confidence limit (P = 0.95) of the systems.
estimated potency is not less than 2.0 IU per single human The production method is validated to demonstrate that the
dose. product, if tested, would comply with the test for abnormal
toxicity for immunosera and vaccines for human use (2.6.9)
Poliomyelitis component
and also with the test for pyrogens (2.6. (ร), carried out as
D-antigen content As a measure of consistency of production,
follows: inject per kilogram of the rabbit's mass a quantity of
determine the D-antigen content for human poliovirus
the vaccine equivalent to: 1 pg of PRP for a vaccine with
types 1, 2 and 3 by a suitable immunochemical method
diphtheria toxoid or CRM 197 diphtheria protein as carrier;
(2.7.7) following desorption, using a reference preparation
0.1 pg of PRP for a vaccine with tetanus toxoid as carrier;
calibrated in European Pharmacopoeia Units of D-antigen.
0.025 pg of PRP for a vaccine with OMP (meningococcal
For each type, the content, expressed with reference to the
group B outer membrane protein complex) as carrier.
approved for the particular product. Poliomyelitis vaccine During development studies and wherever revalidation of the
(inactivated) BRP is calibrated in European Pharmacopoeia manufacturing process is necessary, it shall be demonstrated
Units and intended for use in the assay of D-antigen. by tests in animals that the vaccine consistently induces a
The European Pharmacopoeia Unit and the International T-cell-dependent B-cell immune response.
Unit are equivalent. The stability of the final lot and relevant intermediates is
In vivo test The vaccine complies with the in vivo assay of evaluated using one or more indicator tests. Such tests may
poliomyelitis vaccine (inactivated) (2.7.20). include determination of molecular size, determination of free
PRP in the conjugate and the immunogenicity test in mice.
LABELLING Taking account of the results of the stability testing, release
The label states: requirements are set for these indicator tests to ensure that
— the minimum number of International Units of diphtheria the vaccine will be satisfactory at the end of the period of
and tetanus toxoid per single human dose; validity.
— the minimum number of International Units of pertussis
BACTERIAL SEED LOTS
vaccine per single human dose;
The seed lots of H. influenzae type b are shown to be free
— the nominal amount of poliovirus of each type (1, 2 and
from contamination by methods of suitable sensitivity. These
3), expressed in European Pharmacopoeia Units of
may include inoculation into suitable media, examination of
D-antigen, per single human dose;
colony morphology, microscopic examination of Gram-
— the type of cells used for production of the poliomyelitis stained smears and culture agglutination with suitable specific
component;
antisera.
No complex products of animal origin are included in the Residual reagents

medium used for preservation of strain viability, either for Where applicable, tests are carried out to determine residues
treeze-drying or for frozen storage. of reagents used during inactivation and purification.
It is recommended that PRP produced by the seed lot be An acceptable value for each reagent is established for the
characterised using nuclear magnetic resonance spectrometry particular product and each batch of PRP must be shown to
comply with this limit. Where validation studies have
H. INFLUENZAE TYPE B POLYSACCHARIDE (PRP) demonstrated removal of a residual reagent, the test on PRP
H influenzae Type b is grown in a liquid medium that does may be omitted.
not contain high-molecular-mass polysaccharides; if any CARRIER PROTEIN
ingredient of the medium contains blood-group substances, The production and characteristics of the carrier proteins are
the process shall be validated to demonstrate that after the described in general chapter 5.2.77. Carrier proteins for the
purification step they are no longer detectable. The bacterial production of conjugated polysaccharide vaccines for human use.
purity of the culture is verified by methods of suitable Only a carrier protein that complies with the requirements of
sensitivity. These may include inoculation into suitable this chapter may be used in the preparation of the conjugate.
media, examination of colony morphology, microscopic BULK CONJUGATE
examination of Gram-Stained smears and culture PRP is chemically modified to enable conjugation; it is
agglutination with suitable specific antisera. The culture may usually partly depolymerised either before or during this
be inactivated. PRP is separated from the culture medium procedure. Reactive functional groups or spacers may be
and purified by a suitable method. Volatile matter, including introduced into the appropriate carrier protein or PRP prior
water, in the purified polysaccharide is determined by a to conjugation. As a measure of consistency, the extent of
suitable method; the result is used to calculate the results of derivatisation is monitored. The conjugate is obtained by the
certain tests with reference to the dried substance, as covalent binding of PRP and the appropriate carrier protein.
prescribed below. Where applicable, unreacted but potentially reactogenic
Only PRP that complies with the following requirements may functional groups are made unreactive by means of capping
be used in the preparation of the conjugate. agents; the conjugate is purified to remove reagents.
Identification Only a bulk conjugate that complies with the following
PRP is identified by an immunochemical method (2.7.7) or requirements may be used in the preparation of the final bulk
other suitable method, for example 'll nuclear magnetic vaccine. For each test and for each particular product, limits
resonance spectrometry (2.235). of acceptance are established and each batch of conjugate
must be shown to comply with these limits. For a freeze-
Molecular-size distribution dried vaccine, some of the tests may be carried out on the
The percentage of PRP eluted before a given Kq value or final lot rather than on the bulk conjugate where the freeze-
within a range of Kq values is determined by size-exclusion drying process may affect the component being tested.
chromatography (2.2.50); an acceptable value is established
PRP
for the particular product and each batch of PRP must be
The PRP content is determined either by assay of ribose
shown to comply with this limit. Where applicable, the
(2.537) or phosphorus (2.5.18), by an immunochemical
molecular-size distribution is also determined after chemical
method (2.7.7) or by anion-exchange liquid chromatography
modification of the polysaccharide.
(2.2.29) with pulsed amperometric detection.
Liquid chromatography (2.2.29) with multiple-angle laser
light-scattering detection may also be used for determination
Protein
The protein content is determined by a suitable chemical
of molecular-size distribution.
method (for example, 2.5.16).
A validated determination of the degree of polymerisation or
of the weight-average molecular weight and the dispersion of PRP-to-protein ratio
molecular masses may be used instead of the determination Determine the ratio by calculation.
of molecular size distribution. Molecular-size distribution
Ribose (2.5.57) Molecular-size distribution is determined by size-exclusion
Within the limits approved by the competent authority for chromatography (2.2.30).
the particular product, calculated with reference to the dried Free PRP
substance. A number of methods have been used to separate free PRP
Phosphorus (2.5.18) from the conjugate, including precipitation, gel filtration,
Within the limits approved by the competent authority for size-exclusion, anion exchange and hydrophobic
chromatography, ultrafiltration and ultracentrifugation.
the particular product, calculated with reference to the dried
substance. The free PRP can then be quantified by a range of
techniques, including high-performance anion-exchange
Protein (2.5.76) chromatography with pulsed amperometric detection
Maximum 1.0 per cent, calculated with reference to the dried (HPAEC-PAD) and immunoassays with anti-PRP
substance. Use sufficient PRP to allow detection of proteins antibodies.
at concentrations of 1 per cent or greater.
Free carrier protein
Nucleic acid (2.5.17) Determine the content by a suitable method, either directly
Maximum 1.0 per cent, calculated with reference to the dried or by deriving the content by calculation from the results of
substance. other tests. The amount is within the limits approved for the
Bacterial endotoxins (2.6.14) particular product.
Less than 10 ru per microgram of PRP. 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 Water (2.5.12)
the subsequent manufacturing process (for example, owing to Maximum 3.0 per cent for freeze-dried vaccines.
short half-life).
Sterility (2.6.1)
Residual reagents It complies with the test for sterility.
Removal of residual reagents such as cyanide, EDAC
(ethyldimethylaminopropylcarbodiimide) and phenol is The content is within die limits approved by the competent
confirmed by suitable tests or by validation of the process. authority for the particular product. If any components of the
Sterility (2.6.1) vaccine prevent the determination of endotoxin, a test for
Carry out the test using for each medium 10 mL or the pyrogens is carried out as described under General
equivalent of 100 doses, whichever is less. provisions.
FINAL BULK VACCINE
LABELLING
An adjuvant, an antimicrobial preservative and a stabiliser
The label stares:
may be added to the bulk conjugate before dilution to the — the number of micrograms of PRP per single human
final concentration with a suitable diluent.
dose;
Only a final bulk vaccine that complies with the following — the type and nominal amount of carrier protein per single
requirements may be used in preparation of the final lot. human dose.
Antimicrobial preservative _______________ PhEtr
method. The content is not less than 85 per cent and not
Sterility (2.6. 7) Haemophilus Type b and Meningococcal
It complies with the test for sterility, carried out using 10 mL Group c Conjugate Vaccine
for each medium.
FINAL LOT
The label may state ‘Hib/MenC’.
Only a final lot that is satisfactory with respect to each of the DEFINITION
following requirements and the requirements given below Haemophilus Type b and Meningococcal Group c
under Identification and Tests may be released for use. Conjugate Vaccine is a combined vaccine. It is prepared from
Provided the test for antimicrobial preservative has been purified polysaccharides derived from a suitable strain of
carried out on the final bulk vaccine, it may be omitted on Neisseria meningitidis group c and from a suitable strain of
the final lot. Haemophilus influenzae, type b covalcndy bound to a earner
pH (2.2.5) protein.
The pH of the vaccine, reconstituted if necessary, is within The product is presented as a combined lyophilisate of the
the limits approved for the particular product. haemophilus type b and meningococcal group c components
Free PRP together with the solvent in a separate container. The final
A number of methods have been used to separate free PRP product is prepared by mixing the contents of the two
from the conjugate, including precipitation, gel filtration, containers immediately before use.
size-exclusion, anion exchange and hydrophobic PRODUCTION
chromatography, ultrafiltration and ultra centrifugation. GENERAL PROVISIONS
The free PRP can then be quantified by a range of The production method shall have been shown to yield
techniques, including HPAEC-PAD and immunoassays with consistently vaccines comparable with the vaccine of proven
anti-PRP antibodies. The amount of free PRP is not greater clinical efficacy and safety in man.
than that approved for the particular product. The production method is validated to demonstrate that the
IDENTIFICATION product, if tested, would comply with the test for abnormal
The vaccine is identified by a suitable immunochemical toxicity for immunosera and vaccines, Appendix XIV E.
method (2.7. I) for PRP. The stability of the final lot and relevant intermediates is
evaluated using one or more indicator tests. Such tests may
TESTS include determination of molecular size, determination of free
PRP saccharides in the conjugate or an immunogenicity test in
Minimum 80 per cent of the amount of PRP stated on the animals. Taking account of the results of the stability testing,
label. PRP is determined either by assay of ribose (2.5.57) or release requirements are set for these indicator tests to ensure
phosphorus (2.5.18), by an immunochemical method (2.7.7) that the vaccine will be satisfactory at the end of the period
or by anion-exchange liquid chromatography (2.2.29) with of validity.
pulsed amperometric detection.
During development studies and wherever revalidation of the
Aluminium (2.5.75) manufacturing process is necessary, it shall be demonstrated
Maximum 1.25 mg per single human dose, if aluminium by tests in animals that the vaccine consistently induces a
hydroxide or hydrated aluminium phosphate is used as the T-cell-dependent B-cell immune responses to PRP and to
adsorbent. meningococcal group c polysaccharides.
Antimicrobial preservative PRODUCTION OF THE COMPONENTS
Where applicable, determine the amount of antimicrobial The production of the vaccine components complies with the
preservative by a suitable chemical or physico-chemical requirements of the monographs for Haemophilus Type b
method. The content is not less than the minimum amount Conjugate Vaccine and Meningococcal Group c Conjugate
shown to be effective and not greater than 115 per cent of Vaccine.
the quantity stated on the label.
FINAL bulk vaccine Antimicrobial preservative

A final bulk may be prepared by mixing suitable quantities of Where applicable, determine the amount of antimicrobial
the haemophilus and meningococcal c bulk conjugates and preservative by a suitable chemical or physico-chemical
dilution to the final concentration with a suitable diluent. method. The content is not less than the minimum amount
Different methods of preparation may be used. A suitable shown to be effective and not greater than 115% of the
adjuvant, a suitable antimicrobial preservative and a stabiliser quantity stated on the label.
may be added to the components bulk conjugates.
Bacterial endotoxins
Only a final bulk vaccine that complies with the following Carry out the test for bacterial endotoxins, Appendix XVI c.
requirements may be used in the preparation of the final lot. Less than 25 IU per single human dose.
Antimicrobial preservative Sterility
Where applicable, determine the amount of antimicrobial Complies with the test for sterility, Appendix XVI A.
method. The content is not less than the minimum amount LABELLING
shown to be effective and not greater than 115% of the The label states per human dose: (1) the number of
intended amount. micrograms of PRP; (2) the number of micrograms of
meningococcal group c polysaccharide; (3) the type and
Sterility
nominal amount of carrier protein.
Carry out the test for sterility, Appendix XVI A, using 10 mL
for each medium.
FINAL LOT
requirements given below under Identification and Tests may
Inactivated Hepatitis A Vaccine * *
be released for use. Provided the test for antimicrobial (Hepatitis A Vaccine (Inactivated, Adsorbed), ***
preservative has been carried out on the final bulk vaccine, it Ph. Eur. monograph 1107)
may be omitted on the final lot. The label may state ‘HepA’
Acidity or alkalinity Ph Eur______________________________________________________________
pH of the reconstituted vaccine is within the range approved
by the competent authority for the particular product, DEFINITION
Appendix V L. Hepatitis A vaccine (inactivated, adsorbed) is a suspension
consisting of a suitable strain of hepatitis A virus grown in
Free PRP
cell cultures, inactivated by a validated method and adsorbed
Unbound PRP is determined after removal of the conjugate, on a mineral carrier.
for example by anion-exchange, size-exclusion or
hydrophobic chromatography, ultrafiltration or other PRODUCTION
validated methods. The amount of free PRP is not greater GENERAL PROVISIONS
than that approved by the competent authority for the Production of the vaccine is based on a virus seed-lot system
particular product. and a cell-bank system. The production method shall have
been shown to consistently yield vaccines that comply with
Free meningococcal c saccharide
the requirements for immunogenicity, safety and stability.
Unbound saccharide is determined after removal of the
conjugate, for example by anion-exchange liquid The production method is validated to demonstrate that the
chromatography, size-exclusion or hydrophobic product, if tested, would comply with the test for abnormal
chromatography, ultrafiltration or other validated methods. toxicity for immunosera and vaccines for human use (2.6.9).
The amount of free meningococcal c saccharide is not Unless otherwise justified and authorised, the virus in the
greater than that approved for the particular product. final vaccine shall not have undergone more passages from
The vaccine complies with the requirements stated under Vaccines the master seed lot than were used to prepare the vaccine
and with the following requirements. shown in clinical studies to be satisfactory with respect to
safety and efficacy.
IDENTIFICATION Reference preparation A part of a batch shown to be at least as
The haemophilus and menigococcal components are immunogenic in animals as a batch that, in clinical studies in
identified by a suitable immunochemical method, young healthy adults, produced not less than 95 per cent
Appendix xrv B. seroconversion, corresponding to a level of neutralising
TESTS antibody accepted to be protective, after a full-course primary
PRP immunisation is used as a reference preparation. An antibody
Not less than 80% of the amount of PRP stated on the label. level of 20 mIU/mL determined by enzyme-linked
PRP is determined either by assay of ribose or phosphorus, immunosorbent assay is recognised as being protective.
Appendix XV G, by an immunochemical method, SUBSTRATE FOR VIRUS PROPAGATION
Appendix XIV B, or by anion-exchange liquid The virus is propagated in a human diploid cell line (5.2.3)
chromatography, Appendix ITT D, with pulsed-amperometric or in a continuous cell line approved by the competent
detection. authority.
Meningococcal saccharide SEED LOTS
Not less than 80% of the amount of meningococcal group c The strain of hepatitis A virus used to prepare the master
polysaccharide stated on the label. The saccharide content is seed lot shall be identified by historical records that include
determined by a suitable validated assay, for example sialic information on the origin of the strain and its subsequent
acid assay, Appendix XV G or anion-exchange liquid manipulation.
chromatography, Appendix in D, with pulsed-amperometric Only a seed lot that complies with the following requirements
detection. may be used for virus propagation.
FV-592 Vaccines 2016
Identification Virus concentration

Each master and working seed lot is identified as hepatitis A The concentration of infectious virus in the purified harvest
virus using specific antibodies. is determined by a suitable cell culture method to monitor
Virus concentration production consistency and as a starting point for monitoring
The virus concentration of each master and working seed lot the inactivation curve.
is determined to monitor consistency of production. Antigenitotal protein ratio
Extraneous agents Determine the hepatitis A virus antigen content by a suitable
The working seed lot complies with the requirements for immunochemical method (2.7.1'). Determine the total protein
seed lots for virus vaccines (2.6.76). In addition, if primary by a validated method. The ratio of hepatitis A virus antigen
monkey cells have been used for isolation of the strain, content to total protein content is within the limits approved
measures are taken to ensure that the strain is not for the particular product.
contaminated with simian viruses such as simian Bovine serum albumin
immunodeficiency virus and filoviruses. Not more than 50 ng in the equivalent of a single
VIRUS PROPAGATION AND HARVEST
human dose, determined by a suitable immunochemical
All processing of the cell bank and subsequent cell cultures is method (2.7.7). Where appropriate in view of the
done under aseptic conditions in an area where no other cells manufacturing process, other suitable protein markers may
are being handled. Animal serum (but not human serum) be used to demonstrate effective purification.
may be used in the cell culture media. Serum and trypsin Residual host-cell DNA
used in the preparation of cell suspensions and media are If a continuous cell line is used for virus propagation, the
shown to be free from extraneous agents. The cell culture content of residual host-cell DNA, determined using a
media may contain a pH indicator, such as phenol red, and suitable method, is not greater than 100 pg in the equivalent
antibiotics at the lowest effective concentration. Not less than of a single human dose.
500 mL of the cell cultures employed for vaccine production Residual chemicals
is set aside as uninfected cell cultures (control cells). Multiple If chemical substances are used during the purification
harvests from the same production cell culture may be process, tests for these substances are carried out on the
pooled and considered as a single harvest. purified harvest (or on the inactivated harvest), unless
Only a single harvest that complies with the following validation of the process has demonstrated total clearance.
requirements may be used in the preparation of the vaccine. The concentration must not exceed the limits approved for
When the determination of the ratio of virus concentration to the particular product.
antigen content has been carried out on a suitable number of INACTIVATION AND INACTIVATED HARVEST
single harvests to demonstrate production consistency, it may Several purified harvests may be pooled before inactivation.
subsequently be omitted as a routine test. In order to avoid interference with the inactivation process,
Identification virus aggregation must be prevented or aggregates must be
The test for antigen content also serves to identify the single removed immediately before and/or during the inactivation
harvest. process. The virus suspension is inactivated by a validated
method; the method shall have been shown to be consistently
Bacterial and fungal contamination capable of inactivating hepatitis A virus without destroying
The single harvest complies with the test for sterility (2.6.7), the antigenic and immunogenic activity; for each inactivation
carried out using 10 mL for each medium. procedure, an inactivation curve is plotted representing
Mycoplasmas (2.6.7) residual live virus concentration measured at not fewer than
The single harvest complies with the test for mycoplasmas, 3 points in time (for example, on days 0, 1 and 2 of the
carried out using 1 mL for each medium. inactivation process). If formaldehyde is used for inactivation,
Control cells the presence of excess free formaldehyde is verified at the
The control cells of the production cell culture comply with a end of the inactivation process.
test for identification and the requirements for extraneous Only an inactivated harvest that complies with the following
agents (2.6.76). requirements may be used in the preparation of the final bulk
Antigen content vaccine.
Determine the hepatitis A antigen content by a suitable Inactivation
immunochemical method (2.7.7) to monitor production Carry out an amplification test for residual infectious
consistency; the content is within the limits approved for the hepatitis A virus by inoculating a quantity of the inactivated
particular product. harvest equivalent to 5 per cent of the batch or, if the harvest
contains the equivalent of 30 000 doses or more, not less
Ratio of virus concentration to antigen content
than 1500 doses of vaccine into cell cultures of the same type
The consistency of the ratio of the concentration of infectious
as those used for production of the vaccine; incubate for a
virus, determined by a suitable cell culture method, to
total of not less than 70 days making not fewer than one
antigen content is established by validation on a suitable
passage of cells within that period. At the end of the
number of single harvests.
incubation period, carry out a test of suitable sensitivity for
PURIFICATION AND PURIFIED HARVEST residual infectious virus. No evidence of hepatitis A virus
The harvest, which may be a pool of several single harvests, multiplication is found in the samples taken at the end of the
is purified by validated methods. If continuous cell lines are inactivation process. Use infectious virus inocula concurrently
used for production, the purification process shall have been as positive controls to demonstrate cellular susceptibility and
shown to reduce consistently the level of host-cell DNA. absence of interference.
Only a purified harvest that complies with the following Sterility (2.6.7)
requirements may be used in the preparation of the The inactivated viral harvest complies with the test for
inactivated harvest. sterility, carried out using 10 mL for each medium.

Less than 2 ru in the equivalent of a single human dose. Hepatitis A Vaccine (Inactivated, *****
Antigen content Virosome) *****
Determine the hepatitis A virus antigen content by a suitable (Ph. Eur. monograph 1935)
immunochemical method (2.7.1). The label may state ‘HepA’
Residual chemicals Ph Eur______________________________________________________________
See under Purification and purified harvest.
DEFINITION
FINAL bulk vaccine
Hepatitis A vaccine (inactivated, virosome) is a suspension of
The final bulk vaccine is prepared from one or more
a suitable strain of hepatitis A vims grown in cell cultures
inactivated harvests. Approved adjuvants, stabilisers and
and inactivated by a validated method. Virosomes composed
antimicrobial preservatives may be added.
of influenza proteins of a strain approved for the particular
Only a final bulk vaccine that complies with the following product and phospholipids are used as adjuvants.
PRODUCTION
Sterility (2.6./)
GENERAL PROVISIONS
The final bulk vaccine complies with the test for sterility,
earned out using 10 mL for each medium.
Antimicrobial preservative clinical efficacy and safety in man.
Where applicable, determine the amount of antimicrobial The production method is validated to demonstrate that the
preservative by a suitable chemical or physico-chemical product, if tested, would comply with the test for abnormal
method. The amount is not less than 85 per cent and not toxicity for immunosera and vaccines for human use (2.6.9).
Reference preparation A reference preparation of inactivated
FINAL LOT hepatitis A antigen is calibrated against a batch of hepatitis A
The final bulk vaccine is distributed aseptically into sterile vaccine (inactivated, virosome) that, in clinical studies in
containers. The containers arc then closed so as to avoid young healthy adultร, produced not less than 95 per cent
contamination. seroconversion, corresponding to a level of neutralising
Only a final lot that complies with each of the requirements antibody accepted to be protective, after a full-course primary
given below under Identification, Tests and Assay may be immunisation. An antibody level not less than 20 mIU/mL
released for use. Provided that the tests for free formaldehyde determined by enzyme-linked immunosorbent assay is
(where applicable) and antimicrobial preservative content recognised as being protective.
(where applicable) have been carried out on the final bulk PREPARATION OF HEPATITIS A ANTIGEN
vaccine with satisfactory results, these tests may be omitted Production of the hepatitis A antigen is based on a virus
on the final lot. If the assay is carried out using mice or other seed-lot system and a cell-bank system. The production
animals, then provided it has been carried out with method shall have been shown to consistently yield vaccines
satisfactory results on the final bulk vaccine, it may be that comply with the requirements for immunogenicity, safety
omitted on the final lot. and stability.
IDENTIFICATION Unless otherwise justified and authorised, the virus in the
The assay (2.7.14) serves also to identify the vaccine. final vaccine shall not have undergone more passages from
the master seed lot than were used to prepare the vaccine
TESTS
shown in clinical studies to be satisfactory with respect to
Aluminium (2.5.13)
hydroxide or hydrated aluminium phosphate is used as the SUBSTRATE FOR PROPAGATION OF HEPATITIS A VIRUS
adsorbent. The virus is propagated in a human diploid cell line (5.2.3).
Free formaldehyde (2.4.18) SEED LOTS OF HEPATITIS A VIRUS
^Maximum 0.2 g/L. The strain of hepatitis A virus used to prepare the master
seed lot shall be identified by historical records that include
Antimicrobial preservative information on the origin of the strain and its subsequent
manipulation.
method. The amount is not less than the minimum amount
shown to be effective and is not greater than 115 per cent of may be used for virus propagation.
that stated on the label. Identification
Each master and working seed lot is identified as hepatitis A
Sterility (2.6.1)
virus using specific antibodies.
The vaccine complies with the test for sterility.
Virus concentration
ASSAY The virus concentration of each master and working seed lot
The vaccine complies with the assay of hepatitis A vaccine is determined to monitor consistency of production.
.
(2.7.14)
Extraneous agents
LABELLING The working seed lot complies with the requirements for
The label states the biological origin of the cells used for the seed lots for virus vaccines (2.6.16).
preparation of the vaccine.
Ph Eur
PROPAGATION AND HARX'EST OF HEPATITIS A VIRUS Bovine serum albumin

All processing of the cell bank and subsequent cell cultures is Maximum 50 ng per single human dose if foetal bovine
done under aseptic conditions in an area where no other cells serum is used, determined by a suitable immunochemical
are handled. Animal serum (but not human serum) may be method (2.7./). Where appropriate in view of the
used in the cell culture media. Serum and trypsin used in the manufacturing process, other suitable protein markers may
preparation of cell suspensions and media are shown to be be used to demonstrate effective purification.
free from extraneous agents. The cell culture media may Residual chemicals
contain a pH indicator such as phenol red and antibiotics at If chemical substances are used during the purification
the lowest effective concentration. Not less than 500 mL of process, tests for these substances are earned out on the
the cell cultures employed for vaccine production is set aside purified harvest (or on the inactivated harvest), unless
as uninfected cell cultures (control cells). Multiple harvests validation of the process has demonstrated total clearance.
from the same production cell culture may be pooled and The concentration must not exceed the limits approved for
considered as a single harvest. the particular product.
Only a single harvest that complies with the following INACTIVATION AND INACTIVATED HARVEST OF
requirements may be used in the preparation of the vaccine. HEPATITIS A VIRUS
When the determination of the ratio of virus concentration to Several purified harvests may be pooled before inactivation.
antigen content has been carried out on a suitable number of In order to avoid interference with the inactivation process,
single harvests to demonstrate consistency, it may virus aggregation must be prevented or aggregates must be
subsequently be omitted as a routine test. removed immediately before and/or during the inactivation
Identification process. The virus suspension is inactivated by a validated
The test for antigen content also serves to identify the single method; the method shall have been shown to be consistently
harvest. capable of inactivating hepatitis A virus without destroying
Bacterial and fungal contamination the antigenic and immunogenic activity; for each inactivation
The single harvest complies with the test for sterility (2.6./), procedure, an inactivation curve is plotted representing
carried out using 10 mL for each medium. residual live virus concentration measured on at least
3 occasions (for example, on days 0, 1 and 2 of the
Mycoplasmas (2.6.7) inactivation process). If formaldehyde is used for inactivation,
The single harvest complies with the test for mycoplasmas.
the presence of excess free formaldehyde is verified at the
Control cells end of the inactivation process.
The control cells of the production cell culture comply with a Only an inactivated harvest that complies with the following
test for identity and the requirements for extraneous agents requirements may be used in the preparation of the final bulk
(2.6.16). vaccine.
Antigen content Inactivation
Determine the hepatitis A antigen content by a suitable Carry out an amplification test for residual infectious
immunochemical method (2.7./) to monitor production hepatitis A virus by inoculating a quantity of the inactivated
consistency; the content is within the limits approved for the harvest equivalent to 5 per cent of the batch or, if the harvest
particular product. contains the equivalent of 30 000 doses or more, not less
Ratio of virus concentration to antigen content than 1500 doses of vaccine into cell cultures of the same type
The consistency of the ratio of the concentration of infectious as those used for production of the vaccine; incubate for a
virus, as determined by a suitable cell culture method, to total of not less than 70 days making not fewer than 1
antigen content is established by validation on a suitable passage of cells within that period. At the end of the
number of single harvests. incubation period, carry out a test of suitable sensitivity for
PURIFICATION AND PURIFIED HARVEST OF HEPATITIS A residual infectious virus. No evidence of hepatitis A virus
VIRUS multiplication is found in the samples taken at the end of the
The harvest, which may be a pool of several single harvests, inactivation process. Use infective virus inocula concurrently
is purified by validated methods. If continuous cell lines are as positive controls to demonstrate cellular susceptibility and
used for production, the purification process shall have been absence of interference.
shown to reduce consistently the level of host-cell DNA. Sterility (2.6./)
Only a purified harvest that complies with the following The inactivated viral harvest complies with the test for
requirements may be used in the preparation of the sterility, carried out using 10 mL for each medium.
inactivated harvest. Bacterial endotoxins (2.6.14)
Virus concentration Less than 2 IU of endotoxin in the equivalent of a single
The concentration of infective virus in the purified harvest is human dose.
determined by a suitable cell culture method to monitor Antigen content
production consistency and as a starting point for monitoring Determine the hepatitis A virus antigen content by a suitable
the inactivation curve. immunochemical method (2.7.1).
Ratio of antigen to total protein Residual chemicals
Determine the hepatitis A virus antigen content by a suitable See under Purification and purified harvest.
immunochemical method (2.7./). Determine the total protein PREPARATION OF INACTIVATED INFLUENZA VIRUS
by a validated method. The ratio of hepatitis A virus antigen The production of influenza viruses is based on a seed-lot
content to total protein content is within the limits approved system. Working seed lots represent not more than
for the particular product. 15 passages from the approved reassorted virus or the
approved virus isolate. The final production represents 1
passage from the working seed lot. The strain of influenza and incubate at 33-37 °C for 3 days. The test is not valid
Virus to be used is approved by the competent authority. unless at least 8 of the 10 embryos survive. Harvest about
SUBSTRATE FOR PROPAGATION OF INFLUENZA VIRUS 0.1 mL of the allantoic fluid from each surviving embryo and
frifluenza virus seed to be used in the production of vaccine examine each individual harvest by a haemagglutination test.
is propagated in fertilised eggs from chicken flocks free from If haemagglutination is found for any of the fluids, carry out
specified pathogens (5.2.2) or in suitable cell cultures (5.2.4), for that fluid a further passage in eggs and test for
such as chick-embryo fibroblasts or chick kidney cells haemagglutination; no haemagglutination occurs.
obtained from chicken flocks free from specified pathogens Ovalbumin
(5.2.2). For production, the virus is grown in the allantoic Maximum 1 pg of ovalbumin in the equivalent of 1 human
cavity of fertilised hens' eggs from healthy flocks. dose, determined by a suitable technique using a suitable
SEED LOTS OF INFLUENZA VIRUS reference preparation of ovalbumin.
The haemagglutinin and neuraminidase antigens of each seed Antimicrobial preservative
lot are identified as originating from the correct strain of Where applicable, determine the amount of antimicrobial
influenza virus by suitable methods. preservative by a suitable chemical method. The content is
Only a working virus seed lot that complies with the not less than 85 per cent and not greater than 115 per cent
following requirements may be used in the preparation of the of the intended amount.
monovalent pooled harvest.
Residual chemicals
Bacterial and fungal contamination Tests are carried out on the monovalent pooled harvest for
Carry out the test for sterility (2.6.7), using 10 mL for each the chemicals used for inactivation, the limits being approved
medium. by the competent authority.
Mycoplasmas (2.6.7) PREPARATION OF VIROSOMES
Carry out the test for mycoplasmas, using 10 mL. Inactivated influenza virions are solubilised using a suitable
PROPAGATION AND HARVEST OF INFLUENZA VIRUS detergent and are purified by high-speed centrifugation in
An antimicrobial agent may be added to the inoculum. After order to obtain supernatants containing mainly influenza
incubation at a controlled temperature, the allantoic fluids antigens. After the addition of suitable phospholipids,
are harvested and combined to form the monovalent pooled virosomes are formed by removal of the detergent either by
harvest. An antimicrobial agent may be added at the time of adsorption chromatography or another suitable technique
harvest. At no stage in the production is penicillin or Only virosomes that comply with the following requirements
streptomycin used. may be used in the preparation of the final bulk vaccine.
POOLED HARVEST OF INFLUENZA VIRUS Haemagglutinin content
To limit the possibility of contamination, inactivation is Determine the content of haemagglutinin antigen by an
initiated as soon as possible after preparation. The virus is immunodiffusion test (2.7.7), by comparison with a
inactivated by a method that has been demonstrated on haemagglutinin antigen reference preparation or with an
3 consecutive batches to be consistently effective for the antigen preparation calibrated against it.
manufacturer. The inactivation process shall have been Phospholipids
shown to be capable of inactivating the influenza virus The content and identity of the phospholipids are determined
without destroying antigenicity of haemagglutinin. by suitable immunochemical or physico-chemical methods.
The inactivation process shall also have been shown to be
Ratio of phospholipid to haemagglutinin
capable of inactivating avian leucosis viruses and
The ratio of phospholipid content to haemagglutinin content
mycoplasmas. If the monovalent pooled harvest is stored
is within the limits approved for the particular product.
after inactivation, it is held at a temperature of 5 ± 3 °C.
If formaldehyde solution is used, the concentration does not Residual chemicals
exceed 0.2 g/L of CH2O at any time during inactivation; Tests are carried out for the chemicals used during the
if betapropiolactone is used, the concentration does not process. The concentration of each residual chemical is
exceed 0.1 per cent VIV at any time during inactivation. within the limits approved for the particular product.
Only a pooled harvest that complies with the following FINAL BULK VACCINE
requirements may be used in the preparation of the The bulk vaccine is prepared by adding virosomes to
virosomes. inactivated hepatitis A viruses to yield an approved
hepatitis A antigemhaemagglutinin ratio. Several bulks may
Haemagglutinin antigen
be pooled, and approved stabilisers and antimicrobial
Determine the content of haemagglutinin antigen by an
preservatives may be added.
immunodiffusion test (2.7.7), by comparison with a
haemagglutinin antigen reference preparation or with an Only a final bulk vaccine that complies with the following
antigen preparation calibrated against it. Carry out the test at requirements may be used in the preparation of the final lot.
20-25 °C Protein content
Sterility (2.6.7) The amount of protein is determined using a suitable
Carry out the test for sterility, using 10 mL for each technique, the limits being approved by the competent
medium. authorithy.
Viral inactivation Phospholipids

The content and identity of the phospholipids are determined
Inoculate 0.2 mL of the harvest into the allantoic cavity of
by suitable immunochemical or physico-chemical methods.
each of 10 fertilised eggs and incubate at 33-37 °C for
The amount of phospholipids complies with the limits
3 days. The test is not valid uni ess at least 8 of the
10 embryos survive. Harvest 0.5 mL of the allantoic fluid approved for the particular product.
from each surviving embryo and pool the fluids. Inoculate
0.2 mL of the pooled fluid into a further 10 fertilised eggs
Haemagglutinin content ASSAY

Determine the content of haemagglutinin antigen by an The vaccine complies with the assay of hepatitis A vaccine
immunodiffusion test (2.7.7). The amount of haemagglutinin (2.7.14, Method A).
must not exceed the limits approved for the particular
product. LABELLING
The label states:
Hepatitis A antigen content — the biological origin of the cells used for the preparation
Determine the hepatitis A antigen content by a suitable of the vaccine,
immunochemical method. The amount of antigen must not — that the carrier contains influenza proteins prepared in
exceed the limits approved for the particular product.
eggs,
Ratio of hepatitis A antigen to haemagglutinin — that the vaccine is not to be frozen,
The ratio of hepatitis A antigen content to haemagglutinin — that the vaccine is to be shaken before use.
content is within the limits approved for the particular ____________ PnEur
product.
Ovalbumin
Maximum 1 pg of ovalbumin per human dose, determined
by a suitable technique using a suitable reference preparation
of ovalbumin. Hepatitis A (Inactivated, Adsorbed) ** ***
Virosome size and Typhoid Polysaccharide *****
The size distribution of the virosome-hepatitis A virus
mixture is within the limits approved for the particular Vaccine
product. (Ph. Eur. monograph 2597)
Sterility (2.6.1) The label may state ‘HepA/Typhoid’
The final bulk vaccine complies with the test for sterility, Ph Eur------------------------------------------------------------------------ --- -------------------------------
carried out using 10 mL for each medium.
DEFINITION
Antimicrobial preservative Hepatitis A (inactivated, adsorbed) and typhoid
Where applicable, determine the amount of antimicrobial polysaccharide vaccine is a suspension consisting of a suitable
preservative by a suitable chemical or physico-chemical strain of hepatitis A virus, grown in cell cultures and
method. The amount is not less than 85 per cent and not inactivated by a validated method, and of purified Vi capsular
greater than 115 per cent of the intended amount. polysaccharide obtained from Salmonella typhi Ty 2 strain or
Residual chemicals some other suitable strain that has the capacity to produce
If chemical substances are used during the formulation Vi polysaccharide.
process, tests for these substances are carried out, the limits The hepatitis A antigen is adsorbed on a mineral carrier,
being approved by the competent authorithy. such as aluminium hydroxide, and the Vi capsular
FINAL LOT polysaccharide consists of partly 3-O-acetylated
The final bulk vaccine is distributed aseptically into sterile repeated units of 2-acetylamino-2-deoxy-D-
containers. The containers are then closed so as to avoid galactopyranuronic acid with a-(l->4) linkages.
contamination. The product is presented cither as a liquid mixture
Only a final lot that complies with each of the requirements containing the hepatitis A component and the typhoid
given below under Identification, Tests and Assay may be Vi polysaccharide component or as 2 separate liquids, one
released for use. Provided that ±e tests for free formaldehyde containing the hepatitis A component and the other the
(where applicable) and antimicrobial preservative content typhoid Vi polysaccharide component, which are mixed
(where applicable) have been carried out on the final bulk together immediately before use.
vaccine with satisfactory results, these tests may be omitted PRODUCTION
on the final lot. If the assay is carried out in vivo, provided it GENERAL PROVISIONS
has been carried out with satisfactory results on the final bulk The 2 components are prepared as described in the
vaccine, it may be omitted on the final lot. monographs Hepatitis A vaccine (inactivated, adsorbed) (1107)
IDENTIFICATION and Typhoid polysaccharide vaccine (1160) and comply with
The assay (2.7.14) serves also to identify the vaccine. the requirements prescribed therein.
TESTS The production method is validated to demonstrate that the
toxicity for immunosera and vaccines for human use (2.6.9).
Maximum 0.2 g/L.
Reference preparation The hepatitis A 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, produced not less than 95 per cent
method. The amount is not less than the minimum amount
seroconversion, corresponding to a level of neutralising
shown to be effective and is not greater than 115 per cent of
antibody accepted to be protective, after a full-course primary
that stated on the label.
immunisation. An antibody level not less than 20 mIU/mL
Sterility (2.6.1) determined by enzyme-linked immunosorbent assay is
The vaccine complies with the test for sterility. recognised as being protective.
Less than 2 IU of endotoxin per human dose.
FINAL bulks pH, free formaldehyde, osmolality and sterility are carried out
The hepatitis A final bulk is prepared from 1 or more immediately after mixing both components. If the vaccine is
inactivated harvests of hepatitis A virus. Approved adjuvants, presented as a liquid mixture, the test for O-acetyl groups is carried
stabilisers and antimicrobial preservatives may be added. out before the 2 components are mixed.
The Vi polysaccharide final bulk is prepared from 1 or more pH (2.2.3)
batches of purified Vi polysaccharide which are dissolved in a 6.8 to 7.8 for the hepatitis A component and 6.5 to 7.5 for
suitable solvent, which may contain an antimicrobial the typhoid Vi polysaccharide component; 6.6 to 7.6 for the
preservative, so that the volume corresponding to 1 dose vaccine presented as a liquid mixture or immediately after
contains 25 pg of polysaccharide and the solution is isotonic mixing both components if the vaccine is presented as
with blood (250-350 mosmol/kg). 2 separate liquids.
Where the vaccine is presented as a liquid mixture of both Aluminium (2.5.73)
components, the final bulk is prepared by addition of a Maximum 1.25 mg per single human dose, if aluminium
suitable quantity of the Vi capsular polysaccharide bulk to hydroxide is used as the adsorbent.
the hepatitis A bulk.
Only final bulks that comply with the following requirements Maximum 0.2 g/L.
may be used in the preparation of the final lot.
Antimicrobial preservative Where applicable, determine the amount of antimicrobial
Where applicable, determine the amount of antimicrobial preservative by a suitable chemical or physico-chemical
preservative by a suitable chemical or physico-chemical method. The amount is not less than the minimum amount
method. The amount is not less than 85 per cent and not shown to be effective and is not greater than 115 per cent of
greater than 115 per cent of the intended amount. the amount stated on the label.
Sterility (2.6.7) Sterility (2.6.7)
Carry out the test for sterility using 10 mL for each medium. The vaccine complies with the test for sterility.
FINAL LOT Osmolality (2.2.35)
The final bulks are distributed aseptically into sterile Where applicable, the osmolality of the vaccine is within the
containers. The containers are then closed so as to avoid limits approved for the particular product.
contamination.
Only a final lot that complies with each of the requirements The bacterial endotoxins content is less than 2 IU per
given below under Identification, Tests and Assay may be human dose for the hepatitis A component and within the
released for use. Provided that the tests for free formaldehyde limit approved for the typhoid Vi polysaccharide component.
(where applicable), antimicrobial preservative (where If the vaccine is presented as a liquid mixture of hepatitis A
applicable) and bacterial endotoxins have been carried out on component and typhoid Vi polysaccharide component the
the final bulks with satisfactory results, they may be omitted bacterial endotoxins content is within the limit approved for
on the final lot. If the assay of the hepatitis A component is the specific product.
carried out in vivo, then provided it has been carried out with
O-Acetyl groups (2.5.79)
satisfactory results on the final bulk containing the
0.085 pmol (±25 per cent) per dose (25 pg of
hepatitis A component, it may be omitted on the final lot.
polysaccharide).
CHARACTERS
ASSAY
If the vaccine is presented as 2 separate liquids test A is carried out
Hepatitis A component
using the hepatitis A component and test B is carried out using the
The vaccine complies with the assay of hepatitis A vaccine
typhoid Vi polysaccharide component. Test c is carried out if the
.
(2.7.14)
vaccine is presented as a liquid mixture of both components or
immediately after mixing both components if the vaccine is Typhoid Vi polysaccharide component
presented as 2 separate liquids. Determine Vi polysaccharide by a suitable immunochemical
method (2.7.7), using a reference purified polysaccharide.
A. Whitish, cloudy suspension.
The estimated amount of polysaccharide per dose is
B. Clear, colourless liquid, free from visible particles. 80 per cent to 120 per cent of the content stated on the
c. Turbid liquid with a slow settling white deposit. label. The confidence limits (P = 0.95) of the estimated
IDENTIFICATION amount of polysaccharide are not less than 80 per cent and
If the vaccine is presented as 2 separate liquids, identification not more than 120 per cent.
test A is carried out using the hepatitis A component and LABELLING
identification test B is carried out using the typhoid Vi The label states:
polysaccharide component. If the vaccine is presented as a liquid — the amount of hepatitis A virus antigen per human dose;
mixture, tests A and B are carried out. — the number of micrograms of polysaccharide per human
K. The assay (2.7. IT) serves also to identify the vaccine. dose (25 pg);
B. Typhoid Vi polysaccharide is identified by a suitable — the total quantity of polysaccharide in the container;
immunochemical method (2.7. 7) using specific antibodies. — the type of cells used for production of the vaccine;
— the name and amount of the adsorbent used;
TESTS — ±at the vaccine must be shaken before use;
If the vaccine is presented as 2 separate liquids, the tests for pH, — that the vaccine must not be frozen.
antimicrobial preservative and bacterial endotoxins are carried out _______________Phi
on both components; the test for aluminium is carried out using the
hepatitis A component and the test for O-acetyl groups is carried
out using the typhoid Vi polysaccharide component; the tests for
Hepatitis A (Inactivated) and ★* *★ Hepatitis B component

The assay (2.7.75) or, where applicable, the electrophoretic
Hepatitis B (rDNA) Vaccine ***** profile, serves also to identify the vaccine.
(Hepatitis A (Inactivated) and Hepatitis B (rDNA) TESTS
Vaccine (Adsorbed), Ph. Eur. monograph 1526) Aluminium (2.5.13)
The label may state ‘HepA/HepB’ Maximum 1.25 mg per single human dose, if aluminium
PhEir___________________________________________________________ hydroxide or hydrated aluminium phosphate is used as the
adsorbent.
DEFINITION
Hepatitis A (inactivated) and hepatitis B (rDNA) vaccine Free formaldehyde (2.4.18)
(adsorbed) is a suspension consisting of a suitable strain of Maximum 0.2 g/L.
hepatitis A virus, grown in cell cultures and inactivated by a Antimicrobial preservative
validated method, and of hepatitis B surface antigen Where applicable, determine the amount of antimicrobial
(HBsAg), a component protein of hepatitis B virus obtained preservative by a suitable chemical or physico-chemical
by recombinant DNA technology; the antigens are adsorbed method. The amount is not less than the minimum amount
on a mineral carrier, such as aluminium hydroxide or shown to be effective and is not greater than 115 per cent of
hydrated aluminium phosphate. that stated on the label.
PRODUCTION Sterility (2.6.1)
GENERAL PROVISIONS The vaccine complies with the test for sterility.
The two components are prepared as described in the Bacterial endotoxins (2.6.14)
monographs on Hepatitis A vaccine (inactivated, Less than 2 IU per human dose.
adsorbed) (1107) and Hepatitis B vaccine (rDNA) (1056) and
comply with the requirements prescribed therein. ASSAY
The production method is validated to demonstrate that the Hepatitis A component
The vaccine complies with the assay of hepatitis A vaccine
(2.7.14)
.
Reference preparation The reference preparation is part of a Hepatitis B component
representative batch shown to be at least as immunogenic in The vaccine complies with the assay of hepatitis B vaccine
animals as a batch that, in clinical studies in young healthy (rDNA) (2.7.15).
adults, produced not less than 95 per cent seroconversion, LABELLING
corresponding to a level of neutralising antibody recognised The label states:
to be protective, after a full-course primary' immunisation. — the amount of hepatitis A virus antigen and hepatitis B
For hepatitis A, an antibody level not less than 20 mIU/mL surface antigen per container,
determined by enzyme-linked immunosorbent assay is — the type of cells used for production of the vaccine,
recognised as being protective. For hepatitis B, an antibody — the name and amount of the adsorbent used,
level not less than 10 mIU/mL against HBsAg is recognised — that the vaccine must be shaken before use,
as being protective. — that the vaccine must not be frozen.
FINAL BULK VACCINE PfiEu
The final bulk vaccine is prepared from one or more
inactivated harvests of hepatitis A virus and one or more
batches of purified antigen.
requirements may be used in the preparation of the final lot. Hepatitis B Vaccine (rDNA)
preservative by a sdtable chemical or physico-chemical The label may state ‘HepB’.
method. The amount is not less than 85 per cent and not Ph Eur______________________________ ___ _____
DEFINITION
Sterility (2.6.1) Hepatitis B vaccine (rDNA) is a preparation of hepatitis B
The final bulk vaccine complies with the test for sterility, surface antigen (HBsAg), a component protein of hepatitis B
carried out using 10 mL for each medium. virus; the antigen may be adsorbed on a mineral earner such
FINAL LOT as aluminium hydroxide or hydrated aluminium phosphate.
Only a final lot that complies with each of the requirements The vaccine may also contain the adjuvant 3-O-desacyl-4'-
given below under Identification, Tests and Assay may be monophosphoryl lipid A. The antigen is obtained by
released for use. Provided that the tests for free formaldehyde recombinant DNA technology.
(where applicable) and antimicrobial preservative content
PRODUCTION
(where applicable) have been carried out on the final bulk
GENERAL PROVISIONS
vaccine with satisfactory results, they may be omitted on the
The vaccine shall have been shown to induce specific,
final lot. If the assay of the hepatitis A and/or the hepatitis B
protective antibodies in man. The production method shall
component is carried out in vivo, then provided it has been
have been shown to yield consistently vaccines that comply
with the requirements for immunogenicity and safety.
it may be omitted on the final lot.
IDENTIFICATION product, if tested, would comply with the test for abnormal
Hepatitis A component toxicity for immunosera and vaccines for human use (2.6.9).
The assay (2.7.14) serves also to identify the vaccine.
Hepatitis B vaccine (rDNA) is produced by the expression of suitable methods such as SDS-PAGE with staining by acid
the viral gene coding for HBsAg in yeast (Saccharomyces blue 92 and silver. A suitable method is sensitive enough to
cerevisiae) or mammalian cells (Chinese hamster ovary detect a potential contaminant at a concentration of
(CHO) cells or other suitable cell lines), purification of the 1 per cent of total protein. Not less than 95 per cent of the
resulting HBsAg and the rendering of this antigen into an total protein consists of hepatitis B surface antigen.
immunogenic preparation. The suitability and safety of the Composition
cells are approved by the competent authority.
The content of proteins, lipids, nucleic acids and
The vaccine may contain the product of the ร gene (major carbohydrates is determined.
protein), a combination of the ร gene and pre-S2 gene
products (middle protein) or a combination of the ร gene,
the pre-S2 gene and pre-Sl gene products (large protein). If mammalian cells are used for production, not more than
10 pg of DNA in the quantity of purified antigen equivalent
Reference preparation Part of a representative batch shown to
to a single human dose of vaccine.
be at least as immunogenic in animals as a batch that, in
clinical studies in young, healthy adults, produced not less Caesium
than 95 per cent seroconversion, corresponding to a level of If a caesium salt is used during production, a test for residual
HBsAg neutralising antibody recognised to be protective, caesium is carried out on the purified antigen. The content is
after a full-course primary immunisation. An antibody level within the limits approved for the specific product.
not less than 10 mIU/mL is recognised as being protective. Sterility (2.6.7)
CHARACTERISATION OF THE SUBSTANCE The purified antigen complies with the test, carried out using
Development studies are carried out to characterise the 10 mL for each medium.
antigen. The complete protein, lipid and carbohydrate Additional tests on the purified antigen may be required
structure of the antigen is established. The morphological depending on the production method used: for example, a
characteristics of the antigen particles are established by test for residual animal serum where mammalian cells are
electron microscopy. The mean buoyant density of the used for production or tests for residual chemicals used
antigen particles is determined by a physico-chemical during extraction and purification.
method, such as gradient centrifugation. The antigenic ADSORBED 3-O-DESACYL-4'-MONOPHOSPHORYL LIPID A
epitopes are characterised. The protein fraction of the antigen BULK
is characterised in terms of the primary structure (for If 3-O-desacyl-4'-monophosphoryl lipid A is included in the
example, by determination of the amino-acid composition, by vaccine it complies with the monograph 3-O-desacyl-4'-
partial amino-acid sequence analysis and by peptide monophosphoryl lipid A (2537). Where 3-O-desacyl-4'-
mapping). monophosphoryl lipid A liquid bulk is adsorbed prior to
CULTURE AND HARVEST inclusion in the vaccine, the adsorbed 3-O-desacyl-4'-
Identity, microbial purity, plasmid retention and consistency monophosphoryl lipid A bulk complies with the following
of yield are determined at suitable production stages. requirements.
If mammalian cells are used, tests for extraneous agents and
Degree of adsorption of 3-O-desacyl-4'-
mycoplasmas are performed in accordance with general
monophosphoryl lipid A
chapter 2.6.16. Tests for extraneous agents in viral vaccines for The content of non-adsorbed 3-O-desacyl-4'-
human use, but using 200 mL of harvest in the test in cell monophosphoryl lipid A in the adsorbed 3-O-desacyl-4'-
culture for other extraneous agents. monophosphoryl lipid A bulk is determined by a suitable
PURIFIED ANTIGEN method, for example gas chromatographic quantification of
Only a purified antigen that complies with the following the 3-O-desacyl-4'-monophosphoryl lipid A (2537) fatty
requirements may be used in the preparation of the final bulk acids in the supernatant, evaporated to dryness, after
vaccine. centrifugation.
Total protein pH (2.2.3)
The total protein is determined by a validated method. The pH is within the limits approved for the particular
The content is within the limits approved for the specific preparation.
product.
Sterility (2.6.7)
Antigen content and identification It complies with the test, carried out using 10 mL for each
The quantity and specificity of HBsAg is determined in medium.
comparison with the International Standard for HBsAg
subtype ad or an in-house reference, by a suitable FINAL BULK VACCINE
immunochemical method (2.7.7) such as radio-immunoassay An antimicrobial preservative, a mineral carrier, such as
(RIA), enzyme-linked immunosorbent assay (ELISA), aluminium hydroxide or hydrated aluminium phosphate, and
the adjuvant 3-O-desacyl-4'-monophosphoryl lipid A may be
immunoblot (preferably using a monoclonal antibody
directed against a protective epitope) or single radial included in the formulation of the final bulk.
diffusion. The antigen/protein ratio is within the limits Only a final bulk vaccine that complies with the following
approved for the specific product. requirements may be used in the preparation of the final lot.
The molecular weight of the major band revealed following Antimicrobial preservative
sodium dodecyl sulfate polyacrylamide gel electrophoresis Where applicable, determine the amount of antimicrobial
(SDS-PAGE) performed under reducing conditions preservative by a suitable chemical or physico-chemical
corresponds to the.value expected from the known nucleic method. The amount is not less than 85 per cent and not
acid and polypeptide sequences and possible glycosylation. greater than 115 per cent of the intended amount.
Antigenic purity Sterility (2.6.7)
The purity of the antigen is determined by comparison with The final bulk vaccine complies with the test, earned out
a reference preparation using liquid chromatography or other using 10 mL for each medium.
FINAL LOT
Only a final lot that complies with each of the requirements Inactivated Influenza Vaccine ** \
given below under Identification, Tests and Assay may be (Whole Virion) *****
released for use. Provided that the tests for free formaldehyde (Influenza Vaccine (Whole Virion, Inactivated),
(where applicable) and antimicrobid preservative content Ph Eur monograph 0159)
(where applicable) have been carried out on the final bulk
The label may state ‘Flu’.
vaccine Hath satisfactory results, they may be omitted on the
final lot. If the assay is earned out in vivo, then provided it When Inactivated Influenza Vaccine or Influenza Vaccine is
has been carried out with satisfactory’ results on the final bulk prescribed or demanded and the form is not stated,
vaccine, it may be omitted on the final lot. Inactivated Influenza Vaccine (Whole Virion), Inactivated
Influenza Vaccine (Split Virion) or Inactivated Influenza
Degree of adsorption
Vaccine (Surface Antigen) may be dispensed or supplied.
The degree of adsorption of the antigen and, where
Ph Eur_______________ ._________
applicable, 3-O-desacyl-4'-monophosphoryl lipid A is
assessed. DEFINITION
IDENTIFICATION Influenza vaccine (whole virion, inactivated) is a sterile,
The assay or, where applicable, the electrophoretic profile, aqueous suspension of a strain or strains of influenza virus,
serves also to identify’ the vaccine. In addition, where type A or B, or a mixture of strains of the 2 types grown
applicable, the test for 3-O-desacyl-4'-monophosphoryl lipid individually in fertilised hens' eggs and inactivated in such a
A content also serves to identify’ the 3-O-desacyl-4'- manner that their antigenic properties are retained.
monophosphoryl lipid A-containing vaccine. The stated amount of haemagglutinin antigen for each strain
present in the vaccine is 15 pg per dose, unless clinical
TESTS evidence supports the use of a different amount.
Aluminium (2.5.13)
The vaccine is a slightly opalescent liquid.
hydroxide or hydrated aluminium phosphate is used as the PRODUCTION
adsorbent. The production method is validated to demonstrate that the
3-0-Desacyl-4'-monophosphoryl lipid A product, if tested, would comply with the test for abnormal
toxicity for immunoscra and vaccines for human use (2.6.9).
Minimum 80 per cent and maximum 120 per cent of the
intended amount. CHOICE OF VACCINE STRAIN
Where applicable, determine the content of 3-O-desacyI-4'- The World Health Organization reviews the world
monophosphoryl lipid A by a suitable method, for example epidemiological situation annually and if necessary
gas chromatography (2.2.28). recommends the strains that correspond to this
epidemiological evidence.
Maximum 0.2 g/L. Such strains are used in accordance with the regulations in
force in the signatory States of the Convention on the
Antimicrobial preservative Elaboration of a European Pharmacopoeia. It is now
Where applicable, determine the content of antimicrobial common practice to use reassorted strains giving high yields
preservative by a suitable chemical or physico-chemical of the appropriate surface antigens. The origin and passage
method. The amount is not less than the minimum amount history of virus strains shall be approved by the competent
shown to be effective and is not greater than 115 per cent of authority.
that stated on the label.
SUBSTRATE FOR VIRUS PROPAGATION
Sterility (2.6.1) Influenza virus seed to be used in the production of vaccine
The vaccine complies with the test. is propagated in fertilised eggs from chicken flocks free from
Pyrogens (2.6.8) specified pathogens (SPF) (5.2.2) or in suitable cell cultures
The vaccine complies with the test for pyrogens. Inject the (5.2.4), such as chick-embryo fibroblasts or chick kidney cells
equivalent of one human dose into each rabbit or, if the obtained from SPF chicken flocks (5.2.2). For production,
vaccine contains 3-O-desacyl-4'-monophosphoryl lipid A, the virus of each strain is grown in the allantoic cavity of
inject per kilogram of the rabbit's mass an amount of the fertilised hens' eggs from healthy flocks.
vaccine containing 2.5 |ig of 3-O-desacyl-4'-monophosphoryl VIRUS SEED LOT
lipid A. The production of vaccine is based on a seed-lot system.
ASSAY Working seed lots represent not more than 15 passages from
The vaccine complies with the assay of hepatitis B vaccine the approved reasserted virus or the approved virus isolate.
(rDNA) (2.7.15). The final vaccine represents 1 passage from the working seed
lot. The haemagglutinin and neuraminidase antigens of each
LABELLING seed lot are identified as originating from the correct strain of
The label states: influenza virus by suitable methods.
— the amount of HBsAg per container;
Only a working virus seed lot that complies with the
— the type of cells used for production of the vaccine;
following requirements may be used in the preparation of the
— the name and amount of the adjuvant and/or adsorbent
used;
— that the vaccine must be shaken before use; Bacterial and fungal contamination
— that the vaccine must not be frozen. Carry out the test for sterility (2.6. /), using 10 mL for each
medium.
__ ______________________________________________ ___________ Ph Eur
Mycoplasmas (2.6.7)
Carry out the test for mycoplasmas, using 10 mL.
VIRUS PROPAGATION AND HARVEST Only a final lot that is satisfactory with respect to each of the
An antimicrobial agent may be added to the inoculum. After requirements given below under Tests and Assay may be
incubation at a controlled temperature, the allantoic fluids released for use. Provided that the test for residual infectious
ะ!re harvested and combined to form a monovalent pooled virus has been performed with satisfactory results on each
harvest. An antimicrobial agent may be added at the time of monovalent pooled harvest and that the tests for free
harvest. At no stage in the production is penicillin or formaldehyde, ovalbumin and total protein have been
streptomycin used. performed with satisfactory results on the final bulk vaccine,
MONOVALENT POOLED HARVEST they may be omitted on the final lot.
To limit the possibility of contamination, inactivation is IDENTIFICATION
initiated as soon as possible after preparation. The virus is The assay serves to confirm the antigenic specificity of the
inactivated by a method that has been demonstrated on vaccine.
3 consecutive batches to be consistently effective for the
manufacturer. The inactivation process shall have been TESTS
shown to be capable of inactivating the influenza virus Residual infectious virus
without destroying its antigenicity; the process should cause Inoculate 0.2 mL of the vaccine into the allantoic cavity of
minimum alteration of the haemagglutinin and each of 10 fertilised eggs and incubate at 33-37 °C for
neuraminidase antigens. The inactivation process shall also 3 days. The test is not valid unless at least 8 of the
have been shown to be capable of inactivating avian leucosis 10 embryos survive. Harvest 0.5 mL of the allantoic fluid
viruses and mycoplasmas. If the monovalent pooled harvest is from each surviving embryo and pool the fluids. Inoculate
stored after inactivation, it is held at 5 ± 3 °C. 0.2 mL of the pooled fluid into a further 10 fertilised eggs
If formaldehyde solution is used, the concentration docs not and incubate at 33-37 °C for 3 days. The test is not valid
exceed 0.2 g/L of CH2O at any time during inactivation; unless at least 8 of the 10 embryos survive. Harvest about
if betapropiolactone is used, the concentration does not 0.1 mL of the allantoic fluid from each surviving embryo and
exceed 0.1 per cent VIV at any time during inactivation. examine each individual harvest for live virus by a
haemagglutination test. If haemagglutination is found for any
Before or after the inactivation process, the monovalent
of the fluids, carry out for that fluid a further passage in eggs
pooled harvest is concentrated and purified by high-speed and test for haemagglutination; no haemagglutination occurs.
centrifugation or other suitable method.
Only a monovalent pooled harvest that complies with the
final bulk vaccine.
Haemagglutinin antigen is not greater than 115 per cent of the quantity stated on the
Determine the content of haemagglutinin antigen by an label.
immunodiffusion test (2.7.7), by comparison with a
haemagglutinin antigen reference preparation or with an
Maximum 0.2 g/L, where applicable.
antigen preparation calibrated against it(1). Carry out the test
at 20-25 °C. Ovalbumin
Not more than the quantity stated on the label and in any
Neuraminidase antigen
case not more than 1 pg per human dose, determined by a
The presence and type of neuraminidase antigen are
suitable immunochemical method (2.7.1) using a suitable
confirmed by suitable enzymatic or immunological methods
reference preparation of ovalbumin.
on the first 3 monovalent pooled harvests from each working
seed lot. Total protein
Not more than 6 times the total haemagglutinin content of
Sterility (2.6.7)
the vaccine as determined in the assay, but in any case, not
Carry out the test for sterility, using 10 mL for each
more than 100 pg of protein per virus strain per human dose
medium.
and not more than a total of 300 pg of protein per human
Residual infectious virus dose.
Carry out the test described below under Tests.
Sterility (2.6.7)
FINAL BULK VACCINE It complies with the test for sterility.
Appropriate quantities of the monovalent pooled harvests are
blended to make the final bulk vaccine.
Less than 100 IU per human dose.
requirements may be used in the preparation of the final lot. ASSAY
Antimicrobial preservative immunodiffusion test (2.7.1) 3 by comparison with a
Where applicable, determine the amount of antimicrobial haemagglutinin antigen reference preparation or with an
preservative by a suitable chemical method. The content is antigen preparation calibrated against it. Carry out the test at
not less than 85 per cent and not greater than 115 per cent 20-25 °C. The confidence limits (P = 0.95) are not less than
of the intended amount. 80 per cent and not more than 125 per cent of the estimated
Sterility (2.6.7) haemagglutinin antigen content. The lower confidence limit
Carry out the test for sterility using 10 mL for each medium. (P ะ= 0.95) is not less than 80 per cent of the amount stated
FINAL LOT on the label for each strain.
The final bulk vaccine is distributed aseptically into sterile, LABELLING
tamper-proof containers. The containers are closed so as to The label states:
prevent contamination. — that the vaccine has been prepared on eggs,
— the strain or strains of influenza virus used to prepare the SUBSTRATE FOR VIRUS PROPAGATION
vaccine, Influenza virus seed and all vaccine batches are propagated in
— the method of inactivation, fertilised eggs from chicken flocks free from specified
— the haemagglutinin content in micrograms per virus strain pathogens (SPF) (5.2.2).
per dose,
VIRUS SEED LOTS
— the maximum amount of ovalbumin,
The production of vaccine is based on a seed-lot system.
— the season during which the vaccine is intended to
The attenuated donor virus strains and the wild type virus
protect.
strains used for the production of the attenuated master seed
___________________________________________________________ Ph Eur lots are identified by historical records that include
1 Reference haemagglutinin antigens are available from the National information on their origins and the tests used in their
Institute for Biological Standards and Control, Blanche Lane, South characterisations.
Mimms, Potters Bar, Hertfordshire EN6 3QG, Great Britain.
Only an attenuated master donor virus strain that has been
demonstrated by a suitable method (e.g. multiplex PCR
assay) to be free from human respiratory pathogens which
are able to replicate in eggs could be used for the production
of attenuated master virus seed lots. This assay is omitted if
Influenza Vaccine (Live, Nasal) ; * reverse genetics method (e.g. plasmid rescue) is used.
(Ph Eur monograph 2772) * ** The production of the attenuated master virus seed lot has to
Ph Elf___________________________________________________________ be approved by the competent authority. The attenuated
master virus seed lot must have the same characteristics as
DEFINITION the attenuated donor virus strain. The number of passages
Influenza vaccine (live, nasal) is an aqueous suspension of a required to produce the attenuated master virus seed lot from
live attenuated strain or strains of influenza virus, type A the attenuated donor virus strain is limited and approved by
or B, or a mixture of strains of the 2 types grown individually the competent authority. Unless otherwise justified and
in fertilised hens' eggs. The vaccine is presented in a form authorised, the inoculum for infecting the eggs used in the
suitable for nasal administration. The vaccine is a colourless production of a vaccine lot shall be a virus harvest without
slightly opalescent liquid and may contain white particles. intermediate passage, so that no vaccine virus is more than 1
PRODUCTION passage from an attenuated master virus seed lot that has
GENERAL PROVISIONS passed all safety tests.
Production of the vaccine is based on a virus seed-lot system. Each virus seed lot used for propagation must have been
The production method shall have been shown to filtrated through a bacteria' retentive filter.
consistently yield influenza vaccine (live) that complies with The attenuated master virus seed lot has to express the
the requirements for immunogenicity, safety and stability. haemagglutinin and the neuraminidase from the wild type
CHOICE OF VACCINE STRAIN virus strain and other proteins from the attenuated donor
The World Health Organization reviews the world virus strain.
epidemiological situation annually and if necessary The attenuated master virus seed lot characterisation shall
recommends new strains corresponding to this include the following tests:
epidemiological evidence. — genotype analyses using validated nucleic acid
Such strains are used in accordance with the regulations in amplification techniques (2.6.21);
force in the signatory States of the Convention on the — virus sequencing of the seed lot and comparison of the
Elaboration of a European Pharmacopoeia. coding sequences as follows; the sequences of the
The attenuated donor virus strain and the attenuated vaccine haemagglutinin and neuraminidase genes with those of
virus strain may be generated by the manufacturer itself by the recommended strains and the sequences of the 6
classical reassortant methods or reverse genetics (e.g. plasmid remaining genes with those of the attenuated donor
rescue). The wild type virus strains used for the production strain.
of the attenuated vaccine virus seed lots must have been — genetic stability by sequencing, cold adapted and
approved by the competent authority. temperature sensitive phenotypes determination and
attenuation test upon several passages in the substrate.
The complete history of production of the attenuated vaccine
virus strain including description of the derivation of the Only an attenuated master virus seed lot that complies with
seeds from the attenuated donor virus strain(s) and the the following requirements may be used in the preparation of
WHO recommended wild virus strain(s) shall be approved by the harvest.
the competent authority. Identification
During development studies and whenever a new HA For each attenuated master virus seed lot the haemagglutinin
subtype of influenza A virus (i.e. non-Hl, non-H3 subtype) and the neuraminidase antigens are identified using suitable
or a new influenza B virus type differing from the currently methods.
circulating genetic lineages is included in the vaccine, the Cold adapted and temperature sensitive phenotype
neurovirulence of the master virus seed lots is assessed using For each attenuated master virus seed lot a test is carried out
suitable animal models (e.g. in mice) with the attenuated in cell cultures to demonstrate the cold adapted and
donor virus strain as a comparator. The new strain shall not temperature sensitive phenotypes of the seed lot.
be more neurovirulent than the comparator. The attenuated master virus seed lot complies with the test:
Genotypic and phenotypic characterisations of attenuated — For the cold adaptation if the loss of virus titre between
donor virus strain(ร) are undertaken using techniques for the incubation at 4- 25 °C and + 33 °C is not more than
identification of attenuation markers and nucleotide 2.0 log10 of infectious units as expressed in Fluorescent
sequences. Focus Unit (FFU).
For the temperature sensitivity if the loss of virus titre to determine the total viable aerobic count and to verify the
between the incubation at + 33 °C and 4 37 °C (for absence of yeast and mould using selective media. The total
strains B) or 39 °C (for strains A) is not less than viable aerobic count is within the limit approved by the
2.0 logio of infectious units as expressed in Fluorescent competent authority. Verification of absence of Vibrio,
Focus Unit (FFU). Shigella and Salmonella is carried out using supplementary
Attenuation specific validated techniques approved by the competent
For each attenuated master virus seed lot, an in vivo authority.
attenuation test is carried out on ferrets. The conditions of MONOVALENT BULK
the test such as inoculation dose and observation period are Monovalent bulks are prepared by pooling a number of
established in validation studies. The attenuation test is satisfactory single harvests or monovalent pooled harvests of
performed by intranasal inoculation of ferrets, free from the same virus type. The monovalent bulk is concentrated
antibodies against influenza virus, with the attenuated master and purified by high-speed centrifugation or other suitable
virus seed lot. The animals are monitored for a defined method then filtered through a bacteria retentive filter.
number of days post-inoculation for signs of influenza-like Only a monovalent bulk that complies with the following
illness, including nasal discharge, frequent sneezing, severe requirements may be used in the preparation of the final bulk
lethargy, or fever. vaccine.
At the conclusion of the monitoring period, animals are Identification
euthanized. Nasal turbinate and lung tissues are collected Each monovalent bulk is identified as influenza virus of the
and analysed for the presence of infectious virus using a given type using suitable haemagglutinin type specific assay.
suitable infectivity assay.
Virus concentration
For a master virus seed lot to be identified as attenuated, the
The virus concentration of each monovalent bulk is
virus must be detected in samples of nasal turbinate tissues
determined by titration using a suitable validated in vitro
and samples from lung tissues from individual animals, and
assay (e.g. fluorescent focus assay).
must demonstrate that the virus growth is restricted or shows
no virus replication. In addition, there are no signs of Cold adapted and temperature sensitive phenotype
influenza-like illness in the inoculated animals. Each monovalent bulk complies with the test as described
under Virus seed lots.
Virus concentration
The virus concentration of each attenuated master virus seed Attenuation test
lot is determined by titration in cell cultures using a suitable The attenuation test is performed by intranasal inoculation of
validated in vitro cell based assay (e.g. fluorescent focus ferrets, free from antibodies against influenza virus, with each
assay) to monitor the consistency of production. monovalent bulk test sample as described under Virus seed
lots.
Extraneous agents (2.6.76)
If sufficient consistency data are available, and approved by
Each attenuated master virus seed lot complies with the
the competent authority, only the first 3 monovalent bulks
requirements for virus seed lots.
following the introduction of a new attenuated master virus
Avian leucosis viruses (2.6.24) seed lot are tested on ferrets.
Each attenuated master virus seed lot complies with the test
Wherever possible in accordance with the provisions of the
for avian leucosis viruses.
European Convention for the Protection of Vertebrate
PROPAGATION AND HARVEST Animals used for Experimental and Other Scientific
All processing of the fertilised eggs is done under aseptic Purposes, manufacturers are encouraged to develop validated
conditions in an area where no other infectious agents or in vitro alternative methods to the animal test for monovalent
cells are handled at the same time. After inoculation and bulks using appropriate tools such as molecular methods or
incubation at a controlled temperature, only eggs containing other suitable methods for determination of viral attenuation
living and typical chick embryos are harvested. markers.
The percentage of rejected eggs is recorded. After
Genotyping
homogenisation and clarification by centrifugation, the
The genotype of each monovalent bulk is verified using
clarified allantoic fluid is tested as described below and kept
suitable validated nucleic acid amplification techniques
at -70 °C or colder until further processing. No human
(2.6.21).
protein is added to the virus suspension at any stage during
production. If stabilisers are added, they shall have been Bacterial and fungal contamination
shown to have no antigenic or sensitising properties for man. Each monovalent bulk complies with the test for sterility
(2.6.1), carried out using 10 mL of each medium.
Only a single harvest or a monovalent pooled harvest that
comply with the following requirements may be used in the Total protein content
preparation of the monovalent bulk. Maximum 0.25 mg per human dose before the addition of
Extraneous agents (2.6.16) any stabiliser.
Each single harvest or monovalent pooled harvests comply FINAL BULK VACCINE
with the tests for extraneous agents with the exception of the A final bulk vaccine is formulated aseptically from
tests for mycobacteria and sterility which are not required at appropriate quantities of the monovalent bulks of each virus
this stage of production. strain. The final bulk vaccine is distributed aseptically into
sterile, tamper-proof containers. Where a final bulk vaccine is
Avian leucosis viruses (2.6.24)
formulated as a release intermediate, it complies with the
Each single harvest or a monovalent pooled harvest comply
following requirements and is within the limits approved for
with the test for avian leucosis viruses.
the particular product. A suitable stabiliser may be added.
Microbiological contamination
The bioburden test using a membrane filtration is carried out
on each single harvest or on each monovalent pooled harvest
Bacterial and fungal contamination — for each virus strain, the virus concentration of the
The final bdk vaccine complies with the test for sterility reference preparation differs by more than 0.5 logio
(2.6./), earned out using 10 mL for each medium. infectious units as expressed in FFU from the established
FINAL LOT value.
An approved minimum virus concentration for release of the The assay is repeated if the confidence interval (P = 0.95) of
product is established for each virus strain to ensure, in light the combined virus concentration of the vaccine is greater
of stability data, that the minimum concentration stated on than ± 0.3 log10 infectious units as expressed in FFU; data
the label will be present at the end of the period of validity. obtained from valid assays only are combined by the usual
Only a final lot that is satisfactory with respect to each of the statistical methods (for example, 5.3) to calculate the virus
requirements given below under Identification, Tests and concentration of the sample. The confidence interval
Assay may be released for use. (P = 0.95) of the combined virus concentration is not greater
than ± 0.3 log10 infectious units as expressed in FFU.
Thermal stability
Maintain not fewer than 3 containers of the final lot at an LABELLING
elevated temperature for a defined period of time, using The label states:
conditions found suitable for the particular product as — that the vaccine has been prepared on eggs,
approved by the competent authority. Determine the virus — the strain or strains of influenza virus used in preparation
concentration as described under Assay in parallel for the of the vaccine,
heated vaccine and for vaccine maintained at the temperature — the minimum and maximum virus strain concentration
recommended for storage. For each virus strain, the virus per human dose,
concentration of the containers that have been heated does — the maximum amount of ovalbumin,
not decrease by more than an approved amount during ±e — the season during which the vaccine is intended to
period of exposure. protect.
_____________ Pt) Eur
IDENTIFICATION
The assay serves to confirm the antigenic specificity of the
vaccine.
TESTS
Ovalbumin Influenza Vaccine (Whole Virion, ** **
Not more than the quantity' stated on the label and in any
case not more than 1 pg per human dose, determined by a Inactivated, Prepared in Cell *****
suitable immunochemical method (2.7. /) using a suitable Cultures)
reference preparation of ovalbumin.
Total protein The label may state ‘Flu’ or ‘Flu(adj)’ as appropriate.
Not more than the quantity' stated on the label and in any
Ph Eur_______________________________________________ _________________
case not more than 2.2 mg per human dose.
Bacterial and fungal contamination DEFINITION
It complies with the test for sterility (2.6./). Influenza vaccine (whole virion, inactivated, prepared in cell
cultures) is a sterile, aqueous suspension of a strain or strains
of influenza virus, type A or B, or a mixture of strains of the
Less than 6 ru per single human dose.
2 types grown individually in cell cultures and inactivated in
ASSAY such a manner that their antigenic properties are retained.
Titrate the vaccine for infective virus in cell cultures using at The stated amount of haemagglutinin antigen for each strain
least 3 separate containers of vaccine and inoculating a present in the vaccine is 15 pg per dose, unless clinical
suitable number of wells for each dilution step. evidence supports the use of a different amount. The vaccine
Titrate 1 container of an appropriate virus reference is a slightly opalescent or opalescent liquid. The vaccine may
preparation in triplicate to validate each assay. The virus contain an adjuvant. This monograph applies to vaccines
concentration of the reference preparation is monitored using produced in diploid or continuous cell lines of mammalian
a control chart and a titre is established for each virus strain origin.
on a historical basis by each laboratory. If the vaccine PRODUCTION
contains more than one influenza virus strain, titrate each
GENERAL PROVISIONS
virus strain separately, using an appropriate type-specific
Production of the vaccine is based on a virus seed-lot system
antiserum.
and a cell-bank system. The production method shall have
Calculate the individual virus concentration for each been shown to yield consistently vaccines that comply with
container of vaccine and for each replicate of the reference the requirements for immunogenicity, safety and stability.
preparation as well as the corresponding combined virus
concentrations, using the usual statistical methods (for product, if tested, would comply with the test for abnormal
example, 5.3). For each virus strain, the combined virus
concentration for the 3 containers of vaccine is within the
The production method is validated to demonstrate suitable
range stated on the label.
reduction of residual host-cell protein. With the agreement of
The assay is not valid if:
the competent authority and for each specific product,
— for each virus strain, the confidence interval (P ะะะ 0.95) of routine testing for residual host-cell proteins may be omitted
the estimated virus concentration of the reference based on the results of validation studies for the product.
preparation for the 3 replicates combined is greater than Guidance on the principles of such validation studies is
± 0.3 log10 infectious units as expressed in FFU; given, for example, in the monograph Products of recombinant
DNA technology (0784), in particular in the sections
'Validation of the production process - Extraction and reviewed when new information becomes available on
purification and ‘Production consistency - Host-cell-derived potential viral contaminants, and the justification of the
proteins’. chosen PCR panel of extraneous agents tested for is provided
CHOICE OF VACCINE STRAIN to the competent authority within the annual update. This
The 7°rHealth Organization reviews the world update also includes vaccine strain-specific aspects such as
epidemiological situation annually and if necessary specific PCR inhibitory effects.
recommends new strains corresponding to this If an agent is detected in a virus seed and the mammalian
epidemiological evidence. cells used for production are shown to be susceptible to this
Such strains are used in accordance with the regulations in agent, the virus seed is not used for vaccine production.
force 1110 signatory states °fthc Convention on the If an agent is detected in a virus seed and the mammalian
Elaboration of a European Pharmacopoeia. It is now cells are not susceptible to the agent, validation of the
common practice to use reasserted strains giving high yields production process to demonstrate removal or inactivation of
of the appropriate surface antigens. The origin and passage the agent is carried out. If removal or inactivation cannot be
history of virus strains shall be approved by the competent demonstrated, the inactivated monovalent harvest is tested to
authority. demonstrate absence of any contaminant identified in the
SUBSTRATE FOR VIRUS PROPAGATION virus seed.
Influenza virus used in the preparation of seed lots is PROPAGATION AND SINGLE HARVEST
propagated in fertilised eggs from chicken flocks free from All processing of the cell bank and subsequent cell cultures is
specified pathogens (SPF) (5.2.2) or in suitable cell cultures done under aseptic conditions in an area where no other cells
(5.2.3) , such as chick-embryo fibroblasts, chick kidney cells are being handled at the same time. Approved animal serum
obtained from SPF chicken flocks (5.2.2), or a diploid or (but not human serum) may be used in the cell culture
continuous cell line. The final passage for establishment of media. Serum and trypsin used in the preparation of cell
the working seed lot is prepared in the cell line used for suspensions or media are shown to be free from extraneous
routine production. For this production, the virus of each agents. The cell culture media may contain a pH indicator,
strain is propagated in a diploid or continuous cell line such as phenol red, and antibiotics at the lowest effective
(5.2.3) . concentration. A sufficient quantity of the cell cultures
VIRUS SEED LOT employed for vaccine production are set aside as uninfected
The production of vaccine is based on a seed-lot system. cell cultures (control cells).
Each of the strains of influenza virus used shall be identified Only a single harvest that complies with the following
by historical records that include information on the origin of requirements may be used in the preparation of the vaccine.
the strain and its subsequent manipulation. Working seed Identification
lots represent not more than 15 passages from the approved The test for antigen content also serves to identify the single
reasserted virus or the approved virus isolate. The final harvest.
vaccine represents 1 passage from the working seed lot.
Only a seed lot that complies with tile following requirements Carry out the test for sterility (2.6.7), using 10 mL for each
may be used for virus propagation. medium.
Identification Mycoplasmas (2.6.7)
The haemagglutinin and neuraminidase antigens of each Carry out the test for mycoplasmas, using 10 mL for each
master and working seed lot are identified as originating from medium.
the correct strain of influenza virus by suitable methods.
Control cells
Virus concentration The control cells of the production cell culture comply with a
The virus concentration of each working seed lot is test for identification and the requirements for extraneous
determined. Where applicable, the virus concentration of agents (2.6.76).
each master seed lot is determined.
Extraneous agents (2.6.76) Determine the haemagglutinin antigen content by a suitable
The working seed lots comply with the requirements for seed immunochemical method (2.7.7).
lots. It is recognised that due to a seasonal change in one or
INACTIVATED AND PURIFIED MONOVALENT HARVEST
more of the influenza vaccine strains, timely testing of a virus
The harvest, which may be a pool of several single harvests
seed for extraneous agents according to general
of the same strain, is inactivated and purified by validated
chapter 2.6.16 may be problematic (e.g. duration of in vivo
methods. Before or after the inactivation process, the
tests, timely availability of specific neutralising antisera).
monovalent harvest is concentrated and purified by high
In agreement with the competent authority, and in light of a
speed centrifugation or another suitable method.
risk assessment, rapid assays (e.g. multiplex PCR) may be The influenza virus is inactivated by a method that has been
applied as alternatives to general chapter 2.6.16 following demonstrated on 3 consecutive batches to be consistently
validation. effective for the manufacturer. The inactivation process shall
Such risk assessment and validation includes more general have been shown to be capable of inactivating the influenza
considerations on potential contaminants of the virus isolates, virus without destroying its antigenicity; the process is
the susceptibility of the cell substrate to such viruses and the designed so as to cause minimum alteration of the
capacity of the production process for viral removal or haemagglutinin and neuraminidase antigens.
inactivation; validation includes also comparative data on If continuous cell lines are used for production, the
testing of seeds according to general chapter 2.6.16 and the purification process shall have been validated to reduce
proposed rapid assays. Each applied PCR/NAT test (2.6.21) consistently host-cell DNA to a suitable level.
must be shown to be suitable for its intended use by
appropriate analytical validation. The risk assessment is
Only an inactivated, purified monovalent harvest that TESTS

complies with the following requirements may be used in the Residual infectious virus
preparation of the final bulk vaccine. Carry out an amplification test for residual infectious
Haemagglutinin antigen influenza virus by inoculating not less than 4 mL of the
Determine the haemagglutinin antigen content by a suitable vaccine into cell cultures of the same type as used for
immunochemical method (2.7./). production of the vaccine; incubate for not less than 7 days
at 32 ± 2 °C. Inoculate not less than 10. mL of the cell
Antigen/total protein ratio
culture harvested medium into a new semi-confluent cell
Determine the haemagglutinin antigen content by a suitable
culture and incubate as before. At the end of the incubation
immunodiffusion test. Determine the total protein by a
period, examine for live virus by a haemagglutination test
validated method. The ratio of haemagglutinin antigen
If haemagglutination is found for any of the fluids, carry7 out
content to total protein content is within the limits approved
for the particular product. for that fluid a further passage on cell cultures and test for
haemagglutination; no haemagglutination occurs.
on the first 3 monovalent harvests from each working seed
lot.
Sterility' (2.6. /) label.
Cany7 out the test for sterility, using 10 mL for each
medium.
Maximum 0.2 g/L, where applicable.
Residual infectious virus
/Maximum 50 ng per human dose, determined by a suitable
FINAL BULK VACCINE immunochemical method (2.7.1).
Appropriate quantities of the inactivated, purified monovalent
Total protein
pooled harvests are blended to make the final bulk vaccine.
Not more than 6 times the total haemagglutinin content of
An adjuvant may be added.
the vaccine as determined in the assay, but in any case, not
Only a final bulk vaccine that complies with the following more than 100 pg of protein per virus strain per human dose.
Sterility (2.6.1)
Antimicrobial preservative It complies with die test for sterility7.
preservative by a suitable chemical method. The content is Bacterial endotoxins (2.6.14)
not less than 85 per cent and not greater than 115 per cent Less than 25 IU per human dose.
of the intended amount. ASSAY
Sterility (2.6./) Determine the content of haemagglutinin antigen by an
Carry out the test for sterility, using 10 mL for each immunodiffusion test (2.7.1), by comparison with a
medium. haemagglutinin antigen reference preparation1 or with an
antigen preparation calibrated against it. Carry out the test at
Residual host-cell DNA
20-25 °C. The confidence limits (P = 0.95) are not less than
If a continuous cell line is used for virus propagation, the
content of residual host-cell DNA, determined using a
content. The lower confidence limit (P = 0.95) is not less
suitable method, is not greater than 10 ng in ±e equivalent
than 80 per cent of the amount stated on the label for each
of a single human dose.
strain.
FINAL LOT
The final bulk vaccine is distributed aseptically into sterile, LABELLING
tamper-proof containers. The containers are closed so as to The label states:
prevent contamination. — the biological origin of the cells used for the preparation
of the vaccine;
— the strain or strains of influenza virus used to prepare the
requirements given below under Tests and Assay may be
vaccine;
released for use. Provided that the test for residual infectious
— the method of inactivation;
virus has been performed with satisfactory results on each
— the haemagglutinin antigen content in micrograms per
inactivated and purified monovalent harvest and that the tests
virus strain per dose;
for free formaldehyde, bovine serum albumin and total
protein have been performed with satisfactory results on the
protect;
final bulk vaccine, they may be omitted on the final lot. — where applicable, the name and the quantity of adjuvant
If the vaccine contains an adjuvant, suitable tests for identity used.
and other relevant quality criteria are carried out on the final ________________________________________________________ ■ PnEs
lot. These tests may include chemical and physical analysis,
1 Reference haemagglutinin antigens are available from the National
determination of particle size and determination of the Institute for Biological Standards and Control, Blanche Lane, South
number of particles per unit volume. Mimms, Potters Bar, Hertfordshire EN6 3QG, Great Britain.
IDENTIFICATION
The assay serves to confirm the antigenic specificity of the
vaccine.
Inactivated Influenza Vaccine ** * Mycoplasmas (2.6.7)

(Split Virion) **** VIRUS PROPAGATION AND HARVEST
(Influenza Vaccine (Spilt Virion, Inactivated), An antimicrobial agent may be added to the inoculum. After
Pn Eur monograph 0158) incubation at a controlled temperature, the allantoic fluids
The label may state ‘Flu’. are harvested and combined to form a monovalent pooled
When Inactivated Influenza Vaccine or Influenza Vaccine is harvest. An antimicrobial agent may be added at the time of
prescribed or demanded and the form is not stated, harvest. At no stage in the production is penicillin or
Inactivated Influenza Vaccine (Whole Virion), Inactivated streptomycin used.
Influenza Vaccine (Split Virion) or Inactivated Influenza MONOVALENT POOLED HARVEST
Vaccine (Surface Antigen) may be dispensed or supplied. To limit the possibility of contamination, inactivation is
initiated as soon as possible after preparation. The virus is
inactivated by a method that has been demonstrated on
definition 3 consecutive batches to be consistently effective for the
Influenza vaccine (split virion, inactivated) is a sterile, manufacturer. The inactivation process shall have been
aqueous suspension of a strain or strains of influenza virus, shown to be capable of inactivating the influenza virus
type A or B, or a mixture of strains of the 2 types grown without destroying its antigenicity; the process should cause
individually in fertilised hens7 eggs, inactivated and treated so minimum alteration of the haemagglutinin and
that the integrity of the virus particles has been disrupted neuraminidase antigens. The inactivation process shall also
without diminishing tile antigenic properties of the have been shown to be capable of inactivating avian leucosis
haemagglutinin and neuraminidase antigens. The stated viruses and mycoplasmas. If the monovalent pooled harvest is
amount of haemagglutinin antigen for each strain present in stored after inactivation, it is held at 5 ± 3 °C.
the vaccine is 15 pg per dose, unless clinical evidence If formaldehyde solution is used, the concentration does not
supports the use of a different amount. exceed 0.2 g/L of CH2O at any time during inactivation;
The vaccine is a slightly opalescent liquid. if betapropiolactone is used, the concentration does not
PRODUCTION exceed 0.1 per cent V/V at any time during inactivation.
The production method is validated to demonstrate that the Before or after the inactivation procedure, the monovalent
product, if tested, would comply with the test for abnormal pooled harvest is concentrated and purified by high-speed
toxicity for immunosera and vaccines for human use (2.6.9). centrifugation or other suitable method and the virus
particles are disrupted into component subunits by the use of
CHOICE OF VACCINE STRAIN
approved procedures. For each new strain, a validation test is
The World Health Organization reviews the world carried out to show that the monovalent bulk consists
epidemiological situation annually and if necessary predominantly of disrupted virus particles.
recommends the strains that correspond to this
epidemiological evidence. Only a monovalent pooled harvest that complies with the
Such strains are used in accordance with the regulations in final bulk vaccine.
force in the signatory States of the Convention on the
Elaboration of a European Pharmacopoeia. It is now Haemagglutinin antigen
common practice to use reasserted strains giving high yields Determine the content of haemagglutinin antigen by an
of the appropriate surface antigens. The origin and passage immunodiffusion test (2.7J), by comparison with a
history of virus strains shall be approved by the competent haemagglutinin antigen reference preparation or with an
authority. antigen preparation calibrated against itci). Carry out the test
at 20-25 °C.
For some vaccines, the physical form of the haemagglutinin
Influenza virus seed to be used in the production of vaccine
particles prevents quantitative determination by
is propagated in fertilised eggs from chicken flocks free from
immunodiffusion after inactivation of the virus. For these
specified pathogens (SPF) (5.2.2) or in suitable cell cultures
vaccines, a determination of haemagglutinin antigen is made
(5.2.4)
, such as chick-embryo fibroblasts or chick kidney cells
on the monovalent pooled harvest before inactivation.
obtained from SPF chicken flocks (5.2.2). For production,
The production process is validated to demonstrate suitable
the virus of each strain is grown in the allantoic cavity of
conservation of haemagglutinin antigen and a suitable tracer
fertilised hens' eggs from healthy flocks.
is used for formulation, for example, protein content.
VIRUS SEED LOT
Working seed lots represent not more than 15 passages from
the approved reassorted virus or the approved virus isolate.
The final vaccine represents 1 passage from the working seed
seed lot.
lot. The haemagglutinin and neuraminidase antigens of each
seed lot are identified as originating from the correct strain of Sterility (2.6. 1)
influenza virus by suitable methods. Carry out the test for sterility, using 10 mL for each
medium.
Only a working virus seed lot that complies with the
following requirements may be used in the preparation of the Residual infectious virus
monovalent pooled harvest. Carry out the test described below under Tests.
Bacterial and fungal contamination Chemicals used for disruption
Carry out the test for sterility (2.6.1)3 using 10 mL for each Tests are carried out on the monovalent pooled harvest for
medium. the chemicals used for disruption, the limits being approved
by the competent authority.
พ-608 Vaccines 2016
FINAL BULK VACCINE Sterility (2.6.1)

Appropriate quantities of the monovalent pooled harvests are It complies with the test for sterility.
blended to make the final bulk vaccine.
Only a final bulk vaccine that complies with the following Less than 100 IU per human dose.
ASSAY
Antimicrobial preservative Determine the content of haemagglutinin antigen by an
Where applicable, determine the amount of antimicrobial immunodiffusion test (2.7.1), by comparison with a
preservative by a suitable chemical method. The content is haemagglutinin antigen reference preparation or with an
not less than 85 per cent and not greater than 115 per cent antigen preparation calibrated against itr;>). Carry out the test
of the intended amount. at 20-25 °C. The confidence limits (P = 0.95) are not less
Sterility (2.6. /) than 80 per cent and not more than 125 per cent of the
Carry out the test for sterility, using 10 mL for each estimated haemagglutinin antigen content. The lower
medium. confidence limit (P = 0.95) is not less than 80 per cent of
FINAL LOT the amount stated on the label for each strain.
The final bulk vaccine is distributed aseptically into sterile, For some vaccines, quantitative determination of
tamper-proof containers. The containers are closed so as to haemagglutinin antigen with respect to available reference
prevent contamination. preparations is not possible. An immunological identification
Only a final lot that is satisfactory with respect to each of the of the haemagglutinin antigen and a semi-quantitativc
requirements given below under Tests and Assay may be determination are carried out instead by suitable methods.
released for use. Provided that the test for residual infectious LABELLING
virus has been performed with satisfactory results on each The label states:
monovalent pooled harvest and that the tests for free — that the vaccine has been prepared on eggs,
formaldehyde, ovalbumin and total protein have been — the strain or strains of influenza virus used to prepare the
performed with satisfactory results on the final bulk vaccine, vaccine,
they may be omitted on the final lot. — the method of inactivation,
IDENTIFICATION — the haemagglutinin content in micrograms per vims strain
The assay serves to confirm the antigenic specificity of the per dose,
vaccine. — the maximum amount of ovalbumin,
TESTS protect.
Inoculate 0.2 mL of the vaccine into ±e allantoic cavity of
1 Reference haemagglutinin antigens are available from the National
Institute for Biological Standards and Control, Blanche Lane, South
3 days. The test is not valid unless at least 8 of the Mimms, Potters Bar, Hertfordshire EN6 3QG, Great Britain.
10 embryos survive. Harvest 0.5 mL of the allantoic fluid
from each surviving embryo and pool the fluids. Inoculate
0.2 mL of the pooled fluid into a further 10 fertilised eggs
and incubate at 33-37 °C for 3 days. The test is not valid
unless at least 8 of the 10 embryos survive. Harvest about Inactivated Influenza Vaccine * โ
0.1 mL of the allantoic fluid from each surviving embryo and
examine each individual harvest for live virus by a (Surface Antigen) *****
haemagglutination test. If haemagglutination is found for any (Influenza Vaccine (Surface Antigen, Inactivated),
of the fluids, carry out for that fluid a further passage in eggs Ph Eur monograph 0869)
and test for haemagglutination; no haemagglutination occurs. The label may state ‘Flu’ or ‘Flu(adj)’ as appropriate.
Antimicrobial preservative When Inactivated Influenza Vaccine or Influenza Vaccine is
Where applicable, determine the amount of antimicrobial prescribed or demanded and the form is not stated,
preservative by a suitable chemical method. The content is Inactivated Influenza Vaccine (Whole Virion), Inactivated
not less than the minimum amount shown to be effective and Influenza Vaccine (Split Virion) or Inactivated Influenza
is not greater than 115 per cent of the quantity stated on the Vaccine (Surface Antigen) may be dispensed or supplied.
label.
Ph Eur_______________________________________ _________________ ______
Maximum 0.2 g/L, where applicable. DEFINITION
Influenza vaccine (surface antigen, inactivated) is a sterile
Ovalbumin suspension of a strain or strains of influenza virus, type A
Not more than the quantity stated on the label and in any or B, or a mixture of strains of the 2 types grown individually
case not more than 1 pg per human dose, determined by a in fertilised hens' eggs, inactivated and treated so that the
suitable immunochemical method (2.7.1) using a suitable preparation consists predominantly of haemagglutinin and
reference preparation of ovalbumin. neuraminidase antigens, without diminishing the antigenic
Total protein properties of these antigens. The stated amount of
Not more than 6 times the total haemagglutinin content of haemagglutinin antigen for each strain present in the vaccine
the vaccine as determined in the assay, but in any case, not is 15 pg per dose, unless clinical evidence supports the use of
more than 100 pg of protein per virus strain per human dose a different amount. The vaccine may contain an adjuvant.
and not more than a total of 300 pg of protein per human
dose.
production Before or after the inactivation process, the monovalent

The production method is validated to demonstrate that the pooled harvest is concentrated and purified by high-speed
product, if tested, would comply with the test for abnormal centrifugation or other suitable method. Virus particles are
toxicity for immunosera and vaccines for human use (2.6.9). disrupted into component subunits by approved procedures
CHOICE OF VACCINE STRAIN and further purified so that the monovalent bulk consists
The World Health Organization reviews the world mainly of haemagglutinin and neuraminidase antigens.
epidemiological situation annually and if necessary Only a monovalent pooled harvest that complies with the
recommends the strains that correspond to this following requirements may be used in the preparation of the
epidemiological evidence. final bulk vaccine.
Such strains are used in accordance with the regulations in Haemagglutinin antigen
force in the signatory states of the Convention on the Determine the content of haemagglutinin antigen by an
Elaboration of a European Pharmacopoeia. It is now immunodiffusion test (2.7./), by comparison with a
common practice to use reassorted strains giving high yields haemagglutinin antigen reference preparation or with an
of the appropriate surface antigens. The origin and passage antigen preparation calibrated against it. Carry out the test at
history of virus strains shall be approved by the competent 20-25 °C.
authority.
SUBSTRATE FOR VIRUS PROPAGATION The presence and type of neuraminidase antigen are
Influenza virus seed to be used in the production of vaccine confirmed by suitable enzymatic or immunological methods
is propagated in fertilised eggs from chicken flocks free from on the first 3 monovalent pooled harvests from each working
specified pathogens (SPF) (5.2.2) or in suitable cell cultures seed lot.
(5.2.4)
, such as chick-embryo fibroblasts or chick kidney cells Sterility (2.6. /)
obtained from SPF chicken flocks (5.2.2). For production, Carry out the test for sterility, using 10 mL for each
the virus of each strain is grown in the allantoic cavity of medium.
fertilised hens' eggs from healthy flocks.
VIRUS SEED LOT
Working seed lots represent not more than 15 passages from Purity
The purity of the monovalent pooled harvest is examined by ■
the approved reassorted virus or the approved virus isolate.
polyacrylamide gel electrophoresis or by other approved
The final vaccine represents one passage from the working
techniques. Mainly haemagglutinin and neuraminidase
seed lot. The haemagglutinin and neuraminidase antigens of
antigens shall be present.
each seed lot are identified as originating from the correct
strain of influenza virus by suitable methods. Chemicals used for disruption and purification
Only a working virus seed lot that complies with the Tests are carried out on the monovalent pooled harvest for
following requirements may be used in the preparation of the the chemicals used for disruption and purification, the limits
monovalent pooled harvest. being approved by the competent authority.
FINAL BULK VACCINE
Carry out the test for sterility (2.6./), using 10 mL for each Appropriate quantities of the monovalent pooled harvests are
medium. blended to make the final bulk vaccine. An adjuvant may be
added.
Mycoplasmas (2.6.7)
VIRUS PROPAGATION AND H.ARVEST
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 is penicillin or
streptomycin used. Sterility (2.6. /)
Carry out the test for sterility, using 10 mL for each
MONOVALENT POOLED HARVEST
medium.
To limit the possibility of contamination, inactivation is
initiated as soon as possible after preparation. The virus is FINAL LOT
inactivated by a method that has been demonstrated on The final bulk vaccine is distributed aseptically into sterile,
3 consecutive batches to be consistently effective for the tamper-proof containers. The containers are closed so as to
manufacturer. The inactivation process shall have been prevent contamination.
shown to be capable of inactivating the influenza virus Only a final lot that is satisfactory with respect to each of the
without destroying its antigenicity; the process should cause requirements given below under Tests and Assay may be
minimum alteration of the haemagglutinin and released for use. Provided that the test for residual infectious
neuraminidase antigens. The inactivation process shall also virus has been performed with satisfactory results on each
have been shown to be capable of inactivating avian leucosis monovalent pooled harvest and that the tests for free
viruses and mycoplasmas. If the monovalent pooled harvest is formaldehyde, ovalbumin and total protein have been
stored after inactivation, it is held at 5 ± 3 °C. performed with satisfactory results on the final bulk vaccine,
If formaldehyde solution is used, the concentration does not they may be omitted on the final lot.
exceed 0.2 g/L of CH2O at any time during inactivation; If the ovalbumin and formaldehyde content cannot be
if betapropiolactone is used, the concentration does not determined on the final lot, owing to interference from the
exceed 0.1 per cent VIV at any time during inactivation. adjuvant, they are determined on the monovalent pooled
harvest, the acceptance limits being set to ensure that the — the haemagglutinin content in micrograms per virus strain
limits for the final product wall not be exceeded. per dose,
If the vaccine contains an adjuvant, suitable tests for identity — the season during which the vaccine is intended to
and other relevant quality criteria are carried out on the final protect,
lot. These tests may include chemical and physical analysis, — the maximum amount of ovalbumin,
determination of particle size and determination of the — where applicable, the name and the quantity of adjuvant
number of particles per unit volume. used.
IDENTIFICATION ______________________________________________________________ PhE'j
The assay serves to confirm the antigenic specificity of the 1 Reference haemagglutinin antigens are available from the National
vaccine. Institute for Biological Standards and Control, Blanche Lane, South
Mimms, Potters Bar, Hertfordshire EN6 3QG, Great Britain.
TESTS
Inoculate 0.2 mL of the vaccine into the allantoic cavity of
3 days. The test is not valid unless at least 8 of the Influenza Vaccine (Surface ** *
10 embryos survive. Harvest 0.5 mL of the allantoic fluid
from each surviving embryo and pool the fluids. Inoculate Antigen, Inactivated, Prepared in *****
0.2 mL of the pooled fluid into a further 10 fertilised eggs Cell Cultures)
and incubate at 33-37 °C for 3 days. The test is not valid (Ph. Eur. monograph 2149)
unless at least 8 of the 10 embryos survive. Harvest about
The label may state ‘Flu’ or ‘Flu(adj)’ as appropriate.
0.1 mL of the allantoic fluid from each surviving embryo and
examine each individual harvest for live virus by a Ph Eur________________________________________________ _ _____________
haemagglutination test. If haemagglutination is found for any DEFINITION

of the fluids, carry out for that fluid a further passage in eggs Influenza vaccine (surface antigen, inactivated, prepared in
and test for haemagglutination; no haemagglutination occurs. cell cultures) is a sterile, aqueous suspension of a strain or
Antimicrobial preservative strains of influenza virus, type A or B, or a mixture of strains
Where applicable, determine the amount of antimicrobial of the 2 types grown individually in cell cultures, inactivated
preservative by a suitable chemical method. The content is and treated so that the preparation consists predominantly of
not less than the minimum amount shown to be effective and haemagglutinin and neuraminidase antigens, preserving
is not greater than 115 per cent of the quantity stated on the adequate antigenic properties of these antigens. The stated
label. amount of haemagglutinin antigen for each strain present in
Free formaldehyde (2.4.18) the vaccine is 15 pg per dose, unless clinical evidence
Maximum 0.2 g/L, where applicable. supports the use of a different amount. The vaccine is a clear
or slightly opalescent liquid. The vaccine may contain an
Ovalbumin
adjuvant. This monograph applies to vaccines produced in
Not more than the quantity stated on the label and in any
diploid or continuous cell lines of mammalian origin.
case not more than 1 pg per human dose, determined by a
suitable immunochemical method (2.7.1) using a suitable PRODUCTION
reference preparation of ovalbumin. GENERAL PROVISIONS
Total protein Production of the vaccine is based on a virus seed-lot system
Not more than 40 pg of protein other than haemagglutinin and a cell-bank system. The production method shall have
per virus strain per human dose and not more ±an a total of been shown to yield consistently vaccines that comply with
120 pg of protein other than haemagglutinin per the requirements for immunogenicity, safety and stability.
human dose. The production method is validated to demonstrate that the
Sterility
It complies with the test for sterility (2.6.1).
The production method is validated to demonstrate suitable
reduction of residual host-cell protein. With the agreement of
Less than 100 IU per human dose.
the competent authority and for each specific product,
ASSAY routine testing for residual host-cell proteins may be omitted
Determine the content of haemagglutinin antigen by an based on the results of validation studies for the product.
immunodiffusion test (2.7.1), by comparison with a Guidance on the principles of such validation studies is
haemagglutinin antigen reference preparation or with an given, for example, in the monograph Products of recombinant
antigen preparation calibrated against it1. Cany out the test DNA technology (0784), in particular in the sections
at 20-25 °C. The confidence lifnits (P = 0.95) are not less ‘Validation of the production process - Extraction and
than 80 per cent and not more than 125 per cent of the purification’ and ‘Production consistency - Host-cell-derived
estimated content. The lower confidence limit (P ะ= 0.95) proteins’.
haemagglutinin antigen is not less than 80 per cent of the CHOICE OF VACCINE STRAIN
amount stated on the label for each strain. The World Health Organization reviews the world
LABELLING epidemiological situation annually and if necessary
The label states: recommends new strains corresponding to this
— that the vaccine has been prepared on eggs, epidemiological evidence.
— the strain or strains of influenza virus used to prepare the Such strains are used in accordance with the regulations in
vaccine, force in the signatory states of the Convention on the
— the method of inactivation, Elaboration of a European Pharmacopoeia. It is now
2016 Vaccines rV-611
common practice to use reasserted strains giving high yields production process to demonstrate removal or inactivation of
of the appropriate surface antigens. The origin and passage the agent is carried out. If removal or inactivation cannot be
history of virus strains shall be approved by the competent demonstrated, the inactivated monovalent harvest is tested to
authority. demonstrate absence of any contaminant identified in the
SUBSTRATE FOR VIRUS PROPAGATION virus seed.
Influenza virus used in the preparation of seed lots is PROPAGATION AND SINGLE HARVEST
propagated in fertilised eggs from chicken flocks free from All processing of the cell bank and subsequent cell cultures is
specified pathogens (SPF) (5.2.2) or in suitable cell cultures done under aseptic conditions in an area where no other cells
(5.2.3)3 such as chick-embryo fibroblasts, chick kidney cells are being handled at the same time. Approved animal serum
obtained from SPF chicken flocks (5.2.2), or a diploid or (but not human serum) may be used in ±e cell culture
continuous cell line. The final passage for establishment of media. Serum and trypsin used in the preparation of cell
the working seed lot is prepared in the cell line used for suspensions or media are shown to be free from extraneous
routine production. For this production, the virus of each agents. The cell culture media may contain a pH indicator,
strain is propagated in a diploid or continuous cell line such as phenol red, and antibiotics at the lowest effective
.
(5.2.5) concentration. Not less than 500 mL of the cell cultures
VIRUS SEED LOT employed for vaccine production are set aside as uninfected
The production of vaccine is based on a seed-lot system. cell cultures (control cells).
Each of the strains of influenza virus used shall be identified Only a single harvest that complies with the following
by historical records that include information on the origin of requirements may be used in the preparation of the vaccine.
the strain and its subsequent manipulation. Working seed Identification
lots represent not more than 15 passages from the approved The test for antigen content also serves to identify the single
reassorted virus or the approved virus isolate. The final harvest.
vaccine represents 1 passage from the working seed lot.
Only a seed lot that complies with the following requirements Carry out the test for sterility (2.6. /), using 10 mL for each
may be used for virus propagation. medium.
Identification Mycoplasmas (2.6.7)
The haemagglutinin and neuraminidase antigens of each Carry out the test for mycoplasmas, using 10 mL for each
master and working seed lot are identified as originating from medium.
the correct strain of influenza virus by suitable methods.
Control cells
Virus concentration The control cells of the production cell culture comply with a
The virus concentration of each working seed lot is test for identification and the requirements for extraneous
determined. Where applicable, the virus concentration of agents (2.6.16).
each master seed lot is determined.
Extraneous agents (2.6.76) Determine the haemagglutinin antigen content by a suitable
The working seed lots comply with the requirements for seed immunochemical method (2.7.1).
lots. It is recognised that due to a seasonal change in one or
INACTIVATED AND PURIFIED MONOVALENT HARVEST
more of the influenza vaccine strains, timely testing of a virus
The harvest, which may be a pool of several single harvests
seed for extraneous agents according to general
of the same strain, is inactivated and purified by validated
chapter 2.6.16 may be problematic (e.g. duration of in vivo
methods. Before or after the inactivation process, the
tests, timely availability of specific neutralising antisera).
monovalent harvest is concentrated and purified by high
In agreement with the competent authority, and in light of a
speed centrifugation or another suitable method.
risk assessment, rapid assays (e.g. multiplex PCR) may be
The influenza virus is inactivated by a method that has been
applied as alternatives to general chapter 2.6.16 following
demonstrated on 3 consecutive batches to be consistently
validation.
effective for the manufacturer. The inactivation process shall
Such risk assessment and validation includes more general have been shown to be capable of inactivating the influenza
considerations on potential contaminants of the virus isolates, virus without destroying its antigenicity; the process is
the susceptibility of the cell substrate to such viruses and the designed so as to cause minimum alteration of the
capacity of the production process for viral removal or haemagglutinin and neuraminidase antigens.
inactivation; validation includes also comparative data on
Virus particles are disrupted into component subunits by
testing of seeds according to general chapter 2.6.16 and the
approved procedures and further purified so that the
proposed rapid assays. Each applied PCR/NAT test (2.6.21)
monovalent bulk consists mainly of haemagglutinin and
must be shown to be suitable for its intended use by
neuraminidase antigens.
appropriate analytical validation. The risk assessment is
reviewed when new information becomes available on If continuous cell lines are used for production, the
potential viral contaminants, and the justification of the purification process shall have been validated to reduce
chosen PCR panel of extraneous agents tested for is provided consistently host-cell DNA to a suitable level.
to the competent authority within the annual update. This Only an inactivated, purified monovalent harvest that
update also includes vaccine strain-specific aspects such as complies with the following requirements may be used in the
specific PCR inhibitory effects. preparation of the final bulk vaccine.
If an agent is detected in a virus seed and the mammalian Haemagglutinin antigen
cells used for production are shown to be susceptible to this Determine the haemagglutinin antigen content by a suitable
agent, the virus seed is not used for vaccine production. immunochemical method (2.7.1).
If an agent is detected in a virus seed and the mammalian
cells are not susceptible to the agent, validation of the
Antigen/total protein ratio IDENTIFICATION

Determine the haemagglutinin antigen content by a suitable The assay serves to confirm the antigenic specificity of the
immunodiffusion test. Determine the total protein by a vaccine.
validated method. The ratio of haemagglutinin antigen
TESTS
content to total protein content is within the limits approved
for the particular product. Residual infectious virus
Carty7 out an amplification test for residual infectious
Neuraminidase antigen influenza virus by inoculating not less than 0.2 mL of the
The presence and type of neuraminidase antigen are vaccine into cell cultures of the same type as used for
confirmed by suitable enzymatic or immunological methods production of the vaccine; incubate for not less than 4 days
on the first 3 monovalent harvests from each working seed at 37 °C. Inoculate not less than 0.2 mL of the cell culture
lot. harvested medium into a new semiconfluent cell culture and
Sterility (2.6. /) incubate as before. At the end of the incubation period,
Carry out the test for sterility, using 10 mL for each examine for live virus by a haemagglutination test.
medium. If haemagglutination is found for any of the fluids, carry out
Residual infectious virus for that fluid a further passage on cell cultures and test for
Carty7 out the test described below under Tests. haemagglutination; no haemagglutination occurs.
Purity Antimicrobial preservative
The purity of the monovalent harvest is examined by Where applicable, determine the amount of antimicrobial
polyacrylamide gel electrophoresis or by other approved preservative by a suitable chemical method. The content is
techniques. Mainly haemagglutinin and neuraminidase not less than the minimum amount shown to be effective and
antigens are present. is not greater than 115 per cent of the quantity stated on the
label.
Chemicals used for disruption and purification
Tests are carried out on the monovalent harvest for the Free formaldehyde {2.4.18}
chemicals used for disruption and purification, unless Maximum 0.2 g/L, where applicable.
validation of the process has demonstrated total clearance. Bovine serum albumin
The concentration must not exceed the limits approved by Maximum 50 ng per human dose, determined by a suitable
the competent authority for the particular product. immunochemical method (2.7./).
FINAL BULK VACCINE Total protein
Appropriate quantities of the inactivated, purified monovalent Maximum 40 pg of protein other titan haemagglutinin per
pooled harvests are blended to make the final bulk vaccine. virus strain per human dose.
An adjuvant may be added. Sterility (2.6./)
Only a final bulk vaccine that complies with the following It complies with ±e test for sterility.
requirements may be used in the preparation of the final lot. Bacterial endotoxins (2.6. / 4)
Antimicrobial preservative Less than 25 IU per human dose.
ASSAY
immunodiffusion test {2.7.1}, by comparison with a
haemagglutinin antigen reference preparation* 1 or with an
Sterility (2.6./) antigen preparation calibrated against it. Carry out the test at
Carry out the test for sterility, using 10 mL for each 20-25 °C. The confidence limits {P = 0.95) are not less than
medium. 80 per cent and not more than 125 per cent of the estimated
Residual host-cell DNA content. The lower confidence limit {P = 0.95) is not less
If a continuous cell line is used for virus propagation, the than 80 per cent of the amount stated on the label for each
content of residual host-cell DNA, determined using a strain.
suitable method, is not greater than 10 ng in the equivalent LABELLING
of a single human dose.
The label states:
FINAL LOT — the biological origin of the cells used for the preparation
The final bulk vaccine is distributed aseptically into sterile, of the vaccine;
tamper-proof containers. The containers are closed so as to — the strain or strains of influenza virus used to prepare the
prevent contamination. vaccine;
Only a final lot that is satisfactory with respect to each of the — the method of inactivation;
requirements given below under Tests and Assay may be — the haemagglutinin antigen content in micrograms per
released for use. Provided that the test for residual infectious virus strain per dose;
virus has been performed with satisfactory results on each — the season during which the vaccine is intended to
inactivated and purified monovalent harvest and that the tests protect;
for free formaldehyde, bovine serum albumin and total — where applicable, the name and the quantity of adjuvant
protein have been performed with satisfactory results on the used.
final bulk vaccine, they may be omitted on the final lot. _____________________________________________________ PnE-J
If the vaccine contains an adjuvant, suitable tests for identity 1 Reference haemagglutinin antigens are available from the National
and other relevant quality criteria are carried out on the final Institute for Biological Standards and Control, Blanche Lane, South
lot. These tests may include chemical and physical analysis, Mimms, Potters Bar, Hertfordshire EN6 3QG, Great Britain.
determination of particle size and determination of the
number of particles per unit volume.
Influenza Vaccine (Surface VIRUS PROPAGATION AND HARVEST

An antimicrobial agent may be added to the inoculum. After
Antigen, Inactivated, Virosome) incubation at a controlled temperature, the allantoic fluids
(Ph. Eur. monograph 2053) are harvested and combined to form a monovalent pooled
The label may state ‘Flu’. harvest. An antimicrobial agent may be added at the time of
harvest.
MONOVALENT POOLED HARVEST
definition To limit the possibility of contamination, inactivation is
Influenza vaccine (surface antigen, inactivated, virosome) is a initiated as soon as possible after preparation. The virus is
sterile, aqueous suspension of a strain or strains of influenza inactivated by a method that has been demonstrated on
virus, type A or B, or a mixture of strains of the 2 types 3 consecutive batches to be consistently effective for the
grown individually in fertilised hens’ eggs, inactivated and manufacturer. The inactivation process shall have been
treated so that the preparation consists predominantly of shown to be capable of inactivating the influenza virus
haemagglutinin and neuraminidase antigens reconstituted to without destroying its antigenicity; the process is designed so
virosomes and without diminishing the antigenic properties of as to cause minimum alteration of the haemagglutinin and
the antigens. The stated amount of haemagglutinin antigen neuraminidase antigens. The inactivation process shall also
for each strain present in the vaccine is 15 I-Ig per dose, have been shown to be capable of inactivating avian leucosis
unless clinical evidence supports the use of a different viruses and mycoplasmas. If the monovalent pooled harvest is
amount. stored after inactivation, it is held at a temperature of
The vaccine is a slightly opalescent liquid. 5 ± 3 °C. If formaldehyde solution is used, the
concentration does not exceed 0.2 g/L of CH2O at any time
PRODUCTION
during inactivation; if betapropiolactone is used, the
GENERAL PROVISIONS
concentration does not exceed 0.1 per cent VIV at any time
during inactivation.
toxicity for immunosera and vaccines for human use (2.6.9). Before or after the inactivation process, the monovalent
pooled harvest is concentrated and purified by high-speed
CHOICE OF VACCINE STRAIN centrifugation or another suitable method.
The World Health Organization reviews the world
Only a monovalent pooled harvest that complies with the
epidemiological situation annually and if necessary
following requirements may be used for the preparation of
recommends the strains that correspond to this
virosomes.
epidemiological evidence.
Provided the tests for haemagglutinin antigen, neuraminidase
Such strains are used in accordance with the regulations in
antigen and residual infectious virus have been carried out
force in the signatory states of the Convention on the
with satisfactory results on the monovalent virosomal
Elaboration of a European Pharmacopoeia. It is now
preparation, they may be omitted on the monovalent pooled
common practice to use reassorted strains giving high yields
harvest when the manufacturing process is continuous
of the appropriate surface antigens. The origin and passage
between the monovalent pooled harvest and the monovalent
history of virus strains shall be approved by the competent
virosomal preparation.
authority.
Influenza virus seed to be used in the production of vaccine
immunodiffusion test (2.7J), by comparison with a
is propagated in fertilised eggs from chicken flocks free from
haemagglutinin antigen reference preparation1 or with an
specified pathogens (SPF) (5.2.2) or in suitable cell cultures
antigen preparation calibrated against it. Carry out the test at
(5.2.4), such as chick-embryo fibroblasts or chick kidney cells
20-25 °C.
obtained from SPF chicken flocks (5.2.2). For production,
the virus of each strain is grown in the allantoic cavity of Neuraminidase antigen
fertilised hens’ eggs from healthy flocks. The presence and type of neuraminidase antigen are
VIRUS SEED LOT
The production of vaccine is based on a seed lot system.
seed lot.
Working seed lots represent not more than 15 passages from
the approved reasserted virus or the approved virus isolate. Residual infectious virus
The final vaccine represents 1 passage from the working seed Carry out the test described under Tests.
lot. The haemagglutinin and neuraminidase antigens of each PREPARATION OF MONOVALENT VIROSOMES
seed lot are identified as originating from the correct strain of Virus particles are disrupted into component subunits by
influenza virus by suitable methods. approved procedures and further purified so that the
Only a working virus seed lot that complies with the monovalent bulk consists mainly of haemagglutinin and
following requirements may be used in the preparation of the neuraminidase antigens. Additional phospholipids may be
monovalent pooled harvest. added and virosomes may be formed by removal of the
detergent either by adsorption chromatography or another
Bacterial and fungal contamination suitable technique. Several monovalent virosomal
Carry out the test for sterility (2.6. /), using 10 mL for each
preparations may be pooled.
medium.
Only a monovalent virosomal preparation that complies with
Mycoplasmas (2. 6.7) the following requirements may be used in the preparation of
Carry out the test for mycoplasmas, using 10 mL. the final bulk vaccine.
Haemagglutinin antigen ovalbumin and total protein have been performed with
Determine the content of haemagglutinin antigen by an satisfactory results on the final bulk vaccine, they may be
immunodiffusion test (2.7J), by comparison with a omitted on the final lot.
haemagglutinin antigen reference preparation or with an IDENTIFICATION
antigen preparation calibrated against it. Carry out the test at The assay serves to confirm the antigenic specificity of the
20-25 °C.
vaccine.
The presence and type of neuraminidase antigen are TESTS
confirmed by suitable enzymatic or immunological methods Residual infectious virus
Inoculate 0.2 mL of the vaccine into the allantoic cavity of
on the first 3 virosomal preparations from each working seed
lot. each of 10 fertilised eggs and incubate at 33-37 c for
3 days. The test is not valid unless at least 8 of the
Residual infectious virus 10 embryos survive. Harvest 0.5 mL of the allantoic fluid
Carr}7 out the test described under Tests. Provided this test from each surviving embryo and pool the fluids. Inoculate
has been carried out with satisfactory results on the 0.2 mL of the pooled fluid into a further 10 fertilised eggs
monovalent pooled harvest, it may be omitted on the and incubate at 33-37 °C for 3 days. The test is not valid
preparation of monovalent virosomes. unless at least 8 of the 10 embryos survive. Harvest about
Sterility (2.6. I) 0.1 mL of the allantoic fluid from each surviving embryo and
Cany out the test for sterility, using 10 mL for each examine each individual harvest for live virus by a
medium. haemagglutination test. If hacmagglutination is found for any
Purity of the fluids, carry out for that fluid a further passage in eggs
The purity of the monovalent virosomal preparation is and test for haemagglutination; no haemagglutination occurs.
examined by polyacrylamide gel electrophoresis (2.2.31) or pH (2.2.5)
by other approved techniques. Mainly haemagglutinin and 6.5 to 7.8
neuraminidase antigens are present. Phospholipids
Chemicals used for disruption and purification The content and identity of the phospholipids is determined
Tests for the chemicals used for disruption and purification by a suitable immunochemical or physico-chemical method.
are carried out on the monovalent virosomal preparation, the Ratio of haemagglutinin to phospholipid
limits being approved by the competent authority. The ratio of haemagglutinin content to phospholipid content
Phospholipids is within the limits approved for the particular product.
The content and identity of the phospholipids are determined Antimicrobial preservative
by suitable immunochemical or physico-chemical methods. Where applicable, determine the amount of antimicrobial
Ratio of haemagglutinin to phospholipid preservative by a suitable chemical or physico-chemical
The ratio of haemagglutinin content to phospholipid content method. The content is not less than the minimum amount
is within the limits approved for the particular product. shown to be effective and is not greater than 115 per cent of
Virosome size the quantity stated on the label.
The average virosome diameter, determined by a suitable Free formaldehyde (2.4.18)
method such as photon-correlation spectroscopy, is not less Maximum 0.2 g/L, where applicable.
than 100 nm and not greater than 300 nm. Ovalbumin
The polydispersity index is not greater than 0.4. Not more than the quantity stated on the label and in any
FINAL BULK VACCINE case not more than 1 pg per human dose, determined by a
Appropriate quantities of the monovalent virosomal suitable immunochemical method (2.7.1) using a suitable
preparations are blended to make the final bulk vaccine. reference preparation of ovalbumin.
Only a final bulk vaccine that complies with the following Total protein
requirements may be used in the preparation of the final lot. Not more than 40 pg of protein other than haemagglutinin
Antimicrobial preservative per virus strain per human dose, and not more than a total of
Where applicable, determine the amount of antimicrobial 120 pg of protein other than hemagglutinin per human dose.
preservative by a suitable chemical or physico-chemical Sterility (2.6.1)
method. The content is not less than 85 per cent and not It complies with the test for sterility.
greater ±an 115 per cent of the intended amount. Virosome size
Sterility (2.6.1) The average virosome diameter, determined by a suitable
Carry out the test for sterility, using 10 mL for each method such as photon-correlation spectroscopy, is not less
medium. than 100 nm and not greater than 300 nm.
FINAL LOT The polydispersity index is not greater than 0.4.
The final bulk vaccine is distributed aseptically into sterile, Bacterial endotoxins (2.6.14)
tamper-proof containers. The containers are closed so as to Less than 100 IU per human dose.
ASSAY
Only a final lot that is satisfactory with respect to each of the Determine the content of haemagglutinin antigen by an
requirements given under Tests and Assay may be released immunodiffusion test (2.7.1) 3 by comparison with a
for use. Provided that the test for residual infectious virus has haemagglutinin antigen reference preparation or with an
been performed with satisfactory results on each monovalent antigen preparation calibrated against it. Carry out the test at
pooled harvest or, where appropriate, on the monovalent 20-25 °C. The confidence limits (P = 0.95) are not less than
virosomal preparations, and that the tests for phospholipids, 80 per cent and not more than 125 per cent of the estimated
ratio of haemagglutinin to phospholipid, free formaldehyde,
haemagglutinin antigen content. The lower confidence limit are prepared in large quantities and stored at temperatures
(P = 0.95) is not less than 80 per cent of the amount stated below -20 °C if freeze-dried, or below -60 °C if not freeze-
on the label for each strain. dried.
LABELLING Only a seed lot that complies with the following requirements
The label states: may be used for virus propagation.
that the vaccine has been prepared on eggs; Identification
the strain or strains of influenza virus used to prepare the The master and working seed lots are identified as measles
vaccine; virus by serum neutralisation in cell culture, using specific
— the method of inactivation; antibodies.
the haemagglutinin content, in micrograms per virus Virus concentration
strain per dose;
The virus concentration of the master and working seed lots
— the maximum amount of ovalbumin;
is determined to monitor consistency of production.
the season during which the vaccine is intended to
protect. Extraneous agents (2.6.16)
The working seed lot complies with the requirements for
-—— -------------------- - -----------------------------------------------------------Ph Eur
seed lots.
Reference haemagglutinin antigens are available from the National
Institute for Biological Standards and Control, Blanche Lane, South PROPAGATION AND HARVEST
Aiimms, Potters Bar, Hertfordshire EN6 3QG, Great Britain. All processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cells
are handled during production. Suitable animal (but not
human) serum may be used in the growth medium, but the
final medium for maintaining cells during virus multiplication
Measles Vaccine, Live ** ** does not contain animal serum. Serum and trypsin used in
the preparation of cell suspensions and culture media are
(Measles Vaccine (Live), Ph Eur monograph 0213) *** shown to be free from extraneous agents. The cell culture
The label may state ‘Measles’. medium may contain a pH indicator such as phenol red and
Ph Eur__________ suitable antibiotics at the lowest effective concentration. It is
preferable to have a substrate free from antibiotics during
DEFINITION production. Not less than 500 mL of the production cell
Measles vaccine (live) is a freeze-dried preparation of a cultures is set aside as uninfected cell cultures (control cells).
suitable attenuated strain of measles virus. The vaccine is The viral suspensions are harvested at a time appropriate to
reconstituted immediately before use, as stated on the label, the strain of virus being used.
to give a clear liquid that may be coloured owing to the Only a single harvest that complies with the following
presence of a pH indicator. requirements may be used in the preparation of the final bulk
PRODUCTION vaccine.
The production of vaccine is based on a virus seed-lot system Identification
and, if the virus is propagated in human diploid cells, a cell The single harvest contains virus that is identified as measles
bank system. The production method shall have been shown virus by serum neutralisation in cell culture, using specific
to yield consistendy live measles vaccines of adequate antibodies.
immunogenicity and safety in man. Unless otherwise justified
Virus concentration
and authorised, the virus in the final vaccine shall have
The virus concentration in the single harvest is determined as
undergone no more passages from the master seed lot than
prescribed under Assay to monitor consistency of production
were used to prepare the vaccine shown in clinical studies to
and to determine the dilution to be used for the final bulk
be satisfactory with respect to safety and efficacy; even with
vaccine.
authorised exceptions, the number of passages beyond the
level used for clinical studies shall not exceed 5. Extraneous agents (2.6.16)
The single harvest complies with the tests for extraneous
The potential neurovirulence of the vaccine strain is
agents.
considered during preclinical development, based on available
epidemiological data on neurovirulence and neurotropism, Control cells
primarily for the wild-type virus. In light of this, a risk If human diploid cells are used for production, the control
analysis is carried out. Where necessary and if available, a cells comply with a test for identification. They comply with
test is carried out on the vaccine strain using an animal the tests for extraneous agents (2.6.16).
model that differentiates wild-type and attenuated virus; tests FINAL BULK VACCINE
on strains of intermediate attenuation may also be needed. Virus harvests that comply with the above tests are pooled
The production method is validated to demonstrate that the and clarified to remove cells. A suitable stabiliser may be
product, if tested, would comply with the test for abnormal added and the pooled harvests diluted as appropriate.
toxicity for immunosera and vaccines for human use (2.6.9). Only a final bulk vaccine that complies with the following
SUBSTRATE FOR VIRUS PROPAGATION requirement may be used in the preparation of the final lot.
The virus is propagated in human diploid cells (5.2.2) or in Bacterial and fungal contamination
cultures of chick-embryo cells derived from a chicken flock The final bulk vaccine complies with the test for sterility
free from specified pathogens (5.2.2). (2.6J), carried out using 10 mL for each medium.
SEED LOT FINAL LOT
The strain of measles virus used shall be identified by A minimum virus concentration for release of the product is
historical records that include information on the origin of established such as to ensure, in light of stability data, that
the strain and its subsequent manipulation. Virus seed lots
the minimum concentration stated on the label will be sample. The confidence interval (P = 0.95) of the combined
present at the end of the period of validity. virus concentration is not greater than ± 0.3 logio CCIDy).
Only a final lot that complies with the requirements for Measles vaccine (live) BRP is suitable for use as a reference
minimum virus concentration for release, with the following preparation.
requirement for thermal stability and with each of the Where justified and authorised, different assay designs may
requirements given below under Identification and Tests may be used; this may imply the application of different validity
be released for use. Provided that the test for bovine serum and acceptance criteria. However, the vaccine must comply if
albumin has been carried out with satisfactory' results on the tested as described above.
LABELLING
Thermal stability'
The label states:
Maintain at least 3 vials of the final lot of freeze-dried
— the strain of virus used for the preparation of the vaccine;
vaccine in the dry state at 37 ± 1 °C for 7 days. Determine
— the ty'pe and origin of the cells used for the preparation of
the virus concentration as described under Assay in parallel
the vaccine;
for the heated vaccine and for vaccine stored at the
— the minimum virus concentration;
temperature recommended for storage. The virus
— that contact between the vaccine and disinfectants is to be
concentration of the heated vaccine is not more than
avoided.
1.0 logio lower than that of the unheated vaccine.
__________________________________________________________ ____ PnEtf
IDENTIFICATION
When the vaccine reconstituted as stated on the label is
mixed with specific measles antibodies, it is no longer able to
infect susceptible cell cultures.
TESTS Measles, Mumps and Rubella * *
Bacterial and fungal contamination Vaccine, Live *****
The reconstituted vaccine complies with the test for sterility
(Measles, Mumps and Rubella Vaccine (Live),
(2.6.7).
Bovine serum albumin The label may state ‘MMR’.
Not more than 50 ng per single human dose, determined by
Ph Eur__________________________________________ __________________ -—
a suitable immunochemical method (2.7.7).
Water (2.5.72) DEFINITION
Not more than 3.0 per cent, determined by the semi-micro Measles, mumps and rubella vaccine (live) is a freeze-dried
determination of water. preparation of suitable attenuated strains of measles virus,
mumps virus and rubella vims.
ASSAY
The vaccine is reconstituted immediately before use, as
Titrate the vaccine for infective virus, using at least
stated on the label, to give a clear liquid that may be
3 separate vials of vaccine and inoculating a suitable number
coloured owing to the presence of a pH indicator.
of wells for each dilution step. Titrate 1 vial of an
appropriate virus reference preparation in triplicate to PRODUCTION
validate each assay. The virus concentration of the reference The 3 components are prepared as described in the
preparation is monitored using a control chart and a titre is monographs Measles vaccine (live) (0213), Mumps vaccine
established on a historical basis by each laboratory. (live) (0538) and Rubella vaccine (live) (0162) and comply
The relation with the appropriate European Pharmacopoeia with the requirements prescribed therein.
Biological Reference Preparation is established and The production method is validated to demonstrate that the
monitored at regular intervals if a manufacturer's reference product, if tested, would comply with the test for abnormal
preparation is used. Calculate the individual virus toxicity for immunosera and vaccines for human use (2.6.9).
concentration for each vial of vaccine and for each replicate
FINAL BULK VACCINE
of the reference preparation as well as the corresponding
Vims harvests for each component are pooled and clarified to
combined virus concentrations, using the usual statistical
remove cells. A suitable stabiliser may be added and the
methods (for example, 5.3). The combined estimate of the
pooled harvests diluted as appropriate. Suitable quantities of
virus concentration for the 3 vials of vaccine is not less than
the pooled harvest for each component are mixed.
that stated on the label; the minimum virus concentration
stated on the label is not less than 3.0 log10 CCID50 per Only a final bulk vaccine that complies with the following
single human dose. requirement may be used in the preparation of the final lot.
The assay is not valid if: Bacterial and fungal contamination
— the confidence interval (P = 0.95) of the estimated virus Carry out the test for sterility (2.6.7), using 10 mL for each
concentration of the reference preparation for the medium.
3 replicates combined is greater than ± 0.3 FINAL LOT
logio CCID50; For each component, a minimum vims concentration for
— the virus concentration of the reference preparation differs release of the product is established such as to ensure, in
by more than 0.5 logio CCID50 from ±e established light of stability data, that the minimum concentration stated
value. on the label will be present at the end of the period of
The assay is repeated if the confidence interval (P = 0.95) of validity.
the combined virus concentration of the vaccine is greater Only a final lot that complies with the requirements for
than ± 0.3 logio CCID50; data obtained from valid assays minimum vims concentration of each component for release,
only are combined by the usual statistical methods (for with the following requirement for thermal stability and with
example, 5.3) to calculate the virus concentration of the each of the requirements given below under Identification
and Tests may be released for use. Provided that the tests for The assay is not valid if:
bovme serum albumin and, where applicable, for ovalbumin — the confidence interval (P = 0.95) of the estimated virus
ha\e been carried out with satisfactory results on the final concentration of the reference preparation for the
bulk vaccine, they may be omitted on the final lot? 3 replicates combined is greater than
Thermal stability ± 0.3 logio CCID50;
Maintain at least 3 vials of the final lot of freeze-dried — the virus concentration of the reference preparation differs
vaccine in the dry state at 37 i 1 °C for 7 days. Determine by more than 0.5 logio CCID50 from the established
the virus concentration as described under Assay in parallel value.
for the heated vaccine and for vaccine stored at the The assay is repeated if the confidence interval (P = 0.95) of
temperature recommended for storage. For each component, the combined virus concentration of the vaccine is greater
the virus concentration of the heated vaccine is not more than ± 0.3 logio CCID50; data obtained from valid assays
than 1.0 logio lower than that of the unheated vaccine. only are combined by the usual statistical methods (for
IDENTIFICATION example, 5.3) to calculate the virus concentration of the
sample. The confidence interval (P = 0.95) of the combined
virus concentration is not greater than ± 0.3 log10 CCID50.
mixed with antibodies specific for measles virus, mumps virus
and rubella virus, it is no longer able to infect cell cultures Measles vaccine (live) BRP is suitable for use as a reference
susceptible to these viruses. When the vaccine reconstituted preparation.
as stated on the label is mixed with quantities of specific Mumps vaccine (live) BRP is suitable for use as a reference
antibodies sufficient to neutralise any 2 viral components, the preparation.
3rd viral component infects susceptible cell cultures. Rubella vaccine (live) BRP is suitable for use as a reference
TESTS preparation.
Bacterial and fungal contamination Where justified and authorised, different assay designs may
The reconstituted vaccine complies with the test for sterility be used; this may imply the application of different validity
(2.6./). and acceptance criteria. However, the vaccine must comply if
tested as described above.
Not more than 50 ng per single human dose, determined by LABELLING
a suitable immunochemical method (2.7./). The label states:
Ovalbumin — the strains of virus used in the preparation of the vaccine;
If the mumps component is produced in chick embryos, the — where applicable, that chick embryos have been used for
vaccine contains not more than 1 pg of ovalbumin per single the preparation of the vaccine;
human dose, determined by a suitable immunochemical — the type and origin of the cells used for the preparation of
method (2.7.1'). the vaccine;
— the minimum virus concentration for each component of
Water (2.5.12) the vaccine;
Not more than 3.0 per cent, determined by the semi-micro — that contact between the vaccine and disinfectants is to be
determination of water. avoided.
ASSAY ______________________________________________________________ PhEur
The cell lines and/or neutralising antisera are chosen to
ensure that each component is assayed without interference
from the other 2 components.
Titrate the vaccine for infective measles, mumps and rubella
virus, using at least 3 separate vials of vaccine and Measles, Mumps, Rubella and * *
inoculating a suitable number of wells for each dilution step.
Titrate 1 vial of the appropriate virus reference preparation in
Varicella Vaccine (Live) *****
triplicate to validate each assay. The virus concentration of (Ph. Eur. monograph 2442)
the reference preparation is monitored using a control chart The label may state ‘MMRVar’.
and a titre is established on a historical basis by each PhEur____ ___________________
laboratory. The relation with the appropriate European
DEFINITION
Pharmacopoeia Biological Reference Preparation is
Measles, mumps, rubella and varicella vaccine (live) is a
established and monitored at regular intervals if a
freeze-dried preparation of suitable attenuated strains of
manufacturer's reference preparation is used. Calculate ±e
measles virus, mumps virus, rubella virus and human
individual virus concentration for each vial of vaccine and for
herpesvirus 3. The vaccine is reconstituted immediately
each replicate of the reference preparation as well as the
before use, as stated on the label, to give a clear liquid that
corresponding combined virus concentrations, using the usual
may be coloured owing to the presence of a pH indicator.
statistical methods (for example, 5.3).
The combined estimates of the measles, mumps and rubella PRODUCTION
virus concentrations for the 3 vials of vaccine are not less The 4 components are prepared as described in the
than that stated on the label; the minimum measles virus monographs Measles vaccine (live) (0213) 3 Mumps vaccine
concentration stated on the label is not less than (live) (0538)3 Rubella vaccine (live) (0162) and Varicella
3.0 log10 CCID50 per single human dose; the minimum vaccine (live) (0648) and comply with the requirements
mumps virus concentration stated on the label is not less prescribed therein.
than 3.7 logio CCID50 per single human dose; the minimum The production method is validated to demonstrate that the
rubella virus concentration stated on the label is not less than product, if tested, would comply with the test for abnormal
3.0 logio CCID50 per single human dose. toxicity for immunosera and vaccines for human use (2.6.9).
FINAL BULK VACCINE Titrate the vaccine for infective measles virus, mumps virus,
Virus harvests for each component are pooled and clarified to rubella virus and human herpesvirus 3 using at least
remove cells. A suitable stabiliser may be added and for each 3 separate containers of vaccine and inoculating a suitable
component the pooled harvests diluted as appropriate. number of wells for each dilution step. Titrate 1 container of
Suitable quantities of the pooled harvest for each component the appropriate virus reference preparation in triplicate to
are mixed. validate each assay. The virus concentration of the reference
Only a final bulk vaccine that complies with the following preparation is monitored using a control chart and a titre is
requirement may be used in the preparation of the final lot. established on a historical basis by each laboratory. Unless
otherwise justified and authorised, for the measles, mumps,
rubella and human herpesvirus 3 viruses the relation with the
appropriate European Pharmacopoeia Biological Reference
medium.
Preparation is established and monitored at regular intervals
FINAL LOT if a manufacturer's reference preparation is used. Calculate
For each component, a minimum virus concentration for the individual virus concentration for each container of
release of the product is established such as to ensure, in vaccine and for each replicate of the reference preparation as
light of stability data, that the minimum concentration stated well as the corresponding combined virus concentrations,
on the label will be present at the end of the period of using the usual statistical methods (for example, 5.3).
validity. The final bulk vaccine is distributed aseptically into
The combined estimates of the measles virus, mumps virus,
sterile, tamper-proof containers and freeze-dried to a
rubella virus and human herpesvirus 3 concentrations for the
moisture content shown to be favourable to the stability of
3 containers of vaccine are not less than that stated on the
the vaccine. The containers are then closed so as to prevent
label; the minimum measles virus concentration stated on the
contamination and the introduction of moisture.
label is not less than 3.0 logic CCID50 per single human
Only a final lot that complies with the requirements for dose; the minimum mumps virus concentration stated on the
minimum virus concentration of each component for release, label is not less than 3.7 log10 CCID50 per single human
with the following requirements for thermal stability, bovine dose; the minimum rubella virus concentration stated on the
serum albumin and water, and with each of the requirements label is not less than 3.0 log10 CCID50 per single human
given under Identification and Tests may be released for use. dose.
Provided that the test for bovine serum albumin has been
— the confidence interval (P = 0.95) of the estimated virus
concentration of the reference preparation for the
Thermal stability 3 replicates combined is greater than ± 0.3
For the measles, mumps and rubella components maintain at log10 CCID50 (measles virus, mumps virus and rubella
least 3 containers of the final lot of freeze-dried vaccine in virus) or + 0.3 logio PFU (human herpesvirus 3);
the dry state at 37 ± 1 °C for 7 days. Determine the virus — the virus concentration of the reference preparation differs
concentration as described under Assay in parallel for the by more than 0.5 logic CCID50 (measles virus, mumps
heated vaccine and for vaccine stored at the temperature virus and rubella virus) or 0.5 log10 PFU (human
recommended for storage. For each component, the virus herpesvirus 3) from the established value.
concentration of the heated vaccine is not more than 1.0 The assay is repeated if the confidence interval (P = 0.95) of
log 10 lower than that of the unheated vaccine. the combined virus concentration of the vaccine is greater
Bovine serum albumin than ± 0.3 logio CCID50 (measles virus, mumps virus and
Not more than the amount approved by the competent rubella virus) or ± 0.3 logic PFU (human herpesvirus 3);
authority, determined by a suitable immunochemical method data obtained from valid assays only are combined by using
(2.7./). the usual statistical methods (for example, 5.3) to calculate
Water (2.5./2) the virus concentration of the sample. The confidence
Not more than the amount shown to ensure stability of the interval (P = 0.95) of the combined virus concentration is
vaccines as approved by the competent authority, determined not greater than ± 0.3 logio CCID50 (measles virus, mumps
by the semi-micro determination of water. virus and rubella virus) or ± 0.3 logio PFU (human
herpesvirus 3).
IDENTIFICATION
Measles vaccine (live) BRP is suitable for use as a reference
preparation.
mixed with antibodies specific for measles virus, mumps
virus, rubella virus and human herpesvirus 3, it is no longer Mumps vaccine (live) BRP is suitable for use as a reference
able to infect cell cultures susceptible to these viruses. When preparation.
the vaccine reconstituted as stated on the label is mixed with Rubella vaccine (live) BRP is suitable for use as a reference
quantities of specific antibodies sufficient to neutralise any preparation.
3 viral components, the 4th viral component infects Varicella vaccine (live) BRP is suitable for use as a reference
susceptible cell cultures. preparation.
TESTS Where justified and authorised, different assay designs may
Bacterial and fungal contamination be used; this may imply the application of different validity
The reconstituted vaccine complies with the test for sterility and acceptance criteria. However, the vaccine must comply if
(2.6./). tested as described above.
ASSAY LABELLING
The cell lines and/or neutralising antisera are chosen to The label states:
ensure that each component is assayed without interference — the strains of virus used in the preparation of the vaccine;
from the other 3 components.
the type and origin of the cells used for the preparation of MENINGOCOCCAL GROUP c POLYSACCHARIDE
the vaccine; N. meningitidis is grown in a liquid medium that does not
the minimum virus concentration for each component of contain high-molecular-mass polysaccharides and is free from
the vaccine; ingredients that will form a precipitate upon addition of
that contact between the vaccine and disinfectants is to be cetyltrimethylammonium bromide (CTAB). The culture may
avoided. be inactivated by heat and filtered before the polysaccharide
------------------------------------------------ - -------------------------------------------------------Ph Eur is precipitated by addition of CTAB. The precipitate is
further purified using suitable methods to remove nucleic
acids, proteins and lipopolysaccharides and the final
purification step consists of ethanol precipitation.
An O-deacetylation step may also be included. Volatile
Meningococcal Group c matter, including water, in the purified polysaccharide is
determined by a suitable method such as thermogravimetry
Conjugate Vaccine (2.2.54). The value is used to calculate the results of other
(Ph. Eur. monograph 2112) tests with reference to the dried substance, as prescribed
The label may state ‘MenC(conj)’. below.
Only meningococcal group c polysaccharide that complies
with the following requirements may be used in the
DEFINITION
preparation of the conjugate.
Meningococcal group c conjugate vaccine is a liquid or
freeze-dried preparation of purified capsular polysaccharide Protein (2.5.76)
derived from a suitable strain of Neisseria meningitidis group c Maximum 1.0 per cent, calculated with reference to the dried
covalently linked to a carrier protein. Meningococcal group c substance.
polysaccharide consists of partly O-acetylated or Nucleic acid {2.5.17)
O-deacetylated repeating units of sialic acids, linked with Maximum 1.0 per cent, calculated with reference to the dried
2ot—*9 glycosidic bonds. The carrier protein, when substance.
conjugated to group c polysaccharide, is capable of inducing O-acetyl groups
a T-cell-depcndent B-cell immune response to the Examine by a suitable method (for example 2.5.19).
polysaccharide. The vaccine may contain an adjuvant. An acceptable value is established for the particular product
PRODUCTION and each batch of meningococcal group c polysaccharide
GENERAL PROVISIONS must be shown to comply with this limit.
The production method shall consistently have been shown Sialic acid (2.5.25)
to yield meningococcal group c conjugate vaccines of Minimum 0.800 g of sialic acid per gram of meningococcal
satisfactory immunogenicity and safety in man. group c polysaccharide using N-acetyIncuraminic acid R to
The production of meningococcal group c polysaccharide prepare the reference solution.
and of the carrier protein are based on seed-lot systems. Residual reagents
During development studies and wherever revalidation is Where applicable, tests are carried out to determine residues
necessary, a test for pyrogens in rabbits (2.6. ร) is carried out of reagents used during inactivation and purification.
by injection of a suitable dose of the final lot. The vaccine is An acceptable value for each reagent is established for the
shown to be acceptable with respect to absence of pyrogenic particular product and each batch of meningococcal group c
activity. polysaccharide must be shown to comply with this limit.
The production method is validated to demonstrate that the Where validation studies have demonstrated removal of a
vaccine, if tested, would comply with the test for abnormal residual reagent, the test on purified meningococcal group c
toxicity for immunosera and vaccines for human use (2.6.9). polysaccharide may be omitted.
During development studies and wherever revalidation of the Molecular-size distribution
manufacturing process is necessary, it shall be demonstrated Examine by size-exclusion chromatography {2.2.30).
by tests in animals that the vaccine consistently induces a An acceptable value is established for the particular product
T-cell-dependent B-cell immune response. and each batch of meningococcal group c polysaccharide
The stability of the final lot and relevant intermediates is must be shown to comply with this limit. Where applicable,
evaluated using 1 or more indicator tests. Such tests may the molecular-size distribution is also determined after
include determination of molecular size, determination of free chemical modification of the meningococcal group c
saccharide in the conjugate or an immunogenicity test in polysaccharide.
animals. Identification and serological specificity
The identity and serological specificity are determined by a
BACTERIAL SEED LOTS
suitable immunochemical method (2.7.1) or other suitable
The bacterial strains used for master seed lots shall be
method, for example JH nuclear magnetic resonance
identified by historical records that include information on
their origin and the tests used to characterise the strain. spectrometry (2.2.55).
Cultures from the working seed lot shall have the same Bacterial endotoxins {2.6.14)
characteristics as the strain that was used to prepare the Less than 100 IU per microgram of meningococcal group c
master seed lot. polysaccharide.
Purity of bacterial' cultures is verified by methods of suitable CARRIER PROTEIN
sensitivity. These may include inoculation into suitable The production and characteristics of the carrier proteins are
media, examination of colony morphology, microscopic described in general chapter 5.2.11. Carrier proteins for the
examination of Gram-stained smears and culture production of conjugated polysaccharide vaccines for human use.
agglutination with suitable specific antisera.
พ-620 Vaccines 2016
Only a earner protein that complies with the requirements of Only a final bulk vaccine that complies with the following
this chapter may be used in the preparation of the conjugate. requirement and is within the limits approved for the
BULK CONJUGATE particular product may be used in the preparation of the final
Meningococcal group c polysaccharide is chemically lot.
modified to enable conjugation; it is usually partly Sterility (2.6.1)
depolymerised either before or during this procedure. It complies with the test for sterility, carried out using 10 mL
The conjugate is obtained by the covalent binding of for each medium.
activated meningococcal group c oligosaccharide and the FINAL LOT
appropriate carrier protein. The conjugate purification Only a final lot that is within the limits approved for the
procedures are designed to remove residual reagents used for particular product and is satisfactory with respect to each of
conjugation. The removal of residual reagents and reaction the requirements given below under Identification, Tests and
by-products is confirmed by suitable tests or by validation of Assay may be released for use.
the purification process.
IDENTIFICATION
Only a bulk conjugate that complies with the following
The vaccine is identified by a suitable immunochemical
requirements may be used in the preparation of the final bulk
vaccine. For each test and for each particular product, limits method (2.7.7).
of acceptance are established and each batch of conjugate TESTS
must be shown to comply with these limits. pH (2.2.3)
Molecular-size distribution The pH of the vaccine, reconstituted if necessary, is within
Examine by size-exclusion chromatography (2.2.30. the limits approved for the particular product.
An acceptable value is established for the particular product Aluminium {2.5.13)
and each batch of bulk conjugate must be shown to comply Maximum 1.25 mg per single human dose, if aluminium
with this limit. hydroxide or hydrated aluminium phosphate is used as the
Saccharide adsorbent.
The saccharide content is determined by a suitable validated Water (2.5.72)
assay (for example 2.5.23). Anion-exchange liquid Maximum 3.0 per cent for freeze-dried vaccines.
chromatography (2.2.29) with pulsed amperometric detection Free saccharide
may also be used for determination of saccharide content. Unbound saccharide is determined after removal of the
An acceptable value is established for the particular product conjugate, for example by anion-exchange liquid
and each batch of bulk conjugate must be shown to comply chromatography, size-exclusion or hydrophobic
with this limit. chromatography, ultrafiltration or other validated methods.
Protein An acceptable value consistent with adequate
The protein content is determined by a suitable chemical immunogenicity, as shown in clinical trials, is established for
method (for example 2.5.16). An acceptable value is the particular product and each final lot must be shown to
established for the particular product and each batch of bulk comply with this limit.
conjugate must be shown to comply with this limit. Sterility (2.6.1)
Saccharide-to-protein ratio It complies with the test for sterility.
Determine the ratio by calculation. Bacterial endotoxins (2.6.74)
Free saccharide Less than 25 IU per single human dose.
Unbound saccharide is determined after removal of the
ASSAY
conjugate, for example by anion-exchange liquid
Saccharide
chromatography, size-exclusion or hydrophobic
Minimum 80 per cent of the amount of meningococcal
chromatography, ฟtrafiltration or other validated methods.
group c polysaccharide stated on the label. The saccharide
An acceptable value is established for the particular product
content is determined by a suitable validated assay, for
and each batch of bulk conjugate must be shown to comply
example sialic acid assay (2.5.23) or anion-exchange liquid
with this limit.
chromatography (2.2.29) with pulsed amperometric
Free carrier protein detection.
Determine the content, either directly by a suitable method
or by deriving the content by calculation from the results of LABELLING
other tests. An acceptable value is established for the The label states:
particular product and each batch of bulk conjugate must be — the number of micrograms of meningococcal group c
shown to comply with this limit. polysaccharide per human dose;
— the type and number of micrograms of carrier protein per
Residual reagents human dose.
Removal of residual reagents such as cyanide is confirmed by
______ PhEj
suitable tests or by validation of the process.
Sterility (2.6.1)
for each medium or the equivalent of 100 doses, whichever is
less.
FINAL BULK VACCINE
An adjuvant and a stabiliser may be added to the bulk
conjugate before dilution to the final concentration with a
suitable diluent.
mammalian origin. The inoculum may undergo 1 or more

Meningococcal Polysaccharide subcultures in liquid medium before being used for
Vaccine inoculating the final medium. The liquid media used and the
(Ph. Eur. monograph 0250) final medium are semisynthetic and free from substances
The label may state ‘Men’ plus relevant antigen. precipitated by cetrimonium bromide
For example, ‘MenAC’. (hexadecyltrimethylammonium bromide) and do not contain
blood-group substances or high-molecular-mass
polysaccharides.
DEFINITION The bacterial purity of the culture is verified by methods of
Meningococcal polysaccharide vaccine is a freeze-dried suitable sensitivity. These may include inoculation into
preparation of one or more purified capsular polysaccharides suitable media, examination of colony morphology,
obtained from one or more suitable strains of Neisseria microscopic examination of Gram-Stained smears and culture
meningitidis group A, group c, group Y and group พ 135 that agglutination with suitable specific antisera.
are capable of consistently producing polysaccharides. The cultures are centrifuged and the polysaccharides
Ar meningitidis Group A polysaccharide consists of partly precipitated from the supernatant by addition of cetrimonium
O-acetylated repeating units of 7V-acetylmannosamine, linked bromide. The precipitate obtained is harvested and may be
with la-*6 phosphodiester bonds. stored at —20 °C awaiting further purification.
iV meningitidis Group c polysaccharide consists of partly PURIFIED POLYSACCHARIDES
O-acetylated repeating units of sialic acid, linked with The polysaccharides are purified, after dissociation of the
2a-»9 glycosidic bonds. complex of polysaccharide and cetrimonium bromide, using
N meningitidis Group Y polysaccharide consists of partly suitable procedures to remove successively nucleic acids,
O-acetylated alternating units of sialic acid and D-glucose, proteins and lipopolysaccharides.
linked with 2a-*6 and la-*4 glycosidic bonds. The final purification step consists of ethanol precipitation of
N meningitidis Group พ 135 polysaccharide consists of partly the polysaccharides which are then dried and stored at
O-acetylated alternating units of sialic acid and D-galactose, -20 °C. The loss on drying is determined by
linked with 2a-*6 and la-»4 glycosidic bonds. thermogravimetry (2.2.34) and the value is used to calculate
The polysaccharide component or components stated on the the results of the other chemical tests with reference to the
label together with calcium ions and residual moisture dried substance.
account for over 90 per cent of the mass of the preparation. Only purified polysaccharides that comply with the following
PRODUCTION
vaccine.
Production of the meningococcal polysaccharides is based on
a seed-lot system. The production method shall have been Protein (2.5.16)
shown to yield consistently meningococcal polysaccharide Not more than 10 mg of protein per gram of purified
vaccines of satisfactory immunogenicity and safety in man. polysaccharide, calculated with reference to the dried
substance.
The production method is validated to demonstrate that ±e
product, if tested, would comply with the test for abnormal Nucleic acids (2.5.17)
toxicity for immunosera and vaccines for human use (2.6.9). Not more than 10 mg of nucleic acids per gram of purified
polysaccharide, calculated with reference to the dried
SEED LOTS
substance.
The strains of N. meningitidis used for the master seed lots
shall be identified by historical records ±at include O-Acetyl groups (2.5.19)
information on their origin and by their biochemical and Not less than 2 mmol of O-acetyl groups per gram of
serological characteristics. purified polysaccharide for group A, not less than 1.5 mmol
per gram of polysaccharide for group c, not less than
Cultures from each working seed lot shall have the same 0.3 mmol per gram of polysaccharide for groups Y and
characteristics as the strain that was used to prepare the พ135, all calculated with reference to the dried substance.
master seed lot. The strains have the following
characteristics:
Phosphorus (2.5.18)
Not less than 80 mg of phosphorus per gram of group A
— colonies obtained from a culture are rounded, uniform in
purified polysaccharide, calculated with reference to the dried
shape and smooth with a mucous, opalescent, greyish
substance.
appearance,
— Gram staining reveals characteristic Gram-negative Sialic acid (2.5.23)
diplococci in “coffee-bean” arrangement, Not less than 800 mg of sialic acid per gram of group c
— the oxidase test is positive, polysaccharide and not less than 560 mg of sialic acid per
— the culture utilises glucose and maltose, gram of purified polysaccharide for groups Y and พ135, all
— suspensions of the culture agglutinate with suitable calculated with reference to the dried substance. Use the
specific antisera. following reference solutions.
Purity of bacterial strains used for the seed lots is verified by Group c polysaccharide: a 150 mg/L solution of
methods of suitable sensitivity. These may include N-acetylneuraminic acid R.
inoculation into suitable media, examination of colony Group Y polysaccharide: a solution containing 95 mg/L of
morphology, microscopic examination of Gram-Stained N-acetylneuraminic acid R and 55 mg/L of glucose R.
smears and culture agglutination with suitable specific Group พ135 polysaccharide: a solution containing 95 mg/L
antisera. of N-acetylneuraminic acid R and 55 mg/L of galactose R.
PROPAGATION AND HARVEST Calcium
The working seed lots are cultured on solid media that do If a calcium salt is used during purification, a determination
not contain blood-group substances or ingredients of of calcium is carried out on the purified polysaccharide;
the content is within the limits approved for the particular For a divalent vaccine (group A + group C), use cross-linked
product. agarose for chromatography R. The vaccine complies with the
Distribution of molecular size test if:
Examine by size-exclusion chromatography (2.2.30) using — 65 per cent of group A polysaccharide is eluted before
agarose for chromatography R or cross-linked agarose for Kq = 0.50,
chromatography R. Use a column about 0.9 m long and — 75 per cent of group c polysaccharide is eluted before
16 mm in internal diameter equilibrated with a solvent Kq = 0.50.
having an ionic strength of 0.2 mol/kg and a pH of 7.0-7.5. For a tetravalent vaccine (group A + group C -r group Y
Apply to the column about 2.5 mg of polysaccharide in T group พ 135), use cross-linked agarose for chromatography R1
a volume of about 1.5 mL and elute at about 20 mL/h. and apply a suitable immunochemical method (2.7. /) to
Collect fractions of about 2.5 mL and determine the content establish the elution pattern of the different polysaccharides.
of polysaccharide by a suitable method. At least 65 per cent The vaccine complies with tile test if Kq for the principal
of group A polysaccharide, 75 per cent of group c peak is:
polysaccharide, 80 per cent of group Y polysaccharide and — not greater than 0.70 for group A and group c
80 per cent of group พ 135 polysaccharide is eluted before a polysaccharide,
distribution coefficient (Kq) of 0.50 is reached. In addition, — not greater than 0.57 for group Y polysaccharide,
the percentages eluted before this distribution coefficient are — not greater than 0.68 for group พ135 polysaccharide.
within the limits approved for ±e particular product. Water (2.5.12)
Identification and serological specificity Not more than 3.0 per cent, determined by the semi-micro
The identity and serological specificity are determined by a determination of water.
suitable immunochemical method (2.7./). Identity and purity Sterility (2.6. /)
of each polysaccharide shall be confirmed; it shall be shown It complies with the test for sterility.
that there is not more than 1 per cent m/m of group- Pyrogens (2.6.5)
heterologous N. meningitidis polysaccharide. It complies with the test for pyrogens. Inject per kilogram of
Pyrogens (2.6.5) the rabbit's mass 1 mL of a solution containing:
The polysaccharide complies with the test for pyrogens. — 0.025 pg of polysaccharide for a monovalent vaccine,
Inject into each rabbit per kilogram of body mass 1 mL of a — 0.050 pg of polysaccharide for a divalent vaccine,
solution containing 0.025 pg of purified polysaccharide per — 0.10 pg of polysaccharide for a tetravalent vaccine.
millilitre. ASSAY
FINAL BULK VACCINE Carry out an assay of each polysaccharide present in the
One or more purified polysaccharides of 1 or more vaccine.
A7. meningitidis groups are dissolved in a suitable solvent that For a divalent vaccine (group A -r group C), use
may contain a stabiliser. When dissolution is complete, the measurement of phosphorus (2.5.18) to determine the
solution is filtered through a bacteria-retentive filter. content of polysaccharide A and measurement of sialic acid
Only a final bulk vaccine that complies with the following (2.5.23) to determine the content of polysaccharide c.
requirement may be used in the preparation of the final lot. To determine sialic acid, use as reference solution a
150 mg/L solution of N-acetylneuraminic acid R.
Sterility (2.6./)
The final bulk vaccine complies with the test for sterility, For a tetravalent vaccine (group A + group c + group Y
+ group พ 135) a suitable immunochemical method (2.7.1)
is used with a reference preparation of purified
FINAL LOT
polysaccharide for each group.
The final bulk vaccine is distributed aseptically into sterile
The vaccine contains not less than 70 per cent and not more
containers. The containers are then closed so as to avoid than 130 per cent of the quantity of each polysaccharide
contamination.
stated on the label.
LABELLING
requirements prescribed below under Identification, Tests
and Assay may be released for use. The label states:
— the group or groups of polysaccharides (A, c, Y or
CHARACTERS พ135) present in the vaccine,
A white or cream-coloured powder or pellet, freely soluble in — the number of micrograms of polysaccharide per
water. human dose.
IDENTIFICATION
Carry out an identification test for each polysaccharide
present in the vaccine by a suitable immunochemical method
.
(2.7.7) Mumps Vaccine, Live ★ J
TESTS
(Mumps Vaccine (Live)J Ph Eur monograph 0538)
Distribution of molecular size
Examine by size-exclusion chromatography (2.2.30). Use a The label may state ‘Mumps’.
column about 0.9 m long and 16 mm in internal diameter Ph Eur----------------------------------------------------------------------------- -----------------------------
equilibrated with a solvent having an ionic strength of DEFINITION

0.2 mol/kg and a pH of 7.0-7.5. Apply to the column about Mumps vaccine (live) is a freeze-dried preparation of a
2.5 mg of each polysaccharide in a volume of about 1.5 mL suitable attenuated strain of mumps virus. The vaccine is
and elute at about 20 mL/h. Collect fractions of about reconstituted immediately before use, as stated on the label,
2.5 mL and determine the content of polysaccharide by a to give a clear liquid that may be coloured owing to the
suitable method. presence of a pH indicator.
PRODUCTION Identification
The production of vaccine is based on a virus seed-lot system The single harvest contains virus that is identified as mumps
and, if the virus is propagated in human diploid cells, a cell virus by serum neutralisation in cell culture, using specific
bank system. The production method shall have been shown antibodies.
to yield consistently live mumps vaccines of adequate Virus concentration
immunogenicity and safety in man. Unless otherwise justified The virus concentration in the single harvest is determined as
and authorised, the virus in the final vaccine shall have prescribed under Assay to monitor consistency of production
undergone no more passages from the master seed lot than and to determine the dilution to be used for the final bulk
were used to prepare the vaccine shown in clinical studies to vaccine.
be satisfactory with respect to safety and efficacy.
The potential neurovirulence of the vaccine strain is
The single harvest complies with the tests for extraneous
considered during preclinical development, based on available agents.
epidemiological data on neurovirulence and neurotropism,
primarily for the wild-type virus. In light of this, a risk Control cells or eggs
analysis is carried out. Where necessary and if available, a If human diploid cells are used for production, the control
test is earned out on the vaccine strain using an animal cells comply with a test for identification; the control cells
model that differentiates wild-type and attenuated virus; tests and the control eggs comply with the tests for extraneous
on strains of intermediate attenuation may also be needed. agents (2.6.76).
The production method is validated to demonstrate that the FINAL BULK VACCINE
product, if tested, would comply with the test for abnormal Single harvests that comply with the above tests are pooled
toxicity for immunosera and vaccines for human use (2.6.9). and clarified to remove cells. A suitable stabiliser may be
added and the pooled harvests diluted as appropriate.
The virus is propagated in human diploid cells (5.2.3) or in Only a final bulk vaccine that complies with the following
chick-embryo cells or in the amniotic cavity of chick embryos requirement may be used in the preparation of the final lot.
derived from a chicken flock free from specified pathogens Bacterial and fungal contamination
(5.2.2). The final bulk vaccine complies with the test for sterility
SEED LOT (2.6.7) , carried out using 10 mL for each medium.
The strain of mumps virus used shall be identified by FINAL LOT
historical records that include information on the origin of A minimum virus concentration for release of the product is
the strain and its subsequent manipulation. Virus seed lots established such as to ensure, in light of stability data, that
are prepared in large quantities and stored at temperatures the minimum concentration stated on the label will be
below -20 °C if freeze-dried, or below -60 °C if not freeze- present at the end of the period of validity.
dried. Only a final lot that complies with the requirements for
Only a seed lot that complies with the following requirements minimum virus concentration for release, with the following
may be used for virus propagation. requirement for thermal stability and with each of the
Identification requirements given below under Identification and Tests may
be released for use. Provided that ±e tests for bovine serum
The master and working seed lots are identified as mumps
albumin and, where applicable, for ovalbumin have been
virus by serum neutralisation in cell culture, using specific
antibodies. carried out with satisfactory results on the final bulk vaccine,
Virus concentration
Thermal stability
The virus concentration of the master and working seed lots
Maintain at least 3 vials of the final lot of freeze-dried
is determined to ensure consistency of production.
vaccine in the dry state at 37 ± 1 °C for 7 days. Determine
Extraneous agents (2.6.76) the virus concentration as described under Assay in parallel
The working seed lot complies with the requirements for for the heated vaccine and for vaccine stored at the
seed lots. temperature recommended for storage. The virus
PROPAGATION AND HARVEST concentration of the heated vaccine is not more than 1.0
All processing of the cell bank and subsequent cell cultures is log10 lower than ±at of the unheated vaccine.
done under aseptic conditions in an area where no other cells IDENTIFICATION
are handled during the production. Suitable animal (but not When the vaccine reconstituted as stated on the label is
human) serum may be used in the culture media. Serum and mixed with specific mumps antibodies, it is no longer able to
trypsin used in the preparation of cell suspensions and infect susceptible cell cultures.
culture media are shown to be free from extraneous agents.
The cell culture medium may contain a pH indicator such as TESTS
phenol red and suitable antibiotics at the lowest effective Bacterial and fungal contamination
concentration. It is preferable to have a substrate free from The reconstituted vaccine complies with the test for sterility
antibiotics during production. Not less than 500 mL of the (2.6.7) .
production cell cultures is set aside as uninfected cell cultures Bovine serum albumin
(control cells). If the virus is propagated in chick embryos, Not more than 50 ng per single human dose, determined by
2 per cent but not less than 20 eggs are set aside as a suitable immunochemical method (2.7.7).
uninfected control eggs. The viral suspensions are harvested Ovalbumin
at a time appropriate to the strain of virus being used. If the vaccine is produced in chick embryos, it contains not
Only a single harvest that complies with the following more than 1 pg of ovalbumin per single human dose,
requirements may be used in the preparation of the final bulk determined by a suitable immunochemical method (2.7.7).
vaccine.
Water (2.5.12)
Not more than 3.0 per cent, determined by the semi-micro Human Papillomavirus Vaccine ; *.
determination of water. (rDNA)
ASSAY (Ph. Eur. monograph 2441)
Titrate the vaccine for infective virus, using at least The label may state ‘HPV’.
3 separate vials of vaccine and inoculating a suitable number Ph Eur________________________________________ ___ _ ________________
of wells for each dilution step. Titrate 1 vial of an
appropriate virus reference preparation in triplicate to DEFINITION
validate each assay. The virus concentration of the reference Human papillomavirus vaccine (rDNA) is a preparation of
preparation is monitored using a control chart and a titre is purified virus-like particles (VLPs) composed of the major
established on a historical basis by each laboratory. capsid protein (LI) of one or more human papillomavirus
The relation with the appropriate European Pharmacopoeia (HPV) genotypes; the antigens may be adsorbed on a
Biological Reference Preparation is established and mineral carrier such as aluminium hydroxide or hydrated
monitored at regular intervals if a manufacturer's reference aluminium phosphate. The vaccine may also contain the
preparation is used. Calculate the individual virus adjuvant 3-O-desacyl-4'-monophosphoryl lipid A.
concentration for each vial of vaccine and for each replicate The antigens are obtained by recombinant DNA technolog}’.
of the reference preparation as well as the corresponding PRODUCTION
combined virus concentrations, using the usual statistical GENERAL PROVISIONS
methods (for example, 5.3). The combined estimate of the The vaccine shall have been shown to induce specific
virus concentration for the 3 vials of vaccine is not less than neutralising antibodies in man. The production method shall
that stated on the label; the minimum virus concentration have been shown to yield consistently vaccines comparable in
stated on the label is not less than 3.7 log10 CCID50 per quality with the vaccine of proven clinical efficacy and safety
single human dose. in man.
The assay is not valid if: The production method is validated to demonstrate that the
— the confidence interval (P = 0.95) of the estimated virus product, if tested, would comply with the test for abnormal
concentration of the reference preparation for the toxicity for immunosera and vaccines for human use (2.6.9).
3 replicates combined is greater than ± 0.3
The vaccine is produced by the expression of the viral genes
logio CCID50;
coding for the capsid proteins in yeast or in an insect
— the virus concentration of the reference preparation differs
cell/baculoviruร expression vector system, purification of the
by more than 0.5 log10 CCID50 from the established
resulting VLPs and the rendering of these particles into an
value.
immunogenic preparation. The suitability and safety of the
The assay is repeated if the confidence interval (P = 0.95) of expression systems are approved by the competent authority.
the combined virus concentration of the vaccine is greater Production of the vaccine is based on a seed lot/cell bank
than ± 0.3 logI0 CCID50; data obtained from valid assays system. Unless otherwise justified and authorised, the virus
only are combined by ±e usual statistical methods (for and cells used for vaccine production shall not have
example, 5.3) to calculate the virus concentration of the undergone more passages from the master seed lot/cell bank
sample. The confidence interval (P = 0.95) of the combined than was used to prepare the vaccine shown in clinical
virus concentration is not greater than ± 0.3 logi0 CCID50. studies to be satisfactory' with respect to safety and efficacy.
Mumps vaccine (live) BRP is suitable for use as a reference Reference preparation A batch of vaccine shown to be effective
preparation. in clinical trials or a batch representative thereof is used as a
‘Where justified and authorised, different assay designs may reference vaccine. The reference vaccine is preferably
be used; this may imply the application of different validity stabilised and the stabilisation method shall have been shown
and acceptance criteria. However, the vaccine must comply if to have no significant effect on the assay validity.
tested as described above. CHARACTERISATION
LABELLING Characterisation of the VLPs is performed on lots produced
The label states: during vaccine development, including the process validation
— the strain of virus used for the preparation of the vaccine; batches. Characterisation includes protein composition, for
— that the vaccine has been prepared in chick embryos or example using techniques such as sodium dodecyl sulfate
the type and origin of cells used for the preparation of the polyacrylamide gel electrophoresis (SDS-PAGE) and Western
vaccine; blotting or mass spectrometry, peptide mapping and/or
— the minimum virus concentration; terminal amino acid sequence analysis. Morphological
— that contact between the vaccine and disinfectants is to be characteristics of the VLPs and degree of aggregation are
avoided. determined to confirm the presence of the conformational
epitopes that are essential for efficacy. VLP characterisation
----------------------------------------------------------------------------------------- -- Ph Eur
may be done by atomic force microscopy and transmission
electron microscopy, dynamic light scattering, epitope
mapping and reactivity with neutralising monoclonal
antibodies. In addition, the protein, lipid, nucleic acid and
carbohydrate content are measured where applicable.
The level of residual host-cell protein derived from insect
cells meets acceptable safety criteria as set by the competent
authority.
CELL BANKS AND SEED LOTS
Production in recombinant yeast cells Only cell banks that have
been satisfactorily characterised for identity, microbial purity.
growth characteristics and stability shall be used for Extraneous agents (2.6. / 6)
production. Gene homogeneity is studied for the master and The working seed lot complies with the requirements for
working cell banks. A full description of the biological seed lots and control cells. Special attention is given to
characteristics of the host cell and expression vectors is given. Spiroplasma spp. and insect-bome viruses, in particular
The physiological measures used to promote and control the insect-borne potential human pathogens (e.g. arboviruses).
expression of the cloned gene in the host cell are described in PROPAGATION AND HARVEST
detail. This includes genetic markers of the host cell, the All processing of the cell banks and baculovirus seed lots and
construction, genetics and structure of the expression vector, subsequent cell cultures is done under aseptic conditions in
and the origin and identification of the gene that is being an area where no other cells are being handled.
cloned. The nucleotide sequence of the gene insert and of
Where justified and authorised for production in an insect
adjacent segments of the vector and restriction-enzyme
cell/baculovirus expression vector system, a stored virus
mapping of the vector containing the gene insert are
intermediate culture that complies with the 5 following tests
provided. Data that demonstrates the stability of the
may be used for virus propagation.
expression system during storage of the recombinant working
cell bank up to or beyond the passage level used for Identification
production is provided. Each stored virus intermediate culture is identified by HPV
Production m an insect celUbaculovirus expression vector system type, by an immunological assay using specific antibodies or
by a molecular identity test such as NAT (2.6.2/).
Insect cell substrate. Only cell banks that have been
satisfactorily characterised for identity, purity, growth Bacterial and fungal contamination
characteristics, stability, extraneous agents and Each stored virus intermediate culture complies with the test
tumorigenicity shall be used for production. Such for sterility (2.6. /), carried out using 10 mL for each
characterisation is performed at suitable stages of medium.
production in accordance with general chapters 5.2.3. Cell Virus concentration
substrates for the production of vaccines for human use and The virus concentration of each stored baculovirus
2.6. ไ 6. Tests for extraneous agents in viral vaccines for intermediate culture is determined by a suitable method such
human use. Special attention is given to insect-borne as plaque assay or NAT (2.6.2/) in order to monitor
viruses, in particular insect-bomc potential human consistency of production.
pathogens (e.g. arboviruses). Adventitious infectious
Extraneous agents (2.6. / 6)
agents of insect cells may be without cytopathic effect. Each stored virus intermediate culture complies with the tests
Tests therefore include nucleic acid amplification
for extraneous agents.
techniques, and other tests such as electron microscopy
and co-cultivation. Control cells
— Recombinant baculovirus. 'rhe use of the recombinant The control cells of the production cell culture from which
baculovirus vector is based on a seed-lot system with a each stored virus intermediate is derived comply with a test
defined number of passages between the original virus for identity and with the requirements for extraneous agents
and the master and the working seed-lots, as approved by (2.6.16).
the competent authorities. The recombinant baculovirus Production in recombinant yeast cells Identity, microbial purity,
expression vector contains the coding sequence of the plasmid retention and consistency of yield are determined at
HPV LI antigen. Segments of the expression construct suitable production stages.
are analysed using nucleic acid amplification techniques Production in an insect celUbaculovirus expression vector system
in conjunction with other tests performed on the purified Insect cell cultures are inoculated with recombinant
recombinant protein for assuring the quality and baculovirus at a defined multiplicity of infection as approved
consistency of the expressed HPV LI antigens. by the competent authority. Several single harvests may be
The recombinant baculovirus used in the production of pooled before testing. No antibiotics are added at the time of
HPV vaccines is identified by historical records, which harvesting or at any later stage of manufacturing.
include information on the origin and identity of the gene SINGLE HARVESTS
being cloned as well as the construction, genetics and Only a single harvest or a pool of single harvests that
structure of the baculovirus expression vector(s). complies with the following requirements may be used in the
The genetic stability of the expression construct is preparation of the purified monovalent antigen.
demonstrated from the baculovirus master seed up to at
least the highest level used in production and preferably Identification
Each single harvest is identified as the appropriate HPV type
beyond this level.
by immunological assay or by a molecular biology-based
Recombinant baculovirus seed lots are prepared in large assay, for example hybridisation or polymerase chain
quantities and stored at temperatures favourable for stability.
reaction (PCR).
In case of production in an insect cell/baculovirus expression
Identification vector system the single harvest complies with the test for
The master and working seed lots are identified by the HPV sterility (2.6./). In case of production in yeast cells the single
type of the inserted gene of origin, by an appropriate method harvest is tested for culture purity by inoculation of suitable
such as nucleic acid amplification techniques (NAT) medium to ensure no growth other than the host organism.
(2.6.2/). Extraneous agents (2.6.16)
Virus concentration In case of production in an insect cell/baculovirus expression
The virus concentration of the master and working seed lots vector system the single harvest complies with the tests for
is determined to monitor consistency of production. extraneous agents. Special attention is given to insect-bome
viruses as mentioned under Cell banks and seed lots.
Control cells Chemicals used for disruption and purification

In case of production in an insect cell/baculovirus expression Tests for the chemicals used for purification or other stages
vector system the control cells comply with a test for of production are carried out. The content is within the
identification and with the requirements for extraneous limits approved for the particular products.
agents (2.6.76). Special attention is given to insect-bome Sterility (2.6.1)
viruses as mentioned under Cell banks and seed lots. Each purified monovalent antigen complies with the test,
PURIFIED MONOVALENT ANTIGEN carried out using 10 mL for each medium.
Harvests are purified using validated methods. When an ADSORBED /MONOVALENT ANTIGEN
insect cell/baculovirus expression vector system substrate is The purified monovalent antigens may be adsorbed onto a
used, the production process is validated for its capacity to mineral vehicle such as an aluminium salt.
eliminate (by removal and/or inactivation) adventitious
Only an adsorbed monovalent antigen that complies with the
viruses and recombinant baculoviruses.
Only a purified monovalent antigen that complies with the final bulk.
final bulk. In agreement with the competent authority one or Sterility (2.6.7)
Each adsorbed monovalent antigen complies with the test,
more of the tests mentioned below may be omitted if
performed on the adsorbed monovalent antigen.
Total protein Bacterial endotoxins (2.6.14}
Each adsorbed monovalent antigen is tested for bacterial
The total protein is determined by a validated method.
endotoxins. The content is within the limits approved for the
The content is within the limits approved for the particular
particular product.
product.
Antigen content and identification Antigen content and identification
Each antigen type is identified by a suitable immunochemical
The quantity and specificity of each antigen type is
method (2.7.1} such as radio-immunoassay (RIA), enzyme-
determined by a suitable immunochemical method (2.7.7)
linked immunosorbent assay (ELISA), immunoblot
such as radio-immunoassay (RIA), enzyme-linked
(preferably using a monoclonal antibody directed against a
immunosorbent assay (ELISA), immunoblot (preferably
protective epitope) or single radial diffusion.
using a monoclonal antibody directed against a protective
The antigen/protein ratio is determined.
epitope) or single radial diffusion. The antigen/protein ratio
may be determined and is within the limits approved for the Mineral vehicle concentration.
particular product. Where applicable, each adsorbed monovalent antigen is
tested for the content of mineral vehicle. The content is
Antigenic purity
within the limits approved for the particular product.
The purity of each purified monovalent antigen is determined
by a suitable method, such as SDS-PAGE with quantification ADSORBED 3-O-DESACYL-4'-MONOPHOSPHORYL LIPID A
by densitometric analysis, the limit of detection being BULK
1 per cent of impurities or better with respect to total If 3-O-desacyl-4'-monophosphoryl lipid A is included in the
protein. A reference preparation is used to validate each test. vaccine it complies with the monograph 3-O-desacyl-4'-
The protein purity is calculated as the ratio of the Ll monophosphoryl lipid A (2537). Where 3-O-desacyl-4'-
protein-related bands relative to the total protein bands, monophosphoryl lipid A is adsorbed prior to inclusion in the
expressed as a percentage. For the genotypes included in the vaccine, the adsorbed 3-O-desacyI-4'-monophosphoryl
vaccine, the value calculated for purity is within the limits lipid A bulk complies with the following requirements.
approved for the particular product. Degree of adsorption of 3-O-desacyl-4'-
Percent intact Ll monomer monophosphoryl lipid A
The antigenic purity assay serves also to assess the integrity The content of non-adsorbed 3-O-desacyl-4'-
of the Ll monomer. The percent intact Ll monomer is the monophosphoryl lipid A in the adsorbed 3-O-desacyl-4'-
ratio of the intact Ll monomer to the total protein, monophosphoryl lipid A bulk is determined by a suitable
expressed as a percentage. method, for example gas chromatographic quantification of
the 3-O-desacyl-4'-nionophosphoryl lipid A (2537) fatty acids in
VLP size and structure
the supernatant, evaporated to dryness, after centrifugation.
The size and structure of the VLPs is established and
monitored by a suitable method such as dynamic light pH (2.2.3}
scattering. The size is within the limits approved for the The pH is within the limits approved for the particular
particular product. preparation.
Composition Sterility (2.6.7)
The protein, lipid, nucleic acid and carbohydrate contents It complies with the test, carried out using 10 mL for each
are determined, where applicable. medium.
Host-cell and vector-derived DNA FINAL BULK VACCINE
Maximum 10 ng of DNA in a quantity of purified antigen The final bulk vaccine is prepared directly from each purified
equivalent to a single human dose of vaccine, determined in monovalent antigen HPV type or adsorbed purified
each monovalent purified antigen by sensitive methods. monovalent antigen HPV type. An antimicrobial preservative,
a mineral vehicle such as an aluminium salt and the adjuvant
Residual host-cell proteins 3-O-desacyl-4'-monophosphoryl lipid A may be included in
Tests for residual host-cell proteins are carried out.
the formulation of the final bulk.
The content is within the limits approved for the particular
product. Only a final bulk that complies with the following
requirements may be used in the preparation of the final loL
Antimicrobial preservative of a reference vaccine preparation, using for each dilution a

Where applicable, determine the amount of antimicrobial group of a suitable number of female mice of a suitable
preservative by a suitable chemical or physico-chemical strain. The vaccine is diluted in a solution of sodium
method. The amount is not less than 85 per cent and not chloride R containing the aluminium adjuvant used for the
greater than 115 per cent of the intended content. vaccine production. The mice are 6-8 weeks old at the time
Sterility (2.6.7) of injection, and each mouse is given a 0.5 mL injection.
The final bulk vaccine complies with the test, carried out A preimmunisation serum sample is taken prior to
using 10 mL for each medium. inoculation, and a final serum sample is taken at a defined
FINAL LOT time between days 21 and 28. Assay the individual sera for
Only a final lot that complies with each of the requirements specific neutralising antibodies against each HPV type by a
given below under Identification, Tests and Assay may be suitable immunochemical method (2.7.7).
released for use. Provided that the test for antimicrobial The test is not valid unless:
preservative content (where applicable) has been carried out — for both the vaccine to be examined and the reference
with satisfactory results on the final bulk vaccine, it may be vaccine, ±e ED50 lies between the smallest and the
omitted on the final lot. If an in vivo assay is carried out, largest doses given to the animals;
then provided it has been carried out with satisfactory results — the statistical analysis shows no significant deviation from
on the final bulk vaccine, it may be omitted on the final lot. linearity or parallelism;
Adjuvants — the confidence limits (P = 0.95) are within the limits
It the vaccine contains an adjuvant, the amount is approved for the particular product.
determined and shown to be within acceptable limits with In vitro test
respect to the expected amount. A suitable method for 3-0- Carry out an immunochemical determination (2.7.7) of each
desacyl-4'-monophosphoryl lipid A is, for example, gas antigen genotype content. Enzyme-linked immunosorbent
chromatography. assay (ELISA) and radio-immunoassay (RIA) using
Degree of adsorption monoclonal antibodies specific for protection-inducing
The degree of adsorption of each antigen and, where epitopes of the LI protein have been shown to be suitable.
applicable, 3-O-desacyl-4'-monophosphoryl lipid A is Suitable numbers of dilutions of the vaccine to be examined
assessed. and a manufacturer's reference preparation are used and a
suitable model is used to analyse the data. For each type, the
IDENTIFICATION
antigen content is within the limits approved for the
The vaccine is shown to contain the different types of HPV particular product.
antigen by a suitable immunochemical method (2.7.7).
The in vitro assay may serve to identify the vaccine. LABELLING
In addition, where applicable, the test for 3-O-desacyl-4'- The label states:
monophosphoryl lipid A content also serves to identify the — the amount of LI proteins and the genotype of HPV
3-O-desacyl-4'-monophosphoryl lipid A-containing vaccine. contained in the vaccine;
TESTS — the cell substrate used for production of the vaccine;
— the name and amount of the adjuvant and/or adsorbent
Aluminium (2.5.72)
used;
— that the vaccine must not be frozen.
adsorbent. ______________________________________________________________ Ph Eur
3-O-Desacyl-4'-monophosphoryl lipid A
Minimum 80 per cent and maximum 120 per cent of the
intended amount.
Where applicable, determine the content of 3-O-desacyl-4'- Pertussis Vaccine (Whole Cell, ******
monophosphoryl lipid A by a suitable method, for example Adsorbed) *****
gas chromatography (2.2.25).
The label may state ‘wP’.
Where applicable, determine the content of antimicrobial
preservative by a suitable chemical or physico-chemical Ph Eur_______________________________________________________________
method. The amount is not less than the minimum amount DEFINITION
shown to be effective and is not greater than 115 per cent of Pertussis vaccine (whole cell, adsorbed) is a sterile suspension
that stated on the label. of inactivated whole cells of one or more strains of Bordetella
Sterility (2.6.7) pertussis, treated to minimise toxicity and retain potency.
The vaccine complies with the test. The vaccine contains a mineral adsorbent such as hydrated
Bacterial endotoxins (2.6.14) aluminium phosphate or aluminium hydroxide.
iMaximum 5 IU per single human dose. If the adjuvant PRODUCTION
prevents the determination of endotoxin, a suitable in-process GENERAL PROVISIONS
test is carried out. The production process shall have been shown to yield
ASSAY consistently vaccines comparable with the vaccine of proven
The assay is performed by an in vivo test or an in vitro test clinical efficacy and safety in man.
having acceptance criteria established by correlation studies Levels of pertussis toxin, active heat-labile toxin
against an in vivo test. (dermonecrotic toxin) or tracheal cytotoxin must be
In vivo test comparable to the levels present in the vaccine of proven
A suitable in vivo assay method consists of the injection of clinical efficacy and safety in man and be approved by the
not fewer than 3 dilutions of the vaccine to be examined and competent authority.
CHOICE OF VACCINE STRAIN INACTIVATION AND DETOXIFICATION OF B. PERTUSSIS

The vaccine consists of a mixture of one or more strains of SUSPENSION
B. pertussis. Strains of B. pertussis used in preparing vaccines Inactivation is initiated as soon as possible after taking
are well characterised and chosen in such a way that the final samples of single harvests for purity control and opacity
vaccine contains predominantly phase I cells that display measurement. The bacteria are killed and detoxified in
fimbriae 2 and 3, as determined by an agglutination test or controlled conditions by means of a suitable chemical agent
other suitable immunochemical method (2.7.1). or by heating or by a combination of these methods.
SEED LOTS The suspension is maintained at 5 i 3 °C for a suitable
The production of pertussis vaccine is based on a seed-lot period to diminish its toxicity.
system. Only an inactivated monovalent cell bulk that complies with
The strains of B. pertussis used are identified by a full established specifications for the following tests may be used
historical record, including information on the origin of the in the preparation of the final bulk.
strain and its subsequent manipulation, characteristics on Residual live B. pertussis
isolation, and particularly on all tests carried out periodically Inactivation of the whole cells of B. pertussis is verified by a
to verify the strain's characters. suitable culture medium.
The media chosen for growing B. pertussis are carefully Pertussis toxin
selected and enable the micro-organism to retain phase I Presence of pertussis toxin is measured by a CHO cell
characteristics. culture assay using a semi-quantitative technique and range
When animal blood or animal blood products are used, they determined for the particular product.
are removed by washing ±e harvested bacteria. pH (2.2.3)
Human blood or human blood products are not used in any Within the range approved for the particular product.
culture media for propagating bacteria, either for seed or for Identification
vaccine. Verified by agglutination assay or suitable immunodiffusion
PROPAGATION AND HARVEST assay.
Each strain is grown separately from the working seed lot. Sterility (2.6.1)
Cultures are checked at different stages of fermentation It complies with the test for sterility, carried out using 10 mL
(subcultures and main culture) for purity, identity, cell for each medium.
opacity and pH. Unsatisfactory cultures must be discarded. Opacity
Production cultures are shown to be consistent in respect of The opacity of each single harvest is measured in the final
growth rate, pH and yield of cells or cell products. phase, at the end of the main fermentation process, by
The bacteria are harvested and may be washed to remove comparison with the International Reference Preparation of
substances derived from the medium and suspended in a Opacity. The equivalence in International Units of the
9 g/L solution of sodium chloride or other suitable isotonic International Reference Preparation is stated by the World
solution. Health Organization. The absorbance, for example measured
MONOVALENT CELL HARVEST
at 600 nm (2.2.25), is within the range approved for the
Consistency of production is monitored in respect of growth particular product.
rate, pH, yield and demonstration of characteristics of FINAL BULK
phase I organisms in the culture, such as presence of The final bulk vaccine is prepared by aseptically mixing
fimbriae 2 and 3 and haemolytic activity. Single harvests are suitable quantities of the inactivated single harvests.
not used for the final bulk vaccine unless they have been If 2 or more strains of B. pertussis are used, the composition
shown to contain B. pertussis cells with the same of consecutive lots of the final bulk vaccine shall be
characteristics with regard to growth and agglutinogens as the consistent with respect to the proportion of each strain as
parent strain, and to be free from contaminating bacteria and measured in opacity units. The bacterial concentration of the
fungi. final bulk vaccine does not exceed that corresponding to an
Only a monovalent harvest that complies with the following opacity of 20 IU per single human dose. The opacity
requirements may be used in further production. measured on the single harvests is used to calculate the
bacterial concentration in the final bulk. A mineral adsorbent
Purity
such as hydrated aluminium phosphate or aluminium
Samples of single harvests taken before inactivation are
hydroxide is added to the cell suspension. Suitable
examined by microscopy of Gram-Stained smears or by
antimicrobial preservatives may be added. Phenol is not used
inoculation into appropriate culture media or by another
suitable procedure. as a preservative.
Only a final bulk that complies with the following
Opacity
The opacity of each single harvest is measured not later than
2 weeks after harvest and before the bacterial suspension has Fimbriae
been subjected to any process capable of altering its opacity, Each bulk is examined, before adsorbent is added, for the
by comparison with the International Reference Preparation presence of fimbriae 2 and 3 to ensure that appropriate
of Opacity, and used as the basis of calculation for expression has occurred during bacterial growth.
subsequent stages in vaccine preparation. The equivalence in Sterility (2.6.1)
International Units of the International Reference It complies with the test for sterility, carried out using 10 mL
Preparation is stated by the World Health Organization. for each medium.
A spectrophotometric method validated against the opacity Antimicrobial preservative
reference preparation may be used and absorbance may, for Where applicable, determine the amount of antimicrobial
example, be measured at 600 nm (2.2.25). preservative by a suitable chemical or physico-chemical
method. The amount is not less than 85 per cent and not estimated potency is not less than 2.0 IU per single human
greater than 115 per cent of the intended amount. dose.
FINAL lot
LABELLING
The final bulk is mixed to homogeneity and filled aseptically The label states:
into suitable containers. — the minimum number of International Units per single
Only a final lot that is within the limits approved for the human dose;
particular product and is satisfactory with respect to each of — the method used for inactivation;
the requirements given below under Identification, Tests and — the name and the amount of the adsorbent;
Assay may be released for use. Provided the tests for specific — that the vaccine must be shaken before use;
toxicity, free formaldehyde and antimicrobial preservative and — that the vaccine is not to be frozen.
the determination of potency have been carried out with _____________________________________________________________ Ph Eur
satisfactory results on the final bulk vaccine, they may be
IDENTIFICATION
citrate R to give a 100 g/L solution. Maintain at 37 °C for Adsorbed Pertussis Vaccine * *
about 16 h and centrifuge to obtain a bacterial precipitate. (Acellular Component) *****
Identity of pertussis vaccine is based on an immunological
(Pertussis Vaccine (Acellular, Component, Adsorbed),
reaction, for example agglutination of the resuspended
bacteria with a specific anti-pertussis serum or another Ph Eur monograph 1356)
suitable immunochemical method {2.7.7). The label may state ‘aP’.
TESTS PhEir______________________________________________________________
Specific toxicity DEFINITION

Use not fewer than 5 healthy mice each weighing 14-16 g for Pertussis vaccine (acellular, component, adsorbed) is a
the vaccine group and for the saline control. Use mice of the preparation of individually prepared and purified antigenic
same sex or distribute males and females equally between the components of BordeteUa pertussis adsorbed on a mineral
groups. Inject each mouse of the vaccine group carrier such as aluminium hydroxide or hydrated aluminium
intraperitoneally with 0.5 mL, containing a quantity of the phosphate.
vaccine equivalent to not less than half the single human The vaccine contains either pertussis toxoid or a pertussis
dose. Inject each mouse of the control group with 0.5 mL of toxin-like protein free from toxic properties, produced by
a 9 g/L sterile solution of sodium chloride R, preferably expression of a genetically modified form of the
containing, where applicable, the same amount of corresponding gene. Pertussis toxoid is prepared from
antimicrobial preservative as that injected with the vaccine. pertussis toxin by a method that renders the latter harmless
Weigh the groups of mice immediately before the injection while maintaining adequate immunogenic properties and
and 72 h and 7 days after the injection. The vaccine avoiding reversion to toxin. The vaccine may also contain
complies with the test if: (a) at the end of 72 h the average filamentous haemagglutinin, pertactin (a 69 kDa outer
weight of the group of vaccinated mice is not less than that membrane protein) and other defined components of
preceding the injection; (b) at the end of 7 days the average B. pertussis such as fimbrial-2 and fimbrial-3 antigens.
increase in mass per vaccinated mouse is not less than The latter 2 antigens may be co-purified. The antigenic
60 per cent of that per control mouse; and (c) not more than composition and characteristics are based on evidence of
5 per cent of the vaccinated mice die during the test. If ±e protection and freedom from unexpected reactions in the
rest is carried out using 5 mice and 1 vaccinated mouse dies, target group for which the vaccine is intended.
the test may be repeated using 15 mice and the results of
both tests combined.
PRODUCTION
GENERAL PROVISIONS
Aluminium {2.5.13) The production method shall have been shown to yield
Maximum 1.25 mg per single human dose, if aluminium consistently vaccines comparable with the vaccine of proven
hydroxide or hydrated aluminium phosphate is used as the clinical efficacy and safety in man.
adsorbent.
Where a genetically modified form of B. pertussis is used,
Free formaldehyde {2.4.18) production consistency and genetic stability shall be
Maximum 0.2 g/L, where applicable. established in conformity with the requirements of the
Antimicrobial preservative monograph Products of recombinant DNA technology (0784).
Where applicable, determine the amount of antimicrobial Reference vaccine A batch of vaccine shown to be effective in
preservative by a suitable chemical method. The content is clinical trials or a batch representative thereof is used as a
not less than the minimum amount shown to be effective and reference vaccine. For the preparation of a representative
is not greater than 115 per cent of the quantity stated on the batch, strict adherence to the production process used for the
label. batch tested in clinical trials is necessary. The reference
Sterility (2.6.1) vaccine is preferably stabilised by a method that has been
It complies with the test for sterility. shown to have no significant effect on the assay procedure
when the stabilised and non-stabilised batches are compared.
ASSAY
CHARACTERISATION OF COMPONENTS
Carry out the assay of pertussis vaccine (whole cell) (2.7.7).
During development of the vaccine, the production process
The estimated potency is not less than 4.0 IU per single shall be validated to demonstrate that it yields consistently
human dose and the lower confidence limit {P = 0.95) of the individual components that comply with the following
requirements; after demonstration of consistency, the tests Only purified components that comply with the following
need not be applied routinely to each batch. requirements may be used in the preparation of the final bulk
Adenylate cyclase vaccine.
Not more than 500 ng in the equivalent of 1 dose of the final Sterility (2.6.1)
vaccine, determined by immunoblot analysis or another Carry out the test for sterility using for each medium a
suitable method. quantity of purified component equivalent to not less than
Tracheal cytotoxin 100 doses.
Not more than 2 pmol in the equivalent of 1 dose of the final Residual pertussis toxin (2.6.33)
vaccine, determined by a suitable method such as a biological It complies with the test.
assay or liquid chromatography (2.2.29). A validated test based on the clustering effect of the toxin for
Absence of residual dermonecrotic toxin Chinese hamster ovary (CHO) cells may be used instead of
Inject intradermally into each of 3 unweaned mice, in the test in mice.
a volume of 0.1 mL, the amount of component or antigenic Residual detoxifying agents and other reagents
fraction equivalent to 1 dose of the final vaccine. Observe for The content of residual detoxifying agents and other reagents
48 h. No dermonecrotic reaction is demonstrable. is determined and shown to be below approved limits unless
Specific properties validation of the process has demonstrated acceptable
The components of the vaccine are analysed by one or more clearance.
of the methods shown below in order to determine their Antigen content
identity and specific properties (activity per unit amount of Determine the antigen content by a suitable
protein) in comparison with reference preparations. immunochemical method (2.7.1) and protein nitrogen by
Pertussis toxin Chinese hamster ovary (CHO) cell-clustering sulfuric acid digestion (2.5.9) or another suitable method.
effect and haemagglutination as in vitro methods; The ratio of antigen content to protein nitrogen is within the
lymphocytosis-promoting activity, histamine-sensitising limits established for the product.
activity and insulin secretory activity as in vivo methods. FINAL BULK VACCINE
The toxin shows ADP-ribosyl transferase activity using The vaccine is prepared by adsorption of suitable quantities
transducin as the acceptor. of purified components, separately or together, onto
Filamentous haemagglutinin Haemagglutination and inhibition aluminium hydroxide or hydrated aluminium phosphate.
by a specific antibody. A suitable antimicrobial preservative may be added.
Pertactin, fimbrial-2 and fimbrial-3 antigens Reactivity with Only a final bulk vaccine that complies with the following
specific antibodies. requirements may be used in the preparation of the final lot.
Pertussis toxoid The toxoid induces in animals production of Antimicrobial preservative
antibodies capable of inhibiting all the properties of pertussis Where applicable, determine the amount of antimicrobial
toxin. preservative by a suitable chemical or physico-chemical
PURIFIED COMPONENTS method. The amount is not less than 85 per cent and not
Production of each component is based on a seed-lot system. greater than 115 per cent of the intended content.
The seed cultures from which toxin is prepared are managed Sterility (2.6.1)
to conserve or, where necessary, restore toxinogenicity by Carry out the test for sterility using 10 mL for each medium.
deliberate selection.
FINAL LOT
None of ±e media used at any stage contains blood or blood Only a final lot that is satisfactory with respect to each of the
products of human origin. Media used for the preparation of requirements given below under Identification, Tests and
seed lots and inocula may contain blood or blood products of Assay may be released for use. Provided that the tests for
animal origin. residual pertussis toxin and irreversibility of pertussis toxoid,
Pertussis toxin and, where applicable, filamentous antimicrobial preservative, free formaldehyde and the assay
haemagglutinin and pertactin are purified and, after have been carried out with satisfactory results on the final
appropriate characterisation, detoxified using suitable bulk vaccine, these tests may be omitted on the final lot.
chemical reagents, by a method that avoids reversion of the
IDENTIFICATION
toxoid to toxin, particularly on storage or exposure to heat.
Subject the vaccine to a suitable desorption procedure such
Other components such as fimbrial-2 and fimbrial-3 antigens
as the following: dissolve in the vaccine to be examined
are purified either separately or together, characterised and
sufficient sodium citrate R to give a 10 g/L solution; maintain
shown to be free from toxic substances. The purification
at 37 °C for about 16 h and centrifuge until a clear
procedure is validated to demonstrate appropriate clearance
supernatant is obtained. Examined by a suitable
of substances used during culture or purification.
immunochemical method (2.7.1), the clear supernatant reacts
The content of bacterial endotoxins (2.6.14) is determined to with specific antisera to the components stated on the label.
monitor the purification procedure and to limit the amount
in the final vaccine. The limits applied for the individual TESTS
components are such that the final vaccine contains less than Residual pertussis toxin and irreversibility of pertussis
100 IU per single human dose. toxoid (2.6.33)
The final lot complies with the test.
Before detoxification, the purity of the components is
determined by a suitable method such as polyacrylamide gel Aluminium (2.5.13)
electrophoresis (PAGE) or liquid chromatography. SDS- Maximum 1.25 mg per single human dose, if aluminium
PAGE or immunoblot analysis with specific monoclonal or hydroxide or hydrated aluminium phosphate is used as the
polyclonal antibodies may be used to characterise subunits. adsorbent.
Requirements are established for each individual product.
Free formaldehyde {2.4.18) Reference vaccine A batch of vaccine shown to be effective in

Maximum 0.2 g/L. clinical trials or a batch representative thereof is used as a
•Antimicrobial preservative reference vaccine. For the preparation of a representative
Where applicable, determine the amount of antimicrobial batch, strict adherence to the production process used for the
preservative by a suitable chemical or physico-chemical batch tested in clinical trials is necessary. The reference
method. The amount is not less than the minimum amount vaccine is preferably stabilised, by a method that has been
shown to be effective and is not greater than 115 per cent of shown to have no significant effect on the assay procedure
the quantity stated on the label. when the stabilised and non-stabilised batches are compared.
Sterility (2.6.1) CHARACTERISATION OF COMPONENTS
It complies with the test for sterility. During development of the vaccine, the production process
shall be validated to demonstrate that it yields consistently an
assay
antigenic fraction that complies with the following
Carry out one of the prescribed methods for the assay of requirements; after demonstration of consistency, the tests
pertussis vaccine (acellular) {2.7.16). need not be applied routinely to each batch.
The capacity of the vaccine to induce antibodies for each
Adenylate cyclase
included acellular pertussis antigen is not significantly Not more than 500 ng in the equivalent of 1 dose of the final
(P= 0.95) less than that of the reference vaccine.
vaccine, determined by immunoblot analysis or another
LABELLING suitable method.
The label states: Tracheal cytotoxin
the names and amounts of the components present in the Not more than 2 pmol in the equivalent of 1 dose of the final
vaccine; vaccine, determined by a suitable method such as a biological
where applicable, that the vaccine contains a pertussis assay or liquid chromatography (2.2.29).
toxin-like protein produced by genetic modification;
Absence of residual dermonecrotic toxin
— the name and amount of the adsorbent;
Inject intradermally into each of 3 unweaned mice, in a
that the vaccine must be shaken before use;
volume of 0.1 mL, the amount of antigenic fraction
— that the vaccine is not to be frozen.
equivalent to 1 dose of the final vaccine. Observe for 48 h.
————— ------------------------------------------------------------------------ Ph Eur No dermonecrotic reaction is demonstrable.
Specific properties
The antigenic fraction is analysed by one or more of the
methods shown below in order to determine the identity and
specific properties (activity per unit amount of protein) of its
Adsorbed Pertussis Vaccine components in comparison with reference preparations.
(Acellular, Co-purified) Pertussis toxin Chinese hamster ovary (CHO) cell-clustering
(Pertussis Vaccine (Acellular, Co-purified, Adsorbed), effect and haemagglutination as in vitro methods;
Ph Eur monograph 1595) lymphocytosis-promoting activity, histamine-sensitising
The label may state ‘aP’. activity and insulin secretory activity as in vivo methods.
The toxin shows ADP-ribosyl transferase activity using
transducin as the acceptor.
DEFINITION Filamentous haemagglutinin Haemagglutination and inhibition
Pertussis vaccine (acellular, co-purified, adsorbed) is a by a specific antibody.
preparation of antigenic components of BordeteUa pertussis Pertactin, fimbrial-2 and fimbrial-3 antigens Reactivity with
adsorbed on a mineral carrier such as aluminium hydroxide specific antibodies.
or hydrated aluminium phosphate.
Pertussis toxoid The toxoid induces in animals the production
The vaccine contains an antigenic fraction purified without of antibodies capable of inhibiting all the properties of
separation of the individual components. The antigenic pertussis toxin.
fraction is treated by a method that transforms pertussis toxin
PURIFIED ANTIGENIC FRACTION
to toxoid, rendering it harmless while maintaining adequate
immunogenic properties of all the components and avoiding Production of the antigenic fraction is based on a seed-lot
reversion to toxin. The antigenic fraction is composed of system. The seed cultures are managed to conserve or, where
pertussis toxoid, filamentous haemagglutinin, pertactin (a necessary, restore toxinogenicity by deliberate selection.
69 kDa outer-membrane protein) and other defined None of the media used at any stage contains blood or blood
components of B. pertussis such as fimbrial-2 and fimbrial-3 products of human origin. Media used for the preparation of
antigens. It may contain residual pertussis toxin up to a seed batches and inocula may contain blood or blood
maximum level approved by the competent authority. products of animal origin.
The antigenic composition and characteristics are based on The antigenic fraction is purified and, after appropriate
evidence of protection and freedom from unexpected characterisation, detoxified using suitable reagents by a
reactions in the target group for which the vaccine is method that ensures minimal reversion of toxoid to toxin,
intended. particularly on storage or exposure to heat. The purification
procedure is validated to demonstrate appropriate clearance
PRODUCTION
of substances used during culture or purification.
general provisions
The production method shall have been shown to yield The content of bacterial endotoxins {2.6.14) is determined to
Consistently vaccines comparable with the vaccine of proven monitor the purification procedure and to limit the amount
clinical efficacy and safety in man. in the final vaccine. The limits applied are such that the final
vaccine contains not more than 100 IU per single human immunochemical method (2.7.1), the clear supernatant reacts
dose. with specific antisera to the components in the vaccine.
Before detoxification, the purity of the antigenic fraction is TESTS
determined by a suitable method such as polyacrylamide gel Residual pertussis toxin and irreversibility of pertussis
electrophoresis (PAGE) or liquid chromatography. SDS- toxoid (2.6.33)
PAGE or immunoblot analysis with specific monoclonal or The final lot complies with the test.
polyclonal antibodies may be used to characterise subunits.
Requirements are established for each individual product. Antimicrobial preservative
Only a purified antigenic fraction that complies with the preservative by a suitable chemical or physico-chemical
following requirements may be used in the preparation of the method. The amount is not less than the minimum amount
final bulk vaccine.
shown to be effective and is not greater than 115 per cent of
Sterility the quantity stated on the label.
Carry out the test for sterility (2.6. ไ) using for each medium
Aluminium (2.5.13)
a quantity of purified antigenic fraction equivalent to not less Maximum 1.25 mg per single human dose, if aluminium
than 100 doses of the final vaccine.
Residual pertussis toxin (2.6.33) adsorbent.
The purified antigenic fraction complies with the test. Free formaldehyde (2.4.18)
A validated test based on the clustering effect of the toxin for Maximum 0.2 g/L.
Chinese hamster ovary (CHO) cells may be used instead of
Sterility
the test in mice.
It complies with the test for sterility (2.6.1).
Residual detoxifying agents and other reagents
The content of residual detoxifying agents and other reagents ASSAY
is determined and shown to be below approved limits unless Carry out one of the prescribed methods for the assay of
validation of the process has demonstrated acceptable pertussis vaccine (acellular) (2.7.16).
clearance. The capacity of the vaccine to induce antibodies for each
Antigen content
Determine the complete quantitative antigen composition of
the antigenic fraction by suitable immunochemical methods LABELLING
(2.7.7) and protein nitrogen by sulfuric acid digestion (2.5.9) The label states:
or another suitable method. The ratio of total antigen — the names and amounts of the antigenic components
content to protein nitrogen is within the limits established for present in the vaccine,
±e product. — the maximum amount of residual pertussis toxin present
FINAL BULK VACCINE in the vaccine,
The vaccine is prepared by adsorption of a suitable quantity — the maximum degree of reversion of toxoid to toxin
of the antigenic fraction onto aluminium hydroxide or during the period of validity,
hydrated aluminium phosphate. A suitable antimicrobial — the name and amount of the adsorbent,
preservative may be added. — that the vaccine must be shaken before use,
requirements may be used in the preparation of the final lot. _________________________________________________ ______________ Pn E-r
method. The amount is not less than 85 per cent and not Pneumococcal Polysaccharide ** *
greater than 115 per cent of the intended content.
Vaccine *****
Sterility
The final bulk vaccine complies with the test for sterility
(2.6.7) , carried out using 10 mL for each medium. The label may state 'Pneumo7.
Ph Eur________________________________________________ ___———
FINAL LOT
Only a final lot that is satisfactory with respect to each of the DEFINITION
requirements given below under Identification, Tests and Pneumococcal polysaccharide vaccine consists of a mixture of
Assay may be released for use. equal parts of purified capsular polysaccharide antigens
Provided that the tests for residual pertussis toxin and prepared from suitable pathogenic strains of Streptococcus
irreversibility of pertussis toxoid, antimicrobial preservative, pneumoniae whose capsules have been shown to be made up
free formaldehyde and the assay have been carried out with of polysaccharides that are capable of inducing satisfactory
satisfactory results on the final bulk vaccine, these tests may levels of specific antibodies in man. It contains the
be omitted on the final lot. 23 immunochemically different capsular polysaccharides
listed in Table 0966-1.
IDENTIFICATION
The vaccine is a clear, colourless liquid.
Subject the vaccine to a suitable desorption procedure such
as the following: dissolve in the vaccine to be examined PRODUCTION
sufficient sodium curate R to give a 10 g/L solution; maintain Production of the vaccine is based on a seed-lot system for
at 37 °C for about 16 h and centrifuge until a clear each type. The production method shall have been shown to
supernatant is obtained. Examined by a suitable yield consistently pneumococcal polysaccharide vaccines of
adequate safety and immunogenicity in man.
2016 Vaccines rV-633
Table 0966.-1 - Percentage contents of components of monovalent bulk polysaccharides

Molecular Nucleic Total Molecular size Uronic Hexos Methyl O-acetyl
type* Protein Phosphorus
acids nitrogen (JQ acids amines pentoses Groups
•• •••
1 < 2 <2 3.5-6 0-1.5 ร 0.15 £ 45 £ 1.8
< 2 <2 0-1 0-1.0 $0.15 £ 15 £ 38
< 5 $ 2 0-1 0-1.0 $0.15 £ 40
4 £3 < 2 4-6 0-1.5 $ 0.15 > 40
5 $ 7.5 < 2 2.5-6.0 $2 $0.60 £ 12 £ 20
6B $2 < 2 0-2 2.5-5.0 $0.50 £ 15
7F $5 $ 2 1.5-4.0 0-1.0 $0.20 £13
8 $2 $2 0-1 0-1.0 $0.15 £ 25
9N $2 $ 1 2.2-4 0-1.0 $0.20 £ 20 £ 28
9V $2 < 2 0.5-3 0-1.0 $ 0.45 £ 15 £ 13
10A $า $ 2 0.5-3.5 1.5-3.5 $0.65 £ 12
11A < 3 < 2 0-2.5 2.0-5.0 $0.40 £9

12F ร 3 < 2 3-5 0-1.0 $0.25 £ 25
14 $ 5 ร 2 1.5-4 0-1.0 $ 0.30 £ 20

15B £ 3 ร 2 1-3 2.0-4.5 $0.55 £ 15
17A orl7F $2 < 2 0-1.5 0-3.5 $0.45 £ 20
18C $ 3 $ 2 0-1 . 2.4-4.9 $ 0.15 £ 14
19A <1 2 < 2 0.6-3.5 3.0-7.0 $0.45 £ 12 £ 20
19F <. 3 $2 1.4-3.5 3.0-5.5 $ 0.20 £ 12.5 £ 20
20 $2 $2 0.5-2.5 1.5-4.0 $0.60 £ 12
22F <2 $2 0-2 0-1.0 $ 0.55 £ 15 £ 25
23F < 2 < 2 0-1 3.0-4.5 $0.15 £ 37
33F $ 2.5 £2 0-2 0-1.0 $0.50
• The different types are indicated using the Danish nomenclature.
•’ Cross-linked agarose for chromatography R.
•** Cross-linked agarose for chromatography Rl.
The production method is validated to demonstrate that the Only a monovalent bulk polysaccharide that complies with
product, if tested, would comply with the test for abnormal the following requirements may be used in the preparation of
toxicity for immunosera and vaccines for human use (2.6.9) the final bulk vaccine. Percentage contents of components,
modified as follows for the test in guinea-pigs: inject 10 determined by the methods prescribed below, are shown in
human doses into each guinea-pig and observe for 12 days. Table 0966-1.
MONOVALENT BULK POLYSACCHARIDES Protein (2.5.16)
The bacteria are grown in a suitable liquid medium that does Nucleic acids (2.5.17)
not contain blood-group substances or high-molecular-mass
Total nitrogen (2.5.9)
polysaccharides. The bacterial purity of the culture is verified
and the culture is inactivated with phenol. Impurities are Phosphorus (2.5.18)
removed by such techniques as fractional precipitation, Molecular size
enzymatic digestion and ultrafiltration. The polysaccharide is Determine by size-exclusion chromatography (2.2.30) using
obtained by fractional precipitation, washed, and dried in a cross-linked agarose for chromatography R or cross-linked agarose
vacuum to a residual moisture content shown to be for chromatography Rl.
favourable to the stability of the polysaccharide. The residual Uronic acids (2.5.22)
moisture content is determined by drying under reduced
pressure over diphosphorus pentoxide or by Hexosamines (2.5.20)
therm ©gravimetric analysis and the value obtained is used to Methylpentoses (2.5.21)
calculate the results of the tests shown below with reference O-Acetyl groups (2.5.19)
to the dried substance. The monovalent bulk polysaccharide
Identification (2.7.1)
is stored at a suitable temperature in conditions that avoid
Confirm the identity of the monovalent bulk polysaccharide
the uptake of moisture.
by double immunodiffusion or electroimmunodiffusion
(except for polysaccharides 7F, 14 and 33F), using specific each polysaccharide contained in the vaccine, including factor
antisera. sera for types within groups, and purified polysaccharides of
Specificity each type as standards.
No reaction occurs when the antigens are tested against all The vaccine contains not less than 70 per cent and not more
the antisera specific for the other polysaccharides of the than 130 per cent of the quantity stated on the label for each
vaccine, including factor sera for distinguishing types within polysaccharide. The confidence limits (P = 0.95) are not less
groups. The polysaccharides are tested at a concentration of than 80 per cent and not more than 120 per cent of the
50 pg/mL using a method capable of detecting 0.5 pg/mL. estimated content.
FINAL BULK VACCINE LABELLING
The final bulk vaccine is obtained by aseptically mixing the The label states:
different polysaccharide powders. The uniform mixture is — the number of micrograms of each polysaccharide per
aseptically dissolved in a suitable isotonic solution so that one human dose,
human dose of 0.50 mL contains 25 pg of each — the total amount of polysaccharide in the container.
polysaccharide. An antimicrobial preservative may be added. _______ Ph Elf
The solution is sterilised by filtration through a bacteria-
retentive filter.
Only a final bulk vaccine that complies with the folloxring
Antimicrobial preservative Pneumococcal Polysaccharide * \
Where applicable, determine the amount of antimicrobial Conjugate Vaccine (Adsorbed) *****
not less than 85 per cent and not greater than 115 per cent (Ph. Eur. monograph 2150)
of the intended amount. The label may state ‘Pneumo(conj)’.
Sterility (2.6. /) Ph Eur_______________________________________ __ __________________ __
The final bulk vaccine complies with the test for sterility, DEFINITION
using 10 mL for each medium. Pneumococcal polysaccharide conjugate vaccine (adsorbed) is
FINAL LOT a sterile suspension of purified capsular polysaccharides
The final bulk vaccine is distributed aseptically into sterile, obtained from Streptococcus pneumoniae serotypes individually
tamper-proof containers. conjugated to a carrier protein. The carrier protein used may
Only a final lot that is satisfactory with respect to each of the vary for the various polysaccharide conjugates contained in a
requirements given below under identification, tests and assay multivalent vaccine. The vaccine may be adsorbed on a
may be released for use. Provided that the tests for phenol suitable adjuvant or adsorbant.
and for antimicrobial preservative have been carried out with Each serotype, produced from suitable pathogenic strains of
satisfactory results on the final bulk vaccine, they may be ร. pneumoniae, is grown in an appropriate medium.
omitted on the final lot. When consistency of production has The individual polysaccharides are purified through suitable
been established on a suitable number of consecutive purification methods (for example centrifugation,
batches, the assay may be replaced by a qualitative test that precipitation, ultrafiltration and column chromatography).
identifies each polysaccharide, provided that an assay has Each polysaccharide has a defined composition and a defined
been performed on each monovalent bulk polysaccharide molecular size range.
used in the preparation of the final lot.
The choice of polysaccharide depends on the frequency of
IDENTIFICATION the serotypes responsible for acute pathologies and their
The assay serves also to identify the vaccine. geographical distribution. The vaccine contains
TESTS immunochemically different capsular polysaccharides.
pH (2.2.5) PRODUCTION
The pH of the vaccine is 4.5 to 7.4. GENERAL PROVISIONS
Antimicrobial preservative The production method shall have been shown to yield
Where applicable, determine the amount of antimicrobial consistently ร. pneumoniae conjugate vaccines of adequate
preservative by a suitable chemical method. The content is safety and immunogenicity in man. The production of
not less than the minimum amount shown to be effective and polysaccharides and of the carrier(s) is based on a seed-lot
is not greater than 115 per cent of the quantity stated on the system.
label. During development studies and wherever revalidation is
Phenol (2.5.75) necessary, a test for pyrogens in rabbits (2.6.8) is carried out.
Not more than 2.5 g/L. The vaccine is shown to be acceptable with respect to
absence of pyrogenic activity.
Sterility (2.6.7)
Pyrogens (2.6. ร) toxicity for immunosera and vaccines for human use (2.6.9).
It complies wi± the test for pyrogens. Inject per kilogram of During development studies and whenever revalidation of the
the rabbit's mass 1 mL of a dilution of the vaccine manufacturing process is necessary, it shall be demonstrated
containing 2.5 pg/mL of each polysaccharide. by tests in animals that the vaccine consistently induces a
ASSAY T-cell-dependent B-cell immune response.
Determine the content of each polysaccharide by a suitable The stability of the conjugated bulk and/or final lot and
immunochemical method (2.7.1) 3 using antisera specific for pneumococcal saccharide is evaluated using suitable indicator
tests. Such tests may include determination of molecular size, An acceptable value for each reagent is established for the
quantification of saccharide content and free polysaccharide particular product and each batch of polysaccharide must be
content in the conjugate. shown to comply with this limit. Where validation studies
bacterial seed lots have demonstrated removal of residual reagents, the test on
The bacterial strains used for master seed lots shall be polysaccharides may be omitted.
identified by historical records that include information on Water
their origin and the tests used to characterise the strain. Where applicable, the values are within the limits approved
Cultures obtained from the working seed lot shall have the for each serotype, determined by a suitable method.
same characteristics as the strain that was used to prepare the Depending on the chemical composition of a pneumococcal
master seed lot. polysaccharide serotype, not all of the following tests may be
fhinty of bacterial cultures is verified by methods of suitable applicable. The values are within the limits approved.
sensitivity. These may include inoculation into suitable Suitable limits for some pneumococcal polysaccharide
media, examination of colony morphology, microscopic serotypes are given in the monograph Pneumococcal
examination of Gram-Stained smears and culture polysaccharide vaccine (0966).
agglutination with suitable specific antisera. Total nitrogen (2.5.9)
PNEUMOCOCCAL POLYSACCHARIDES Phosphorus (2.5.18)
Each strain of ร. pneumoniae serotypes is individually grown
Uronic acids (2.5.22)
in a liquid medium that does not contain high-molecular-
mass polysaccharides; if any ingredient of the medium Hexosamines (2.5.20)
contains blood-group substances, the process is validated to Methylpentoses (2.5.21)
demonstrate that after the purification step they are no longer O-Acetyl groups (2.5.19)
detectable. The bacterial purity of the culture is verified by MODIFIED PNEUMOCOCCAL POLYSACCHARIDES
suitable methods. The culture is then inactivated. Each Before conjugation, the polysaccharide can be depolymerised
polysaccharide is separated from the liquid culture and by chemical or mechanical means followed by a
purified by suitable methods. Volatile matter, including concentration step to obtain polysaccharides of a desired
water, in the purified polysaccharide is determined by a molecular size range. Polysaccharides or depolymerised
suitable method such as thermogravimetry (2.2.34)) semi polysaccharides are modified by an activation process.
micro determination of water (2.5.12) or, where applicable,
Only modified polysaccharides that comply with the
determination of solvent and/or alcohol content by
spectrometry. The values are used to calculate the results of
conjugate.
other chemical tests with reference to the dried substance, as
prescribed below. Molecular size
In the case of a size-reduced modified pneumococcal
Only polysaccharides that comply with the following
polysaccharide, the molecular size is evaluated by liquid
requirements may be used in the preparation of the
conjugate. chromatography (2.2.29) with MALLS detection or other
suitable methods, such as size-exclusion chromatography
Identification (2.2.30) using cross-linked agarose for chromatography R or
Each polysaccharide is identified by an immunochemical cross-linked agarose for chromatography Rl. The values are
method (2.7.1) or other suitable methods, for example within the limits approved for each serotype.
lH nuclear magnetic resonance spectrometry (2.2.33).
Degree of oxidation
Protein (2.5.16) Where applicable, the degree of oxidation is represented by
Depending on the serotype used, not more than the limit the ratio of moles of saccharide repeat unit per mole of
approved for the product, calculated with reference to ±e aldehyde and determined by a suitable method. The values
dried substance. are within the limits approved for each serotype.
Nucleic acid (2.5.17) CARRIER PROTEIN
Depending on the serotype used, not more than the limit The production and characteristics of the carrier proteins are
approved for the product, calculated with reference to the described in general chapter 5.2.11. Carrier proteins for the
dried substance. production of conjugated polysaccharide vaccines for human use.
Molecular size Only a carrier protein that complies with the requirements of
'rhe molecular size is evaluated by liquid chromatography this chapter may be used in the preparation of the conjugate.
(2.2.29) with multiple-angle laser light scattering detection MONOVALENT BULK CONJUGATE
(MALLS) or other suitable methods, such as size-exclusion The conjugate is obtained by the covalent binding of
chromatography (2.2.30) using cross-linked agarose for activated polysaccharides to the appropriate carrier protein.
chromatography R or cross-linked agarose for chromatography Rl. The conjugate purification procedures are designed to
The values are within the limits approved for each serotype. remove residual reagents used for conjugation. The removal
A validated determination of the degree of polymerisation or of residual reagents is confirmed by suitable tests or by
of the weight-average molecular weight and the dispersion of validation of the purification process.
molecular masses may be used instead of the determination
Only a bulk conjugate that complies with the following
of molecular-size distribution.
Bacterial endotoxins (2.6.14) vaccine. For each test, limits of acceptance are established
Less than 0.5 IU per microgram of polysaccharide. and each batch of conjugate must be shown to comply with
Residual reagents these limits.
Where applicable, suitable tests are carried out to determine
residues of reagents used during inactivation and purification.
Saccharide method (2.7./). The value complies with the requirement

The polysaccharide content is determined by a suitable approved for each serotype.
physical or chemical method or by an immunochemical Free saccharide
method (2.7./). The value complies with the requirement Centrifuge the adsorbed monovalent bulk conjugate. In the
approved for each serotype. supernatant the unbound polysaccharide is determined after
Protein removal of the conjugate, for example by anion-exchange,
The protein content is determined by a suitable physical or size-exclusion or hydrophobic liquid chromatography,
chemical method (for example, 2.5.16). The value complies ultrafiltration, or other validated methods. An acceptable
with the requirement approved for each serotype. value consistent with adequate immunogenicity as shown in
Saccharide-to-protein ratio clinical trials is established for each serotype and each lot
Determine the saccharide-to-protein ratio by calculation. must be shown to comply with this limit.
The value complies with the requirement approved for each Degree of adsorption
serotype. The degree of adsorption of each polysaccharide conjugate is
Free saccharide assessed.
Unbound polysaccharide is determined after removal of the FINAL BULK VACCINE
conjugate, for example by anion-exchange, size-exclusion or A final bulk vaccine may be formulated from the individually
hydrophobic chromatography, ultrafiltration, or other adsorbed monovalent bulk conjugates, or from the mixture of
validated methods. A value consistent with adequate the monovalent bulk conjugates that is adsorbed on an
immunogenicity as shown in clinical trials is established for aluminium-containing adjuvant.
each serotype and each lot must be shown to comply with Where a final bulk vaccine is formulated as a release
this limit. intermediate, it complies with the following requirements and
Free carrier protein is within the limits approved for the particular product.
Determine the content by a suitable method, either directly Only a final bulk vaccine that complies with the following
or by deriving the content by calculation from the results of requirements may be used in the preparation of the final lot.
other tests. The value complies with the requirement
Sterility (2.6.1)
approved for each serotype.
Molecular size or the equivalent of 100 doses for each medium, whichever is
The molecular size is evaluated by liquid chromatography less.
(2.2.29) with MALI'S detection or other suitable methods.
FINAL LOT
The values are within the limits approved for each serotype.
Only a final lot that is within the limits approved for the
Residual reagents particular product and is satisfactory with respect to each of
Where applicable, suitable tests are carried out to determine the requirements given below under Identification, Tests and
residues of reagents used during inactivation and purification. Assay may be released for use.
An acceptable value for each reagent is established for the
particular product and each batch of conjugate must be
IDENTIFICATION
shown to comply with this limit. Where validation studies Each polysaccharide present in the vaccine is identified by a
have demonstrated removal of residual reagents, the test on suitable immunochemical method (2.7.1).
conjugate polysaccharides may be omitted. TESTS
Sterility (2.6. /) Aluminium (2.5.13)
It complies with the test for sterility, carried out using 10 mL Maximum 1.25 mg per single human dose.
for each medium or the equivalent of 100 doses for each Sterility (2.6. /)
medium, whichever is less. It complies with the test for sterility.
Bacterial endotoxins (2.6.14) Bacterial endotoxins (2.6.14)
Less than 0.75 IU per microgram of polysaccharide. Less than 12.5 IU per single human dose, unless otherwise
ADSORBED MONOVALENT BULK CONJUGATE justified and authorised.
An aluminium-containing adjuvant may be added to each of ASSAY
the monovalent bulk conjugates prior to formulation of the Saccharide content
final bulk. Once the conjugates are adsorbed on a sterile The polysaccharide content for each serotype is determined
adjuvant, sterility is assured by aseptic processing. by a suitable immunochemical method (for example,
Only an adsorbed monovalent bulk conjugate that complies nephelometry assay or enzyme-linked immunosorbent assay
with the following requirements may be used in the (ELISA)). The vaccine contains not less than 70 per cent
preparation of the final bulk vaccine. and not more than 130 per cent of the quantity stated on the
Identification label for each polysaccharide. The confidence
Each adsorbed polysaccharide conjugate is identified by an limits (P = 0.95) are not less than 80 per cent and not more
immunochemical method (2.7.1) or other suitable methods. than 120 per cent of the estimated content.
Sterility (2.6.1) LABELLING
It complies with the test for sterility, carried out using 10 mL The label states:
or the equivalent of 100 doses for each medium, whichever is — the pneumococcal serotype and carrier protein present in
less. each single human dose;
— the number of micrograms of each polysaccharide per
Saccharide
single human dose;
The polysaccharide content is determined by a suitable
— the number of micrograms of carrier protein per single
physical or chemical method or by an immunochemical
human dose;
if applicable, the name and amount of adsorbent; imposed before entering the colony, and then during its stay
— if applicable, that the vaccine must be shaken before use; in the colony.
if applicable, that the vaccine must not be frozen. The monkeys used are shown to be tuberculin-negative and
- ----------------------------------------------------- ---------------------------------------------- Ph Eur free from antibodies to simian virus 40 (SV40) and simian
immunodeficiency virus. The blood sample used in testing
for SV40 antibodies must be taken as close as possible to the
time of removal of the kidneys. If Macaca sp. monkeys are
used for production, the monkeys are also shown to be free
Inactivated Poliomyelitis Vaccine ***** from antibodies to herpesvirus B (cercopithecine
herpesvirus 1) infection. Human herpesvirus 1 has been used
(Poliomyelitis Vaccine (Inactivated), ***
as an indicator for freedom from herpesvirus B antibodies on
account of the danger of handling herpesvirus B
The label may state ‘IPV’. (cercopithecine herpesvirus 1).
Monkeys from which kidneys are to be removed are
definition thoroughly examined, particularly for evidence of tuberculosis
Poliomyelitis vaccine (inactivated) is a liquid preparation of and herpesvirus B (cercopithecine herpesvirus 1) infection.
suitable strains of human poliovirus types 15 2 and 3 grown If a monkey shows any pathological lesion relevant to the use
in suitable cell cultures and inactivated by a validated of its kidneys in the preparation of a seed lot or vaccine, it is
method. It is a clear liquid that may be coloured owing to not to be used nor are any of the remaining monkeys of the
the presence of a pH indicator. group concerned unless it is evident that their use will not
impair the safety of the product.
PRODUCTION
All the operations described in this section are conducted
The production method shall have been shown to yield outside the area where the vaccine is produced.
consistently vaccines of acceptable safety and immunogenicity
in man. Monkey cell cultures for vaccine production Kidneys that show
no pathological signs are used for preparing cell cultures.
Production of the vaccine is based on a vims seed-lot system. Each group of cell cultures derived from a single monkey
Cell lines are used according to a cell-bank system. forms a separate production cell culture giving rise to a
If primary, secondary or tertiary monkey kidney cells are separate single harvest.
used, production complies with the requirements indicated
below. The primary monkey kidney cell suspension complies with
the test for mycobacteria (2.6.2); disrupt the cells before
Unless otherwise justified and authorised, the vims in the carrying out the test.
final vaccine shall not have undergone more passages from
If secondary or tertiary cells are used, it shall be
the master seed lot than was used to prepare the vaccine
demonstrated by suitable validation tests that cell cultures
shown in clinical studies to be satisfactory with respect to
beyond the passage level used for production are free from
tumorigenicity.
SEED LOTS
toxicity for immunosera and vaccines for human use (2.6.9). Each of the 3 strains of poliovirus used shall be identified by
historical records that include information on the origin of
SUBSTRATE FOR VIRUS PROPAGATION the strain and its subsequent manipulation.
The vims is propagated in a human diploid cell line (5.2.2),
Only a working seed lot that complies with the following
in a continuous cell line (5.2.2) or in primary, secondary or
requirements may be used for virus propagation.
tertiary monkey kidney cells.
Identification
Primary, secondary or tertiary monkey kidney cells
Each working seed lot is identified as human poliovirus
The following special requirements for the substrate for vims
types 1, 2 or 3 by virus neutralisation in cell cultures using
propagation apply to primary, secondary or tertiary monkey
specific antibodies.
kidney cells.
Monkeys used in the preparation of kidney cell cultures for
Virus concentration
The virus concentration of each working seed lot is
production and control of the vaccine The animals used are of a
determined to define the quantity of virus to be used for
species approved by the competent authority, in good health
inoculation of production cell cultures.
and, unless otherwise justified and authorised, have not been
previously employed for experimental purposes. Kidney cells Extraneous agents
used for vaccine production and control are derived from The working seed lot complies with the requirements for
monitored, closed colonies of monkeys bred in captivity, not seed lots for virus vaccines (2.6J6). In addition, if primary,
from animals caught in the wild; a previously approved seed secondary or tertiary monkey kidney cells have been used for
lot prepared using virus passaged in cells from wild monkeys isolation of the strain, measures are taken to ensure that the
may, subject to approval by the competent authority, be used strain is not contaminated with simian viruses such as simian
for vaccine production if historical data on safety justify this. immunodeficiency virus, simian virus 40, filoviruses and
herpesvirus B (cercopithecine herpesvirus 1). A working seed
Monitored, closed colonies of monkeys The monkeys are kept in
lot produced in primary, secondary or tertiary monkey kidney
groups in cages. Freedom from extraneous agents is achieved
cells complies with the requirements given below under Virus
by the use of animals maintained in closed colonies that are
propagation and harvest for single harvests produced in such
subject to continuous and systematic veterinary and
laboratory monitoring for the presence of infectious agents. cells.
The supplier of animals is certified by the competent PROPAGATION AND HARVEST
authority. Each monkey is tested serologically at regular All processing of the cell bank and cell cultures is done under
intervals during a quarantine period of not less than 6 weeks aseptic conditions in an area where no other cells or viruses
are being handled. Approved animal serum (but not human Mycoplasmas (2.6.7)
serum) may be used in the cell culture media. Serum and The single harvest complies with the test for mycoplasmas,
trypsin used in the preparation of cell suspensions and media carried out using 10 mL.
are shown to be free from extraneous agents. The cell culture Test in rabbit kidney cell cultures
media may contain a pH indicator such as phenol red and Where primary, secondary or tertiary monkey kidney cells are
approved antibiotics at the lowest effective concentration. used for production, test a sample of at least 10 mL of the
Not less than 500 mL of the cell cultures employed for single harvest for the absence of herpesvirus B
vaccine production is set aside as uninfected cell cultures (cercopithecine herpesvirus 1) and other viruses by
(control cells); where continuous cell lines in a fermenter are inoculation onto rabbit kidney cell cultures as described
used for production, 200 X 1 o6 cells are set aside to prepare above for the control cells.
control cells; where primary, secondary or tertiary monkey
kidney cells are used for production, a cell sample equivalent Test in cercopithecus kidney cell cultures
to at least 500 mL of the cell suspension, at the Where primary, secondary or tertiary monkey kidney cells are
concentration employed for vaccine production, is taken to used for production, test a sample of at least 10 mL of the
prepare control cells. single harvest for the absence of SV40 virus and other
extraneous agents. Neutralise the sample by a high-titre
Only a single harvest that complies with the following
antiserum against the specific type of poliovirus. Test the
requirements may be used in the preparation of the vaccine.
sample in primary cercopithecus kidney cell cultures or cells
The tests for identification and bacterial and fungal
that have been demonstrated to be at least as susceptible for
contamination may be carried out instead on the purified,
SV40. Incubate the cultures at 37 °C and observe for
pooled monovalent harvest. After demonstration of
14 days. At the end of tins period, make at least one
consistency of production at the stage of the single harvest,
subculture of fluid in the same cell culture system and
the test for virus concentration may be carried out instead on
observe both primary cultures and subcultures for an
the purified, pooled monovalent harvest.
additional 14 days.
Control cells
PURIFICATION AND PURIFIED MONOVALENT HARVEST
The control cells of the production cell culture comply with a
Several single harvests of the same type may be pooled and
test for identification (if a cell-bank system is used for
may be concentrated. The monovalent harvest or pooled
production) and with the requirements for extraneous agents
monovalent harvest is purified by validated methods.
(2.6.16; where primary, secondary or tertiary monkey kidney
If continuous cell lines are used for production, the
cells are used, the tests in cell cultures are carried out as
purification process shall have been shown to reduce
shown below under Test in rabbit kidney cell cultures and
consistently the content of substrate-cell DNA to not more
Test in cercopithecus kidney cell cultures).
than 100 pg per single human dose.
Test in rabbit kidney cell cultures Test a sample of at least
Only a purified monovalent harvest that complies with the
10 mL of the pooled supernatant fluid from the control
following requirements may be used for the preparation of
cultures for the absence of herpesvirus B (cercopithecine
the inactivated monovalent harvest.
herpesvirus 1) and other viruses by inoculation onto rabbit
kidney cell cultures. The dilution of supernatant in the Identification
nutrient medium is not greater than 1/4 and the area of the The virus is identified by virus neutralisation in cell cultures
cell layer is at least 3 cm2 per millilitre of inoculum. Set aside using specific antibodies or by determination of D-antigen.
one or more containers of each batch of cells with the same Virus concentration
medium as non-inoculated control cells. Incubate the The virus concentration is determined by titration of
cultures at 37 °C and observe for at least 2 weeks. The test is infectious virus.
not valid if more than 20 per cent of the control cell cultures Specific activity
are discarded for non-specific, accidental reasons. The ratio of the virus concentration or the D-antigen
Test in cercopithecus kidney cell cultures Test a sample of at content, determined by a suitable immunochemical method
least 10 mL of the pooled supernatant fluid from the control (2.7./), to the total protein content (specific activity) of the
cultures for the absence of SV40 virus and other extraneous purified monovalent harvest is within the limits approved for
agents by inoculation onto cell cultures prepared from the the particular product.
kidneys of cercopi±ecus monkeys, or other cells shown to be INACTIVATION AND INACTIVATED MONOVALENT
at least as sensitive for SV40, by the method described under HARVEST
Test in rabbit kidney cell cultures. The test is not valid if Several purified monovalent harvests of the same type may
more than 20 per cent of the control cell cultures are be mixed before inactivation. To avoid failures in inactivation
discarded for non-specific, accidental reasons. caused by the presence of virus aggregates, filtration is
Identification carried out before and during inactivation; inactivation is
The single harvest is identified as containing human started within a suitable period, preferably not more than
poliovirus types 1, 2 or 3 by virus neutralisation in cell 24 h and in any case not more than 72 h, of the prior
cultures using specific antibodies. filtration. The virus suspension is inactivated by a validated
Virus concentration method that has been shown to inactivate poliovirus without
The virus concentration of each single harvest is determined destruction of immunogenicity; during validation studies, an
by titration of infectious virus in cell cultures. inactivation curve with at least 4 points (for example, time
0 h, 24 h, 48 h and 96 h) is established showing the decrease
Bacterial and fungal contamination in concentration of live virus with time. If formaldehyde is
The single harvest complies with the test for sterility (2.6. /), used for inactivation, the presence of an excess of
carried out using 10 mL for each medium. formaldehyde at the end of the inactivation period is verified.
The inactivation kinetics tests mentioned below are earned
out on each batch to ensure consistency of the inactivation released for use. Provided that the tests for free formaldehyde
process. and antimicrobial preservative and the in vivo assay have
Only an inactivated monovalent harvest that complies with been performed with satisfactory results on the final bulk
the following requirements may be used in the preparation of vaccine, they may be omitted on the final lot.
a trivalent pool of inactivated monovalent harvests or a final The in vivo assay may be omitted once it has been
bulk vaccine. demonstrated for a given product and for each poliovirus
Test for effective inactivation type that the acceptance criteria for the D-antigen
After neutralisation of the formaldehyde with sodium bisulfite determination are such that it yields the same result as the in
(where applicable), verify the absence of residual live vivo assay in terms of acceptance or rejection of a batch. This
poliovirus by inoculation on suitable cell cultures of demonstration must include testing of subpotent batches,
2 samples of each inactivated monovalent harvest, produced experimentally if necessary, for example by heat
corresponding to at least 1500 human doses. Cells used for treatment or other means of diminishing the immunogenic
the test must be of optimal sensitivity regarding residual activity. Where there is a significant change in the
infectious poliovirus, for example kidney cells from certain manufacturing process of the antigens or their formulation,
monkey species (Macaca, Cercopithecus or Papio), or Hep-2 any impact on the in vivo and in vitro assays must be
cells. If other cells are used, they must have been shown to evaluated, and the need for revalidation considered.
possess at least the same sensitivity as those specified above. Provided that the protein content has been determined on
Take one sample not later than 3/4 of the way through the the purified monovalent harvests or on the inactivated
inactivation period and the other at the end. Inoculate the monovalent harvests and that it has been shown that the
samples in cell cultures such that the dilution of vaccine in content in the final lot will not exceed 10 pg per single
the nutrient medium is not greater than 1/4 and the area of human dose, the test for protein content may be omitted on
the cell layer is at least 3 cm2 per millilitre of inoculum. the final lot.
Set aside one or more containers with the same medium as Provided that the test for bovine serum albumin has been
non-inoculated control cells. Observe the cell cultures for at performed with satisfactory results on the trivalent pool of
least 3 weeks. Make not fewer than 2 passages from each inactivated monovalent harvests or on the final bulk vaccine,
container, one at the end of the observation period and the it may be omitted on the final lot.
other 1 week before; for the passages, use cell culture
IDENTIFICATION
supernatant and inoculate as for the initial sample. Observe
The vaccine is shown to contain human poliovirus types 1, 2
the subcultures for at least 2 weeks. No sign of poliovirus
and 3 by a suitable immunochemical method (2.7. 7) such as
multiplication is present in the cell cultures. At the end of the
the determination of D-antigen by enzyme-linked
observation period, test the susceptibility of the cell culture
immunosorbent assay (ELISA).
used by inoculation of live poliovirus of the same type as that
present in the inactivated monovalent harvest. TESTS
Inactivation kinetics Free formaldehyde (2.4.18)
Kinetics of inactivation are established and approved by the Maximum 0.2 g/L.
competent authority. Adequate data on inactivation kinetics Antimicrobial preservative
are obtained and consistency of the inactivation process is Where applicable, determine the amount of antimicrobial
monitored. preservative by a suitable chemical or physicochemical
Sterility (2.6. 7) method. The amount is not less than the minimum amount
The inactivated monovalent harvest complies with the test for shown to be effective and is not greater than 115 per cent of
sterility, carried out using 10 mL for each medium. that stated on the label.
D-antigen content Protein content (2.5.35, Method 2)
The content of D-antigen determined by a suitable Maximum 10 pg per single human dose.
immunochemical method (2.7.7) is within the limits Bovine serum albumin
approved for the particular preparation. Maximum 50 ng per single human dose, determined by a
FINAL BULK VACCINE suitable immunochemical method (2.7.7).
The final bulk vaccine is prepared directly from the Sterility (2.6.7)
inactivated monovalent harvests of human poliovirus types 1, It complies with the test.
2 and 3 or from a trivalent pool of inactivated monovalent Bacterial endotoxins (2.6.14)
harvests. A suitable stabiliser and a suitable antimicrobial Less than 5 IU per single human dose.
preservative may be added.
ASSAY
D-antigen content
As a measure of consistency of production, determine the
Sterility (2.6. 7) D-antigen content for human poliovirus types 1, 2 and 3 by
The final bulk vaccine complies with the test for sterility, a suitable immunochemical method (2.7.7) using a reference
carried out using 10 mL for each medium. preparation calibrated in European Pharmacopoeia Units of
Antimicrobial preservative D-antigen. For each type, the content, expressed with
Where applicable, determine the amount of antimicrobial reference to the amount of D-antigen stated on the label, is
preservative by a suitable chemical or physicochemical within the limits approved for the particular product.
method. The amount is not less than 85 per cent and not Poliomyelitis vaccine (inactivated) BRP is calibrated in
greater than 115 per cent of the intended amount. European Pharmacopoeia Units and intended for use in the
assay of D-antigen. The European Pharmacopoeia Unit and
final lot
the International Unit are equivalent.
Only a final lot that complies with each of the requirements
given below under Identification, Tests and Assay may be
In vivo test Primary monkey kidney cell cultures

The vaccine complies with the in vivo assay of poliomyelitis The following special requirements for the substrate for virus
vaccine (inactivated) (2.7.20). propagation apply to primary monkey kidney cell cultures.
LABELLING Monkeys used for preparation of primary monkey kidney cell
The label states: cultures and for testing of virus If the vaccine is prepared in
— the typcs of poliovirus contained in the vaccine; primary monkey kidney cell cultures, animals of a species
— the nominal amount of virus of each type (1,2 and 3), approved by the competent authority, in good health, kept in
expressed in European Pharmacopoeia Units of closed or intensively monitored colonies and not previously
D-antigen, per single human dose; employed for experimental purposes shall be used.
— the cell substrate used to prepare the vaccine. The monkeys shall be kept in well-constructed and
----------------------------------------------------------------------.——--------------------------- Ph Eur 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
2 monkeys shall be housed per cage and cage-mates shall not
be interchanged. The monkeys shall be kept in the country’ of
Poliomyelitis Vaccine, Live (Oral) ***** manufacture of the vaccine in quarantine groups for a period
(Poliomyelitis Vaccine (Oral), *** of not less than 6 weeks before use. A quarantine group is a
Ph Eur monograph 0215) colony of selected, healthy monkeys kept in one room, with
separate feeding and cleaning facilities, and having no contact
The label may state ‘OPV’. with other monkeys during the quarantine period. If at any
Ph Eur____________________________________________________ _________ time during the quarantine period the overall death rate of a
DEFINITION shipment consisting of one or more groups reaches
วิ per cent (excluding deathร from accidents or where the
Oral poliomyelitis vaccine is a preparation of approved strains
of live attenuated poliovirus type 1, 2 or 3 grown in in vitro cause was specifically determined not to be an infectious
cultures of approved cells, containing any one type or any disease), monkeys from that entire shipment shall continue in
combination of the 3 types of Sabin strains, presented in a quarantine from that time for a minimum of 6 weeks.
form suitable for oral administration. The groups shall be kept continuously in isolation, as in
quarantine, even after completion of the quarantine period,
The vaccine is a clear liquid that may be coloured owing to
until the monkeys are used. After the last monkey of a group
the presence of a pH indicator.
has been taken, the room that housed the group shall be
PRODUCTION thoroughly cleaned and decontaminated before being used
The vaccine strains and the production method shall have for a fresh group. If kidneys from near-term monkeys arc
been shown to yield consistently vaccines ±at are both used, the mother is quarantined for the term of pregnancy.
immunogenic and safe in man. Monkeys from which kidneys are to be removed shall be
The production of vaccine is based on a virus seed-lot anaesthetised and thoroughly examined, particularly for
system. Cell lines are used according to a cell-bank system. evidence of tuberculosis and cercopithccid herpesvirus 1
If primary monkey kidney cell cultures are used, production (B virus) infection.
complies with the requirements indicated below. Unless If a monkey shows any pathological lesion relevant to the use
otherwise justified and authorised, the virus in the final of its kidneys in the preparation of a seed lot or vaccine, it
vaccine shall not have undergone more than 2 passages from shall not be used, nor shall any of the remaining monkeys of
the master seed lot. the quarantine group concerned be used unless it is evident
REFERENCE STANDARDS that their use-will not impair the safety of the product.
Poliomyelitis vaccine (oral) BRP is suitable for use as a virus All the operations described in this section shall be
reference preparation for the assay. conducted outside the areas where the vaccine is produced.
The International Standards for poliovirus type 2 (Sabin) for The monkeys used shall be shown to be free from antibodies
MAPREC (Mutant Analysis by PCR and Restriction Enzyme to simian virus 40 (SV40), simian immunodeficiency virus
Cleavage) assays and poliovirus, type 3 (Sabin) synthetic and spumaviruses. The blood sample used in testing for
DNA for MAPREC assays are suitable for use in the tests for SV40 antibodies must be taken as close as possible to the
genetic markers and the molecular tests for consistency of time of removal of the kidneys. If Macaca spp. are used for
production. production, the monkeys shall also be shown to be free from
Reference preparations of each poliovirus type at the Sabin antibodies to cercopithecid herpesvirus 1 (B virus). Human
Original + 2 passage level, namely WHO (SO + 2)/I for herpesvirus has been used as an indicator for freedom from
type 1 virus, WHO (SO 4- 2)/II for type 2 virus and WHO B virus antibodies on account of the danger of handling
(SO + 2)/m for type 3 virus are available for comparison of cercopithecid herpesvirus 1 (B virus). Monkeys used for the
the in vivo neurovirulence with that of homotypic vaccines. production of new seed lots are shown to be free from
Requests for the WHO reference preparations for in vivo antibodies to simian cytomegalovirus (sCMV).
neurovirulence tests are to be directed to WHO, Biologicals, Primary monkey kidney cell cultures for vaccine production
Geneva, Switzerland. Kidneys that show no pathological signs are used for
A suitable reference preparation is to be included in each preparing cell cultures. If the monkeys are from a colony
test. maintained for vaccine production, serially passaged monkey
kidney cell cultures from primary monkey kidney cells may
be used for virus propagation, otherwise the monkey kidney
The virus is propagated in human diploid cells (5.2.2), in cells are not propagated in series. Virus for the preparation of
continuous cell lines (5.2.5) or in primary monkey kidney cell
vaccine is grown by aseptic methods in such cultures.
cultures (including serially passaged cells from primary
If animal serum is used in the propagation of the cells, the
monkey kidney cells).
maintenance medium after virus inoculation shall contain no VIRUS PROPAGATION AND HARVEST
added serum. All processing of the cell banks and subsequent cell cultures
Each group of cell cultures derived from a single monkey or is done under aseptic conditions in an area where no other
from foetuses from no more than 10 near-term monkeys is cells are handled during the production. Suitable animal (but
prepared and tested as an individual group. not human) serum may be used in the culture media, but the
VIRUS SEED LOTS final medium for maintaining cell growth during virus
The strains of poliovirus used shall be identified by historical multiplication does not contain animal serum. Serum and
records that include information on the origin and trypsin used in the preparation of cell suspensions and media
subsequent manipulation of the strains. are shown to be free from live extraneous agents. The cell
culture medium may contain a pH indicator such as phenol
Working seed lots are prepared by a single passage from a
red and suitable antibiotics at the lowest effective
master seed lot and at an approved passage level from the concentration. It is preferable to have a substrate free from
ongmal Sabin virus. Virus seed lots are prepared in large antibiotics during production. On the day of inoculation with
quantities and stored at a temperature below —60 °C. the virus working seed lot, not less than 5 per cent or
Only a virus seed lot that complies with the following 1000 mL, whichever is the less, of the cell cultures employed
requirements may be used for virus propagation. for vaccine production are set aside as uninfected cell
Identification cultures (control cells), special requirements, given below,
Each working seed lot is identified as poliovirus of the given apply to control cells when the vaccine is produced in
type, using specific antibodies. primary monkey kidney cell cultures. The virus suspension is
Virus concentration harvested not later than 4 days after virus inoculation. After
Determined by the method described below, the virus inoculation of the production cell culture with the virus
working seed lot, inoculated cells are maintained at a fixed
concentration is the basis for the quantity of virus used in the
neurovirulence test. temperature, shown to be suitable, within the range
33-35 °C; the temperature is maintained constant to
Extraneous agents (2.6.16) ± 0.5 °C; control cell cultures are maintained at 33-35 °C
If the working seed lot is produced in human diploid cells or for the relevant incubation periods.
in a continuous cell line, it complies with the requirements
Only a single virus harvest that complies with the following
for seed lots for virus vaccines. If the working seed lot is
requirements may be used in the preparation of the
produced in primary' monkey kidney cell cultures, it complies
with the requirements given below under Virus Propagation
and Harvest and Monovalent Pooled Harvest and with the Virus concentration
tests in adult mice, suckling mice and guinea-pigs given in The virus concentration of virus harvests is determined as
chapter 2.6.16. prescribed under Assay to monitor consistency of production
and to determine the dilution to be used for the final bulk
In addition to the requirements in chapter 2.6.76, for
vaccine.
vaccines produced in cell lines and when the seed lot was
produced in primary monkey kidney cell cultures, a validated Molecular tests for consistency of production
test for sCMV is performed. The MAPREC assay is performed on each virus harvest.
Working seed lots shall be free from detectable DNA The acceptance/rejection criteria for consistency of
sequences from simian virus 40 (SV40). production are determined for each manufacturer and for
each working seed by agreement with the competent
Neuro virulence authority. These criteria are periodically reviewed and
Each master and working seed lot complies with the test for updated to the satisfaction of the competent authority.
neurovirulence of poliomyelitis vaccine (oral) in monkeys An investigation of consistency occurs if a virus harvest gives
(2.6.79). In addition, at least the first 4 consecutive batches results that are inconsistent with previous production history.
of monovalent pooled harvest prepared from these seed lots
shall be shown to comply with the test for neurovirulence of
Control cells
The control cells of the production cell culture from which
poliomyelitis vaccine (oral) in monkeys (2.6.79) before the
the virus harvest is derived comply with a test for identity
seed lot is deemed suitable for use. Furthermore, the seed lot
and with the requirements for extraneous agents (2.6.76) or,
shall cease to be used in vaccine production if the frequency
where primary monkey kidney cell cultures are used, as
of failure of the monovalent pooled harvests produced from it
shown below.
is greater than predicted statistically. This statistical
prediction is calculated after each test on the basis of all the Primary monkey kidney cell cultures
monovalent pooled harvests tested; it is equal to the The following special requirements apply to virus propagation and
probability of false rejection on the occasion of a first test harvest in primary monkey kidney cell cidtures.
(i.e.l per cent), the probability of false rejection on retest Cell cultures On the day of inoculation with the virus working
being negligible. If the test is carried out only by the seed lot, each cell culture is examined for degeneration
manufacturer, the test slides are provided to the control caused by an infective agent. If, in this examination, evidence
authority for assessment. is found of the presence in a cell culture of any extraneous
Genetic markers agent, the entire group of cultures concerned shall be
Each working seed lot is tested for its replicating properties at rejected.
temperatures ranging from 36 °C to 40 °C as described On the day of inoculation with the virus working seed lot, a
under Monovalent pooled harvest. A profile (i.e. percentage sample of at least 30 mL of the pooled fluid removed from
of mutant) of the Seed virus using the MAPREC assay is the cell cultures of ±e kidneys of each single monkey or from
prepared. Type 3 virus seed lots comply with the MAPREC foetuses from not more than 10 near-term monkeys is
assay as described under Monovalent pooled harvest. divided into 2 equal portions. 1 portion of the pooled fluid 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 portion of the pooled fluid is, where test for B virus, which may be held at 4 °C, provided that the
necessary, tested in monkey kidney cell cdtures from another test is done not more than 7 days after it has been taken.
species so that tests on the pooled Adds are done in cell Control cell cultures On the day of inoculation with the virus
cdtures from at least 1 species known to be sensitive to working seed lot, 25 per cent (but not more than 2.5 L) of
SV40. The pooled fluid is inoculated into bottles of these cell the cell suspension obtained from the kidneys of each single
cdtures in such a way ±at the dilution of the pooled Add in monkey or from not more than 10 near-term monkeys is
the nutrient medium does not exceed 1 in 4. The area of the taken to prepare uninoculated control cell cdtures. These
cell sheet is at least 3 cm2/mL of pooled Add. At least control cell cultures are incubated in the same conditions as
1 bottle of each type of cell cdture remains uninocdated to the inoedated cdtures for at least 2 weeks and are examined
serve as a control. If the monkey species used for vaccine during this period for evidence of cytopathic changes.
production is known to be sensitive to SV40, a test in a The tests are not valid if more than 20 per cent of the
2nd species is not required. Animal serum may be used in the control cell cdtures have been discarded for non-specific,
propagation of the cells, provided that it does not contain accidental reasons. At the end of the observation period, the
SV40 antibody, but the maintenance medium after control cell cdtures are examined for degeneration caused by
inoedation of test material contains no added serum except an infectious agent. If this examination or any of the tests
as described below. reqdred in this section shows evidence of the presence in a
The cdtures are incubated at a temperature of 35-37 °C and control cdture of any extraneous agent, the poliovirus grown
are observed for a total period of at least 4 weeks. During in the corresponding inoculated cultures from the same
this observation period and after not less than 2 weeks' group shall be rejected.
incubation, at least 1 subculture of fluid is made from each Tests for haemadsorbing viruses At the time of harvest or within
of these cultures in the same cell cdture system. 4 days of inoculation of the production cultures with the
The subcultures are also observed for at least 2 weeks. virus working seed lot, a sample of 4 per cent of the control
Serum may be added to the original cdture at the time of cell cultures is taken and tested for haemadsorbing viruses.
subedturing, provided that the serum does not contain SV40 At the end of the observation period, the remaining control
antibody. cell cultures are similarly tested. The tests are carried out as
Fluorescent-antibody techniques may be useful for detecting described in chapter 2.6.16.
SV40 virus and other viruses in the cells. Tests for other extraneous agents At the time of harvest, or
A further sample of at least 10 mL of the pooled Add is within 7 days of the day of inoculation of the production
tested for cercopithecid herpesvirus 1 (B virus) and other cdtures with the working seed lot, a sample of at least
viruses in rabbit kidney cell cultures. Serum used in the 20 mL of the pooled Add from each group of control
nutrient medium of these cultures shall have been shown to cdtures is taken and tested in 2 kinds of monkey kidney cell
be free from inhibitors of B virus. Human herpesviruร has cdture, as described above.
been used as an indicator for freedom from B virus inhibitors At the end of the observation period for the original control
on account of the danger of handling cercopithecid cell cultures, similar samples of the pooled fluid are taken
herpesvirus 1 (B virus). The sample is inoculated into bottles and the tests referred to in this section in the 2 kinds of
of these cell cdtures in such a way that the dilution of the monkey kidney cell culture and in the rabbit cell cultures are
pooled Add in the nutrient medium does not exceed 1 in 4. repeated, as described above under Cell cultures.
The area of the cell sheet is at least 3 cm2/mL of pooled If the presence of cercopithecid herpesvirus 1 (B virus) is
Add. At least 1 bottle of the cell cultures remains demonstrated, the production cell cultures shall not be used
uninoculated to serve as a control. and the measures concerning vaccine production described
The cdtures are incubated at a temperature of 35-37 °C and above must be undertaken.
observed for at least 2 weeks. The Adds collected from the control cell cultures at the lime
A further sample of 10 mL of the pooled fluid removed from of virus harvest and at the end of the observation period may
the cell cultures on the day of inoculation with the seed lot be pooled before testing for extraneous agents. A sample of
virus is tested for the presence of extraneous agents by 2 per cent of the pooled fluid is tested in each of the cell
inoedation into human cell cdtures sensitive to measles cdture systems specified.
virus. Single harvests
The tests are not valid if more than 20 per cent of the Tests for neutralised single harvests in primary monkey kidney cell
cdture vessels have been discarded for non-specific cultures K sample of at least 10 mL of each single harvest is
accidental reasons by the end of the respective test periods. neutralised by a type-specific poliomyelitis antiserum
If, in these tests, evidence is found of the presence of an prepared in animals other than monkeys. In preparing
extraneous agent, the single harvest from the whole group of antisera for this purpose, the immunising antigens used shall
cell cdtures concerned is rejected. be prepared in non-simian cells.
If the presence of cercopithecid herpesvirus 1 (B virus) is Half of the neutralised suspension (corresponding to at least
demonstrated, the manufacture of oral poliomyelitis vaccine 5 mL of single harvest) is tested in monkey kidney cell
shall be discontinued and the competent authority shall be cdtures prepared from the same species, but not the same
informed. Manufacturing shall not be resumed und a animal, as that used for vaccine production. The other half of
thorough investigation has been completed and precautions the neutralised suspension is tested, if necessary, in monkey
have been taken against any reappearance of the infection, kidney cell cdtures from another species so that the tests on
and then ody with the approval of the competent authority. the neutralised suspension are done in cell cdtures from at
If these tests are not done immediately, the samples of least 1 species known to be sensitive to SV40.
pooled cell-culture fluid shall be kept at a temperature of The neutralised suspensions are inoculated into bottles of
-60 °C or below, with the exception of the sample for the 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/mL of neutralised The MAPREC analysis of poliovirus type 3 (Sabin) is carried
suspension. At least 1 bottle of each type of cell culture out using a standard operating procedure approved by the
remains uninoculated to serve as a control and is maintained competent authority. A suitable procedure (Mutant analysis
by nutrient medium containing the same concentration of the by PCR and restriction enzyme cleavage (MAPREC) for oral
specific antiserum used for neutralisation. poliovirus (Sabin) vaccine) is available from WHO, Quality
•Animal serum may be used in the propagation of the cells, and Safety of Biologicals (QSB), Geneva. A laboratory must
provided that it does not contain SV40 antibody, but the demonstrate to the competent authority that it is competent
maintenance medium, after the inoculation of the test to perform the assay. The manufacturer and the competent
material, contains no added serum other than the poliovirus authority shall agree on the procedure and the criteria for
neutralising antiserum, except as described below. deciding whether a monovalent pooled harvest contains
The cultures are incubated at a temperature of 35-37 °C and significantly more 472-C than the International Standard.
observed for a total period of at least 4 weeks. During this Acceptance/rcjection criteria for assessment of consistency of
observation period and after not less than 2 weeks' production are determined for each manufacturer and for
incubation, at least 1 subculture of fluid is made from each each working seed lot by agreement with the competent
of these cultures in the same cell-culture system. authority. These criteria are updated as each new bulk is
The subcultures are also observed for at least 2 weeks. prepared and analysed. An investigation of consistency occurs
Serum may be added to the original cultures at the time of if a monovalent pooled harvest gives results that are
subculturing, provided that the serum does not contain SV40 inconsistent with previous production history.
antibody. As the MAPREC assay for type 3 poliovirus (Sabin) is highly
Additional tests are made for extraneous agents on a further predictive of in vivo neurovirulence, if a filtered monovalent
sample of the neutralised single harvests by inoculation of pooled harvest of type 3 poliovirus (Sabin) fails the
10 mL into human cell cultures sensitive to measles virus. MAPREC assay then this triggers an investigation of the
This test is also validated for the detection of sCMV. consistency of the manufacturing process. This investigation
also includes a consideration of the suitability of the working
Fluorescent-andbody techniques may be useful for detecting seed lot.
SV40 virus and other viruses in the cells.
Monovalent pooled harvests passing the MAPREC assay are
The tests are not valid if more than 20 per cent of the subsequently tested for in vivo neurovirulence.
culture vessels have been discarded for non-specific
accidental reasons by the end of the respective test periods. For poliovirus type 3, results from the MAPREC assay and
the monkey neurovirulence test (2.6.19) are used
If any cytopathic changes occur in any of the cultures, the concomitantly to assess the impact of changes in the
causes of these changes are investigated. If the cytopathic production process or when a new manufacturer starts
changes are shown to be due to unneutralised poliovirus, the production.
test is repeated. If there is evidence of the presence of SV40
Pending validation of MAPREC assays for poliovirus types
or other extraneous agents attributable to the single harvest,
that single harvest is rejected. 1 and 2, for these viruses filtered bulk suspension is tested
for the property of reproducing at temperatures of 36 °C and
MONOVALENT POOLED HARVEST 40 °C. A ratio of the replication capacities of the virus in the
Monovalent pooled harvests are prepared by pooling a monovalent pooled harvest is obtained over a temperature
number of satisfactory single harvests of the same virus type. range between 36 °C and 40 °C in comparison with the seed
Monovalent pooled harvests from continuous cell lines may lot or a reference preparation for the marker tests and with
be purified. Each monovalent pooled harvest is filtered appropriate rct/40- and rct/40-r strains of poliovirus of the
through a bacteria-retentive filter. same type. The incubation temperatures used in this test are
Only a monovalent pooled harvest that complies with the controlled to within ±0.1 °C. The monovalent pooled
following requirements may be used in the preparation of the harvest passes the test if, for both the virus in the harvest and
final bulk vaccine. the appropriate reference material, the titre determined at
Identification 36 °C is at least 5.0 logio greater than that determined at
Each monovalent pooled harvest is identified as poliovirus of 40 °C. If growth at 40 °C is so low that a valid comparison
the given type, using specific antibodies. cannot be established, a temperature in the region of
39.0-39.5 °C is used, at which temperature the reduction in
Virus concentration
titre of the reference material must be in the range
The virus concentration is determined by the method
3.0-5.0 logio of its value at 36 °C; the acceptable minimum
described below and serves as the basis for calculating the
reduction is determined for each virus strain at a given
dilutions for preparation of the final bulk, for the quantity of temperature. If the titres obtained for 1 or more of the
virus used in the neurovirulence test and to establish and reference viruses are not concordant with the expected
monitor production consistency. values, the test must be repeated.
Genetic markers Neurovirulence (2.6.19)
For Sabin poliovirus type 3, a validated MAPREC assay is Each monovalent pooled harvest complies with the test for
performed. In this analysis the amount of the mutation at neurovirulence of poliomyelitis vaccine (oral). If the monkey
position 472 of the genome (472-C) is estimated and neurovirulence test is carried out only by the manufacturer,
expressed as a ratio relative to the International Standard for the test slides are provided to the competent authority for
MAPREC analysis of poliovirus type 3 (Sabin). A poliovirus assessment. The TgPVR21 transgenic mouse model provides
type 3 monovalent pooled harvest found to have significantly a suitable alternative to the monkey neurovirulence test for
more 472-C than the International Standard for MAPREC neurovirulence testing of types 1, 2 or 3 vaccines once a
analysis of poliovirus type 3 (Sabin) fails in the MAPREC laboratory qualifies as being competent to perform the test
assay. 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. FINAL BULK VACCINE

A suitable procedure (Neurovirulence test of type 1, 2 or 3 live The final bulk vaccine is prepared from one or more
poliomyelitis vaccines (oral) in transgenic mice susceptible to satisfactory monovalent pooled harvests and may contain
poliovirus') is available from WHO, Quality and Safety of more than one virus type. Suitable flavouring substances and
Biologicals, Geneva. stabilisers may be added.
Primary monkey kidney cell cultures Only a final bulk vaccine that complies with the following
The following special requirements apply to monovalent pooled requirement may be used in the preparation of the final lot.
harvests derived from primary monkey kidney cell cultures. Bacterial and fungal contamination
Retroviruses The monovalent pooled harvest is examined Carry out the test for sterility (2.6. ใ), using 10 mL for each
using a reverse transcriptase assay. No indication of the medium.
presence of retroviruses is found. FINAL LOT
Test in rabbits A sample of the monovalent pooled harvest is Only a final lot that complies with the following requirement
tested for cercopithecid herpesvirus 1 (B virus) and other for thermal stability and is satisfactory with respect to each of
viruses by injection of not less than 100 mL into not fewer the requirements given below under Identification, Tests and
than 10 healthy rabbits each weighing 1.5-2.5 kg. Each rabbit Assay may be released for use.
receives not less than 10 mL and not more than 20 mL, of
Thermal stability
which 1 mL is given intradermally at multiple sites since the Maintain not fewer than 3 containers of the final lot at
maximum volume to be given intradermally at each site is
37 ± 1 °C for 48 h. Determine the total virus concentration
0.1 mL, and the remainder subcutaneously. The rabbits are as described under Assay in parallel for the heated vaccine
observed for at least 3 weeks for death or signs of illness.
and for vaccine maintained at the temperature recommended
All rabbits that die after the first 24 h of the test and those for storage. The total virus concentration of the heated
showing signs of illness are examined by autopsy, and the vaccine is not more than 0.5 logio lower than that of the
brain and organs removed for detailed examination to unheated vaccine.
establish the cause of death.
IDENTIFICATION
The test is not valid if more than 20 per cent of the
The vaccine is shown to contain poliovirus of each type
inoculated rabbits show signs of intercurrent infection during
stated on the label, using specific antibodies.
the observation period. The monovalent pooled harvest
passes the test if none of the rabbits shows evidence of TESTS
infection with B virus or with other extraneous agents or Bacterial and fungal contamination
lesions of any kind attributable to the bulk suspension. The vaccine complies with the test for sterility (2.6./).
If the presence of B virus is demonstrated, the measures ASSAY
concerning vaccine production described above under Cell Titrate the vaccine for infectious virus, using not fewer than
cultures are taken. 3 separate containers of vaccine, following the method
Test in guinea-pigs If the primary monkey kidney cell cultures are described below. Titrate 1 container of an appropriate virus
not derived from monkeys kept in a closed colony, the monovalent reference preparation in triplicate to validate each assay.
pooled harvest shall be shown to comply with the following test The virus concentration of the reference preparation is
Administer to each of not fewer than 5 guinea-pigs, each monitored using a control chart and a titre is established on a
weighing 350-450 g, 0.1 mL of the monovalent pooled historical basis by each laboratory'. If the vaccine contains
harvest by intracerebral injection (0.05 mL in each cerebral more than one poliovirus type, titrate each type separately,
hemisphere) and 0.5 mL by intraperitoneal injection. using an appropriate type-specific antiserum (or preferably a
Measure the rectal temperature of each animal on each monoclonal antibody) to neutralise each of the other types
working day for 6 weeks. At the end of the observation present.
period carry out autopsy on each animal. Calcdate the individual virus concentration for each
In addition, administer to not fewer than 5 guinea-pigs container of vaccine and for each replicate of the reference
0.5 mL by intraperitoneal injection and observe as described preparation as well as the coiTcsponding combined virus
above for 2-3 weeks. At the end of the observation period, concentrations, using the usual statistical methods (for
carry out a passage from these animals to not fewer than example, 5.2).
5 guinea-pigs using blood and a suspension of liver or spleen For a trivalent vaccine, the combined estimated virus titres
tissue. Measure the rectal temperature of the latter guinea- per single human dose must be:
pigs for 2-3 weeks. Examine by autopsy all animals that, after — not less than 6.0 log 10 infectious virus units (CCID50) for
the first day of the test, die or are eufoanised because they type 1;
show disease, or show on 3 consecutive days a body — not less than 5.0 log10 infectious virus units (CCID50) for
temperature higher than 40.1 °C; carry out histological type 2; and
examination to detect infection with filoviruses; in addition, — not less than 5.5 log10 infectious virus units (CCID50) for
inject a suspension of liver or spleen tissue or of blood type 3.
intraperitoneally into not fewer than 3 guinea-pigs. If any For a monovalent or divalent vaccine, the minimum virus
signs of infection with filoviruses are noted, confirmatory titres are decided by the competent authority.
serological tests are carried out on the blood of the affected
Method Inoculate a suitable number of wells in a microtitre
animals. The monovalent pooled harvest complies with the
plate with a suitable volume of each of the selected dilutions
test if not fewer than 80 per cent of the guinea-pigs survive
of virus followed by a suitable volume of a cell suspension of
to the end of the observation period and remain in good
the Hep-2 (Cincinnati) line. Examine the cultures
health, and no animal shows signs of infection with
between days 7 and 9.
filoviruses.
the confidence interval (P = 0.95) of the estimated virus SUBSTRATE FOR VIRUS PROPAGATION
concentration of the reference preparation for the 3 The virus is propagated in a human diploid cell line, or in a
replicates combined is greater than ±03 logio CCID50; continuous cell line (5.2.2) approved by the competent
the virus concentration of the reference preparation differs authority, or in cultures of chick-embryo cells derived from a
by more than 0.5 log10 CCID50 from the established flock free from specified pathogens (5.2.2).
value. The relation with the appropriate European SEED LOTS
Pharmacopoeia Biological Reference Preparation is The strain of rabies virus used shall be identified by historical
established and monitored at regular intervals when a records that include information on the origin of the strain
manufacturer's reference preparation is used. and its subsequent manipulation.
The assay is repeated if the confidence interval (P = 0.95) of Working seed lots are prepared by not more than 5 passages
the combined virus concentration of the vaccine is greater from the master seed lot.
than ± 0.3 log10 CCID50; data obtained from valid assays
Only a working seed lot that complies with the following tests
only are combined by the usual statistical methods (for
example, 5.3) to calculate the virus concentration of the
sample. The confidence interval (P = 0.95) of the combined Identification
virus concentration is not greater than ± 0.3 logio CCID50. Each working seed lot is identified as rabies virus using
Poliomyelitis vaccine (oral) BRP is suitable for use as a specific antibodies.
reference preparation. Virus concentration
Where justified and authorised, different assay designs may The virus concentration of each working seed lot is
be used; this may imply the application of different validity determined by a cell-culture method using
and acceptance criteria. However, the vaccine must comply if immunofluorescence, to ensure consistency of production.
tested as described above. Extraneous agents (2.6.76)
LABELLING
virus seed lots. If the virus has been passaged in mouse brain,
The label states:
specific tests for murine viruses are carried out.
— the types of poliovirus contained in the vaccine;
— the minimum amount of virus of each type contained in a VIRUS PROPAGATION AND HARVEST
single human dose; All processing of the cell bank and subsequent cell cultures is
— the cell substrate used for the preparation of the vaccine. done under aseptic conditions in an area where no other cells
are handled. Approved animal (but not human) serum may
- ----------------------------- -------------------------------------------------------------------------- Ph Eur
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 and trypsin used in the preparation of cell suspensions
and media are shown to be free from extraneous agents.
Rabies Vaccine ** * The cell culture media may contain a pH indicator such as
(Rabies Vaccine for Human Use Prepared in Cell *** phenol red and approved antibiotics at the lowest effective
Cultures, Ph Eur monograph 0216) concentration. Not less than 500 mL of the cell cultures
The label may state ‘Rab’. employed for vaccine production are set aside as uninfected
cell cultures (control cells). The virus suspension is harvested
Ph Elf______________________________________________________________
on one or more occasions during incubation. Successive
DEFINITION harvests from the same production cell culture may be
Rabies vaccine for human use prepared in cell cultures is a pooled and considered as a single harvest.
freeze-dried preparation of a suitable strain of fixed rabies Only a single harvest that complies with the following
virus grown in cell cultures and inactivated by a validated requirements may be used in the preparation of the
method. inactivated viral harvest.
The vaccine is reconstituted immediately before use as stated Identification
on the label to give a clear or opalescent liquid that may be The single harvest contains virus that is identified as rabies
coloured owing to the presence of a pH indicator. virus using specific antibodies.
PRODUCTION Virus concentration
GENERAL PROVISIONS Titrate for infective virus in cell cultures; the titre is used to
The production of the vaccine is based on a virus seed-lot monitor consistency of production.
system and, if a cell line is used for virus propagation, a cell Control cells
bank system. The production method shall have been shown The control cells of the production cell culture from which
to yield consistently vaccines that comply with the the single harvest is derived comply with a test for
requirements for immunogenicity, safety and stability. Unless identification and with the requirements for extraneous
otherwise justified and authorised, the virus in the final agents (2.6.76).
vaccine must not have undergone more passages from the
PURIFICATION AND INACTIVATION
master seed lot than were used to prepare the vaccine shown
The virus harvest may be concentrated and/or purified by
in clinical studies to be satisfactory with respect to safety and
suitable methods; the virus harvest is inactivated by a
efficacy; even with authorised exceptions, the number of
validated method at a fixed, well-defined stage of the process,
passages beyond the level used for clinical studies must not
which may be before, during or after any concentration or
exceed 5.
purification.
In order to ensure that the virus inactivation process is immunodiffusion, enzyme-linked immunosorbent assay or an
effective, conditions that could lead to virus aggregation antibody-binding test. The content is within the limits
should be avoided at process steps preceding virus approved for the particular product.
inactivation. Any aggregates present in the preparation to be Sterility (2.6. /)
inactivated must be removed immediately before the The final bulk vaccine complies with the test for sterility,
inactivation process, for example by a suitable filtration carried out using 10 mL for each medium.
method. It shall have been demonstrated in process
validation studies that the inactivation process is consistently FINAL LOT
effective in inactivating rabies virus in such a way that it The final bulk vaccine is distributed aseptically into sterile
assures consistent protective immunogenic activity. containers and freeze-dried to a moisture content shown to
be favourable to the stability of the vaccine. The containers
The demonstration of consistency must be based on the
are then closed so as to avoid contamination and the
following: introduction of moisture.
— the inactivation kinetics are showท to be consistent using
at least 5 consecutive batches. Samples of virus, collected Only a final lot that complies with each of the requirements
at appropriate times, are inoculated into a sensitive given below under Identification, Tests and Assay may be
released for use. Provided that the test for bovine serum
substrate to establish the inactivation curve. If necessary,
the agent used for inactivation is neutralised prior to albumin has been carried out with satisfactory results on the
inoculation;
— the time needed to achieve complete inactivation is IDENTIFICATION
determined in order to define the required inactivation The vaccine is shown to contain rabies virus antigen by a
time. The test for residual infectious virus is used for this suitable immunochemical method (2.7. /) using specific
purpose. The total inactivation time used in routine antibodies, preferably monoclonal; alternatively, the assay
production must be at least twice the time needed for serves also to identify the vaccine.
complete virus inactivation.
TESTS
If betapropiolactone is used, the concentration shall at no Glycoprotein content
time exceed 1:3500 VIV. Determine the glycoprotein content by a suitable
Only an inactivated viral suspension that complies with the immunochemical method (2.7.7), for example, single-radial
following requirements may be used in the preparation of the immunodiffusion, enzyme-linked immunosorbent assay or an
final bulk vaccine. antibody-binding test. The content is within the limits
Residual infectious virus approved for the particular product.
Carry out an amplification test for residual infectious rabies Bovine serum albumin
virus immediately after inactivation or using a sample frozen Maximum 50 ng per single human dose, determined by a
immediately after inactivation and stored at -70 °C. suitable immunochemical method (2.7.1).
Inoculate a quantity of inactivated viral suspension equivalent
Sterility (2.6. 7)
to not less than 25 ml of bulk vaccine corresponding to at
least 25 human doses of vaccine into cell cultures of the
same type as those used for production of the vaccine or of Bacterial endotoxins (2.6.14)
another approved cell type. Cells used for the test must be of Less than 25 IU per single human dose.
optimal sensitivity regarding residual infectious rabies virus, Pyrogens (2.6.8)
for example, Veto, BHK-21 or neuroblastoma cells that are The test for pyrogens is performed only in cases of evidence
known to be highly sensitive to rabies virus may be used. of the presence of non-endotoxin pyrogenic substances.
If other cells are used, they must have been shown to possess It complies with the test. Unless otherwise justified and
at least the same sensitivity as those specified. A passage may authorised, inject into each rabbit 1 mL of a single human
be made after 7 days. Maintain the cultures for a total of dose of the vaccine diluted to 10-100 times its volume.
21 days and then examine the cell cultures for rabies virus Water (2.5.12)
using an immunofluorescence test or another suitable method Maximum 3.0 per cent.
of comparable sensitivity. The inactivated virus harvest
complies with the test if no replicating infectious rabies virus ASSAY
is detected. The potency of rabies vaccine is determined by comparing
the dose necessary to protect mice against the effects of a
Residual host-cell DNA lethal dose of rabies virus, administered intracerebrally, with
If a continuous cell line is used for virus propagation, the the quantity of a reference preparation of rabies vaccine
content of residual host-cell DNA, determined using a necessary to provide the same protection. For this
suitable method, is not greater than 10 ng per single human
comparison a reference preparation of rabies vaccine,
dose.
calibrated in International Units, and a suitable preparation
FINAL BULK VACCINE of rabies virus for use as the challenge preparation are
The final bulk vaccine is prepared from one or more necessary. Alternatively, in the interest of animal welfare, a
inactivated viral suspensions. An approved stabiliser may be validated serology potency assay or an immunochemical assay
added to maintain ±e activity of the product during and (2.7.1) for a native glycoprotein content is recommended.
after freeze-drying. The alternative method is validated against a challenge assay
Only a final bulk vaccine that complies with the following and approved for a given product by the competent
requirements may be used in ±e preparation of the final lot. authority.
Glycoprotein content The International Unit is the activity contained in a stated
Determine the glycoprotein content by a suitable quantity of the International Standard. The equivalence in
immunochemical method (2.7.7), for example, single-radial International Units of the International Standard is stated by
The challenge test described below uses a parallel-line model — the titration of the challenge suspension shows that
with at least 3 points for the vaccine to be examined and the 0.03 mL of the suspension contained not less than 10
reference preparation. Once the analyst has experience with LD50;
the method for a given vaccine, it is possible to carry out a — the statistical analysis shows a significant slope and no
simplified test using a single dilution of the vaccine to be significant deviations from linearity or parallelism of the
examined and of the reference preparation. Such a test dose-response curves;
enables the analyst to determine that the vaccine has a — the confidence limits (P = 0.95) are not less than
potency significantly higher than the required minimum, but 25 per cent and not more than 400 per cent of the
does not give full information on the validity of each estimated potency.
individual potency determination. The use of a single dilution The vaccine complies with the test if the estimated potency is
allows a considerable reduction in the number of animals not less than 2.5 ru per human dose.
required for the test and must be considered by each
Application of alternative end-points Once a laboratory has
laboratory in accordance with the provisions of the European
established the above assay for routine use, the lethal
Convention for the Protection of Vertebrate Animals Used
end-point is replaced by an observation of clinical signs and
for Experimental and Other Scientific Purposes.
application of an end-point earlier than death to reduce
Selection and distribution of the test animals Use healthy female animal suffering. The following is given as an example.
mice, about 4 weeks old, each weighing 11-1 ว g, and from
The progress of rabies infection in mice following
the same stock. Distribute the mice into 6 groups of a size
intracerebral injection can be represented by 5 stages defined
suitable to meet the requirements for validity of the test and,
by typical clinical signs:
for titration of the challenge suspension, 4 groups of 5.
Stage 1 ะ ruffled fur, hunched back;
Preparation of the challenge suspension Inoculate mice
intracerebrally with the Challenge Virus Standard (CVS) Stage 2: slow movements, loss of alertness (circular
strain of rabies virus and when the mice show signs of rabies, movements may also occur);
but before they die, euthanise them, then remove the brains Stage 3: shaky movements, trembling, convulsions;
and prepare a homogenate of the brain tissue in a suitable Stage 4: signs of paresis or paralysis;
diluent. Separate gross particulate matter by centrifugation Stage 5: moribund state.
and use the supernatant as the challenge suspension. Mice are observed at least twice daily from day 4 after
Distribute the suspension in small volumes in ampoules, seal
challenge. Clinical signs are recorded using a chart such as
and store at a temperature below -60 °C. Thaw 1 ampoule that shown in Table 0216.-1. Experience has shown that
of the suspension and make serial dilutions in a suitable using stage 3 as an end-point yields assay results equivalent
diluent. Allocate each dilution to a group of 5 mice and to those found when a lethal end-point is used. This must be
inject intracerebrally into each mouse 0.03 mL of the dilution verified by each laboratory by scoring a suitable number of
allocated to its group. Observe the mice for 14 days. assays using both the clinical signs and the lethal end-point.
Calculate the LD50 of the undiluted suspension using the
number in each group that, between the 5th and 14th days,
die or develop signs of rabies. Table 0216.-1. - Example of a chart used to record clinical signs
in the rabies vaccine potency test
Determination of potency of the vaccine Prepare 3 fivefold serial
dilutions of the vaccine to be examined and 3 fivefold serial Days after challenge
dilutions of the reference preparation. Prepare the dilutions Clinical signs 4 5 6 7 8 9 10 11
such that the most concentrated suspensions may be
Ruffled fur
expected to protect more than 50 per cent of the animals to Hunched back
which they are administered and the least concentrated
suspensions may be expected to protect less than 50 per cent
of the animals to which they are administered. Allocate the Slow movements
Loss of alertness
6 dilutions, 1 to each of the 6 groups of mice, and inject by
Circular movements
the intraperitoneal route into each mouse 0.5 mL of the
dilution allocated to its group. After 7 days, prepare
3 identical dilutions of the vaccine to be examined and of the Shaky movements
reference preparation and repeat the injections. Seven days Trembling
after the second injection, prepare a suspension of the Convulsions
challenge virus such that, on the basis of the preliminary

titration, 0.03 mL contains about 50 LD50. Inject Paresis
intracerebrally into each vaccinated mouse 0.03 mL of this Paralysis
suspension. Prepare 3 suitable serial dilutions of the
challenge suspension. Allocate the challenge suspension and Moribund state
the 3 dilutions, 1 to each of the 4 groups of 5 control mice,
and inject intracerebrally into each mouse 0.03 mL of ±e
suspension or dilution allocated to its group. Observe the
LABELLING
animals in each group for 14 days and record the number in
The label states the biological origin of the cells used for the
each group that die or show signs of rabies in the period
preparation of the vaccine.
5-14 days after challenge.
_____________________________________________ —_______________ Ph Eur
The test is not valid unless:
— for both the vaccine to be examined and the reference
preparation the 50 per cent protective dose lies between
the largest and smallest doses given to the mice;
LV-648 Vaccines 2016
Rotavirus Vaccine (Live, Oral) ★* *★ sequences of polymerase chain reaction (PCR)-amplified

VP7 gene segments.
(Ph. Eur. monograph 2417) * ** Virus concentration
The label may state ‘Rotavirus (live, oral)’. The virus concentration of the master and working seed lots
PhEir_______________________________________________ is determined to monitor consistency of production. Direct
cell-culture based methods and nucleic acid amplification
DEFINITION techniques (NAT) (2.6.2/) such as PCR quantification of
Rotavirus vaccine (live, oral) is a preparation of one or more virus replication in cell culture may be used.
suitable virus serotypes, grown in an approved cell substrate
and presented in a form suitable for oral administration. Extraneous agents (2.6.76)
Each working seed lot complies with the requirements for
The vaccine is a clear liquid or it may be a freeze-dried virus seed lots.
preparation to be reconstituted immediately before use, as
stated on the label, to give a slightly turbid liquid. VIRUS PROPAGATION, SINGLE HARVEST, MONOVALENT
The vaccine ready for administration may be coloured owing POOLED HARVEST
to the presence of a pH indicator. All processing of the cell bank and subsequent cell cultures is
PRODUCTION are being handled. Suitable animal (but not human) serum
GENERAL PROVISIONS may be used in the culture media, but the final medium for
The vaccine strains and the production method shall have maintaining cell growth during virus multiplication does not
been shown to yield consistently vaccines comparable with contain animal serum. Serum and trypsin used in the
the vaccine of proven clinical efficacy and safety in man. preparation of cell suspensions and culture media are shown
The vaccine is formulated so as to avoid inactivation by to be free from extraneous agents. The cell culture medium
gastric fluids. Where the vaccine is freeze-dried, the antacid may contain a pH indicator such as phenol red and suitable
capacity of the solvent and its stability are established. antibiotics at the lowest effective concentration. It is
The production of vaccine is based on a virus seed-lot system preferable to have a substrate free from antibiotics during
and a cell-bank system. Unless otherwise justified and production.
authorised, the virus in the final vaccine shall have undergone STORED VIRUS INTERMEDIATE CULTURE
no more passages from the master seed lot than were used to Where a stored virus intermediate culture, prepared from the
prepare the vaccine shown in clinical studies to be working seed lot, is used for inoculation, on the day of
satisfactory with respect to safety and efficacy. inoculation not less than 5 per cent or 500 mL of the cell
If purification steps are present, the reduction of selected cultures employed, whichever is greater, are set aside as
process-related impurities and residuals such as residual host uninfected cell cultures (control cells). Stored virus
cell proteins, residual cellular DNA, endotoxins, bovine intermediate cultures are harvested at a time appropriate to
serum, trypsin, and antibiotics is monitored to establish the strain of vims and stored at temperatures below - 60°C.
consistency of the purification process. Only a stored virus intermediate culture that complies with
REFERENCE PREPARATION the following requirements may be used for virus
A suitable reference preparation that is representative of propagation.
batches of vaccine shown to be effective in clinical trials is Identification
established for use in tests to determine virus concentration. Each stored vims intermediate culture is identified by
The differences in the composition and characteristics of rotavirus type by an immunological assay using specific
rotavirus vaccines mean that there will be a specific reference antibodies or by a molecular identity test such as NAT
preparation for each one. (2.6.2/).
SUBSTRATE FOR VIRUS PROPAGATION Bacterial and fungal contamination
The virus is propagated in a suitable cell line (5.2.2). Each stored vims intermediate culture complies with the test
VIRUS SEED LOTS for sterility (2.6./), carried out using 10 mL for each
The strain(s) of rotavirus used shall be identified by historical medium.
records that include information on the origin of each strain Virus concentration
and its subsequent manipulation including the method of The vims concentration of each stored vims intermediate
attenuation, whether the strains have been biologically cloned culture is determined as prescribed under Assay to monitor
prior to generation of the master seed lot, genetic sequence consistency of production. Both direct cell-culture based
information, the phenotypic and genotypic stability of the methods and NAT (2.6.2/) such as PCR quantification of
master and working seed lots when passaged up to the single vims replication in cell culture may be used.
harvest level, and the passage level at which attenuation for
humans was demonstrated by clinical trials. Virus seed lots
Each stored vims intermediate culture complies with the tests
are stored at temperatures below -20 cc if freeze-dried, or
for extraneous agents.
below -60 °C if not freeze-dried.
Control cells
The control cells of the production cell culture from which
each stored vims intermediate culture is derived comply with
Identification a test for identity and with the requirements for extraneous
The master and working seed lots are shown to be of the agents (2.6.16).
required rotavirus type by an immunological assay using
VIRUS PROPAGATION AND SINGLE HARVEST
specific antibodies or by a molecular identity test such as
On the day of inoculation with the vims working seed lot or
polyacrylamide gel electrophoresis of RNA, RNA/RNA
stored vims intermediate culture, cell cultures employed for
hybridisation, or restriction-enzyme mapping of genetic
vaccine production are set aside as uninfected cell cultures
^control cells). If bioreactor technology is used, the size and Only a final bulk vaccine that complies with the following
handling of the cell sample to be examined is approved by requirement may be used in the preparation of the final lot.
the competent authority. The virus suspensions are harvested
at a time appropriate to the strain of virus being used.
The final bulk vaccine complies with the test for sterility
Only a single virus harvest that complies with the following (2.6./), carried out using 10 mL for each medium.
requirements may be used for further processing.
FINAL LOT
Bacterial and fungal contamination The final bulk vaccine is distributed aseptically into sterile
Each single virus harvest complies with the test for sterility containers and may be freeze-dried to a moisture content
(2.6./), earned out using 10 mL for each medium. shown to be favourable to the stability of the vaccine.
Control cells The containers are then closed so as to avoid contamination
The control cells of the production cell culture from which and the introduction of moisture.
each single harvest is derived comply with a test for identity An approved minimum virus concentration for release of the
and with the requirements for extraneous agents (2.6.76). product is established for each virus type to ensure, in light
MONOVALENT POOLED HARVEST of stability data, that the minimum concentration stated on
Monovalent pooled harvests are prepared by pooling a the label will be present at the end of the period of validity.
number of single harvests of the same virus type. If no For freeze-dried vaccines, tests for identity, pH, volume,
monovalent pooled harvest is prepared, the tests below are sterility and content of key components are carried out on
earned out on each single harvest. the solvent.
Only a single harvest or a monovalent pooled harvest that Only a final lot that complies with the following requirement
complies with the following requirements may be used in the for thermal stability and is satisfactory with respect to each of
preparation of the purified monovalent harvest. the requirements given below under Identification, Tests and
Identification Assay may be released for use.
Each single harvest or monovalent pooled harvest is Thermal stability
identified by rotavirus type by an immunological assay using Maintain not fewer than 3 containers of the final lot at an
specific antibodies or by a molecular identity test such as elevated temperature for a defined time period, using
NAT (2.6.2/). conditions found suitable for the particular product as
Bacterial and fungal contamination approved by the competent authority. Determine the virus
Each single harvest or monovalent pooled harvest complies concentration as described under Assay in parallel for the
with the test for sterility (2.6. /), carried out using 10 mL for heated vaccine and for vaccine maintained at the temperature
each medium. recommended for storage. The virus concentration of the
containers that have been heated does not decrease by more
Virus concentration than an approved amount during the period of exposure.
The virus concentration of each single harvest or monovalent For a multivalent vaccine, if there is no significant difference
pooled harvest is determined as prescribed under Assay to in the virus loss between serotypes, the loss may be
monitor consistency of production. Both direct cell-culture determined from total virus concentration.
based methods and NAT (2.6.2/) such as PCR
quantification of virus replication in cell culture may be used. IDENTIFICATION
The vaccine is shown to contain rotavirus of each type stated
on the label by an immunological assay using specific
Each single harvest or monovalent pooled harvest complies
antibodies or by a molecular identity test. If PCR is used for
with the tests for extraneous agents.
the assay, this may serve as the identity test.
PURIFIED MONOVALENT HARVEST
The purified monovalent harvest is prepared from a single TESTS
harvest or a pooled monovalent harvest. The single harvest or Bacterial and fungal contamination
pooled monovalent harvest is clarified to remove cell debris The vaccine complies with the test for sterility (2.6./).
and may be further purified. Water (2.5.72)
Only a purified monovalent harvest that complies with the Maximum 3.0 per cent for each final lot of freeze-dried
following requirements may be used in the preparation of the vaccine.
final bulk vaccine. ASSAY
Bacterial and fungal contamination The assay of rotavirus vaccine is carried out by inoculation of
The purified monovalent harvest complies with the test for suitable cell cdtures with dilutions of the vaccine and
sterility (2.6./), carried out using 10 mL for each medium. evaluation of the rotavirus concentration, either by
Virus concentration visualisation of infected areas of a cell monolayer or by
The virus concentration of the purified monovalent harvest is comparison of the capacity of the vaccine to produce viral
determined as prescribed under Assay to monitor consistency RNA following infection of cells with the corresponding
of production. Both direct cell-culture based methods and capacity of an approved reference preparation.
NAT (2.6.2/) such as PCR quantification of virus replication For the assay based on visualisation of infected areas of a cell
in cell culture may be used. monolayer, titrate the vaccine for infective virus using at least
3 separate containers. Titrate the contents of 1 container of
Residual cellular DNA
an appropriate virus reference preparation in triplicate to
Maximum 100 pg of cellular DNA per human dose for
validate each assay. If the vaccine contains more than
viruses grown in continuous cells lines.
1 rotavirus type, titrate each type separately using a method
FINAL BULK VACCINE of suitable specificity. The virus concentration of the
The final bulk vaccine is prepared from one or more reference preparation is monitored using a control chart and
satisfactory purified monovalent harvests and may contain a titre is established on a historical basis by each laboratory.
more than one virus type. Suitable stabilisers may be added.
Calculate the individual virus concentration for each example, 5.3) to calculate the virus concentration of the
container of vaccine and for each replicate of the reference sample. The confidence interval (P = 0.95) of the combined
preparation as well as the corresponding combined virus virus concentration is not greater than + 0.3 logio
concentrations, using the usual statistical methods (for infectious units.
example, 5.3).
LABELLING
The assay is not valid if: The label states:
— the confidence interval (P = 0.95) of the estimated virus — the type or types of rotavirus contained in the vaccine;
concentration of the reference preparation for the 3 — the minimum amount of each type of virus contained in
replicates combined is greater than ± 0.3 log]0 CCID50 1 single human dose;
(or an equivalent value expressed with a unit suitable for — the cell substrate used for the preparation of the vaccine.
the method used for the assay);
__________ _ PhEir
by more than 0.5 log10 CCID50 (or an equivalent value
expressed with a unit suitable for the method used for the
assay) from the established value.
The assay is repeated if the confidence interval (P = 0.95) of Rubella Vaccine, Live ** **
the combined virus concentration of the vaccine is greater
than ± 0.3 log]0 CCID50 (or an equivalent value expressed (Ph. Eur. monograph 0162) *
with a unit suitable for the method used for the assay); data The label may state ‘Rubella’.
generated from valid assays only are combined by the usual
Ph Eur________________________________________ _____ ________________
statistical methods (for example, 5.3) to calculate the vims
concentration of the sample. The confidence interval DEFINITION
(P = 0.95) of the combined virus concentration is not greater Rubella vaccine (live) is a frcezc-dricd preparation of a
than ± 0.3 logio CCID50 (or an equivalent value expressed suitable attenuated strain of rubella virus. The vaccine is
with a unit suitable for the method used for the assay). reconstituted immediately before use, as stated on the label,
Where justified and authorised, different assay designs may to give a clear liquid that may be coloured owing to the
be used; this may imply the application of different validity presence of a pH indicator.
and acceptance criteria. However, the vaccine must comply if PRODUCTION
tested as described above. The production of vaccine is based on a virus seed-lot system
For the assay based on comparison of the capacity of the vaccine and a cell-bank system. The production method shall have
to produce viral RNA Following infection of cells with the been shown to yield consistently live rubella vaccines of
corresponding capacity of an approved reference preparation, adequate immunogenicity and safety in man. Unless
a suitable number of cell cultures in a microtitre plate are otherwise justified and authorised, the virus in the final
infected in parallel with serial dilutions of the vaccine and the vaccine shall have undergone no more passages from the
reference preparation. After incubation to allow virus master seed lot than were used to prepare the vaccine shown
replication, viral RNA in the individual wells is released from in clinical studies to be satisfactory with respect to safety and
the cells and quantified by NAT (2.6.2/), such as real-time efficacy.
quantitative reverse-transcriptase polymerase chain reaction The potential neurovirulence of the vaccine strain is
(RT-PCR) technology. considered during preclinical development, based on available
Not fewer than 3 separate containers of the vaccine are epidemiological data on neurovirulence and neurotropism,
assayed against a container of the reference preparation primarily for the wild-type virus. In light of this, a risk
titrated in triplicate. analysis is carried out. Where necessary and if available, a
Calculate the individual virus concentration for each test is carried out on the vaccine strain using an animal
container of vaccine against the reference preparation as well model that differentiates wild-type and attenuated virus; tests
as the corresponding combined virus concentrations, using on strains of intermediate attenuation may also be needed.
the usual statistical methods (for example, 5.3). The production method is validated to demonstrate that the
The combined estimate of the virus concentration for the product, if tested, would comply with the test for abnormal
3 containers of vaccine is not less than that stated on the toxicity for immunosera and vaccines for human use (2.6.9).
label. SUBSTRATE FOR VIRUS PROPAGATION
The assay is not valid unless: The virus is propagated in human diploid cells (5.2.2).
— the negative external NAT control is unambiguously SEED LOT
negative; The strain of rubella virus used shall be identified by
— the positive external NAT control is unambiguously historical records that include information on the origin of
positive; the strain and its subsequent manipulation. Virus seed lots
— the negative matrix control (uninfected cells) is are prepared in large quantities and stored at temperatures
unambiguously negative; below -20 °C if freeze-dried, or below -60 °C if not freeze-
— the positive matrix control (cells spiked with viral RNA) is dried.
unambiguously positive; Only a seed lot that complies with the following requirements
— the statistical analysis shows a significant slope and no may be used for virus propagation.
significant deviations from linearity or parallelism of the
dose-response curves. Identification
The master and working seed lots are identified as rubella
The assay is repeated if the confidence interval (P = 0.95) of virus by serum neutralisation in cell culture, using specific
the combined virus concentration of the vaccine is greater antibodies.
than ± 0.3 Iog10 infectious units; data generated from valid
assays only are combined by the usual statistical methods (for
Virus concentration albumin has been carried out with satisfactory results on the
The virus concentration of the master and working seed lots final bulk vaccine, it may be omitted on the final lot.
IS determined to ensure consistency of production.
Thermal stability
Extraneous agents (2.6.76) Maintain at least 3 vials of the final lot of freeze-dried
The working seed lot complies with the requirements for vaccine in the dry state at 37 ± 1 °C for 7 days. Determine
seed lots. the virus concentration as described under Assay in parallel
PROPAGATION AND HARVEST for the heated vaccine and for vaccine stored at the
All processing of the cell bank and subsequent cell cultures is temperature recommended for storage. The virus
done under aseptic conditions in an area where no other cells concentration of the heated vaccine is not more than 1.0
are handled during the production. Suitable animal (but not logio lower than that of the unheated vaccine.
human) serum may be used in the growth medium, but the IDENTIFICATION
nnal medium for maintaining cell growth during virus When the vaccine reconstituted as stated on the label is
multiplication does not contain animal serum. Serum and mixed with specific rubella antibodies, it is no longer able to
trypsin used in the preparation of cell suspensions and infect susceptible cell cultures.
culture media are shown to be free from extraneous agents.
The cell culture medium may contain a pH indicator such as TESTS
phenol red and suitable antibiotics at the lowest effective Bacterial and fungal contamination
concentration. It is preferable to have a substrate free from The reconstituted vaccine complies with the test for sterility
antibiotics during production. Not less than 500 mL of the (2.6.7).
production cell cultures is set aside as uninfected cell cultures Bovine serum albumin
(control cells). The temperature of incubation is controlled Not more than 50 ng per single human dose, determined by
during the growth of the virus. The virus suspension is a suitable immunochemical method (2.7.7).
harvested, on one or more occasions, within 28 days of Water (2.5.72)
inoculation. Multiple harvests from the same production cell Not more than 3.0 per cent, determined by the semi-micro
culture may be pooled and considered as a single harvest. determination of water.
ASSAY
vaccine. Titrate the vaccine for infective virus, using at least
3 separate vials of vaccine and inoculating a suitable number
Identification of wells for each dilution step. Titrate 1 vial of an
The single harvest contains virus that is identified as rubella appropriate virus reference preparation in triplicate to
virus by serum neutralisation in cell culture, using specific validate each assay. The virus concentration of the reference
antibodies. preparation is monitored using a control chart and a titre is
Virus concentration established on a historical basis by each laboratory.
The virus concentration in the single harvest is determined as The relation with the appropriate European Pharmacopoeia
prescribed under Assay to monitor consistency of production Biological Reference Preparation is established and
and to determine the dilution to be used for the final bulk monitored at regular intervals if a manufacturer's reference
vaccine. preparation is used. Calculate the individual virus
Extraneous agents (2.6.16) concentration for each vial of vaccine and for each replicate
The single harvest complies with the tests for extraneous of the reference preparation as well as the corresponding
agents. combined virus concentrations, using the usual statistical
methods (for example, 5.3). The combined estimate of the
Control cells virus concentration for the 3 vials of vaccine is not less than
The control cells comply with a test for identification and that stated on the label; the minimum virus concentration
with the tests for extraneous agents (2.6.76). stated on the label is not less than 3.0 log10 CCED50 per
FINAL BULK VACCINE single human dose.
Single harvests that comply with the above tests are pooled The assay is not valid ifi
and clarified to remove cells. A suitable stabiliser may be — the confidence interval (P = 0.95) of the estimated virus
added and the pooled harvests diluted as appropriate. concentration of ±e reference preparation for the
Only a final bulk vaccine that complies with the following 3 replicates combined is greater than ± 0.3
requirement may be used in the preparation of the final lot. logio CCID50;
Bacterial and fungal contamination — the virus concentration of the reference preparation differs
The final bulk vaccine complies with the test for sterility by more than 0.5 logio CCID50 from the established
(2.6.7), carried out using 10 mL for each medium. value.
FINAL LOT
The assay is repeated if the confidence interval (P = 0.95) of
A minimum virus concentration for release of the product is
than ± 0.3 logio CCID50; data obtained from valid assays
established such as to ensure, in light of stability data, that
only are combined by the usual statistical methods (for
the minimum concentration stated on the label will be
example, 5.3) to calcdate the virus concentration of the
present at the end of the period of validity.
Only a final lot ±at complies with the requirements for virus concentration is not greater than ± 0.3 logio CCID50.
minimum virus concentration for release, with the following
Rubella vaccine (live) BRP is suitable for use as a reference
requirement for thermal stability and with each of the
preparation.
requirements given below under Identification and Tests may
be released for use. Provided that the test for bovine serum Where justified and authorised, different assay designs may
be used; this may imply the application of different validity
and acceptance criteria. However, the vaccine must comply if Identification

tested as described above. The master and working seed lots arc identified as human
LABELLING herpesvirus 3 by serum neutralisation in cell culture, using
The label states: specific antibodies.
— the strain of virus used for the preparation of the vaccine; Virus concentration
— the type and origin of the cells used for the preparation of The virus concentration of the master and working seed lots
the vaccine; is determined as prescribed under Assay to monitor
— the minimum virus concentration; consistency of production.
— that contact between the vaccine and disinfectants is to be Extraneous agents (2.6.76)
avoided. The working seed lot complies with the requirements for
------------------------------------------------------------------------ ------------------------------ PhEur seed lots for live virus vaccines; a sample of 50 mL is taken
for the test in cell cultures.
VIRUS PROPAGATION AND HARVEST
All processing of the cell bank and subsequent cell cultures is
Shingles (Herpes Zoster) Vaccine ****** or virus are being handled. Approved animal (but not
(Live) ***** human) serum may be used in the 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
The label may state ‘Shingles (live)’. medium may contain a pH indicator such as phenol red and
PhEur_____________________________________________________________ approved antibiotics at the lowest effective concentration.
DEFINITION It is preferable to have a substrate free from antibiotics
during production. 5 per cent, but not less than 50 mL, of
Shingles (herpes zoster) vaccine (live) is a freeze-dried
the cell cultures employed for vaccine production is set aside
preparation of a suitable attenuated strain of human
as uninfected cell cultures (control cells). The infected cells
herpesvirus 3. The vaccine is reconstituted immediately
constituting a single harvest arc washed, released from the
before use, as stated on the label, to give a clear or slightly
support surface and pooled. The cell suspension is disrupted
opalescent liquid, almost white suspension or pale yellow
by sonication.
liquid that may be coloured owing to the presence of a pH
indicator. It is intended for administration to adults. Only a virus harvest that complies with the following
PRODUCTION vaccine.
The production of vaccine is based on a virus seed-lot system
and a cell-bank system. The production method shall have
Identification
The virus harvest contains virus that is identified as human
been shown to yield consistently live shingles vaccines of
herpesvirus 3 by serum neutralisation in cell culture, using
adequate immunogenicity and safety in man. The virus in the
specific antibodies.
final vaccine shall not have been passaged in cell cultures
beyond a defined number of passages approved by the Virus concentration
competent authority from the original isolated virus. The concentration of infective virus in virus harvests is
The potential neurovirulence of the vaccine strain is determined as prescribed under Assay to monitor consistency
considered during preclinical development, based on available of production and to determine the dilution to be used for
epidemiological data on neurovirulence and neurotropism, the final bulk vaccine.
primarily for the wild-type virus. In fight of this, a risk Extraneous agents (2.6.76)
analysis is carried out. Where necessary and if available, a Use 50 mL for the test in cell cultures.
test is carried out on the vaccine strain using an animal Control cells
model that differentiates wild-type and attenuated virus; tests The control cells of the production cell culture from which
on strains of intermediate attenuation may also be needed. the single harvest is derived comply with a test for identity
The production method is validated to demonstrate that the and with the requirements for extraneous agents (2.6.76).
product, if tested, would comply with the test for abnormal FINAL BULK VACCINE
toxicity for immunosera and vaccines for human use (2.6.9). Virus harvests that comply with the above tests are pooled
SUBSTRATE FOR VIRUS PROPAGATION and clarified to remove cells. A suitable stabiliser may be
The virus is propagated in human diploid cells (5.2.2). added and the pooled harvests diluted as appropriate.
VIRUS SEED LOT Only a final bulk vaccine that complies with the following
The strain of human herpesvirus 3 shall be identified as requirements may be used in the preparation of the final lot.
being suitable by historical records that include information Bacterial and fungal contamination
on the origin of the strain and its subsequent manipulation. Carry out the test for sterility (2.6.7) using 10 mL for each
The virus shall at no time have been passaged in continuous medium.
cell lines. Seed lots are prepared in the same kind of cells as FINAL LOT
those used for the production of the final vaccine. Virus seed The final bulk vaccine is distributed aseptically into sterile,
lots are prepared in large quantities and stored at tamper-proof containers and freeze-dried to a moisture
temperatures below -20 °C if freeze-dried, or below -60 °C content shown to be favourable to the stability of the vaccine.
if not freeze-dried. The containers are then closed so as to prevent
Only a virus seed lot that complies with the following contamination and the introduction of moisture.
requirements may be used for virus propagation. Only a final lot that is satisfactory with respect to the test for
water and each of the requirements given below under
Identification, Tests and Assay may be released for use.

Provided that the test for bovine serum albumin has been Smallpox Vaccine (Live) * ♦
earned out with satisfactory results on the final bulk vaccine, (Ph. Eur. monograph 0164) *★*
The label may state ‘SMV(live)’.
Water (2.5.12)
Ph Eur--------------------------------------------------------------------------------------------------------
Not more than the amount shown to ensure stability of the
vaccine as approved by the competent authority, determined DEFINITION
by the semi-micro determination of water. Smallpox vaccine (live) is a liquid or freeze-dried preparation
of live vaccinia virus grown in ovo in the membranes of the
IDENTIFICATION
chick embryo, in cell cultures or in the skin of living animals.
mixed with specific human herpesvirus 3 antibodies, it is no This monograph applies to vaccines produced using strains of
longer able to infect susceptible cell cultures. confirmed efficacy in man, in particular those used during
eradication of smallpox, for example the Lister strain
TESTS (sometimes referred to as the Lister/Elstree strain) and the
Bacterial and fungal contamination New York City Board of Health (NYCBOH) strain. It does
The reconstituted vaccine complies with the test for sterility not apply to non-replicative strains such as Modified Virus
(2.6./). Ankara (MVA).
Bovine serum albumin PRODUCTION
Maximum 0.65 pg per human dose, determined by a suitable GENERAL PROVISIONS
immunochemical method (2.7. /). The production method shall have been shown to yield
ASSAY consistently smallpox vaccines of adequate safety and
Titrate the vaccine for infective virus, using at least immunogenicity in man. The strain used shall have been
3 separate vials of vaccine. Titrate 1 vial of an appropriate shown to produce typical vaccinia skin lesions in man.
virus reference preparation in triplicate to validate each assay. Production is based on a seed-lot system.
The virus concentration of tile reference preparation is The production method is validated to demonstrate that the
monitored using a control chan and a titre is established on a product, if tested, would comply with the test for abnormal
historical basis by each laboratory. Calculate the individual toxicity of immunosera and vaccines for human use (2.6.9).
virus concentration for each vial of vaccine and for each The International Reference Preparation for smallpox vaccine
replicate of the reference preparation as well as the is suitable for use as a reference preparation in virus titration.
corresponding combined virus concentrations, using the usual
statistical methods (for example, 5.3). The combined
estimate of the virus concentration for the 3 vials of vaccine Animals used for production of skin-derived vaccines
is not less than that stated on the label. If the vaccine is prepared in animals skins, the animals used
are of a species approved by the competent authority, are in
good health, are kept in closed or intensively monitored
— the confidence interval (P = 0.95) of the estimated virus
colonies, and have not previously been employed for
concentration of the reference preparation for the
experimental purposes. Only animals susceptible to infection
3 replicates combined is greater than + 0.3 loglo PFU;
by dermal inoculations with vaccinia virus may be used for
vaccine production.
by more than 0.5 logic PFU from the established value.
The animals are kept in well-constructed and adequately
The assay is repeated if the confidence interval (P = 0.95) of
ventilated animals rooms with cages spaced as far apart as
possible. Adequate precautions are taken to prevent cross
than ± 0.3 log10 PFU; data obtained from valid assays only
infection between cages. Not more than 1 large animal is
are combined by the usual statistical methods (for
housed per stall. Not more than 2 small animals are housed
example, 5.3) to calculate the virus concentration of the
per cage and cage-mates must not be interchanged.
The animals must be kept in the country of production of
virus concentration is not greater than ± 0.3 log10 PFU.
the vaccine in quarantine groups for a period of not less than
Where justified and authorised, different assay designs may 6 weeks before use.
be used; this may imply the application of different validity
If at any time during the quarantine period the overall death
and acceptance criteria. However, the vaccine must comply if
rate of the group reaches 5 per cent, no animals from that
tested as described above.
entire group may be used for vaccine production.
LABELLING The groups are kept continuously in isolation, as in
The label states: quarantine, even after completion of the quarantine period,
— the strain of virus used for the preparation of the vaccine; until the animals are used. After the last animal of a group
— the type and origin of the cells used for the preparation of has been taken, the room that housed the group is
the vaccine; thoroughly cleaned and decontaminated before receiving a
— the minimum virus concentration; new group.
— that contact between the vaccine and disinfectants is to be Animals that are to be inoculated are anaesthetised and
avoided; thoroughly examined. If an animal shows any pathological
— that the vaccine is not to be administered to pregnant lesion, it is not used in the preparation of a seed lot or a
women. vaccine, nor are any of the remaining animals of the
__________ ________ _ _________________________________________ Ph Eur quarantine group concerned unless it is evident that their use
will not impair the safety of the product.
The prophylactic and diagnostic measures adopted to
exclude the presence of infectious disease are approved by
the competent authority. According to the species of animals Virus from the working seed lot must have the same
used and the diseases to which that animal is liable in the characteristics as the strain that was used to prepare the
country where the vaccine is being produced, these measures master seed lot. The number of passages required to produce
may vary. Consideration must also be given to the danger of single harvests from the original isolate is limited and
spreading diseases to other countries to which the vaccine approved by the competent authority. Vaccine is produced
may be shipped. Special attention must always be given to from the working seed with a minimum number of
foot-and-mouth disease, brucellosis, Q fever, tuberculosis and intervening passages.
dermatomycosis, and it may also be necessary to consider Since cell culture production and clonal selection (for
diseases such as contagious pustular dermatitis (orf), anthrax, example, plaque purification) may lead to altered
rinderpest, haemorrhagic septicaemia, Rift valley fever and characteristics of the virus, the master seed virus must be
others. characterised as fully as possible, for example by comparing
Embryonated eggs the safety profile and biological characteristics of the strain
Embryonated eggs used for production are obtained from a with that of the parental isolate. The characterisation shall
flock free from specified pathogens (SPF) (5.2.2). include the following:
Human diploid cells, continuous cell lines — antigenic analyses using specific antisera and/or
Human diploid cells and continuous cell lines comply with monoclonal antibodies;
the requirements for cell substrates (5.2. ร). — biological studies such as infectivity titre, chorioallantoic
membrane (CAM) assay, in vitro yield and in vivo growth
Primary chick embryo cells characteristics in a suitable animal model;
Pnman,' chick embryo cells are derived from an SPF flock — genetic analyses such as restriction mapping/southem
(5.2.2). blotting, PCR analyses and limited sequencing studies;
Primary rabbit kidney cells — phenotypic and genetic stability upon passage in the
Only healthy rabbits derived from a closed colony approved substrate;
by the competent authority are used as a source. — neurovirulence testing and immunogenicity studies.
The animals, preferably 2-4 weeks old, are tested to ensure The characterisation tests arc also carried out on each
freedom from specified pathogens or their antibodies. working seed lot and on 3 batches of vaccine from the first
Where new animals are introduced into the colony, they are working seed lot to verify genetic stability of the vaccine
maintained in quarantine for a minimum of 2 months and strain.
shown to be free from specified pathogens. Animals to be Only a virus seed that complies with the following
used to provide kidneys shall not have been previously requirements may be used for virus propagation.
employed for experimental purposes, especially those
involving infectious agents. The colony is monitored for
Identification
Each working seed lot is identified as vaccinia virus using
zoonotic viruses and markers of contamination at regular
specific antibodies and molecular tests. Suitable tests are
intervals.
conducted to exclude the presence of variola virus and other
At the time the colony is established, all animals are tested to orthopoxviruses.
determine freedom from antibodies to possible viral
contaminants for which there is evidence of capacity for Virus concentration
infecting humans or evidence of capacity to replicate in vitro Determine by the CAM assay or by a suitable validated m
in cells of human origin. A test for retroviruses using a vitro assay (plaque assay or CCID50 assay). The virus
sensitive polymerase chain reaction (PCR)-based reverse concentration is the basis for the quantity of virus used in the
transcriptase assay is also included. Nucleic acid neurovirulence test.
amplification tests (2.6.2/) for retroviruses may also be used. Extraneous agents (2.6.16)
After the colony is established, it is monitored by testing a If the working seed lot is produced in embryonated eggs,
representative group of at least 5 per cent of the animals, human diploid cells, or in a continuous cell line, it complies
which are then bled at suitable (for example monthly) with the requirements for seed lots for virus vaccines. Seed
intervals. In addition, the colony is screened for pathogenic lots produced in embryonated eggs and seed lots produced in
micro-organisms, including mycobacteria, fungi and primary cell cultures comply with the additional requirements
mycoplasmas. The screening programme is designed to described below.
ensure that all animals are tested within a given period of Where the tests prescribed cannot be carried out because
time. complete neutralisation of the seed virus is not possible, the
Any animal that dies is examined to determine the cause of seed lot may be diluted to a concentration equivalent to that
death. If the presence of a causative infectious agent is of the dilution used as inoculum for production of vaccine
demonstrated in the colony, the production of smallpox prior to testing for extraneous viruses. Supplementary specific
vaccine is discontinued. testing for extraneous viruses using validated nucleic acid
amplification techniques (2.6.2/) or immunochemical
At the time of kidney harvest, the animals are examined for
methods (2.7./) may be envisaged. Where the indicator cell
the presence of abnormalities and, if any are noted, the
culture method for mycoplasma detection (2.6.7) cannot be
animals are not used for vaccine production.
carried out, nucleic acid amplification testing is performed
Each set of control cultures derived from a single group of instead.
animals used to produce a single virus harvest must remain
Seed lots to be used for embryonated egg or cell culture
identifiable as such until all testing, especially for extraneous
production are in addition to be tested for carry-over of
agents, is completed.
potential extraneous agents from the original seed. Given that
VIRUS SEED LOT the complete passage history of the original seed is unlikely to
The vaccinia virus isolate used for the master seed lot is be known and that more than one species may have been
identified by historical records that include information on its used, this additional testing must at least cover important
origin and the tests used in its characterisation. extraneous agents of concern.
The bioburden of master and working seed lots prepared in described below and kept at -70 °C or below until further
animal skins is limited by meticulous controls of facilities, processing. Virus harvests that comply with the prescribed
personnel, and animals used for production, and by specific tests may be pooled. No human protein is added to the virus
tests on the seeds. However, it may be difficult to ensure that suspension at any stage during production. If stabilisers are
seed lots produced in animal skins are totally free from added, they shall have been shown to have no antigenic or
extraneous agents, and consideration must be given to sensitising properties for man.
production procedures which remove or reduce them. Such Only a single harvest that complies with the following
lots must comply with the requirements indicated below. requirements may be used in the preparation of the final bulk
The absence of specific human pathogens is confirmed by vaccine.
additional testing procedures, for example, bacterial and
fungal cultures, virus culture, nucleic acid amplification
Control eggs
Control eggs comply with the tests for extraneous agents
testings (2.6.21} for viral agents.
(2.6.16). A sample of 2 per cent of uninoculated
Neurovirulence embryonated eggs (not less than 20 and not more than 50)
The neurovirulence of master and working seed lots is from the batch used for vaccine production shall be
assessed using a suitable animal model, for example in incubated under the same conditions as the inoculated eggs.
monkeys or mice. The parental isolate is used as comparator. At the time of virus harvest the uninoculated eggs are
Where the original isolate is not available for this purpose, processed in the same manner as the inoculated eggs.
equivalent materials may be used.
Sterility (2.6./)
VIRUS PROPAGATION AND HARVEST It complies with the test for sterility, carried out using 10 mL
Vaccine produced in living animals for each medium.
Before inoculation the animals are cleaned and thereafter Vaccine produced in cell cultures (primary chick
kept in scrupulously clean stalls until the vaccinia material is embryo cells, primary rabbit kidney cells, human
harvested. For 5 days before inoculation and during diploid cells or continuous cell lines)
incubation the animals remain under veterinary supervision All processing of the cell bank and subsequent cell cultures is
and must remain free from any sign of disease; daily rectal done under aseptic conditions in an area where no other cells
temperatures are recorded. If any abnormal rise in are handled at the same time during production. Suitable
temperature occurs or any clinical sign of disease is observed, animal (but not human) serum may be used in the culture
the production of vaccine from the group of animals media, but the final medium for maintaining cell growth
concerned must be suspended until the cause has been during virus multiplication does not contain animal serum.
resolved. Serum and trypsin used in the preparation of cell suspensions
The inoculation of seed virus is carried out on such parts of and media are shown to be free from extraneous agents.
the animal that are not liable to be soiled by urine and The cell culture medium may contain a pH indicator such as
faeces. The surface used for inoculation is shaved and phenol red and suitable antibiotics at the lowest effective
cleaned so as to achieve conditions that are as close as concentration. It is preferable to have a substrate free from
possible to surgical asepsis. If any antiseptic substance antibiotics during production. On the day of inoculation with
deleterious to the virus is used in the cleaning process it is the virus working seed lot, not less than 5 per cent or
removed by thorough rinsing with sterile water prior to 1000 mL, whichever is the least, of the cell cultures
inoculation. During inoculation the exposed surface of the employed for vaccine production are set aside as uninfected
animal not used for inoculation is covered with a sterile cell cultures (control cells); special requirements, given
covering. By historical experience the ventral surface of below, apply to control cells when the vaccine is produced in
female animals is appropriate for inoculation and inoculation primary rabbit kidney cell cultures.
of male animals is more appropriate on the flank. After inoculation of the production cell culture with the
Before the collection of the vaccinia material, any antibiotic is working seed lot, inoculated cells are maintained at a suitable
removed and the inoculated area is cleaned. fixed temperature, and the virus suspension is harvested after
The uninoculated surfaces are covered with a sterile covering. a suitable incubation period.
Before harvesting the animals are euthanised and Only a single harvest ±at complies with the following
exsanguinated to avoid heavy mixtures of the vaccinia requirements may be used in the preparation of the
material with blood. The vaccinia material from each animal monovalent pooled harvest.
is collected separately with aseptic precautions. All animals
Control cells
used in the production of vaccine are examined by autopsy. The control cells of the production cell culture from which
If evidence of any generalised or systemic disease other than the virus harvest is derived comply with a test for identity
vaccinia is found, the vaccinia material from that animal is and with the requirements for extraneous agents (2.6.16) or,
discarded. If the disease is considered to be a communicable where primary rabbit kidney cells cultures are used, with
one, the harvest from the entire group of animals exposed specific tests as mentioned hereafter. The test is invalid if
must be discarded unless otherwise justified and authorised. more than 20 per cent of the control cell cultures have been
Vaccine produced in eggs discarded at the end of the observation period.
All processing of embryonated eggs is done under aseptic Extraneous agents (2.6.16}
conditions in an area where no other infectious agents or The single harvest complies with the tests for extraneous
cells are handled at the same time. After inoculation and agents. Complete neutralisation of vaccinia virus may be
incubation at a controlled temperature only living and difficult to achieve at high virus concentration. In this case
suitable chick embryos are harvested. The age of the embryos specific tests such as nucleic acid amplification (2.6.2/) and
at the time of virus harvest is reckoned from the initial immunochemical tests (2.7. /) can replace non-specific testing
introduction of the egg into the incubator and shall be not in cell culture or eggs. To save biological reagents such as
more than 12 days. After homogenisation and clarification by vaccinia neutralising antisera, testing for extraneous agents
centrifugation, the extract of embryonic pulp is tested as
may be performed on the final bulk instead of on the single Virus concentration
harvests. The vaccinia virus concentration of the pooled harvest is
Vaccine prepared in primary chick embryo cells A sample of determined by chick egg CAM assay or in cell cultures.
fluids pooled from the control cultures is tested for A reference preparation is assayed in the same system in
adenoviruses and for avian retroviruses such as avian leukosis parallel for validation of die pooled harvest titration.
virus. In addition, a volume of each neutralised virus pool The virus concentration serves as the basis for the quantity of
equivalent to 100 human doses of vaccine or 10 mL, virus used in the neurovirulcnce test in mice.
whichever is the greater, is tested in a group of fertilised eggs Consistency of virus characteristics
by the allantoic route of inoculation, and a similar sample is Vaccinia virus in the pooled harvest or the final bulk is
tested in a separate group of eggs by the yolk-sac route of examined by tests that are able to determine that the
inoculation. In both cases 0.5 mL of inoculum is used per phenotypic and genetic characteristics of the vaccinia virus
egg. The virus pool passes the test if, after 3-7 days, there is have not undergone changes during the multiplication in the
no evidence of the presence of any extraneous agent. production system. The master seed or an equivalent
Vaccine prepared in primary rabbit kidney' cell cultures The preparation is used as a comparator in these tests and the
following special requirements apply to virus propagation, comparator and the tests to be used are approved by the
harvest and testing. On the day of inoculation with virus competent authority.
working seed, a sample of at least 30 mL of the pooled fluid Neurovirulence
is removed from the cell cultures of the kidneys of each The neurovirulcnce of the pooled harvest is assessed versus a
group of animals used to prepare the primary cell suspension. comparator original seed (or equivalent) by intracerebral
The pooled fluid is inoculated in primary kidney cell cultures inoculation into suckling mice. Other tests may be useful to
in such a way that the dilution of the pooled fluid does not discriminate between acceptable and unacceptable batches.
exceed 1 in 4. The cultures are incubated at a temperature of
Residual DNA
34-36 °C and observed for a period of at least 4 weeks.
For viruses grown in continuous cells the pooled harvest is
During this observation period and after not less than
tested for residual DNA. The production process
2 weeks of incubation, at least 1 subculture of fluid is made
demonstrates a level of cellular DNA of less than 10 ng per
from each of these cultures and observed also for a period of
human dose.
2 weeks. The test is invalid if more than 20 per cent of the
cultures are discarded. If evidence is found of the presence of Bacterial and fungal contamination
an extraneous agent, no cell cultures from the entire group For vaccines other than those prepared on animal skins, the
may be used for vaccine production. final bulk complies with the test for sterility (2.6./) using
— Control cell cultures. Cultures prepared on the day of 10 mL for each medium.
inoculation with the working virus seed lot from Mycoplasma (2.6.7)
25 per cent of the cell suspensions obtained from the For vaccines other than those prepared on animal skins, the
kidneys of each group of animals are maintained as final bulk complies with the test for mycoplasma, carried out
controls. These control cell cultures are incubated under using 10 mL.
the same conditions as the inoculated cultures for at least FINAL BULK VACCINE
2 weeks. The test is invalid if more than 20 per cent of A minimum virus concentration for release of the product is
the control cell cultures are discarded for non-specific established such as to ensure, in the light of stability data,
reasons. that the minimum concentration stated on the label will be
— Test for haemadsorbing viruses. At the time of harvest or present at the end of the period of validity.
not more than 4 days after the day of inoculation of the
production cultures with the virus working seed, a sample Vaccine produced in living animals
of 4 per cent of the control cell cultures is tested for The pooled harvest is centrifuged. If the vaccine is intended
haemadsorbing viruses by addition of guinea-pig red for issue in the liquid form, treatment to reduce the presence
blood cells. of extraneous agents may consist of the addition of glycerol
— Test for other extraneous agetits. At the time of harvest or or another suitable diluent, with or without an antimicrobial
not more than 7 days after the day of inoculation of the substance, and temporary storage at a suitable temperature.
production cultures with the virus working seed, a sample If the vaccine is intended for issue in the dried form, the
of at least 20 mL of the pooled fluid from each group of treatment may consist of the addition of a suitable
control cultures is tested for other extraneous agents. antimicrobial substance. The following special requirements
— Tests of neutralised single harvest in primary rabbit kidney cell apply to the bulk vaccine for vaccines produced in living
cultures. Each neutralised single harvest is additionally animals.
tested in primary kidney cell cultures prepared from a Only a final bulk vaccine that complies with the following
different group of animals to that used for production. requirements may be used in the preparation of the final lot.
POOLED HARVEST Total bacterial count
Only a pooled harvest that complies with the following For vaccines produced on animal skins only,
requirements and is within the limits approved for the maximum 50 per millilitre, determined by plate count using
product may be used in the preparation of the final lot. a suitable volume of the final bulk vaccine.
Identity Escherichia coll
The vaccinia virus in the pooled harvest is identified by At least 1 mL samples of a 1:100 dilution of the final bulk
serological methods, which may be supplemented by vaccine is cultured on plates of a medium suitable for
molecular methods. Molecular tests such as restriction differentiating E. coli from other bacteria. The plates are
fragment length polymorphism or partial sequencing, incubated at 35-37 °C for 48 h. If E. coli is detected the final
especially of terminal DNA sequences which show die bulk is discarded or, subject to approval by the competent
greatest variation between vaccinia strains, may be useful. authority, processed further.
Haemolytic streptococci, coagulase-positive the temperature recommended for storage. The virus
staphylococci or any other pathogenic micro-organisms concentration of the heated vaccine is not more than
which are known to be harmful to man by vaccination 1.0 logio lower than that of the unheated vaccine.
At least 1 mL samples of a 1:100 dilution of the final bulk
IDENTIFICATION
vaccine are cultured on blood agar. The plates are incubated
at 35-37 °C for 48 h. If micro-organisms are detected, the The vaccinia virus is identified by an appropriate method.
final bulk vaccine is discarded. TESTS
Bacillus anthracis Antimicrobial preservative
Any colony seen on any of the plates that morphologically Where applicable determine the amount of antimicrobial
resembles B. anthracis is examined. If the organisms preservative by a suitable chemical method. The content is
contained in the colony are non-motile, further tests for the not less than the minimum amount shown to be effective and
cultural character of B. anthracis are carried out, including is not greater than 115 per cent of the quantity stated on the
pathogenicity tests in suitable animals. If B. anthracis is found label.
to be present, the final bulk vaccine and any other associated Phenol (2.5J5)
bulks are discarded. Additional validated molecular testing Maximum 0.5 per cent, if phenol is used.
may be performed.
Protein content
Clostridium tetani and other pathogenic spore-forming The protein content of each filling lot, if not done on the
anaerobes final bulk, is determined and is within the limits approved by
A total volume of not less than 10 mL of the final bulk the competent authority.
vaccine is distributed in equal amounts into 10 tubes, each Bovine serum albumin
containing not less than 10 mL of suitable medium for the Maximum 50 ng per single human dose, determined by a
growth of anaerobic micro-organisms. The tubes are kept at suitable immunochemical method (2.7.7), where bovine
65 °C for 1 h in order to reduce the content of non-spore- serum albumin is used during cell culture.
forming organisms, after which they are anaerobically
incubated at 35-37 °C for at least 1 week. From every tube Ovalbumin
or plate showing growth, subcultures are made on plates of a For vaccines produced in embryonated eggs, the ovalbumin
suitable medium. Tubes and plates are incubated content is within the limits approved by the competent
anaerobically at the same temperature. All anaerobic colonies authority.
are examined and identified and if c. tetani or other Residual moisture
pathogenic spore-forming anaerobes are present, the final The residual moisture content of each final lot of freeze-dried
bulk is discarded. vaccines is within the limits approved by the competent
Vaccine produced in eggs authority.
The pooled harvest is clarified and may be further purified. Bacterial count
For skin-derived vaccines, examine the vaccine by suitable
Vaccine produced in cell cultures (primary chick
microscopic and culture methods for micro-organisms
embryos fibroblasts, human diploid cells or continuous
cell lines) pathogenic for man and, in particular, haemolytic
streptococci, staphylococci, pathogenic spore-bearing
The pooled harvest is clarified to remove cells and may be
further purified. organisms, especially B. anthracis, and E. coli. The vaccine is
free from such contaminants. The total number of non-
FINAL LOT pathogenic bacteria does not exceed 50 per millilitre.
Only a final lot that complies with the requirements for
Sterility (2.6. 7)
minimum virus concentration for release, with the following
Except for skin-derived vaccines, the vaccine complies with
requirement for thermal stability and with each of the
the test for sterility.
Assay may be released for use. Provided that the tests for Bacterial endotoxins (2.6.14)
antimicrobial preservative, protein content, bovine serum The vaccine complies with the specification approved by the
albumin and ovalbumin have been carried out with competent authority.
satisfactory results on the final bulk vaccine, they may be ASSAY
omitted on the final lot. Reconstitue the vaccine if necessary and titrate for infectious
Thermal stability virus using at least 3 separate containers of vaccine. Titrate
For liquid products, maintain not fewer than 3 containers of 1 container of an appropriate virus reference preparation in
the final lot at an elevated temperature for a defined time triplicate to validate each assay. The virus concentration of
period, using conditions found suitable for the particular the reference preparation is monitored using a control chart
product as approved by the competent authority. Determine and a titre is established on a historical basis by each
the virus concentration as described under Assay in parallel laboratory. Calculate the individual virus concentration for
for the heated vaccine and for vaccine stored at the each container of vaccine and for each replicate of the
temperature recommended for storage. The virus reference preparation as well as the corresponding combined
concentration of the containers that have been heated does virus concentrations, using the usual statistical methods (for
not decrease by more than an approved amount during the example, 5.3). The combined virus concentration for the
period of exposure. The conditions of the test and the 3 containers of vaccine is not less than 8.0 logio pock
requirements are approved by the competent authority. forming units per millilitre or the validated equivalent in
For freeze-dried products, maintain at least 3 containers of plaque-forming units or 50 per cent cell culture infective
the final lot in the dry state at 37 ± 1 °C for 28 days. doses, unless a lower titre is justified by clinical studies.
Determine the virus concentration as described under Assay The assay is not valid if:
in parallel for the heated vaccine and for vaccine stored at
— the confidence interval (P = 0.95) of the estimated virus toxinogenic strain of Clostridium letani with known origin and
concentration of the reference preparation for the 3 history is grown in a suitable liquid medium. At the end of
replicates combined is greater than ± 0.5 log]0 cultivation, the purity of each culture is tested and
infectious units; contaminated cultures are discarded. Toxin-containing
— the virus concentration of the reference preparation differs culture medium is collected aseptically. The toxin content
by more than 0.5 log]0 infectious units from the (Lf per millilitre) is checked (2.7.27) to monitor consistency
established value. of production. Single harvests may be pooled to prepare the
The assay is repeated if the confidence interval (P = 0.95) of bulk purified toxoid. The toxin is purified to remove
the combined virus concentration of the vaccine is greater components likely to cause adverse reactions in humans.
than ± 0.5 logio infectious units; data obtained from valid The purified toxin is detoxified with formaldehyde by a
assays only are combined by the usual statistical methods (for method that avoids destruction of the immunogenic potency
example, 5.5) to calculate the virus concentration of the of the toxoid and reversion of toxoid to toxin, particularly on
sample. The confidence interval (P = 0.95) of the combined exposure to heat. Alternatively, purification may be carried
virus concentration is not greater than ± 0.5 log10 out after detoxification.
infectious units. Only bulk purified toxoid that complies with the following
Where justified and authorised, different assay designs may requirements may be used in the preparation of the final bulk
be used; this may imply the application of different validity vaccine.
and acceptance criteria. However, the vaccine must comply if Sterility (2.6.7)
tested as described above. Carry out the test for sterility using 10 mL for each medium.
LABELLING Absence of toxin and irreversibility of toxoid
The label states: Using the same buffer solution as for the final vaccine,
— the designation of the vaccinia virus strain; without adsorbent, prepare a solution of bulk purified toxoid
— the minimum amount of virus per millilitre; at the same concentration as in the final vaccine. Divide the
— the substrate used for the preparation of the vaccine; dilution into 2 equal parts. Keep one of them at 5 + 3 °C
— the nature and amount of stabiliser, preservative or and the other at 37 °C for 6 weeks. Test both dilutions as
additive present in the vaccine and/or in the diluent. described below. Use 15 guinea-pigs, each weighing
_____________________________________________________ _________Ph Eur
250-350 g and that have not previously been treated with any
material that will interfere with the test. Inject
subcutaneously into each of 5 guinea-pigs 5 mL of the
dilution incubated at 5 ± 3 °C. Inject subcutaneously into
each of 5 other guinea-pigs 5 mL of the dilution incubated at
Adsorbed Tetanus Vaccine * * 37 °C. Inject subcutaneously into each of 5 guinea-pigs at
least 500 Lf of the non-incubated bulk purified toxoid in a
(Tetanus Vaccine (Adsorbed)3 *** volume of 1 mL. The bulk purified toxoid complies with the
Ph Eur monograph 0452) test if during the 21 days following the injection no animal
The label may state ‘Tef. shows signs of or dies from tetanus. If more than 1 animal
When Tetanus Vaccine is prescribed or demanded and the dies from non-specific causes, repeat the test; if more than
form is not stated, Adsorbed Tetanus Vaccine may be 1 animal dies in the second test, the toxoid does not comply
dispensed or supplied. with the test.
Ph Etr______________________________________________________________ Antigenic purity (2.7.27)
Not less than 1000 Lf per milligram of protein nitrogen.
DEFINITION
FINAL BULK VACCINE
Tetanus vaccine (adsorbed) is a preparation of tetanus formol
The final bulk vaccine is prepared by adsorption of a suitable
toxoid with a mineral adsorbent. The formol toxoid is
quantity of bulk purified toxoid onto a mineral carrier such
prepared from the toxin produced by the growth of
as hydrated aluminium phosphate or aluminium hydroxide;
Clostridium letani.
the resulting mixture is approximately isotonic with blood.
PRODUCTION Suitable antimicrobial preservatives may be added. Certain
GENERAL PROVISIONS antimicrobial preservatives, particularly those of the phenolic
Specific toxicity type, adversely affect the antigenic activity and must not be
The production method is validated to demonstrate that the used.
product, if tested, would comply with the following test: Only final bulk vaccine that complies with the following
inject subcutaneously 5 times the single human dose stated requirements may be used in the preparation of the final lot.
on the label into each of 5 healthy guinea-pigs, each weighing Antimicrobial preservative
250-350 g, that have not previously been treated with any Where applicable, determine the amount of antimicrobial
material that will interfere with the test. If within 21 days of preservative by a suitable chemical method. The amount is
the injection any of the animals shows signs of or dies from not less than 85 per cent and not greater than 115 per cent
tetanus, the vaccine does not comply with the test. If more of the intended amount.
than 1 animal dies from non-specific causes, repeat the test
Sterility (2.6. /)
once; if more than 1 animal dies in the second test, the
vaccine does not comply with the test.
FINAL LOT
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
2016 Vaccines TV-659
Only a final lot that is satisfactory with respect to each of the PRODUCTION
requirements given below under Identification, Tests and GENERAL PROVISIONS
Assay may be released for use. Provided the test for Production of the vaccine is based on a virus seed-lot system.
antimicrobial preservative and the assay have been carried The production method shall have been shown to yield
out with satisfactory results on the final bulk vaccine, they consistently vaccines comparable with the vaccine of proven
may be omitted on the final lot. clinical efficacy and safety in man. Unless otherwise justified
Provided the free formaldehyde content has been determined and authorised, the virus in the final vaccine shall not have
on the bulk purified toxoid or on the final bulk and it has undergone more passages from the master seed lot than the
been shown that the content in the final lot will not exceed virus in the vaccine used in clinical trials.
0.2 g/L, the test for free formaldehyde may be omitted on the The production method is validated to demonstrate that the
final lot. product, if tested, would comply with the test for abnormal
IDENTIFICATION toxicity for immunosera and vaccines for human use (2.6.9).
Tetanus toxoid is identified by a suitable immunochemical SUBSTRATE FOR VIRUS PROPAGATION
method (2.7.7). The following method, applicable to certain The virus is propagated in chick embryo cells prepared from
vaccines, is given as an example. Dissolve in the vaccine to eggs derived from a chicken flock free from specified
be examined sufficient sodium citrate R to give a 100 g/L pathogens (5.2.2) or in other suitable cell cultures (5.2.2).
solution. Maintain at 37 °C for about 16 h and centrifuge SEED LOTS
until a clear supernatant is obtained. The clear supernatant The strain of virus used is identified by historical records that
reacts with a suitable tetanus antitoxin, giving a precipitate. include information on the origin of the strain and its
TESTS subsequent manipulation. Virus seed lots are stored at or
Aluminium (2.5.72) below -60 °C.
Maximum 1.25 mg per single human dose, if aluminium Only a seed lot that complies with the following requirements
hydroxide or hydrated aluminium phosphate is used as the may be used for virus propagation.
adsorbent.
Identification
Free formaldehyde (2.4.18) Each seed lot is identified as containing the vaccine strain of
Maximum 0.2 g/L. tick-borne encephalitis virus by a suitable immunochemical
Antimicrobial preservative method (2.7.7), preferably using monoclonal antibodies.
Where applicable, determine the amount of antimicrobial Virus concentration
preservative by a suitable chemical method. The content is The virus concentration of each seed lot is determined by
not less than the minimum amount shown to be effective and titration in suitable cell cultures to monitor consistency of
is not greater than 115 per cent of the quantity stated on the production.
label.
Sterility (2.6.7) Each seed lot complies with the requirements for extraneous
The vaccine complies with the test for sterility. agents in viral vaccines for human use. For neutralisation of
ASSAY the vaccine virus, the use of monoclonal antibodies is
Carry out one of the prescribed methods for the assay of preferable.
tetanus vaccine (adsorbed) (2.7.8). VIRUS PROPAGATION AND HARVEST
The lower confidence limit (P = 0.95) of the estimated If the virus has been passaged in mouse brain during
potency is not less than 40 IU per single human dose. preparation of the master seed lot, not fewer than 2 passages
of the master seed virus in cell culture are made before
LABELLING
inoculation of the production cell culture.
The label states:
— the minimum number of International Units per single All processing of the cell cultures is performed under aseptic
human dose, conditions in an area where no other cells are being handled.
Serum and trypsin used in the preparation of cell suspensions
— the name and the amount of the adsorbent,
and media used must be shown to be free from extraneous
— that the vaccine must be shaken before use,
agents. The cell culture media may contain a pH indicator
such as phenol red and approved antibiotics at the lowest
_______________________________________________________________ Ph Eur
effective concentration. At least 500 mL of the cell cultures
employed for vaccine production is set aside as uninfected
cell cultures (control cells).
Tick-borne Encephalitis Vaccine, ***** requirements may be used in the preparation of the
Inactivated ***** inactivated harvest.
(Tick-borne Encephalitis Vaccine (Inactivated)> Identification
Ph Eur monograph 1375) The single harvest is shown to contain tick-bome encephalitis
virus by a suitable immunochemical method (2.7.7),
The label may state ‘Tic/enceph’.
preferably using monoclonal antibodies, or by virus
neutralisation in cell cultures.
DEFINITION Bacterial and fungal contamination
Tick-borne encephalitis vaccine (inactivated) is a liquid The single harvest complies with the test for sterility (2.6.7),
preparation of a suitable strain of tick-borne encephalitis carried out using 10 mL for each medium.
virus grown in cultures of chick-embryo cells or other
suitable cell cultures and inactivated by a suitable, validated
method.
Mycoplasmas (2.6.7) Sterility (2.6.1)

The single harvest complies with the test for mycoplasmas The final bulk vaccine complies with the test for sterility,
carried out using 1 mL for each medium. carried out using 10 mL for each medium.
Control cells FINAL LOT
The control cells comply with the tests for extraneous agents Only a final lot that is satisfactory with respect to each of the
(2.6.16). If the vaccine is produced using a cell-bank system, requirements given below under Identification, Tests and
the control cells comply with a test for identification. Assay may be released for use. Provided that the tests for free
Virus concentration formaldehyde, bovine serum albumin (where applicable) and
Determine the virus concentration by titration in suitable cell pyrogens and the assay have been carried out with
cultures to monitor consistency of production. satisfactory results on the final bulk vaccine, they may be
INACTIVATION
To avoid interference, viral aggregates are removed, where IDENTIFICATION
necessary, by filtration immediately before the inactivation The vaccine is shown to contain tick-bome encephalitis virus
process. The virus suspension is inactivated by a validated antigen by a suitable immunochemical method (2.7. /) using
method; the me±od shall have been shown to be consistently specific antibodies. The assay also serves to identify the
capable of inactivating tick-bome encephalitis virus without vaccine.
destroying the antigenic and immunogenic activity; as part of TESTS
the validation studies, an inactivation curve is plotted
Aluminium (2.5.13)
representing residual live virus concentration measured on Maximum 1.25 mg per single human dose, if aluminium
not fewer than 3 occasions. If formaldehyde is used for hydroxide or hydrated aluminium phosphate is used as the
inactivation, the presence of an excess of free formaldehyde is adsorbent.
verified at ±e end of the inactivation process.
Only an inactivated harvest that complies with the following
Maximum 0.1 g/L.
vaccine. Bovine serum albumin
Maximum 50 ng per single human dose, determined by a
Residual infective virus
suitable immunochemical method (2.7./), if bovine serum
Inoculate a quantity of the inactivated harvest equivalent to
albumin has been used during production.
not less than 10 human doses of vaccine in the final lot into
primary chicken fibroblast cell cultures, or other cells shown Sterility (2.6.1)
to be at least as sensitive to tick-bome encephalitis virus, with The vaccine complies with the test for sterility.
not less than 3 cm2 of cell sheet per millilitre of inoculum. Pyrogens (2.6.8)
Incubate at 37 ± 1 °C for 14 days. No cytopathic effect is The vaccine complies with the test for pyrogens. Inject into
detected at the end of the incubation period. Collect the each rabbit, per kilogram of body mass, 1 dose of vaccine.
culture fluid and examine for the presence of infective tick-
ASSAY
bome encephalitis virus by the following test in mice or by a
The potency is determined by comparing the dose necessary’
validated in vitro method: inoculate 0.03 mL intracerebrally
to protect a given proponion of mice against the effects of a
into each of not fewer than 10 mice about 4 weeks old.
lethal dose of tick-bome encephalitis virus, administered
Observe the mice for 14 days. They show no evidence of
intraperitoneally, with the quantity of a reference preparation
tick-bome encephalitis virus infection.
of tick-bome encephalitis vaccine necessary to provide the
PURIFICATION same protection. For this comparison an approved reference
Several inactivated single harvests may be pooled before preparation and a suitable preparation of tick-bome
concentration and purification by suitable methods, encephalitis virus from an approved strain for use as the
preferably by continuous-flow, sucrose density-gradient challenge preparation are necessary.
centrifugation.
The following is cited as an example of a method that has been
Several purified inactivated harvests may be pooled. found suitable for a given vaccine.
Only a purified, inactivated harvest that complies with the Selection and distribution of test animals Use healthy mice
following requirements may be used in the preparation of the weighing 11-17 g and derived from the same stock.
final bulk vaccine. Distribute the mice into not fewer than 6 groups of a suitable
Sterility (2.6./) size to meet the requirements for validity of the test;
The purified, inactivated harvest complies with the test for for titration of the challenge suspension, use not fewer than
sterility carried out using 10 mL for each medium. 4 groups of 10 mice. Use mice of the same sex or distribute
Specific activity males and females equally between groups.
Determine the antigen content of the purified, inactivated Determination of potency of the vaccine Prepare not fewer than
harvest by a suitable immunochemical method (2.7.1). 3 suitable dilutions of the vaccine to be examined and of the
Determine the total protein content by a suitable method. reference preparation; in order to comply with validity criteria
The specific activity, calculated as the antigen content 4 or 5 dilutions will usually be necessary. Prepare dilutions
per unit mass of protein, is within ±e limits approved for the such that the most concentrated suspension is expected to
specific product. protect more than 50 per cent of the animals and the least
concentrated suspension less than 50 per cent. Allocate each
FINAL BULK VACCINE
dilution to a different group of mice and inject
The final bulk vaccine is prepared from one or more purified,
subcutaneously into each mouse 0.2 mL of the dilution
inactivated harvests.
allocated to its group. 7 days later make a second injection
Only a final bulk vaccine that complies with the following using the same dilution scale. 14 days after the second
requirement may be used in the preparation of the final lot. injection prepare a suspension of the challenge virus
containing not less than 100 LD50 in 0.2 mL. Inject 0.2 mL BACTERIAL SEED LOTS
of this virus suspension intraperitoneally into each vaccinated The strain of ร. typhi used for the master seed lot shall be
mouse. To verify the challenge dose, prepare a series of not identified by historical records that include information on its
fewer than 3 dilutions of the challenge virus suspension at origin and by its biochemical and serological characteristics.
not greater than one-hundredfold intervals. Allocate the Cultures from the working seed lot shall have the same
challenge suspension and all of the dilutions, one to each of characteristics as the strain that was used to prepare the
the groups of 10 mice, and inject intraperitoneally into each master seed lot.
mouse 0.2 mL of the challenge suspension or the dilution Only a strain ±at has the following characteristics may be
allocated to its group. Observe the animals for 21 days after used in the preparation of the vaccine: (a) stained smears
the challenge and record the number of mice that die in the from a culture are typical of enterobacteria; (b) the culture
period between 7 and 21 days after the challenge. Humane utilises glucose without production of gas; (c) colonies on
endpoints may be used to avoid unnecessary suffering of agar are oxidase-negative; (d) a suspension of the culture
animals after the virulent challenge. agglutinates specifically with a suitable Vi antiserum or
Calculations Calculate the results for an assay with quantal colonies form haloes on an agar plate containing a suitable Vi
responses by the usual statistical methods (for example, 5.3). antiserum.
Validity criteria The test is not valid unless: Purity of bacterial strain used for the seed lot is verified by
the concentration of the challenge virus is not less than methods of suitable sensitivity. These may include
100 LD50, inoculation into suitable media, examination of colony
for both the vaccine to be examined and the reference morphology, microscopic examination of Gram-Stained
preparation the 50 per cent protective dose (PD50) lies smears and culture agglutination with suitable specific
between the largest and smallest doses given to the mice, antisera.
the statistical analysis shows a significant slope and no CULTURE AND HARVEST
significant deviation from linearity and parallelism of The working seed lot is cultured on a solid medium, which
the dose-response lines, may contain blood-group substances, or a liquid medium;
the confidence limits (P = 0.95) are not less than the inoculum obtained is transferred to a liquid medium
33 per cent and not more than 300 per cent of the which is used to inoculate the final medium. The liquid
estimated potency. medium used and the final medium are semi-synthetic, free
Potency requirement Include all valid tests to estimate the from substances that are precipitated by cetrimonium
mean potency and the confidence limits (P ะ= 0.95) for the bromide and do not contain blood-group substances or high-
mean potency; compute weighted means with the inverse of molecular-mass polysaccharides, unless it has been
the squared standard error as weights. The vaccine complies demonstrated that they are removed by the purification
with the test if the estimated potency is not less than that process.
approved by the competent authority, based on data from The bacterial purity of the culture is verified by methods of
chnical efficacy trials. suitable sensitivity. These may include inoculation into
LABELLING suitable media, examination of colony morphology,
The label states: microscopic examination of Gram-Stained smears and culture
— the strain of virus used in preparation, agglutination with suitable specific antisera.
— the type of cells used for production of the vaccine. The culture is then inactivated at the beginning of the
----- ----------------------------------------------------------------------------------------------------- Ph Eur stationary phase by the addition of formaldehyde. 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
Typhoid Polysaccharide Vaccine ****** -20 °C before purification.
PURIFIED VI POLYSACCHARIDE
The polysaccharide is purified, after dissociation of the
The label may state ‘Typhoid’. polysaccharide/cetrimonium bromide complex, using suitable
Ph Eur _____________________________________________________________ procedures to eliminate successively nucleic acids, proteins
and lipopolysaccharides. The polysaccharide is precipitated as
DEFINITION
the calcium salt in ±e presence of ethanol and dried at
Typhoid polysaccharide vaccine is a preparation of purified
2-8 °C; the powder obtained constitutes the purified Vi
Vi capsular polysaccharide obtained from Salmonella typhi
polysaccharide. The loss on drying is determined by
Ty 2 strain or some other suitable strain that has the capacity
thermogravimetry (2.2.34) and is used to calculate the results
to produce Vi polysaccharide. of the chemical tests shown below with reference to the dried
Capsular Vi polysaccharide consists of partly 3-O-acetylated substance.
repeated units of 2-acetylamino-2-deoxy-D- Only a purified Vi polysaccharide that complies with the
galactopyranuronic acid with a-(l->4) linkages. following requirements may be used in the preparation of the
PRODUCTION final bulk.
The production of Vi polysaccharide is based on a seed-lot Protein (2.5.16)
system. The method of production shall have been shown to Maximum 10 mg per gram of polysaccharide, calculated with
yield consistently typhoid polysaccharide vaccines of adequate reference to the dried substance.
immunogenicity and safety in man.
Nucleic acids (2.5.17)
The production method is validated to demonstrate that the Maximum 20 mg per gram of polysaccharide, calculated with
product, if tested, would comply with the test for abnormal reference to the dried substance.
O-Acetyl groups (2.5.19) O-Acetyl groups

Minimum 2 mmol per gram of polysaccharide, calculated 0.085 (±25 per cent) pmol per dose (25 pg of
with reference to the dried substance. polysaccharide).
Molecular size Test solution Place 3 mL of the vaccine in each of 3 tubes
Examine by size-exclusion chromatography (2.2.30) using (2 reaction solutions and 1 correction solution).
cross-linked agarose for chromatography R. Use a column 0.9 m Reference solutions Dissolve 0.150 g of acetylcholine chloride R
long and 16 mm in internal diameter equilibrated with a in 10 mL of water R (stock solution containing 15 g/L of
solvent having an ionic strength of 0.2 mol/kg and a pH acetylcholine chloride). Immediately before use, dilute
of 7.0-7.5. Apply about 5 mg of polysaccharide in a volume 0.5 mL of the stock solution to 50 mL with water R (working
of 1 mL to the column and elute at about 20 mL/h. Collect dilution containing 150 pg/mL of acetylcholine chloride).
fractions of about 2.5 mL. Determine the point In 10 tubes, place in duplicate (reaction and correction
corresponding to Ko = 0.25 and make 2 pools consisting of solutions) 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL and 1.5 mL of
fractions eluted before and after this point. Determine the working dilution.
O-acetyl groups on the 2 pools (2.5.19). Not less than
Prepare a blank using 3 mL of water R.
50 per cent of the polysaccharide is found in the pool
containing fractions eluted before Ko = 0.25. Make up the volume in each tube to 3 mL with water R.
Add 0.5 mL of a mixture of 1 volume of water R and
Identification 2 volumes of dilute hydrochloric acid R to each of the
Carry out an identification test using a suitable correction tubes and to the blank. Add 1.0 mL of alkaline
immunochemical method (2.7.1). hydroxylamine solution R to each tube. Allow the reaction to
Bacterial endotoxins proceed for exactly 2 min and add 0.5 mL of a mixture of
The content of bacterial endotoxins determined by a suitable 1 volume of water R and 2 volumes of dilute hydrochloric
method (2.6.14) is within the limits approved for the specific acid R to each of the reaction tubes. Add 0.5 mL of a
product. 200 g/L solution of feme chloride R in 0.2 M hydrochloric acid
FINAL BULK VACCINE to each tube, stopper the tubes and shake vigorously to
One or more batches of purified Vi potysaccharide are remove bubbles.
dissolved in a suitable solvent, which may contain an Measure the absorbance (2.2.25) of each solution at 540 nm
antimicrobial preservative, so that the volume corresponding using the blank as the compensation liquid. For each reaction
to 1 dose contains 25 pg of polysaccharide and the solution solution, subtract the absorbance of the corresponding
is isotonic with blood (250 mosmol/kg to 350 mosmol/kg). correction solution. Draw a calibration curve from the
Only a final bulk vaccine that complies with the following corrected absorbances for the 5 reference solutions and the
tests may be used in the preparation of the final lot. corresponding content of acetylcholine chloride and read
from the curve the content of acetylcholine chloride in the
Sterility (2.6.1) test solution for each volume tested. Calculate the mean of
The final bulk vaccine complies with the test for sterility,
the 2 values.
1 mole of acetylcholine chloride (181.7 g) is equivalent to
Antimicrobial preservative 1 mole of O-acetyl (43.05 g).
preservative by a suitable physicochemical method. Free formaldehyde (2.4.18)
The amount is not less than 85 per cent and not greater than Maximum 0.2 g/L.
115 per cent of the intended amount. Antimicrobial preservative
FINAL LOT
preservative by a suitable physicochemical method.
The final bulk vaccine is distributed aseptically into sterile
The content is not less than the minimum amount shown to
tamper-proof containers that are then closed so as to prevent
contamination. be effective and not more than 115 per cent of the content
stated on the label. If phenol has been used in the
Only a final lot that is satisfactory with respect to each of the preparation, the content is not more than 2.5 g/L (2.5.15).
requirements prescribed below under Identification, Tests
and Assay and with the requirement for bacterial endotoxins Sterility (2.6.1)
may be released for use. Provided the tests for free The vaccine complies with the test for sterility.
formaldehyde and antimicrobial preservative have been ASSAY
carried out on the final bulk vaccine, they may be omitted on Determine Vi polysaccharide by a suitable immunochemical
the final lot. method (2.7J), using a reference purified polysaccharide.
Bacterial endotoxins The estimated amount of polysaccharide per dose is
The content of bacterial endotoxins determined by a suitable 80 per cent to 120 per cent of the content stated on the
method (2.6.14) is within the limit approved for the specific label. The confidence limits (P ะ= 0.95) of the estimated
product. amount of polysaccharide are not less than 80 per cent and
not more than 120 per cent.
CHARACTERS
Clear colourless liquid, free from visible particles. LABELLING
The label states:
IDENTIFICATION — the number of micrograms of polysaccharide per human
Cany out an identification test using a suitable dose (25 pg),
immunochemical method (2.7.1). — the total quantity of polysaccharide in the container.
TESTS ____ ______________________________________ . PnEif
pH (2.2.3)
6.5 to 7.5.
Typhoid Vaccine ;* *★ PRODUCTION

The vaccine is prepared using a seed-lot system from a
(Ph. Eur. monograph 0156) *** suitable strain, such as Ty 2 , of ร. typhi. The final vaccine
The label may state ‘Typhoid’. 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
DEFINITION by acetone or by formaldehyde or by heat. Phenol is not used
Typhoid vaccine is a sterile suspension of inactivated in the preparation. The vaccine is distributed into sterile
Salmonella typhi containing not less than 5 X 108 and not containers and freeze-dried to a moisture content favourable
more than 1 X 109 bacteria (5. typhi) per human dose. to the stability of the vaccine. The containers are then closed
The human dose does not exceed 1.0 mL. so as to exclude contamination.
PRODUCTION The production method is validated to demonstrate that the
The vaccine is prepared using a seed-lot system from a product, if tested, would comply with the test for abnormal
suitable strain, such as Ty 2(1), of ร. typhi. The final vaccine toxicity for immunosera and vaccines for human use (2.6.9)
represents not more than 3 subcultures from the strain on modified as follows: inject 0.5 mL of the vaccine into each
which were made the laboratory and clinical tests that mouse and 1.0 mL into each guinea pig.
showed it to be suitable. The bacteria are inactivated by IDENTIFICATION
acetone, by formaldehyde, by phenol or by heating or by a The vaccine reconstituted as stated on the label is identified
combination of the last 2 methods. by specific agglutination.
The production method is validated to demonstrate that the TESTS
Phenol (2.5.75)
toxicity for immunosera and vaccines for human use (2.6.9)
If phenol has been used in the preparation, the concentration
modified as follows: inject 0.5 mL of the vaccine into each
mouse and 1.0 mL into each guinea pig.
Antigenic power
IDENTIFICATION When injected into susceptible laboratory animals, the
It is identified by specific agglutination. reconstituted vaccine elicits anti-O, anti-H and, to a lesser
TESTS extent, anti-Vi agglutinins.
Phenol (2.5.75) Sterility (2.6.7)
If phenol has been used in the preparation, die concentration The reconstituted vaccine complies with the test for sterility.
LABELLING
Antigenic power The label states:
When injected into susceptible laboratory animals, it elicits — the method used to inactivate the bacteria,
anti-O, anti-H and, to a lesser extent, anti-Vi agglutinins. — the number of bacteria per human dose,
Sterility (2.6.7) — that the vaccine should be used within 8 h of
It complies with the test for sterility. reconstitution.
LABELLING ______________________________________________________________ Ph Eur
The label states: 1 This strain is issued by the Whorld Health Organisation
— the method used to inactivate the bacteria, Collaborating Centre for Reference and Research on Bacterial
Vaccines, Human Serum and Vaccine Institute, Szallas Utea 5,
— the number of bacteria per human dose.
H-1107, Budapest, Hungary
______________________________________________________________ Ph Eur
1 This strain is issued by the Whorld Health Organisation
Collaborating Centre for Reference and Research on Bacterial
Vaccines, Human Serum and Vaccine Institute, Szallas Utea 5,
H-1107, Budapest, Hungary
Typhoid (Strain Ty 21a) Vaccine,
Live (Oral) *****
(Typhoid Vaccine (Live, Oral, Strain Ty 21a),
Typhoid Vaccine, Freeze-dried The label may state ‘Typhoid (live/oral)’.
(Ph. Eur. monograph 0157) PhEu_________________________ _ ___________________________________
The label may state ‘Typhoid’. DEFINITION

Ph Ell____________________________________ ________ Typhoid vaccine (live, oral, strain Ty 21a) is a freeze-dried
preparation of live Salmonella typhi strain Ty 21a grown in a
DEFINITION
suitable medium. When presented in capsules, the vaccine
Freeze-dried typhoid vaccine is a freeze-dried preparation of
complies with the monograph Capsules (0016).
inactivated Salmonella typhi. The vaccine is reconstituted as
stated on the label to give a uniform suspension containing PRODUCTION
not less than 5 X 108 and not more ±an 1 X 109 bacteria CHOICE OF VACCINE STRAIN
(ร. typhi) per human dose. The human dose does not exceed The main characteristic of the strain is the defect of the
1.0 mL of the reconstituted vaccine. enzyme uridine diphosphate-galactose-4-epimerase.
The activities of galactopennease, galactokinase and
galactose-1-phosphate uridyl-transferase are reduced by
50 per cent to 90 per cent. Whatever the growth conditions,
the strain does not contain Vi antigen. The strain be favourable to the stability of the vaccine. No antimicrobial
agglutinates to anti-0:9 antiserum only if grown in medium preservative is added to the vaccine.
containing galactose. It contains the flagellar H:d antigen and Only a freeze-dried harvest that complies with the following
does not produce hydrogen sulfide on Kligler iron agar. tests may be used for the preparation of the final bulk.
The strain is nonvirulent for mice. Cells of strain Ty 21a lyse
if grown in the presence of 1 per cent of galactose. Identification
Culture bacteria are examined on an agar medium containing
BACTERIAL SEED LOTS 1 per cent of galactose and bromothymol blue. Light blue,
The vaccine is prepared using a seed-lot system. The working concave colonies, transparent due to lysis of cells, are
seed lots represent not more than one subculture from the formed. No yellow colonies (galactose-fermenting) are found.
master seed lot. The final vaccine represents not more than
four subcultures from the original vaccine on which were
Number of live bacteria
Not fewer than 1 X 1011 live ร. typhi strain Ty 21a per
made the laboratory’ and clinical tests showing the strain to
be suitable. gram.
Only a master seed lot that complies with the following Water (2.5.12)
requirements may be used in the preparation of working seed 1.5 per cent to 4.0 per cent, determined by the semi-micro
lots. determination of water.
Galactose metabolism FINAL BULK VACCINE
In a spectrophotometric assay, no activity of the enzyme The final bulk vaccine is prepared by mixing under suitable
uridine diphosphate-galactose-4-epimerase is found in the conditions one or more freeze-dried harvests with suitable
cytoplasm of strain Ty 21a compared to strain Ty 2. excipients.
Only a final bulk that complies with the following
Biosynthesis of lipopolysaccharide
requirement may be used in the preparation of the final lot.
Lipopolysaccharides are extracted by the hot-phenol method
and examined by size-exclusion chromatography. Strain Number of live bacteria
Ty 21a grown in medium free of galactose shows only the Not fewer than 40 X 109 live ร. typhi strain Ty 21a per
rough (R) type of lipopolysaccharide. gram.
Serological characteristics FINAL LOT
Strain Ty 21a grown in a synthetic medium without The final bulk vaccine is distributed under suitable
galactose does not agglutinate to specific anti-O:9 antiserum. conditions into capsules with a gastro-resistant shell or into
Whatever the growth conditions, strain Ty 21a does not suitable containers.
agglutinate to Vi antiserum. Strain Ty 21a agglutinates to Only a final lot that is satisfactory with respect to each of the
H:d flagellar antiserum. requirements given below under Identification, Tests and
Biochemical markers Number of live bacteria may be released for use, except that
Strain Ty 21a does not produce hydrogen sulfide on Kligler in the determination of the number of live bacteria each
iron agar. This property serves to distinguish Ty 21a from dosage unit must contain not fewer than 4 X 109 live
other galactose-epimerase-negative ร. typhi strains. bacteria.
Cell growth IDENTIFICATION
Strain Ty 21a cells lyse when grown in the presence of Culture bacteria from the vaccine to be examined on an agar
1 per cent of galactose. medium containing 1 per cent of galactose and bromothymol
BACTERIAL PROPAGATION AND HARVEST
blue. Light blue, concave colonies, transparent due to lysis of
cells, are formed. No yellow colonies (galactose-fermenting)
The bacteria from the working seed lot are multiplied in a
preculture, subcultured once and are then grown in a suitable are found.
medium containing 0.001 per cent of galactose at 30 °C for TESTS
13 h to 15 h. The bacteria are harvested. The harvest must Microbial contamination
be free from contaminating micro-organisms. Inoculate at least a total of 2 X 109 live ร. typhi Ty 21a on
Only a single harvest that complies with the following at least 10 plates of casein soya bean digest agar and on at
requirements may be used for the preparation of ±e freeze- least 10 plates of Sabouraud-dextrose agar. Incubate in
dried harvest. aerobic conditions the plates of casein soya bean digest agar
at 30-35 °C for 3-5 days and the plates of Sabouraud-
pH
dextrose agar at 20-25 °C for 5-7 days. Observe the plates
The pH of the culture is 6.8 to 7.5.
for conspicuous colonies of micro-organisms in a lawn of
Optical density weakly growing ร. typhi Ty 21a. The number of
The optical density of the culture, measured at 546 nm, is contaminating micro-organisms per dosage unit is not greater
6.5 to 11.0. Before carrying out ±e measurement, dilute the than 1 o2 bacteria and 20 fungi.
culture so that a reading in the range 0.1 to 0.5 is obtained
Carry out the test for specified micro-organisms on selective
and correct the reading to take account of the dilution. media and under cultivation conditions as indicated in
Identification Table 1055.-1. Test a dose equivalent to at least
Culture bacteria on an agar medium containing 1 per cent of 2 X 109 live ร. typhi Ty 21a.
galactose and bromothymol blue. Light blue, concave Growth-promoting and inhibitory properties of the media
colonies, transparent due to lysis of cells, are formed. used and suitability of the testing conditions are
No yellow colonies (galactose-fermenting) are found. demonstrated.
FREEZE-DRIED HARVEST
The harvest is mixed with a suitable stabiliser and freeze-
dried by a process that ensures the survival of at least
10 per cent of the bacteria and to a water content shown to
Table 1055.-1. - Test for specified micro-organisms:

Bromothymol blue galactose agar
pre-incubation and subculture conditions
Heart infusion agar 40.0 g
Micro Pre-incubation Pre-incubation Subculture
Sodium chloride 5.0 g
organism 2 Purified water 930 mL
Sterilise the medium and cool to 45 °C, ±en add 1.0 g of
Staphylococcus Mannitol salt Mannitol salt
broth
sodium thiosulfate, 40 mL of bromothymol blue solution and
agar*
30-35 °C
30 mL of galactose solution.
40-48 h 18-72 h Bromothymol blue solution
Pseudomonas
Bromothymol blue 2.0 g
Acetamide Cetrimide agar*
aeruginosa broth 0.1 M sodium hydroxide 50 mL
30-35 °C
Purified water 950 mL
18-72 h
40-48 h Galactose solution
Escherichia coli Casein soya MacConkey MacConkey
D-galactose 33.0 g
bean digest broth* agar* Purified water 100 mL
broth’ 41-45 °C 30-35 °C The product complies with the test if no colonies of
18-24 h 18-72 h
18-24 h Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa
or Clostridia, and no salmonella other than strain Ty 21a, are
Salmonella spp. Bromothymol Bromothymol found.
blue galactose blue galactose
broth agar Water (2.5.12)
1.5 per cent to 4.0 per cent, determined on the contents of
18-24 h 18-72 h the capsule or of the container by the semi-micro
Clostridia
determination of water.
Reinforced Columbia agar*
medium for NUMBER OF LIVE BACTERIA
Clostridia*
18-72 h Carry out the test using not fewer than 5 dosage units.
Anaerobic Homogenise the contents of the dosage units in a 9 g/L
40-48 h conditions
Unheated and solution of sodium chloride R at 4 ๐ c using a mixer in a cold
heated at 80 °C room with sufficient glass beads to emerge from the liquid.
for 10 min
in buffered
Immediately after homogenisation prepare a suitable dilution
sodium of the suspension using cooled diluent and inoculate brain
chloride- heart infusion agar, incubate at 36 ± 1 °C for 20 h to 36 h.
peptone
solution pH 7.0’ The vaccine contains not fewer than 2 X 109 live ร. typhi
Anaerobic Ty 21a bacteria per dosage unit.
conditions
LABELLING
•Culture media recommended for identification of specified The label states:
micro-organisms (see general chapter 2.6.13 for composition) — the minimum number of live bacteria per dosage unit;
— that the vaccine is for oral use only.
Composition of culture media listed in Table 1055.-1 that
are not described in general chapter 2.6.13. Microbiological
examination of non-sterile products: test for specified micro
organisms.
Mannitol salt broth
Proteose peptone 10.0 g
Varicella Vaccine (Live) * *
Beef extract 1.5 g (Ph. Eur. monograph 0648) ***
Sodium chloride 75.0 g The label may state ‘Var(live)\
D-Mannitol 10.0 g
Ph Elf_______________________________________________________________
Purified water 1000 mL
Acetamide broth DEFINITION
Acetamide 2.0 g Varicella vaccine (live) is a freeze-dried preparation of a
Magnesium sulfate 0.2 g suitable attenuated strain of human herpesvirus 3.
Sodium chloride 0.2 g The vaccine is reconstituted immediately before use, as
Ferric sulfate 0.0005 g stated on the label, to give a clear liquid that may be
Potassium dihydrogen phosphate 1.0 g coloured owing to the presence of a pH indicator.
Sodium molybdate 0.005 g PRODUCTION
Purified water 1000 mL The production of vaccine is based on a virus seed-lot system
Bromothymol blue galactose broth and a cell-bank system. The production method shall have
Heart infusion broth 25.0 g been shown to yield consistently live varicella vaccines of
Sodium chloride 5.0 g adequate immunogenicity and safety in man. The virus in the
Purified water 930 mL final vaccine shall not have been passaged in cell cultures
Sterilise the medium and cool to 45 °C, then add 1.0 g of beyond a defined number of passages approved by the
sodium thiosulfate, 40 mL of bromothymol blue solution and competent authority from the original isolated virus.
30 mL of galactose solution. The potential neurovirulence of the vaccine strain is
considered during preclinical development, based on available
epidemiological data on neurovirulence and neurotropism,
primarily for the wild-type virus. In light of this, a risk Extraneous agents (2.6.16)
analysis is carried out. Where necessary and if available, a Use 50 mL for the test in cell cultures.
test is carried out on the vaccine strain using an animal Control cells
model that differentiates wild-type and attenuated virus; tests The control cells of the production cell culture from which
on strains of intermediate attenuation may also be needed. the single harvest is derived comply with a test for identity
The production method is validated to demonstrate that the and with the requirements for extraneous agents (2.6.76).
FINAL BULK VACCINE
Virus harvests that comply with the above tests are pooled
SUBSTRATE FOR VIRUS PROPAGATION and clarified to remove cells. A suitable stabiliser may be
The virus is propagated in human diploid cells (5.2.3). added and the pooled harvests diluted as appropriate.
VIRUS SEED LOT Only a final bulk vaccine that complies with the following
The strain of human herpesvirus 3 used shall be identified as requirements may be used in the preparation of the final lot.
being suitable by historical records that include information Bacterial and fungal contamination
on the origin of the strain and its subsequent manipulation. Carry out the test for sterility (2.6.7) using 10 mL for each
The virus shall at no time have been passaged in continuous medium.
cell lines. Seed lots are prepared in the same kind of cells as
FINAL LOT
those used for the production of the final vaccine. Virus seed
lots are prepared in large quantities and stored at The final bulk vaccine is distributed aseptically into sterile,
temperatures below -20 °C if freeze-dried, or below -60 °C tamper-proof containers and freeze-dried to a moisture
if not freeze-dried. content shown to be favourable to the stability of the vaccine.
The containers are then closed so as to prevent
Only a virus seed lot that complies Hath the following
contamination and the introduction of moisture.
requirements may be used for virus propagation.
Identification water and each of the requirements given below under
The master and working seed lots are identified as human Identification, Tests and Assay may be released for use.
herpesvirus 3 by serum neutralisation in cell culture, using Provided that the test for bovine serum albumin has been
specific antibodies. carried out with satisfactory results on the final bulk vaccine,
Virus concentration it may be omitted on the final lot.
The virus concentration of the master and working seed lots Water (2.5.72)
is determined as prescribed under Assay to monitor Not more than the amount shown to ensure stability of the
consistency of production. vaccines as approved by the competent authority, determined
Extraneous agents (2.6.76) by the semi-micro determination of water.
IDENTIFICATION
seed lots for live virus vaccines; a sample of 50 mL is taken
for the test in cell cultures.
mixed with specific human herpesvirus 3 antibodies, it is no
VIRUS PROPAGATION AND HARVEST longer able to infect susceptible cell cultures.
All processing of the cell bank and subsequent cell cultures is
done under aseptic conditions in an area where no other cells TESTS
or viruses are being handled. Approved animal (but not Bacterial and fungal contamination
human) serum may be used in the culture media. Serum and The reconstituted vaccine complies with the test for sterility
trypsin used in the preparation of cell suspensions and media .
(2.6.7)
are shown to be free from extraneous agents. The cell culture Bovine serum albumin
medium may contain a pH indicator such as phenol red and Maximum 0.5 pg per human dose, determined by a suitable
approved antibiotics at the lowest effective concentration. immunochemical method (2.7.1).
It is preferable to have a substrate free from antibiotics ASSAY
during production. 5 per cent, but not less than 50 mL, of
Titrate the vaccine for infective virus, using at least
the cell cultures employed for vaccine production is set aside 3 separate vials of vaccine. Titrate 1 vial of an appropriate
as uninfected cell cultures (control cells). The infected cells virus reference preparation in triplicate to validate each assay.
constituting a single harvest are washed, released from the The virus concentration of the reference preparation is
support surface and pooled. The cell suspension is disrupted monitored using a control chart and a titre is established on a
by sonication. historical basis by each laboratory. The relation with the
Only a virus harvest that complies with the following appropriate European Pharmacopoeia Biological Reference
requirements may be used in the preparation of the final bulk Preparation is established and monitored at regular intervals
vaccine. if a manufacturer'ร reference preparation is used. Calculate
Identification the individual virus concentration for each vial of vaccine and
The virus harvest contains virus that is identified as human for each replicate of the reference preparation as well as the
herpesvirus 3 by serum neutralisation in cell culture, using corresponding combined virus concentrations, using the usual
specific antibodies. statistical methods (for example, 5.3). The combined
estimate of the virus concentration for the 3 vials of vaccine
Virus concentration
is not less than that stated on the label.
The concentration of infective virus in virus harvests is
determined as prescribed under Assay to monitor consistency The assay is not valid if:
of production and to determine the dilution to be used for — the confidence interval (P — 0.95) of the estimated virus
the final bulk vaccine. concentration of the reference preparation for the
3 replicates combined is greater than ± 0.3 logio PFU;
the virus concentration of the reference preparation differs SUBSTRATE FOR VIRUS PROPAGATION
by more than 0.5 log!0 PFU from the established value. Virus for the preparation of master and working seed lots and
The assay is repeated if the confidence interval (P = 0.95) of of all vaccine batches is grown in the tissues of chick embryos
the combined virus concentration of the vaccine is greater from a flock free from specified pathogens (SPF) (5.2.2).
than ± 0.3 log10 PFU; data obtained from valid assays only SEED LOTS
■ire combined by the usual statistical methods (for The 17D strain shall be identified by historical records that
example, 5.5) to calculate the virus concentration of the include information on the origin of the strain and its
sample. The confidence interval (P = 0.95) of the combined subsequent manipulation. Virus seed lots are prepared in
virus concentration is not greater than ± 0.3 logio PFU. large quantities and stored at a temperature below -60 °C.
Varicella vaccine (live) BRP is suitable for use as a reference Master and working seed lots shall not contain any human
preparation. protein, added serum or antibiotics.
Where justified and authorised, different assay designs may Unless otherwise justified and authorised, the virus in the
be used; this may imply the application of different validity final vaccine shall be between passage levels 204 and 239
and acceptance criteria. However, the vaccine must comply if from the original isolate of strain 17D. A working seed lot
tested as described above. shall be only 1 passage from a master seed lot. A working
seed lot shall be used without intervening passage as the
labelling
inoculum for infecting the tissues used in the production of a
The label states:
vaccine lot, so that no vaccine virus is more than 1 passage
the strain of virus used for the preparation of the vaccine;
from a seed lot that has passed all the safety tests.
the type and origin of the cells used for the preparation of
the vaccine; Only a virus seed lot that complies with the following
— the minimum virus concentration; requirements may be used for virus propagation.
that contact between the vaccine and disinfectants is to be Identification
avoided. The master and working seed lots are identified as containing
--------------------------------------- ------------------------------------------------------------------ Ph Eur
yellow fever virus by serum neutralisation in cell culture
using specific antibodies, or by molecular methods
(e.g. nucleic acid amplification techniques (NAT),
sequencing).
Yellow Fever Vaccine, Live ***** Each master seed lot complies with the following tests:
— bacterial and fungal sterility (as described in chapter
(Yellow Fever Vaccine (Live), *** 2.6.16 under Virus seed lot and virus harvests);
Ph Eur monograph 0537) — mycoplasmas (as described in chapter 2.6.16 under Virus
The label may state ‘Yel(live).’ seed lot and virus harvests);
— mycobacteria (as described in chapter 2.6.16 under Virus
seed lot and virus harvests).
DEFINITION
Yellow fever vaccine (live) is a freeze-dried preparation of Avian leucosis viruses (2.6.24)
yellow fever virus derived from the 17D strain and grown in Each master seed lot complies with the test for avian leucosis
fertilised hen eggs. The vaccine is reconstituted immediately viruses.
before use, as stated on the label, to give a clear liquid. Extraneous agents (2.6.16)
Each working seed lot complies with the following tests:
PRODUCTION
— test in adult mice (intraperitoneal inoculation only) (as
The production of vaccine is based on a virus seed-lot
described in chapter 2.6.16 under Virus seed lot);
system. The production method shall have been shown to — test in guinea-pigs (as described in chapter 2.6.16 under
yield consistently yellow fever vaccine (live) of acceptable
Virus seed lot);
immunogenicity and safety for man. — bacterial and fungal sterility (as described in chapter
The production method is validated to demonstrate that the 2.6.16 under Virus seed lot and virus harvests);
product, if tested, would comply with the test for abnormal — mycoplasmas (as described in chapter 2.6.16 under Virus
toxicity for immunosera and vaccines for human use (2.6.9) seed lot and virus harvests);
modified as follows for the test in guinea-pigs: inject — mycobacteria (as described in chapter 2.6.16 under Virus
10 human doses into each guinea-pig at 2 different injection seed lot and virus harvests);
sites and observe for 21 days. — test in cell culture for other extraneous agents: a
Reference preparation In the test for neurotropism, a suitable neutralised sample of 5 mL of working seed lot,
batch of vaccine known to have satisfactory properties in man representing at least 500 000 (5.7 logio) IUj is tested for
is used as the reference preparation. the presence of extraneous agents by inoculation into
A reference preparation calibrated in International Units per continuous simian kidney and human cell cultures as well
ampoule is used to verify the titre of the virus inoculum in as into primary chick-embryo-fibroblast cells; the cells are
the tests for viraemia (viscerotropism) and immunogenicity, incubated at 36 ± 1 °C and observed for a period of
and to titrate the vaccine batch in the potency assay. 14 days; the working seed lot passes the test if there is no
evidence of the presence of any extraneous agents;
The International Unit is the activity contained in a stated
the test is not valid unless at least 80 per cent of the cell
quantity of the International Standard. The equivalence in
cultures remain viable;
International Units of the International Standard is stated by
— avian viruses: a neutralised sample of 1 mL of working
seed lot, representing at least 100 000 (5.0 log10) IU, is
tested for the presence of avian viruses by inoculation by
the allantoic route into a group of at least 20 fertilised,
9- to 11-day-old, SPF eggs (5.2.2), and by inoculation Not more than 10 per cent of the test monkeys have serum
into the yolk sac of a group of at least 20 fertilised, 5- to that fails to reduce ±e number of plaques by 50 per cent at
7-day-old, SPF eggs (5.2.2); incubate for 7 days; the 1:10 dilution.
the working seed lot complies if the allantoic and yolk sac Neurotropism Neurotropism is assessed from clinical evidence
fluids show no signs of haemagglutinating agents and if of encephalitis, from incidence of clinical manifestations and
the embryos and chorio-allantoic membranes examined to by evaluation of histological lesions, in comparison with
detect an}7 macroscopic pathology are typical; the test is 10 monkeys injected with the reference preparation. The seed
not valid unless at least 80 per cent of the inoculated eggs lot is not acceptable if either the onset and duration of the
survive during the 7-day observation period. febrile reaction or the clinical signs of encephalitis and
Avian leucosis viruses (2.6.24) pathological findings are such as to indicate a change in the
Each working seed lot complies with the test for avian properties of the virus.
leucosis viruses. Clinical evaluation
Tests in monkeys The monkeys are examined daily for 30 days by personnel
Each master and working seed lot complies with the familiar with clinical signs of encephalitis in primates (if
following tests in monkeys for viraemia (viscerotropism), necessary, the monkeys are removed from their cage and
immunogenicity and neurotropism. examined for signs of motor weakness or spasticity).
The monkeys shall be Macaca sp. susceptible to yellow fever The seed lot is not acceptable if in the monkeys injected with
virus and shall have been shown to be non-immune to yellow it the incidence of severe signs of encephalitis, such as
fever at the time of injecting the seed virus. They shall be paralysis or inability to stand when stimulated, or mortality is
healthy and shall not have received previously intracerebral or greater than for the reference vaccine. These and other signs
intraspinal inoculation. Furthermore, they shall not have of encephalitis, such as paresis, incoordination, lethargy,
been inoculated by other routes with neurotropic viruses or tremors or spasticity are assigned numerical values for the
with antigens related to yellow fever virus. Not fewer than severity of symptoms by a grading method. Each day each
10 monkeys are used for each test. monkey in the test is given a score based on the following
Use a test dose of 0.25 mL containing the equivalent of not scale:
less ±an 5000 (3.7 log10) IU and not more than — grade 1 ะ rough coat, not eating;
50 000 (4.7 logio) ru> determined by an in vitro titration for — grade 2: high-pitched voice, inactive, slow moving;
infectious virus in cell culture. Inject the test dose into 1 — grade 3: shaky movements, tremors, incoordination, limb
frontal lobe of each monkey under anaesthesia and observe weakness;
the monkeys for not less ±an 30 days. — grade 4: inability to stand, limb paralysis or death (a dead
monkey receives a daily score of 4 from the day of death
Viraemia (Viscerotropism) Viscerotropism is indicated by the
until day 30).
amount of virus present in serum. Take blood from each of
the test monkeys on ±e 2nd, 4th and 6th days after A clinical score for a particular monkey is the average of its
inoculation and prepare serum from each sample. daily scores; the clinical score for the group is the arithmetic
Prepare 1:10, 1:100 and 1:1000 dilutions from each serum mean of the individual monkey scores. The seed lot is not
and inoculate each dilution into a group of at least 4 cell acceptable if the mean of the clinical severity scores for the
culture vessels used for the determination of the virus group of monkeys inoculated with it is significantly greater
concentration. The seed lot complies with the test if none of (P = 0.95) than the mean for the group of monkeys injected
the sera contains more than the equivalent of with the reference preparation. In addition, special
500 (2.7 log!0) IU in 0.03 mL and at most 1 serum contains consideration is given to any animal showing unusually severe
more than the equivalent of 100 (2.0 logio) ru in 0.03 mL signs when deciding on the acceptability of the seed lot.
Immunogenicity Take blood from each monkey 30 days after Histological evaluation
the injection of the test dose and prepare serum from each 5 levels of the brain are examined including:
sample. The seed lot complies with the test if at least — block I: the corpus striatum at the level of the optic
90 per cent of the test monkeys are shown to be immune, as chiasma;
determined by examining their sera in the test for — block II: the thalamus at the level of the mamillary
neutralisation of yellow fever virus described below. bodies;
It has been shown that a low dilution of serum (for — block ni: the mesencephalon at the level of the superior
example, 1:10) may contain non-specific inhibitors that colliculi;
— block IV: the pons and cerebellum at the level of the
influence this test; such serum shall be treated to remove
superior olives;
inhibitors. Mix dilutions of at least 1:10, 1:40 and 1:160 of
— block V: the medulla oblongata and cerebellum at the
serum from each monkey with an equal volume of
level of the mid-inferior olivary nuclei.
17D vaccine virus at a dilution that will yield an optimum
number of plaques with the titration method used. Incubate Cervical and lumbar enlargements of the spinal cord are each
the serum-virus mixtures in a water-bath at 37 °C for 1 h divided equally into 6 blocks; 15 pm sections are cut from
and then cool in iced water; add 0.2 mL of each serum-virus the tissue blocks embedded in paraffin wax and stained with
mixture to each of 4 cell-culture plates and proceed as for gallocyanin. Numerical scores are given to each hemisection
the determination of virus concentration. Inoculate similarly of the cord and to structures in each hemisection of the brain
10 plates with the same amount of virus, plus an as listed below. Lesions are scored as follows:
equal volume of a 1:10 dilution of monkey serum known to — grade 1 - minimal: 1 to 3 small focal inflammatory
contain no neutralising antibodies to yellow fever virus. infiltrates; degeneration or loss of a few neurons;
At the end of the observation period, compare the mean — grade 2 - moderate: 4 or more focal inflammatory
number of plaques in the plates receiving virus plus non- infiltrates; degeneration or loss of neurons affecting not
immune serum with the mean number of plaques in the more than one third of cells;
plates receiving virus plus dilutions of each monkey serum.
" severe: moderate focal or diffuse inflammatory obtain a control embryonic pulp. The age of the embryo at
infiltration; degeneration or loss of 33-90 per cent of the the time of virus harvest is reckoned from the initial
neurons; introduction of the egg into the incubator and shall be not
grade 4 - overwhelming: variable but often severe more than 12 days. After homogenisation and clarification by
inflammatory reaction; degeneration or loss of more than centrifugation, the extract of embryonic pulp is tested as
90 per cent of neurons. described below and kept at -70 °C or colder until further
It has been found that inoculation of yellow fever vaccine processing. Virus harvests may be pooled. No human protein
into the monkey brain causes histological lesions in different is added to the virus suspension at any stage during
anatomical formations of the central nervous system with production. If stabilisers are added, they shall have been
vaiying frequency and severity (I. ร. Levenbook et al., Journal shown to have no antigenic or sensitising properties for man.
of Biological Standardization, 1987, 15, 305-313). Based on Only a single harvest or, where applicable, a pool of single
these 2 indicators, the anatomical structures can be divided harvests that complies with the following requirements may
into target, spared and discriminator areas. Target areas are be used in the preparation of the final bulk vaccine.
those that show more severe specific lesions in a majority of
Identification
monkeys irrespective of the degree of neurovirulence of the The single harvest or pool of single harvests contains virus
seed lot. Spared areas are those that show only minimal ±at is identified as yellow fever virus by serum neutralisation
specific lesions and in a minority of monkeys. Discriminator in cell culture using specific antibodies, or by molecular
areas are those where there is a significant increase in the methods (e.g. NAT, sequencing).
frequency of more severe specific lesions with seed lots
having a higher degree of neurovirulence. Discriminator and Bacterial and fungal contamination
target areas for Macaca cynotnolgus and Macaca rhesus The single harvest complies with the test for sterility (2.6./),
monkeys are shown in the table below. carried out using 10 mL for each medium.
Mycoplasmas (2.6.7)
The single harvest or pool of single harvests complies with
Type of monkey Discriminator areas Target areas
the test for mycoplasmas, carried out using 10 mL
Macaca cynotnolgus Globus pallidus Substantia nigra
Mycobacteria (2.6.2)
Putamen A 5 mL sample of the single harvest or pool of single
Anterior/median harvests is tested for the presence of Mycobacterium spp.
thalamic nucleus by culture methods known to be sensitive for the detection of
Lateral thalamic these organisms.
nucleus
Macaca rhesus Caudate nucleus Substantia nigra
Embryonic pulp of control eggs
The extract of the control eggs shows no evidence of the
Globus pallidus Cervical enlargement presence of any extraneous agents in the tests described
Putamen Lumbar enlargement below.
Anterior/median Test in cell culture for other extraneous agents Inoculate a 5 mL
thalamic nucleus sample of embryonic pulp of the control eggs into continuous
Lateral thalamic simian kidney and human cell cultures as well as into
nucleus
primary chick-embryo-fibroblast cells. The cells are incubated
Cervical enlargement
at 36 ± 1 °C and observed for a period of 14 days.
Lumbar enlargement The embryonic pulp of the control eggs passes the test if
there is no evidence of the presence of any extraneous agents.
The test is not valid unless at least 80 per cent of the cell
Scores for discriminator and target areas are used for the cultures remain viable.
final evaluation of the seed lot. The individual monkey score
Avian viruses Using 0.1 mL per egg, inoculate the embryonic
is calculated from the sum of individual target area scores in
pulp of control eggs: by the allantoic route into a group of
each hemisection divided by the number of areas examined.
10 fertilised, 9- to 11-day-old, SPF eggs (5.2.2); and into the
A separate score is calculated similarly for the discriminator
areas. yolk sac of a group of 10 fertilised, 5- to 7-day-old, SPF eggs
(5.2.2). Incubate for 7 days. The embryonic pulp lot of the
Mean scores for the test group are calculated in 2 ways: control eggs complies if the allantoic and yolk sac fluids show
(1) by dividing the sum of the individual monkey no signs of haemagglutinating agents and if the embryos and
discriminator scores by the number of monkeys; and (2) by chorio-allantoic membranes examined to detect any
dividing the sum of the individual monkey target and macroscopic pathology are typical. The test is not valid
discriminator scores by the number of monkeys. These unless at least 80 per cent of the inoculated eggs survive
2 mean scores are taken into account when deciding on the during the 7 day observation period.
acceptability of the seed lot. The seed lot is not acceptable if
either of the mean lesion scores is significantly greater Virus concentration
In order to calculate the dilution for formulation of the final
(P = 0.95) than for the reference preparation.
bulk, each single harvest is titrated as described under Assay.
propagation and harvest
FINAL BULK VACCINE
All processing of the fertilised eggs is done under aseptic
conditions in an area where no other infectious agents or Single harvests or pools of single harvests that comply with
the tests prescribed above are pooled and clarified again.
cells are handled at the same time. At least 2 per cent but
A test for protein nitrogen content is carried out. A suitable
not fewer than 20 and not more than 80 eggs are maintained
stabiliser may be added and the pooled harvests diluted as
as uninfected control eggs. After inoculation and incubation
appropriate.
at a controlled temperature, only living and typical chick
embryos are harvested. At the time of harvest, the control Only a final bulk vaccine that complies with the following
eggs are treated in the same way as the inoculated eggs to requirements may be used in the preparation of the final lot.
Bacterial and fungal contamination — the confidence interval (P = 0.95) of the estimated virus
The final bulk vaccine complies with the test for sterility concentration of the reference preparation for the
,
(2.6.7) carried out using 10 mL for each medium. 3 replicates combined is greater than ± 0.3 logio IU;
Protein nitrogen content — the virus concentration of the reference preparation differs
Maximum 0.25 mg per human dose before the addition of by more than 0.5 log10 IU from the established value.
any stabiliser. The assay is repeated if the confidence interval (p = 0.95) of
FINAL LOT the combined virus concentration of the vaccine is greater
The final bulk vaccine is distributed aseptically into sterile, than ± 0.3 log10 IU; data obtained from valid assays only
tamper-proof containers and freeze-dried to a moisture are combined by the usual statistical methods (for example,
content shown to be favourable to the stability of the vaccine. 5.3) to calculate the virus concentration of the sample.
The containers are then closed so as to prevent The confidence interval (P = 0.95) of the combined virus
contamination and the introduction of moisture. concentration is not greater than + 0.3 logio IU-
Only a final lot that is satisfactory with respect to thermal Where justified and authorised, different assay designs may
stability and each of the requirements given below under be used; this may imply the application of different validity
Identification, Tests and Assay may be released for use. and acceptance criteria. However, the vaccine must comply if
Provided that the test for ovalbumin has been performed tested as described above.
with satisfactory results on the final bulk vaccine, it may be LABELLING
omitted on the final lot. The label states:
Thermal stability — the strain of virus used in preparation of the vaccine;
Maintain at least 3 containers of the final lot of freeze-dried — that the vaccine has been prepared in chick embryos;
vaccine in the dry state at 37 ± 1 °C for 14 days. Determine — the minimum virus concentration;
the virus concentration as described under Assay in parallel — that contact between the vaccine and disinfectants is to be
for the heated vaccine and for vaccine stored at the avoided.
temperature recommended for storage. The virus ___________________________________________________ ___________ Ph Elf
concentration of the heated vaccine is not more than 1.0
logio lower than that of the unheated vaccine.
IDENTIFICATION
mixed with specific yellow fever virus antibodies, there is a
significant reduction in its ability to infect susceptible cell
cultures. Alternatively, the vaccine reconstituted as stated on
the label contains virus that is identified as yellow fever virus
by molecular methods (e.g. NAT, sequencing).
TESTS
Ovalbumin
Maximum 5 Jig of ovalbumin per human dose, determined
by a suitable immunochemical method (2.7.7).
Water (2.5.12)
The reconstituted vaccine complies with the test for sterility
.
(2.6.7)
Less than 5 IU per single human dose.
ASSAY
Titrate for infective virus in cell cultures using at least
3 separate containers of vaccine. Titrate 1 container of an
appropriate virus reference preparation in triplicate to
validate each assay. The virus concentration of the reference
preparation is monitored using a control chart and a titre is
established on a historical basis by each laboratory. Calculate
the individual virus concentration for each container of
vaccine and for each replicate of the reference preparation as
well as the corresponding combined virus concentrations
using the usual statistical methods (for example, 5.3).
The combined virus concentration for the 3 containers of
vaccine is compared to the results of the reference
preparation titrated in parallel, to obtain results in
International Units. The combined virus concentration of the
vaccine is not less than 3.0 logio IU per human dose and not
more than the upper limit approved for the particular
product by the competent authority.
2016 Diagnostic Preparations IV-671
or another suitable antimicrobial preservative that does not

DIAGNOSTIC PREPARATIONS give rise to false positive reactions may be added.
Only a concentrated harvest that complies with the following
tuberculin.
Old Tuberculin ***** pH (2.2.3)
(Tuberculin for Human Use, Old, The pH of the concentrated harvest is 6.5 to 8.
Ph Eur monograph 0152) Glycerol
rh Elf _ ________ _______________________ Where applicable, determine the glycerol content of the
concentrated harvest. The amount is within the limits
definition approved for the particular product.
Old tuberculin for human use consists of a filtrate,
concentrated by heating, containing the soluble products of
the culture and lysis of one or more strains of Mycobacterium
boots and/or Mycobacterium tuberculosis that is capable of
demonstrating a delayed hypersensitivity in an animal
more than 115 per cent of the intended amount. If phenol
sensitised to micro-organisms of the same species.
has been used in the preparation, the concentration is not
Old tuberculin for human use in concentrated form is a more than 5 g/L (2.5.15).
transparent, viscous, yellow or brown liquid.
Sensitisation
PRODUCTION Carry out the test described under Tests.
GENERAL PROVISIONS
Sterility (2.6.1)
The production of old tuberculin is based on a seed-lot The concentrated harvest complies with the test for sterility,
system. The production method shall have been shown to carried out using 10 mL for each medium.
yield consistently old tuberculin of adequate potency and
safety in man. A batch of old tuberculin, calibrated in Potency
International Units by the method described under Assay Determine the potency as described under Assay.
and for which adequate clinical information is available as to FINAL BULK TUBERCULIN
its activity in man, is set aside to serve as a reference The concentrated harvest is diluted aseptically.
preparation. Only a final bulk tuberculin that complies with the following
The International Unit is the activity of a stated quantity of requirement may be used in the preparation of the final lot.
the International Standard. The equivalence in International Sterility (2.6.1)
Units of the International Standard is stated by the World The final bulk tuberculin complies with the test for sterility,
Health Organization. carried out using 10 mL for each medium.
SEED LOTS FINAL LOT
The strains of mycobacteria used shall be identified by The final bulk tuberculin is distributed aseptically into sterile
historical records that include information on their origin and containers which are then closed so as to prevent
subsequent manipulation. contamination.
The working seed lots used to inoculate the media for the Only a final lot that is satisfactory with respect to each of the
production of a concentrated harvest shall not have requirements given below under Identification, Tests and
undergone more than 4 subcultures from the master seed lot. Assay may be released for use.
Only seed lots that comply with the following requirements The following tests may be omitted on the final lot if they
may be used for propagation. have been carried out at the stages indicated:
Identification — live mycobacteria: concentrated harvest,
The species of mycobacterium of the master and working — sensitisation: concentrated harvest,
seed lots is identified. — toxicity: concentrated harvest or final bulk tuberculin,
— antimicrobial preservative: final bulk tuberculin.
Carry out the test for sterility (2.6.1), using 10 mL for each IDENTIFICATION
medium. The working seed lot complies with the test for Inject increasing doses of the preparation to be examined
sterility except for the presence of mycobacteria. intradermally into healthy, white or pale-coloured guinea-
PROPAGATION AND HARVEST pigs, specifically sensitised (for example, as described under
The bacteria are grown in a liquid medium which may be a Assay). A reaction varying from erythema to necrosis is
glycerolated broth or a synthetic medium. Growth must be produced at the site of the injection. Similar injections
typical for the strain. The culture is inactivated by a suitable administered to non-sensitised guinea-pigs do not stimulate a
method, such as treatment in an autoclave (121 ๐c for not reaction. The assay may also serve as identification.
less than 30 min) or in flowing steam at a temperature not TESTS
less than 100 °C for at least 1 h. The culture liquid, from Old tuberculin for human use in concentrated form
which the micro-organisms may or may not have been (> 100 000 lUhnL) complies with each of the tests prescribed
separated by filtration, is concentrated by evaporation, below; the diluted product complies with the tests for antimicrobial
usually to one-tenth of its initial volume. The preparation is preservative and sterility.
free from live mycobacteria. The concentrated harvest is Toxicity
shown to comply with the test for mycobacteria (2.6.2) Inject a quantity equivalent to 50 000 IU subcutaneously
before addition of any antimicrobial preservative or other into each of two healthy guinea-pigs weighing 250gto 350g
substance that might interfere with the test. Phenol (5 g/L) and which have not been subjected to any treatment likely to
IV-672 Diagnostic Preparations 2016
interfere with the test. Observe the animals for 7 days. STORAGE
No adverse effect is produced. Store protected from light.
Sensitisation LABELLING
Use 3 guinea-pigs that have not been subjected to any The label states:
treatment likely to interfere with the test. On 3 occasions at — the number of International Units per millilitre,
intervals of 5 days, inject intradermally into each guinea-pig — the species of mycobacterium used to prepare the
about 500 ru of the preparation to be examined in a volume product,
of 0.1 mL. 2 to 3 weeks after the third injection, administer — the name and quantity’ of any antimicrobial preservative
the same dose intradermally to the same animals and to a or any other excipient,
control group of 3 guinea-pigs of the same mass that have — the expiry date,
not previously received injections of tuberculin. After 24 h to — where applicable, that old tuberculin is not to be injected
72 h, the reactions in the 2 groups of animals are not in its concentrated form but diluted so as to administer
substantially different. not more than 100 IU per dose.
Antimicrobial preservative ______________________________________________________________ Ph Elf
Where applicable, determine ±e amount of antimicrobial
method. The content is not less than the minimum amount
shown to be effective and not more than 115 per cent of the
amount stated on the label. If phenol has been used in the Tuberculin Purified Protein ** *
preparation, the concentration is not more than 5 g/L
(2.5.15). Derivative *****
Live mycobacteria (2.6.2) Tuberculin P.P.D.
It complies with the test for mycobacteria. (Tuberculine Purified Protein Derivative for Human Use,
Sterility (2.6. /) Ph Eur monograph 0151)
It complies with the test for sterility. Ph Eur_______________________________________________ _______________
ASSAY DEFINITION
The potency of old tuberculin is determined by comparing Tuberculin purified protein derivative (tuberculin PPD) for
the reactions produced by the intradermal injection of human use is a preparation obtained by precipitation from
increasing doses of the preparation to be examined into the heated products of the culture and lysis of Mycobacterium
sensitised guinea-pigs with the reactions produced by known bovis and/or Mycobacterium tuberculosis and capable of
concentrations of the reference preparation. demonstrating a delayed hypersensitivity in an animal
Prepare a suspension containing a suitable amount (0.1 mg sensitised to micro-organisms of the same species.
to 0.4 mg/mL) of heat-inactivated, dried mycobacteria in Tuberculin PPD is a colourless or pale-yellow liquid;
mineral oil with or without emulsifier; use mycobacteria of a the diluted preparation may be a freeze-dried powder which
strain of the same species as that used in the preparation to upon dissolution gives a colourless or pale-yellow liquid.
be examined. Sensitise not fewer than 6 pale-coloured PRODUCTION
guinea-pigs weighing not less than 300 g by injecting GENERAL PROVISION'S
intramuscularly or intradermally a total of about 0.5 mL of The production of tuberculin PPD is based on a seed-lot
the suspension, divided between several sites if necessary. system. The production method shall have been shown to
Carry out the test after the period of time required for yield consistently tuberculin PPD of adequate potency and
optimal sensitisation which is usually 4 to 8 weeks after safety in man. A batch of tuberculin PPD, calibrated in
sensitisation. Depilate the flanks of the animals so that it is International Units by method A described under Assay and
possible to make at least three injections on each side and for which adequate clinical information is available as to its
not more than a total of 12 injection points per animal. activity in man, is set aside to serve as a reference
Use at least three different doses of the reference preparation preparation.
and at least 3 different doses of the preparation to be
The International Unit is the activity of a stated quantity of
examined. For both preparations, use doses such that the
the International Standard. The equivalence in International
highest dose is about 10 times the lowest dose. Choose
the doses such that when they are injected the lesions
produced have a diameter of not less than 8 mm and not
more than 25 mm. In any given test, the order of the SEED LOTS
dilutions injected at each point is chosen at random in a The strains of mycobacteria used shall be identified by
Latin square design. Inject each dose intradermally in a historical records that include information on their origin and
constant volume of 0.1 mL or 0.2 mL. Measure the subsequent manipulation.
diameters of the lesions 24 h to 48 h later and calculate the The working seed lots used to inoculate the media for
results of the test by the usual statistical methods, assuming production of a concentrated harvest shall not have
that the diameters of the lesions are directly proportional to undergone more than 4 subcultures from the master seed lot.
the logarithm of the concentration of the preparation. Only seed lots that comply with the following requirements
The estimated potency is not less than 80 per cent and not may be used for propagation.
more than 125 per cent of the stated potency. Identification
The confidence limits (P = 0.95) are not less than The species of mycobacterium of the master and working
64 per cent and not more than 156 per cent of the stated seed lots is identified.
potency.
2016 Diagnostic Preparations IV-673
Bacterial and fungal contamination IDENTIFICATION

Carry out the test for sterility (2.6. 7), using 10 mL for each Inject increasing doses of the preparation to be examined
medium. The working seed lot complies with the test for intradermally into healthy, white or pale-coloured guinea-
sterility except for the presence of mycobacteria. pigs, specifically sensitised (for example as described under
propagation and harvest Assay). A reaction varying from erythema to necrosis is
The bacteria are grown in a liquid synthetic medium. produced at the site of the injection. Similar injections
Growth must be typical for the strain. The culture is administered to non-sensitised guinea-pigs do not stimulate a
inactivated by a suitable method such as treatment in an reaction. The assay may also serve as identification.
autoclave (121 °C for not less than 30 min) or in flowing TESTS
steam at a temperature not less than 100 °C for at least 1 h Tuberculin purified protein derivative for human use in
and filtered. The active fraction of the filtrate, consisting concentrated form (> 100 000 lUImL) complies with each of the
mainly of protein, is isolated by precipitation, washed and tests prescribed below; the diluted product complies with the tests for
ท?-dissolved. The preparation is free from mycobacteria. pH, antimicrobial preservative and sterility.
The concentrated harvest is shown to comply with the test
pH (2.2.3)
lor mycobacteria (2.6.2) before addition of any antimicrobial The pH of the preparation, reconstituted if necessary as
preservative or other substance that might interfere with the stated on the label, is 6.5 to 7.5.
test. Phenol (5 g/L) or another suitable antimicrobial
preservative that does not give rise to false positive reactions Toxicity
may be added; a suitable stabiliser intended to prevent Inject subcutaneously 50 000 IU of the preparation to be
adsorption on glass or plastic surfaces may be added. examined into each of two healthy guinea-pigs weighing
The concentrated harvest may be freeze-dried. Phenol is not 250 g to 350 g and which have not been subjected to any
added to preparations that are to be freeze-dried. treatment likely to interfere with the test. Observe the
animals for 7 days. No adverse effect is produced.
Only a concentrated harvest that complies with the following
requirements may be used in the preparation of the final bulk Sensitisation
tuberculin PPD. Use 3 guinea-pigs that have not been subjected to any
treatment likely to interfere with the test. On 3 occasions at
intervals of 5 days, inject intradermally into each guinea-pig
about 500 IU of the preparation to be examined in a volume'
of 0.1 mL. 2 to 3 weeks after the third injection, administer
the same dose intradermally to the same animals and to a
more than 115 per cent of the intended amount. If phenol
control group of three guinea-pigs of the same mass that
has been used in the preparation, the concentration is not
have not previously received injections of tuberculin. After
more than 5 g/L (2.5.75).
24 h to 72 h, the reactions in the 2 groups of animals are not
Sensitisation substantially different.
Carry out the test described under Tests.
Sterility (2.6. 7) Where applicable, determine the amount of antimicrobial
The concentrated harvest, reconstituted if necessary, preservative by a suitable chemical or physico-chemical
complies with the test for sterility, carried out using 10 mL method. The content is not less than the minimum amount
for each medium. shown to be effective and not more than 115 per cent of the
Potency amount stated on the label. If phenol has been used in the
Determine the potency as described under Assay. preparation, the concentration is not more than 5 g/L
(2.5.75)?
FINAL BULK TUBERCULIN PPD
The concentrated harvest is diluted aseptically, after Live mycobacteria (2.6.2)
reconstitution if necessary. It complies with the test for mycobacteria.
Only a final bulk tuberculin PPD that complies with the Sterility (2.6.7)
following requirement may be used in the preparation of the It complies with the test for sterility.
final lot. ASSAY
Sterility (2.6.7) Use method A or, where the preparation contains 1 ru to
The final bulk tuberculin PPD complies with the test for 2 IU, use method B.
sterility, carried out using 10 mL for each medium. METHOD A
FINAL LOT The potency of tuberculin PPD is determined by comparing
The final bulk tuberculin PPD is distributed aseptically into the reactions produced by the intradermal injection of
sterile containers which are then closed so as to prevent increasing doses of the preparation to be examined into
contamination. It may be freeze-dried. sensitised guinea-pigs with the reactions produced by known
Only a final lot that is satisfactory with respect to each of the concentrations of the reference preparation.
requirements given below under Identification, Tests and Prepare a suspension containing a suitable amount
Assay may be released for use. (0.1 mg/mL to 0.4 mg/mL) of heat-inactivated, dried
The following tests may be omitted on the final lot if they mycobacteria in mineral oil with or without emulsifier;
have been carried out at the stages indicated: use mycobacteria of a strain of the same species as that used
— live mycobacteria: concentrated harvest in the preparation to be examined. Sensitise not fewer than
— sensitisation: concentrated harvest six pale-coloured guinea-pigs weighing not less than 300 g by
— toxicity: concentrated harvest or final bulk tuberculin injecting intramuscularly or intradermally a total of about
PPD 0.5 mL of the suspension, divided between several sites if
— antimicrobial preservative: final bulk tuberculin PPD. necessary. Carry out the test after the period of time required
IV-674 Diagnostic Preparations 2016
for optimal sensitisation which is usually 4 to 8 weeks after 64 per cent and not more than 156 per cent of the stated
sensitisation. Depilate the flanks of the animals so that it is potency.
possible to make at least 3 injections on each side but not
STORAGE
more than a total of 12 injection points per animal. Prepare
Store protected from light.
dilutions of the preparation to be examined and of the
reference preparation using isotonic phosphate-buffered saline LABELLING
(pH 6.5 to 7.5) containing 50 mg/L of polysorbate 80 R. If the The label states:
preparation to be examined is freeze-dried and does not — the number of International Units per container,
contain a stabiliser, reconstitute it using the liquid described — the species of mycobacteria used to prepare the product,
above. Use at least 3 different doses of the reference — the name and quantity of any antimicrobial preservative
preparation and at least 3 different doses of the preparation or any other excipient,
to be examined. For both preparations, use doses such that — the expiry date,
the highest dose is about 10 times the lowest dose. Choose — for freeze-dried products, a statement that the product is
the doses such that when they are injected the lesions to be reconstituted using the liquid provided by the
produced have a diameter of not less than 8 mm and not manufacturer,
more than 25 mm. In any given test, the order of the — where applicable, that tuberculin PPD is not to be
dilutions injected at each point is chosen at random in a injected in its concentrated form but diluted so as to
Latin square design. Inject each dose intradermally in a administer not more than 100 IU per dose.
constant volume of 0.1 mL or 0.2 mL. Measure the If the package does not contain a leaflet warning that the
diameters of the lesions 24 h to 48 h later and calculate the inhalation of concentrated tuberculin PPD may produce
results of the test by the usual statistical methods, assuming toxic effects, this warning must be shown on the label on the
that the diameters of the lesions are directly proportional to container together with a statement that the powder must be
the logarithm of the concentration of the preparation. handled with care.
The estimated potency’ is not less than 80 per cent and not __________________________________________________________ ■- PnEur
64 per cent and not more than 156 per cent of the stated
potency.
METHOD B
The potency of tuberculin PPD is determined by comparing
the reactions produced by the intradermal injection of the
preparation to be examined into sensitised guinea-pigs with
the reactions produced by known concentrations of the
Prepare a suspension in mineral oil with or without emulsifier
and containing a suitable amount (0.1 mg/mL to 0.4 mg/mL)
of heat-inactivated, dried mycobacteria; use mycobacteria of
a strain of the same species as that used in the preparation to
be examined. Sensitise not fewer than 6 pale-coloured
guinea-pigs weighing not less than 300 g by injecting
intramuscularly or intradermally a total of about 0.5 mL of
the suspension, divided between several sites if necessary.
Carry out the test after the period of time required for
optimal sensitisation which is usually 4 to 8 weeks after
sensitisation. Depilate the flanks of the animals so that it is
possible to make at least 3 injections on each side but not
more than a total of 12 injection points per animal. Prepare
dilutions of the reference preparation using isotonic
phosphate-buffered saline (pH 6.5 to 7.5) containing
50 mg/L of polysorbate 80 R. Use at least 3 different doses of
the reference preparation such that the highest dose is about
10 times the lowest dose and the median dose is the same as
that of the preparation to be examined. In any given test, the
order of the dilutions injected at each point is chosen at
random in a Latin square design. Inject the preparation to be
examined and each dilution of the reference preparation
intradermally in a constant volume of 0.1 mL or 0.2 mL.
Measure the diameters of the lesions 24 h to 48 h later and
calculate the results of the test by the usual statistical
methods, assuming that the areas of the lesions are directly
proportional to the logarithm of the concentration of the
preparation to be examined. (This dose relationship applies
to this assay and not necessarily to other test systems.)
Monographs
Radiopharmaceutical
Preparations
2016 Radiopharmaceutical Preparations IV-677
Radiopharmaceutical Preparations ***** Radioactivity

Generally the term ‘radioactivity’ is used both to describe the
(Ph. Eur. monograph 0125) *** phenomenon of radioactive decay and to express the physical
Ph Fir__________________________________________________________ quantity of this phenomenon.
The radioactivity of a preparation is the number of nuclear
DEFINITIONS
disintegrations or transformations per unit time.
Radiopharmaceutical preparations or radiopharmaceuticals
are medicinal products which, when ready for use, contain 1 In the International System (SI), radioactivity is expressed in
or more radionuclides (radioactive isotopes) included for a becquerel (Bq), which is 1 nuclear transformation per
medicinal purpose. second. Absolute radioactivity measurements require a
specialised laboratory but identification of radioactivity and
For the purpose of this general monograph,
quantitative measurement of radioactivity can be carried out
radiopharmaceutical preparations also cover: relatively by comparing the measured samples with
— radionuclide generators: any system incorporating a fixed
standardised preparations provided by laboratories recognised
parent radionuclide from that is produced a daughter by the competent authority or by using a calibrated
radionuclide that is to be obtained by elution or by any instrument.
other method and used in a radiopharmaceutical
preparation; Radioactive decay
— kits for radiopharmaceutical preparation: any preparation Any radionuclide decays at an exponential rate with its
to be reconstituted or combined with radionuclides in the characteristic decay constant. The curve of exponential decay
final radiopharmaceutical preparation, usually prior to its (decay curve) is described by the following expression:
administration;
— radionuclide precursors: any radionuclide produced for At — Aoe~Xt
radiolabelling of another substance prior to
administration; At = the radioactivity at time r,
— chemical precursors: non-radioactive substances for Aq = the radioactivity at time t = 0;
combination with a radionuclide. X = the decay constant, characteristic of each
Radionuclide precursors may be supplied as solutions for radionuclide;
radiolabelling. e = the base of natural logarithms.
A nuclide is a species of atom characterised by the number of The half-life (T1Z2) is the time in which a given radioactivity
protons and neutrons in its nucleus (and hence by its atomic (amount) of a radionuclide decays to half its initial value.
number z, and mass number A) and also by its nuclear It is related to the decay constant (X) by the following
energy state. Isotopes of an element are nuclides with the equation:
same atomic number but different mass numbers. Nuclides
containing an unstable arrangement of protons and neutrons _ _ In 2
will transform spontaneously to either a stable or another Tz/2 =
unstable combination of protons and neutrons with a
constant statistical probability. Such nuclides are said to be The equation of exponential decay can thus be expressed
radioactive and are called radionuclides. The initial unstable also in the following way, useful for the fast estimation of the
nuclide is referred to as the parent radionuclide and the radioactivity left after elapsing time r.
resulting nuclide as the daughter nuclide.
Decay or transformation of radionuclides may involve the
emission of charged particles, electron capture (EC) or
isomeric transition (IT). The charged particles emined from
nuclei may be alpha particles (nuclei of 4He) or beta particles
(negatively charged, generally called electrons, or positively The penetrating power of each radiation varies considerably
charged, generally called positrons). Alpha decay usually according to its nature and its energy. Alpha particles are
concerns heavy' nuclei (Z > 82). Radionuclides with a deficit completely absorbed in a thickness of a few micrometres to
of protons usually decay by emitting electrons. Radionuclides some tens of micrometres of matter. Beta particles are
with a deficit of neutrons usually decay by electron capture completely absorbed in a thickness of several millimetres to
or by emitting positrons. In the latter case, radionuclides are several centimetres of matter. Gamma rays are not
called positron emitters. Positrons are annihilated after completely absorbed but only attenuated and a tenfold
interaction with electrons in the surrounding matter. reduction may require, for example, several centimetres of
The annihilation results in the emission of 2 gamma photons, lead. The denser the absorbent, the shorter the range of
each with energy of 0.511 MeV, generally emitted at 180° to alpha and beta particles and the greater the attenuation of
each other (annihilation radiation). All decay modes may be gamma rays.
accompanied by an emission of gamma rays. The emission of Each radionuclide is characterised by an invariable half-life,
gamma rays may be partly or completely replaced by the expressed in units of time and by the nature and energy of its
ejection of electrons, known as internal conversion electrons. radiation or radiations. The energy is expressed in
This phenomenon, like, the process of electron capture, electronvolts (eV), kilo-electronvolts (keV) or mega
causes a secondary emission of X-rays (due to a electronvolts (MeV).
reorganisation of the electrons in the atom). This secondary Radionuclidic purity
emission may itself be partly replaced by the ejection of The ratio, expressed as a percentage, of the radioactivity of
electrons, known as Auger electrons. the radionuclide concerned to the total radioactivity of the
radiopharmaceutical preparation. The relevant potential
radionuclidic impurities are listed with their limits in the
individual monographs.
IV-678 Radiopharmaceutical Preparations 2016
Radiochemical purity — in reactions of charged particles (target irradiation using

The ratio, expressed as a percentage, of the radioactivity of accelerators, in particular cyclotrons);
the radionuclide concerned which is present in the — by its separation from radionuclide generators.
radiopharmaceutical preparation in the stated chemical form, The probability of nuclear reaction occurrence depends on
to the total radioactivity of that radionuclide present in the the nature and energy of the incident particles (protons,
radiopharmaceutical preparation. The relevant potential neutrons, deuterons etc.) and on the nature of the nucleus
radiochemical impurities are listed with their limits in the that is irradiated by them. The rate of production (yield) of a
individual monographs. given radionuclide resulting from the irradiation depends in
Chemical purity addition on the isotopic composition of the target material
In monographs on radiopharmaceutical preparations, and its chemical purity, and in the case of neutrons on their
chemical purity is controlled by specifying limits for chemical flux, and in the case of charged particles on beam current.
impurities. In addition to the desired nuclear reaction, simultaneous
Isotopic carrier transformations usually occur. Probability of their occurrence
A stable isotope of the element concerned either present in or is given by the same factors as mentioned in the previous
added to the radioactive preparation in the same chemical paragraph. Such simultaneous transformations may give rise
form as that in which the radionuclide is present. to radionuclidic impurities.
Carrier-free preparation The nuclear reaction (transformation) can be written in the
A preparation free from stable isotopes of the same element form: target nucleus (incident particle, emitted particle)
as the radionuclide concerned present in the preparation in produced nucleus.
the stated chemical form or at the position of the
radionuclide in the molecule concerned. 58Fe(n,y)59Fe
No-carrier-added preparation
18O(p,ท)18F
A preparation to which no stable isotopes of the same
element as the radionuclide concerned are intentionally
added in the stated chemical form or at the position of the NEUTRON IRRADIATION
radionuclide in the molecule concerned. Irradiation of stable radionuclides in nuclear reactors usually
Specific radioactivity results in proton-deficient nuclei, i.e. electron emitters that
The radioactivity of a radionuclide per unit mass of the are formed in (ท,y) reactions (so-called radiative capture).
element or of the chemical form concerned, e.g. becquerel The product is isotopic with the target nucleus and it may
per gram or becquerel per mole. thus contain a considerable amount of carrier.
Radioactivity concentration A number of nuclides with high atomic number are
The radioactivity of a radionuclide per unit volume or unit fissionable by neutrons. Nuclear fission, denoted as (ท, f)
reaction, results in a large number of radionuclides of various
mass of the preparation. For radiopharmaceutical solutions, it
is expressed as radioactivity per unit volume of the masses and half-lives. The most frequently used fission is that
preparation. of 235u. Iodine-131, molybdenum-99 and xenon-133 can be
produced by irradiation of 235u in nuclear reactors and by
Total radioactivity their separation from more than 200 radionuclides formed in
The radioactivity of the radionuclide, expressed per unit (vial, that process.
capsule, ampoule, generator, etc).
CHARGED PARTICLE IRRADIATION
Chemical precursors for synthesis of radioactive
Irradiation of stable radionuclides with charged particles
substances
usually results in neutron-deficient nuclei that decay either by
If the active substance of a radiopharmaceutical preparation
electron capture or by positron emission. They are formed in
is not isolated, the chemical precursor for its synthesis is
particular in (p, xn) reactions (where X is the number of
considered as a substance for pharmaceutical use.
emitted neutrons). The product is not isotopic with the
It is recommended to test each batch of chemical precursor target nucleus and its specific radioactivity might be close to
material in production runs before its use for the that of a carrier-free preparation.
manufacture of radiopharmaceutical preparations to ensure
that, under specified production conditions, the substance RADIONUCLIDE GENERATORS
yields the radiopharmaceutical preparation in the desired Radionuclide generator systems use a parent radionuclide
quantity and of the quality specified. which decays to a daughter radionuclide with a shorter half
life.
Period of validity
The time during which specifications described in the By separating the daughter radionuclide from the parent
monograph must be fulfilled. radionuclide by a chemical or physical process, it is possible
to use the daughter radionuclide at a considerable distance
PRODUCTION from the production site of the generator despite its short
A radiopharmaceutical preparation contains its radionuclide: half-life.
— as an element in atomic or molecular form, e.g. 133Xe,
[,5O]O2; TARGET MATERIALS
— as an ion, e.g. [131I]iodide, ["๓Tc] pertechnetate; The isotopic composition and purity of the target material
together with other factors such as the nature and energy of
— included in, adsorbed on or attached to molecules by
chelation, e.g. [J11 In]indium oxine, or by covalent incident particles will determine the relative percentages of
bonding, e.g. 2-[I8F]fluoro-2-deoxy-d-glucose. the principal radionuclide and radionuclidic impurities
produced by irradiation. The use of isotopically enriched
Radionuclides can be produced in the following ways: target material in which the abundance of the required target
— in reactions of neutrons (target irradiation in nuclear
nuclide has been artificially increased, can improve the
reactors);
production yield and the purity of the desired radionuclide.
The chemical form, the purity and the physical state of the TESTS
target material and the chemical additives, as well as the It is sometimes difficidt to cany out some of the following tests
irradiation conditions and the direct physical and chemical before releasing the batch for use when the half-life of the
environment, determine the chemical state and chemical radionuclide in the preparation is short. The individual monograph
purity of the radionuclides that are produced. In the indicates the tests that need not be completed before release for use.
production of radionuclides, and particularly of radionuclides These tests then constitute a control of the quality of production.
with a short half-life, it may not be possible to determine any Non-radioactive substances and related substances
of these quality criteria before further processing and This section prescribes the determination of non-radioactive
manufacture of radiopharmaceutical preparations. Therefore substances and related substances that can be present.
the quality of each batch of target material is assessed before
its use in routine radionuclide production and manufacture Residual solvents
of radiopharmaceutical preparations. Residual solvents are limited according to general chapter
ร.4.Residual solvents, using the methods given in general
The target material is contained in a holder in gaseous, liquid
chapter 2.4.24. Identification and control of residual solvents or
or solid state, in order to be irradiated by a beam of particles.
another suitable method.
For neutron irradiation, the target material is commonly
contained in quartz ampoules or high-purity aluminium or RADIONUCLIDIC PURITY
titanium containers. It is necessary to ascertain that no Radionuclidic impurities may arise during the production and
interaction can occur between the container and its contents decay of a radionuclide. Potential radionuclidic impurities
under the irradiation conditions. may be mentioned in the monographs and their
For charged particle irradiation, the holder for target material characteristics are described in general chapter 5.7. Table of
is constructed of an appropriate metal, possibly with inlet physical characteristics of radionuclides mentioned in the European
and outlet ports, a surrounding cooling system and usually a Pharmacopoeia.
thin metal foil target window. In most cases, to establish the radionuclidic purity of a
To evaluate all effects on the efficiency of the production of radiopharmaceutical preparation, the identity of every
the radionuclide in terms of quality and quantity, the radionuclide present and its radioactivity must be known.
production procedure must clearly describe and take into Generally, the most useful method for examination of the
consideration: the target material, the construction of the radionuclidic purity of gamma- and X-ray emitting
holder for target material, method of irradiation and radionuclides is gamma-ray spectrometry. The use of sodium
separation of the desired radionuclide. iodide detectors may cause a problem: the peaks due to
gamma-ray emitting impurities may be concealed in the
CHARACTERS spectrum of the principal radionuclide or left unresolved
The Table of physical characteristics of radionuclides (ร.7) from peaks of other radionuclidic impurities in the
summarises the most commonly accepted physical preparation. Alpha- and beta-particle emitting impurities that
characteristics of radionuclides used in preparations that are do not emit gamma- or X-rays cannot be detected in this
the subject of monographs in the European Pharmacopoeia. way. For alpha- and beta-emitters other methods must be
In addition, the Table states the physical characteristics of employed.
the main potential radionuclidic impurities of the The individual monographs prescribe the radionuclidic purity
radionuclides mentioned in the monographs. required and may set limits for specific radionuclidic
The term ‘transition probability’ means the probability of the impurities (for example, molybdenum-99 in technetium-
transformation of a nucleus in a given energy state, via the 99m). While these requirements are necessary, they are not in
transition concerned. Instead of ‘probability’ the term themselves sufficient to ensure that the radionuclidic purity of
‘abundance’ is also used. a preparation is sufficient for its clinical use.
The term ‘emission probability’ means the probability that an The manufacturer must examine the product in detail and
atom of a radionuclide gives rise to the emission of the especially must examine preparations of radionuclides with a
particles or radiation concerned. short half-life for impurities with a long half-life after a
Irrespective of which meaning is intended, probability is suitable period of decay. In this way, information on the
usually stated as a percentage. suitability of the manufacturing processes and the adequacy
of the testing procedures is obtained. In cases where 2 or
IDENTIFICATION more positron-emitting radionuclides need to be identified
A radionuclide is generally identified by its half-life or by the and/or differentiated, for example the presence of op
nature and energy of its radiation or radiations or by both, as impurities in 13N-preparations, half-life determinations need
prescribed in the monograph. to be carried out in addition to gamma-ray spectrometry.
Approximate half-life Due to differences in the half-lives of the different
The half-life as determined over a relatively short time period radionuclides present in a radiopharmaceutical preparation,
to allow release for use of radiopharmaceutical preparations. the radionuclidic purity changes with time.
The calculated approximate half-life is within the range of RADIOCHEMICAL PURITY
the values stated in the individual monograph. Radiochemical impurities may originate from:
Determination of the nature and energy of the — radionuclide production;
radiation — subsequent chemical procedures;
The nature and energy of the radiation emitted are — incomplete preparative separation;
determined using spectrometry. The nature and energy of the — chemical changes during storage.
radiation of positron emitters is usually not determined; their The determination of radiochemical purity requires
identification is performed by determination of their half-life separation of the different chemical substances containing the
and gamma-ray spectrum. radionuclide and determination of the percentage of
radioactivity of the radionuclide concerned associated with
the stated chemical form. The radiochemical purity section of injection or revealed by subsequent assay of tissue
an individual monograph may include limits for specified radioactivity). In that case the test may be repeated.
radiochemical impurities, including isomers. Sterility
In principle, any method of analytical separation may be used Radiopharmaceutical preparations for parenteral
in the determination of radiochemical purity. For example, administration comply with the test for sterility. They must
the monographs for radiopharmaceutical preparations may be prepared using precautions designed to exclude microbial
include paper chromatography (2.2.26), thin-layer contamination and to ensure sterility. The test for sterility is
chromatography (2.2.27), electrophoresis (2.2.2/), size earned out as described in the general method (2.6.1).
exclusion chromatography (2.2.20), gas chromatography Special difficulties arise with radiopharmaceutical
(2.2.25) and liquid chromatography (2.2.29). The technical preparations because of the short half-life of some
description of these analytical methods is set out in the radionuclides, the small size of batches and the radiation
monographs. Moreover, certain precautions special to hazards. In the case that the monograph states that the
radiopharmaceuticals must also be considered, such as preparation can be released for use before completion of the
radiation protection, measurement geometry, detector test for sterility, the sterility test must be started as soon as
linearity, use of carriers, dilution of the preparation. practically possible in relation to the radiation. If not started
Specific radioactivity immediately, samples are stored under conditions that are
Specific radioactivity is usually calculated taking into account shown to be appropriate in order to prevent false negative
the radioactivity concentration and the concentration of the results. Parametric release (5. /. /) of the product
chemical substance being studied, after verification that the manufactured by a fully validated process is the method of
radioactivity is attributable only to the radionuclide choice in such cases. When aseptic manufacturing is used,
(radionuclidic purity) and the chemical species the test for sterility has to be performed as a control of the
(radiochemical purity) concerned. quality of production.
Specific radioactivity changes with time. The statement of the When the size of a batch of the radiopharmaceutical
specific radioactivity therefore includes reference to a date preparation is limited to 1 or a few samples, sampling the
and, if necessary, time. batch for sterility testing according to the recommendations
of the general method (2.6.1) may not be applicable.
Physiological distribution
Tests involving animals should be avoided wherever possible. When the half-life of the radionuclide is less than 5 min, the
Where the tests for identity and for radiochemical purity are administration of the radiopharmaceutical preparation to the
not adequate to completely define and control the patient is generally on-line with a validated production
radiochemical species in a radiopharmaceutical preparation, a system.
physiological distribution test may be required. For safety reasons (high level of radioactivity) it is not
The distribution pattern of radioactivity observed in specified possible to use the quantity of radiopharmaceutical
organs, tissues or other body compartments of an appropriate preparations as required in the test for sterility (2.6./).
animal species can be a reliable indication of the suitability The method of membrane filtration is preferred to limit
for the intended purpose. irradiation of personnel.
Alternatively, a physiological distribution test can serve to Notwithstanding the requirements concerning the use of
establish the biological equivalence of the preparation under antimicrobial preservatives in the monograph Parenteral
test with similar preparations known to be clinically effective. preparations (0520) 5 their addition to radiopharmaceutical
The individual monograph prescribes the details concerning preparations in multidose containers is not obligatory, unless
the conduct of the test and the physiological distribution prescribed in the monograph.
requirements that must be met. Bacterial endotoxins - pyrogens
In general, the test is performed as follows. Radiopharmaceuticals for parenteral administration comply
with the test for bacterial endotoxins (2.6.14) or with the test
Each of 3 animals is injected intravenously with the
preparation. In some cases, dilution immediately before for pyrogens (2.6.8).
injection may be necessary. Eluates of radionuclide generators, solutions for radiolabelling
and kits for radiopharmaceutical preparations also comply
Immediately after injection each animal is placed in a
with the test for bacterial endotoxins if they are intended for
separate cage for collection of excreta and prevention of
the preparation of radiopharmaceuticals for parenteral
contamination of the body surface of the animal. At the
administration without further purification.
specified time after injection, the animals are euthanised by
an appropriate method and dissected. Selected organs and Radionuclide precursors and chemical precursors comply
tissues are assayed for their radioactivity. The physiological wi± the test for bacterial endotoxins if intended for use in
distribution is then calculated and expressed in terms of the the manufacture of parenteral preparations without a further
percentage of the administered radioactivity that is found in appropriate procedure for the removal of bacterial
each of the selected organs or tissues, taking into account endotoxins.
corrections for radioactive decay. For some The test for bacterial endotoxins is carried out as described
radiopharmaceutical preparations it is necessary to determine in the general method (2.6.14), taking the necessary'
the ratio of the radioactivity in weighed samples of selected precautions to limit irradiation of the personnel carrying out
tissues (radioactivity/mass). the test. The limit for bacterial endotoxins is indicated in the
A preparation meets the requirements of the test if the individual monograph or calculated according to general
distribution of radioactivity in at least 2 of the 3 animals chapter 5.1.10. Guidelines for using the test for bacterial
complies with all the specified criteria. endotoxins.
Disregard the results from any animal showing evidence of When the nature of the radiopharmaceutical preparation or
extravasation of the injection (observed at the time of the precursor results in an interference in the test for
bacterial endotoxins by inhibition or activation and it is not
possible to eliminate the interfering factor(s), the test for

pyrogens (2.6.8) may be specifically prescribed.
STORAGE
Store preparations containing radioactive substances in an
airtight container that is sufficiently shielded to protect
personnel from irradiation by primary or secondary emissions
and that complies with national and international regulations
concerning the storage of radioactive substances. During
storage, containers may darken due to irradiation. Such
darkening does not necessarily involve deterioration of the
preparations.
LABELLING
The labelling of radiopharmaceutical preparations complies
with the relevant national and European legislation.
For preparations prepared at the site of use, the labelling can
be modified.
The radioactivity of a preparation is stated at a given date.
If the half-life is less than 70 days the time is also indicated,
with reference to a time zone. The radioactivity at
other times may be calculated from the decay equation or
from tables.
In addition to the above, the label on the container, the
package, a leaflet accompanying the package or a certificate
of analysis accompanying the radiopharmaceutical
preparation states:
— if applicable, the maximum recommended dose in
millilitres;
— the name and concentration of any added antimicrobial
preservative;
— where applicable, any special storage conditions.
For chemical precursors, the accompanying information
recommends testing the substance in 1 or more production
runs before its use for the manufacture of
radiopharmaceutical preparations to ensure that, under
specified production conditions, the substance yields the
radiopharmaceutical preparation in the desired quantity and
of the quality specified.
DETECTION AND MEASUREMENT OF
RADIOACTIVITY
Detection and measurement of radioactivity are carried out
according to general chapter 2.2.66. Detection and
measurement of radioactivity.
______________________________________________________________ Ph Eur
The uncertainty of the half-lives are given in parentheses.

Physical Characteristics of * In principle the digits in parentheses are the standard
Radionuclides Mentioned in the *** uncertainty of the corresponding last digits of the indicated
numerical value (‘Guide to the Expression of Uncertainty in
European Pharmacopoeia Measurement', International Organization for
(Ph Eur general texts 5.7) Standardization (ISO), 1993, ISBN 92-67-10188-9).
The following table is given to complete the general The following abbreviations are used:
monograph Radiopharmaceutical preparations (0125). eA = Auger electrons,
The values are obtained from the database of the National ce = conversion electrons,
Nuclear Data Center (NNDC) at Brookhaven National p- = electrons,
Laboratory, Upton. N.Y., USA, directly accessible via p+ = positrons,
Internet at the address: Y = gamma rays,
‘http://www.nndc.bnl.gov/nndc/nudat/radform.html' . X = X-rays.
In case another source of information is preferred (more
recent values), this source is explicitly mentioned.
Other data sources:
* DAMRI (Departement des Applications et de la
Metrologie des Rayonnements lonisants, CEA Gif-sur-
Yvette, France),
** PTB (Physikalisch-Technische Bundesanstalt,
Braunschweig, Germany),
*** NPL (National Physical Laboratory, Teddington,
Middlesex, UK).
Electronic emission Photon emission
Emission Emission
probability probability
Radionuclide Half-life Type Energy (MeV) Type Energy (MeV) (per 100 dis
(per 100 dis
integrations) integrations)
Tritium (3H) •12.33 (6) years •p- •0.006 ๓ (max; 0.019) •100
Carbon-11 (”C) 20.385 (20) min p‘ 0.386 (I) (max: 0.960) 99.8 Y 0.511 199.5 (n)
Nitrogen-13 (”N) 9.965 (4) min P’ 0.492 (I) (max 1.198) 99.8 Y 0.511 199.6 00
ร 99.9 Y 0.511 199.8 m

Oxygen-15 (,$O) 122.24 (16) p- 0.735 ๓ (max: 1.732)
Fluorine-18 (*’F) 109.77 (5) min p* 0.250 m (max: 0.633) 96.7 Y 0.511 193.5 ™
Phosphonis-32 (”P) 14.26 (4) days p- 0.695 ๓ (max: 1.71) 100
Phosphorus-33 (33p) 25.34 (12) days p- 0.076 (l) (max: 0.249) 100
Sulfur-35 (35S) 87.51 (12) days p- 0.049 (,) (max: 0.167) 100
27.7025 (24) days ex 0.004 67 X 0.005 22.3

Chromium-51 (5,Cr)
0.320 9.9
าา:27 (3) days ex 0.006 47 X 0.006-0.007 25
0.179 ๓ 0.9 Y 0.511 38.0 <n)

p-
0.631 ๓ 18.1 0.847 100.0
1.038 14.1
1.175 22
1.238 66.1
Cobalt-56 (MCo) 4.3
1.360
1.771 15.5
2.015 3.0
2.035 7.8
2.598 17.0
3.202 3.1
3.253 7.6
(I) Mean energy of the p spectrum.

(II) Maximum emission probability corresponding to a total annihilation in the source per 100 disintegrations.

Emission Emission
Radionuclide probability probability
Half-life Type Energy (MeV) Type Energy (MeV)
(per 100 dis (per 100 dis
271.79 (9) days ex+ce 0.006-0.007 177.4 X 0.006-0.007 57
ce 0.014 Y 0.014 9.2

Cobalt-57 (57Co)
0.115 1.8 0.122 85.6
0.129 1.3 0.136 10.7
0.692 0.15
70.86 (7) days eA 0.006 49.4 X 0.006-0.007 26.3
p* 0.201 <° 14.9 Y 0.511 29.9 ๓

Cobalt-58 (“Co)
0.811 99.4
0.864 0.7
1.675 0.5
5.2714 (5) years 0- 0.096 (l) (max: 0.318) 99.9 Y 1.173 100.0
Cobalt-60 (“Co)
1333 100.0
9.49 (7) hours ex 0.008 21 X 0.009-0.010 19.1
0- 0.157 <•> 1 Y 0311 112 ๓
0.331 (l) 0.7 0.834 5.9
0.397 » 3.8 1.039 37
0.782 m 0.3 1333 12
1.90 ๓ 50 1.919 2.1
2.190
Gallium-66 (“Ga)
2.423 1.9
2.752 23.4
3.229 1.5
3.381 1.5
3.792 1.1
4.086 1.3
4.295 4.1
4.807 1.8
3.2612 (6) days ex 0.008 62 X 0.008-0.010 57
ce 0.082-0.084 30.4 Y 0.091-0.093 42.4
0.090-0.092 3.6 0.185

Gallium-67 (67Ga)
0.175 0.3 0.209 2.4
0.300 16.8
0.394 4.7
0.888 0.15
270.82 (27) days ex 0.008 42.4 X 0.009-0.010 44.1
Germanium-68 (“Ge)
in equilibrium with
Gallium-68 (“Ga) p* 0353 ๓ Y 0.511 1783
(“Ga: 67.629 88.0 1.077 3.0
0.836 ro
(24) min)
Emission Emission
(per 100 dis
67.629 (24) min ex 0.008 5.1 X 0.009-0.010 4.7
Gallium-68 (“Ga)
0.511 1783
p- 0.353 ๓ 1.2 Y
0.836 ๓ 88.0 1.077 3.0
13.10 (3) ร ce 0.176 26.4 X 0.012-0.014 17.0
Krypton-81m (,lmKr) 0.189 4.6
Y 0.190 67.6
4.576 (5) hours ex 0.011 31.3 X 0.013-0.014 57.2
ce 0.176 25.0 Y 0.190 64
Rubidium-81 C'Rb) 0.188 4.3 0.446 23.2

in equilibrium with
Krypton-81m(,1BKr) 0.457 3.0
p- 0.253 ๓ 1.8 0.510 5.3
0.447 ๓ 25.0 0.511 54.2
0.538 2.2
(”BKr: 13.10(3) ร)
50.53 (7) days p- 0.583 ๓ (max: 1.492) 99.99 Y 0.909 0.01
Strontium-89 (*’Sr)
in equilibrium with
Yttrium-89m (,9mY)
(”BY: 16.06 (4) ร)
28.74 (4) years p 0.196 ๓ (max: 0.546) 100
Strontium-90 (wSr)
in equilibrium with
Yttrium-90 C°Y)
f°Y: 64.10 (8) hours)
64.10 (8) hours P’ 0.934 (D (max: 2.280) 100
Yttrium-90 (*°Y)
65.94 (1) hours p- 0.133 ๓ 16.4 X 0.018-0.021 3.6
0.290 m 1.1
0.443 ๓ 82.4 Y 0.041 1.1
Molybdene-99 C*Mo) 0.141 4.5

in equilibrium with
Technetium-99m 0.181 6
(*Tc)
0.366 1.2
(WmTc: 6.01 (1) hours) 0.740 12.1
0.778 4.3
6.01 (1) hours ce 0.002 74 X 0.018-0.021 7.3
ex 0.015 2.1 Y 0.141 89.1

Technetium-99m
r-Tc)
ce 0.120 9.4
0.137-0.140 1.3
2.11 X 10s years p" 0.085 ๓ (max: 0.294) 100

Technetium-99 (”Tc)

Emission Emission
(per 100 dis- (per 100 dis-
39.26 (2) days ex+ce 0.017 12 X 0.020-0.023 9.0
Ruthenium-103 (,0JRu) ce 0.030-0.039 88.3 Y 0.497 91

in equilibrium with
Rhodium-103m 0.610 5.8
(103raRh)
p- 0.031 ๓ 6.6
(’ojMRh: 56.114 (20)
0.064 ๓ 92.2
min)
4.9 (1) hours ex 0.019 13.4 X 0.023-0.026 70.5
Y 0.642 25.9
Indium-110 (ll0In) 0.658 983
0.885 92.9
0.938 68.4
0.997 10.5
69.1 (5) min CA 0.019 5.3 X 0.023-0.026 27.8
Indium-110m (ll0mIn) p’ 1.015 ๓ 61 Y 0.511 123 4 ๓1
0.658 97.8
2.129 2.1
2.8047 (5) days ex 0.019 15.6 X 0.003 6.9
0.023-0.026 823
ce 0.145 7.8
Indium-111 (’’’In)
0.167-0.171 13 Y 0.171 90.2
0.219 4.9 0.245 94.0
0.241-0245 1.0
49.51 (1) days ce 0.162 40 X 0.023-0.027 36.3
0.186-0.190 40
Indium-114m (IHmIn)
in equilibrium with Y 0.190 15.6
Indium-114 (IHln)
’P 0.777 ๓ (max: 1.985) 95 0.558 3.2
(luIn: 71.9(1) ร) 0.725 3.2
154.0 (7) days ex 0.003 88.0 X 0.026-0.031 50.5
0.022-0.023 7.4
Tellurium-121m Y 0.212 81.4

(l,lmTe) in equilibrium
ce 0.050 332 1.102 2.5
with Tellure-121 (u,Te)
(12,Te: 19.16 (5) days) 0.077 40.0
0.180 6.1
*•19.16(5) days ex 0.022 11.6 X 0.026-0.030 75.6
Y 0.470 1.4
Tellunum-121 (niTe)
0.508 17.7
0.573 80.3

Emission Emission
(per 100 dis
13.27 (8) hours ex 0.023 12.3 X 0.004 9.3
0.027-0.031 86.6
ce 0.127 13.6
0.154 1.8 Y 0.159 83.3
Iodine-123 (1UI) 0.158 0.4 0.346 0.1
0.440 0.4
0.505 0.3
0.529 1.4
0.538 0.4
59.402 (14) days ex+ce 0.004 80 X 0.004 15.5
0.023-0.035 33 0.027 114
Iodine-125 (,UI) 0.031 26
Y 0.035 6.7
13.11 (5) days ex 0.023 6 X 0.027-0.031 42.2
ce 0.354 0.5 Y 0.388 34
0.634 0.1 0.491 2.9
2 3 (lh
Iodine-126 (*MI)
p- 0.109 ๓ 3.6 0.666
0.290 32.1 0.754 4.2
0.459 (,) 8.0 0.880 0.8
1.420 0.3
p- 0.530 ๓ 1
8.02070 (11) days ce 0.46 3.5 X 0.029-0.030 3.9
0.330 1.6
Y 0.080 2.6
0.069 ๓ 2.1 0.284 6.1

Iodine-131 (’”1) p-
0.097 ๓ 7.3 0.365 81.7
0.192 ๓ 89.9 0.637 7.2
0.723 1.8
11.84 (7) days cx 0.025 6.8 X 0.004 8.3
0.030 44.0
ce 0.129 61 0.034 10.2

Xenon-131m (”,MXe)
0.159 28.5
0.163 8.3 Y 0.164 2.0
0.140 ๓ 3.8 Y 0.530 87

20.8 (1) hours P‘
Iodine-133 (*”I) 0.162 ® 3.2 0.875
(decays to radioactive 2.4
0.299 ๓ 4.2 1.298
Xenon-133)
0.441 (0 83

Electronic emission Photon emlsaio ท

5.243 (1) days 0.026 0.004
2 3 3
0.031
ce 0.045 55.1 0535
Xenon-133 C”Xe)
0.075-0.080
ร
0.101 ๓
2.19 (1) days 0.025
0.030
Xenon-133m (IJJ“Xe)
(decays to radioactive ce 0.199 0.034 10.6
Xenon-133)
0.228 20.7
0.232 0233 10.0

6.57 (2) hours 0.140 ® •0.527
0.237 ® 0547
0.307 ๑ 0537
0.352 ® 21.9 1.039 8.0
Iodine-135 C”I)
(decays to radioactive 0.399 ®
Xenon-135)
1260 28.9
0529®
1.678
1.791
9.14 (2) hours 0.214 0.031-0.035 5.0
Xenon-135 C”Xe)
p- 0.171 90.2
0.308 96.0
30.04 (3) years 0.026 0.8 0.005
0.032-0.036
0.624 8.0
Caesium-137 (*”Cs)
in equilibrium with 0.656 Y 0.662 85.1
Barium-137m (*”“Ba)
p- 0.174 ®
(’’’“Ba: 2.552 (1) min)

0.416® 5.6
26.1 (1) hours 0.285 0.010
0.353 0.069-0.071 633
0.08
r 0.495 ๓ 0.3
Y 0368
0579
Thallium-2 00 ฯๆ) 0.828 103
1206
1226
4.0
(I) Mean energy of the spectrum.

Emission Emission
Radionuclide Half-life Type Energy (MeV) Type Energy (MeV)
(per 100 dis (per 100 dis
9.33 (3) hours ex 0.055 3 X 0.070-0.073 69
0.083 . 19
ce 0.246 8.5
0.276 2 Y 0.331 79
0.316 2.3 0.361 9.9
0.406 2.0
Lead-201 («*Pb) 0.585 3.6

(decays to radioactive
Thallium-201) 0.692
0.767 3.2
0.826 2.4
0.908 5.7
0.946 7.9
1.099 1.8
1.277 1.6
72.912 (17) hours ce 0.016-0.017 17.7 X 0.010 46.0
0.027-0.029 4.1 0.069-0.071 73.7
0.052 7.2 0.080 20.4

Th allium-201 (M,TI)
0.084 15.4
0.153 2.6 Y 0.135 2.6
0,167 10.0
12.23 (2) days ex 0.054 2.8 X 0.010 31.0
0.069-0.071 61.6
ce 0.357 2.4 0.080 17.1

Thallium-202 (M2T1)
Y 0.440 91.4
51.873 (9) hours ex 0.055 3.0 X 0.010 37.0
0.071-0.073 69.6
ce 0.194 13.3 0.083 19.4

Lead-203 (“JPb)
Y 0.279 80.8
0.401 3.4

lobenguane Sulfate for ;* ** — resolution: minimum 4.0 between the peaks due to
Radiopharmaceutical Preparations ***** impurity A and iobenguane.
lobenguane Sulphate for Radiopharmaceutical Limit:
Preparations — impurity A: not more than ±e area of the corresponding
(Ph. Eur. monograph 2351) peak in the chromatogram obtained with reference
solution (d) (1.0 per cent).
Maximum 3.0 per cent, determined on 0.100 g by drying in
H2SO4 an oven at 105 °C.
Less than 10 lU/mg, if intended for use without a further
appropriate procedure for the removal of bacterial
Ci6H22I2N6O4S 648 87862-25-7 endotoxins.
Pn Eur_______________
ASSAY
DEFINITION Liquid chromatography (2.2.29) as described in the test for
Bis[(3-iodobenzyl)guanidine] sulfate. related substances with ±e following modification.
Content Injection Test solution and reference solution (a).
98.0 per cent to 102.0 per cent (dried substance). Calculate the percentage content of c 16H22I2N6O4S from
the declared content of iobenguane sulfate CRS.
CHARACTERS
Appearance STORAGE
White or almost white crystals. Protected from light, at a temperature below 25 °C.
IDENTIFICATION LABELLING
A. Infrared absorption spectrophotometry (2.2.24). The label recommends testing the substance in a production
Comparison Ph. Eur. reference spectrum of iobenguane sulfate. test before its use for the manufacture of radiopharmaceutical
preparations. This ensures that, under specified production
B. Dissolve about 10 mg in 1 mL of water R with gende
conditions, the substance yields the radiopharmaceutical
heating. The solution gives reaction (a) of sulfates (2.3.1}.
preparation in the desired quantity and quality specified.
TESTS
IMPURITIES
Impurity A
Specified impurities A
Liquid chromatography (2.2.29). Prepare the solutions and the
mobile phase immediately before use.
Test solution Dissolve 10.0 mg of the substance to be
examined in 1 mL of the mobile phase and dilute to 5.0 mL
with the mobile phase.
Reference solution (a) Dissolve 10.0 mg of iobenguane A. l-(3-iodophenyl)methanamine.
sulfate CRS in 1 mL of the mobile phase and dilute to ________ _______________________________________________________ Ph Eur
Reference solution (b) Dissolve 23.1 mg of 3-
iodobenzylammonium chloride R (salt of impurity A) in 1 mL
of the mobile phase and dilute to 10.0 mL with the mobile
phase.
Medronic Acid for * *
Reference solution (c) Mix 1 mL of reference solution (a) and Radiopharmaceutical Preparations *****
1 mL of reference solution (b). (Ph. Eur. monograph 2350)
Reference solution (d) Dilute 0.1 mL of reference solution (b)
o o
to 10.0 mL with the mobile phase.
HO-P^^P^-OH
Column'. ho' oh
— size: I = 0.25 m, 0 = 4.0 mm;
— stationary phase: silica gel for chromatography R (5 pm);
CHaO6p2 176.0 1984-15-2
— temperature: maintain at a constant temperature between
20 °C and 30 °C. Action and use
Mobile phase Mix 40 mL of an 80 g/L solution of ammonium Bisphosphonate; used for bone scanning.
nitrate R, 80 mL of dilute ammonia R2 and 1080 mL of
methanol R. Ph Elf-------------------------------------------------------------------------------------- ------------------------
Flow rate 1 mL/min. DEFINITION

Detection spectrophotometer at 254 nm. Methylenediphosphonic acid.
Injection 20 ||T ■ of the test solution and reference solutions (c) Content
and (d). 99.0 per cent to 101.0 per cent (dried substance).
Run time 15 min. CHARACTERS
Relative retention With reference to iobenguane (retention Appearance
time = about 7 min): impurity A = about 0.2. White or almost white, amorphous or crystalline, hygroscopic
powder.
Solubility ASSAY
Ver}7 soluble in water, very slightly soluble in anhydrous Dissolve 75 mg in water R and dilute to 50 mL with the
ethanol, practically insoluble in methylene chloride. same solvent. Titrate with 0.1 M sodium hydroxide, using
IDENTIFICATION 0.1 mL of bromocresol green solution R as indicator.
First identification A 1 mL of 0.1 M sodium hydroxide is equivalent to 8.80 mg of
Second identification B CH6O6p 2.
A. ’H Nuclear magnetic resonance spectrometry (2.2.33). STORAGE
Preparation 100 g/L solution in deuterium oxide R. In an airtight container, protected from light.
Comparison 100 g/L solution of medronic acid CRS in LABELLING
deuterium oxide R. The label recommends testing the substance in a production
B. Infrared absorption spectrophotometry (2.2.24). test before its use for the manufacture of radiopharmaceutical
Comparison medronic acid CRS.
conditions, the substance yields the radiopharmaceutical
TESTS preparation in the desired quantity and quality specified.
Impurities A and B
!H Nuclear magnetic resonance spectrometry (2.2.33).
IMPURITIES
Specified impurities A, B
Test solution To 1.0 g of the substance to be examined add
10 mL of deuterated chloroform R. Stir for 1 hour. Pass the h3c^ch3
resulting solution through a sintered-glass filter to remove the o
precipitate containing medronic acid. Evaporate the filtrate to
about 0.5 mL. H3C o' "o^CH3
Reference solution (a) Mix 10 pL of medronic acid ch3 ch3

impurity A CRS with 1.0 mL of deuterated chloroform R.
Reference solution (b) Mix 10 pL of medronic acid A. tris(l-methylethoxy)phosphanc,
impurity B CRS with 1.0 mL of deuterated chloroform R. ch3 _ _o ch3
T o T
Reference solution (c) After recording the NMR spectrum of
h3c o- p- o ch3
the test solution, add 10 pL of medronic acid impurity A CRS
o o
and 10 pL of medronic acid impurity B CRS to the test
solution.
h3c—(
ch3 h3c
y— ch3
Apparatus NMR spectrometer operating at minimum

250 MHz. B. tetrakis(l-methylethyl) methylencdiphosphonatc.
Record the ]H NMR spectra of the test solution and the
reference solutions, if necessary using tetramethylsilane R as a
chemical shift internal reference compound.
Position of the signals Deuterated chloroform =
about 7.3 ppm; impurity A = about 4.4 ppm and 1.3 ppm;
impurity B = about 4.7 ppm, 2.4 ppm and 1.3 ppm.
Pentetate Sodium Calcium for ** โ
System suitability’. Radiopharmaceutical Preparations *****
— the positions of the signals due to impurities A and B in (Ph. Eur. monograph 2353)
the spectrum obtained with reference solution (c) do not
differ significantly from those in the spectra obtained with
reference solutions (a) and (b).
Limits’.
— integration’, integrate the multiplet at 4.4 ppm due to
impurity A and the multiplet at 2.4 ppm due to
impurity B in the spectra obtained with the test solution
and reference solution (c) to obtain the areas of the peaks
used in the comparison of impurity contents;
— impurities A} B: for each impurity, not more than
0.5 times the area of the corresponding peak in the
spectrum obtained with reference solution (c) C14H18CaN3Na3OI0>xH2O 497.4 (anhydrous substance)
(1 per cent).
Ph Eur__________________________________________ ______________ _______
Phosphates (2.4.11}
Trisodium [1,1',1 ",1 "'-[[(carboxylatomethyOiminoJbis
Dissolve 0.100 g in 10 mL of water R and dilute to
(ethylenenitrilo)] tetraacetate] calciate(3-).
100.0 mL wi± the same solvent. Dilute 1.0 mL of this
solution to 100.0 mL with water R. It is a starting material for the preparation of technetium
(99mTc) pentetate injection.
Loss on drying (2.2.32}
Maximum 0.5 per cent, determined on 0.500 g by drying in Content
an oven at 105 °C. 98.0 per cent to 102.0 per cent (anhydrous substance).
Bacterial endotoxins (2.6.14) CHARACTERS

Less than 2.0 lU/mg. Appearance
White or almost white, hygroscopic powder or crystals.
Solubility Limit:
Freely soluble in water, practically insoluble in ethanol — impurity A: not more than the area of the corresponding
(96 per cent). peak in the chromatogram obtained with reference
IDENTIFICATION solution (b) (0.1 per cent).
A. Infrared absorption spectrophotometry (2.2.24). Impurity B
Comparison pentetate sodium calcium CRS. Maximum 1.0 per cent.
B. Ignite. The residue gives reaction (b) of calcium {2.3.1). Dissolve 5.0 g of the substance to be examined in 250 mL of
water R. Add 10 mL of ammonium chloride buffer solution
c. The substance to be examined gives reaction (a) of
pH 10.0 R and 50 mg of mordant black 11 triturate R.
sodium {2.3.1).
Not more than 1.3 mL of 0.1 M magnesium chloride is
TESTS required to change the colour of the indicator to violet.
Solution ร Chlorides
Dissolve 5.0 g in carbon dioxide-free water R and dilute to Maximum 0.1 per cent
Dissolve 0.7 g in water R and dilute to 20 mL with the same
Appearance of solution solvent. Add 30 mL of dilute nitric acid R, allow to stand for
Solution ร is clear {2.2.1) and colourless (2.2.2, Method II). 30 min and filter. Dilute 10 mL of the filtrate to 50 mL with
pH {2.2.3) water R. Use this solution as the test solution. Prepare the
8.0 to 9.5 for solution ร. reference solution using 0.40 mL of 0.01 M hydrochloric acid,
Impurity A add 6 mL of dilute nitric acid R and dilute to 50 mL with
water R. Filter both solutions if necessary. Add 1 mL of silver
Liquid chromatography (2.2.29). Cany out the test protected
from light. nitrate solution R2 to the test solution and the reference
solution. Mix and allow to stand for 5 min protected from
Solvent mixture Dissolve 10 g of ferric sidfate pentahydrate R in light. Any opalescence in the test solution is not more intense
20 mL of 0.5 M sulfuric acid and add 780 mL of water R. than that in the reference solution.
Adjust to pH 2.0 with 1 M sodium hydroxide and dilute to
1000 mL with water R. Iron {2.4.9)
Maximum 20 ppm.
Test solution Dissolve 0.100 g of the substance to be
Dilute 2.5 mL of solution s to 10 mL with water R.
examined in the solvent mixture and dilute to 25.0 mL with
the solvent mixture. Add 0.25 g of calcium chloride R to the test solution and the
standard before the addition of the thioglycoUic acid R.
Reference solution (a) Dissolve 0.100 g of sodium calcium
edetate R in the solvent mixture and dilute to 25.0 mL with Heavy metals {2.4. ร)
the solvent mixture. Maximum 20 ppm.
Reference solution (b) Dissolve 40.0 mg of nitrilotriacetic acid R 1.0 g complies with test F. For the digestion replace sulfuric
(impurity A) in the solvent mixture and dilute to 100.0 mL acid R by nitric acid R. Prepare the reference solution using
with the solvent mixture. To 10.0 mL of the solution add 2 mL of lead standard solution (10 ppm Pb) R.
1 mL of reference solution (a) and dilute to 100.0 mL with Water (2.5.12)
the solvent mixture. Dilute 1.0 mL of this solution to Maximum 15.0 per cent, determined on 0.100 g.
10.0 mL with the solvent mixture. Bacterial endotoxins {2.6.14)
Column’. Less ±an 0.1 IU/mg, if intended for use in the manufacture
— size', z = 0.10 m, 0 = 4.6 mm; of parenteral preparations without a further appropriate
— stationary phase’, spherical graphitised carbon for procedure for the removal of bacterial endotoxins.
chromatography Rl {5 Jim) with a specific surface area of
120 m2/g and a pore size of 25 nm.
ASSAY
Dissolve 0.100 g in water R and dilute to 50.0 mL with the
Mobile phase Dissolve 50 mg of ferric sulfate pentahydrate R in same solvent To 25.0 mL of this solution add 80 mL of
50 mL of 0.5 M sulfuric acid and add 750 mL of water R; water R and adjust to pH 2.3 with dilute nitric acid R. Titrate
adjust to pH 1.5 with 0.5 M sulfuric acid or 1 M sodium with 0.01 M bismuth nitrate using 0.1 mL of a 1 g/L solution
hydroxide, add 20 mL of ethylene glycol R and dilute to of xylenol orange R as indicator. The colour of the solution
1000 mL with water R. changes from yellow to red.
Flow rate 1 mL/min. 1 mL of 0.01 M bismuth nitrate is equivalent to 4.974 mg of
Detection Spectrophotometer at 273 nm. Ci4Hi8CaN3Na3Oio.
Injection 20 pL of the test solution and reference solution (b); STORAGE
filter the solutions and inject immediately.
Run time 4 times the retention time of the iron complex of
impurity A. LABELLING
The label recommends testing the substance in a production
Retention time Iron complex of impurity A = about 5 min;
test before its use for the manufacture of radiopharmaceutical
iron complex of edetic acid = about 10 min; the iron
complex of pentetic acid elutes with the void volume. conditions, the substance yields the radiopharmaceutical
System suitability’, reference solution (b): preparation in the desired quantity and of the quality
— resolution’, minimum 7 between the peaks due to the iron specified.
complex of impurity A and the iron complex of edetic
acid; IMPURITIES
— signal-to-noise ratio', minimum 50 for the peak due to the Specified impurities A, B
iron complex of impurity A.
co2h
— stationary phase: end-capped polar-embedded octadecylsilyl
HO.C^N^CC^H amorphous organosilica polymer R (5 pm).
Mobile phase acetic acid R, methanol R> water R
A. nitrilotriacetic add, (1:50:50 VIVIV).
Flow rate 1 mUmin.
HOjC co2h
ho2c N N CO2H Injection 20 pL.
Run time 7 times the retention time of 2-iodohippuric acid.
co2h Identification of impurities Use the chromatogram obtained
with reference solution (b) to identify the peak due to
B. [[(carboxymethyl)imino]bis(ethylenenitrilo)]tetraacetic acid impurity A.
(pentetic acid). Relative retention With reference to 2-iodohippuric acid
------------------------------------------------------------------------------- --------------------- Ph Eur (retention time = about 4.5 min): benzoic acid = about 1.6;
impurity A ะ= about 2.1.
2-iodohippuric acid and benzoic acid.
Sodium lodohippurate Dihydrate ****** Limits:
for Radiopharmaceutical ***** — impurity A: not more than 5 times the area of the
principal peak in the chromatogram obtained with
Preparations reference solution (a) (0.5 per cent);
(Ph Eur monograph 2352) — unspecified impurities: for each impurity, not more than the
area of the principal peak in the chromatogram obtained
o with reference solution (a) (0.10 per cent);
— total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent);
— disregard limit: 0.5 times the area of the principal peak in
C9H7INNaO3,2H2O 363.1 5990-94-3 the chromatogram obtained with reference solution (a)
Ph Fir_______________________________________________________ (0.05 per cent).
Water (2.5.72)
DEFINITION
8.0 per cent to 12.0 per cent, determined on 0.100 g.
Sodium (2-iodobenzamido) acetate dihydrate.
Content
Less than 2 lU/mg, if intended for use in the manufacture of
99.0 per cent to 101.0 per cent (anhydrous substance).
parenteral preparations without a further appropriate
CHARACTERS procedure for the removal of bacterial endotoxins.
Appearance
ASSAY
White or almost white, crystalline powder.
Dissolve 0.250 g in 20 mL of glacial acetic acid R. Titrate
Solubility with 0.1 M perchloric acid, determining the end-point
Soluble in water and in ethanol (96 per cent), practically potentiometrically (2.2.20).
insoluble in methylene chloride. 1 mL of 0.1 M perchloric acid is equivalent to 32.71 mg of
IDENTIFICATION C9H7INNaO3
A. Infrared absorption spectrophotometry (2.2.24). STORAGE
Comparison sodium iodohippurate CRS. Protected from light.
B. It gives reaction (b) of sodium (2.3.1). LABELLING
TESTS The label recommends testing the substance in a production
Related substances test before its use for the manufacture of radiopharmaceutical
Liquid chromatography (2.2.29), preparations. This ensures that, under specified production
Test solution Dissolve 0.100 g of the substance to be conditions, the substance yields the radiopharmaceutical
examined in the mobile phase and dilute to 10.0 mL with preparation in the desired quantity and of the quality
the mobile phase. specified.
Reference solution (a) Dilute 1.0 mL of the test solution to IMPURITIES
100.0 mL with the mobile phase. Dilute 1.0 mL of this Specified impurities A
solution to 10.0 mL with the mobile phase.
Reference solution (b) Dissolve 10 mg of 2-iodobenzoic acid R
(impurity A) in methanol R and dilute to 100.0 mL with the
same solvent.
Reference solution (c) Dissolve 10 mg of benzoic acid R in the
mobile phase, add 1 mL of the test solution and dilute to A. 2-iodobenzoic acid.
100 mL with the mobile phase.
Column-.
— size-. I = 0.25 m, 0 = 4.6 mm;
Test solution (a) Dissolve 0.200 g of the substance to be

Tetra-0-Acetyl-Mannose Tritiate examined in acetonitrile R and dilute to 2.0 mL with the same
for Radiopharmaceutical solvent.
Preparations Test solution (b) Dissolve 10.0 mg of the substance to be
examined in acetonitrile R and dilute to 5.0 mL with the same
solvent.
Reference solution (a) Dissolve 10.0 mg of tetra-O-acetyl-
mannose triflate CRS in acetonitrile R and dilute to 5.0 mL
Reference solution (b) Dilute 1.0 mL of test solution (a) to
10.0 mL with acetonitrile R. Dilute 1.0 mL of this solution to
100.0 mL with acetonitrile R.
Reference solution (c) Dissolve 10 mg of 1,3,4,6-tetra-O-acetyl-
p-D-mannopyranose R (impurity A) in 5 mL of acetonitrile R.
Mix 1 mL of this solution and 1 mL of reference
CI5H19F3O12S 480.4 92051-23-5 solution (a).
Ph Eur.__________ Column:
— size: I = 0.25 m, 0 = 4.0 mm;
DEFINITION — stationary phase: octadecylsilyl silica gel for chromatography R
1,3,4,6-Tetra-0-acetyl-2-0-trifluoromethanesulfonyl-[3-D- (5 เนท);
mannopyranose. — temperature: 25 °C.
Content Mobile phase:
97.0 per cent to 102.0 per cent (dried substance). — mobile phase A: water R->
— mobile phase B: acetonitrile Rlj
CHARACTERS
Appearance
White or almost white, crystalline, hygroscopic powder. Time Mobile phase A Mobile phase B
Solubility
0 - 1 80 20
Practically insoluble in water, very soluble in acetonitrile,
freely soluble in methylene chloride, slightly soluble in 1 - 20 80 -> 55 20 -> 45
ethanol (96 per cent). 20 - 35
IDENTIFICATION 55 -> 0 45 -> 100
Infrared absorption spectrophotometry (2.2.24).
45 - 50 0 100
Comparison tetra-O-acetyl-mannose inflate CRS.
TESTS
Specific optical rotation (2.2.7) Flow rate 1 mL/min.
-12.0 to —16.0 (dried substance), measured at 20 °C. Detection Spectrophotometer at 220 nm.
Dissolve 0.250 g in acetonitrile R and dilute to 10.0 mL with Injection 20 |1L of test solution (a) and reference solutions (b)
the same solvent. and (c).
Impurity B Relative retention With reference to tetra-O-acetyl-mannose
l9F Nuclear magnetic resonance spectrometry (2.2.33). triflate (retention time = about 29 min):
Prepare the solutions immediately before use. impurity A = about 0.2.
Test solution Dissolve 20.0 mg of the substance to be System suitability: reference solution (c) ะ
examined in deuterated acetonitrile R and dilute to 1.0 mL — resolution: minimum 5.0 between the peaks due to
with the same solvent. impurity A and tetra-O-acetyl-mannose triflate.
Reference solution (a) Dissolve 20.0 mg of tetra-O-acetyl- Limits:.
— impurity A: not more than twice the area of the principal
mannose triflate CRS in deuterated acetonitrile R and dilute to
peak in the chromatogram obtained with reference
solution (b) (0.2 per cent);
Reference solution (b) Dissolve 4.0 mg of lithium — any other impurity, for each impurity, not more than the
trifluoromethanesulfonate R (lithium salt of impurity B) in area of the principal peak in the chromatogram obtained
deuterated acetonitrile R and dilute to 1.0 mL with the same with reference solution (b) (0.10 per cent);
solvent. — total: not more than 5 times the area of the principal peak
Reference solution (c) Mix 1.0 mL of reference solution (a) in the chromatogram obtained with reference solution (b)
and 10 pL of reference solution (b). (0.5 per cent);
Limit The peak area identified in the spectrum obtained with — disregard limit: 0.5 times the area of the principal peak in
the test solution at about -78 ppm is smaller than the peak the chromatogram obtained with reference solution (b)
area identified in the spectrum obtained with reference (0.05 per cent).
solution (c) at the same chemical shift (0.2 per cent). Loss on drying
Related substances Maximum 0.6 per cent, determined on 25 mg by
Liquid chromatography (2.2.29). Prepare the solutions thermogravimetry (2.2.34). Heat to 80 °C at a rate of
immediately before use. 2.5 °c/min.
ASSAY CHARACTERS
Liquid chromatography (2.2.29) as described in the test for Appearance
related substances with the following modification. Clear, colourless to yellowish solution.
Injection Test solution (b) and reference solution (a). Half-life and nature of radiation of iodine-125
Calculate the percentage content of C15H19F3012ร from the See general chapter 5.7. Table of physical characteristics of
declared content of tetra-O-acetyl-mannose inflate CRS. radionuclides.
STORAGE IDENTIFICATION
In an airtight container, protected from light, at a A. Gamma-ray and X-ray spectrometry.
temperature of 2 °C to 8 °C. Comparison Standardised iodine-125 solution, or by using a
LABELLING calibrated instrument. Standardised iodine-125 solutions
The label recommends testing the substance in a production and/or standardisation senices are available from the
run before its use for the manufacture of radiopharmaceutical competent authority.
preparations. This ensures that, under specified production Results The spectrum obtained with the preparation to be
conditions, the substance yields the radiopharmaceutical examined does not differ significantly from that obtained
preparation in the desired quantity and of the quality with a standardised iodine-125 solution, apart from any
specified. differences attributable to the presence of iodine-126.
The most prominent photon has an energy of 0.027 MeV,
IMPURITIES
corresponding to the characteristic X-ray of tellurium,
Specified impurities A, B
gamma photons of an energy of 0.035 MeV arc also present.
Iodine-126 has a half-life of 13.11 days and its most
prominent gamma photons have energies of 0.388 MeV and
0.666 MeV
B. Examine by a suitable immunoelectrophoresis technique
(2.7.1). Using antiserum to normal human serum, compare
normal human serum and the preparation to be examined,
both diluted if necessary. The main component of the
preparation to be examined corresponds to the main
component of the normal human serum. The diluted
solution may show the presence of small quantities of other
A. 1,3,4,6-tetra-0-acetyl-P-D-mannopyranose,
plasma proteins.
HO CF3 TESTS
pH (2 2.3)
o o
5.0 to 9.0.
B. trifluoromethanesulfonic acid.
Albumin
Reference solution Dilute human albumin solution R with a
____________________________________________ ___________ _____ Ph Eur 9 g/L solution of sodium chloride R to a concentration of 5 mg
of albumin per millilitre.
To 1.0 mL of the preparation to be examined and to 1.0 mL
of the reference solution add 4.0 mL of biuret reagent R and
Iodinated (125l) Albumin Injection ***** mix. After exactly 30 min, measure the absorbance (2.2.25)
of each solution at 540 nm, using as the compensation liquid
(Human Albumin Injection Iodinated (1251)3 * ** a 9 g/L solution of sodium chloride R treated in the same
Ph Eur mottograph 1922) manner. From the absorbances measured, calculate the
Ph Eur_____________________________________________________________ _ content of albumin in the injection to be examined in
milligrams per millilitre.
DEFINITION
Sterile, endotoxin-free solution of human albumin labelled Sterility
with iodine-125. It may contain a suitable buffer and an It complies with the test for sterility prescribed in the
antimicrobial preservative. The human albumin used monograph on Radiopharmaceutical preparations (0125).
complies with the requirements of the monograph on Human Bacterial endotoxins (2.6.14)
albumin solution (0255). Less than 175/l7IU/mL, 1Z being the maximum
Content recommended dose in millilitres.
90 per cent to 110 per cent of the declared iodine-125 RADIONUCLIDIC PURITY
radioactivity at the date stated on the label. Iodine-125
Purity: Minimum 99.0 per cent of the total radioactivity.
— minimum of 99.0 per cent of the total radioactivity Gamma-ray and X-ray spectroscopy.
corresponds to iodine-125, Comparison Standardised solution of iodine-125.
— minimum of 80 per cent of the total radioactivity is
Determine the relative amounts of iodine-125 and iodine-126
associated with the albumin fractions II to V,
present.
— maximum of 5 per cent of the total radioactivity
corresponds to unbound iodide. RADIOCHEMICAL PURITY
Content of albumin 95 per cent to 105 per cent of the Iodine-125 in albumin fractions II to V, iodine-125
declared albumin content stated on the label. corresponding to unbound iodide
Size-exclusion chromatography (2.2.30).
Test solution Mix 0.25 mL of the preparation to be examined

with 0.25 mL of the mobile phase. Use immediately after
Alovudine (18F) Injection
mixing. (Ph. Eur. monograph 2460)
Reference solution Human albumin solution R or another
appropriate human albumin standard diluted with the mobile
phase to a suitable albumin concentration.
Column'.
— size'. I = 0.6 m, 0 ะ= 7.5 mm,
— stationary phase', silica gel for size-exclusion
chromatography R}
Mobile phase Dissolve 11.24 g of potassium dihydrogen
phosphate R3 42.0 g of disodium hydrogen phosphate R, 11.70 g
Cl0H13,8FN2O4 243.2
of sodium chloride R in 2000 mL of water R.
Ph Elf_______________________________________________________________
Detection Spectrophotometer at 280 nm and radioactivity DEFINITION
detector set for iodine-125 connected in series. Sterile solution containing l-[(2R,4S,5R)-4-[l8F]fluoro-5-
Injection Loop injector. (hydroxymethyl)tetrahydrofuran-2-yl]-5-methylpyrimidine-
2,4( 1 H,?>H)-dione (3 '-deoxy-3'- [18F]fluorothymidine,
Run time 85 min. [18F]fluorodeoxythymidine, [l8F]FLT). It may contain a
Retention times: suitable buffer.
Content
Peak Fraction Description of the compound Retention — fluorine-18: 90 per cent to 110 per cent of the declared
No. time fluorine-18 radioactivity at the date and time stated on
(min) the label;
1 18 - 20
— alovudine: maximum 0.1 mg per maximum recommended
I High molecular mass compound
dose in millilitres.
2 II Poly III albumin 23 - 24
CHARACTERS
3 III Poly II albumin 25 - 26 Appearance
Clear, colourless or slightly yellow solution.
4 IV Poly I albumin 28
Half-life and nature of radiation of fluorine-18
5 V Human serum albumin 29 - 31 See general chapter 5.7. Table of physical characteristics of
radionuclides.
6 VI Iodide 43 - 45
IDENTIFICATION
A. Gamma-ray spectrometry.
The main peak in the chromatogram obtained with the Result The principal gamma photons have an energy of
reference solution corresponds to fraction V. 0.511 MeV and, depending on the measurement geometry, a
Limits: sum peak of 1.022 MeV may be observed.
— radioactivity in fractions II to V: minimum 80 per cent of B. Determine the approximate half-life by no fewer than
the total radioactivity applied to the column, 3 measurements of the activity of a sample in the same
— iodine-125 in fraction VI: maximum 5 per cent of the total geometrical conditions within a suitable period of time (for
radioactivity. example, 30 min).
RADIOACTIVITY Result 105 min to 115 min.
Measure the radioactivity using suitable equipment by c. Examine the chromatograms obtained in the test for
comparison with a standardised iodine-125 solution or by [18F]alovudine under radiochemical purity (see Tests).
measurement with a calibrated instrument.
Result The principal peak in the radiochromatogram obtained
LABELLING with the test solution is similar in retention time to the
The label states: principal peak in the chromatogram obtained with reference
— the amount of albumin, solution (a).
— the maximum volume to be injected. TESTS
_ Ph Elf
pH
4.5 to 8.5, using a pH indicator strip R.
Impurity A
Spot test.
Test solution To 100 pL of the preparation to be examined
add 400 pL of water R and mix.
Reference solution (a) water R.
Reference solution (b) Dissolve 11.0 mg of aminopolyether R
(impurity A) in water R and dilute to 25.0 mL with the same
solvent. Dilute 1.0 mL of the solution to V with water R> V
being the maximum recommended dose in millilitres.
Plate TLC silica gel plate for aminopolyether test R.
Application 2.5 pL; as an additional spot, apply 2.5 |1L of the Reference solution (c) Mix 1 mL of reference solution (a) and
test solution and ±en 2.5 pL of reference solution (b) at the 1 mL of reference solution (b).
same place. Blank solution Prepare a solution containing each excipient at
Detection Visually compare the spots 1 min after application. the concentration used in the preparation.
System suitability: Column:
— the spot due to the application of both the test solution — size: I = 0.25 m, 0 = 4.6 mm;
and reference solution (b) is similar in appearance to the — stationary phase: end-capped polar-embedded octadecylsilyl
spot due to reference solution (b), which is characterised amorphous organosilica polymer R (5 pm).
by a number of concentric circles; the darker innermost Mobile phase:
circle (of intensity proportional to the concentration of — mobile phase A ะ carbon dioxide-free water R, protected from
impurity A) may be surrounded by a bluish-black ring, the atmosphere during chromatography;
outside of which is a lighter circle surrounded by a — mobile phase B: acetonitrile R’f
peripheral dark edge;
— the spot due to reference solution (a) has a more diffuse Time Mobile phase A Mobile phase B
inner circle, which is brownish-pink and without a distinct (per cent V/V) (per cent V/V) _
margin between it and the surrounding lighter zone; 0 - 10 90 10
— the spot due to reference solution (b) is clearly different
10 - 20 90 -> 5 10-» 95
from the spot due to reference solution (a).
20 - 30 5 95
Limit’.
— the central portion of the spot due to the test solution is
not more intense than that of the spot due to reference Flow rate 1 mL/min.
solution (b) (2.2 mg/L).
Detection Spectrophotometer at 270 nm and radioactivity
Impurity B detector connected in series.
Injection 20 pL.
Test solution The preparation to be examined. Relative retention With reference to alovudine (retention
Reference solution (a) Dissolve 0.170 g of tetrabutylammonium time = about 8 min): impurity c = about 0.6.
hydroxide R in water R and dilute to 20.0 mL with the same System suitability Reference solution (c) using the
solvent. Dilute 1.0 mL of the solution to V with water R3 V spectrophotometer:
being the maximum recommended dose in millilitres. — resolution: minimum 5.0 between the peaks due to
Reference solution (b) Dissolve 80.0 mg of tetrabutylammonium impurity c and alovudine.
hydroxide R in water R and dilute to 10.0 mL with the same Limits In the chromatogram obtained with the
solvent. Dilute 1.0 mL of the solution to 25.0 mL with spectrophotometer:
water R. — alovudine: not more than the area of the corresponding
Column: peak in the chromatogram obtained with reference
size: I = 0.1 m, 0 = 4.6 mm; solution (a) (0.1 mg/17);
— stationary phase: octadecylsilyl silica gel for chromatography R — impurity C: not more than the area of the corresponding
(3 pm). peak in the chromatogram obtained with reference
Mobile phase 0.95 g/L solution of toluenesulfonic acid R, solution (b) (0.1 mg/L);
acetonitrile R (25:75 V/V). — any other impurity, for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 mg/L);
Detection spectrophotometer at 254 nm. — total: not more than 5 times the area of the principal peak
Injection 20 pL. in the chromatogram obtained with reference solution (a)
Run time Twice the retention time of impurity B. (0.5 mg/L);
Retention time Impurity B = about 3.3 min. — disregard limit: 0.3 times the area of the principal peak in
the chromatogram obtained with reference solution (a)
(0.03 mg/L).
signal-to-noise ratio: minimum 10 for the principal peak;
Ethanol (2.4.24)
symmetry factor, maximum 1.8 for the principal peak.
Limit.
or another suitable, validated method): maximum
10 per cent VIV and maximum 2.5 g per administration,
— impurity B: not more than the area of the corresponding taking the density (2.2.5) to be 0.790 g/mL.
peak in the chromatogram obtained with reference
solution (a) (2.6 mg/ V). Residual solvents
Limited according to the principles defined in general
Alovudine and related substances chapter 5.4. The preparation may be released for use before
Liquid chromatography (2.2.29). completion of the test.
Test solution The preparation to be examined.
Sterility
Reference solution (a) Dissolve 5.0 mg of alovudine R in It complies with the test for sterility prescribed in the
water R and dilute to 50.0 mL with the same solvent. Dilute monograph Radiopharmaceutical preparations (0125).
1.0 mL of the solution to V with waler R> V being the The preparation may be released for use before completion
maximum recommended dose in millilitres. of the test.
Reference solution (b) Dissolve 5.0 mg of stavudine R Bacterial endotoxins (2.6.14)
(impurity C) in water R and dilute to 50.0 mL with the same Less than 175/yiU/mL, V being the maximum
solvent. Dilute 1.0 mL of the solution to V with water R, V recommended dose in millilitres. The preparation may be
being the maximum recommended dose in millilitres. released for use before completion of the test.
RADIONUCLIDIC PURITY
The preparation may be released for use before completion
of test B.
Fluorine-18
Minimum 99.9 per cent of the total radioactivity.
Limit Peaks in the gamma spectrum corresponding to A. 4,7,13,16,21,24-hexaoxa-l,10-
photons with an energy different from 0.511 MeV or diazabicyclo [8.8.8] hexacosane (aminopolyether),
1.022 MeV represent not more than 0.1 per cent of the total
radioactivity. h3c CH;
B. Gamma-ray spectrometry.
Determine the amount of fluorine-18 and radionuclidic H3C ร๙^
impurities with a half-life longer than 2 h. For the detection
and quantification of impurities, retain the preparation to be B. N,N,N-tributylbutan-l-aminium (tetrabutylammonhim),
examined for at least 24 h to allow the fluorine-18 to decay
to a level that permits the detection of impurities.
Result The total radioactivity due to radionuclidic impurities
IS not more than 0.1 per cent.
RADIOCHEMICAL PURITY
[18F] Alovudine
alovudine and related substances. If necessary, dilute the test
solution with water R to obtain a radioactivity concentration c. l-[(27?,5S)-5-(hydroxymethyl)-2,5-dihydrofuran-2-yl]-5-
suitable for the radioactivity detector. methylpyrimidine-2,4 (177,3/7) -dione (stavudine),
Examine the chromatogram recorded using the. radioactivity D. [18F]fluoride,
detector and locate the peak due to [18F]alovudine by
comparison with the chromatogram obtained with reference
solution (a) using the spectrophotometer.
Limit'.
— ['8F]alovudine: minimum 95 per cent of the total
radioactivity due to fluorine-18.
Impurity D
E. tert-butyl 3-[(2R,4R,5R)-5-
Mobile phase water R, acetonitrile R (5:95 VIV). [ [bis(4-methoxyphenyl)phenylmethoxy] methyl] -4-
Application About 5 pL. [[(4-nitrophenyl) sulfonyl] oxy] tetrahydro furan-2-yl]-5-rnethyl-
Development Over 2/3 of the plate. 2,6-dioxo-3,6-dihydropyrimidine-1 (2/7)-carboxylate,
Detection Suitable detector to determine the distribution of
radioactivity.
Retardation factors Impurity D = about 0;
[,8F]alovudine = about 0.7.
Limit:
— impurity D: maximum 5 per cent of the total radioactivity
due to fluorine-18.
RADIOACTIVITY
Determine the radioactivity using a calibrated instrument. F. (2R,3R,5R)-3-
LABELLING [[bis(4-methoxyphenyl)phenylmethoxy]methyl]-8-methyl-2,3-
The label states the percentage content of ethanol in the dihydro-9/7-2,5-methanopyrimido [2,1 -6] [ 1,5,3] dioxazepin-9-
preparation. one.
_ -_____________________________________________________________ Ph Ea
IMPURITIES
Specified impurities A, B, c, D
Other detectable impuritites (the following substances would, if
present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities. It is
therefore not necessary to identify these impurities for
demonstration of compliance).- E3 F.
FV-698 Radiopharmaceutical Preparations 2016
impurities, which should, where possible, be identified and

Ammonia (13N) Injection ****** quantified.
(Ph. Eur. monograph 1492) *** RADIOCHEMICAL PURITY
Ph Fir____________________________________________________________ [13N]Ammonia
DEFINITION Liquid chromatography {2.2.29}.
Sterile solution of [13N]ammonia for diagnostic use. The preparation may be released for use before completion
Nitrogen-13 90 per cent to 110 per cent of the declared of the test.
nitrogen-13 radioactivity at the date and time stated on the Test solution The preparation to be examined.
label. Reference solution Dilute 1.0 mL of dilute ammonia R2 to
CHARACTERS 10.0 mL with water R.
Appearance Column’.
Clear, colourless solution. — size: I -= 0.04 m, 0 = 4.0 mm;
— stationary phase: cation-exchange resin R (10 pm);
Half-life and nature of radiation of nitrogen-13 — temperature: constant at 20-30 °C.
See general chapter 5.7. Table of physical characteristics of
radionuclides. Mobile phase 0.002 M nitric acid.
Flow rate 2 mL/min.
IDENTIFICATION
Detection Suitable radioactivity detector and conductivity
detector.
Results The only gamma photons have an energy of
รุ)’stem suitability The chromatogram obtained with the test
0.511 MeV and, depending on the measurement geometry, a
solution and the radioactivity detector shows a principal peak
sum peak of 1.022 MeV may be observed.
with approximately the same retention time as the peak in
B. Test A for radionuclidic purity (see Tests). the chromatogram obtained with the reference solution and
c. Examine the chromatograms obtained in the test for the conductivity detector.
radiochemical purity (see Tests). Limit:
Result The principal peak in the radiochromatogram obtained — f13N]ammonia: minimum 99 per cent of the total
with the test solution has approximately the same retention radioactivity due to nitrogen-13.
time as the principal peak in the radiochromatogram
RADIOACTIVITY
Determine the radioactivity using a calibrated instrument.
TESTS
IMPURITIES
pH {2.2.3}
5.5 to 8.5.
A. [13N]O2 ,
Sterility
B. [13N]O3-,
It complies with the test for sterility prescribed in the c. [18F"]}
monograph Radiopharmaceutical preparations (0125). D. H2[15O].
The preparation may be released for use before completion _______________________ Pn Ej
of the test.
Bacterial endotoxins {2.6.14}
Less than 175/ V IU/mL, V being the maximum
recommended dose in millilitres. The preparation may be Carbon Monoxide (15O) *** ***
released for use before completion of the test.
Aluminium
Maximum 2 ppm. The preparation may be released for use Ph Eur---------------------------------------------------------------------- —--------------------------------
before completion of the test. DEFINITION

Test solution In a test-tube about 12 mm in internal diameter, Mixture of carbon [15O]monoxide in the gaseous phase and a
mix 1 mL of acetate buffer solution pH 4.6 R and 2 mL of a 1 suitable vehicle such as Medicinal air (1238)5 for diagnostic
in 20 dilution of the preparation to be examined in water R. use.
Add 0.05 mL of a 10 g/L solution of chromazurol ร R. Purity:
Reference solution Prepare at the same time and in the same — minimum 99 per cent of the total radioactivity
manner as the test solution using 2 mL of a 1 in 20 dilution corresponds to oxygen-15,
of aluminium standard solution (2 ppm Al) R. — minimum 97 per cent of the total radioactivity
After 3 min, the colour of the test solution is not more corresponds to oxygen-15 in the form of carbon
intense than that of the reference solution. monoxide (CO).
RADIONUCLIDIC PURITY PRODUCTION
The preparation may be released for use before completion RADIONUCLIDE PRODUCTION
of tests A and B. Oxygen-15 is a radioactive isotope of oxygen which may be
A. Half-life. The half-life is between 9 min and 11 min. produced by various nuclear reactions such as proton
irradiation of nitrogen-15 or deuteron irradiation of nitrogen-
B. Gamma emitting impurities', maximum 1.0 per cent of the
14.
total radioactivity.
Gamma-ray spectrometry Retain a sample of the preparation to RADIOCHEMICAL SYNTHESIS
In order to recover oxygen-15 as molecular oxygen from the
be examined for 2 h. Examine the gamma-ray spectrum of
nitrogen target gas, carrier oxygen is added at concentrations
the decayed material for the presence of radionuclidic
generally ranging from 0.2 per cent v/v to 1.0 per cent VIV.
After irradiation, the target gas is usually reacted with Test sample Carbon [15O] monoxide as described under
activated charcoal at a temperature of about 950 °C. radiochemical synthesis.
The activated charcoal is preconditioned before use by Reference gas (a) Nitrogen gas mixture R.
flushing an inert gas at the production flow rate at a
Reference gas (b) Nitrogen Ry containing 2.0 per cent VIV of
temperature of about 950 °C for not less than 1 h.
carbon monoxide Rl.
The carbon [15O] monoxide obtained is purified by passage
through a carbon dioxide scavenger, such as soda lime, Column'.
before mixing with the vehicle. — size". 1= 1.8 m, 01 = 6.3 mm and 02 = 3.2 mm,
— stationary phase: GC concentrical column R,
CHARACTERS Carrier gas helium for chromatography R.
Appearance
Colourless gas. Flow rate 65 mL/min.
Temperature:
Half-life and nature of radiation of oxygen-15 — column: 40 °C,
See general chapter 5.7. Table of physical characteristics of — injection port. 40 °C,
radionuclides. — thermal conductivity detector. 70 °C.
IDENTIFICATION Detection Thermal conductivity detector and radioactivity
A. Gamma spectrometry. detector connected in series.
Results The only gamma photons have an energy of Injection Loop injector.
0.511 MeV and, depending on the measurement geometry, a Run time 10 min.
Retention times Oxygen, nitrogen and carbon monoxide
B. Radionuclidic purity (see Tests). eluting from the inner column = about 0.4 min; carbon
c. Examine the chromatograms obtained in the test for dioxide eluting from ±e inner column = about 0.8 min;
radiochemical purity. oxygen eluting from the outer column = about 2.1 min;
Results The principal peaks in the chromatogram obtained nitrogen eluting from the outer column = about 3.1 min;
with the test gas using the radioactivity detector are similar in carbon monoxide eluting from the outer column = about
retention times to the principal peaks corresponding to 6.2 min.
carbon monoxide in the chromatogram obtained with System suitability: reference gas (a):
reference gas (a) using the thermal conductivity detector. — 5 clearly separated principal peaks are observed in the
TESTS chromatogram obtained using the thermal conductivity
detector,
The following tests are performed on carbon j15 O]monoxide as
— resolution: minimum of 1.5 between the peaks due to
described under radiochemical synthesis before mixing with the
carbon dioxide eluting from the inner column and oxygen
vehicle.
eluting from the outer column, in the chromatogram
Carbon monoxide obtained using the thermal conductivity detector.
Gas chromatography (2.2.25) as described in the test for Limits Examine the chromatogram obtained with the
radiochemical purity.
radioactivity detector and calculate the percentage content of
The concentration of carbon monoxide in the test sample is oxygen-15 substances from the peak areas.
determined before administration and is used to calculate the — carbon [ไ 5O]monoxide". minimum 97 per cent of the total
amount of carbon monoxide to be administered to the radioactivity.
patient. — disregard the first peak corresponding to components
Injection Test sample, reference gas (b). co-eluting from the inner column.
Examine the chromatogram obtained with ±e thermal RADIOACTIVITY
conductivity detector and calculate the content of carbon The radioactive concentration is determined before
monoxide. administration.
RADIONUCLIDIC PURITY Measure the radioactivity using suitable equipment by
Oxygen-15 comparison with a standardised fluorine-18 solution or by
Minimum 99 per cent of the total radioactivity. measurement in an instrument calibrated with the aid of such
A. Gamma spectrometry. a solution.
Comparison Standardised fluorine-18 solution, or by using an _______________________________________________________________ Ph Eur
instrument calibrated with the aid of such a solution.

Standardised fluorine-18 solutions and/or standardisation
services are available from the competent authority.
Results The spectrum obtained with the solution to be
examined does not differ significantly from ±at obtained
with a standardised fluorine-18 solution.
B. Half-life: 1.9 min to 2.2 min.
of the test.
Carbon [15O]monoxide
procedure.
Chromium (51Cr) Edetate Injection ***** B. Gamma-ray spectrometry.

Determine the relative amount of radionuclidic impurities.
(Ph. Eur. monograph 0266) *** Results The total radioactivity due to radionuclidic impurities
is not more than 0.1 per cent.
[51Cr]Chromium edetate
Descending paper chromatography (2.2.26).
Reference solution Use the reference solution from the test for
Ph Elf___________________________________________ _ _______________ chromium.
DEFINITION Chromate earner solution Dissolve 0.1 g of potassium
Sterile solution containing chromium-51 in the form of a chromate R in 1 mL of concentrated ammonia Rl and dilute to
complex of chromiumfin) with (ethylenedinitrilo)tetraacetic 100 mL with water R.
acid, the latter being present in excess. It may be made Paper paper for chromatography R.
isotonic by the addition of sodium chloride and may contain Mobile phase concentrated ammonia Rl, ethanol (96 per cent) R,
a suitable antimicrobial preservative such as benzyl alcohol. water R (1:2:5 VIVA').
Chromium-51 90 per cent to 110 per cent of the declared Application Apply a band of a 50 g/L solution of lead
chromium-51 radioactivity at the date and time stated on the acetate R to the paper at about 4 cm from the origin and dry
label. in hot air. Apply 10 pL of the chromate carrier solution at
Chromium Maximum 1 mg/mL. the origin, followed by 10 pL of the test solution on the same
spot. On a separate sheet, repeat the above procedure,
CHARACTERS applying 10 pL of the reference solution instead of the test
Appearance solution.
Clear, violet solution.
Development Immediately, over a path of 14 cm.
Half-life and nature of radiation of chromium-51
Drying In air.
radionuclides. Detection Suitable detector to determine the distribution of
radioactivity.
IDENTIFICATION Retardation factors Impurity A = 0; impurity B = 0.2 to 0.4;
A. Radionuclidic purity (see Tests). [51Cr]chromium edetate = 0.8 to 0.9.
B. Examine the chromatograms obtained in the test for System suitability The band of lead acetate turns yellow due
radiochemical purity (see Tests). to reaction with the chromate carrier solution.
Result The principal peak in the radiochromatogram obtained The retardation factor of the radioactive spot due to
with the test solution is similar in retardation factor to the [51Cr] chromium edetate in the radiochromatogram obtained
principal peak in the chromatogram obtained with the with the test solution is similar to that of the violet spot in
reference solution. the chromatogram obtained with the reference solution.
TESTS Limit’.
pH (2.2.3) — ^Cr]chromium edetate’. minimum 97.0 per cent of the
3.5 to 6.5. total radioactivity due to chromium-51.
Chromium RADIOACTIVITY
Maximum 1 mg/mL. Determine the radioactivity using a calibrated instrument.
Ultraviolet and visible absorption spectrophotometry (2.2.25). IMPURITIES
Test solution The preparation to be examined. A. [51Cr] chromium (III) ion,
Reference solution Dissolve 0.96 g of chromic potassium sulfate R B. [51Cr] chromate ion.
and 2.87 g of sodium edetate R in 50 mL of water R, boil for ___________. FhEj
10 min, cool, adjust to pH 3.5-6.5 with dilute sodium
hydroxide solution R and dilute to 100.0 mL with water R.
Measure the absorbance of the test solution and the reference
solution at the absorption maximum at 560 nm.
Result The absorbance of the test solution is not greater than
Cyanocobalamin (57Co) Capsules *** **
that of the reference solution. (Ph. Eur. monograph 0710)
Sterility Ph Eur----------------------------------------------------------------- --------------------------- --------------
It complies with the test for sterility prescribed in the
monograph Radiopharmaceutical preparations (0125). DEFINITION
The preparation may be released for use before completion Capsules containing [57Co]-a-(5,6-dimethylbenzimidazol-l-
of the test. yl)cobamide cyanide; they may contain suitable excipients.
The capsules comply with the requirements for hard capsules
RADIONUCLIDIC PURITY
prescribed in the monograph Capsules (0016), unless
Chromium-51
otherwise justified and authorised.
Cobalt-57
90 per cent to 110 per cent of the declared cobalt-57
Results The only gamma photons have an energy of radioactivity at the date stated on the label.
0.320 MeV.
CHARACTERS RADIOACTIVITY
Appearance Determine the radioactivity using a calibrated instrument.
Hard, gelatin capsules.
STORAGE
Half-life and nature of radiation of cobalt-57 In an airtight container, protected from light, at a
See general chapter 5.7. Table of physical characteristics of temperature of 2 °C to 8 °C.
radionuclides.
IMPURITIES
IDENTIFICATION A. cobalt-56,
B. cobalt-58.
Result The most prominent gamma photon of cobalt-57 has __________ _ __________________________________________________ Ph Eur
an energy of 0.122 MeV.
radiochemical purity (see Tests).
Result The principal peak in the radiochromatogram obtained Cyanocobalamin (57Co) Solution ******
with the test solution is similar in retention time to the (Ph. Eur. monograph 0269) * **
principal peak in the chromatogram obtained with the
reference solution. Ph Elf__________ ___ ._________________________________________________
TESTS DEFINITION
Disintegration Solution of [57Co]-a-(5,6-dimethylbenzimidazol-l-
The capsules comply with the test for disintegration of tablets yl)cobamide cyanide and may contain a stabiliser and an
and capsules (2.9.7), except that 1 capsule is used in the test antimicrobial preservative.
instead of 6. Cobalt-57 90 per cent to 110 per cent of the declared cobalt-
57 radioactivity at the date stated on the label.
Uniformity of content
Determine, by measurement in a suitable counting assembly CHARACTERS
and under identical geometrical conditions, the radioactivity Appearance
of each of not fewer than 10 capsules. Calculate the average Clear, colourless or slightly pink solution.
radioactivity per capsule. The radioactivity of no capsule Half-life and nature of radiation of cobalt-57
differs by more than 10 per cent from the average. See general chapter 5.7. Table of physical characteristics of
The relative standard deviation is less than 3.5 per cent. radionuclides.
RADIONUCLIDIC PURITY IDENTIFICATION
Cobalt-57 A. Gamma-ray spectrometry.
Result The most prominent gamma photon of cobalt-57 has
Gamma-ray spectrometry. an energy of 0.122 MeV.
Determine the relative amounts of cobalt-57, cobalt-56 and B. Examine the chromatograms obtained in the test for
cobalt-58 present. radiochemical purity (see Tests).
RADIOCHEMICAL PURITY Result The principal peak in the radiochromatogram obtained
[57Co]Cyanocobalamin with the test solution is similar in retention time to the
Liquid chromatography (2.2.29). principal peak in the chromatogram obtained with the
Test solution Dissolve the contents of a capsule in 1.0 mL of reference solution.
water R and allow to stand for 10 min. Centrifuge at TESTS
2000 r/min for 10 min. Use the supernatant. pH (2.2.5)
Reference solution Dissolve 10 mg of cyanocobalamin CRS in 4.0 to 6.0.
the mobile phase and dilute to 100 mL wi± the mobile RADIONUCLIDIC PURITY
phase. Dilute 2 mL of this solution to 100 mL with the Cobalt-57
mobile phase. Use within 1 h of preparation. Minimum 99.9 per cent of the total radioactivity.
Column:
Gamma-ray spectrometry.
— size: I = 0.25 m, 0 = 4.0 mm;
— stationary phase: octylsilyl silica gel for chromatography R Determine the relative amounts of cobalt-57, cobalt-56 and
(5 pm). cobalt-58 present.
Mobile phase 26.5 volumes of methanol R and 73.5 volumes of RADIOCHEMICAL PURITY
a 10 g/L solution of disodium hydrogen phosphate R adjusted to [57Co]Cyanocobalamin
pH 3.5 using phosphoric acid R. Use within 2 days of Liquid chromatography (2.2.29).
preparation. Test solution The preparation to be examined.
Flow rate 1.0 mUrnin. Reference solution Dissolve 10 mg of cyanocobalamin CRS in
Detection Radioactivity detector adjusted for cobalt-57 and the mobile phase and dilute to 100 mL with the mobile
spectrophotometer at 361 nm. phase. Dilute 2 mL of this solution to 100 mL with the
mobile phase. Use within 1 h after preparation.
Injection 100 pL.
Column:
Run time 3 times the retention time of cyanocobalamin for
— size: l ะ= 0.25 m, 0 = 4.0 mm;
the test solution; 30 min for the reference solution.
Limit: (5 rim).
— เ57Co]cyanocobalamin: minimum 90 per cent of the total
Mobile phase 26.5 volumes of methanol R and 73.5 volumes of
radioactivity due to cobalt-57.
a 10 g/L solution of disodium hydrogen phosphate R adjusted to
pH 3.วิ using phosphoric acid R (use within 2 days after of each of not less than 10 capsules. Calculate the average
preparation). radioactivity per capsule. The radioactivity of no capsule
Flow rate 1.0 mUmin. differs by more than 10 per cent from the average.
Detection Radioactivity’ detector adjusted for cobalt-57 and The relative standard deviation is less than 3.5 per cent
spectrophotometer at 361 nm. RADIONUCLIDIC PURITY
Injection 100 pL. Cobalt-58
Run time 3 times the retention time of cyanocobalamin for Minimum 98 per cent of the total radioactivity.
the test solution; 30 min for the reference solution. Gamma-ray spectrometry.
Limit: Determine the relative amounts of cobalt-58, cobalt-57 and
— [5/Co]cyanocobalamin: minimum 90 per cent of the cobalt-60 present.
radioactivity due to cobalt-57. Result:
RADIOACTIVITY — cobalt-60: maximum 1 per cent of the total radioactivity’.
Determine the radioactivity using a calibrated instrument. RADIOCHEMICAL PURITY
STORAGE [5sCo]Cyanocobalamin
Protected from light, at a temperature of 2 °C to 8 °C. Liquid chromatography (2.2.29).
Test solution Dissolve the contents of a capsule in 1.0 mL of
IMPURITIES
water R and allow to stand for 10 min. Centrifuge at
A. cobalt-56, 2000 r/min for 10 min. Use the supernatant.
B. cobalt-58. Reference solution Dissolve 10 mg of cyanocobalamin CRS in
__________________________________________________________ __ Ph Eur the mobile phase and dilute to 100 mL with the mobile
phase. Dilute 2 mL of this solution to 100 mL with the
mobile phase. Use within 1 h after preparation.
Column:
Cyanocobalamin (58Co) Capsules ****** — size: I = 0.25 m, 0 = 4.0 mm;
(Ph. Eur. monograph 1505) *** (5 pm).
Ph Eur_____________________________________________________________ Mobile phase 26.5 volumes of methanol R and 73.5 volumes of
a 10 g/L solution of disodium hydrogen phosphate R, adjusted
DEFINITION to pH 3.5 with phosphoric acid R (use within 2 days).
Capsules containing [58Co]-a-(5,6-dimethylbenzimidazol-l-
yl)cobamide cyanide; they may contain suitable excipients.
Detection Radioactivity detector adjusted for cobalt-58 and
The capsules comply with the requirements for hard capsules
spectrophotometer at 361 nm.
in the monograph Capsules (0016), unless otherwise justified
and authorised. Injection 100 pL.
Cobalt-58 Average between 90 per cent and 110 per cent of Run time 3 times the retention time of cyanocobalamin for
the declared cobalt-58 radioactivity at the date stated on the the test solution; 30 min for the reference solution.
label. Limit:
— [58Co]cyanocobalamin: minimum 84 per cent of the total
CHARACTERS
radioactivity due to cobalt-58.
Appearance
Hard gelatin capsules. RADIOACTIVITY
Half-life and nature of radiation of cobalt-58
See general chapter 5.7. Table of physical characteristics of STORAGE
radionuclides. In an airtight container, protected from light, at a
IDENTIFICATION temperature of 2 °C to 8 °C.
A. Gamma-ray spectrometry. IMPURITIES
Results The most prominent gamma photons of cobalt-58 A. cobalt-57,
have energies of 0.511 MeV (annihilation radiation) and B. cobalt-60.
0.811 MeV. ______________________ PnEj
Residt The principal peak in the radiochromatogram obtained
with the test solution is similar in retention time to the
principal peak in the chromatogram obtained with the
Cyanocobalamin (58Co) Solution *** **
reference solution. (Ph. Eur. monograph 0270) *
TESTS Ph Eur---------------------------------------------------------------------- -------- -------------- - ------------
Disintegration
DEFINITION
The capsules comply wi± the test for disintegration of tablets
Solution of [58Co]-a-(5,6-dimethylbenzimidazol-l-
and capsules (2.9.1) except that 1 capsule is used in the test
yl)cobamide cyanide and may contain a stabiliser and an
instead of 6.
antimicrobial preservative.
Uniformity of content Cobalt-58 90 per cent to 110 per cent of the declared cobalt-
Determine by measurement in a suitable counting assembly
58 radioactivity at the date stated on the label.
and under identical geometrical conditions the radioactivity
CHARACTERS
Appearance Fludeoxyglucose (18F) Injection
Clear, colourless or slightly pink solution. (Ph. Eur. monograph 1325)
Half-life and nature of radiation of cobalt-58
radionuclides.
IDENTIFICATION
Results The most prominent gamma photons of cobalt-58
have energies of 0.511 MeV (annihilation radiation) and
0.811 MeV. C6H11'8FO5 181.1
B. Examine the chromatograms obtained in the test for Ph Eur-----------------------------------------------------------------------------------------------------------
DEFINITION
Result The principal peak in the radiochromatogram obtained Sterile solution containing 2-[l8F]fluoro-2-deoxy-D-
with the test solution is similar in retention time to the glucopyranose (2-[18F]fluoro-2-deoxy-D-glucose) prepared by
principal peak in the chromatogram obtained with the nucleophilic substitution. It may also contain 2-[18F]fluoro-2-
reference solution. deoxy-D-mannose.
TESTS Content
pH (2.2.3) — fluorine-18: 90 per cent to 110 per cent of the declared
4.0 to 6.0. fluorine-18 radioactivity at the date and time stated on
RADIONUCLIDIC PURITY the label.
Cobalt-58 — 2-fluoro-2-deoxy-D-glucose: maximum 0.5 mg per
Minimum 98 per cent of the total radioactivity. maximum recommended dose in millilitres.
Gamma-ray spectrometry. CHARACTERS
Determine the relative amounts of cobalt-58, cobalt-57 and Appearance
cobalt-60 present. Clear, colourless or slightly yellow solution.
Reside. Half-life and nature of radiation of fluorine-18
— cobalt-60: maximum 1 per cent of the total radioactivity. See general chapter 5.7. Table of physical characteristics of
radionuclides.
[58Co]Cyanocobalamin IDENTIFICATION
Liquid chromatography (2.2.29). A. Gamma-ray spectrometry.
Test solution The preparation to be examined. Result The principal gamma photons have an energy of
Reference solution Dissolve 10 mg of cyanocobalamin CRS in 0.511 MeV and, depending on the measurement geometry, a
the mobile phase and dilute to 100 mL with the mobile sum peak of 1.022 MeV may be observed.
phase. Dilute 2 mL of this solution to 100 mL with the B. Determine the approximate half-life by no fewer than
mobile phase. Use within 1 h after preparation. 3 measurements of the activity of a sample in the same
Column: geometrical conditions within a suitable period of time (for
— size: I = 0.25 m, 0 ะ= 4.0 mm; example, 30 min).
— stationary phase: octylsilyl silica gel for chromatography R Result 105 min to 115 min.
(5 pm). c. Examine the chromatograms obtained in test A for
Mobile phase 26.5 volumes of methanol R and 73.5 volumes of radiochemical purity (see Tests).
a 10 g/L solution of disodium hydrogen phosphate R adjusted to Result The principal peak in the radiochromatogram obtained
pH 3.5 using phosphoric acid R (use within 2 days). with the test solution is similar in retention time to the
Flow rate 1.0 mUmin. principal peak in the chromatogram obtained with reference
Detection Radioactivity detector adjusted for cobalt-58 and solution (a).
spectrophotometer at 361 nm. TESTS
Injection 100 |1L. Particular tests for chemical impurities may be omitted if the
Run time 3 times the retention time of cyanocobalamin for substances mentioned are not used or cannot be formed in the
the test solution; 30 min for the reference solution. production process.
Limit: pH (2.2.3)
— f8Co]cyanocobalamin: minimum 90 per cent of the 4.5 to 8.5.
radioactivity due to cobalt-58. 2-Fluoro-2-deoxy-D-glucose and impurity A
RADIOACTIVITY Liquid chromatography (2.2.29).
Determine the radioactivity using a calibrated instrument. Test solution The preparation to be examined.
STORAGE Reference solution (a) Dissolve 1.0 mg of 2-fluoro-2-deoxy-D-
glucose R in water R and dilute to 2.0 mL with the same
Protected from light, at a temperature of 2 °C to 8 °C.
solvent. Dilute 1.0 mL of the solution to V with water R) V
IMPURITIES being the maximum recommended dose in millilitres.
A. cobalt-57, Reference solution (b) Dissolve 1.0 mg of 2-chloro-2-deoxy-D-
B. cobalt-60. glucose R (impurity A) in water R and dilute to 2.0 mL with
_________________ _____________________________________________ Ph Eur the same solvent. Dilute 1.0 mL of the solution to V with
FV-704 Radiopharmaceutical Preparations 2016
water R} V being the maximum recommended dose in — the spot due to reference solution (a) has a more diffuse
millilitres. inner circle, which is brownish-pink and without a distinct
Reference solution (c) Dissolve 1.0 mg of 2-fluoro-2-deoxy-D- margin between it and the surrounding lighter zone;
mannose R in water R and dilute to 20.0 mL with the same — the spot due to reference solution (b) is clearly different
solvent. Mix 0.5 mL of this solution with 0.5 mL of from the spot due to reference solution (a).
reference solution (a). Limit:
Column'. — the central portion of the spot due to the test solution is
— size: I = 0.25 m, 0 = 4.0 mm; not more intense than that of the spot due to reference
— stationary phase: strongly basic anion-exchange resin for solution (b) (2.2 mg/P).
chromatography 7? (10 pm); Impurity c
— temperature: 25 °C. Liquid chromatography (2.2.29).
Mobile phase 4 g/L solution of sodium hydroxide R in carbon Test solution The preparation to be examined.
dioxide-free water R} protected from the atmosphere during Reference solution (a) Dissolve 0.170 g of tetrabutylammomum
chromatography. hydroxide R in water R and dilute to 20.0 mL with the same
Flow rate 1 mUmin. solvent. Dilute 1.0 mL of the solution to V with zuater R, V
Detection Detector suitable for carbohydrates in the required being the maximum recommended dose in millilitres.
concentration range, such as a pulsed amperometric detector Reference solution (b) Dissolve 80.0 mg of tetrabutylammomum
and radioactivity detector connected in series. hydroxide R in zuater R and dilute to 10.0 mL with the same
Injection 20 pL. solvent. Dilute 1.0 mL of the solution to 25.0 mL with
Run time Twice the retention time of 2-fluoro-2-deoxy-D- water R.
glucose. Column:
Relative retention With reference to 2-fluoro-2-deoxy-D- — size: I = 0.10 m, 0 = 4.6 mm;
glucose (retention time = about 12 min): 2-fluoro-2-deoxy-D- — stationary phase: octadecylsilyl silica gel for chromatography R
mannose = about 0.9; impurity A = about 1.1. (3 pm).
System suitability Reference solution (c) using the Mobile phase 25 volumes of a 0.95 g/L solution of
carbohydrate detector: toluenesulfonic acid R and 75 volumes of acetonitrile R.
— resolution: minimum 1.5 between the peaks due to Flow rate 0.6 mUmin.
2-fluoro-2-deoxy-D-mannose and 2-fluoro-2-deoxy-D- Detection spectrophotometer at 254 nm.
glucose; Injection 20 |1L.
— signal-to-noise ratio: minimum 10 for the peak due to
Run time Twice the retention time of impurity c.
2-fluoro-2-deoxy-D-glucose.
Retention time Impurity c = about 3.3 min.
Limits In the chromatogram obtained with the carbohydrate
detector: System suitability: reference solution (b):
— 2-fluoro-2-deoxy-D-glucose: not more than the area of the — signal-to-noise ratio: minimum 10 for the principal peak;
corresponding peak in the chromatogram obtained with — symmetry factor, maximum 1.8 for the principal peak.
reference solution (a) (0.5 mg/F); Limit:
— impurity A: not more than the area of the corresponding — impurity C: not more than the area of the corresponding
peak in the chromatogram obtained with reference peak in the chromatogram obtained with reference
solution (b) (0.5 mg/F). solution (a) (2.6 mg/L).
Impurity B Impurity D
Spot test. Maximum 0.02 mg/17.
Test solution To 100 pL of the preparation to be examined Ultraviolet and visible absorption spectrophotometry (2.2.25).
add 400 pL of water R and mix. Test solution The preparation to be examined.
Reference solution (a) water R. Reference solution Dissolve 20.0 mg of 4-(4-methylpiperidin-l-
Reference solution (b) Dissolve 11.0 mg of aminopolyether R yl)pyridine R (impurity D) in water R and dilute to 100.0 mL
(impurity B) in พaier R and dilute to 25.0 mL with the same wi± the same solvent. Dilute 0.1 mL of the solution to T7
solvent. Dilute 1.0 mL of the solution to V with water R) V with พater R, V being the maximum recommended dose in
being the maximum recommended dose in millilitres. millilitres.
Plate TLC silica gel plate for aminopolyether test R. Measure the absorbance of the test solution and the reference
solution at the absorption maximum of 263 nm.
Application 2.5 pL; in addition, apply 2.5 pL of the test
solution and then 2.5 pL of reference solution (b) at the Result The absorbance of the test solution is not greater than
same place. that of the reference solution.
Detection Visually compare the spots 1 min after application. Residual solvents
System suitability: Limited according to the principles defined in general
— the spot due to the successive application of the test chapter 5.4. The preparation may be released for use before
solution and reference solution (b) is similar in completion of the test.
appearance to the spot due to reference solution (b), Sterility
which is characterised by a number of concentric circles; It complies with the test for sterility prescribed in the
the darker innermost circle (of intensity proportional to monograph Radiopharmaceutical preparations (0125).
the concentration of impurity B) may be surrounded by a The preparation may be released for use before completion
bluish-black ring, outside of which is a lighter circle of the test.
surrounded by a peripheral dark edge;
Bacterial endotoxins {2.6.14) Retardation factors [18F]fluoride = about 0; 2-[,8F]fluoro-2-

Less than 175/K IU/mL, I7 being the maximum deoxy-D-glucose and 2-[18F]fluoro-2-deoxy-D-
recommended dose in millilitres. The preparation may be mannose - about 0.45; partially or fully acetylated
released for use before completion of the test. derivatives of 2-[18F]fluoro-2-deoxy-D-glucose and
radionuclidic purity 2-[18F]fluoro-2-deoxy-D-mannose = about 0.8 to 0.95.
The preparation may be released for use before completion System suitability: reference solution:
of test B. — the chromatogram shows 2 clearly separated spots.
Fluorine-18 Limits'.
— [,8F]fluorine in the form of 2-[I8F]fluoro-2-deoxy-D-
glucose and 2-[I8F]fluoro-2-deoxy-D-mannose: minimum
A. Gamma-ray spectrometry. 95 per cent of the total radioactivity due to fluorine-
Limit Peaks in the gamma spectrum corresponding to 18;
photons with an energy different from 0.511 MeV or — [I8F]fluorine in the form offluoride and partially or fiiUy
1.022 MeV represent not more than 0.1 per cent of the total acetylated derivatives of 2-j18F]fluoro-2-deoxy-D-glucose
radioactivity. and 2-[18FJfluoro-2-deoxy-D-mannose: maximum
B. Gamma-ray spectrometry. 5 per cent of the total radioactivity due to fluorine-18.
Determine the amount of fluorine-18 and radionuclidic RADIOACTIVITY
impurities with a half-life longer than 2 h. For the detection Determine the radioactivity using a calibrated instrument.
and quantification of impurities, retain the preparation to be
examined for at least 24 h to allow the fluorine-18 to decay IMPURITIES
to a level that permits the detection of impurities. Specified impurities A, By c, Dy E.
Results The total radioactivity due to radionuclidic impurities
radiochemical purity
A. Liquid chromatography (2.2.29) as described in the test
for 2-fluoro-2-deoxy-D-glucose and impurity A. If necessary,
dilute the test solution with water R to obtain a radioactivity
concentration suitable for the radioactivity detector.
Injection Test solution and reference solutions (a) and (c). A. 2-chloro-2-deoxy-D-glucopyranose (2-chloro-2-deoxy-D-
glucose),
Relative retention With reference to 2-[18F]fluoro-2-deoxy-D-
glucose (retention time = about 12 min): 2-[18F]fluoro-2-
deoxy-D-mannose = about 0.9. Partially or fully acetylated
derivatives of both compounds hydrolyse under the
chromatographic conditions and therefore elute as
2-[I8F]fluoro-2-deoxy-D-glucose and 2-[18F]fluoro-2-deoxy-D-
mannose.
Locate the peaks due to 2-[/5F]fluoro-2-deoxy-D-glucose and
2-[/<SF]fluoro-2-deoxy-D-mannose using the chromatograms
obtained with the carbohydrate detector and reference B. 4,7,13,16,21,24-hexaoxa-l,10-
solutions (a) and (c). diazabicyclo[8.8.8]hexacosane (aminopolyether)3
Limits:
— [t8F]fluorine in the form of 2-[I8F]ftuoro-2-deoxy-D-
glucose and 2-[I8F]fluoro-2-deoxy-D-mannose: minimum
95 per cent of the total radioactivity due to fluorine-
18;
— 2-[18F]fluoro-2-deoxy-D-mannose: maximum 10 per cent c. N,N,N-tributylbutan-l-aminium (tetrabutylammonium),
of the total radioactivity due to 2-[18F]fluoro-2-deoxy-
D-glucose and 2-[18F]fluoro-2-deoxy-D-mannose.
Reference solution Dissolve, with gentle heating, 30 mg of
1,2,3,4-tetra-O-acetyl-p-D-glucopyranose R and 20 mg of
glucose R in 1 mL of water R. D. 4-(4-methylpiperidin-l-yl)pyridine,
Plate TLC silica gel plate R. E. [18F]fluoride.
Mobile phase water Rf acetonitrile R (5:95 VIV). _____________________________________________________________ ___ Ph tor
Application About 5 pL.
Detection Suitable detector to determine the distribution of
radioactivity; immerse the plate in a 75 g/L solution of
sulfuric acid R in methanol R and dry with a heat gun or at
150 °C until the appearance of dark spots in the
Infrared absorption spectrophotometry (2.2.24).

Flumazenil (/U[11C]methyl)
Comparison Ph. Eur. reference spectrum of demethylflumazenil.
Injection
CHARACTERS
Appearance
Clear, colourless solution.
Half-life and nature of radiation of carbon-11
radionuclides.
IDENTIFICATION
Results The only gamma photons have an energy' of
0.511 MeV and, depending on the measurement geometry, a
PhEtf______________________________________________ _________ _____
B. It complies with test B for radionuclidic purity (see Tests),
DEFINITION
Sterile solution of ethyl 8-fluoro-5-[11C]me±yl-6-oxo-5,6-
radiochemical purity'.
dihydro-4H-imidazo[ 1,5-u] [ 1,4] benzodi azepine-3-carboxylate
which may contain a stabiliser such as ascorbic acid. Results The principal peak in the radiochromatogram
obtained with the test solution is similar in retention time to
Content
the principal peak in the chromatogram obtained with
90 per cent to 110 per cent of the declared carbon-11
radioactivity at the date and time stated on the label.
Content of flumazenil TESTS
Maximum 50 pg in the maximum recommended dose in pH (2.23)
millilitres. 6.0 to 8.0.
Sterility
PRODUCTION
It complies with the test for sterility prescribed in the
RADIONUCLIDE PRODUCTION monograph on Radiopharmaceutical preparations (0125).
Carbon-11 is a radioactive isotope of carbon which is most The injection may be released for use before completion of
commonly produced by proton irradiation of nitrogen. the test.
Depending on the addition of either trace amounts of oxygen
or small amounts of hydrogen, the radioactivity is obtained as Bacterial endotoxins (2.6.14)
[HC]carbon dioxide or [11C]methane, respectively. Less than 175/T IU/mL, V being the maximum
recommended dose in millilitres. The injection may be
RADIOCHEMICAL SYNTHESIS released for use before completion of the test.
[5-Me±yl-nC] flumazenil may be prepared by N-alkylation of
Flumazenil and impurity A
ethyl 8-fluoro-6-oxo-5,6-dihydro-4H-imidazo [1,5-
a][l,4]benzodiazepine-3-carboxylate (demethylflumazenil)
with iodo[11C]methane or [HC] methyl Test solution The preparation to be examined.
trifluoromethanesulfonate. Reference solution (a) Dissolve 2.5 mg offlumazenil R in 5 mL
Synthesis of iodo[nC]methane of methanol R.
Iodo[HC]methane may be produced from [HC]carbon Reference solution (b) Dissolve 2.5 mg of demethylflumazenil R
dioxide or from [11C]methane. The most frequently used in 50 mL of methanol R.
method is reduction of [1JC]carbon dioxide with lithium Reference solution (c) To 0.1 mL of reference solution (a) add
aluminium hydride. The [!1C]methanolate formed is reacted 0.1 mL of reference solution (b) and dilute to V with a
with hydriodic acid. Alternatively [’’cjmethane, either 0.9 g/L solution of sodium chloride R, V being the maximum
obtained directly in the target or by on-line processes from recommended dose in millilitres.
["C] carb on dioxide, is reacted with iodine. Reference solution (d) Dilute 0.1 mL of reference solution (a)
Synthesis of [11C]methyl trifluoromethanesulfonate to 50 mL with methanol R. Dilute 1.0 mL of this solution
[nC] methyl trifluoromethanesulfonate may be prepared from to V with a 0.9 g/L solution of sodium chloride R> /being the
iodo[HC]methane using a solid support such as graphitised maximum recommended dose in millilitres.
carbon, impregnated with silver trifluoromethanesulfonate. Column:
Synthesis of [5-methyl-11C]flumazenil — size: z = 0.15 m, 0 = 3.9 mm,
The most widely used method to obtain — stationary phase: spherical octadecylsilyl silica gel for
[5-methyl-,1C]flumazenil is the N-alkylation of chromatography R (5 pm) with a specific surface area of
demethylflumazenil with iodo[HC]methane in alkaline 440 m2/g, a pore size of 100 nm and a carbon loading of
conditions in a solvent such as dimethylformamide or 19 per cent,
acetone. The resulting [5-methyl-nC]flumazenil can be — temperature: maintain at a constant temperature between
purified by semi-preparative liquid chromatography. 20-30 °C.
For example, a column packed with octadecylsilyl silica gel Mobile phase methanol R> water R (45:55 VIV).
for chromatography eluted with a mixture of ethanol and Flow rate 1 mUmin.
water is suitable. Detection Spectrophotometer at 260 nm and radioactivity
PRECURSOR FOR SYNTHESIS detector connected in series.
Demethylflumazenil Injection 100 pL.
Melting point (2.2.14): 286 °C to 289 °C. Run time 10 min.
Relative retention With reference to flumazenil:

impurity A = about 0.74. Fluoride (18F) Solution for ;****
System suitability: reference solution (c): Radiolabelling *****
resolution: minimum 2.5 between the peaks due to (Ph. Eur. monograph 2390)
flumazenil and impurity A.
Ph Elf_______________________________________________________________
Limits Examine the chromatogram obtained with the
spectrophotometer: DEFINITION
flumazenil: not more than the area of the corresponding Alkaline solution containing fluorine-18 in the form of
peak in the chromatogram obtained with reference [18F] fluoride.
solution (c) (50 Jig/P) 5 Content
impurity A: not more than the area of the corresponding 90 per cent to 110 per cent of the declared fluorine-18
peak in the chromatogram obtained with reference radioactivity at the date and time stated on the label.
solution (c) (5 pg/V)> CHARACTERS
any other impurity: not more than the area of the principal Appearance
peak in the chromatogram obtained with reference Clear, colourless solution.
solution (d) (1 ng/ไ^.
Residual solvents See general chapter 5.7. Table of physical characteristics of
Are limited according to the principles defined in the general radionuclides.
chapter (5.4), using the general method (2.4.24).
IDENTIFICATION
of the test. A. Gamma-ray spectrometry.
Result The principal photons have an energy of 0.511 MeV
RADIONUCLIDIC PURITY and, depending on the measurement geometry, a sum peak
Carbon-11 of 1.022 MeV may be observed.
Minimum 99 per cent of the total radioactivity.
B. Determine the approximate half-life by no fewer than 3
The preparation may be released for use before completion measurements of the activity of a sample in the same
of the test. geometrical conditions within a suitable period of time (for
A. Gamma-ray spectrometry. example, 30 min).
Results The spectrum obtained with the solution to be Result 105 min to 115 min.
examined does not differ significantly from that obtained c. Examine the chromatograms obtained in the test for
with a standardised fluorine-18 solution. radiochemical purity (see Tests).
B. Half-life: 19.9 min to 20.9 min. Results The principal peak in the radiochromatogram
RADIOCHEMICAL PURITY obtained with the test solution is similar in retention time to
Liquid chromatography (2.2.29) as described in the test for the principal peak in the chromatogram obtained with the
flumazenil and impurity A, with the following modifications. reference solution; in the chromatogram obtained with the
reference solution, the signal due to fluoride is negative.
Injection Test solution and reference solution (a); if necessary,
dilute the test solution to a radioactivity concentration TESTS
suitable for the detector. pH
Limit Examine the chromatogram obtained with the 8.0 to 14.0, using a pH indicator strip R.
radioactivity detector: Bacterial endotoxins (2.6.14)
— [5-methyl-11C]flumazenil: minimum 95 per cent of the Less than 20 IU/mL, if intended for use in the manufacture
total radioactivity. of parenteral preparations without a further appropriate
procedure for the removal of bacterial endotoxins.
RADIOACTIVITY
of the test.
LABELLING RADIONUCLIDIC PURITY
The label states the maximum recommended dose in The preparation may be released for use before completion
millilitres. of test B.
IMPURITIES Fluorine-18
A. Gamma-ray spectrometry. Preliminary test.
Limit Peaks in the gamma spectrum corresponding to
photons with an energy different from 0.511 MeV or
1.022 MeV represent not more than 0.1 per cent of the total
radioactivity.
o
B. Gamma-ray spectrometry.
Determine the amount of fluorine-18 and radionuclidic
A. R = H: ethyl 8-fluoro-6-oxo-5,6-dihydro-4//-imidazo[l,5-
impurities with a half-life longer than 2 h. For the detection
[l,4]benzodiazepine-3-carboxylate (demethylflumazenil),
and quantification of impurities, retain the preparation to be
B. R = CH2-CO-CH3: ethyl 8-fluoro-6-oxo-9-(2-oxopropyl)- examined for at least 24 h to allow the fluorine-18 to decay
5,6-dihydro-4H-imidazo [ 1,5-a] [1,4] benzodiazepine-3- to a level that permits the detection of impurities.
carboxylate (acetone addition compound of
Result The total radioactivity due to radionuclidic impurities
demethylflumazenil).
Ph Elf
RADIOCHEMICAL PURITY Content

[lsF]fluonde — fluorine-18: 90 per cent to 110 per cent of the declared
Liquid chromatograph}' (2.2.29). fluorine-18 radioactivity at the date and time stated on
Test solution Dilute the preparation to be examined with the label;
water R to obtain a radioactivity concentration suitable for — dopa: maximum 1 mg per maximum recommended dose
the radioactivity detector. in millilitres;
Reference solution Dissolve 10 mg of potassium fluoride R in — 6-fluorolevodopa: maximum 15 mg per maximum
water R and dilute to 10 mL with the same solvent. recommended dose in millilitres.
Column: PRODUCTION
— size: I = 0.25 m, 0 = 4.0 mm; RADIONUCLIDE PRODUCTION
— stationary' phase: strongly basic anion-exchange resin for Fluorine-18 is a radioactive isotope of fluorine that may be
chromatography 7? (10 |im). produced by various nuclear reactions induced by proton
Mobile phase 4 g/L solution of sodium hydroxide R in carbon irradiation of oxygen-18, deuteron irradiation of neon-20, or
dioxide-free water R, protected from atmospheric carbon helium-3 or helium-4 irradiation of oxygen-16.
dioxide. In order to obtain fluorine-18 in a chemical form suitable for
electrophilic substitution reactions, such as fluorine gas or
Flow rate 1 mlVmin.
gaseous acetylhypofluorite, a small amount of non-radioactive
Detection Spectrophotometer at 220 nm and a radioactivity fluorine gas (0.3-0.8 per cent of the target gas volume) must
detector connected in series. be added as a carrier at some step in the production process.
Injection 20 pL. RADIOCHEMICAL SYNTHESIS
Run lime 12 min. 6-[1SF]Fluorolevodopa may be prepared by various
System suitability: reference solution: radiochemical syทthetic pathways, which lead to different
— signal-to-noise ratio: minimum 10 for the principal peak; products in terms of yield, specific radioactivity, by-products
— retention lime offluoride: minimum 3 times the hold-up and possible impurities. Electrophilic pathways for
time. production of 6-[18F]fluorolevodopa may proceed by
Examine the chromatogram obtained with the test solution fluorodemetallation of a stannylated derivative of levodopa,
using the radioactivity detector and locate the peak due to with molecular [18F] fluorine or [1SF] acetylhypofluorite,
fluoride by comparison with the chromatogram obtained with followed by hydrolysis of protecting groups and final
the reference solution using the spectrophotometer. purification by semipreparative liquid chromatography.
Limit: Pathways using demercuration or dethallation must not be
— [! 8F]fluoride: minimum 98.5 per cent of the total used.
radioactivity due to fluorine-18. CHARACTERS
RADIOACTIVITY Appearance
Clear, colourless solution.
Determine the radioactivity' using a calibrated instrument.
LABELLING See general chapter 5.7. Table of physical characteristics of
The label states: radionuclides.
— that the solution is not for direct administration to
humans; IDENTIFICATION
— where applicable, that the substance is suitable for use in A. Test A for radionuclidic purity (see Tests).
the manufacture of parenteral preparations. B. Determine the approximate half-life by at least
3 measurements of the activity' of a sample in the same
--------------------------------------------------------------------------------------------- ----- ----- Ph Eur
geometrical conditions over a suitable period of time, for
example 30 min.
Results 105 min to 115 min.
Fluorodopa (18F) Injection radiochemical purity' (see Tests).
Results The principal peak in the radiochromatogram
(Fluorodopa (18F) (Prepared By Electrophilic obtained with the test solution is similar in retention time to
Substitution) Injection, Ph Eur monograph 1918) the peak due to 6-fluorolevodopa in the chromatogram
obtained with reference solution (a).
D. Examine the chromatograms obtained in the test for
impurities c and D (see Tests).
Results The principal peak in the radiochromatogram
obtained with the test solution is similar in retardation factor
to the peak due to 6-fluorolevodopa in the chromatogram
obtained with reference solution (b).
Ph Elf_____________________________________________________________
TESTS
DEFINITION pH (2.2.2)
Sterile solution of (25)-2-amino-3-(2-([18F]fluoro)-4,5- 4.0 to 5.5.
dihydroxyphenyl)propanoic acid (6-[18F]fluorolevodopa). Sterility
It may contain stabilisers such as ascorbic acid and edetic It complies with the test for sterility prescribed in the
acid. monograph Radiopharmaceutical preparations (0125).
This monograph applies to an injection containing

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British

Incorporating the requirements of the 8th edition of the

Effective date: 1 January 2016

London: The Stationery Office

First Published 2015

ISBN 978 011 3230 006

BRITISH PHARMACOPOEIA COMMISSION

Contents of Volume III

Formulated Preparations: Specific Monographs

Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal Products

Monographs of the European Pharmacopoeia are distinguished by a chaplet

CONTENTS OF THE GENERAL NOTICES

Part I Implementation of Pharmacopoeial Methods

Grade of Materials with other Units

European Monographs of the European Pharmacopoeia are reproduced in this edition

monograph. Any statement containing the word 'should’ constitutes non­

Expression of Where: the standard for the content of a substance described in a

Where the result of an assay or test is required to be calculated with

Temperature The Celsius thermometric scale is used in expressing temperatures.

When the concentration of a solution is expressed in molarity designated

titles. A cumulative list of such Approved Synonyms is provided in

Chemical Formulae When the chemical composition of an official substance is known or

Me -ch3 Bu1 -CH(CH3)CH2CH3

Additional statements concerning the definition of formulated

Manufacture of Attention is drawn to the need to observe adequate hygienic precautions

(1) compliance with all of the requirements stated in the monograph;

Methods of The methods of sterilisation used in preparing the sterile materials

Excipients Where an excipient for which there is a pharmacopoeial monograph is used

Colouring Agents If in a monograph for a formulated preparation defined by means of a full

Characteristics Statements given under the heading Characteristics are not to be

Descriptive term Approximate volume of

The term ‘partly soluble’ is used to describe a mixture of which only

be present and the quantity of the preparation to be taken are calculated

Reference Certain monographs require the use of a reference substance, a reference

Storage Statements under the side-heading Storage constitute non-mandatory

the monographs. Further precautions may be necessary when some

Labelling The labelling requirements of the Pharmacopoeia arc not comprehensive,

In determining the content of the active constituents or the suitable

Homoeopathic Homoeopathic medicines are classed as medicines under European Directive

Unlicensed The General Monograph for Unlicensed Medicines applies to those

A formulated preparation must comply throughout its assigned shelf-life

monographs would be achieved if the official methods were used. In the

General Substances and preparations that are the subject of an individual

General monographs and individual monographs are complementary. If

Implementation of When implementing a pharmacopoeial method, the user must assess

comply with a requirement using an interchangeable method from one of

References to Monographs and general chapters may contain references to documents

Reagents The proper conduct of the analytical procedures described in the

Expression of In defining content, the expression ‘per cent’ is used according to

Temperature Where an analytical procedure describes temperature without a figure, the

13. GENERAL CHAPTERS

Definition Statements under the heading Definition constitute an official definition of

Production Statements under the heading Production draw attention to particular

testing; or to testing that is to be carried out by the manufacturer on the

Potential Due to the increasing number of fraudulent activities and cases of

Descriptive term Approximate volume of solvent in millilitres

Freely soluble from 1 to 10

Sparingly soluble . from 30 to 100

Slightly soluble from 100 to 1000

Very slightly soluble from : 1000 to . 10 000

Practically insoluble more than 10 000

expressed as percentages or as absolute values, are considered significant to

in an outer cover that provides such protection, or is stored in a place from

Labelling In general, labelling of medicines is subject to supranational and national

monograph. Any statement containing the word 'should’ constitutes non